Cardiometabolic diseases, including cardiovascular disease, obesity, and diabetes, are the leading cause of mortality and morbidity worldwide. Cardiometabolic diseases are associated with many overlapping metabolic syndromes such as hypertension, hyperlipidemia, insulin resistance, and central adiposity. However, the underlying causes of cardiometabolic diseases and associated syndromes remain poorly understood. Within the past couple of decades, considerable progresses have been made to understand the role of inflammatory signaling in the pathogenesis of cardiometabolic diseases. The transcription factor, NF-κB, a master regulator of the innate and adaptive immune responses, is highly active in cardiometabolic diseases. IκB kinase β (IKKβ), the predominant catalytic subunit of the IKK complex, is required for canonical activation of NF-κB, and has been implicated as the critical molecular link between inflammation and cardiometabolic diseases. Recent studies have revealed that IKKβ has diverse and unexpected roles in mediating adiposity, insulin sensitivity, glucose homeostasis, vascular function, and atherogenesis through complex mechanisms. IKKβ has been demonstrated as a critical player in the development of cardiometabolic diseases and is implicated as a promising therapeutic target. This review summarizes current knowledge of the functions of IKKβ in mediating the development and progression of cardiometabolic diseases. Cardiometabolic diseases such as atherosclerosis, obesity, and diabetes are related to several risk factors termed cardiometabolic syndromes , 2. CardWhile IKKβ and IKKα, have a similar structure, they have different functions as IKKα contains a putative nuclear localization signal and IKKβ contains a ubiquitin binding domain. In addition, IKKβ activation is necessary for canonical NF-κB pathway activation, while IKKα is not , 9. The Atherosclerosis is the major contributing risk factor for the development of cardiovascular disease (CVD). It is a very complex disease involving the development of plaques in large arteries causing narrowing of the vessel lumen leading to various clinical manifestations, including stroke, ischemic heart disease, chronic kidney disease, and peripheral artery disease. The plaques are characterized by accumulating lipids and immune cells into the sub-endothelial space –18. AtheAs a significant player in atherosclerosis initiation and progression, studies have suggested that the inflammatory response in endothelial dysfunction can be driven by IKKβ/NF-κB signaling , 21, 26.−/−) mice. They found that the mice receiving IKKβ-deficient macrophages exhibited enhanced atherosclerotic lesion development and increased necrosis, which suggest a protective role of bone marrow-derived macrophage IKKβ against atherosclerosis development  also play an important role in atherogenesis. In the early stages of atherosclerosis, VSMCs undergo a phenotypic switch from contractile to synthetic where they gain the ability to proliferate and migrate into the intimal layer. This provides a beneficial effect as these VSMCs proliferate and migrate to the cap of the plaque and reinforces its stability, lowering the risk for plaque rupture . An earl−/− mice . Deficieelopment . Since i−/− mice , these s−/− mice when fed a low-fat diet . In. In−/− mdiposity , 55. Wntdiposity , 57 and diposity , 55. Thudiposity .While studies have suggested a pro-obesogenic role of progenitor IKKβ, the function of IKKβ in mature adipocytes is apparently more complicated. Constitutive activation of IKKβ in adipocytes has been demonstrated to increased energy expenditure in mice, leading to protective effects against diet-induced obesity and insulin resistance . HoweverInsulin resistance is a very complex syndrome and IKKβ has been shown to regulate insulin resistance by directly interfering with the insulin signaling pathway . Once stThe IKKβ/NF-κB pathway has been demonstrated to be active in both obesity-dependent and independent insulin resistance , 53. InhInflammation is an important contributor of insulin resistance, and adipose tissue is one of the important tissues for this high-fat feeding-elicited inflammatory response . Adiposein vitro  may be able to abrogate local insulin resistance, it may not be sufficient for systemic inflammation-induced insulin resistance under obese conditions. For example, it is reported that myeloid-specific IKKβ deficiency can improve obese-dependent systemic insulin resistance , 87, indNon-alcoholic fatty liver disease or non-alcoholic steatohepatitis is also associated with metabolic syndrome. The activation of the IKKβ/NF-κB pathway has been shown to promote fatty liver disease, or hepatic steatosis , whereasAlthough there have been strong links between IKKβ and metabolic diseases within the periphery, more recently, inflammatory activation has been seen within the central nervous system (CNS). Specifically, IKKβ in the hypothalamus can be activated in obesity and obesity-related metabolic dysregulation such as energy, body weight, and glucose dysregulation –98. A stWhile it is critical to study the effects of hypothalamic inflammation on obesity and metabolic syndromes, it is also important to investigate the upstream targets mediating hypothalamic inflammation. For example, astrocytes play essential roles in neuronal development; regulation of blood flow; fluid, ion, pH, and transmitter homeostasis; the regulation of synaptic transmission; and regulate immune response . Under pHypertension, a chronic elevation in arterial blood pressure, is one of the major risk factors for developing CVD such as myocardial infarction, stroke, and heart failure. Although there are therapeutic interventions aimed to target and treat hypertension, it is still a prevalent contributor to cardiometabolic disease burden . IKKβ inRecent research advancements have expanded our knowledge on the function of IKKβ in cardiometabolic diseases. A summary of the role of IKKβ in cardiometabolic diseases is listed in RH: conceptualized, wrote, and edited the manuscript. CZ: reviewed, edited, and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by National Institutes of Health grants (R01HL131925 and R01ES023470) and American Heart Association grant (19TPA34890065) to CZ. RH was supported by an NIH T32 training grant (T32ES018827).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The absence of editing causes amino acid residue changes in CcmFC-267 (Ser to Pro) and Nad2-226 (Phe to Ser), respectively. As CcmFC functions in cytochrome c (Cytc) maturation, the amount of Cytc and Cytc1 protein is drastically reduced in emp17, suggesting that the CcmFC-267 (Ser to Pro) change impairs the CcmFC function. As a result, the assembly of complex III is strikingly decreased in emp17. In contrast, the assembly of complex I appears less affected, suggesting that the Nad2-226 (Phe to Ser) change may have less impact on Nad2 function. Together, these results indicate that EMP17 is required for the C-to-U editing at several sites in mitochondrial transcripts, complex III biogenesis, and seed development in maize.The conversion of cytidines to uridines (C-to-U) at specific sites in mitochondrial and plastid transcripts is a post-transcriptional processing event that is important to the expression of organellar genes. Pentatricopeptide repeat (PPR) proteins are involved in this process. In this study, we report the function of a previously uncharacterized PPR-DYW protein, Empty pericarp17 (EMP17), in the C-to-U editing and kernel development in maize. EMP17 is targeted to mitochondria. The loss-function of EMP17 arrests maize kernel development, abolishes the editing at  The mitochondrion is a semi-autonomous organelle that provides energy and metabolites for cell activity. Plant mitochondrial genome inherits ∼5% genes from its prokaryotic ancestor, which encodes proteins of the respiratory complexes and ribosome, transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) . Post-trbona fide P-motifs with 35 amino acids, while the PLS-class proteins harbor P-, L- (35–36 amino acids), and S-motifs (31 amino acids), and often carry an E, E+, and/or DYW domain at the C-terminus. As ubiquitous RNA binding factors, PPR proteins were found to take part in almost all of the post-transcriptional processing in mitochondria and plastids   . ORRM prd ORRM6) , 2016. Od ORRM6) . A recend ORRM6) .c-type cytochromes: Cytochrome c (Cytc) and Cytochrome c1 (Cytc1). They are the essential factors of the oxidative phosphorylation (OXPHOS) chain. Cytc1, anchored in the inner membrane, is a core subunit of complex III. On the other hand, Cytc, peripherally associated with the inner membrane, shuttles electrons from complex III to complex IV  are involved in the cytochrome c maturation (CCM), referred to as the CCM pathway or system I  by the mitochondrial genes , in maize. The results demonstrate that EMP17 is required for the editing at four sites of emp17 mutant was isolated from the UniformMu mutagenic population in nearly isogenic W22 background (The ckground . The plaMu specific primer TIR8 (TIR8a:TIR8b:TIR8c:TIR8d = 1:2:2:1) were used to detect the Mu insertion in Emp17, and the Emp17-F1/Emp17-R1 primer pair was used to amplify the wild type Emp17.Genomic DNA (gDNA) was extracted by using a urea-phenol-chloroform-based method as described by Emp17 complementary DNA (cDNA) was placed downstream of the maize ubiquitin 1 (Ubi-1) promoter in the pUNTF vector. This pUNTF-Emp17 construct was transformed into maize inbred KN5585 via callus transformation. Positive transgenic plants of Emp17 were screened by using the Bar gene, and further verified by PCR using the Ubi-F primer anchored to the ubiquitin sequence in the vector and the Emp17 specific primer Emp17-R1.The protein-coding region of Emp17 encoding the 140 amino acids of the N-terminus region was cloned into pENTR/D-TOPO vector , and then transferred to the pBI221 vector to create the Emp17N140-GFP fusion by the Gateway site-specific recombination. The resulting construct, pBI221-Emp17N140-GFP, was introduced into Arabidopsis protoplasts. MitoTracker Red was used as a marker for mitochondria. The fluorescence signals were detected under a ZEISS LSM 880 confocal microscope.The 420 bp, 5′-sequence of emp17 kernels were harvested at 10 and 14 days after pollination (DAP) from the selfed ears of emp17 (+/−) heterozygous plants. The kernels were fixed in 4% paraformaldehyde at 4°C for 24 h. The fixed kernels were dehydrated, cleared, infiltrated, embedded, sectioned, stained, and observed as described by Wild type and http://www.qiagen.com) according to the manufacturer’s instruction. The potential gDNA contamination was removed through Dnase I  treatment. The single-stranded cDNA was obtained by reverse transcription reaction using the Transcriptor First Strand cDNA Synthesis kit . Quantitative real-time PCR (qRT-PCR) with SYBR green  was performed in a Roche Light Cycler 96. The relative transcript level was calculated as described in previous studies (ZmActin (GRMZM2G126010) was used as a control in RT-PCR and qRT-PCR. Detailed information of the primers was listed in Total RNA was extracted from developing kernels and other tissues of maize with the RNeasy Plant Mini Kit . For Western detection of complex proteins, 8 μg protein from emp17, and 8 μg, 4 μg (1/2), and 2 μg (1/4) protein from wild type were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes and detected by anti-Nad9, anti-Cytc1, anti-Cytc, anti-Cox2, and anti-Atp1 as described previously  in a reaction buffer containing 10 mM HEPES, 10 mM MES, and 2 mM CaCl2 (where pH was adjusted to 6.8 with KOH). Total respiration rate (Vt) was defined by the O2 consumption before adding any respiration inhibitor. The cytochrome pathway capacity (Vcyt) was determined by Vt minus the O2 consumption in the presence of 2 mM salicylhydroxamic acid (SHAM) and the alternative pathway capacity  was defined by Vt minus the O2 consumption in the presence of 2 mM potassium cyanide (KCN). All three respiration rates were expressed as nmol O2 min–1 g–1 fresh weight.The respiration rates were determined according to the protocol described by Emp17”  and then transiently expressed in Arabidopsis protoplasts. GFP signals were detected in punctated dots that merged with the mitochondria stained by the MitoTracker Red  insertion mutant (emp17) was isolated from the UniformMu mutagenic population (Mu3 element was confirmed to be inserted at +660 bp from the translation start codon of Emp17 (emp17 (+/−) heterozygous plants produced about 1/4 kernels with an empty pericarp phenotype  in the inbred line KN5585 by placing Emp17 under the Ubi-1 promoter. Eight independent transgenic lines (Emp17-OE1 to OE8) were obtained, and the expression level of Emp17 in these lines was 5–9.5 times higher than that in the wild type as detected by qRT-PCR (Emp17-OE1 with the emp17 (+/−) heterozygous plants and selfed the F1 to obtain the F2 progeny. The F2 seedlings were genotyped by PCR. To distinguish between the endogenous Emp17 and transgene Emp17, the Emp17-F1 primer was anchored to the 5’-UTR of the endogenous Emp17, ensuring that it could not anneal to the transgene that lacks this 5′-UTR sequence. The ubiquitin specific primer Ubi-F and Mu primer TIR8 were then used. Thus, the Emp17-F1/R1 primer pair amplified the endogenous Emp17, the Ubi-F/Emp17-R1 pair amplified the transgene, and the TIR8/Emp17-F1 pair detected the Mu3 insertion in Emp17. Genotyping 32 of the F2 seedlings identified 5 seedlings that were homozygous for emp17 harboring the Emp17 transgene (emp17 (−/−) seedlings, which were proven to contain the Mu3 element. These five seedlings showed normal growth and development compared with the wild type  is the causal gene for the empty pericarp phenotype of maize kernels in emp17.To confirm whether  qRT-PCR . We crosransgene . The Empild type , indicatemp17 kernels were substantially smaller than the wild type siblings throughout kernel development. At 14 DAP, the emp17 embryo and endosperm was smaller than the wild type , shoot apical meristem (SAM), and root apical meristem (RAM) were clearly developed in the wild type embryo  seedlings, the editing at nad2-677, ccmFC-799, -906, and -966 sites was restored , dicots , and monocots  (C and Nad2 proteins.The deficient editing at ectively . Alignme sativa) . The conC is involved in the maturation of Cytc and Cytc1, which are components of mitochondrial complex III  staining showed that the level of complex I was comparable between emp17 and wild type, but the levels of complex III and supercomplex I + III2 were remarkably decreased in emp17   . As indiy ZmAOX2 . Westernin emp17 .Vt, Valt, and Vcyt by using a Chlorolab II liquid-phase oxygen electrode, and specific inhibitors SHAM and KCN, respectively  in emp17 is higher than that by about twofold (43%) in wild type. These data confirm that mitochondrial complex III dysfunction severely reduces the cytochrome respiration and induces the alternative respiration pathway in emp17.To assess the physiological impact, we then measured the ectively . The ratectively . The ratA. trichopoda and mono- and dicotyledonous species. EMP17 shared an over 50% sequence identity with most of its orthologous proteins , and hypothetical protein (accession number KAG4215874) in G. hirsutum. All of these proteins share a less than 38% sequence identity with EMP17, much lower than that with the A. trichopoda homolog (54.7%). The results suggest that these species may have lost the Emp17 gene in the genome.Putative orthologous proteins of EMP17 can be found in many sequenced plant species in the NCBI and Uniport database . Phylogeproteins , i.e., 5g plants . HoweverC shows that the C-ccmF799 site is “T” in A. trichopoda and eudicots. However, in monocots, both “T” and “C” present   . In the  be lost . A consi as well . These rccmFC-799 and nad2-677 sites while reducing the editing at ccmFC-906 and ccmFC -966 sites  that are essential for the CCM and biogenesis of complex III   . CcmFC h in wtf9 . Consisttectable , and thee  in Arabidopsis (ccmFN and ccmFC) in maize  of CcmFC (267 in various CcmFC proteins is highly conserved across species (C function. It is known that Pro residue is a disruptor of protein α-helix and not favored in the β-sheet structures, as it has structure is limited and cannot complete the H-bonding network. For example, the Leu-to-Pro change in Nad7-279 and Atp6-213 and in the α-helix region is attributed to the destruction of the Nad7 and Atp6 function in the maize mutant emp18 and smk1 (267 in CcmFC was predicted in a β-sheet by the Swiss-model algorithm4 (267-to-Pro267 mutation in CcmFC in the β-sheet may negatively impact the structural stability of D6 that leads to non-functional CcmFC. The exact function of the D6 intermembrane space loop in CcmFC is unknown, however, these results imply that the Ser267 residue in D6 plays a critical role for the function of CcmFC.The lack of editing at apo-cytc . In planbidopsis , and twoin maize . Structu domains . Transmeof CcmFC . Alignme species , suggestand smk1 , 2019. Tgorithm4 . ProlineThe original contributions presented in the study are included in the article/YW and B-CT designed the research and analyzed the data. YW and X-YL conducted most of the experiments. FS performed the BN gel assay. Z-QH, Y-YL, and Z-QG participated in the linkage and genetic complementarity analysis. XW performed the respiration rate assay. YW, X-YL, Y-ZY, AS, and B-CT wrote the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, tumor cells adapt to survive by inducing autophagy, limiting the therapy effect. Therefore, anti-cell adaptation via autophagy inhibition could be used as a troubleshooting method to enhance tumor starvation therapy. Herein, we introduce an anti-cell adaptation strategy based on dendritic mesoporous organosilica nanoparticles (DMONs) loaded with GOx and 3-methyladenine (3-MA) (an autophagy inhibition agent) to yield DMON@GOx/3-MA. This formulation can inhibit cell adaptative autophagy after starvation therapy. Our in vitro and in vivo results demonstrate that autophagy inhibition enhances the efficacy of starvation therapy, leading to tumor growth suppression. This anti-cell adaptation strategy will provide a new way to enhance the efficacy of starvation cancer therapy.Cell autophagy is a well-known phenomenon in cancer, which limits the efficacy of cancer therapy, especially cancer starvation therapy. Glucose oxidase (GOx), which is considered as an attractive starvation reagent for cancer therapy, can effectively catalyze the conversion of glucose into gluconic acid and hydrogen peroxide (H These Mg2Si nanoparticles produced SiO2, which aggregates in tumor capillaries, causing extensive devascularization, and thus achieving significant tumor growth inhibition [2O2) in the presence of O2, leading to the simultaneous consumption of oxygen and glucose, and consequently starvation [Deriving from an ancient concept of anti-angiogenesis, cancer starvation therapy implemented via ensuring an insufficient supply of nutrition and oxygen by obstructing blow vessels, has been proposed as a new antitumor approach ,2. With hibition . Subsequhibition ,5. Notabhibition . In conthibition ,8. Therearvation ,10,11. Harvation . Therefoarvation , photodyarvation , phototharvation , and chearvation .3O4 for fabrication of the sequential nanocatalyst, which exhibits excellent catalytic performances for efficient tumor therapy [Autophagy is a ubiquitous process that induces the breakdown of abnormal cellular constituents within lysosome in response to a variety of stress conditions, including DNA damage, chemotherapy, protein folding errors, microbial infection, organelle injury, radiotherapy, or starvation ,18,19, a therapy . Thus, i2O2 through the following reaction:Motivated by these facts, we constructed DMON nanoparticles for co-delivery GOx and 3-MA, creating the nanoparticles DMON@GOx/3-MA, which releases its 3-MA to enhance GOx-induced cancer starvation. As shown in Equation (1), GOx catalyzed the oxidation process of glucose to gluconic acid and HThe change in the intracellular environment caused by the lowered levels of oxygen and glucose activates autophagy to protect cells from energy starvation. However, this process is efficiently inhibited by 3-MA, sensitizing GOx-induced tumor starvation therapy . OverallCetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), 1,2-bis(triethoxysilyl)ethane (BTEE), triethanolamine (TEA), glucose oxidase (GOx), 3-methyladenine (3-MA) and sodium salicylate  were purchased from Sigma-Aldrich . 1-Ethyl-3(3-dimethylpropylamine) carbodiimide (EDCI) and N-hydroxysulfosuccinimide (NHS) were purchased from Macklin . Ethanol and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. . All chemicals were used as received without further purification.g, 10 min), washed three times with 20 mL ethanol to remove the residual reactants, and finally redispersed in ultrapure ethanol. Next, DMON nanoparticles were then functionalized with amino (-NH2) groups. First, the DMON nanoparticles were dispersed in ethanol  and placed in an oil bath (78 °C) with magnetic stirring. Then 3-aminopropyltriethoxysilane (APTES) (50 μL) was added into the above solution and kept stirring for another 12 h. Finally, the amino decorated DMONs (DMON-NH2) were collected by centrifugation , washed three times with ethanol (20 mL) and deionized water (20 mL) before being redispersed in deionized water (50 mL) for further use.In a typical synthesis of DMONs, TEA (0.068 g) was added to water (25 mL) and stirred at 80 °C in an oil bath for 0.5 h. Then, CTAB (380 mg) and NaSal (126 mg) were added to the above solution with stirring for 5 h. Subsequently, a mixture of BTEE (1.6 mL) and TEOS (2 mL) was added to the abovementioned solution with vigorous stirring for 12 h. Then, the products were collected by high-speed centrifugation  water solution with vigorous magnetic stirring. Then, EDCI  and NHS  were added to the resulting solution. After 12 h of reaction, the GOx decorated DMON-NH2 (DMON@GOx) nanoparticles were collected by centrifugation  and washed three times with ethanol (20 mL) and deionized water (50 mL) before being redispersed in deionized water for further use.For the synthesis of DMON@GOx nanoparticles, GOx powder (2 mg) was added to DMON-NHg, 10 min).For the synthesis of DMON@GOx nanoparticles, 3-MA (2 mg) was added into DMON/GOx  water solution with vigorous magnetic stirring. After 12 h of reaction, the 3-MA was loaded onto the dendritic pores of DMON@GOx nanoparticles. Then, DMON@GOx/3-MA nanoparticles were collected by centrifugation  and washed three times with deionized water (20 mL) to remove the unloaded FITC.For the synthesis of fluorescein isothiocyanate (FITC)-labeled DMON@GOx/3-MA nanoparticles, FITC (1 mg) was added into DMON/GOx/3-MA  water solution under vigorous magnetic stirring. After 8 h, FITC was loaded on the dendritic pores of DMON@GOx/3-MA nanoparticles. Then, FITC-labeled DMON@GOx/3-MA nanoparticles were collected by centrifugation  patterns were analyzed on an Ultima IV X-ray diffractometer  utilizing Cu Kα (λ = 1.5405 Å) radiation along with scan range from 10° to 80°. Transmission electron microscopy (TEM) images were captured on a JEOL JEM-2100 microscope operated at 200 kV . All the TEM samples were obtained by dropping dilute nanomaterials onto carbon coated copper grids. The particle size and zeta potential analyzers were measured on Microtrac Nanotrac Wave II Dynamic light scattering . Ultraviolet Visible (UV-VIS) spectra were recorded on a UV-3600 Plus spectrophotometer . Fourier transform infrared (FTIR) spectra were recorded on a TENSOR II FT-IR spectrophotometer  using KBr pellets. CLSM images were obtained on a Nikon A1Rt-980 confocal microscope .To measure the catalytic ability of DMON@GOx nanoparticles, different concentrations of glucose solutions  were added to DMON@GOx solution (GOx = 0.5 mg/mL) in an open 96-well plate. After 2 h of sufficient catalytic reaction, 50 μL of titanous sulfate reagent (20 mM) was added to each well. Then the absorbance of each well at 415 nm was recorded using a Spark microplate reader .In order to release the performance of 3-MA in simulated body fluid (SBF) solution, one milliliter of DMON@GOx/3-MA (10 mg/mL) was added to the dialysis bag  and then transfer to a beaker with 20 mL of SBF buffer (pH = 7.4 or 5.4). At different time points, one milliliter of SBF solution was collected and measured by high phase liquid chromatography (HPLC). HPLC parameter settings—Mobile phase: ammonium acetate (30 mM): acetonitrile = 95:5 (by volume); Determine wavelength = 240 nm. Chromatographic column: C-18 reverse-phase column.2 atmosphere.4T1 cells were obtained from the Shanghai Institute of Cells, Chinese Academy of Sciences. The 4T1 cells were cultured in Dulbecco’s modified Eagle medium (DMEM)  with 10% heat-inactivated fetal bovine serum (FBS), streptomycin (100 mg/mL) and penicillin (100 U/mL). The cells were incubated in standard incubators at 37 °C, in a 5% CO4 per well, then treated with different concentrations of DMON@GOx, DMON@GOx/3-MA, or glucose for 24 h. Next, the culture medium was discarded, and 100 μL of MTT medium solution (0.8 mg/mL) was added to each well. Each plate was then incubated at 37 °C for 4 h. After incubation, the MTT medium solution was aspirated, and 100 μL of DMSO solution was added to each well. The OD490 value of each well was then measured on a Bio-Tel ELx800 microplate reader using a detection wavelength of 490 nm. The cell survival rate was calculated using the following formula: Cell viability (%) =  × 100%.The cytotoxicity of the DMON@GOx, DMON@GOx/3-MA and glucose on the 4T1 cells were evaluated using the standard methyl thiazolyl tetrazolium (MTT) method, and each data point was described using the mean and standard deviation of 6 replicates. The specific experimental procedure was as follows: The 4T1 cells were seeded into 96-well plates at a density of 1 × 104T1 cells plated into 6-well plates were treated with DMON@GOx and DMON@GOx/3-MA for 24 h. The cells were lysed with 100 μL of RIPA lysis buffer  supplemented with protease inhibitor (PMSF 0.5 mM). The equal amounts of protein added with 2× Laemmli loading buffer  were denatured by boiling the sample at 100 °C for 10 min. After that, the proteins were separated on 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes . After blocking with 5% nonfat milk, the PVDF membrane was incubated with a primary antibody, such as anti-P62, anti-LC3B, anti-caspase3, Actin and GAPDH  at 4 °C overnight. Then the membrane was incubated with a related secondary antibody (Alexa Fluor 680 Goat anti-Rabbit IgG (H + L) and Alexa Fluor 690 Goat anti-Mouse IgG (H + L), Jackson, West Grove, PA, USA) for 1 h at room temperature, and finally scanned using an Odyssey infrared imager .6/dish) treated with 400 μg/mL of DMON@GOx/3-MA were placed at 37 °C incubator for 24 h. The cells were then washed twice with PBS buffer and digested with 0.25% trypsin. The cells were collected by centrifugation  and fixed with 1 mL glutaraldehyde at room temperature. Then, washed with 1 mL PBS buffer, the cells were dehydrated with a gradient of ethanol  and washed with 1 mL propylene oxide. The cells were embedded in epoxy resin EPOM812 and polymerized at 37 °C for 12 h, 45 °C for 12 h and 60 °C for 48 h. The epoxy resin was cut into 70 nm sections, poststained with 4% uranyl acetate and 2.5% glutaraldehyde for 10 min and imaged at 100 kV on a transmission electron microscope (TEM).First, 4T1 cells (104/well) were treated with PBS, DMON@GOx, and DMON@GOx/3-MA for 24 h. The suspended and adherent cells in each well were collected by centrifugation, washed twice with PBS, and stained with 2 μL of fluorescein isothiocyanate (FITC)-labeled annexin V (Annexin V-FITC) and 2 μL of propidium iodide at room temperature for 30 min in the dark. The apoptotic cells were detected by flow cytometry and analyzed using the FlowJo software.4T1 cells seeded in 6-well plates  and LC3B (1 μg/mL) at 4 °C overnight. Cells were incubated with secondary antibody (1:500) at room temperature for 1 h. Then incubated with phalloidins (1:400) at room temperature for 1 h. Finally, all samples were incubated with DAPI for 10 min and observed by CLSM.4T1 cells were seeded in confocal dishes, then infected with Ad-virus expressing mRFP-GFP-LC3 (tfLC3)  2 μL/dish, refresh the medium after 6 h. After infected 24 h, treated with PBS, DMON@GOx, DMON@GOx/3-MA for 24 h. The cells were examined under CLSM.All animal experiments were performed according to the Principles of Laboratory Animal Care (China) and the Guidelines of the Animal Investigation Committee. Animal testing procedures followed the guidelines of the Animal Protection and Use Committee.6 cell/site) were injected subcutaneously into the female Balb/c nude mice (~20 g). In vivo therapy experiments were performed when the tumor reached 6 mm in average diameter (7 days after implant). The mice were divided into five groups. The first group of mice received PBS, as the control group; the second group was intravenously injected with DMON@GOx once a day for day 1–3 , as DMON@Gox (i.v.) group; the third group was intravenously injected with DMON@GOx/3-MA once a day for day 1–3 , as DMON@GOx/3-MA (i.v.) group; the fourth group was intratumorally injected with DMON@GOx at day 1 , as DMON@GOx (i.t.) group; the fifth group was intratumorally injected with DMON@GOx/3-MA at day 1 , as DMON@GOx/3-MA (i.t.) group. After the corresponding treatments, the volume of each tumor was measured every other day and calculated by the following equation: V = L × W2/2. Furthermore, the tumors were sectioned into slices for TUNEL staining.4T1 cells  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. . These mice were intravenously injected with DMON@GOx/3-MA at a calculated dose of 75 mg/kg. The mice were sacrificed at day 0, 3 and day 30. The major organs  were dissected, fixed in a 10% formalin solution and stained with hematoxylin and eosin (H&E) for histological analysis. Alanine aspartate aminotransferase (AST), nephric blood urea nitrogen (BUN), aminotransferase (ALT), alkaline phosphatase (AKP) and creatinine (CREA) indicators, together with the complete blood panel parameters including red blood cells (RBC), white blood cells (WBC), red blood cell distribution width-standard deviation (RDW-SD), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), hematocrit (HCT) and lymphocyte (LYM) of all groups by using authoritative standard biochemistry test.2  and 1,2-bis(triethoxysilyl)ethane (BTEE) with the assistance of cetyltrimethylammonium bromide (CTAB) and sodium salicylate . As the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images in 2 groups for the further modification of GOx, as shown in 2 turned negative (−30.4 mV) to positive (+19.7 mV) after the -NH2 functionalization. Then the GOx was loaded on DMON by the condensation reaction of NH2- (in DMON-NH2) and -COOH (in GOx). In the end, the 3-MA was loaded on the dendritic pores of DMON@GOx and formed the final formulation (DMON@GOx/3-MA). As indicated in 2 and DMON@GOx/3-MA were recorded. As we can see from 2, DMON@GOx and DMON@GOx/3-MA were carried out to determine the loading capacities of GOx and 3-MA. By comparing the weight loss curves of different samples  media with different pH (7.4 and 5.4) values were used to imitate the in vivo neutral healthy body fluids and mildly acidic tumor microenvironment. As shown in To determine the biological activity of DMON@GOx/3-MA, the process of endocytosis of DMON@GOx/3-MA was firstly visualized via confocal laser scanning microscopy (CLSM). As shown in To further evaluate the activity of DMON@GOx/3-MA, a cell apoptosis assay was performed. Similar to the result of MTT, PI/Annexin V staining showed that DMON@GOx/3-MA triggered 40.80% cell apoptosis in 4T1 cells, which was nearly three times more than DMON@GOx did  assay, a valuable tool for examining autophagosome maturation and autolysosome formation ,34. LC3 6 cell/mouse) were subcutaneously implanted into female Balb/c nude mice (~20 g) and then intravenous injection (i.v.) or intratumoral injection (i.t.) with PBS, DMON@GOx or DMON@GOx/3-MA when the tumor diameter reached about 6 mm. DMON@GOx and DMON@GOx/3-MA significantly inhibited tumor growth in 4T1 cells no matter i.v. or i.t. injection, however, the tumor growth rate in mice treated with DMON@GOx/3-MA (i.t. or i.v.) was much slower than those tumors treated with DMON@GOx (i.t. or i.v.), respectively, demonstrating autophagy inhibition enhanced GOx-induced starvation therapy . In addition, histological analyses  of the organ tissues, such as heart, liver, spleen, lung and spleen of these groups were performed, and there were no evident physiological and morphological abnormalities in these organs , cancer cells are more sensitive to changes in intracellular glucose and O2 concentration [2 in tumor cells and meanwhile produces a considerable amount of H2O2, which gives GOx a great prospect for application in cancer starvation therapy. However, cancer cells can harness intrinsic protective and adaptative autophagy to eat or digest their own cytoplasmic components for nutritive compensation, limiting the efficiency of starvation therapy. Thus, the combination of tumor starvation therapy with anti-autophagy therapy would deplete exogenous and endogenous nutrition, leading to a remarkable synergetic antitumor outcome. Notably, just as in tumor starvation therapy, tumors cell may also induce adaptative autophagy to counteract other kinds of tumor therapies, such as photothermal therapy, photodynamic therapy, radiotherapy, and part of chemotherapy [Starvation therapy is an effective strategy for suppressing tumor growth and tumor survival by blocking blood flow or depleting nutrients/Ontration . Therefootherapy . TherefoIn this study, we first prepared biodegradable DMONs, and then we aminated DMON using APTES. Through the EDC/NHS chemistry method, we successfully loaded GOx covalently into DMON to prepare DMON@GOx. After that, we successfully loaded the autophagy inhibitor 3-MA into the dendritic pores of DMON to prepare the final formulation-DMON@GOx/3-MA. Through a TGA detection method ,37, we dIn this study, we found that DMON@GOx exhibited higher cytotoxicity than DMON + GOx groups, and DMON@GOx/3-MA exhibited higher cytotoxicity than DMON + GOx + 3-MA groups, demonstrating the cytotoxicity of nanoparticle formulation is better than the mixture of the components. To determine if autophagy is involved in GOx-mediated cell death, autophagy inhibitor 3-MA was used to perform cell proliferation assay (MTT) and cell apoptotic assay (PI/annexin V and Western blot). All these results demonstrate that 3-MA enhanced the GOx-induced starvation therapy. To further investigate how autophagy is involved in this process, two important proteins LC3B and P62 were used as autophagy markers. LC3BII is a marker of autophagosome formation, which is generated by the conjugation of cytosolic LC3BI to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. P62 is a marker of autophagic flux, which is an autophagy substrate that is negatively related to autophagy. The reduction of p62 has been regarded as a marker for the increase of autophagic flux ,39. SurpIt has been reported that 3-MA has dual functions . Wu et a2 are higher in an in vitro culture system than in vivo. There is 4.5 g/L of glucose (25 mM) in a DMEM culture medium, and an adequate concentration of O2 in incubators, which are more than available in physiological conditions (approximately 7 mM glucose and an appropriate amount of O2). This phenomenon is also consistent with the studies published before [2) in vivo, resulting in the higher sensitivity to tumor starvation therapy. Therefore, the starvation therapy efficiency of DMON@GOx/3-MA in vivo is better than in vitro. It has been shown that there is no significant difference in the tumor volume after administration of DMON@GOx/3-MA (i.t. or i.v.) in In this study, DMON@GOx/3-MA shows relatively modest cytotoxicity in vitro. However, the cytotoxicity of DMON@GOx/3-MA in vivo is relatively outstanding. We hypothesize that the levels of glucose/Od before . In addid before , the 4T1In summary, an enhanced tumor starvation therapy was achieved via autophagy inhibition with the GOx and 3-MA co-loaded DMON. After DMON@GOx/3-MA accumulates in tumors, GOx locally consumes glucose and oxygen to starve cells, leading to tumor-associated metabolic abnormalities that activate autophagy. Furthermore, 3-MA severely blocks auto-phagolysosome formation, augmenting the efficacy of tumor starvation. Our study offers an anti-cell adaptation strategy for enhanced tumor starvation therapy via autophagy regulation in cancer cells, which is promising for effective cancer treatment. This anti-cell adaptative strategy would also be beneficial to other autophagy-related cancer therapies such as oxidative stress therapy, hyperthermia therapy, etc.


Various characterization techniques such as XRD, FE-SEM and EDS were used to establish its nano-sized chemical structure. It was determined that Pd-Fe3O4-CWH is a useful nanocatalyst, which can efficiently reduce various nitroarenes, including 4-nitrobenzoic acid (4-NBA), 4-nitroaniline (4-NA), 4-nitro-o-phenylenediamine (4-NPD), 2-nitroaniline (2-NA) and 3-nitroanisole (3-NAS), using NaBH4 in aqueous media and ambient conditions. Catalytic reactions were monitored with the help of high-performance liquid chromatography. Additionally, Pd-Fe3O4-CWH was proved to be a reusable catalyst by maintaining its catalytic activity through six successive runs. Moreover, the nanocatalyst displayed a superior catalytic performance compared to other catalysts by providing a shorter reaction time to complete the reduction in nitroarenes.In the present study, a novel heterogeneous catalyst was successfully fabricated through the decoration of palladium nanoparticles on the surface of designed Fe Due to the increasing demand for sustainable recovery and the exploitation of biowaste, the conversion of biowaste into valuable products has attracted a lot of attention. At the same time, the atmospheric emissions from the use of fossil fuels cause problems such as global warming, climate change and environmental pollution. Furthermore, fossil fuel reserves are rapidly depleting, while energy consumption is increasing dramatically ,2. UntilHydrochar is a carbon-based material that is prepared by the hydrothermal carbonization of high moisture biomass waste, such as sewage sludge, algae or grass, in an aqueous environment at temperatures in the range of 180–260 °C . BiocharSome examples of pollutants of concern today include heavy metals, herbicides, oil spills, pharmaceuticals and fertilizers. Compounds containing nitro groups have been determined in aqueous environments . Due to 3O4 particles, followed by the dispersion of Pd nanoparticles on the magnetic substrate surface. The chemical structure and composition of the nanocatalyst (referred to as Pd-Fe3O4-CWH thereof) were determined by various imaging and spectroscopic methods. Pd-Fe3O4-CWH was then applied as heterogeneous nanocatalyst for the reduction in 4-nitrobenzoic acid (4-NBA), 4-nitroaniline (4-NA), 4-nitro-o-phenylenediamine (4-NPD), 2-nitroaniline (2-NA) and 3-nitroanisole (3-NAS), using NaBH4 as a reducing reagent. The respective aniline products were determined by high performance liquid chromatography. A detailed investigation of the mechanism of reduction in the nitro groups was beyond the scope of this study. Finally, the reusability of the nanocatalyst was investigated by applying it in six successive catalytic runs.In the framework of circular bio-economy, the rationale behind this work is to develop a novel pathway for the utilization of coffee waste and the production of a high added-value material. Therefore, spent coffee grounds were converted to hydrochar through hydrothermal carbonization. This process helps to increase the structural and chemical stability of the coffee grounds. The resultant hydrochar was then used as a substrate for the deposition of Fe4, 99%), FeSO4·7H2O (4.2 g), FeCl3·6H2O, PdCl2, ethanol and methanol were purchased from Merck Chemical . Hydrothermal carbonization was performed in a Berghoff Ins.-Heidolph MR Hei-standard reactor . Reductions in the nitro compounds were monitored by using a PerkinElmer Flexar Series HPLC system . SEM images and EDS of CWH, Fe3O4–CWH and Pd-Fe3O4-CWH were recorded in a Supra 55 field emission (FE) microscope . TEM images of Pd-Fe3O4-CWH were obtained in a JEOL JEM-1011 instrument. A SmartLab SE instrument Rigaku, Tokyo, Japan) was used to obtain the XRD patterns for the nanocatalyst. The exact Pd loading on Pd-Fe3O4-CWH was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) .Spent coffee grounds were collected from a coffee shop. All nitro aromatic compounds, sodium borohydride  and FeCl3·6H2O (6.1 g) were dissolved in 100 mL distilled water and heated to 90 °C. Ammonium hydroxide (10 mL-26%) and a suspension of 1 g of CWH in 200 mL of water were mixed, the mixture was stirred at 90 °C for 40 min and, finally, cooled to 25 °C. Fe3O4–CWH was collected as a black precipitate by filtering, being repeatedly washed with distilled water until a neutral pH was reached, dried at 70 °C for 18 h and stored. The next procedure was applied to load the Pd nanoparticles onto Fe3O4–CWH. A total of 0.25 g of Fe3O4–CWH was suspended in 30 mL water and a specific quantity of Na2PdCl4 (as the Pd precursor) was added, representative of a 5% Pd loading. After 40 min of stirring at 25 °C, an ascorbic acid solution (nascorbicacid:nPd ¼ 2:1) was added and allowed to react for 130 min. After filtration, the solid catalyst was rinsed repeatedly with distilled water. Pd-Fe3O4-CWH was recovered with extremal magnet after drying at 80 °C for 12 h. The preparation of Pd-Fe3O4-CWH nanocatalyst is presented in Feus study . First, 3O4-CWH was transferred into 1 mL of nitro compound (3 × 10−4 M), followed by stirring for 1 min at room temperature. Freshly prepared NaBH4  was then added to the reaction medium and the nitro compound reduction was followed by HPLC. Finally, the nanocatalyst was removed from the reaction media by a magnetic bar and reactivated by washing with water before using it for subsequent runs. Kinetic studies were performed at 25 °C by using 4-NBA as the model substrate and an excess concentration of NaBH4.For the reduction in the nitro compounds to the respective amino derivatives, 20 mg of Pd-Fev/v acetonitrile/water, to which 1% acetic acid was added. The flow rate was set at 1.0 mL·min–1 and the injection volume at 10 μL. UV detection was set at 270 nm.The analysis of the reduced nitro-aromatic compounds was performed by using a PerkinElmer Flexar Series HPLC system . Separation was achieved on a ZORBAX SB Phenyl column  maintained at 25 °C. The mobile phase used was 20/79 3O4-CWH. FE-SEM images of CWH showed an irregular but porous surface morphology , (311), (400), (422), (511) and (440) planes [3O4–CWH surface.) planes   planes ,20 were 3O4-CWH nanocatalyst was performed and the corresponding images are displayed in 3O4-CWH.For a more detailed surface morphology, a TEM analysis of the Pd-Fe3O4-CWH nanocatalyst by the display of Fe 2p (709.4 eV (2p3/2), 723.8 eV (2p1/2)) and Pd 3d (335.3 eV (3d5/2), 341.6 eV (3d3/2)) of metallic Pd(0), respectively, in the spectrum [The XPS analysis  also conspectrum ,22. The 4 as a hydrogen source. To test the catalytic performance of Pd-Fe3O4-CWH and establish the optimal reaction conditions  in the reduction in nitro compounds, 4-NA was selected as a representative substrate and control studies were performed (−4 M of nitro compound and 0.4 mL of NaBH4 (0.08 M). Subsequently, the catalytic behavior of Pd-Fe3O4-CWH was examined in the reduction in 2-NA, 4-NBA, 4-NPD and 3-NAS reductions with the detected optimal conditions and the obtained findings shown in 3O4-CWH nanocatalyst provided a complete reduction in 4-NBA within 60 s. Pd-Fe3O4-CWH catalyzed the 4-NA reduction within 82 s. Furthermore, the reduction in 2-NA proceeded quickly and was completed within 90 s. In a similar manner, the catalytic reductions in 4-NPD and 3-NAS were completed within 168 and 428 s, respectively. On the other hand, the reduction in 4-NA was performed without the Pd-Fe3O4-CWH catalyst, and no product formation was observed after 1 h. This result revealed the importance of the Fe3O4–CWH catalyst against the reduction in nitroarenes. Additionally, based on previous studies, the possible reaction mechanism for the Pd-Fe3O4-CWH catalyzed reduction in nitroarenes was given in The performance of the nanocatalyst was explored by employing it in the catalytic reduction in various nitro compounds in the presence of NaBHerformed . The proerformed . As seen3O4-CWH over other reported catalysts, its catalytic performance for the reduction in 2-NA was compared to that of other catalysts reported in the literature. As seen in To indicate the superiority of Pd-Fe4 meant that the rate constant could be assumed to be independent of the NaBH4 concentration and a pseudo-first-order kinetics model could be applied to the reduction in 4-NBA [k) value was calculated from the slope of the following equation:t and 4-NBA0 are the 4-NBA concentration at time t and initial concentration, respectively. As indicated by the regression coefficient (R2 = 0.9829), the reduction data fitted very well to the pseudo-first-order model (−1, indicating a kinetically unhindered process with no induction period, in contrast to some earlier studies that showed that Pd catalysts have an initial stage of reduced performance during the reduction in nitro compounds [For the kinetic study, a high excess of NaBHin 4-NBA . The pseer model . This obompounds ,39,40.3O4-CWH was evaluated on 4-NBA reduction. The nanocatalyst was readily separated from reaction media magnetically after the 4-NBA reduction and rinsed with water to use in the further reactions (3O4-CWH was reused up to a number six runs without any significant loss of catalytic performance (One of the most important properties of heterogeneous catalysts is their reusability, a valuable attribute for industrial applications. Therefore, the recycling potential of Pd-Feeactions . Reusabiformance .3O4 and Pd nanoparticles were deposited on the hydrochar surface through a simple process and the resultant catalyst exhibited a positive performance in the reduction in nitroarenes. Kinetically, the nanocatalyst showed no induction period, achieving complete reductions for a wide range of nitro compounds in very short times. Furthermore, the stability and reusability of the catalyst was established by collecting it through the use of a magnet and applying it in successive reaction runs. Further work should focus on testing the performance of the catalyst in the reduction in or oxidation reactions of other organic contaminants and investigating the economics of the process in detail, identifying necessary steps before scaling-up application.It was demonstrated that coffee waste hydrochar can be used as a substrate for the development of an efficient nanocatalyst. Overall, our approach combined concepts of waste management, circular bio-economy and wastewater treatment. Therefore, biomass-based materials can provide sustainable building blocks for the production of catalysts for environmental remediation. Fe


Amyand’s hernia was an unusual condition defined by the presence of an appendix located in the inguinal hernia sac. Its prevalence was 1% of all inguinal hernia in children. The clinical manifestation of Amyand’s hernia was depending on hernia’s stage, an incarcerated hernia will present with an inguinal mass following by pain and motility disorder. It could lead to abdominal distention in the late stage. The common location of Amyand’s hernia was on the right side, the left side was uncommon. Early feeding on infants could provoke symptoms of bowel obstruction (SBO). More than 76% of infants in Java, Indonesia was given banana as solid food in infants before six months old. There is a correlation between the early banana diet and SBO. Amyand’s hernia could present as morbidity of early banana diet.We describe a case of two months old infant present with an incarcerated left inguinal hernia and history of early banana diet that performed herniotomy procedure. During the operation, we found left-side incarcerated Amyand’s hernia with appendicitis, excoriation caecum, and sticky banana mass.This case suggest the possibility of early feeding of banana diet may provoke incarceration of an inguinal hernia and if the incarcerated hernia content contains the appendix, then an Amyand's hernia. The inguinal hernia was defined as a protrusion of fascia or visceral organ through the abdominal wall into the inguinal canal. In children, an inguinal hernia is caused by obliteration failure of processus vaginalis during gonad development . Rarely,The clinical manifestation of Amyand’s hernia was difficult to differentiate with another inguinal hernia. In the incarceration stage, it’s common to present with an inguinal mass following by pain and motility disorder. In the late stage could lead to abdominal distention as symptoms of bowel obstruction (SBO). Amyand’s hernia was common to present on the right side based on the appendix vermiform position but in a rare condition, it could find in the left-side. Left-side Amyand’s hernia could be associated with situs inversus, intestinal malrotation, and mobile caecum .The early banana diet is very common in Indonesia, especially in Java. More than 76% of infants in Java, Indonesia is given banana as solid food in infants before six months old. There is a correlation between the early banana diet and SBO .A 2 months old male infant was taken to Emergency Department Soebandi Hospital with abdominal distention and bilious vomiting. The symptoms were present after the left inguinal hernia appear and could not be reduced for 12 h before. A left inguinal hernia was present from two weeks old and could be reduced spontaneously before. An early banana diet was given to the patient one day before the symptoms appear. No other food was given except breast milk.From the physical examination, we found abdominal distention with hypertympanic and increasing bowel sound as a sign of intestinal obstruction Fig. . We alsoFluid resuscitation and nasogastric tube decompression were performing followed by broad-spectrum antibiotics. Then the patient planned to a herniotomy. During the operation, we found left-side incarcerated Amyand’s hernia with appendicitis and erythematous caecum. No perforation was performed. We also found sticky banana mass to support the suggestion of early banana diet as a predisposing factor Fig. ..Fig. 3DThen we perform an appendectomy, reposition of the caecum to the abdomen, and ligation of the sac. It has a good result. There was no postoperative complication. It has a good functional bowel and the patient could take oral breast milk gradually increase. The patient’s parent was satisfied with this treatment.Amyand’s hernia was not easily diagnosed pre-operatively. It was difficult to differentiate with another inguinal hernia. The patient could come to the Emergency Department with inguinal mass associated with pain and SBO or inguinoscrotal swelling as a sign and symptom of an incarcerated hernia. Fever could present at appendicitis but not always present. At late presentation, abdominal distention and vomiting could present. It depends on the condition of appendix vermiform whether inflammation or perforated . RadioloAppendicitis could perform following Amyand’s hernia. The possibility caused was obstruction of incarcerated hernia restricts appendix blood flow and bacterial growth lead to inflammation and perforation. Adhesion and obstruction of the hernia neck caused the appendix trapped in the hernia sac . ClassifThe right-side inguinal hernia was the most common condition of Amyand’s hernia. Left-side was uncommon. Cigsar et al. reported from 11 years of experience to treat Amyand’s hernia, left-side Amyand’s hernia occurs in 4.3%. It could be associated with situs inversus, intestinal malrotation, and mobile caecum. Mobile caecum was the only finding based on the research while situs inversus and intestinal malrotation was still a suggestion based on theory . In oyr The early banana diet was very popular in Indonesia. More than 74% infant was given by early banana diet as solid food before six months old. The culture associated and lack awareness of potential danger has been suggested as a predisposing factor of early banana diet. An early banana diet could induce symptoms of bowel obstruction (SBO). Wiryo et al. reported from 3420 neonates in Indonesia, the relative risk of early banana diet that provokes SBO was 9.18. It was concluded that an early banana diet was an important risk factor of SBO . The carAmyand’s hernia was a rare condition of inguinal hernia and left-side Amyand’s hernia was even rarer. The early banana diet was very common in Indonesia. It could provoke the incarceration of an inguinal hernia that was presented before. This case suggest the possibility of early feeding of banana diet may provoke incarceration of an inguinal hernia and if the incarcerated hernia content contains the appendix, then an Amyand's hernia. This case could be a good example to educate the mothers in Indonesia to prevent giving early banana diet in infants. Therefore, it was necessary to educating not giving an early banana diet to prevent intestinal obstruction in infants.


Tension regulates α-catenin conformation. Actomyosin-generated force stretches the middle (M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry showed that αT-catenin binds the β-catenin–N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates the M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell–cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results show that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes. Cnt cells , 5, 6. Tnt cells , 12, 13.The best characterized member of the α-catenin family of proteins is mammalian epithelial (αE)-catenin. Structurally, it is composed of 5 four-helix bundles connected to a C-terminal five-helix bundle by a flexible linker , 15, 16.Three α-catenin family proteins are expressed in mammals: the ubiquitous αE-catenin, neuronal (αN)-catenin, and testes (αT)-catenin , 37. αE-Despite a growing awareness of its importance in human disease, the molecular properties and ligand interactions of αT-catenin remain poorly understood. Our previous work revealed that αT-catenin, unlike mammalian αE-catenin, is a monomer in the solution that can bind to F-actin with low micromolar affinity in the absence of tension. F-actin binding is also not allosterically regulated, as the β-catenin–αT-catenin complex binds to F-actin with the same affinity as the αT-catenin monomer . Single-in vitro, revealing that they may compete with one another for binding β-catenin at the plasma membrane. We also show that the M-region of αT-catenin is not autoinhibited and can bind the vinculin N-terminus in the absence of tension with strong affinity. Unlike αE-catenin, however, when the N-terminus of αT-catenin is attached to the M-region, the affinity for vinculin drops significantly. This indicates that interdomain interactions between the N-terminus and the M-region of αT-catenin regulate its interaction with vinculin. We measured the recruitment of vinculin to cell–cell contacts and found that, despite the distinct mechanism of regulation, recruitment of vinculin is still tension dependent. Our findings indicate that the way in which tension regulates vinculin binding differs between αE-catenin and αT-catenin. We postulate that this mechanism is important for the ability of αT-catenin to build specific and distinct molecular complexes at AJs.Here we show that αT-catenin associates with the components of the cadherin–catenin complex with an affinity similar to αE-catenin A) for these experiments because it is more stable than full-length αT-catenin and yields sufficiently high protein concentrations for ITC. Past studies revealed that the αE-catenin head region (aa 1–651) binds β-catenin and the β-catenin–E-cadherin tail complex with a similar affinity to full-length αE-catenin  using purified recombinant proteins. We used the head region (comprising the N- and M-domains) of αT-catenin .Figure cyto) influences the αT-catenin–β-catenin interaction by titrating the β-catenin–Ncadcyto complex into αT-catenin head (C). The affinity of this interaction was 5 to 6 nM  and compared them to the V94D mutant. Analytical size-exclusion chromatography of αT-catenin WT N1-N2 and V94D N1-N2 revealed that WT N1-N2 eluted as a single monomer species , whereas the V94D mutant eluted as a dimer species .Full-length αT-catenin is primarily a monomer in the solution, although it does have homodimerization potential in vitro . The besin vitro , 44. We in vitro A and comB). Incubation with BS3 did not affect migration at low concentrations, although at higher concentrations (4 and 8 μM), larger species were detected at both temperatures. In contrast, αT-catenin V94D N1-N2 ran as 50-kDa protein in the presence of BS3 at all concentrations tested (C), indicating a cross-linked dimer. We conclude that the αT-catenin N terminus, similar to full-length protein, is primarily a monomer in the solution.We then analyzed the oligomeric state of the αT-catenin N-terminus by cross-linking. Increasing concentrations of αT-catenin N1-N2 were incubated with or without the cross-linker bis(sulfosuccinimidyl)suberate (BS3) at 4 °C or 37 °C and the resulting products analyzed by SDS-PAGE. As expected, the αT-catenin N1-N2 migrated as a 25-kDa protein in the absence of a cross-linker B. Incubas tested C, indicacyto complex by ITC. The affinity of this interaction was 7 nM . We then probed the M-region flexibility by limited trypsin proteolysis. Tryptic digestion of αE-catenin M1-M3 revealed that it was largely protease resistant: nearly 50% of the fragment was still intact after 120 min of digestion, consistent with it forming a closed, autoinhibited domain . In contrast, αT-catenin M1-M3 was completely digested after 60 min into three stable fragments at 23, 16, and 12 kDa . Note that both M regions contain a similar number of lysine and arginine residues (47 in αE-catenin and 39 in αT-catenin), the majority of which are conserved. N-terminal sequencing revealed that the 23-kDa and 16-kDa fragments both started at aa 379 and represented the M2-M3 and M2 bundles, respectively. The 12-kDa fragment started at aa 485 and corresponded to the M3 bundle . Similar protease-resistant fragments were observed from digest of full-length protein . αT-catenin M1-M3 eluted as a single, discrete peak by size-exclusion chromatography, identical to αE-catenin M1-M3  that contains the first 2 four-helix bundles and binds to αE-catenin with a similar affinity to the vinculin head domain, D1-D4 . As obseFig. 3in vitro single-molecule stretching experiments revealed that force is required to expose the vinculin-binding site in αT-catenin M1-M3  did not weaken vinculin D1 binding relative to M1-M3 . αE-catenin and αT-catenin recruited similar levels of vinculin in dimethyl sulfoxide controls . Importantly, blebbistatin treatment significantly reduced vinculin recruitment to both αE-catenin and αT-catenin containing AJs (C–E). Thus, myosin-based tension is required to recruit vinculin to αT-catenin, similar to αE-catenin.We tested the ability of αT-catenin to restore cell–cell adhesion and recruit vinculin in α-catenin-deficient R2/7 carcinoma cells . We tranning AJs , C–E. ThWe then examined the correlation between EGFP fusion expression and vinculin recruitment. As expected, EGFP–αE-catenin expression levels did not correlate with vinculin levels because vinculin was only recruited when αE-catenin was activated by sufficient tension , 28. HowN). αE-catenin and αT-catenin N1-M3  were both cytosolic and, as expected, unable to form cell–cell contacts and recruit vinculin (J and K). Deletion of M3 in αE-catenin relieves autoinhibition, and αE-catenin N1-M2 (aa 1–510) was able to recruit vinculin and restore cell–cell adhesion, as shown previously  or the head domain minus M3 and ABD (N1-M2). All EGFP-tagged αE-catenin and αT-catenin fragments expressed as soluble proteins of the predicted size N. αE-catvinculin , J and Keviously . Here vicyto complex binds with high, ∼5-nM affinity to αT-catenin similar to 1- to 2-nM affinity previously reported between the β-catenin–cadherin tail complex and αE-catenin . This suggest that αT-catenin does not compete with αE-catenin for binding cytosolic β-catenin. Although there is evidence of a cytosolic αE-catenin–β-catenin complex in epithelial cells  between the αT-catenin N-terminus and M-region promote M1 folding and M-region autoinhibition. Interestingly, the first 56 residues of αE-catenin contribute to the inhibition of vinculin binding in addition to the M3 domain (The αT-catenin head fragment (N1-M3) showed weak, micromolar binding to vinculin, indicating that the N-terminus regulates M-region autoinhibition. The entire N-terminus (N1-N2) is required for autoinhibition because deleting N1 restored strong vinculin binding. Notably, replacing the N-terminus with another well-structured protein moiety, the titin I27 repeats, reduced vinculin binding and caused the binding reaction to switch from exothermic to endothermic, suggesting the M1 was stabilized. This observation explains the force-dependent vinculin binding to the αT-catenin M-region observed in recent biophysical experiments . The abi3 domain .Expression of EGFP–αT-catenin was sufficient to restore cell–cell contacts and recruit vinculin in α-catenin-deficient R2/7 cells. Vinculin binding to αT-catenin requires actin binding by the latter and is tension dependent, similar to αE-catenin. This is consistent with the vinculin-binding site in the αT-catenin M1 domain being occluded in the absence of force. Notably, deletion of M3 in αT-catenin did not relieve autoinhibition, offering additional evidence that the intramolecular interactions required for autoinhibition in αT-catenin differ from αE-catenin.in vivo. Loss of αE-catenin from the mouse heart disrupts AJ formation and causes a marked decrease in vinculin expression and recruitment, despite the presence of αT-catenin, resulting in cardiomyopathy  in pGEX-TEV were described previously (M. musculus αT-catenin N1-N2 (aa 1–259), N1-M3 (aa 1–659), M1-M3 (aa 260–626), N2-M3 (aa 147–626), and αE-catenin M1-M3 (aa 273–651) and M1-M2 (aa 273–510) were cloned into pGEX-TEV. The construct encoding αT-catenin M1-M3 flanked by I27 handles in pET151/D-TOPO was kindly provided to us by Jie Yan  in 0.05 mg/ml sequencing grade trypsin (Roche Applied Science). Digestions were stopped with 2X Laemmli sample buffer and placed on ice until analysis. Samples were boiled and run by SDS-PAGE and then stained with 0.1% Coomassie Brilliant Blue R-250, 40% ethanol, and 10% acetic acid. Gels were scanned on a LI-COR scanner. For N-terminal sequencing, digested peptides were blotted onto polyvinylidene difluoride membrane, stained , destained, and dried. Individual bands were excised from the membrane and sequenced by Edman degradation (Iowa State University Protein Facility).αT-catenin protein fragments were incubated with or without 1-mM BS3 (Thermo Scientific) in 20-mM Hepes, pH 7.4, 150-mM NaCl, and 1-mM DTT for 30 min at 4 °C or 37 °C, separated by SDS-PAGE, stained with the Coomassie dye, and imaged on a LI-COR scanner.cyto complex, or 396- to 600-μM vinculin aa 1 to 259 (D1) into the cell containing 9- to 55-μM of αT-catenin or αE-catenin. For experiments on the MicroCal VP-ITC, the ligand was added with an initial 2-μl injection followed by 32 × 9-μl injections. The concentration of αT-catenin head or αT-catenin head–β-catenin complex in the cell varied between 22 and 56 μM. Vinculin D1 concentrations in the syringe varied between 240 and 546 μM. All calorimetry experiments were performed at 25 °C. All data analyses were performed using Malvern MicroCal ITC analysis software. For baseline correction, a mean baseline value, calculated by averaging the data points at saturation, was subtracted from each data point.Proteins used for ITC were purified as described except the S200 buffer was replaced with the ITC buffer . An identical buffer was used to purify both cell and titrant samples to ensure buffer match. Only fresh, unfrozen proteins were used for ITC. Measurements were performed on a Malvern MicroCal PEAQ-ITC or MicroCal VP-ITC calorimeter . For experiments on the MicroCal PEAQ-ITC, titration occurred by an initial 0.5-μl injection followed by 18 × 2-μl injections of 110- to 150-μM β-catenin, 103- to 158-μM β-catenin–NcadR2/7 carcinoma cells were cultured in Dulbecco's modified Eagle's medium (4.5 g/l glucose), 10% fetal bovine serum, 1-mM sodium pyruvate, and penicillin/streptomycin. Lipofectamine 2000 was used for all transient transfections.R2/7 cells were lysed 48 to 72 h after transfection in RIPA buffer  plus protease inhibitors (Millipore). The lysate protein concentration was measured by bicinchoninic acid protein assay, and 15 μg of each sample was separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked in TBST  + 5% BSA  for 1 h at RT, washed in TBST, and incubated with anti-GFP  and anti-GAPDH  antibodies for 1 h at RT. The membrane was washed twice in TBST and then incubated with anti-rabbit IRDye 680 and anti-mouse IRDye 800 for 1 h at RT, washed twice with TBST, and washed once with PBS. Membranes were scanned on a Bio-Rad ChemiDoc MP imaging system.Cells were fixed in 4% paraformaldehyde in PHEM buffer , washed with PBS, permeabilized with 0.2% Triton X-100 in PBS for 2 min, and then blocked for 1 h at RT in PBS +10% BSA. Samples were washed three times in PBS, incubated with the primary antibody in PBS +1% BSA for 1 h at RT, washed three times in PBS, incubated with the secondary antibody in PBS +1% BSA for 1 h at RT, washed three times in PBS, and mounted on the ProLong Diamond mounting medium. Cells were imaged on a Nikon Eclipse Ti inverted microscope outfitted with a Prairie swept-field confocal scanner, Agilent monolithic laser launch, and Andor iXon3 camera using NIS-Elements imaging software.p < 0.05.To quantify vinculin recruitment to EGFP-tagged αE-catenin and αT-catenin, a maximum projection was created from four planes of the z-stack  where cell–cell contacts were best in focus. IsoJ Dark thresholding was used to create a mask of the GFP channel to define the region of analysis in ImageJ. Vinculin signal intensity was then measured within the masked region. Next, three random intensity measurements of vinculin staining were taken in the cell cytoplasm and these values averaged. Finally, the vinculin intensity within the mask was divided by the cytoplasmic signal to normalize between samples and calculate the contact/cytoplasmic ratio. Colocalization data were plotted with Prism software (GraphPad). A one-way ANOVA with Tukey’s comparisons was performed to determine significance; 2 value, and p value using Prism software (GraphPad).To examine the relationship between EGPF–αE-catenin or EGFP–αT-catenin levels and vinculin recruitment, the vinculin contact/cytoplasmic ratio was plotted against the average GFP intensity of each fusion construct within the masked cell contact region (described above). Linear regression analysis was performed to calculate the slope, 95% confidence intervals, RAll data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


Pharmacological inhibition through XAV-939 as well as si-RNA mediated inhibition of β-catenin is initially effective in re-sensitization to therapy, however, prolonged inhibition shifts dependency from β-catenin to Notch signaling, with particularly high levels of receptors Notch 1 and Notch 2. The results are verifiable in a cohort of T-ALL patients comprising of responders vs. those who have progressed, with β-catenin, Notch 1 and Notch 2 elevated in progressed patients. Further, in patients-derived cells, silencing of Notch 1 or Notch 2 does not counter resistance to β-catenin inhibition, rather pharmacological pan-Notch inhibition is needed to overcome resistance and its effect on in vitro tumor sphere formations as well as in vivo liver metastases. Thus, wnt and Notch signaling are part of a regulatory loop mutually compensating for each other in T-ALL, while ensuring the maintenance of stem cell phenotype.Leukemia-initiating cells play critical role in relapse, resistance to therapies and metastases but the mechanism remains largely elusive. We report that β-catenin is over-expressed in almost all T-ALL patients and flow sorted β-catenin T-cell acute lymphoblastic leukemia (T-ALL) is aggressive, as its name suggests, progresses rather quickly. The T-cell variant of ALL accounts for about 15% ALLs in children and about 25% of ALLs in adults . AlthougThe goal of this study is to mechanistically dissect the role of β-catenin in stemness of T-ALL, particularly in patient-derived samples. We evaluated the role of β-catenin in inducing drug resistance and metastasis of T-ALL. Further, in view of the relapse observed in clinics, we mimicked such conditions to evaluate alternate signaling pathways that can compensate for the effective targeting of β-catenin. Our results indicate an intricate relationship between wnt and Notch signaling that helps maintain the stemness in T-ALL patients.The study involving human subjects was approved by the Institutional Review Board and the Ethics Committee at Sun Yat-sen University. A signed consent form was received from all patients and volunteers. The identifying information for T-ALL patients as well as healthy volunteers was not revealed to the researchers.Cells were treated with VDL  for 10 days. Control cells were left untreated. Cells were then harvested by centrifugation at 500 rcf for 5 min and re-suspended in DPBS  containing FBS at a final concentration of 2.5%. XAV-939 and BMS906024 used in the study were procured from Sigma (China).Active Beta Catenin Pathway Assay Kit  was used to quantitate β-catenin in patient-derived samples, following manufacturer’s suggested protocol. This qualitative ELISA kit is specifically designed to detect the active dephosphorylated form of β-Catenin. Notch family receptors were quantitated using specific ELISA kits for Notch1, Notch2, Notch3 and Notch4 . C-myc and Spi1 were also quantitated using specific ELISA kits purchased from Seajet Scientific, Shanghai, China.Single cell suspensions, made using GentleMACS kit , from patient samples were incubated in phosphate-buffered saline containing 1% FBS with human beta-Catenin APC-conjugated Antibody , and isotype-matched mouse immunoglobulins were used as controls. Samples were analyzed and sorted, using an EPICS ALTRA flow cytometer . For the positive and negative population, only the top 10% most brightly stained cells or the bottom 10% most dimly stained cells were selected, respectively.in vivo experiments, involving NSG mice , was approved by the Animal Research Ethics Committee at the Sun Yat-sen University. All methods were performed in accordance with the relevant guidelines and regulations. All mice were housed in sterilized animal facility. Based on our initial titrations, we injected 50,000 cells into the tail veins of NSG mice and euthanized mice after 7 weeks. The livers were visually inspected for signs of metastases.Our protocol for Samples from hepatic metastases were sectioned, conventionally stained with HE and immunohistochemically labeled with anti-cytokeratin using R-IHC and standard IHC and examined by a trained pathologist.Trypan blue dye exclusion assay was used to evaluate cell viability. Trypan blue  (0.4%) was added in 1:1 ratio with the single cell suspensions and the cells monitored and quantitated under bright field microscope. The live cells did not retain blue stain and their cytoplasm was clear while the dead cells were marked with blue cytoplasm.First, single cell suspensions were ensured enzymatically using trypsin and mechanically using a 22 gauge needle for 2 min. For the generation of tumorospheres, cells were plated in 24-well ultra-low attachment plates  at a density of 5,000 viable cells/well. Average sphere forming efficiency was evaluated after 18–21 days of culture under an inverted Olympus IX70 microscope camera  by counting spheres that were larger than 50 μm in diameter using the ImageJ Software.Trizol reagent  was used to isolate RNA following the manufacturer’s protocol. Quantitative RT-PCR reactions were performed on an ABI 7500 RT-PCR system (Applied Biosystems) using validated primers from BioRad (China). Only RNAse-free water was used throughout the assays.t-test and ANOVA were used to compare tested groups. Differences were considered significant when p < 0.05. Statistical analyses were conducted using Graphpad Prism software.All experiments were performed in triplicates. Two-tailed independent Student’s p < 0.0001) elevated in T-ALL patients, compared to controls , the sensitization was totally lost at 30 days . ClearlyTo ascertain whether increased β-catenin as well as Notch levels are relevant to T-ALL patients who have acquired resistance and whose disease has relapsed and/or progressed, we recruited T-ALL patients currently undergoing treatment at our Hospital. For this investigation, we identified 24 T-ALL patients (mostly diagnosed within the last 6 months) who were still responding to their assigned threrapies and another 28 patients whose disease had progressed and these patients had stopped responding to their last assigned therapy. We found β-catenin, Notch1, Notch2 as well as c-Myc to be significantly over-expressed in patients whose disease had progressed .in vivo in a mice metastasis model. Cells from control condition, si-β-catenin and si-β-catenin + BMS906024 were injected in tail veins of mice and as seen in Next, we identified 2 patients with progressed disease and the highest expression of β-catenin. Tumor cells were isolated, single cell suspensions made and the cells cultured in the lab. We now wished to study the inter relationship between wnt and Notch signaling, with regards to the maintenance of stemness. In assays performed on cells from one patient, VDL resistance was observed as expected (because of increased β-catenin) . FurtherEven though wnt signaling is often de-regulated in hematological malignancies , its rolNotch1 mutations (The relevance of Notch signaling in T-ALL is well regarded . A role utations . Not jusutations .In the report on role of stabilized β-catenin in development of T-ALL, up-regulation of c-Myc was reported as a consistent secondary event . MoreoveBased on our observations, we propose that future investigations should evaluate simultaneous inhibition of multiple signaling pathways, particularly those that are closely related. Even though we investigated Notch signaling as an alternate pathway when β-catenin was inhibited because of its relevance to T-ALL and the probability of taking up almost all the functions, particularly those connected to stemness, our unpublished preliminary results from high throughput assays indicate that Notch family members indeed are among the top potential candidates that can control the proliferation and metastasis of T-ALL cells when β-catenin inhibition is rendered ineffective.A number of drugs that target either Notch signaling or β-catenin are progressing through the clinical trials . This isThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Review Board and the Ethics Committee at Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Research Ethics Committee at the Sun Yat-sen University.LZ, JW, YF, BK, ZF, and J-JL participated in performing experiments, collecting data, and analyzing results. JG, BT, RX, and GP analyzed data and performed statistical analyses. J-JL conceptualized and supervised study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong β-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes β-catenin. Deletion of this exon yields unconstrained β-catenin signalling activity. We also demonstrate that β-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which β-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express  During ectively , 7, 8, aectively . The devectively , 11.Wnt10b whose overexpression in the developing mammary gland induces precocious alveolar development and focal mammary adenocarcinomas [The role of EDAR signalling in tumour development has received little attention, despite its ability to activate NFκB  and the rcinomas . Receptorcinomas .EDAR as a novel candidate mammary oncogene. We assessed expression data from human breast carcinomas, identifying a specific subtype of ductal carcinoma with squamous metaplasia that expressed EDAR strongly. To determine whether high EDAR levels are capable of causing breast cancer, we examined mammary tumour formation in a transgenic mouse line expressing high levels of Edar from its endogenous regulatory elements. We found that breeding female mice develop mammary tumours, and, as observed in the EDAR-high human tumours, these tumours contain extensive squamous metaplasia. Our analysis of the tumours indicated that the modestly increased EDAR signalling in the EdarTg951/951 mice is further increased in transformed cells, as is β-catenin transcriptional activity. All tumours carry exon 3 deletions of CTNNB1, conferring WNT-independent activity on β-catenin. This β-catenin activity is required for tumour cell proliferation, highlighting the interplay between EDAR and WNT pathways in tumourigenesis. Together our data identify EDAR as a novel breast oncogene.Thus, based on its signalling function and its role in mammary development, we evaluated EDAR in a meta-dataset of 1107 invasive human breast cancers classified into the basal-like, luminal A, luminal B, normal breast-like, and HER2 molecular subtypes [EDAR, which almost invariably belonged to the basal-like subgroup , KERATIN1 (KRT1) and INVOLUCRIN (IVL), suggesting the occurrence of squamous metaplasia within the EDAR-high cancers   Fig. , demonstity Fig.  demonstrEdar expression and signalling activity in the EdarTg951/951 mammary gland, we next assessed the tumour incidence in these mice. We followed cohorts of female nulliparous and continuously breeding EdarTg951/951, along with wild-type mice, over a 1-year period. The continuously breeding EdarTg951/951 mice developed solitary mammary gland tumours from as early as 3 months of age and with an average latency of approximately 7 months  expression , together with an Edar expression construct. We found that introduction of high EDAR signalling in these conditions suppressed β-catenin activity , indicating that these tumours had undergone squamous metaplasia, and an activation of the WNT/β-catenin signalling pathway (expression of WNT10B and LEF1). The latter could explain the overexpression of WNT10B that is seen in some human breast cancers [ing mode  and its ing mode , 29. Our cancers .EdarTg951 mouse model which carries a high copy number of Edar expressed from its endogenous regulatory elements [EdarTg951 mice upon continual mating, which was not observed in wild-type mated or transgenic nulliparous females, demonstrated a driving role for EDAR as an oncogene. In this mouse model, the characteristic squamous metaplasia and expression of epidermal differentiation genes in mammary tumours matches the expression phenotype of the human metaplastic tumours with high EDAR expression. Thus in both human and mouse increased EDAR expression is associated with this distinct mammary tumour type.To test whether elevated EDAR signalling is sufficient to lead to mammary tumourigenesis, we employed the elements , 25, 31.EdarTg951/951 mice that is seen in the MMTV-RankL mice or the ductal hyperplasia observed in the transplanted tissue that lacks IκBα [MMTV-c-rel mouse strain display squamous metaplasia [Previous work has highlighted a role for NFκB signalling in mammary gland development and tumourigenesis. In particular, RANK signalling has been shown to play an essential role downstream of Progesterone Receptor in the formation of the milk-producing alveoli during pregnancy , 33 and cks IκBα , 36. Thicks IκBα , 37. IntEdarTg951 gland is consistent with an increase in WNT signalling [Wnt10b expression, suggesting a stimulatory effect of EDAR on WNT pathway signalling over a sustained period. However, an inhibitory effect of EDAR signalling on β-catenin function is revealed by EDAR overexpression over a 48 h period in cultured cells, potentially through the activation of WNT/β-catenin pathway inhibitors such as DKK4 [EdarTg951/951 females of one mammary gland phenotype associated with excess WNT/β-catenin signalling, precocious lobular-alveloar development. This phenotype is seen when the pathway is activated directly by overexpressing a WNT protein or a non-degradable form of β-catenin [EdarTg951/951 mice. If lobular-alveolar development requires a strong WNT/β-catenin signal, then it is reasonable to hypothesise that the signal is simply insufficient in EdarTg951/951 females to drive lobular-alveolar development.During development, EDAR signalling is intertwined with that of the WNT/β-catenin pathway , 11. Botgnalling , 38, and-catenin , 40. In The short-term inhibitory relationship between EDAR and β-catenin is also likely to apply a selective pressure for unrestrained β-catenin activity to drive cell transformation. In tumours, the release of β-catenin activity from this inhibitory EDAR influence is provided by somatic mutation, with cells carrying β-catenin exon 3 deletions growing out to form tumours. The signalling environment caused by EDAR, with very high NFκB activity, likely provides a suitable environment for cells carrying these specific mutations to proliferate rapidly. The occurrence of deletion mutations can be explained by the high frequency of double-strand breaks in the EDAR driven tumours. In non-replicative cells, like the involuting mammary gland, these breaks are repaired by NHEJ which frequently leads to deletions like those observed. It is also interesting to note that mutations in WNT pathway components, although rare in human breast cancer in general, are common in metaplastic breast cancers .The basis for the specificity of squamous metaplasia observed in EDAR driven tumours is unclear. EDAR acts during development of the surface ectoderm to control the patterning and morphogenesis of cutaneous appendages, but does not appear to have a direct influence on epidermal differentiation. Rather, EDAR signalling tends to drive cells away from an epidermal fate and towards that of ectodermal appendage . Thus, iEDAR encoding a receptor with altered signalling potentials exist in human populations, with East Asian and Native American groups having a non-synonymous polymorphism leading to substantially increased signalling [Alleles of gnalling , 43. Thegnalling , 45, witgnalling . Such aghttp://www.ebi.ac.uk/arrayexpress/) experiment number E-NCMF-3. Treeview was used to generate heatmaps (http://rana.lbl.gov/EisenSoftware.htm).A meta-analysis of six Affymetrix gene expression datasets comprising 1107 primary human breast cancers was performed as previously described . CentroiEdarTg951/951 mice were on an inbred FVB/N genetic background and have been described previously [EdarTg951/951 and wild-type FVB/N mice were established to analyse tumour formation. For the phenotypic analysis at least three animals were examined for each genotype and stage, specific numbers used are given in the appropriate figure legends. Randomisation and blinding were not used.eviously . All proWhole mounts of the rostral inguinal (4th) mammary gland were prepared as previously reported . Images Rostral inguinal mammary glands and shaven back skin were fixed in 4% paraformaldehyde and paraffin-embedded. Antigen retrieval was carried out on mounted sections using a Borg Decloaker  and the sections were subsequently stained as described before . ControlIn situ hybridisation was carried out on 8-µm sections from paraffin-embedded mammary tissue using the RNAScope system , according to the manufacturer’s instructions.4 and 1× Protease inhibitor cocktail ) per gram of tissue and incubating for 15 min on ice. Lysates were then spun at 18,000 g to remove nuclei and genomic DNA. Nuclear-enriched fractions were obtained from powdered frozen mammary tissue samples in 1 ml NP-40 lysis buffer  per gram of tissue and incubating for 15 minutes on ice. Lysates were then spun at 18,000 g for 30 min at 4 °C. The nuclear pellets were resuspended in SDS buffer  and genomic DNA was sheared using a probe sonicator. Equal quantities of the protein samples were run on SDS/polyacrylamide gels as described [Cell lysates were obtained from powdered frozen mammary tissue samples in 1 ml 2× RIPA lysis buffer  and treated with RNAse-free DNAse 1 to remove contaminating genomic DNA (New England Biolabs). Two micrograms of total RNA was reverse transcribed using the High Capacity RNA to cDNA kit (Applied Biosystems). The resultant cDNA was diluted tenfold and 2 µl was used as template for subsequent quantitative PCR reactions.Keratin18 as a normalisation control to account for differences in the amount of ductal epithelium in each sample.qPCR was carried out in triplicate using the primers listed in supplementary Table Changes within the sequence of exon 3 of the β-catenin cDNA and genomic locus were screened and sequenced by amplifying the relevant region from either cDNA or the genomic DNA found within the initial RNA extraction prepared from tumours respectively, using the relevant primers listed in supplementary table The cell populations present within the adult mammary gland were analysed as described in .pNFĸB-Luc and pTopFlash to monitor NFκB and WNT signalling respectively. EpH4 cells stably transduced with a pCDH EF1α-Edar-T2A-GFP or a pCDH EF1α-ΔExon3-β-catenin-T2A-RFP lentivirus were generated as described in [The normal mouse mammary epithelial cell lines C57MG and EpH4 were gifts from Dr. Anthony Brown  and Prof. Christine Watson . Luciferase assays were conducted in HEK293T and EpH4 cells as described in , using pribed in . Expressribed in .t-tests, one-way ANOVA followed by a Tukey’s test, or log-rank (Mantel–Cox) test.All data are presented as the mean ± SEM. Statistical significance was evaluated, where appropriate, using unpaired Supplemental materialSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Figure 8