We show that PAR2 is a potent inducer of β-catenin stabilization, a core process in cancer biology, leading to its transcriptional activity. Novel association of low-density lipoprotein-related protein 6 (LRP6), a known coreceptor of Frizzleds (Fz), with PAR2 takes place following PAR2 activation. The association between PAR2 and LRP6 was demonstrated employing co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), and confocal microscopy analysis. The association was further supported by ZDOCK protein-protein server. PAR2-LRP6 interaction promotes rapid phosphorylation of LRP6, which results in the recruitment of Axin. Confocal microscopy of PAR2-driven mammary gland tumors in vivo, as well as in vitro confirms the association between PAR2 and LRP6. Indeed, shRNA silencing of LRP6 potently inhibits PAR2-induced β-catenin stabilization, demonstrating its critical role in the induced path. We have previously shown a novel link between protease-activated receptor-1 (PAR1) and β-catenin stabilization, both in a transgenic (tg) mouse model with overexpression of human PAR1 (hPar1) in the mammary glands, and in cancer epithelial cell lines. Unlike in PAR1-Gα13 axis, both Gα12 and Gα13 are equally involved in PAR2-induced β-catenin stabilization. Disheveled (DVL) is translocated to the cell nucleus through the DVL-PDZ domain. Collectively, our data demonstrate a novel PAR2-LRP6-Axin interaction as a key axis of PAR2-induced β-catenin stabilization in cancer. This newly described axis enhances our understanding of cancer biology, and opens new avenues for future development of anti-cancer therapies.Protease-activated receptor-2 (PAR Despite the growing appreciation of G protein-coupled receptor (GPCR) signal involvement in cancer pathogenesis, very little is known about the role GPCRs play in tumor etiology. GPCRs are seven transmembrane proteins responsible for transducing signals from a diverse range of ligands that affect numerous physiological processes –4. GPCRsFrizzled (Fz) receptors are a subgroup of GPCRs that play a pivotal role in development, tissue homeostasis, and cancer. Activation of the evolutionarily conserved Wnt/β-catenin signaling pathway, also called the canonical Wnt pathway, involves stabilization of β-catenin through binding of Wnt ligands to Fz cell surface receptors and low-density lipoprotein-related protein 5/6 (LRP5/6) coreceptors. In the absence of Wnt, the key effector of this pathway, β-catenin, is continuously degraded by the “degradation complex”. This complex is comprised of Axin, adenomatis polyposis coli (APC), glycogen synthase kinase3β (GSK3β), casein kinase1alpha (CK1α), and the E3 ubiquitin ligase subunit β-TrCP1. Axin provides a scaffolding site for GSK3β to phosphorylate the N-terminus portion of β-catenin (after priming by CK1α), thus generating a phosphorylated form of β-catenin, recognized by the ubiquitin ligase adaptor β-TrCP  −cf.BREThPar2 construct is expressed alone in the same experiment.The correction factor (cf), corresponds to (emission at 515 ± 30nm)/(emission at 410 ± 70nm), which is found when the Rluc-The BRET ratio was measured as mean ± standard deviation (SD) (mean ± SD), derived from three independent experiments using T Test statistical analysis. Data have been expressed as scattered and bar diagrams in excel drawing.5) were plated in 12-well dishes and incubated overnight at 37°C. The cells were transfected with hPar2-wt or hPar2 mutants  plasmids, human Lef-1 TOPflash  reporter plasmid containing two sets (the second set in reverse orientation) of three copies of the Tcf binding site upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame using Fugene 6 transfection reagent (Boehringer-Mannheim). CMV/β-gal plasmid was cotransfected as an internal control for transfection efficiency. After 48 h transfection, the cells were washed in PBS and luciferase assay performed with the Luciferase Reporter System  according to the manufacturer's instructions, and detected on a luminometer Mithras LB940, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany).RKO cells (0.5×10YFP-hPar2 and Lrp6. After overnight serum deprivation (0.2% BSA), the cells were activated by SLIGKV for various periods of time. Samples were then fixed with 4% paraformaldehyde in PBS containing 5% sucrose for 10 min and permeabilized with 0.5% triton X-100 in PBS for 3 min. Samples were fixed and stained with anti-LRP6  and anti GFP . Cy2- and Cy3-conjugated anti-mouse IgG  was used as asecond antibody. Cells were examined using laser confocal microscopy .Either HCT-116 colon cancer or HEK-293 cells were plated on fibronectin (5μg/ml) coated coverslipsand transfected with 2 induced mammary gland tumors were deparaffinized and incubated in 3% H2O2. Antigen retrieval was carried out by heating (20 min) in a microwave oven in 10 mM Tris buffer containing 1 mM EDTA. After blocking the slides were incubated with the following primary antibodies: anti LRP6 , anti-PAR2 (4μg/ml [SAM11] Santa Cruz), Cy2 conjugated anti-rabbit IgG, and Cy3-conjugated anti-mouse IgG antibodies  were used as secondary antibodies. Nuclear staining was performed using DRAQ5 . Images were obtained using a Zeiss LSM 5 confocal microscope and analyzed with Zen software (Zeiss).Paraffin embedded slides derived from PAR


Aberrant activation of the canonical Wingless type (Wnt) signaling pathway plays a key role in the development of hypertrophic scars and keloids, and this aberrant activation of Wnt pathway can be a potential target for the development of novel anti-fibrotic agents. In this study, we evaluated the anti-fibrotic potential of a soluble Wnt decoy receptor (sLRP6E1E2)-expressing non-replicating adenovirus  on human dermal fibroblasts (HDFs), keloid fibroblasts (KFs), and keloid tissue explants. Higher Wnt3a and β-catenin expression was observed in the keloid region compared to the adjacent normal tissues. The activity of β-catenin and mRNA expression of type-I and -III collagen were significantly decreased following treatment with dE1-k35/sLRP6E1E2 in HDFs and KFs. The expression of LRP6, β-catenin, phosphorylated glycogen synthase kinase 3 beta, Smad 2/3 complex, and TGF-β1 were decreased in Wnt3a- or TGF-β1-activated HDFs, following administration of dE1-k35/sLRP6E1E2. Moreover, dE1-k35/sLRP6E1E2 markedly inhibited nuclear translocation of both β-catenin and Smad 2/3 complex. The expression levels of type-I and -III collagen, fibronectin, and elastin were also significantly reduced in keloid tissue explants after treatment with dE1-k35/sLRP6E1E2. These results indicate that Wnt decoy receptor-expressing Ad can degrade extracellular matrix in HDFs, KFs, and primary keloid tissue explants, and thus it may be beneficial for treatment of keloids. Various mechanisms have been proposed to explain keloid pathogenesis4. However, treatment of keloids is extremely difficult as keloids frequently form at the site of injury, recur after excision, and always overgrow beyond the boundaries of original wound. Many conventional treatments for keloids have been unsuccessful6 and thus we explored a novel therapeutic adenovirus (Ad) which can inhibit activities of key pathophysiological pathways of keloid formation.Keloids are skin abnormalities that are unique to humans and characterized by excessive deposition of collagen in the dermis and subcutaneous tissues due to traumatic or surgical injuries7. There is considerable evidence that Wnt signaling and its effector β-catenin functions during fibro-proliferative processes characterized by excessive proliferation of mesenchyme cells, which can form hypertrophic scars or neoplasms11. Wnt proteins are secreted ligands that transmit their signal across the plasma membrane by interacting with Frizzled receptors and low-density lipoprotein receptor-related protein co-receptors (LRP5/6)13. Receptor-bound Wnt proteins recruit disheveled, axin, adenomatosis polyposis coli (APC), and glycogen synthase kinase (GSK)-3β to the plasma membrane, resulting in stabilization of β-catenin. β-catenin accumulates and translocate to the nucleus, where it interacts with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) to induce the transcription of Wnt target genes13.The canonical Wingless protein (Wnt) signaling profoundly affects developmental processes during embryogenesis, but has an important role for tissue homeostasis in adults14. Signaling cross-talk between the Wnt/β-catenin, TGF-β1, and Smads signaling is well-documented, where Wnt/β-catenin signaling activates TGF-β115 and TGF-β1 promotes Wnt/β-catenin signaling15. TGF-β1 also stimulates canonical Wnt signaling by decreasing the expression of the Wnt antagonist Dickkopf protein-17. Therefore, inhibition of the canonical Wnt pathway can be an effective approach to target TGF-β1 signaling which both can synergistically downregulate fibrogenesis.Recently, it has been demonstrated that canonical Wnt signaling is necessary for transforming growth factor (TGF)-β1-mediated fibrosis16. LRP6 consists of four distinct YWTD β-propeller/EGF-like domain pairs; the first and second YWTD domains (E1 and E2) are required for binding to Wnt19. Previously, we have demonstrated that soluble Wnt decoy receptor of LRP6 (sLRP6E1E2)-expressing Ad (dE1-k35/sLRP6E1E2), which expresses LRP6’ E1 and E2 domain, prevents Wnt-mediated stabilization of cytoplasmic β-catenin and decreases Wnt/β-catenin signaling20.LRP6, a member of the LRP superfamily, is required to respond to physiological activation of the canonical Wnt signaling pathway, which leads to the stabilization and nuclear translocation of β-catenin that is essential for fibrogenesisIn this study, we examined the therapeutic potential of dE1-k35/sLRP6E1E2 for treatment of pathologic fibrosis such as keloid. We evaluated the effects of the dE1-k35/sLRP6E1E2 on the expression levels of type-I and -III collagen on human dermal fibroblasts (HDFs), keloid fibroblasts (KFs), and its effect on nuclear translocation of β-catenin and Smad 2/3 complex in HDFs. Additionally, the expression levels of type-I and -III collagen, fibronectin, and elastin in keloid tissue explants transduced with the sLRP6E1E2-expressing Ad were investigated by immunohistochemistry.22.As shown in Supplementary Figure To evaluate the ability of sLRP6E1E2 secreted from dE1-k35/sLRP6E1E2 to inhibit Wnt3a/β-catenin signaling, we used a luciferase reporter system activated by β-catenin or TCF. When canonical Wnt signaling is activated, β-catenin will translocate to the nucleus in order to associate with β-catenin/TCF transcription factors which activates transcription of Wnt target genes. Luciferase expression in KF was 1.8-fold higher than those observed in HDFs. dE1-k35/sLRP6E1E2 reduced luciferase expression by 51% compared to the treatment with dE1-k35/LacZ in KFs Fig. . After tTo evaluate the potential effect of sLRP6E1E2 on the downregulation of extracellular matrix (ECM) production by inhibition of Wnt3a/β-catenin signaling in HDFs and KFs, the change of type-I and -III collagen mRNA expression levels of HDFs and KFs after treatment with dE1-k35/sLRP6E1E2 or dEl-k35/LacZ were examined. After addition of TGF-β1 (20 ng/mL), type-I and –III collagen mRNA levels were significantly increased in HDFs. However, increased type-I (a) and type-III (b) collagen mRNA levels were significantly attenuated by dE1-k35/sLRP6E1E2 treatment, especially at 50 and 100 multiplicity of infection (MOI)s, when compared to dEl-k35/LacZ-transduced HDFs Fig. . SimilarWe examined the intracellular signaling pathway involved in the anti-fibrotic effect of sLRP6E1E2 on canonical Wnt signaling by analyzing β-catenin, pGSK-3β, and Smad 2/3 complex expression. As shown in Fig. 23. It has been previously reported that activation of Wnt pathway elicits stabilization and nuclear translocation of β-catenin protein24. Nuclear β-catenin accumulation in fibroblastic foci of the patients with systemic sclerosis associated advanced fibrosis has also been reported25. Therefore, we examined the extent of nuclear localization of β-catenin and Smad 2/3 by immunofluorescence staining of HDFs after transduction with dE1-k35/LacZ or dE1-k35/sLRP6E1E2  transient therapeutic effect of Ad is not particularly well-suited for the treatment of chronically persistent keloids and (2) immunogenic nature of Ad vector could cause adverse inflammatory response. Further refinements to vector and treatment strategies will be required to achieve more efficient and safe gene transfer in clinical trials.Previous reports report the nuclear accumulation of β-catenin in fibroblast foci of most idiopathic pulmonary fibrosis samples and experimental lung fibrosisKeloid tissues were obtained from the central dermal layer of active-stage keloid patients n = 11) (Supplementary Table 1 (Supple20. The replication-incompetent Ad (dE1-k35/LacZ) was used as a negative control11. All viruses were propagated in human embryonic kidney 293 cells , purified by CsCl density purification, and then stored at −80 °C. Viral particle (VP) numbers were calculated from measurements of optical density at 260 nm (OD260) where 1 absorbency unit is equivalent to 1012 VP per milliliter. The MOI was calculated from VP.The preparation of a replication-incompetent Ad with chimeric 5/35 knob expressing sLRP6E1E2 (dE1-k35/sLRP6E1E2) was described in a previous study48  and fiber-modified Ads (dE1-k35/LacZ and dE1-RGD/LacZ), we transduced HDFs with dE1/LacZ, dE1-k35/LacZ, or dE1-RGD/LacZ at either 5 or 10 MOI. β-galactosidase activity was visualized at 48 h post-transduction by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside staining , as previously described49. HDFs and KFs were seeded (3 × 105 cells) into 6-well plates and transfected with 0.3 µg of TOPFLASH (containing wild-type TCF binding sites) in conjunction with dE1-k35/LacZ or dE1-k35/sLRP6E1E2 (50 MOI) in serum-free DMEM. After 12 h, the medium was replaced with 1% DMEM with or without Wnt3a (100 ng/mL) or TGF-β1 (20 ng/mL), and then cells were incubated for another 24 hr. Cells were lysed with passive lysis buffer , and 20 mL of the cell extract was analyzed by Dual-Luciferase Reporter Assay System (Promega). Experiments were carried out in triplicates and repeated at least three times.TOPFLASH luciferase reporter vectors  were used to measure β-catenin/TCF signaling activity. A reporter plasmid (pTOPFLASH) contains three copies of optimal TCF-binding motif CCTTTGATC upstream of a minimal c-Fos promoter driving luciferase reporter gene5 cells) were transduced with dE1-k35/sLRP6E1E2 or control Ad (dE1-k35/LacZ) in the presence of TGF-β1 (20 ng/mL) at 20, 50, and 100 MOI. At 3 days post-transduction, total RNA was prepared with TRIzol reagent (GIBCO), and complementary DNA was prepared from 0.5 μg of total RNA by random priming using a first-strand cDNA synthesis kit (Promega) as previously described50. Experiments were carried out in triplicates and repeated at least three times.HDFs and KFs  at 50 MOI. At 3 days after transduction, cells were lysed in a solution containing 50 mM Tris-HCl (pH 7.6), 1% Nonidet P-40 (NP-40), 150 mM NaCl, 0.1 mM zinc acetate, and protease inhibitors. Protein concentration was determined by the Lowry method , and 30 μg of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins from the gels were electrotransferred to polyvinylidene fluoride membrane and initially incubated with primary mouse anti-LRP6 , rabbit anti-β-catenin , rabbit anti-pGSK-3β , rabbit anti-Smad 2/3 , or rabbit anti-β-actin antibody , and then secondarily incubated with the horseradish peroxidase-conjugated secondary antibody . The expression patterns were revealed using enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). And the expression levels of LRP6, β-catenin, pGSK-3β, and Smad 2/3 were semi-quantitatively analyzed using ImageJ software .5 cells) in 6-cm culture dishes were transduced with dEl-k35/LacZ or dEl-k35/sLRP6E1E2 at 50 MOI. At 3 days post-infection, the supernatant was collected by centrifugation at 15,000 g for 10 min at 4 °C, and the level of TGF-β1 expression was assessed using an ELISA kit  according to the manufacturer’s recommendations.HDFs and KFs , fixed in 4% paraformaldehyde for 10 min at room temperature, and then permeabilized by incubation for 15 min with 0.1% Triton X-100 in PBS. The samples were blocked with 1% bovine serum albumin followed by incubation with rabbit anti-Smad 2/3 or rabbit anti-β-catenin  primary antibody overnight at 4 °C. One day after incubation, cells were washed with PBS and incubated with Alexa Flour 488-conjugated goat anti-rabbit IgG  or Alexa Flour 633-conjugated goat anti-rabbit IgG (invitrogen) secondary antibody, respectively, for 60 min at room temperature. The final antibody treatment also contained tetramethylrhodamine isothiocyanate-conjugated phalloidin (Sigma) for actin and DAPI for nucleus staining . The slides were mounted with Vectashield mounting medium  and cells were viewed under a confocal laser-scanning microscope .® 24 multi-well plates  after which they were cultured for 4 hr in Isocove’s modified Dulbecco’s medium (GIBCO) supplemented with 5% FBS, 10 mM insulin (Sigma) and 1 mM hydrocortisone (Sigma). For histological analysis, dEl-k35/LacZ or dEl-k35/sLRP6E1E2 at 1 × 1010 VP or 10 mM of commercially available Wnt inhibitor, JW 55 , were added into the plates containing keloid tissue explants, and incubated at 37 °C in 5% CO2 incubator for 3 or 5 days.Keloid tissues were obtained from active-stage keloid patients (n = 4). Keloid tissue explants were prepared by dissecting keloid central dermal tissue into 2-mm diameter pieces with sterile 21-gauge needles. The explants were plated onto HydroCell® image analysis software . Results are expressed as the mean optical density of six different digital images.The transduced keloid tissue explants were then fixed with 10% formalin, paraffin-embedded, and cut into 5-μm-thick sections. Representative sections were stained with Masson’s trichrome or Picrosirius red, and then examined by light microscopy. For immunohistochemical staining, keloid tissue explants sections were incubated at 4 °C overnight with mouse anti-Wnt3a (Abcam), rabbit anti-TGF-β1 (Abcam), mouse anti-collagen type-I (Abcam), mouse anti-collagen type-III (Sigma), mouse anti-elastin (Sigma), mouse anti-fibronectin (Santa Cruz Biotechnology), rabbit anti-MMP-9 (Abcam) primary antibody, and then incubated at room temperature for 20 min with the Dako Envision™ Kit  as a secondary antibody. Diaminobenzidine/hydrogen peroxidase (Dako) was used as the chromogen substrate. All slides were counterstained with Meyer’s hematoxylin. The expression levels of Wnt3a, TGF-β1, collagen type-I, collagen type-III, elastin, and fibronectin were semi-quantitatively analyzed using MetaMorphP < 0.05 was considered statistically significant.Results were expressed as the mean ± standard deviation (SD). Data were analyzed by one-way ANOVA for assays comparing more 3 experimental groups or more and a paired t-test for assays comparing two experimental groups. Supplementary information


In nature, individual cells contain multiple isolated compartments in which cascade enzymatic reactions occur to form essential biological products with high efficiency. Here, we report a cell-inspired design of functional hydrogel particles with multiple compartments, in which different enzymes are spatially immobilized in distinct domains that enable engineered, one-pot, tandem reactions. The dense packing of different compartments in the hydrogel particle enables effective transportation of reactants to ensure that the products are generated with high efficiency. To demonstrate the advantages of micro-environmental modifications, we employ the copolymerization of acrylic acid, which leads to the formation of heterogeneous multi-compartmental hydrogel particles with different pH microenvironments. Upon the positional assembly of glucose oxidase and magnetic nanoparticles, these hydrogel particles are able to process a glucose-triggered, incompatible, multistep tandem reaction in one pot. Furthermore, based on the high cytotoxicity of hydroxyl radicals, a glucose-powered therapeutic strategy to kill cancer cells was approached. Cells contain isolated compartments where cascade enzymatic biochemical reactions occur to form essential biological products with high efficiency. Here the authors produce functional hydrogel particles with multiple compartments via microfluidics that contain spatially immobilized natural enzymes in distinct domains for one-pot, tandem reactions. Moreover, the presence of organelle compartments enables the spatial confinement of incompatible and opposing reagents in different cellular domains, shielding them from each other and allowing their corresponding reactions to occur in optimized microenvironments3. Accordingly, numerous incompatible (and competing) catalytic transformations can be performed simultaneously in a cell with unsurpassed efficiency and specificity, producing system-level essential bioproducts.Living cells contain multiple compartmentalized organelles surrounded by membranes that perform distinct functions to maintain cell physiology. The close confinement and dense packing of different organelles can facilitate cascade enzymatic bioreactions with efficient bioproduct transportation between compartments, minimizing the decomposition of active intermediates7. Soft components, such as liposomes, polymersomes, and polymer capsules, have been investigated for the fabrication of various types of multi-compartmentalized systems, including vesosomes8, polymersome-in-polymersome structures9, and capsosomes10. However, the contradiction between close confinements with dense packing and micro-environmental controls of different compartments remained. For example, the liposomes and polymersomes were developed to closely immobilize enzymes for dense packing confinement, triggering cascade process effectively, but these systems reached the limitations in processing individual reactions in spatial compartmental microenvironments13. The Pickering emulsions and functional polymeric micelles were manipulated to form distinguished spatial compartments to optimize the incompatible tandem reactions, but without well-defined close packing, the product generation efficiency was limited by slow transportations of intermediates between the compartments in Pickering emulsions and polymeric micelles15. Moreover, these systems required organic chemical processes to prepare, causing the challenges in bio-compatibility.Recently, the construction of multi-compartmental systems to perform distinct biochemical reactions in one pot, as in living cellular systems, has attracted substantial attention17. Compared with free enzymes, encapsulated enzymes often show greatly improved catalytic activities, enhanced thermal stabilities and tolerance for both high-pH conditions and organic solvents19. Moreover, hydrogels exhibit reliable mechanical stability and biocompatibility, making them widely applicable in the biomedical industry. In this regard, hydrogels based systems appear promising for the construction of multi-compartmental systems. Several methods have been developed to fabricate structured hydrogels with multiple compartments22 to load different drugs in separate compartments with a programmed, controllable sequential release. However, most such studies focused on controlling drug release from hydrogels, requiring a drug-reloading process for long-term drug delivery. These hydrogels were not used for enzyme-triggered product fabrication, as occurs in cells. Indeed, for the incorporation of multistep enzymatic reactions and the system-level production, it is essential to construct well-defined multi-compartmental cell-like hydrogel systems with immobilized enzymes.Hydrogels are three-dimensionally cross-linked polymeric networks containing  ~ 95% water, similar to natural cells. On the basis of the high porosity of hydrogels, functional enzymes can be immobilized in the network to produce enzymatic products over long periodsIn this study, a heterogeneous multi-compartmental hydrogel particle with distinct microenvironments was designed and fabricated using microfluidics within which incompatible multistep tandem reactions were conducted. The combination of close confinement of hydrogel building blocks and distinguished compartments through microfluidic assembly acted synergistically to perform tandem reactions. The dense package of hydrogels in micro-scaled allowed effective transportation of reactants, while the distinguished compartments offered desirable micro-environment controls to ensure product generation with high efficiency. For example, the copolymerization of acrylic acid (AA) provided an acidic microenvironment for the corresponding hydrogel particles because of the presence of a carboxyl acid group. The incompatible natural enzyme and nanozyme were loaded independently in defined compartments during the formation of these hydrogel particles through microfluidics, allowing the spatial organization and segregation of the incompatible multistep tandem reaction. Accordingly, the incompatible multistep tandem reaction occurred simultaneously within the multi-compartmentalized hydrogel particles without mutual interference. The products generated by the enzymatic reactions in the first compartment were effectively transported to the second compartment, which contained a distinct environment, triggering catalysis reactions to accomplish a system-level tandem reaction with high efficiency in one pot.A two-step sequential gelation process along with droplet assembly in a microfluidic device was conducted to fabricate multi-compartmental hydrogel particles, as shown in Supplementary Fig. 13. The single-compartmental PEG hydrogel particles were prepared as the carriers for enzyme encapsulation. Two different single-compartmental particles containing GOX (GOX@PEG) and HRP (HRP@PEG) were fabricated  and horseradish peroxidase (HRP) were selected as model enzymes for performing tandem reaction, which has already been applied as an extremely popular system in various compartmentalized structures3+-hydroxamine colorimetric assay for GOX  and turnover number (Kcat) to those in free GOX and HRP enzymes. This finding indicates that the intermediates (H2O2) from the GOX reaction freely diffused across the multi-compartmentalized hydrogel particle without requiring artificial transport mediators13, endowing GOX@HRP with the ability to process a tandem reaction in one pot with high efficiency.By separately measuring the total protein  and Km values of GOX@HRP were calculated . Table Km value of GOX@HRP was similar to the Km values of the free GOX/HRP system, the GOX@PEG/HRP@PEG system and GOX&HRP@PEG, indicating their comparable affinity to that of glucose. However, a significantly enhanced Vmax (ranging from 3.7- to 8.5-fold compared with the other three tandem reaction systems) and the associated increase of Kcat were observed in GOX@HRP, demonstrating the highest catalytic performance of GOX@HRP. Since the intermediate (H2O2) could freely move across the hydrogel particle due to its small size ( ~ 0.4 nm in diameter) and charge neutrality, the highest activity of GOX@HRP compared with the other three tandem systems was attributed to its unique multi-compartmental structure in which GOX and HRP were separately confined and closely packaged, facilitating H2O2 transfer among enzymes  and minimizing H2O2 inhibition to HRP activity.A detailed kinetic analysis of GOX@HRP was performed using glucose as the substrate in Supplementary Fig. 2O2 from GOX to solution and then to HRP. As expected, the GOX@PEG/HRP@PEG system without the proximity effect showed a lower catalytic activity than the activity in GOX@HRP , a  ~ 4-fold lower activity in GOX&HRP@PEG was observed Fig. . Previou2O2 on the presented GOX-HRP tandem reactions, a series of glucose conversion experiments catalyzed by changing molar ratios of GOX to HRP from 1:1 to 10:1 were conducted in GOX@HRP systems, as local H2O2 concentrations were determined by GOX concentrations. As the GOX amounts in GOX@HRP increased, the oxidation reactions of Amplex Red were gradually suppressed  in GOX&HRP@PEG was stronger than that in GOX@HRP. Accordingly, a certain distance between GOX and HRP is necessary to ensure that HRP, with high activity, triggers the tandem reaction by minimizing the H2O2 inhibition effect. In addition, the dilution of the local H2O2 concentration at the HRP site due to the free and non-targeted diffusion of H2O2 in the hydrogel particle could also contribute to the enhancement of the GOX@HRP activity.To discuss the inhibition of Htem Fig. . However2O2 produced from the GOX reaction in the inner compartment was effectively transferred to the outer compartment to trigger the HRP reaction, while the HRP activity was not suppressed due to the direct interaction with a high concentration of H2O2, as occurs in living cells.In summary, the combination of the advantages in positional assembly and spatial segregation of GOX and HRP in a micro-scale multi-compartmental system acted synergistically to perform tandem reactions. The H32. To test the biological stability of GOX@HRP against protease, trypsin was employed as a model digesting enzyme. As shown in Supplementary Fig. The enzymes in GOX@HRP displayed excellent stability and recyclability. The ability of the enzyme to be stored for a long period at room temperature (25 °C) was observed in Supplementary Fig. 15. First, close compartmentalization and positional assembly in a micro-sized cell allowed the effective transport of reagents to trigger tandem reactions. Second, different microenvironments (known as organelles) could be generated to spatially confine incompatible opposing reagents in distinguished domains. For example, the pH of the cytosol and the nucleus is  ~ 7.2, whereas the lysosome has a pH of 4.733.As with living cells, there are two main characteristics required to process multistep tandem chemical reactions in one pot2O2 in an acidic environment (pH  ~ 4). Indeed, to demonstrate the advantage of generating compartments with different pH levels, as commonly witnessed in the cell, it is desirable to manipulate microenvironments in the hydrogel particle to construct a heterogeneous multi-compartmental system with distinct microenvironments for selectively confining incompatible catalysts, allowing an incompatible tandem reaction to be performed in one pot.Previously, the GOX@HRP system was studied to demonstrate the advantage of close compartmentalization and positional assembly in a multi-compartmental system. However, the challenges of performing incompatible reactions remained due to the homogeneous compartments in a system. For example, using GOX and nanozyme as peroxidase mimics for coupled tandem reactions required distinguished compartments with different pH values. The GOX reaction was processed in a neutral medium (pH  ~ 7), while nanozymes should react with H36. To characterize the acidic microenvironments generated, a series of poly(PEG-co-AA) hydrogel particles with AA contents ranging from 0 to 45% were prepared. The LysoSensor conjugated with dextran was encapsulated in a hydrogel particle as a pH indicator to test the pH changes of the interior microenvironment. The existence of conjugated dextran effectively prevented the leakage of the LysoSensor from the hydrogel particle. The LysoSensor is a ratiometric pH probe that produced blue fluorescence in a neutral environment but changes to yellow fluorescence in an acidic environment. As shown in Fig. To prepare the hydrogel particles with an acidic microenvironment, AA was co-polymerized with PEG3O4 MNPs with excellent dispersion and stability  hydrogel particle (denoted as MNP@PEG-co-AA) in neutral medium  was investigated to evaluate the feasibility of performing an incompatible catalytic reaction within a poly(PEG-co-AA) hydrogel particle. Carbon-coated Fe2O2, MNP@PEG-co-AA effectively catalyzed the oxidization of TMB in neutral medium, generating oxidized TMB (oxTMB) to show a color change (transparent to blue). In contrast, under the same conditions , no clear reaction was observed in MNP@PEG because of the absence of microenvironment controls of pH  hydrogel as the outer compartment through microfluidics. In addition, to achieve the high-catalytic activity, GOX and MNP were separately confined in the inner and outer compartments of these hydrogel particles, forming an incompatible tandem catalytic system (GOX@MNP)  through the decomposition of H2O2, a byproduct of glucose oxidization -2,5-diphenyltetrazolium bromide (MTT) assay against HeLa cells. To avoid the influence of glucose, the HeLa cells treated with GOX@MNP were incubated in a glucose-free culture medium  demonstrated that the glucose-triggered oxidation of TMB in the presence of GOX@MNP results from the generation of hydroxyl radicals  chips connected by a polytetrafluoroethylene tube, as shown in Supplementary Fig. −1 GOX, whereas pre-gel solution II contained 45% (v/v) PEGDA (Mn = 700), 30% (v/v) AA, 5% (wt%) photoinitiator and 1.2 mg ml−1 MNPs. By exchanging the positions of pre-gel solutions I and II, MNP@GOX can be obtained. The total protein contents in these multi-compartmental hydrogel particles were determined using a colorimetric BCA protein assay. The encapsulated GOX amounts were used to calculate the concentrations of GOX@HRP and GOX@MNP in aqueous solutions.For the homogenous multi-compartmental hydrogel particles, pre-gel solutions I and II contained 20% (v/v) of PEGDA (Mn = 575), 2% (wt%) of the photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and one of the following components: water for pure PEG@PEG, fluorescent materials for Albumin-FITC@Dextran-RhB, and enzymes for GOX@HRP. The heterogeneous multi-compartmental hydrogel particles (GOX@MNP) were obtained using two different pre-gel solutions: pre-gel solution I contained 20% (v/v) PEGDA (Mn = 575), 2% (wt%) photoinitiator and 1.5 mg mlaqueous = 2 μl min−1 and Voil = 20 μl min−1. A UV filter (OmniCure S1500) was used to provide the desired excitation for polymerization.Single-compartmental hydrogel particles were fabricated using one T-type chip .The Amplex Red and TMB colorimetric reactions were used to characterize the overall activities of GOX@HRP and GOX@MNP, respectively. Glucose was used to initiate these two tandem reactions in PBS . The total reaction volumes of these two tandem reactions were 200 μl. After the reactions were complete, the absorption spectra were recorded by a microplate reader. For the detailed procedure for the Amplex Red and TMB colorimetric reactions, please see ν = Km/Vm (1/[S] + 1/Km), where ν is the initial velocity, Vm represents the maximal reaction velocity, [S] corresponds to the substrate concentration, and Km is the Michaelis constant. Each sample was analyzed three times. All of the error bars represent the standard deviations from three repeated experiments.The kinetic behavior of GOX@HRP was studied by monitoring the absorbance in 3-min intervals while varying the glucose concentration. The Michaelis–Menten constant was calculated using Lineweaver-Burk plots of the double reciprocal of the Michaelis–Menten equation—1/2 atmosphere, different amounts of GOX@MNP were added to each well. The plates were incubated for another 24 h, and the cell medium was removed. Subsequently, 10 μl of MTT (12 mM) was added to each well and allowed to react for 4 h at 37 °C. The supernatant in each well was aspirated, and 150 μl of dimethyl sulfoxide was added to each well to dissolve the MTT formazan crystals. Subsequently, the absorbance was determined at 540 nm using a microplate reader . Cell viability was calculated as the percentage of viable cells after treatment with GOX@MNP compared with the percentage of viable cells among untreated cells. To investigate the glucose-powered killing of cancer cells, the same experimental steps and conditions described above were used, except that different concentrations of glucose were added to the wells containing GOX@MNP.The standard MTT assay was used to study cell viability. HeLa cells were employed as the model cells and were incubated in Dulbecco’s modified eagle medium (DMEM) without glucose. Typically, 100 μl of a HeLa cell solution was pipetted into each well of a 96-well plate to provide a cell density of 5000 cells per well. After incubating the cells for 24 h at 37 °C in a 5% CO2 for 24 h. Next, freshly prepared FDA and PI solutions were added and incubated for 5 min at room temperature in the dark. Subsequently, the staining solution was removed, and the cells were washed with PBS. Finally, the as-prepared samples were analyzed using fluorescence microscopy.For cell staining, fluorescein diacetate (FDA) and propidium iodide (PI) were used to stain viable cells and dead cells, respectively. The FDA stock solution was prepared by dissolving 5 mg of FDA in 1 ml of acetone (the stock solution was stored at −20 °C), and the PI stock solution was prepared by dissolving 2 mg of PI in 1 ml of PBS (the stock solution was stored at 4 °C). Briefly, HeLa cells in different wells were first incubated at 37 °C in DMEM without glucose in an atmosphere of 5% COData supporting the findings of this study are available from the corresponding author on reasonable request.Supplementary InformationPeer Review File


This indicated that the particle sparse zone was mainly deformed by dislocation slip while the nanoparticle dense zone may have been deformed by twinning. The yield strength and ultimate tensile strength of the composites were gradually enhanced through increasing the single-pass forging temperature from 300 °C to 400 °C, which demonstrated that initial high forging temperature contributed to the improvement of the mechanical properties. During multi-pass forging with varying temperatures, the grain size of the composite was gradually decreased while the grain size distribution tended to be uniform with reducing the deformation temperature and extending the forging passes. In addition, the amount of precipitated second phases was significantly increased compared with that after multi-pass forging under a constant temperature. The improvement in the yield strength of the developed composite was related to grain refinement strengthening and Orowan strengthening resulting from synergistical effect of the externally applied SiC nanoparticles and internally precipitated second phases.In this study, SiC nanoparticles were added into matrix alloy through a combination of semisolid stirring and ultrasonic vibration while dynamic precipitation of second phases was obtained through multi-pass forging with varying temperatures. During single-pass forging of the present composite, as the deformation temperature increased, the extent of recrystallization increased, and grains were refined due to the inhibition effect of the increasing amount of dispersed SiC nanoparticles. A small amount of twins within the SiC nanoparticle dense zone could be found while the precipitated phases of Mg Light-weight magnesium alloy with high strength is potential choice for the applications in the fuel-intensive automotive and aerospace sectors, as well as for electronic components and biomedical implants, due to its low mass density, high stiffness and environmental friendliness ,2,3. TheWith regard to extensive conducted studies related to the fabrication of nanoparticles reinforced magnesium matrix composite, only a few researches focus on the secondary thermo mechanical processing of as-cast composites ,17,18. LTherefore, the synergistical influence of nanoparticle addition and second phases on tensile strength at room temperature and the microstructure of matrix alloy during multi-pass forging with varying temperatures is investigated in the paper. The nanoparticle reinforced composite were initially prepared with semisolid stirring and ultrasonic infiltration followed by solution treatment and multi-pass forging with varying temperatures. The relationship between microstructures and deformation behaviors of the composites is analyzed, which may open a new approach for further studies on magnesium matrix composite.2/SF6 and mechanical stirring was adopted to add SiC nanoparticles. Secondly, the melt was heated up to 700 °C and processed by applying the ultrasonic vibration. Finally, liquid composite was poured into a preheated mold with a temperature of 450 °C and solidified under pressure. It must be pointed out that there was no ultrasound applied during solidification. In order to minimize the influence of the Mg17Al12 phase, as-cast ingots were solution treated at 415 °C for 24 h before multi-pass forging.The matrix of the composite is AZ91 alloy with nominal composition of Mg-9.07Al-0.68Zn-0.21Mn, which is provided by Northeast Light Alloy Company Limited, Harbin, China. The reinforcement is nano-sized SiC particles with an average dimension of 60 nm, which is supplied by Hefei Kaier Nanometer Energy & Technology Company Limited, Hefei, China. SiC particles with volume fraction of 1% were added into matrix alloy using semisolid stirring and ultrasonic infiltration. A detailed description of the used fabrication process is given by Nie et al. . First, −1. The original shape of the billets was maintained constant on the whole at the end of the multi-pass forging although the direction of the load was rotated 90° between passes. To create a uniform temperature distribution, all of the specimens were heated to the desired deformation temperatures performed in a resistance furnace. The strain for each pass forging was 0.693 [The as-cast composite ingot was cut into rectangular billet specimens with a size of 30 × 30 × 60 mm through the electrical discharge machining method. The multi-pass forging was performed on a press with a load limit of 2000 kN. The deformation temperatures changed from 250 °C to 400 °C while punch velocity was 15 mm sas 0.693 . A graph2O + 100 mL ethanol (95%)). Image-Pro Plus image analysis software  was used to analyze the grain size, and the phase identification of the composites was carried out on an energy dispersive spectroscope (EDS). The thickness of the TEM specimens was reduced to less than 50 μm by manual grinding. Then a disk with diameter of 3 mm was sliced and ion beam thinned. By means of optical microscopy , scanning electron microscopy , and transmission electron microscopy , microstructures of the composites were examined. Samples for metallographic observation and SEM were prepared by the polishing machine and then etched in acetic picral (5 mL acetic acid + 6g picric acid + 10 mL H−4 (s−1). In the present work, the values of tensile properties were calculated according to three repeated tensile tests.In an uniaxial tension experiment, the mechanical properties of the composite reinforced with SiC nanoparticles were determined using an Instron-1186 tension machine. Flat dog-bone samples with gage size of 15 × 6 × 2 mm perpendicular to the last compression axis were machined from billet specimen by numerically controlled wire cutting machine. The initial strain rate for tensile test was 8.33 × 10The optical microstructure of the nano-SiCp/AZ91 composite subjected to single-pass forging at different temperatures is given in 17Al12 by electron diffraction patterns The distribution of SiC nanoparticles is improved while the amount of dispersed nanoparticles is increased with the increase of single-pass forging temperature for the present composite, resulting in an increased DRX degree and refined grains.(2)SiC nanoparticle dense zones with twins may deform by twinning in the matrix while nanoparticle sparse zones with deformation bands and high density dislocations deform mainly by dislocation slip.(3)Yield strength and ultimate tensile strength are gradually enhanced with the increase of the single-pass forging temperature from 300 °C to 400 °C. This indicates that an initial high forging temperature is beneficial to improve the tensile properties.(4)During multi-pass forging with varying temperatures, the value of grain size is gradually reduced and the distribution is more even with increasing the forging passes and lowering the deformation temperature. The amount of the precipitated second phases is significantly increased compared with that after multi-pass forging at a constant temperature.(5)The improvement in yield strength could be attributed to grain refinement strengthening and Orowan strengthening, which are related to synergistical enhancement by precipitated second phases and SiC nanoparticles.The microstructures and tensile properties of nano-SiCp/AZ91 composites containing SiC nanoparticle and second phases Mg


In this study, adoptive (T/B-cell based) immunity-deficient RAG1−/− Sgo1−/+ double mutant mice developed lung adenocarcinomas more aggressively than did Sgo1−/+ or RAG1−/− mice, suggesting immune system involvement in CIN-mediated lung carcinogenesis. To identify molecular causes of the lung adenocarcinoma, we used systems biology approach, comparative RNAseq, to RAG1−/− and RAG1−/− Sgo1−/+. The comparative RNAseq data and follow-up analyses in the lungs of naive Sgo1−/+ mice demonstrate that, (i) glutathione is depleted, making the tissue vulnerable to oxidative stress, (ii) spontaneous DNA damage is increased, (iii) oncogenic Wnt signaling is activated, (iv) both major branches of the immune system are weakened through misregulations in signal mediators such as CD80 and calreticulin and (v) the actin cytoskeleton is misregulated. Overall, the results show multi-faceted roles of CIN in lung carcinoma development in Sgo1−/+ mice. Our model presents various effects of CIN and will help to identify potential targets to prevent CIN-driven carcinogenesis in the lung.Mitotic error-mediated chromosome instability (CIN) can lead to aneuploidy, chromothripsis, DNA damage and/or whole chromosome gain/loss. CIN may prompt rapid accumulation of mutations and genomic alterations. Thus, CIN can promote carcinogenesis. This CIN process results from a mutation in certain genes or environmental challenge such as smoking, and is highly prevalent in various cancers, including lung cancer. A better understanding of the effects of CIN on carcinogenesis will lead to novel methods for cancer prevention and treatment. Previously Shugoshin-1 (Sgo1 CIN can lead to a variety of outcomes in post-mitotic cells, including tetraploidy, chromothripsis, DNA damage and aneuploidy.3 Further, the gain of oncogenes or loss of tumor suppressors may result. Thus, CIN can serve as a mutator.4 In addition, CIN can produce DNA damage and facilitate mutagenesis.5 Micronuclei formation and chromothripsis may also occur, leading to extensive genome shuffling and mutation.7 Thus, CIN is mutagenic on a cellular level. Carcinogenic environmental factors, such as hepatitis virus infection9 and smoking,11 can produce CIN. These cell biological observations and other evidence strongly suggest a role of CIN in carcinogenesis.Aneuploidy has been predicted to cause cancer.3 Most primary lung cancers carry loss of heterozygosity, which is associated with CIN, further implicating CIN in lung carcinogenesis.12 CIN can be caused by mutations in various genes, many of which are bona fide mitotic regulators, such as mitotic spindle checkpoint components BubR1 and Mad2, the mitotic motor Cenpe, and chromosome cohesion regulators such as Sgo1. CIN model mice, transgenic mice with mutations in mitotic regulators, were created to investigate the effects of CIN on carcinogenesis in vivo.16Tumor-mass sequencing data indicate that the genes that are frequently mutated in colon cancer function to prevent CIN, suggesting that progressive CIN is an integrated process in colonic carcinogenesis.in vivo have been identified and characterized using animal models. Aneuploidy causes gross transcriptome changes17 and imbalances in protein generation, producing proteotoxic effects that lead to endoplasmic reticulum (ER) stress, lysosomal stress and overwhelmed autophagy in the cells.18 Mouse studies showed that high rates of CIN and aneuploidy do not always translate directly to high rates of cancer, and that CIN can be both oncogenic and tumor suppressing in different organs.20 Therefore, the relationship between CIN and carcinogenesis is not straightforward and is organ specific. Cell death is a determining factor. A high rate of CIN and aneuploidy can lead to cell death rather than tumorigenesis.21 Another possible determining factor is the involvement of immune system and its surveillance to remove aneuploid cells. G Kroemer‘s group showed that tetraploid cells express specific cell surface antigens, including calreticulin , and proposed that immune surveillance specifically targets the tetraploid cells to suppress cancer.23 Although whether immune surveillance is involved in removing CIN cells in vivo remains to be investigated further, there are reports supportive to the notion linking smoking, CIN and immune function, such as, tobacco smoking is known to decrease immune functions in lung,25 and lung cancers with smoking signatures respond better to immune checkpoint PD1 inhibitor therapy.26The consequences of CIN and aneuploidy 29 Sgo1 expression is abnormal in various human cancers, including colon, lung and liver cancer.33 The inner centromere-Shugoshin mitotic network was shown to be dysfunctional in numerous cancers, suggesting that proper Sgo1 function is crucial to preventing CIN and cancer.34 To further investigate carcinogenesis for cancer prevention, we generated Sgo1−/+ CIN model mice, and have demonstrated the involvement of CIN in carcinogenesis in several organs, including the colon, liver and lungs.37 The colon in particular showed a modified carcinogenesis profile after treatment with the carcinogen Azoxymethane.37 In the Sgo1−/+ CIN model mice, the lungs and liver were also prone to spontaneous carcinogenesis.36 Spontaneous hepatic and lung carcinogenesis were reported in other CIN models, including BubR1−/+ and Mad2−/+ mice,16 suggesting that these cancers may be common consequences of CIN.Shugoshin-1 (Sgo1) regulates chromosome cohesion and centrosome integrity.39 CIN is a marker of poor prognosis for non-small cell lung cancer.40 Inducible Mad2 overexpression CIN model mice, which are defective in the mitotic spindle checkpoint and show CIN, demonstrated a higher rate of recurrence of lung tumor in an experimental setting in which lung tumorigenesis was at first induced through k-ras oncogene addiction then the tumors regressed by the oncogene shutdown.41CIN has shown to be at least correlational, if not causal, to carcinogenesis in the lung. Smoking increases copy number variations and CIN in lungs in humans and animal models.−/− Sgo1−/+ mice and assessed carcinogenesis to investigate the involvement of adoptive immunity and immune surveillance without or with high rate of CIN  in vivo. Recombination activating gene 1 (RAG1) is involved in activation of immunoglobulin V-D-J recombination. The loss (−/−) of RAG1 leads to compromised adoptive immunity,42 yet due to exclusive dependence on another immune system branch mediated by natural killer (NK) cells,43 no lung tumor proneness has been reported. We observed enhanced carcinogenesis in lungs from RAG1−/− Sgo1−/+ mice, suggesting a link between CIN-aggravated lung carcinogenesis and adoptive immunity. To investigate the molecular link, we performed next-generation sequencing/RNAseq in lungs from RAG1−/− and RAG1−/− Sgo1−/+ mice, and identified culprit pathways responsible for the lung carcinogenesis. We validated the pathways in naive Sgo1−/+ mouse lungs, uncovering the multi-faceted carcinogenic effects of CIN in the lung for the first time.As above, the link between CIN and immune function has only been suggestive and the role in carcinogenesis requires further study. In this study, we generated RAG1−/+ mice develop with normal appearance and with modest proneness to spontaneous carcinogenesis in the lungs and liver at 12 months of age and later.36 RAG1−/− mice also develop normally with no apparent tumors up to 12 months, despite their compromised adoptive immunity.43 The relatively normal phenotype of RAG1−/− mice in the laboratory environment is at least partly explained by surveillance of NK cells, another major branch of the immune system.43Sgo1−/+ CIN model, we generated RAG1−/− Sgo1−/+ mice. All animals (RAG1−/− (n=20), Sgo1−/+ [n=32], RAG1−/− Sgo1−/+ (n=16)) developed and lived normally until the ages of 6 and 9 months. However, significantly more RAG1−/− Sgo1−/+ mice  died by the 12 months end point (−/− Sgo1−/+ mice (37.5%) had gross lung carcinomas at the 12 months end point , megamitochondria and binucleation were observed in mice with Sgo1−/+ background. No significant inflammatory infiltrates or steatosis were appreciated in any of the sections examined.  and RAG1−/− Sgo1−/+ (N=3) mice. Using the data set, we identified differentially expressed genes. There were total of 153 hits with a twofold expression difference threshold, P<0.05; 72 upregulated genes and 81 downregulated genes (complete list in −/− and RAG1−/−Sgo1−/+ groups (z score >2) were identified, and indicated as Kyoto Encyclopedia of Genes and Genomes pathways (z score 16.24); phenylalanine metabolism (10.02); alanine, aspartate and glutamate metabolism (7.29); tyrosine metabolism (6.85); and cysteine and methionine metabolism (6.66)), and immune function-related ; graft-vs-host disease (8.79); autoimmune thyroid disease (7.78); systemic lupus erythematosus (4.83); and antigen processing and presentation (4.62)). Downregulated pathways included phenylalanine metabolism (7.23), tyrosine metabolism (4.86) and graft-vs-host disease (4.02). Overall, the pathway analysis indicated significant misregulations in amino-acid metabolism and immune system. Notably, some pathways were commonly found in colonic RNAseq-transcriptome analysis in our recent study:37 diabetes mellitus and graft-vs-host disease. The results indicated that the immune response (graft-vs-host disease) may be a pathway compromised in multiple organs in Sgo1-CIN mice.To investigate the molecular causes that led to high incidence of lung carcinomas in RAG1+ groups . Signifipathways . UpregulS-transferase mu5 (GSTM5)  and Sgo1−/+ mice (n=5), and measured levels of total cellular glutathione (oxidized GSSG and reduced GSH forms combined) in the lungs (P=0.0325). Quantitative real-time PCR with wild-type and Sgo1−/+ mice lungs confirmed that GSTM5 mRNA expression was decreased . Although SFRP4 localized in both alveolar cells and branchiolar cells in control wild-type mice, in Sgo1−/+ mice the localization occurred mainly in branchioli cells (Secreted frizzled-related protein 4 (SFRP4) was among the most downregulated genes (6.05-fold downregulation). SFRP4 is a soluble modulator/inhibitor of Wnt signaling, and its underexpression activates Wnt signaling. P<0.05, , and Wntli cells . To confli cells .45 In zebrafish, Rspo3 knockdown activates Wnt/β-catenin signaling, which is involved in anteroposterior patterning.46 A 2.61-fold downregulation in Rspo3 expression was observed in the RNAseq analysis on the RAG1−/− background, which was confirmed in quantitative RT–PCR in wild-type and Sgo1−/+ (−/+ background.R-spondin3 (Rspo3) is a secreted protein that has been implicated in Wnt signaling. Rspo3 is aberrantly expressed at high levels in approximately half of all Keap1-mutated human lung adenocarcinomas, and may promote cancer aggressiveness. Sgo1−/+ . These f−/− background, we hypothesized that adoptive immunity is involved in suppressing CIN cells in the lung, and that genes or proteins involved in the immune system would be misregulated in Sgo1. The CD80 precursor was among the upregulated genes in RAG1−/−Sgo1−/+ (4.3-fold). CD80 and CD86 work as ligands on the surface of activated B cells and monocytes, and bind to the CD28 receptors on T cells for T-cell activation and survival. However, the RNAseq analyses showed no significant differences in CD86 and CD28 expression (−/+. CD80 mRNA was significantly downregulated (P=0.0031) and the protein was modestly reduced  was reduced  is an NK-cell receptor that is required for activation. Major histocompatibility complex class I recognition receptor  serves as an NK-cell activator48 and is involved in controlling cytomegalovirus infection.49 The reduction in receptors for NK-cell activation would explain the higher degree of lung carcinoma development in RAG1−/− Sgo1−/+ animals, because of reduced NK-cell-mediated surveillance for mutagenic CIN cells. The reduction was confirmed in naive Sgo1−/+ with quantitative reverse transcriptase–PCR, and was not limited to the RAG1−/− background  downregulation in naive Sgo1−/+ lungs, showing an immunocompromised state.In the above results provided, we further tested expression of markers for T-cell activation , thymosin beta  and thymosin beta-like . All of these genes are involved in regulation of the actin cytoskeleton.50 However, comprehensive pathway identification is necessary to formulate an effective strategy without an oversight to antagonizing multiple pathways. For the purpose, CIN models are uniquely valuable. Cellular level CIN, caused by environmental factors or gene mutations, can manifest locally as an initial step of carcinogenesis. The use of tissue/organ-level comparative RNAseq-transcriptomics with CIN mouse models can magnify the molecular effects that occur in cells with CIN, enabling us to identify the cellular effects of CIN in the particular organ. Thus, the use of bioinformatics with a CIN model is an effective way to identify initial molecular changes in an organ of interest.To suppress pre- or early-cancer cells and prevent cancers, various strategies can be utilized. These strategies include inhibition of oncogenic signaling, activation of cell death, activation of tumor suppressors that induce senescence, differentiation and/or non-proliferation, and immune surveillance.The present bioinformatic-based characterization study suggested that CIN is involved in the development of lung adenocarcinomas through the pathways newly identified in the study. We identified reprogramming in transcription in Sgo1-CIN mice lungs, leading to (1) decrease in protection by glutathione , (2) increase in DNA damage possibly with CIN itself and with oxidative stress, (3) activation of Wnt signaling , (4) decrease in T/B-cell activation and immune surveillance , (5) decrease in NK-cell activation and immune surveillance  and (6) others such as actin cytoskeleton disturbance. All can create pro-carcinogenic lung tissue environment .17 However, our results indicated organ-specific and nonspecific  transcriptomic changes.37 Major nonspecific pathways that may be considered CIN signatures are involved in oxidative stress response and immune functions. We speculate that the difference between aneuploid models and CIN models is simply the characteristics of these two models. Aneuploid models carry aneuploid cells uniformly in all cells of the body, whereas CIN models can generate various outcomes after mitosis, such as apoptotic and necrotic cells, senescent cells and aneuploid cells. In CIN models, aneuploid is only one possible outcome. As a result, CIN models only occasionally carry aneuploid cells, and may do so temporarily.Aneuploidy is an outcome of CIN. We anticipated that CIN-mediated transcriptomic changes would show some similarities with those in aneuploid cells. Aneuploid cells in diverse organisms, including yeast, plants, mice and humans, showed highly related gene expression patterns that are conserved between species. In aneuploid cells, genes that were involved in the response to stress were consistently upregulated, and genes associated with the cell cycle and cell proliferation were downregulated.41 in inducible Mad2 overexpression mice may require re-interpretation. Thus far, cells with CIN were interpreted as a source of recurrence, and involvement of the immune system (or the lack thereof) in recurrence was not considered earlier. To capture various processes in vivo as a whole and to elucidate the complex competition among pro- and anti-carcinogenic events, the bioinformatic systems biology approach has demonstrated its usefulness.In light of our results, previous results about lung tumor recurrenceDrosophila mad2 model that CIN causes lethality with metabolic stress as well as oxidative stress,51 suggesting that CIN commonly affects cellular metabolism and oxidative stress pathway over various species.Metabolic misregulation is another aspect of Sgo1-CIN our results indicated. It is consistent with reports from 52 Thus, the rapid development of prevention measures is imperative. This study aided in identifying potential targets for the preventive drug development, and the results may lead to a combinatorial chemoprevention measure simultaneously targeting multiple pathways for better efficacy.Overall, our bioinformatic results indicated that CIN can influence carcinogenesis and possibly cancer recurrence through multiple pathways. The results also suggest possible countermeasures: glutathione supplementation, removal of CIN cells through targeting CIN-specific markers, Wnt signaling attenuation and immune restoration. Lung cancer is the most lethal cancer worldwide: it is predicted to claim 158 080 lives in the United States alone in 2016. −/+ haploinsufficient mice have been described earlier.37 RAG1−/− mice were obtained from Dr Naveena Janakiram (OUHSC). All mice were maintained in the OUHSC BRC rodent barrier facility. Initially, RAG1−/− mice and Sgo1−/+ mice were mated to generate F1; RAG1−/+ Sgo1−/+ mice, then F2; RAG1−/− Sgo1−/+, RAG1−/−, Sgo1−/+ and RAG1−/+ mice that served as controls. Sample size was determined following Mead's resource equation. Group designation was used for randomization: all F2 litters were separated at the weaning (3 weeks of age) to male cage and female cage, and the cage and animals received a number. All animals were genotyped by PCR using genomic DNA from tail. After genotyping, we maintained all mice with regular diet (Purina) for 6, 9 or 12 months without any experimental treatment, then collected samples after killing at the end point based on the birth date/age. All mice were handled equally and both genders were used. Animal's genotype was not informed to the researcher(s) involved in sample collection. All mice were generated with the non-cancer-prone C57BL/6 background. All treatments complied with protocols approved by the OUHSC Institutional Animal Care and Use Committee. Samples from 12-month-old naive Sgo1−/+ and control wild-type mice that were used for confirmatory experiments were obtained from a previous study.36Generation, genotyping and characterization of mouse embryonic fibroblasts (MEF) and the colonic carcinogenesis assay with Azoxymethane injections in Sgo1Lung tissues were fixed in 10% formalin, paraffin embedded and subjected to IHC . The following primary antibodies were used at 1.0 μg/ml: anti-phospho-H2AX , anti-SFRP4 , β-catenin , anti-actin , anti-calreticulin  and anti-CD80 .t-test (unpaired) with Graphpad Prism5 software . Immunofluorescence images were taken with a confocal microscope  in the OUHSC Laboratory for Molecular Biology and Cytometry Research.Four-to-six mice per strain were analyzed. At least 10 IHC images were captured from each tissue. The percentages of IHC-positive cells were calculated. Data were expressed as means±s.d., or as variances. The differences between groups were analyzed using Student's Histopathological characterization was performed in a blinded manner by a histopathologist, with duplicated hematoxylin/eosin-stained slides for each sample.36 Briefly, the lysates were aliquoted and the protein content was determined by Bradford reagent. The aliquots were stored at −80 °C. All primary and secondary antibodies were first standardized for dilution and were then used accordingly. Proteins were resolved on 8–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and probed with specific antibodies overnight at 4 °C. The membranes were washed three times with Tris-buffered saline (pH 7.4) for 15 min and were then incubated with anti-rabbit, anti-goat or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000 dilutions in 2.5% milk) and visualized using chemiluminescence reagent . β-Actin was used as the loading control. Densitometry of various proteins and their respective loading controls from the same blot was performed using ImageJ 1.43 software . Relative optical density was calculated by dividing the densitometry of protein with that of the respective loading control.Our standard procedures were followed.−/− (N=3) and RAG1−/− Sgo1−/+ (N=3) mice. The RNA samples were submitted to the OUHSC Laboratory for Bioinformatics core facility for library construction and RNA sequencing with an Illumina MiSeq next-generation sequencer with each run generating approximately 30 million 2 × 150 bp paired end reads. The readouts were analyzed with Genesifter . Reads were normalized using mapped reads and base-lined to the data set. Analysis of differential gene expression of the normalized signal values between the control and experimental group was done using EdgeR, a moderated t-test, with Benjamini and Hochberg correction. P<0.05 and twofold expression thresholds were used to determine differentially expressed genes. Comparative RNAseq data sets were deposited to GEO-NCBI with accession number GSE84383.We isolated total RNA from lung tissues of RAG1405 measurements and curve plotting were performed with Clariostar .We used the OxiSelect Total Glutathione (GSSG/GSH) Assay Kit  and followed the instructions. OD53 Total RNA from normal and tumor samples was extracted using TRizol reagent for total cellular RNA  per the manufacturer's instructions. Equal amounts of DNA-free RNA were used for reverse transcription (RT) reactions to make complementary DNA using an iScript cDNA synthesis kit  per the manufacturer's protocol. Real-time PCR was carried out in a 12-μl reaction volume containing 5 μl of diluted complementary DNA (50 ng) and FastStart Universal SYBR Green master  and primers (Invitrogen). All PCRs were performed in a Bio-Rad iCycler iQTM5 real-time PCR detection system. The fluorescence threshold values (Ct) were calculated. Relative mRNA levels were assessed by standardization to actin or glyceraldehyde 3-phosphate dehydrogenase. Results are expressed as a relative fold difference in gene expression compared with control. Relative gene expression was calculated using the 2-ΔΔCT formula. PCR conditions were as follows: denaturation at 94 °C for 10 min, followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 45 s. All experiments were performed in triplicate.We followed procedures described previously.


SS genes were cloned and characterized from banana fruit. Among them, MaSSIII-1 showed the highest expression in banana pulp during fruit development at transcriptional levels. Further Western blot analysis suggested that the protein was gradually increased during banana fruit development, but drastically reduced during storage. This expression pattern was highly consistent with changes in starch granules, amylopectin content, and SS activity at the late phase of banana fruit development. Lastly, overexpression of MaSSIII-1 in tomato plants distinctly changed the morphology of starch granules and significantly increased the total starch accumulation, amylopectin content, and SS activity at mature-green stage in comparison to wild-type. The findings demonstrated that MaSSIII-1 is a key gene expressed in banana fruit and responsible for the active amylopectin biosynthesis, this is the first report in a fresh fruit species. Such a finding may enable the development of molecular markers for banana breeding and genetic improvement of nutritional value and functional properties of banana fruit.Soluble starch synthase (SS) is one of the key enzymes involved in amylopectin biosynthesis in plants. However, no information is currently available about this gene family in the important fruit crop banana. Herein, we characterized the function of MaSSIII-1 in amylopectin metabolism of banana fruit and described the putative role of the other MaSS family members. Firstly, starch granules, starch and amylopectin content were found to increase during banana fruit development, but decline during storage. The SS activity started to increase later than amylopectin and starch content. Secondly, four putative  Starch is the most widespread carbohydrate storage molecule in plants and plays a vital role in human nutrition, food industry and chemical manufacturing. The physicochemical properties of starch are strongly affected by its two key components, amylose and amylopectin . In highvia α-1,4-glycosidic linkages, and directly catalyzes the amylopectin biosynthesis  is not only one of the most highly consumed fruits in the world but also widely used as a staple food in the tropical and subtropical regions (MaGBSSI (DBE (SUCROSE PHOSPHATE SYNTHASE (SPS), SUCROSE SYNTHASE (SuSy), and INVERTASE  seedlings were obtained from the banana tissue culture center  and were grown at 28°C with 70% humidity, 200 μmol⋅m-2⋅s-1 light intensity, and long day condition (16 h light/8 h dark cycle). When banana seedlings produced five leaves, they were planted in the Institute of Tropical Bioscience and Biotechnology banana plantation  until harvest. Roots, stems, leaves, bracts, flowers, peels, and pulps at 60 days after emergence from the pseudostem (DAF) were collected separately using tweezers, frozen immediately in liquid nitrogen, and stored at -80°C until expression analysis in different tissues. For each biological replicate, two banana hands having a similar developmental stage from two plants were selected and six fingers from the hands were obtained. Banana pulps at 0, 10, 20, 30, 40, 50, and 60 DAF were collected, immediately frozen in liquid nitrogen, and stored at -80°C until starch/amylopectin quantification, mRNA and protein expression analysis.Banana (Banana hands (60 DAF) were separated into individual fingers representing the same developmental stage. For natural ripening treatment, banana samples were kept at 22°C and allowed to ripen in open air. In accordance with the previously published banana ripening stages , fruits Pulp samples were fixed in stubs using double-faced tape and coated with a 10 nm thick platinum layer using a Bal-tec MED 020 Coating system  before analysis with a FEI Quanta 600 FEG Scanning Electron Microscope . SEM observations were performed using the secondary electron mode operating at 15 kV.3)2, placed in a boiling water bath for 10 min, and centrifuged for 4 min at low speed . The supernatant was then transferred to a 20 mL volumetric flask. The total starch, amylose, and amylopectin contents of the extract were determined following Banana pulp was immersed in 0.5% (w/v) sodium bisulfite solution for 10 min to prevent browning, and then dried at 40°C for 20∼24 h. The dried pulp was milled to powder and suspended in 5 mL 80% (w/v) Ca, SSII (GSMUA_Achr6G23190_001), SSIII-1 (GSMUA_Achr11G18570_001), and SSIII-2 (GSMUA_Achr5G00700_001), were retrieved from the banana DH-Pang (AA group) genome sequence database1. Four primer pairs (Supplementary Table S1) were designed based on these sequences. The SS coding sequences were submitted to BLAST analysis to recover their corresponding genomic DNA sequences. Exon lengths were calculated by alignment of genomic DNA sequences with cDNA sequences, and introns were determined according to the “GC-AG” rule (RNA purification and cDNA synthesis were performed as previously described by AG” rule . The dedSupplementary Table S1). Moreover, PCR products were sequenced to confirm the specificity of primer pairs. Amplification efficiencies of primer pairs ranged from 0.9 to 1.1. The levels of MaSS and SISSIII-1 expression were quantified by qRT-PCR using an iQ5 real-time PCR detection system  with the SYBR ExScript RT-PCR Kit . ACTIN or GAPDH that were verified to be constitutive in expression and hence suitable to be used as internal controls were used as reference genes to normalize transcriptional levels of each MaSS gene and SISSIII-1 (Supplementary Table S1). Relative expression levels of four MaSS genes were analyzed in three technical replicates and calculated using the 2-ΔΔCT method  glycerol  and incubated at 30°C for 10 min. The supernatant (SS enzyme solution) was used to Western blot analysis.Pulp samples (0.5 g) were homogenized with a set of mortar and pestle on ice in an equal volume of solution, which consisted of 50 mM HEPES-NaOH (pH 7.4), 2 mM MgClglycerol . A 20 μLTM-N+ membranes . Membranes were probed with rabbit anti-MaSSIII-1 polyclonal antibody  and banana actin antibody (control) in 1:1,000 dilution in PBS-Tween 20 plus 3% BSA, respectively, followed by alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody  in 1:1,000 dilution. Positive signals on the membranes were detected with a 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) solution .For Western blot analysis, 30 μL of the SS enzyme solution from banana pulps at different developmental stages extracted by the above methods was loaded on each lane and separated on a 12% polyacrylamide gel. Proteins were then transferred onto HybondMaSSIII-1 coding region was inserted into the pCAMBIA-1302 vector under the transcriptional control of CaMV 35S promoter following a double digestion with Nco I and Spe I. The pCAMBIA-1302-MaSSIII-1 was transferred into A. tumefaciens strain LBA4404. Transgenic tomato (Solanum lycopersicum L.) plants were generated using the Agrobacterium-mediated transformation as previously described  agarose gel and transferred onto Hybond-N+ nylon membranes   . Probes t-tests, at significant levels of p < 0.05 (∗), and p < 0.01 (∗∗).Three biological replicates were performed for each sample, unless specified otherwise. Statistical analyses were performed using Microsoft Excel and SPSS . Analysis of variance was used to compare the statistical difference based on Student’s Figure 1A). As observed using SEM, starch granules in banana pulp were barely detectable at the initial sampling time, i.e., 0 DAF, but the oval-shaped starch granules become clearly visible and were significantly increased in size as the fruit developed from 10 (6.0∼8.2 μm) to 60 DAF (28.0∼35.7 μm). Morphological comparison of starch granules during this period of time indicated that the shape and number of starch granules remained consistent, but the size was significantly increased and maximized at 60 DAF .During banana fruit development the fruit size gradually increases and it becomes curled in a crescent shape as fruit develops .Starch granules in mature banana pulp were also observed using SEM at the 30 DPH during storage at 22°C, at 5 days’ intervals. Compared to the first time point (0 DPH), the quantity of starch granules per unit volume decreased rapidly over time. After 15 DPH of storage, starch granules were hardly detectable , implying that after maturity starch and amylopectin may have experienced a rapid degradation process. However, the SS enzyme activity increased later than starch and amylopectin accumulation at maturity and had an abrupt decline at the very beginning of storage .The total starch content and amylopectin content in pulp increased as fruit developed and peaked at 50 DAF  before decline at the 60 DAF time point and continued to decrease gradually during storage  genome sequence database2 revealed that MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 are located on chromosome 3, 6, 11, and 5, respectively . The sequence analysis also revealed markedly different primary structures as MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 contain 25, 6, 5, and 9 exons, respectively. The stop codon usage is also different as TAA was used by MaSSI and MaSSIII-1, but TGA was used by MaSSII and MaSSIII-2 .Full-length cDNAs encoding Supplementary Table S2), as previously identified in SS enzymes from amaranth (Supplementary Table S3), as previously identified in SSIII enzymes from wheat . MaSSIII-1 and MaSSIII-2 are aligned next to each other, forming a side branch in association with other SSIII sequences from monocot species. Similarly, MaSSI and MaSSII are clustered together with their respective orthologs from other species. This may also indicate that the divergence of the SSI, SSII and SSIII occurred prior to the speciation of these plant species, but the separation of SSIII-1 and SSIII-2 might be of a more recent history likely through a gene duplication and subsequent divergence.To elucidate phylogenetic relationship of banana MaSS genes were analyzed by qRT-PCR in seven different tissues, including roots, stems, leaves, bracts, flowers, peels, and pulp (60 DAF) . MaSSI was expressed at low levels in all tissues examined with the highest expression in pulps. MaSSII was also found to express low in all the vegetative tissues, including roots, stems, leaves, and bracts, and particularly low in flowers and fruit tissues. The expression patterns of MaSSIII-1 and MaSSIII-2 were very similar, with highest expression in pulps and leaves (starch content in leaves was approximately 22.67 ± 1.32%), but that MaSSIII-1 had the highest levels of expression.The spatial expression patterns of the four MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 during banana fruit development was also determined by qRT-PCR . Despite of being highly variable, the expression levels of MaSSI and MaSSII genes were found to be generally low throughout the entire banana pulp development. Both MaSSIII-1 and MaSSIII-2 showed a sharp increase of expression during the fruit development, the increase in expression occurred earlier in MaSSIII-1 than in MaSSIII-2, with the highest expression detected at the last sampling stage when banana had reached its maturity. However, the expression of MaSSIII-1 was about 10∼20-fold higher than that of MaSSIII-2 during the fruit development.The temporal expression of MaSS genes were also found to be differentially expressed . MaSSII showed consistently low levels of expression throughout the storage period, but the MaSSI expression was slightly increased during storage. The seemingly negative association between MaSSI expression and starch degradation needs to be further investigated. Although there can be a lack of congruency between transcript and activity, it is possible that there can be some starch biosynthesis occurring even during net degradation . The Western blot results was consistent with changes in starch granules , amylopectin content  and SS activity  during banana development and ripening, suggesting the expression of MaSSIII-1 protein might play an important role in regulating amylopectin metabolism in banana fruit during development and ripening.Western blot analysis with rabbit anti-MaSSIII-1 polyclonal antibody as a probe indicated that the size of MaSSIII-1 is approximately 90.0 kDa, which is consistent with the molecular weight (90.69 kDa) as predicted by the PeptideMass program. The expression of MaSSIII-1 protein was gradually increased during banana fruit development, but drastically reduced from 0 to 30 DPH of storage  were identified by Southern blot analysis . The fruit shape, starch granule morphology, gene expression, total starch content, amylopectin content, and SS activity were investigated in the wild-type (WT) and the MaSSIII-1 overexpressing transgenic plants.To further examine the function of MaSSIII-1 transgenic plants did not change , but the morphology of starch granules was significantly altered from immature green (IG) to orange-breaker (BR) in transgenic tomato plants, severe cracks in the surface of starch granules were observed . For fruit weight and volume, there are no significant differences between MaSSIII-1 transgenic plants and WT . Overexpression of MaSSIII-1 in tomato plants significantly increased the gene expression , SISSIII-1  expression in tomato fruit was lower than that of MaSSIII-1 transgenic plants . At IG and mature-green (MG) stages, total starch content, amylopectin content, and SS activity showed significant differences between transgenic plants and WT . Especially at the MG stage, the gene expression, total starch content, amylopectin content, and SS activity significantly increased 55∼120-fold, 13.47∼15.83 mg⋅g-1, 6.36∼9.17 mg⋅g-1, and 0.54∼0.80 U⋅min-1 in the transgenic fruits compared to WT, respectively.Compared to WT, the fruit shape of Despite of the extensive studies on cereal starch, little information is available regarding the dynamics of starch accumulation (net result of starch synthesis and degradation), SS activity, and amylopectin metabolism during fruit development and storage in fresh starchy fruits, such as banana. In tomato, starch is transiently accumulated during fruit development and degraded within the lifecycle of that organ, while starch synthesis and degradation are occurring simultaneously . HoweverGBSS genes have been cloned and identified in banana  in plants and play important roles in amylopectin biosynthesis . GBSS asn banana . In thisn banana . Moreoven banana . Sequenc SS gene . The MaS SS gene . Domain  SS gene . The ami SS gene .MaSSI and MaSSII were found to express in low levels in vegetative organs, flower and banana fruits, similar to their homologs in kidney bean . Such an observation is consistent with previous studies in rice, wheat, and Arabidopsis , suggesting these parameter increases may be attributed to MaSSIII because of increased transcript and polypeptide. Similar results were reported in wheat (MaSSIII-1 in tomato fruit showed severe cracks in the surface of starch granules and changed the starch granules morphology. This study suggests that MaSSIII-1 is a key gene in the amylopectin biosynthesis and therefore it could be used as a useful tool for marker assisted molecular breeding in banana.SSIII protein as a catalytic factor plays an important role in amylopectin metabolism . SSIII cin maize . Inhibitn potato . SSIII an potato . In fresin wheat  and ricein wheat , but thiSS genes encoding MaSSI, MaSSII, MaSSIII-1, and MaSSIII-2 were cloned and characterized. Expression pattern of only MaSSIII-1 was highly consistent with dynamic changes in starch granules, amylopectin content, and SS activity. MaSSIII-1 transgenic lines distinctly changed the morphology of starch granules. Overexpression of MaSSIII-1 in tomato plants significantly increased the amylopectin accumulation and SS activity in comparison to WT. This is the first report about the MaSSIII-1 gene involved in amylopectin metabolism in a fresh fruit species. These findings establish a solid foundation to further regulate the amylopectin metabolism in banana fruits or other fresh fruits using the MaSSIII-1 or its homologous genes.Starch synthase activity increased along with the amylopectin accumulation at later stages of banana fruit development, but declined during storage. Four ZJ and BX conceived and designed the experiments. HM, PS, QL, CJ, JL, and WH performed the experiments and carried out the analysis. HM, PS, and QL designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.ZJ and BX conceived and designed the experiments. HM, PS, QL, CJ, JL, and WH performed the experiments and carried out the analysis. HM, PS, and QL designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.


In contrast, the same mass fraction of silicon carbide micron particles (SiC MPs) incorporated into epoxy matrix showed less improvement on thermal conduction properties. This is attributed to the formation of effective heat conduction pathways among SiC NWs as well as a strong interaction between the nanowires and epoxy matrix. In addition, the thermal properties of epoxy/SiC NWs composites were also improved. These results demonstrate that we developed a novel approach to enhance the thermal conductivity of the polymer composites which meet the requirement for the rapid development of the electronic devices.In this study, we report a facile approach to fabricate epoxy composite incorporated with silicon carbide nanowires (SiC NWs). The thermal conductivity of epoxy/SiC NWs composites was thoroughly investigated. The thermal conductivity of epoxy/SiC NWs composites with 3.0 wt% filler reached 0.449 Wm Epoxy resins are widely applied in the aerospace, automotive and electronic industries for supporting and adhesive, owing to their excellent mechanical properties, thermal stability, and chemical resistances3. However, intrinsic low thermal conductivity of neat epoxy resins (only the order of 0.10 Wm−1 K−1 at room temperature)4 limits their applications5. In order to overcome this problem, a variety of methods have been developed to improve the thermal conductivity. A traditional method is to introduce high contents of thermally conductive fillers, such as metals11, ceramic particles15, carbon nanotubes20, or graphene nanoplateles25. In particular, the silicon carbides are provided with excellent properties such as high thermal conductivity, high thermal stability, high breakdown field, excellent mechanical properties and chemical inertness32. Therefore, they can be applied in various circumstances such as in high temperatures, high electron density, high frequency and in harsh environments33. The high aspect ratio of silicon carbide nanowires (SiC NWs) makes it favorable to be used as filler in epoxy resin, in order to achieve the desired properties.The rapidly-developing electronic industry focuses on at miniaturizing and lightening of electronic equipment. Meanwhile, performance and versatility of electronic devices have been integrated with a decreasing dimensions. Consequently, a high level of circuit integration cause high heat, which needs to be rapidly dissipated to let the device work efficiently1. Copper and aluminum are traditional heat dissipating materials, which show large mismatch with silicon and insulating ceramics and cannot directly attach to silicon without stress compensating interlayers34. Therefore, control of reinforcing fillers into the polymer can fulfill the high thermal conductivity and the lowest CTE or thermal distortion parameter  potential requirements. It will meet the requirement of thermal management and electronic packaging applications ranging from microelectronic components and devices to supercomputers36.Efficiently cooling of electronic devices requires minimal coefficient of thermal expansion (CTE) mismatch of the heat source and heat sink. Nowadays, the heat source are usually Si-based47. Therefore, detailed investigation of epoxy/SiC NWs for thermal management or heat dissipation is needed, which is still in its infancy. Herein, we proposed a rapid and simple strategy for the preparation of epoxy/SiC NWs composites. The SiC NWs were mixed uniformly with epoxy using ethanol as the solvent. Subsequently, the mixture was stirred in water bath and curing agent was added to obtain the epoxy/SiC NWs composites. Based on this method, the SiC NWs loading can be easily controlled, and the interconnected network of nanowires in the composites results in not only enhanced thermal properties but also retained low CTE performance.It is noted that so far only few works have been reported about the application of epoxy/SiC NWs compositesSilicon carbide nanowires were produced by Changsha Sinet Advanced Materials Co., Ltd. China. Silicon carbide micron particles with the particle size about 1 μm were purchased from Shanghai St-Nano Science and Technology Co., Ltd. China. Anhydrous ethanol was produced by Sinopharm Chemical reagent Co., Ltd. Cycloaliphatic epoxy resin  along with hardener of methyl-hexahydrophthalic anhydride  was used in the present study. Neodymium (III) acetylacetonatetrihydrate (Nd(III)acac) purchased from Aldrich Chemicals was used as latent catalyst.Epoxy composites with different SiC NWs loadings were prepared by the following procedures. Required quantity of Nd(III)acac was added into a cycloaliphatic epoxy resin and subsequently stirred at 80 °C in a three-necked flask for 2 h. The homogeneous solution was then cooled down to ambient temperature. A desired amount  of SiC NWs was dispersed in ethanol in an ultrasonic bath for 0.5 h and then added into the predetermined amount of epoxy resin. The obtained mixture was then placed in a beaker with vigorous mechanical stirring at 80 °C in water bath until complete evaporation of ethanol. Curing agent was added at a ratio of 100:95 (epoxy:curing agent) by weight into the beaker and was stirred for 20 min. It was further degassed in the vacuum oven for 10 min to remove air bubbles. Finally, the mixture of epoxy with the homogeneously dispersed SiC NWs was poured into the mold to cure at 135 °C for 2 h and 165 °C for 14 h. After the curing process, the samples were naturally cooled down to the room temperature and then polished with emery paper for different characterizations. For comparison, the epoxy composites with SiC MPs were also fabricated with the same procedures as described above. For the sake of convenience, the composites containing SiC NWs and SiC MPs were denoted as epoxy/SiC NWs composites and epoxy/SiC MPs composites, respectively. The preparation process of epoxy/SiC NWs composites is illustrated in Fig. −1 at room temperature. The fractured surface of the composites was examined by field emission scanning electron microscopy  at an acceleration voltage of 20 kV. Samples that were broken and the fractured surface was coated with a thin gold layer to avoid accumulation of charge. The microstructures of the samples were obtained from JEOL JEM-2100  instrument with an acceleration voltage of 200 kV. Thermal conductivities of the composites were measured using the light flash apparatus LFA 447 NanoFlash® . The IR-photos were captured by infrared camera . Differential scanning calorimetry was performed by a Pyris Diamond DSC  from 20 to 250 °C at a heating rate of 10 °C/min under a nitrogen atmosphere to study glass transition temperature (Tg) of the composites. The CTE measurements were performed on a thermal mechanical analyzer  from 310 to 460 K at a heating rate of 5 K/min. Thermogravimetric analysis (TGA) was carried out using TG 209 F3 thermo-analyzer . The temperature range was from 50 to 800 °C at a ramp rate of 10 °C/min in nitrogen atmosphere.Atomic force microscope (AFM) measurement was conducted on a multimode SPM from digital instruments with Nanoscope IA controller. X-ray photoelectron spectroscopy (XPS) was carried out with a Kratos AXIS Ultra DLD spectrometer, using AIKα excitation radiation (hγ:1253.6 eV). The X-ray diffraction (XRD) patterns of the samples were recorded on a D8 DISCOVER with GADDS (BRUKER Ltd. Germany) with CuKα radiation (λ = 1.5406 Å). The scanning was performed from 10° to 80° with a speed of 4° minFigure 48. However the diffraction pattern of SiC MPs is complicated. Except for the above the five peaks resulting from β-SiC, several other peaks can be observed, indexing as the (101), (103) and (109) reflections of hexagonal polytypes 6H-SiC (α-SiC), lattice parameters of 6H-SiC calculated by least squares method from the positions of the peaks observed, as follows: α = 3.078 Å, c = 15.12 Å. We speculated that SiC MPs are polycrystalline, which is in good agreement with the previous conclusion49. XPS analysis is employed to further verify the surface composition and investigate the functional groups and the Si2p and C1s high-resolution results are shown in Fig. 2p, the spectrum can be decomposed to three Gaussian components located 100.2, 101.4 and 102.8 eV, originated from Si-Si, Si-C, and Si-O, respectively. The spectra of C1s compose of three bonds centered at 283.3, 285.6, and 287.9 eV, which corresponds to C-Si, C-C, and C=O, respectively. A small amount of C=O can be attributed to the adsorbed CO2 on the SiC NWs surface. According to XPS quantification analysis, Si and C atomic ratio of β-SiC nanowire is approximately 1.0:1.251.Figure To understand the relationship between the structure and properties of epoxy composites, the fracture surfaces of neat epoxy and epoxy composites are characterized by SEM, as illustrated in Fig. 52. In order to fabricate composites with superior thermal conductivity, beside the amount of filler, uniform dispersion in polymer matrix is the critical factor35. We adopted a transient laser flash method to indirectly estimate the thermal conductivity of the neat epoxy and its composites at room temperature. In order to assess the effect of SiC fillers on the thermal properties of composites, thermal diffusivity was measured first and then thermal conductivity was calculated as a function of SiC fillers loading content (0–3 wt%). The dependence of thermal diffusivity and thermal conductivity on SiC loading is presented in Fig. 2/s. With the addition of 0.5 wt% SiC NWs, the thermal diffusivity of epoxy/SiC NWs composite increased to 0.121 mm2/s with about 11% enhancement. As the content of SiC NWs is further increased to 3 wt%, the thermal diffusivity is improved from 0.108 mm2/s to 0.196 mm2/s, and the thermal conductivity was improved from 0.218 Wm−1 K−1 to 0.449 Wm−1 K−1. At 3 wt% SiC NWs loading, the thermal conductivity of epoxy/SiC NWs composite is improved significantly by 106% compared to that of neat epoxy, as illustrated in Fig. −1 K−1 at 3.0 wt%, lower than that of epoxy/SiC NWs composites at the same loading fraction, only enhance 51% comparing to that neat epoxy, as shown in Fig. 27.Recently, it has been reported that the fillers with high aspect ratio, such as nanowires and nanosheets, can form more continuous thermally conductive network in the polymer matrix and therefore are more efficiently in improving the heat transfer53. Neat epoxy cannot obtain an expected thermal conductivity because the low crystallinity and phonon scattering of the randomly entangled moleculechains54. Introducing high thermal conductivity SiC NWs (~100 Wm−1 K−1)55 into epoxy is an effective approach to enhance the thermal conductivity of the composites. In general, the low loading SiC NWs in the polymer matrix are in an isolated state which is analogous to the “sea-island” structure. If the quantity of SiC NWs is further increased and reaches the percolation threshold, local chain and network would mutually bridge to generate the whole network. The formation of efficient network that contributes epoxy and SiC NWs to fabricate continuous phases, resulting in enhancing the thermal conductivity of composites56. In order to better demonstrate the superiority of SiC NWs as compared with SiC MPs, the thermal conduction visualized model of epoxy composites was proposed, as shown in Fig. 57.It is well known that the phonons play a major role in heat conduction of majority solid materials. Meanwhile, both the harmonic or anharmonic phonon-phonon interaction at high temperatures and the scattering of the phonons by the crystal boundaries at low temperatures determine the thermal conductivity of semiconductors−1 K−1 at 50 °C and increased with temperature over the temperature range investigated. Similarly, the thermal conductivity of epoxy composites shows the same trend with the increase of temperature. Under Tg  curves display the same trend that can be observed in Fig. −1, compared to conventional nanowires based composites, this the lowest CTE value at only 3 wt% contents of SiC NWs66. This consequence indicates that the thermal expansion is significantly restrained by the introduction of nanowires. The following relation is used to determine the volume fraction of the filler for a given weight fraction, which is represented by Equation (f, Wf, ρf and ρm are the volume fraction of fillers, the mass fraction of fillers, the density of fillers, and the density of matrix, respectively. The mass fraction of 3 wt% can be converted to 1.1 vol%. Meanwhile, the composite incorporated with 3 wt% SiC MPs show the similar property. Besides, this outstanding performance combining with high thermal conductivity result in lower TDP, which is a prominent indicator at the engineering-scale and be defined as following Equation (1. The lower TDP predicts greater thermal stability, on account of the high thermal conductivity and low CTE of the epoxy/SiC NWs composite. Figure 70. Materials with high thermal conductive and low TDP will meet wide-ranging application in the modern electronics industry.Figure −1 K−1, which is increased by 106% comparing to that of neat epoxy. It is found that the thermal stability also has a certain degree of enhancement and retains the low coefficient of thermal expansion. This composite with remarkable thermal properties is crucial in thermal management, electronic packaging, and other electrical devices applications.In summary, a facile method to prepare epoxy composites has been demonstrated. The thermal conductivity of epoxy/SiC NWs composites with 3 wt% filler was 0.449 WmSupplementary Information