Fusarium oxysporum f. sp. cubense (Foc), the deadly strain that causes Fusarium wilt of bananas than conventional planting material due to the loss of indigenous endophytes. In this study, an endophytic bacterium Bacillus velezensis EB1 was isolated and characterized. EB1 shows remarkable in vitro antagonistic activity against Foc with an inhibition rate of 75.43% and induces significant morphological and ultrastructural changes and alterations in the hyphae of Foc. Colony-forming unit (c.f.u.) counting and scanning electron microscopy (SEM) revealed that EB1 could colonize both the surface and inner tissues of banana tissue culture plantlets. Banana tissue culture plantlets of late rooting stage bioprimed with EB1 could efficiently ward off the invasive of Foc. The bio-priming effect could maintain in the acclimatized banana plants and significantly decrease the disease severity of Fusarium wilt and induce strong disease resistance by manipulating plant defense signaling pathways in a pot experiment. Our results provide the adaptability and potential of native endophyte EB1 in protecting plants from pathogens and infer that banana tissue culture plantlets bio-priming with endophytic microbiota could be a promising biological solution in the fight against the Fusarium wilt of banana.Tissue culture techniques have been routinely used for banana propagation and offered rapid production of planting materials with favorable genotypes and free of pathogenic microorganisms in the banana industry. Meanwhile, extensive scientific work suggests that micropropagated plantlets are more susceptible to  Musa spp.) production contributes significantly to income and food security . Foc is composed of different evolutionary lineages and at least 24 vegetative compatibility groups (VCGs). Foc race 1 wiped out the highly susceptible Gros Michel (Musa AAA) variety in Central America in the mid-twentieth century  as a replacement for Gros Michel , the most destructive and uncontrollable pathogen of banana, to which Cavendish and many other cultivars are highly susceptible, has created havoc on banana production worldwide again  , and other control measures once it penetrates the host plant  how EB1 modulates the resistance of banana plants against Foc by using a banana plant–EB1system created by inoculating banana tissue culture plantlets with EB1 at the end of rooting stage. Our study was designed to lend new insights into the sophisticated mechanisms of host plants–endophytes interactions for coping with environmental stresses and to provide potential strategies to control the Fusarium wilt of banana.Nowadays, the most common application strategies of endophytes in agricultural systems are adding them directly into the soil and preparing them as seed-coating agents, which are rather inefficient in practice. Thus, it is imperative to explore alternative strategies for endophyte application  was cultivated on a potato dextrose agar (PDA) plate at 28°C and used in this study. Isolated endophytic bacteria were inoculated in Luria-Bertani (LB) agar  plates. Basal Murashige and Skoog (MS) medium was used for tissue culture experiments.Wild-type Foc TR4 by a dual-culture experiment  was uniformly inoculated 2.0 cm away from the fungal inoculum. Plates inoculated only with Foc TR4 plug were served as control. Plates were incubated at 28°C for 5 days and recorded with a Canon EOS 77D camera  with the same parameters, and the surface area of the mycelia was measured using the Image J software . The inhibitory effect was evaluated by calculating the percentage of area inhibition using the following formula: (Sc - St)/Sc × 100, where Sc and St represent the growth area of Foc TR4 in the control and treated plates, respectively. The experiment was repeated 3 times, with 4 replicates each time. Strain EB1 was isolated through the above screening and stored at −80°C with glycerol . The antifungal efficiency of EB1 against Foc TR4 was further measured by observing the morphology and ultrastructure characteristics of Foc TR4 in the dual-culture experiment by applying a scanning electron microscope  and a transmission electron microscope .The healthy banana plants used in this study were collected from a wilt-diseased banana field in Dongguan, Guangdong Province, China . Banana plant samples were washed with tap water thoroughly to remove the airborne counterparts and soaked in 75% (v/v) ethanol for 1 min, 0.1% (v/v) NaClO for 15 min, followed by being rinsed 5 times with sterile water to deplete epiphytic microorganisms in aseptic conditions. Ten grams of plant tissue was weighed and ground with 20 ml sterilized distilled water premixed with sterilized quartz sand using a sterilized mortar and pestle for 5 min. Aliquots of 1 ml of the resulting suspension were diluted using a serial dilution method and spread evenly on an LB agar plate and incubated at 28°C for 5 days. All culturable bacterial colonies were purified and selected based on their morphological characteristics. The antagonistic efficacies of these endophytic bacterial isolates were evaluated against Overnight bacterial cultures of EB1 in LB broth were collected, centrifuged at 3,000 rpm for 15 min, and washed two times with sterile PBS buffer . Whole-Genome Sequencing of EB1 was performed using a combination of the Oxford Nanopore Technologies (ONT) GridION platform  and Illumina MiSeq platform  by Gene Denovo Biotechnology Co. . DNA was extracted from Qiagen's DNeasy UltraClean Microbial Kit  and its quality and concentrations were determined using a Nanodrop spectrophotometer  and Qubit Fluorometer . For ONT sequencing, library preparation was conducted according to the manufacturer's protocol of the SQK-LSK109 sequencing kit . For Illumina sequencing, genomic DNA (gDNA) was fragmented and a paired-end library with an average DNA insert size of 300–400 bp was constructed using Illumina TruSeq Nano DNA Library Prep Kit (Illumina). The assembled sequences were deposited in the NCBI (BioProject ID: PRJNA807456). The components of coding genes, noncoding RNA (ncRNA), and functional annotation were analyzed using a range of databases including the non-redundant protein database (Nr), SwissProt, Cluster of Orthologous Groups (COGs), and Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene clusters for the biosynthesis of secondary metabolites were identified by using antiSMASH.https://blast.ncbi.nlm.nih.gov/Blast.cgi), and 16S rRNA gene sequences closest to the isolates (98% sequence homology) were recovered for further phylogenetic analysis. Strain EB1 was subjected to phylogenetic analysis using MEGA version 7  based on a full-length 16S ribosomal RNA (16S rRNA) sequence, and a phylogenetic tree was constructed using the neighbor-joining method. The reliability of this resulting tree was evaluated by the bootstrap method with 1,000 replications.The 16S rDNA sequence of strain EB1 derived from the EB1 genome was aligned with an NCBI 16S ribosomal RNA sequences database by Nucleotide BLAST (600) = 0.2. Each flask was inoculated with 0.1 ml of the bacterial suspension  or 0.1 ml MS by pipetting to the rhizosphere of banana tissue culture plantlets and cultured at 22°C and 16/8 h light/dark cycle. The colonization and reproduction of EB1 on the plantlets were quantified each day over a period of 7 days. Total c.f.u. values of EB1 were quantified per the programs described previously  or 0.1 ml MS to the rhizosphere of banana tissue culture plantlets after prior inoculation with EB1 for 3 days and cultured at 22°C and 16:8 h light/dark cycle for another 4 days. The experiment was conducted in triplicate, with at least eight plants per treatment. After 7 days of successive culture, SEM was used to observe the distributions of EB1 and the interactions between EB1 and Foc on the banana tissue plantlets.Uniformly grown banana tissue culture plantlets [“Cavendish” banana (AAA) cv. “Brazilian”] of rooting stage were surface sterilized in 75% (v/v) ethanol for 1 min and 0.1% (v/v) NaClO for 15 min, rinsed with sterile water for 5 times, air-dried, and then transferred and grown vertically in tissue culture flasks containing 100 ml of cooled-down MS. Four plantlets were transferred to each flask. For EB1 inoculation, overnight culture of EB1 in LB broth was harvested, centrifuged, and washed in liquid MS twice, and resuspended in MS to a final optical density  and fresh weight of shoot and root (g) were conducted after 60 days of planting. The disease index of each plantlet was assessed according to the rating scale of 0–4: 0 = no symptom; 1 = some brown spots in the inner rhizome; 2 = < 25% of the inner rhizome show browning; 3 = up to 75% of the inner rhizome show browning; and 4 = entire inner rhizome and pseudostem show dark brown, dead  after 7 days of successive culture were subjected to hardening for 10 days by transferring into pots with sterilized planting soil . Then the biocontrol efficacy of EB1 on banana plantlets was investigated in greenhouse experiments with the acclimatized banana plants . Three t) following the manufacturer's instructions. HiScript II One Step qRT-PCR SYBR Green kit  was employed for qRT-PCR assays according to the manufacturer's instructions. First-strand cDNA was prepared by reverse transcription from 1 μg of DNA-free total RNA in a final reaction volume of 20 μl. RT-qPCR was conducted using a QuantStudio 5 Real-Time PCR System  in four replicates. The qTUB gene (banana) was used as a reference for data normalization, and the target genes were amplified using the primer sets listed in Ct−ΔΔ method . Data were analyzed using Student's Bacillus species , intact plasma membrane (PM), and normal cytoplasm containing an intact nucleus and all organelles. In reverse, noticeably disruptions such as loss of cellular integrity, thickened CW, evident plasmolysis, serious vacuolation, invaginated PM, abnormal architecture of the nucleus and degenerated organelles were observed in EB1 treated hyphae. The results indicated that the morphology and structural integrity of the treated fungal were dramatically affected during co-culture with EB1.The morphological observation was preliminarily carried out for strain EB1. It was found that the colony of EB1 on LB medium was dry and round with irregular protrusions at the margin, showing the typical characteristics of  species . The cel species . EB1 sho, and this inhibitory effect was dramatically reversed upon co-inoculation with EB1 (TR4 + EB1) (p = 0.002) and 1.49-fold (p = 0.007), respectively. Plants in the EB1 + TR4 treatment group also had increased biomass compared with those treated by Foc TR4 only . Moreover, EB1 did not cause any mortality or disease symptoms in banana plants when inoculated alone and greatly enhanced the survival rates and reduced the disease severity caused by Foc TR4 in banana plants who had been pre-inoculated with EB1 prior to inoculation with Foc TR4 (EB1 + TR4) compared with those inoculated only with Foc TR4  , B. Comp Foc TR4 . OverallNPR1, PR1, LOX2, and MYC2 were analyzed . Whereas, the degree of expression changes in the Foc TR4 treatment group varied from gene to gene. Foc TR4 downregulated the expressions of NPR1 and LOX2 by 0.46- (p = 0.0024) and 0.63-fold (p = 0.016) and upregulated the expressions of PR1 and MYC2 by 16.62- (p < 0.001) and 2.23-fold (p < 0.001), respectively. Notably, both SA and JA signaling pathways were significantly upregulated by 1.40- (p = 0.015), 35.80- (p < 0.001), 1.50- (p = 0.0028), and 2.44-fold (p < 0.001) for NPR1, PR1, LOX2, and MYC2, respectively. The above results have indicated that EB1 primes the plants for enhanced immunity following a subsequent attack by Foc TR4.To further examine whether the EB1 could activate defense signaling in the banana plant, the expression patterns of the defense-related marker genes involved in SA and JA pathways including analyzed . CompareB. velezensis EB1 was isolated and selected for in-depth analysis based on its antifungal activity against Foc TR4 and strong colonization ability in banana plants to support a new approach using EB1 to control Fusarium wilt of banana by introducing into banana tissue culture plantlets at the end of rooting stage.Besides resistant cultivar breeding, BCA comprising endophytic bacteria has been considered another promising control strategy against Fusarium wilt that reached the field-testing stage  understand the underlying molecular mechanisms of the beneficial effect of EB1 on the growth and stress tolerance of banana plants, (2) isolate more efficient, multifunctional, stress tolerant microbes and design an artificial disease suppressive synthetic community (SynCom) which comprised by multiple microbial strains rather than mono-strain inoculums to take advantage of functional complementarity to mimic a natural disease-suppressive community in plants, and (3) develop bioformulations for sustainable application of endophytic microbes in plant tissue culture. New strategies for the successful delivery of BCAs into the plant under field conditions are needed to realize the large-scale implementation of microbial strains in agricultural practice. The introduction of endophytic microbes, as a probiotic material that enhances plant growth as well as induces defense responses of plants to cope with stress, into tissue-cultured banana plantlets, could be a novel and stable biological control method to protect bananas from Foc infection.Our current study focused on providing a comprehensive understanding of the endophytic strain The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/DX and SL: conceptualization. DX, XY, BL, and YC: experimentation. DX, XY, and CL: review and drafting. SL and CL: validation and statistical analysis. All authors contributed to the article and approved the submitted version.


Indocyanine green (ICG), glucose oxidase (GOx), and copper(II) sulfate (Cu)‐installed hybrid gel based on organic nanorod  and inorganic nanodisk (Laponite [LAP]) was developed to perform a combination of starvation therapy (ST), chemodynamic therapy (CDT), and photothermal therapy (PTT) for localized cancers. A hybrid CNC/LAP network with a nematic phase was designed to enable instant gelation, controlled viscoelasticity, syringe injectability, and longer in vivo retention. Moreover, ICG was introduced into the CNC/LAP gel system to induce hyperthermia of tumor tissue, amplifying the CDT effect; GOx was used for glucose deprivation (related to the Warburg effect); and Cu was introduced for hydroxyl radical generation (based on Fenton‐like chemistry) and cellular glutathione (GSH) degradation in cancer cells. The ICG/GOx/Cu‐installed CNC/LAP gel in combination with near‐infrared (NIR) laser realized improved antiproliferation, cellular reactive oxygen species (ROS) generation, cellular GSH degradation, and apoptosis induction in colorectal cancer (CT‐26) cells. In addition, local injection of the CNC/ICG/GOx/Cu/LAP gel into the implanted CT‐26 tumor while irradiating it with NIR laser provided strong tumor growth suppression effects. In conclusion, the designed hybrid nanorod/nanodisk gel network can be efficiently applied to the local PTT/ST/CDT of cancer cells. The combination of ST and CDT can boost the killing efficiency of cancer cells by hunger and improved radical generation. The generation of hydroxyl radical based on the “cascade effect” induced by ST/CDT and its combination with PTT in a single delivery vehicle is expected to overcome the limitations of each therapeutic strategy and provide synergistic outcomes in cancer therapy.In this study, multifunctional therapeutic modalities were entrapped in the CNC/LAP birefringent gel network, and its therapeutic potential against cancer was assessed following intratumoral injection. The following ingredients were added to realize the combined effects of PTT, ST, and CDT: indocyanine green (ICG) was introduced into the CNC/LAP network to realize PTT of localized cancer under near‐infrared (NIR) light exposure.22.14·5H2O), d‐glucose, fluorescein isothiocyanate (FITC), GOx (from Aspergillus niger), GSH, 2,2′‐biquinoline‐4,4′‐dicarboxylic acid dipotassium salt trihydrate (BCA), 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA), 3,3′,5,5′‐tetramethylbenzidine dihydrochloride hydrate (TMB), 5‐sulfosalicylic acid, and 5,5′‐dithiobis (2‐nitrobenzoic acid) (DTNB) were acquired from Sigma–Aldrich . 5,5‐Dimethyl‐1‐pyrroline N‐oxide (DMPO) and ICG were supplied by Tokyo Chemical Industry Co. Ltd. . CNC (lyophilized form) was purchased from Cellulose Lab . LAP (Laponite XLG) was obtained from BYK Additives & Instruments .Ascorbic acid, Cu  according to the manufacturer's guidelines. In addition, the diameter, polydispersity index, and zeta potential values of CNC in distilled water (DW) (1 mg/ml) and LAP in DW (1.5 mg/ml) were measured.−1.Fourier‐transform infrared (FT‐IR) data for CNC, LAP, and CNC/LAP  were obtained using a Frontier FT‐IR spectrometer  and the attenuated total reflectance (ATR) sampling technique. The transmittance values of each specimen were monitored in the wavenumber range 400–4000 cmθ range 10–50°. The scan step size and speed were set to 0.04° and 1 s/step, respectively.The crystalline/amorphous properties of CNC, LAP, and CNC/LAP  were determined using X‐ray diffractometry (XRD)  at 40 mA and 40 kV. Diffraction angle‐dependent intensity values were detected in the 2q) range was set at 0.01–0.10 Å−1. The SAXS patterns were obtained using a Pilatus 300 K detector (Dectris).CNC (50 mg/ml in DW), LAP (75 mg/ml in DW), and CNC/LAP (50/75 mg/ml in DW) were analyzed using a small‐angle X‐ray scattering (SAXS) spectrometer  with a wavelength of 1.542 Å. Each specimen was loaded at a point 2500 mm from the detector, and the scattering vector (CuKα). The scattering wave vector (q) was calculated using the following formula: q = 4πsinθ/λCuKα.CNC, LAP, and freeze‐dried CNC/LAP (50/75 mg/ml) were studied using a wide‐angle X‐ray scattering (WAXS) system . The scattering angle (2θ) range was 2.9–74.7° with 50 kV and 1000 μA generator conditions and a 1.542 Å wavelength  were using simultaneous thermal analysis . The heat flow and weight change data were obtained using thermogravimetric analysis (TGA). To obtain the TGA profiles of CNC, LAP, and CNC/LAP, the temperature was increased at a constant rate of 10 °C/min at 50–800 °C under a 100 ml/min NSubsequently, each specimen (2 ml) was placed in a glass vial and incubated for 6 h at room temperature. After incubation, the specimen contained in the glass vial was placed between the cross‐polarizers for polarized optical observations. The polarization properties of CNC, CNC/LAP, and CNC/ICG/GOx/Cu/LAP were analyzed using polarizing microscopy . A retardation filter (λ plate) was used to enhance the optical path differences in the specimen.The morphological shapes of CNC/LAP (1/1.5 mg/ml), CNC/LAP , and CNC/ICG/GOx/Cu/LAP  were characterized with transmission electron microscopy (TEM) . Lyophilized CNC/LAP (50/75 mg/ml) and CNC/ICG/GOx/Cu/LAP (50/1/0.001/1/75 mg/ml) were dispersed in DW at a 50‐fold dilution. Finally, each specimen was loaded onto a copper grid (with carbon film) and dried. Prior to imaging, each sample was stained with 1% uranyl acetate.2.3Gel networks were designed using a simple blending method.The gelation features of the designed nanomaterial‐associated gel systems were investigated via inversion and injection tests.−1 shear rate) were also obtained. To determine the liquid crystalline phase, the angular frequency (1–100 rad/s)‐dependent complex viscosity profile of CNC/ICG/GOx/Cu/LAP gel was measured.The rheological features of the CNC/LAP, CNC/ICG/LAP, CNC/GOx/Cu/LAP, and CNC/ICG/GOx/Cu/LAP specimens were examined using a modular compact rheometer  after 24 h of incubation. Strain sweep  and frequency sweep  data were acquired at 25 °C. The shear rate‐variable shear stress and viscosity data (at 0.01–100 sThe morphology of freeze‐dried CNC/ICG/GOx/Cu/LAP was examined using variable pressure‐field emission‐scanning electron microscopy (VP‐FE‐SEM) . Prior to SEM imaging, the lyophilized specimen was coated with Au under vacuum.Atomic distribution in the freeze‐dried gel structure was investigated by SEM  linked with energy dispersive spectroscopy (EDS). Lyophilized CNC/Cu/LAP and CNC/ICG/GOx/Cu/LAP specimens were coated with Pt prior to SEM imaging.+, Thermo Fisher Scientific, East Grinstead, UK). The contents of elements in the surface of freeze‐dried CNC/Cu/LAP and CNC/ICG/GOx/Cu/LAP samples were determined by XPS.Atomic composition of the lyophilized gel system was further studied with X‐ray photoelectron spectroscopy  of Cu (1 mg/ml), GOx/Cu (0.001/1 mg/ml), CNC/LAP (50/75 mg/ml), and CNC/GOx/Cu/LAP (50/0.001/1/75 mg/ml) was added to a dialysis bag (molecular weight cut‐off (MWCO):12–14 kDa; Viskase Inc., Chicago, IL) and moved to a conical tube. DTNB (0.476 mg) in DW (4 ml) and/or GSH (0.740 mg) in DW (4 ml) was sequentially placed in a conical tube and incubated for 30 min at 37 °C. In case of CNC/GOx/Cu/LAP group, reaction time‐dependent absorbance profiles were acquired following incubation with the mixture of DTNB and GSH solution for 0, 60, 90, and 120 min. Each specimen was diluted with DW four times and absorbance data at 250–600 nm were obtained using an ultraviolet–visible (UV–Vis) spectrometer .D‐glucose  and further incubated for 1 h. TMB  was added to the conical tube, and the absorbance (at 650 nm) was acquired at 0, 30, and 360 min with a microplate reader .The TMB assay was performed to detect hydroxyl radicals using a colorimetric method. Aliquots (1 ml) of GOx/Cu (0.001/1 mg/ml), CNC/LAP (50/75 mg/ml), and CNC/GOx/Cu/LAP (50/0.001/1/75 mg/ml) were individually added to the dialysis tube  and incubated for 24 h. Samples were then transferred to conical tubes containing d‐glucose (50 mM) solution (2.5 ml) for 30 min at 37 °C. H2O2 solution  was added to that mixture and further incubated for 30 min at 37 °C. Then, dialyzed sample was reacted with DMPO (100 mM) at same volume ratio for 30 min prior to analysis with ESR spectrometer .Cascade production of hydroxyl radical from CNC/ICG/GOx/Cu/LAP gel was investigated with electron spin resonance (ESR) test. Aliquot (0.5 ml) of CNC/ICG/GOx/Cu/LAP gel was inserted to dialysis tube  and that was incubated with GSH (1 mM)/4 in DW  was then placed into the tube and further incubated for 30 min. The absorbance values (at 405 nm) of the dialyzed samples were measured using a microplate reader .Aliquots (0.3 ml) of CNC/GOx/LAP (50/0.001/75 mg/ml) and CNC/GOx/Cu/LAP (50/0.001/1/75 mg/ml) were added to a dialysis tube (MWCO:12–14 kDa) and transferred to a conical tube. DW (2 ml) or d‐glucose (100 mM) in DW (2 ml) was added to a specimen‐loaded dialysis bag and incubated at 37 °C for 6 h. TiOSOThe release profile of Cu from the fabricated gel network was tested in phosphate buffered saline . Cu was analyzed using a modified BCA method as previously reported.5 cells/well) and incubated for 1 day. The determined volume (0.2 ml) of CNC/ICG/GOx/Cu/LAP (5/0.1/0.0001/0.1/7.5 mg/mL) gel was added to the Transwell insert  and transferred to a 12‐well plate. Intracellular fluorescence intensity values were measured at 0, 10, 30, 60, 120, 360, and 1440 min using VISQUE InVivo Smart .ICG release from the gel system and its uptake into cancer cells were evaluated using near‐infrared fluorescence (NIRF) imaging. CT‐26 cells  were cultured in DMEM containing penicillin–streptomycin  and FBS . Cells were seeded in 12‐well plates  in dimethyl sulfoxide  and GOx (10 mg) in 0.1 M carbonate buffer (pH 9) were blended and the mixture was incubated for 4 h at room temperature in the dark. It was then transferred to a dialysis tube (MWCO: 12–14 kDa) and dialyzed against PBS  for 1 day. The dialyzed product was then lyophilized for subsequent use. An aliquot (1 ml) of the CNC/ICG/FITC‐GOx/Cu/LAP gel (including 1 μg of FITC) was added to the conical tube, and PBS  was used as the release medium. At determined time points , the medium (0.2 ml) was collected and the fluorescence intensity values were measured using a microplate reader  at 495 nm (excitation) and 550 nm (emission).2.43 cells/well. ICG , GOx , Cu , CNC/LAP , CNC/ICG/LAP , CNC/GOx/Cu/LAP , CNC/ICG/GOx/LAP , CNC/ICG/Cu/LAP , and CNC/ICG/GOx/Cu/LAP  were treated to the cells and NIR light  was applied to corresponding specimens. Subsequently, the samples were incubated with each specimen for 72 h. After removing each specimen, the cells were treated with CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent , and absorbance values (at 490 nm) were read using a microplate reader .The antiproliferation potentials of ICG (with or without NIR light), GOx, Cu, CNC/LAP, CNC/ICG/LAP, CNC/GOx/Cu/LAP, CNC/ICG/GOx/LAP, CNC/ICG/Cu/LAP, and CNC/ICG/GOx/Cu/LAP (in the presence or absence of NIR light) were tested in CT‐26 cells using a colorimetric assay.5 cell/well in the 6‐well plate. Aliquots (1 ml) of CNC/LAP (1000/1500 μg/ml), GOx/Cu (0.02/20 μg/ml), ICG (20 μg/ml), ICG/GOx/Cu (20/0.02/20 μg/ml), CNC/ICG/LAP (1000/20/1500 μg/ml), CNC/GOx/Cu/LAP (1000/0.02/20/1500 μg/ml), CNC/ICG/GOx/LAP (1000/20/0.02/1500 μg/ml), CNC/ICG/Cu/LAP (1000/20/20/1500 μg/ml), and CNC/ICG/GOx/Cu/LAP (1000/20/0.02/20/1500 μg/ml) were then added to the cells. NIR light  was applied to each well after 6 h of incubation, and the cells were further incubated for 18 h. After removing the specimen, DCFH‐DA (10 μM) was added to the cells and incubated for 20 min at 37 °C. Cell pellets were obtained by centrifugation and the cellular fluorescence signal was detected using a flow cytometer .Cellular ROS levels in the designed gel structures were analyzed using ROS Detection Reagents . CT‐26 cells were incubated for 24 h after seeding at 5.0 × 105 cells/well. Aliquots (1 ml) of Cu (20 μg/ml), CNC/LAP (1000/1500 μg/ml), GOx/Cu (0.02/20 μg/ml), ICG (20 μg/ml), ICG/GOx/Cu (20/0.02/20 μg/ml), CNC/ICG/LAP (1000/20/1500 μg/ml), CNC/GOx/Cu/LAP (1000/0.02/20/1500 μg/ml), CNC/ICG/GOx/LAP (1000/20/0.02/1500 μg/ml), CNC/ICG/Cu/LAP (1000/20/20/1500 μg/ml), and CNC/ICG/GOx/Cu/LAP (1000/20/0.02/20/1500 μg/ml) were added to the cells and further incubated for 24 h. The CNC/ICG/GOx/Cu/LAP gel‐treated cells were placed under NIR light . After eliminating each specimen, 5‐sulfosalicylic acid (5% w/v) was added to the cells. The freeze–thaw cycle was repeated three times to obtain cell lysates. Then, a Glutathione Colorimetric Detection Kit  was used to measure intracellular GSH levels according to the guideline provided by the manufacturer.The intracellular GSH levels were determined using a colorimetric assay. CT‐26 cells were incubated for 24 h after seeding in 6 wells at 5.0 × 105 cells/well and incubated for 24 h. GOx (0.01 μg/ml), CNC/LAP (500/750 μg/ml), and CNC/ICG/GOx/Cu/LAP (500/10/0.01/10/750 μg/ml) were applied to the cells and they were incubated for 24 h. CNC/ICG/GOx/Cu/LAP‐treated cells were placed under NIR laser . After removing the samples, cells were rinsed with cold PBS and they were treated with the reagents of ATP assay kit  according to the manufacturer's protocol. The absorbance of each sample was measured and ATP concentration was calculated. The relative ratio of each group to control group was plotted.ATP assay was conducted to verify the activity of GOx included in the gel system. CT‐26 cells were seeded onto 12‐well plate at 5.0 × 105 cells/well. Aliquots (0.5 ml) of CNC/LAP (500/750 μg/ml), GOx/Cu (0.01/10 μg/ml), ICG (10 μg/ml), ICG/GOx/Cu (10/0.01/10 μg/ml), CNC/ICG/LAP (500/10/750 μg/ml), CNC/GOx/Cu/LAP (500/0.01/10/750 μg/ml), CNC/ICG/GOx/LAP (500/10/0.01/750 μg/ml), CNC/ICG/Cu/LAP (500/10/10/750 μg/ml), and CNC/ICG/GOx/Cu/LAP (500/10/0.01/10/750 μg/ml) in the presence or absence of NIR light  were added to the cells and incubated for 12 h. All specimens were removed, and the cells were washed with PBS. Cells were pelleted by centrifugation and suspended in the FITC Annexin V Apoptosis Detection Kit . Following co‐staining with annexin V‐FITC and propidium iodide (PI), cellular fluorescence intensity values were determined using a flow cytometer (BD Bioscience).The apoptosis induction efficacy of the fabricated gel system was investigated in CT‐26 cells. Cells were incubated for 24 h after seeding in 24‐well plate at 5.0 × 102.52, and 5 min) was irradiated on the prepared specimens. The temperature distribution was scanned at 0, 1, and 5 min using a thermal camera .In vitro photothermal properties of the gel network were assessed by monitoring temperature change patterns following NIR light exposure.2, and 3 min), and whole‐body images were captured using a thermal camera on day 0 and 14.The photothermal properties of the fabricated gel structures were tested in mice following subcutaneous injection.2.6The biodegradation features of the associated gel networks were assessed in mice following subcutaneous injection.2.72) for 3 min. Blood samples were obtained from the heart on day 14, and heparinized blood samples were collected by centrifugation for 5 min at 16,100 g. Serum (~0.2 ml) was analyzed to determine albumin, alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) concentrations using a Cobas 8000 C702 chemical autoanalyzer .The toxicity of the associated gel structures was examined in mice by blood chemistry tests and histological staining of vital organs.2.86 cells in 0.1 ml) was inoculated into the back of BALB/c mice . Tumor size was estimated using the following formula: V (mm3) = (4π/3) × (longest diameter/2) × (shortest diameter/2) × (height/2).3, mice were randomly divided into six groups . Each sample was injected intratumorally on day 0 and 6, at a dose of 5 ml/kg. The tumor size and body weight of the mice were measured on day 0, 2, 6, 8, and 9. NIR light (808 nm and 1 W/cm2) was focused on the gel‐injected region for 3 min on day 0, 2, 3, 6, and 8. The temperature of the gel‐injection site was scanned using a thermal camera during the NIR light application process (for 3 min). The excised tumor mass was weighed on day 9 and fixed in a formaldehyde solution  for histological assays. The fixed specimens were embedded in paraffin blocks and sectioned into thin slices. The cells were then stained with H&E and terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) reagents.The local anticancer efficacy of the associated gel structures was examined in CT‐26 tumor‐implanted mice. CT‐26 cell suspension .All data are presented as mean ± SD. Each experiment was repeated at least thrice. Statistical analyses were performed using a two‐tailed 33.12O2 and Cu+ ions can produce hydroxyl radicals from H2O2 (by Fenton‐like reaction), as previously reported.In this study, liquid crystalline‐based gel network systems aligned with a nanorod (CNC) and nanodisk (LAP) were developed for local cancer therapy Figure . Nanostr+0.7[(Si8Mg5.5Li0.3)O20(OH)4]−0.7.+ and Mg2+ induces a net negative charge on the face of LAP, which can be balanced by the positive charge of sodium ions.The particle characteristics  of the CNC and LAP (dispersion in DW) were studied, as shown in Figures −1.−1 in the spectrum of LAP might indicate Si–O stretching, which was marginally shifted to 983 cm−1 in the spectrum of CNC/LAP (freeze‐dried form),The interactions between CNC and LAP were investigated using FT‐IR spectroscopy Figure . In the The crystalline and amorphous states of the CNC/LAP constructs were studied using XRD Figure . Diffrac−1 of Q range, the exponent of the power law in CNC/LAP group was 2.2, implying the presence of gel‐like characteristics.−1, corresponding to the (11¯0), (110), and (200) planes of cellulose Iβ.The structural information of the CNC/LAP mixture was acquired using SAXS Figure . The CNCThe thermal properties of the fabricated CNC/LAP constructs were investigated using TGA Figure . In the The arrangement of each ingredient in the CNC/LAP structure was observed using polarization microscopy Figure . A retarThe morphology of the CNC/LAP dispersion was observed using TEM Figure . To ensuThe gelation behavior of the prepared hybrid systems was evaluated using an inversion test Figure . The CNCThe viscoelastic features of the nanocomponent mixtures were investigated using a rheometer Figure  and S6. The cross‐sectional structure of the lyophilized CNC/ICG/GOx/Cu/LAP mixture was visualized using FE‐SEM Figure . A similElemental distribution in the lyophilized gel structure was studied by SEM with EDS mapping Figure . In part2+ ions may convert GSH to GSSG and GSH can transform DTNB to 2‐nitro‐5‐thiobenzoic acid (TNB).The effect of copper ions on GSH depletion was investigated using an extracellular GSH assay Figures  and S10.2O2 from glucose, and Cu+ can generate hydroxyl radicals from H2O2 via a Fenton‐like reaction. The hydroxyl radical produced may oxidize TMB (colorless) to its charge‐transfer complex form (blue).Generation of hydroxyl radicals based on the “cascade effect” in the designed system was demonstrated using the TMB assay with glucose Figure . In addi2O2 by GOx and it was further converted to hydroxyl radical by copper ion. ESR data also supported the successful catalytic function of designed CNC/ICG/GOx/Cu/LAP gel system.Cascade production of hydroxyl radical by GOx/Cu combination was further studied with ESR spectroscopy analysis Figure . With th2O2 generation capability of GOx (introduced into the gel network) was assessed using a TiOSO4‐based test  group than in the DW group, probably because of the generation of H2O2 by GOx. Interestingly, there was a significant difference between the CNC/GOx/Cu/LAP and CNC/GOx/LAP groups treated with D‐glucose (p < 0.05). The copper ions in the CNC/GOx/Cu/LAP group consumed H2O2, thus the absorbance of the CNC/GOx/Cu/LAP group was lower than that of the CNC/GOx/LAP group. This indicates that the normal enzymatic activity of GOx entrapped in the gel network was maintained.The Ht Figure . GOx decCu release from the CNC/ICG/GOx/Cu/LAP structure was investigated Figure . The relICG release from the gel system and subsequent entry into cancer cells were assessed by NIRF imaging Figure . To avoiThe GOx release profile from the gel system was evaluated by measuring the fluorescence intensity of the FITC‐GOx Figure . Owing t3.2p < 0.05). Considering that GOx consumes the accumulated glucose in cancer cells and it leads to cancer cell starvation,2O2,2O2 can react with copper ions (Fenton‐like reactions), leading to the production of hydroxyl radicals. This cascade reaction may support the role of ST and CDT in cancer treatment. CNC/ICG/LAP + NIR group data suggest concentration‐dependent antiproliferation potential due to PTT efficacy. CNC/GOx/Cu/LAP group has lower cell viability values in higher sample concentration range which may be induced by ST/CDT combination. In CNC/ICG/GOx/LAP + NIR group, marked reduction in cell viability was seen in high sample concentration, indicating the efficacy of PTT/ST. Similar antiproliferation efficacy was observed in CNC/ICG/Cu/LAP + NIR group which may be from PTT and partial CDT options. The CNC/ICG/GOx/Cu/LAP + NIR group exhibited an enhanced antiproliferation effect in the 100/2/0.002/2/150, 500/10/0.01/10/750, and 1000/20/0.02/20/1500 μg/ml groups, compared with the CNC/ICG/GOx/Cu/LAP group (p < 0.05). Considering the cell viability data of the CNC/LAP group, the antiproliferation effect seems to be supplied by the ICG/GOx/Cu + NIR combination. These findings indicate that the triple combination of ST/CDT (by GOx/Cu) and PTT (by ICG), rather than partial combination, can provide significantly enhanced antiproliferation effects in CT‐26 cells.Antiproliferation efficacy was evaluated by a colorimetric assay in a murine colorectal carcinoma cell line (CT‐26 cell) Figure . ICG, whp < 0.05). These findings indicate that the CNC/ICG/GOx/Cu/LAP gel, when combined with NIR light, accelerates glucose deprivation and hydroxyl radical generation. Higher cellular ROS level in the CNC/ICG/GOx/Cu/LAP + NIR group, compared with the other groups, indicates the maximized local anticancer efficacy based on the integrated PTT/ST/CDT strategy in colorectal cancer cells.Intracellular ROS levels were measured using the cellular fluorescence intensity Figure . Measurep < 0.05). It means that incorporated Cu molecule works appropriately for the conversion of GSH to GSSG. The relative GSH level in the CNC/ICG/GOx/Cu/LAP + NIR group (38.3%) was also lower than that in the CNC/ICG/GOx/Cu/LAP group (57.2%) (p < 0.05). Hyperthermia induction can contribute to elevated GSH consumption, as reported.The intracellular GSH depletion ability of the designed nanocomposite‐based gel construct was evaluated using colorimetric analysis Figure .5 IntrATP assay was conducted for assessing the catalytic potential of designed hybrid gel system Figure . GOx‐trep < 0.05). The introduction of GOx/Cu to CNC/LAP system significantly elevated the apoptosis potential via a cascade of hydroxyl radical production. Moreover, photothermal conversion by ICG with NIR light absorption (shown in the CNC/ICG/GOx/Cu/LAP + NIR group) also strengthened the apoptosis induction capability, as revealed by the antiproliferation, intracellular ROS, and intracellular GSH assays  and inorganic nanodisk (LAP) hybridized gel systems were designed to realize glucose deprivation in cancer cells (for ST), a self‐supplying cascade of hydroxyl radicals (for CDT), and light‐to‐heat conversion (for PTT). Nano‐size particle‐aligned gel structures were designed by optimizing the concentrations of CNC and LAP to tune the rheological properties. As modalities of ST and CDT, GOx and Cu were introduced to the CNC/LAP structure for glucose decomposition and Fenton‐like reaction‐based hydroxyl radical production, respectively. Furthermore, Cu contributed to cellular GSH depletion and enhanced anticancer activity. ICG was entrapped in the CNC/LAP structure to induce hyperthermia in the tumor tissue upon NIR laser irradiation. However, it not only achieved its objective but also further amplified the CDT efficacy of the GOx/Cu combination. In short, the CNC/LAP gel system exhibited higher retention capability at the injection site, and a prolonged PTT/ST/CDT effect was achieved. Thus, the results of this study indicate that the designed nano‐unit‐aligned gel network is effective for local cancer therapy and can ensure a high safety profile.Sungyun Kim: Conceptualization ; formal analysis (lead); investigation (lead); visualization ; writing – original draft (lead); writing – review and editing . ChaeRim Hwang: investigation, formal analysis, visualization, writing—original draft. Da In Jeong: investigation, formal analysis. JiHye Park: investigation, formal analysis. Han‐Jun Kim: investigation, formal analysis. KangJu Lee: investigation, formal analysis. Junmin Lee: investigation, formal analysis. Seung‐Hwan Lee: Funding acquisition (lead); resources ; writing – review and editing (supporting). Hyun‐Jong Cho: conceptualization, supervision, funding acquisition, resources, writing—original draft, writing—review & editing.The authors declare no conflict of interest.https://publons.com/publon/10.1002/btm2.10470.The peer review history for this article is available at APPENDIX S1. Supporting informationClick here for additional data file.


H. pylori infection is the strongest known risk factor for gastric carcinoma. The activation of the yes-associated protein 1 (YAP) and β-catenin pathways has been associated with multiple tumor types. In this study, we investigated the crosstalk between the YAP and β-catenin pathways in H. pylori-associated gastric tumorigenesis. Immunohistochemical analysis of YAP and β-catenin expression was performed in human gastric cancer tissues. The small molecules Super-TDU and KYA1797K, pharmacological inhibitors of YAP and β-catenin, respectively, were used to investigate the role of these signaling pathways in H. pylori-induced gastric carcinogenesis in murine models of infection. The common downstream targets of YAP and β-catenin signaling were evaluated by RNA sequencing (RNA-seq). Western blot, immunofluorescence, luciferase, RT-PCR, immunoprecipitation, cell counting kit-8 (CCK8), EdU and spheroid assays were used. H. pylori infection promoted YAP and β-catenin nuclear accumulation and transcriptional activity in gastric epithelial cells and transgenic insulin–gastrin (INS-GAS) mice, whereas silencing of both YAP and β-catenin synergistically inhibited H. pylori-induced cell proliferation and expansion. In addition, YAP was found to directly interact with β-catenin and knockdown of YAP suppressed H. pylori-induced nuclear translocation of β-catenin. Moreover, downstream genes caudal-type homeobox 2 (CDX2), leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) and RuvB like AAA ATPase 1 (RUVBL1) were shared by both YAP and β-catenin signaling. Furthermore, treatment with the YAP inhibitor Super-TDU or β-catenin inhibitor KYA1797A significantly alleviated gastric inflammation and epithelial DNA damage in H. pylori-infected mice. Finally, the elevation of gastric YAP was positively correlated with β-catenin expression in human gastric cancer tissues. These findings indicate that YAP and β-catenin synergistically promote H. pylori-induced gastric carcinogenesis via their physical interaction and reveal that CDX2, LGR5 and RUVBL1 are the downstream genes shared by both the YAP and β-catenin signaling pathways, and potentially contribute to H. pylori pathogenesis. Helicobacter pylori (H. pylori) has been recognized as a causal factor causing active chronic gastritis and peptic ulcer diseases. Infection with H. pylori is the strongest risk factor for gastric cancer through the histopathological stages of gastritis, intestinal metaplasia, and dysplasia3. H. pylori is usually acquired during childhood and remains in the stomach mucosa for many decades if untreated4. Notably, the prevalence of H. pylori infection is very high in East Asian countries, such as China, Japan, and Korea5. Sufficient evidence indicates that H. pylori eradication treatment significantly reduces the incidence of gastric cancer7. Given the high prevalence of H. pylori infection, screening and eradicating H. pylori in all populations may be impossible. The elucidation of the pathogenic mechanism of H. pylori infection would not only contribute to the identification of high-risk populations of gastric cancer but also provide an important theoretical basis for the eradication of H. pylori.Gastric cancer remains a major health concern as it is the fifth most newly diagnosed cancer and the fourth leading cause of cancer-related deaths worldwide. It is noteworthy that more than half of gastric cancer cases occur in East Asian countries H. pylori infection leads to the activation of YAP through the upregulation of total protein and promotion of nuclear translocation, which induces epithelial–mesenchymal transition and contributes to malignant phenotypes8. YAP, the core effector of the Hippo signaling pathway, plays a critical role in maintaining cell proliferation, apoptosis, and tissue development9. The Hippo cascade phosphorylates YAP on serine residues, leading to its cytoplasmic localization and degradation. Once the Hippo signaling pathway is inactive, YAP translocates to the cell nucleus and interacts with TEA domain transcription factors (TEADs) to initiate the target gene expression11. Accumulating evidence indicates that the aberrant activation of YAP promotes tumorigenesis, such as liver tumorigenesis12 and colorectal tumorigenesis13.Previously, we reported that 15. Accumulating evidence shows that H. pylori infection augments β-catenin expression and nuclear accumulation17. The important role of developmental signaling pathways in tumorigenesis is indisputable. These pathways often function cooperatively, rather than independently. Recently, based on similar biological functions, there have been multiple accumulating lines of evidence suggesting the close interaction of YAP and β-catenin18. Intriguingly, it has been reported that the coactivation of YAP and β-catenin cooperates to induce hepatocarcinogenesis19. In basal-like breast cancers, YAP is required for β-catenin activity, and they cooperate to regulate cancer stem cells (CSCs)20. However, the molecular mechanism of their interaction in the pathogenesis of H. pylori infection remains unknown.The Wnt/β-catenin cascade is also considered to play a central role in cell proliferation, differentiation and apoptosis. Upon Wnt signaling, β-catenin is activated and translocates into the nucleus, where it interacts with LEF/TCF transcription factors and induces the expression of downstream genesH. pylori significantly augmented YAP and β-catenin total expression, nuclear accumulation, and transcriptional activity in gastric epithelial cells. Furthermore, RNA-seq was performed to identify the common downstream genes of YAP and β-catenin. In response to H. pylori infection, YAP strongly interacts with β-catenin and is required for β-catenin activation. Notably, the knockdown of YAP and β-catenin by siRNA synergistically inhibited cell proliferation and expansion induced by H. pylori infection. Intriguingly, we found that treatment with a YAP inhibitor or β-catenin inhibitor could alleviate H. pylori infection-induced gastric inflammation and DNA damage in mice. Our findings revealed a novel molecular mechanism by which the crosstalk between YAP and β-catenin promoted H. pylori-associated gastric tumorigenesis.In this study, we demonstrated previously known overexpression of YAP and β-catenin in human gastric cancer specimens. Infection with H. pylori strains 7.13, ATCC43504 and rodent-adapted CagA+H. pylori strain pre-murine Sydney Strain 1 (PMSS1) were used in this study. Briefly, H. pylori bacteria were cultured on Brucella agar with 5% sheep blood (BD Bioscience) under microaerophilic conditions for in vitro passages as previously described. For an in vitro co-culture with gastric epithelial cells, bacteria were then propagated in Brucella broth (BD Bioscience) supplemented with 10% FBS  overnight. H. pylori 7.13, 43504 and PMSS1 strains were co-cultured with gastric epithelial cells at a multiplicity of infection (MOI) of 100:1. Additionally, PMSS1 strain was used in mice experiments.The wild-type CagA+Human gastric cancer cells AGS and MKN45 were cultured in DMEM/F12 and RPMI/1640 medium, respectively, containing 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2.Gastric cancer and adjacent normal gastric tissues microarray sections were acquired from Wuhan Servicebio Biotech Co. Ltd . The tissue array contains 48 paired paraffin-embedded gastric carcinoma and adjacent tissues. The clinical and pathological information was shown in Supplementary Table S1. Immunohistochemical staining was performed to detect the expression of YAP and β-catenin. The sections were incubated with an anti-YAP antibody (1:600 dilution) and anti-β-catenin antibody (1:100 dilution).21. It also facilitates profiling according to cancer type or pathological stage, patient survival, similar gene detection, correlation, and dimensionality reduction analysis.GEPIA is a newly developed interactive web server, based on the analysis of RNA sequencing expression data from The Cancer Genome Atlas (TCGA) and The Genotype Tissue Expression (GTEx) database. GEPIA provides an array of customizable functions including the ability to analyze differential expression in tumor and normal tissues22 and M50 Super 8× TOPFlash (cat. 12456)23 were obtained from Addgene. Cells were transfected with appropriate plasmid or siRNA using Lipofectamine 3000  according to manufacturer’s instructions.The recombinant plasmid containing the YAP and β-catenin cDNA was purchased from HITRO BioTech . Small interfering (si) RNA duplexes were obtained from GenePharma . The YAP inhibitor Super-TDU and β-catenin KYA1797K were purchased from Selleckchem . The plasmids of 8×GTIIC-luciferase (cat. 34615)All procedures performed on animals were approved by the Ethics Committee of First Affiliated Hospital of Nanchang University. Six- to eight-week male specific-pathogen-free INS-GAS mice (Jackson Lab) were kept with a 12 h light/dark cycle and provided ad libitum access to food and water. The air conditions were controlled at a temperature of 18–26°C with 40–70% relative humidity.H. pylori strain PMSS1 (2 × 109 CFU)/mouse) every other day for five times or challenged with Brucella Broth (BB) as an uninfected control group24. All mice were euthanized at 16 weeks post infection.After 1 week of acclimation, mice were gavaged with the mouse-adapted wild-type Total RNA was extracted using the TRIzol reagent  according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Then, the libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd .n = 42) were purchased from GemPharmatech . Mice were randomly divided into six groups: control group, H. pylori-infected group, Super-TDU (YAP inhibitor) treatment group, H. pylori in combination with Super-TDU treatment, KYA1797K (β-catenin inhibitor) treatment group and H. pylori in combination with KYA1797K treatment group. Super-TDU and KYA1797K were gavaged with sterile Brucella. After 1 month of H. pylori infection, mice were intraperitoneally injected with 500 μg/kg Super-TDU for 9 weeks or 25 mg/kg KYA1797K for 7 weeks. All mice were euthanized at 13 weeks post infection. For gastric tissues collection, the stomach was opened along the lesser curvature extending from the forestomach through the proximal duodenum.Six-week-old male C57BL/6 mice  for 1 h, the cells were incubated with primary antibodies against YAP or β-catenin overnight at 4°C, and then incubated with Alexa Fluor Plus 488 or Alexa Fluor Plus 555  secondary antibody for 1 h in the dark. Cell nuclei were counters with 4′,6-diamidino-2-phenylindole (DAPI). All slides were imaged using a fluorescence microscope (Leica Stellaris 5).25. Briefly, the paraffin-embedded tissues were deparaffinized in xylene and gradient ethanol. Sodium citrate buffer was then used for antigen retrieval. 3% hydrogen peroxide (H2O2) solution was used to block endogenous peroxidase activity. The primary antibody was incubated overnight at 4°C. After washing three times with PBS, the sections were incubated with secondary antibody using PV-6000 kit . The slides were stained with diaminobenzidine (DAB) chromogen and counterstained with hematoxylin. All images were acquired under a microscope (Nikon Eclipse). The immunoreactivity of the samples was evaluated by two trained pathologists and scored for intensity  and frequency . For the statistical analysis, the intensity and frequency of targets were transformed into a composite expression score using the following formula intensity × frequency. The score ranges from 0 to 12.Immunohistochemical staining was performed to detect the expression profiles of YAP, β-catenin, Ki67 and γ-H2A× as described previouslyThe nucleus-cytoplasm separation assay was performed using the Nucleus-cytoplasm Protein Extraction Kit . Extracted nuclear and cytoplasmic proteins were resolved by SDS-PAGE and then immunoblotted with the indicated antibodies.8. Primary anti-YAP (#4912), anti-β-catenin (#37447), anti-β-tubulin (#2128), anti-Histone 3 (#4499), anti-GAPDH (#2118) antibodies were purchased from Cell Signal Technology ; anti-CagA  were from Santa Cruz , anti-β-actin (#20536–1-AP) from proteintech . Briefly, cell proteins were extracted after adding lysis buffer supplemented with protease inhibitor cocktail , and then the protein concentration was assessed using a BCA assay kit . The cell lysates were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% milk blocking buffer, the membranes were incubated with the primary antibodies overnight at 4°C, and then incubated with HRP-conjugated secondary antibodies  for 1 h at room temperature. The protein bands were visualized by SuperSignal West Pico stable peroxide solution (Thermo Scientific) in a darkroom or using an iBright imaging system (Thermo Scientific). β-actin and GAPDH were used as internal controls to normalize protein expression in cells sample and animal tissues sample, respectively.Western blot analyses were performed as previously described25. In brief, total RNA was extracted using TRIzol reagent . The primer primers were listed in the Supplementary Table S2. Then, qPCR assays were performed with a QuantStudio 5 Real-time PCR system (Life Technologies) according to the manufacturer’s protocol. The GAPDH gene was used as an internal control.The qRT-PCR analysis was performed as described in previous studies3 cells/100 μl per well. The cells were infected with H. pylori strain at an MOI of 50 for the indicated time. The CCK-8 assay  was performed according to the manufacturer’s instructions. The optical density values were measured at a wavelength of 450 nm using a Molecular Devices SpectraMax M2e.The cell viability was detected by CCK-8 and EdU. Cells were transfected with an indicated plasmid vector or siRNA and then seeded into 96-well plates at a density of 2 × 10For EdU cell proliferation assays, the relative viability of cells was determined by Cell-Light EDU Apollo 488 in Vitro imaging kit (RiboBio) following the kit protocol. All images were acquired and quantified using the high-content screening (HCS) platform In-Cell Analyzer 2200 .An immunoprecipitation assay was performed to determine the interaction between proteins. Cell lysates were collected in the lysis buffer, then incubated overnight with the mixture of 1 ug of antibodies and beads at 4°C. Then, the beads were washed three times with 1 mL of lysis buffer and then boiled in loading buffer. The samples were subjected to western blot analysis as described above.H. pylori strain. The cell lysates were collected, and luciferase activity was detected by the Dual-Luciferase Reporter Assay System  according to the manufacturer’s protocol. The results were expressed as relative luciferase activity (firefly luciferase/Renilla luciferase).The luciferase reporter assay was used to detect the transcriptional activity of YAP/TEADs and TCF/LEFs. The cells were seeded into 12-well plates and then transfected with plasmids or indicated siRNA. After transfection for 48 h, cells were cocultured with 2 chamber at 37°C for 7 days. The culture medium was changed every 3 days. The diameter and number of tumor spheres in 3 random 100 magnification fields were calculated under Fluorescence Microscope  in the bright light model.After treatment with YAP or β-catenin siRNA, MKN45 cells were seeded into ultra-low attachment 6-well dishes  and cultured in serum-free DMEM/F12 medium supplemented with 20 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 2% B-27 (Life Technologies), and 2 mM L-glutamine (Life Technologies). Cells were incubated in a 5% COp < 0.05 .All statistical analysis was performed using SPSS 21.0 software. The data are presented as mean ± standard deviation (SD) of three independent experiments. A student t test was used to determine the difference between two independent groups. Mann–Whitney tests were used to determine the differences in numerical variables between differently defined groups. Kaplan–Meier survival curves and log-rank (Mantel–Cox) tests were used for survival analysis. Pearson correlation analysis was used for the correlation between YAP and β-catenin in human gastric cancer tissues. The results were considered statistically significant at H. pylori infection upregulates YAP expression and nuclear translocation in gastric epithelial cells8. Consistent with our previous studies, infection with the H. pylori ATCC43504 and 7.13 strains significantly elevated the total expression and nuclear accumulation of YAP. Likewise, the total protein and nuclear protein levels of β-catenin were increased following H. pylori infection . Together, these findings indicate that a YAP is required for H. pylori-induced activation of the β-catenin pathway. Additionally, these data suggested the important role of the common target genes of YAP and β-catenin, including CDX2, LGR5 and RUVBL1, in the pathogenesis of H. pylori infection.Our results indicated that in response to -catenin . Additioby siRNA  and S3a.nfection  and S3b.nfection . The imm control . These fse genes . The othH. pylori-associated gastric pathogenesis, we introduced the YAP inhibitor Super-TDU and the β-catenin inhibitor KYA1797K in C57BL/6 mice following infection with PMSS1 . Histological examination of the gastric mucosa revealed that mice infected with the H. pylori PMSS strain showed significant inflammation and slight Alcian-blue-positive intestinal metaplasia compared with uninfected mice . These discrepancies between in vivo and in vitro data may be attributed to the fact that H. pylori infection induced gastric inflammation in mice rather than intestinal metaplasia or even gastric neoplasm. Accumulating evidence shows that H. pylori infection provokes host cell DNA double-strand breaks (DSBs) to cause genomic instability that drives gastric tumorigenesis35. In accordance with a previous report25, the biomarker of DSBs γH2A× was significantly increased in H. pylori-infected mice at 13 weeks post infection compared with WT mice. Notably, we observed a significant reduction in γH2A× expression in infected mice treated with the YAP inhibitor Super-TDU compared with untreated counterparts, as assessed by Western blotting . The shared gene targets downstream of YAP and β-catenin, including CDX2, LGR5 and RUVBL1, were further examined in gastric tissues. The expression of RUVBL1 was significantly decreased in the gastric tissue of PMSS1-infected mice treated of KYA1797K or Super-TDU compared with the infected mice without this treatment . Howeverblotting  and S5c,blotting  and immu of mice  and S5d.H. pylori infection can synergistically promote malignant transformation. We next examined the clinical relevance of these findings by comparing expressions using immunohistochemical staining in 48 patient-matched stomach tumors and adjacent normal tissues. The clinicopathological characteristics are listed in Supplementary Table S6. As shown in R = 0.52, p = 0). Moreover, the expression patterns and clinical outcomes were confirmed via the analysis of the GEPIA online database  and then slightly decreased at 24 HPI38.In several studies, researchers have investigated the effect of H. pylori infection synergistically regulates the YAP and β-catenin pathways in in vitro and in vivo models. First, herein we show that in response to H. pylori infection, both YAP and β-catenin signaling pathways were activated; upregulation of YAP transcription preceded the activation of β-catenin transcription as evidenced by assays using TEADs and TCF/LEF reporters. Second, our data indicate that there is an intimate crosstalk between YAP and β-catenin in H. pylori-induced gastric tumorigenesis. Knockdown of YAP significantly inhibited the nuclear accumulation of β-catenin induced by H. pylori. Third, infection with H. pylori in gastric epithelial cells enhanced YAP interaction with β-catenin. Although the mechanism underlying this interaction between YAP and β-catenin remains to be defined in the future, various recent studies have confirmed the role of YAP on the regulation of β-catenin signaling pathway40. Recently, the overexpression of YAP has been shown to increase the expression of β-catenin and its transcriptional target genes, which contributed to the self-renewal and regeneration of intestinal epithelial cells in ulcerative colitis41. Intriguingly, although YAP is essential for the nuclear accumulation of β-catenin, inhibition of YAP has no effect on the mRNA levels of β-catenin. This can be explained by our finding that YAP physically interacted with β-catenin (42. However, we found that knockout of YAP caused a slight increase in β-catenin mRNA levels. Additional studies will be needed to address the functional interaction between YAP and β-catenin in response to H. pylori infection.There is no data demonstrating that -catenin , demonstH. pylori-induced gastric carcinogenesis was identified in this study. First, simultaneous activation of YAP and β-catenin was observed in human gastric cancer tissues compared with paired adjacent tissues. Furthermore, our current data showed that H. pylori induces gastric cell proliferation, survival and expansion, which has also been reported in other studies27. Notably, we found that combined knockdown of YAP and β-catenin leads to a strong inhibition of cell proliferation and expansion, compared to knockdown of YAP or β-catenin alone. In support of our findings, concomitant activation of YAP and β-catenin has been reported to drive liver tumorigenesis in mice, while activation of YAP or β-catenin did not lead to any tumor formation19. Consistently, YAP has been found to induce nuclear β-catenin activity and cooperate with β-catenin to drive oncogenesis in basal-like breast cancer20. Mechanistically, we performed transcriptomic analysis to identify the common downstream targets of YAP and β-catenin. Our observations suggested that the overlapping downstream genes, CDX2, LGR5 and RUVBL1, were linked to H. pylori-induced gastric carcinogenesis. CDX2 has been considered as a biological marker for intestinal metaplasia, a precancerous gastric lesion43. We have previously indicated the upregulation of CDX2 induced by H. pylori infection44. Now we show that H. pylori-induced CDX2 is closely associated with YAP and β-catenin. Consistent with our observations, the stem cell fate marker LGR5, defined as the common target of YAP and β-catenin45, was responsible for stem cell proliferation and H. pylori-induced gastric pathology46. Another common target gene for YAP and β-catenin, RUVBL1, has been reported to regulate the pro-inflammatory response of macrophages47. Therefore, we could speculate that RUVBL1 may be involved in H. pylori-associated gastric inflammation and lesions. In summary, we have identified a novel mechanism regarding the relationship and synergistic effect between YAP and β-catenin pathway in H. pylori-induced gastric carcinogenesis.Given the similar biological functions of YAP and β-catenin, the synergistic effect of YAP and β-catenin in H. pylori induced carcinogenesis. The results suggest the potential therapeutic significance of these two small molecules, Super-TDU and KYA1797K, for the treatment of gastric lesions related to H. pylori infection. Consistent with our data, Super-TDU, as a selective YAP inhibitor could effectively suppress gastric cancer growth32. Germline telomere defects have been found to induce DNA damage together with upregulated YAP1 levels. Pharmacological inhibition of YAP1 effectively ameliorated inflammation48. Likewise, there is some evidence showing the potential therapeutic approach of the β-catenin inhibitor KYA1797K in colorectal cancer49 and triple-negative breast cancer50. However, translational research on these small-molecule drugs still needs to be further explored.The critical role of YAP and β-catenin was further substantiated by our in vivo findings. Our data showed that YAP inhibition by Super-TDU or β-catenin inhibition by KYA1797K led to a reduction in gastric pathology, including gastric inflammation and DNA damage, the most accepted mechanism operating in cases of H. pylori infection-induced gastric tumorigenesis. YAP and β-catenin cooperate to activate target genes that promote gastric pathology associated with H. pylori. However, there are some limitations to the present study. Mice only developed gastric inflammation after H. pylori infection in this study. In further studies, a longer period of H. pylori infection in animal models is needed to induce more severe gastric pathology. This would be helpful to investigate the carcinogenic role of H. pylori. These findings suggest the potential therapeutic strategy of YAP or β-catenin inhibitors to reverse gastric precancerous lesions associated with H. pylori infection.In summary, our findings indicated, for the first time, that the crosstalk between YAP and β-catenin signaling pathway contributes to Click here for additional data file.


Cymbopogon winterianus extract (CWE) in controlling the C. musae that caused anthracnose disease in banana fruit. In vitro assay results showed that CWE at 1.5–2.5 gL−1 concentrations controlled the development of the test pathogen. Mycelial blast, cytoplasmic discharge, and spore edema were noticed when CWE was applied. The Minimum Effective Concentration (MEC) of CWE for the in vivo assay was 1.50 gL−1 and can be used as a postharvest treatment on banana fruit to deter anthracnose infection. Moreover, no visible phytotoxicity or changes in aroma were observed on banana fruit treated with CWE, even at the highest concentration of 2.5 gL−1. The GCMS analysis revealed 41 chemical components associated with CWE. The five main compounds were the following: Methyl oleyl ether (40.20%), γ-Sitosterol (15.80%), 6-Methylheptan-3-ol (7.13%), α-Terpineol (5.56%), and n-Pentadecanol (4.05%). The CWE possesses excellent fungicidal effects against C. musae; in the near future, it can be used as an alternative to commercially available traditional fungicides on the market.The genus Cymbopogon (Poaceae) species have been widely cultivated throughout the world for a wide range of uses in the pharmaceutical and agricultural fields. The current work investigates the fungicidal activities of  Philippine banana farming practices have significantly transitioned due to consumer preference for sustainable agricultural products. As an illustration, most of the banana farms in the Philippines have converted land to produce “organic” bananas. Similarly, small-scale farmers cultivate bananas in their backyards without using synthetic fungicides during the growing process or after harvest. Plantation-grown organic and non-chemical bananas are typically exported to various Asian nations. Furthermore, most consumers choose organic bananas because they are free of agricultural synthetic chemicals.1. Colletotrichum species can cause anthracnose, but the most prevalent species is Colletotrichum musae, which results in 30–40% losses in commercial value3. This fungus infects young banana fruit but goes unrecognized until the fruit turns ripe and yellow4.Bananas have been arriving on the market with substandard quality due to postharvest disease, mainly anthracnose, owing to the lack of synthetic fungicide treatments available16. Various Cymbopogon species have been widely cultivated throughout the world for a wide range of uses, which include antifungal17, insecticidal18, antibacterial19, and pharmaceutical20. However, little evidence is available regarding the efficacy of Cymbopogon winterianus extract (CWE) in controlling the C. musae that causes anthracnose disease in bananas. Further, the availability of raw plant materials of C. winterianus in the trading area in tropical and subtropical regions of Asia, America, and Africa was the basis for choosing it as a test plant. Also, the current investigation was to establish the fungicidal impact of CWE in managing anthracnose diseases in banana fruit. The information gathered could be used to treat anthracnose infection in banana fruit and a potential replacement for synthetic fungicides sold commercially soon.On the other hand, earlier studies have shown that natural products  and other edible coatings (chitosan) are effective against pests and diseases of agricultural commoditiesC. musae mycelial growth (in-vitro) were summarized in Fig. C. musae at a minimum rate of 1.50 gL−1  was considerably better than Mancozeb at 2.50 gL−1, which did not affect the growth of C. musae on the banana fruit surface. However, the preventative effects of CWE at a concentration of 1.50 gL−1 demonstrated complete control against anthracnose infection, while the efficacy given by Mancozeb was only 7.72–9.62%. On the other hand, CWE was statistically equal to a synthetic fungicide with a 100% inhibitory effect on a fungal pathogen in a simultaneous assay.The application of CWE 24 h before, after, or simultaneously administered with the fungal pathogen (in-vivo) was summarized in Fig. 8 mL−1C. musae suspension developed black lesions on the fruit's surface  of 1.50 gL−1 in managing anthracnose infection, and it was observed at this dose with no significant phytotoxicity on banana fruit. On the other hand, the C. winterianus is well-known for its strong scent, primarily due to the presence of monoterpene compounds. However, under the phytotoxicity test results, the scent of CWE is only recognizable in the first 48 h after application; thereafter, no CWE aroma was detected. Thus, the quality can be acceptable and recommendable to consumers' preferences. Moreover, these monoterpene compounds were naturally degraded by the environment26.In general, the CWE successfully suppressed the development of −1 was 1.67 times lower than the volume content of the synthetic chemical fungicide (Mancozeb at 2.5 gL−1) which is necessary to achieve control. The main components and minor derivatives of CWE worked together to make it even more toxic, causing the target species to die quickly.The CWE, on the other hand, showed dose dependence, with the higher concentration having a more significant effect. According to the present data, the volume content of CWE at 1.50 gLC. musae29. Based on the results of a preventative assay, C. musae may possibly have developed Mancozeb resistance. Other studies have shown that major fungal pathogens of fruit are also resistant to synthetic chemical fungicides such as Mancozeb30. Meanwhile, the findings of the preventative test outperformed those of the curative assay in terms of treatment efficacy. This could be due to the fact that C. musae had colonized the surface of the banana fruit prior to treatment application.The variations in treatment responses between in vivo and in vitro were explained by the absorption capacity and degree of complexity of two distinct surfaces: agar (in vitro) and banana fruit (in vivo). In an in-vivo test, the fruit's physiological properties cooperate with each other, causing treatments with moderate to high toxicity against 32. So far, no biological activities on microorganisms have been reported for oleyl alcohol.methyl ether, 6-Methylheptan-3-ol, and n-Pentadecanol,Two of CWE's five key components have shown antimicrobial properties: γ-sitosterol and α-Terpineol36. Moreover, there is a decrease in the development of chemical resistance. In some cases, a microorganism can develop resistance to a single compound. However, it is much more difficult for microorganisms to develop resistance to multiple or complex compounds38. Because various microorganisms have different responses to the same compound, and multiple compounds have a significant potential to increase the toxicity they have on the target microorganisms they are trying to kill39. In addition to this, C. winterianus is readily available on the market. On the other hand, solvent and the equipment needed for extraction are also commercially available, and processing does not present any tough challenges. As a result, small-to-medium-scale banana farmers may be able to use it to control anthracnose infection in banana fruit.Meanwhile, there are two significant benefits associated with using plant extracts as fungicides. For example, most extracts derived from plants do not pose health risks, and in most cases, they are beneficial to the environmentMeanwhile, future research should focus on the interactive effect of CWE in combination with other natural products. The CWE storage studies may also be required. Other aspects of these natural products, such as the mechanism of action and formulation, should be addressed to ensure optimal stability and potency.C. winterianus samples were collected in Nueva Ecija, Philippines 15° 35′ N  121° 00′ E. The aerial portion of CWE was extracted using Mangoba and Alvindia's methodology11. The CWE was evaporated using a USA Lab RE-1020 rotary evaporator. The extracts were immediately stored in an ultralow freezer at − 80 °C until biological assays and chemical analyses were performed. On the other hand, the chemical profiling of CWE was carried out according to the Alvindia and Mangoba11 protocol using a Shimadzu GC–MS. Furthermore, Shimadzu Gas Chromatography (GC) and Mass Spectrometer (MS) models GC2010 and QP2010 were used. Furthermore, the individual components of the CWE were characterized using the National Institute of Standards and Technology's system library's fragmentation display of mass spectra and retention time (RT). The peak area of the GC was used to calculate the percentage of each compound in the CWE.The C. musae culture was acquired from the laboratory of Dionysus Enterprises, located in Nueva Ecija, Philippines. The test pathogen was previously isolated from naturally infected Cavendish banana fruit, which exhibits anthracnose lesions11. The pure isolated culture of C. musae was kept at 6 °C on potato dextrose agar (PDA). The pathogenicity of C. musae was maintained and tested on banana fruit by means of artificial inoculation and periodic re-isolation.The C. musae was conducted using the method described by Alvindia and Mangoba10. PDA was homogenized with CWE at dosages ranging from 0.05 to 2.50 gL−1. The PDA plates that had not been treated served as a negative control, whereas the PDA plates that had been treated with a commercial fungicide (Mancozeb at 2.5 gL−1) served as a positive control. Further, the C. musae mycelial plug (5 mm) was put into the center of treated PDA plates for seven days and stored at 25 ± 2 ℃ and 70 ± 5% relative humidity (RH). The growth of C. musae was assessed after seven days by measuring the colony diameter using a digital caliper. The test was done twice, with three replications per treatment, and was set up in a completely randomized design (CRD). The percent efficacy of treatment (%ET) was computed as follows: (CNC-CDT)/CNC X 100, where CNC is the negative control colony diameter, and CDT is the treated plug colony diameter.The efficacy of CWE on the mycelial growth of 11. CWE (0.05–2.50 gL−1) was applied to a 10 μL fungal suspension containing 108 spores mL−1 and evenly dispensed on water agar. Under the compound microscope, 100 spores were counted after 48 h. The percent efficacy of treatment (% ET) for spore germination was calculated using the method recently described by Alvindia and Mangoba11. Spore dilatation and burst were noted throughout the study. The experiment was repeated twice with a tri-replicate for each treatment and was arranged as mentioned earlier.The spore germination assay was conducted following the Alvindia and Mangoba proceduresC. musae [10d old (108 spores mL−1)] and treatments (CWE and Mancozeb) were administered to each inoculating site either 24 h before (preventative test), simultaneously applied or after 24 h (curative test). Control fruit was solely applied with C. musae spore suspension. The treated banana fingers were maintained in a sterile plastic box (25 X 40 cm) at 25 ± 2 ℃ and 70 ± 5% RH. After seven days, the treatment impact on C. musae was evaluated using a digital caliper to measure the lesion diameter on the fruit surface. The treatments' percent effectiveness was calculated as previously stated. Further, the phytotoxicity of CWE and the pathogenicity of C. musae on banana fruit were recorded. The experiment was done twice with six replicates per treatment and arranged using CRD.Cavendish banana samples were collected in Davao del Norte, Philippines 7°21′N 125°42′E. The owner of the banana plantation, Mr. Eduardo Madrid, gave permission to take samples during the study. The fruit (80% maturity) was de-handed and rinsed in sterile water before removing the crown. The fruit was taken from a single banana tree to ensure the homogeneity of the test material. Each treatment is comprised of six banana fingers. The banana finger has three inoculation sites: tip, center, and bottom. Each inoculation site was marked and partially injured with an insect needle (No. 1). The insect needle was stabbed once into the banana fruit skin, leaving a ≦1.50 mm diameter wound. Following that, 10μL of a spore solution of An analysis of variance (ANOVA) was undertaken on all data using SPSS Statistics version 16.0 for IOS. Significant differences between treatments were determined using the Tukey HSD test .


Bioimpedance analysis-assessed extracellular water/total body water (ECW/TBW) ratio may be a marker for mortality and poor nutritional status in hemodialysis patients. In 193 maintenance hemodialysis patients, we retrospectively investigated the relationships among ECW/TBW ratio, mortality, and protein-energy wasting (PEW). Four components—body mass index, normalized protein catabolic rate, normalized serum creatinine level, and serum albumin level—constitute the simple PEW score; this score was calculated based on the positive number of items concerning malnutrition among these four components. A score ≥ 3 indicated PEW. Patients were stratified by an ECW/TBW ratio cut-off value (0.40) and by PEW versus non-PEW status. The simple PEW score, cardiothoracic ratio, and log-transformed C-reactive protein level were independently correlated with the ECW/TBW ratio. Eighty-four patients died during follow-up (median 4.3 years). After adjustments for sex, age, hemodialysis vintage, histories of cardiovascular events and diabetes, and C-reactive protein level, a higher ECW/TBW ratio and PEW were independently related to elevated risks of all-cause death. Adding the ECW/TBW ratio to a baseline risk model including PEW significantly increased C-statistics from 0.788 to 0.835. In conclusion, the ECW/TBW ratio may be an indicator of PEW and may be a predictor of death even accounting for PEW, in hemodialysis patients. A recognized type of malnutrition in this patient group is protein-energy wasting (PEW), which refers to the wasting of fat and muscle due to decreased energy and/or protein consumption and chronic inflammation1. According to the “International Society of Renal Nutrition and Metabolism”, PEW is diagnosed on the basis of four distinct components: biochemical markers, low body fat mass, low muscle mass, and reduced intake of energy and/or protein1. Based on these original components, Moreau-Gaudry et al. proposed a simple PEW score including 4 nutritional indicators: serum albumin (Alb) level, serum creatinine (Cre) level adjusted by the body surface area (BSA) (Cre/BSA), body mass index (BMI), normalized protein catabolic rate (nPCR)4. This simple score was validated as an indicator of death in maintenance hemodialysis patients6. Volume excess is reportedly associated with hypertension and congestive heart failure and leads to cardiovascular death8.Patients receiving hemodialysis frequently experience malnutrition and excessive hydration, which are strongly associated with elevated mortality risks10. We reported that the extracellular water-to-intracellular water ratio (ECW/ICW ratio), a novel marker derived from BIA, was independently associated with the geriatric nutritional risk index (GNRI), a surrogate marker of PEW11. In addition, the combination of the ECW/ICW ratio and GNRI improved the predictability of mortality. Conversely, the extracellular water-to-total body water ratio (ECW/TBW ratio), established as an indicator of volume excess to set dry weight12, is helpful to predict cardiovascular-related and all-cause mortality14. In the dialysis population, recent investigations have demonstrated an independent negative correlation between Alb and nPCR, two elements of the simple PEW score, and the ECW/TBW ratio16. Therefore, we hypothesized that ECW/TBW ratio can indicate PEW; however, relationships among ECW/TBW ratio, mortality, and PEW remain unknown.Recently, body composition assessment using bioimpedance analysis (BIA) is used to evaluate malnutrition and fluid overload simultaneouslyOur study aimed to examine correlations between ECW/TBW ratio and simple PEW score in a baseline hemodialysis patient cohort. In addition, we examined relationships among the ECW/TBW ratio, PEW, and mortality in maintenance hemodialysis patients. We also investigated whether adding ECW/TBW ratio to the PEW-included risk model could predict mortality in these patients.2, 509 ± 162 μmol/L/m2, and 0.77 ± 0.17 g/kg/day, respectively. In this present study, 63 patients (32.6%) were diagnosed with PEW. A significant correlation was found between ECW/TBW ratio and simple PEW score . Moreover, the ECW/TBW ratio of the PEW group was higher (0.419 ± 0.074) than that the of the non-PEW group (0.343 ± 0.073) (p < 0.0001).The present study patients’ characteristics are summarized in Table According to the linear regression univariate analysis, the ECW/TBW ratio was positively correlated with male sex, age, history of diabetes and CVD events, log-transformed CRP (Log CRP), CTR, and simple PEW score and was negatively correlated with phosphorus level, Alb level, Cre/BSA, and nPCR. The multivariate linear regression analysis revealed independent positive associations of ECW/TBW ratio with male sex (β = 0.189), age β = 0.215), history of diabetes (β = 0.142), Log CRP (β = 0.133), CTR (β = 0.213), and simple PEW score (β = 0.256)  during a median follow-up period of 4.3 [1.9–6.8] years. The five-year survival rates in the higher and lower ECW/TBW ratio groups were 25.1% and 79.6%, respectively (p < 0.0001)   when adding ECW/TBW ratio to the baseline risk model, which included age, sex, hemodialysis vintage, history of diabetes and CVD events, CRP, and PEW Table .Table 4TIn patients receiving hemodialysis, the ECW/TBW ratio was independently correlated with simple PEW score, Log-transformed CRP, and CTR. In this population, those with greater ECW/TBW ratios and those who had PEW had higher likelihoods of dying from any cause. Those who had PEW and an elevated ECW/TBW ratio had the highest probability of dying from any cause. Additionally, the prediction of mortality was much improved when adding the ECW/TBW ratio to the simple PEW score-included risk model. The ECW/TBW ratio—a marker of PEW, inflammation, and fluid overload—may therefore be a strong predictor of mortality.11. In addition, the repeated measurements of the ECW/ICW ratio and the GNRI increased the predictability for mortality18. Moreover, the combination of the ECW/ICW ratio and the GNRI improved the predictive accuracy of mortality11. The current study evaluated the association between the ECW/TBW ratio, one of the major established BIA-measured parameters, and PEW. PEW was diagnosed using established diagnostic criteria and not a surrogate marker. The usefulness of the WCW/TBW ratio for predicting all-cause mortality with adjustment for PEW was also investigated.PEW, a wasting of muscle and fat, is common and highly related to mortality in patients undergoing hemodialysis. We have recently proposed that the ECW/ICW ratio, a novel but minor BIA-measured parameter, may be a marker of PEW with the ECW/ICW ratio being independently associated with the GNRI1, body mass is assessed using the BMI, body fat percentage, and unintentional weight loss. As ECW/TBW ratio had a significant association with Cre/BSA but not BMI, this might be an indicator of muscle wasting and not simply an indicator of the obesity paradox20. Furthermore, in this case, the simple PEW score, CRP, and CTR can reflect malnutrition, inflammation, and fluid volume, respectively, and these factors may interact with each other. The possible pathophysiology may be that fluid overload can introduce bowel edema; thereafter, the translocation of bowel endotoxin into the circulation may induce inflammation21. The inflammation can lead to malnutritional status owing to protein catabolism23, and then hypoalbuminemia may exacerbate the systemic edema. Thus, ECW/TBW ratio may indicate PEW, inflammation, and fluid overload. However, the present study was performed in a cross-sectional design; therefore, establishing causal relationships among variables is difficult. A longitudinal study is needed to confirm these findings in the future.In the present study, the ECW/TBW ratio had independent and positive associations with the simple PEW score, log-transformed CRP, and CTR in patients on hemodialysis. To our knowledge, this study is the first to directly examine the association between the ECW/TBW ratio and PEW. In this present study, the ECW/TBW ratio had a significant negative correlation with each item of the simple PEW score except for BMI: Alb, r = − 0.508; Cre/BSA, r = − 0.446; nPCR, r = − 0.416; and BMI, r = 0.031 (p = 0.66). According to the original PEW criteria4. The study divided patients into four groups as follows: (G1) severe wasting, simple PEW score of 0 and 1; (G2) moderate wasting, simple PEW score of 2; (G3) slight wasting, simple PEW score of 3; and (G4) normal nutritional status, simple PEW score of 4. Compared with G1, G3 and G4 (simple PEW score ≥ 3) had an independent correlation with an increased mortality risk. Additionally, the original diagnostic criteria for PEW required at least three of the four components for a diagnosis of PEW. Thus, in this study, patients with a simple PEW score ≥ 3 were diagnosed as having PEW. In the present study, PEW was independently associated with an elevated all-cause death risk. Moreover, patients with a higher ECW/TBW ratio and PEW (G4) were at the highest risk of death; therefore, the combination of the ECW/TBW ratio and PEW may be useful to stratify the risks of death. Interestingly, compared with G1 and G2, G3 and G4 were associated with elevated mortality risks. The predictability of all-cause death significantly improved after the ECW/TBW ratio was added to the simple PEW score-contained baseline risk model. Therefore, the ECW/TBW ratio may be a powerful predictor of all-cause mortality in hemodialysis patients with PEW and without PEW. This might be because ECW/TBW ratio is not merely an indicator of PEW but also an indicator of inflammation and volume overload.In this study, PEW was evaluated using a simple PEW score, and the ARNOS prospective study validated its utility for predicting mortalityThere were some limitations to consider. First, we included only Japanese patients receiving hemodialysis; therefore, our results may not apply to hemodialysis patients of other ethnic groups. Second, this study was a retrospective single-center study with a relatively small number of hemodialysis patients. Third, the ECW/TBW ratio was measured during study enrollment; therefore, the changes in the ECW/TBW ratio during the follow-up periods were not evaluated. To validate our study findings, multicenter prospective studies with large sample sizes and other ethnic groups may be needed in the future.In conclusion, for hemodialysis patients, the simple PEW score, CRP, and CTR were independently correlated with the ECW/TBW ratio. An elevated ECW/TBW ratio was linked to greater risks of all-cause death. Moreover, adding the ECW/TBW ratio to the simple PEW score–containing baseline risk model significantly improved the prediction of death. The ECW/TBW ratio may not only be useful to predict PEW but also to predict inflammation and volume overload and, therefore, may be a powerful predictor of mortality.Patients who had stably undergone hemodialysis  for more than 6 months were retrospectively included. At the Matsunami General Hospital, body composition was measured using BIA between January 2008 and December 2015. Because the current investigation was carried out retrospectively by evaluating data gathered from routine clinical practice, the necessity to obtain prior informed consent was waived by the “Ethics Committee of Matsunami General Hospital”. “Ethics Committee of Matsunami General Hospital” approved the study plan, which was performed adhering to the principles of the Declaration of Helsinki .2 (kg/m2).The baseline information of each participant obtained from medical records was: sex; age; kidney disease that led to end-stage kidney disease, such as diabetic nephropathy, nephrosclerosis, chronic glomerulonephritis, and others; hemodialysis duration; smoking and alcohol habits; and histories of diabetes, hypertension, and cardiovascular disease (CVD) events. Diabetes was defined based on having received a previous diagnosis of diabetic retinopathy and/or a history of using glucose-lowering medication. Hypertension was defined by two parameters: (1) using some anti-hypertensive drugs and/or (2) a pre-hemodialysis blood pressure ≥ 140/90 mmHg. The CVD events included peripheral artery disease, myocardial infarction, heart failure, angina pectoris, and stroke (hemorrhage or infarction). At the start of the week, blood tests were taken in the supine position before and after a hemodialysis session. BIA was performed in the supine position about 30 min after the hemodialysis session, and ICW, ECW, and TBW values were obtained by a body composition analyzer with multiple frequencies (2.5–300 kHz) using the ankle and wrist method . The measurement was performed on Wednesday or Thursday. The CTR was calculated based on chest radiography which was performed on the same day. BMI was calculated from dry weight and height: BMI = dry weight/height4 the positive number of items regarding malnutrition among the four components . The cut-off values of Alb, Cre/BSA, BMI, and nPCR were defined as 3.8 g/dL, 380 μmol/L/m2, 23 kg/m2, and 0.8 g/kg/day, respectively. In this study, accounting for the original version of PEW24, PEW was diagnosed when at least three of the four components were applied.The simple PEW score was defined as12. Moreover, they were also separated into those with PEW and those without PEW (non-PEW). They were further cross-classified into four groups as per the presence or absence of PEW and the cut-off point of the ECW/TBW ratio: non-PEW and lower ECW/TBW ratio (Group 1: G1), PEW and lower ECW/TBW ratio (Group 2: G2), non-PEW and higher ECW/TBW ratio (Group 3: G3), and PEW and higher ECW/TBW ratio (Group 4: G4).ECW/TBW ratios were used to categorize patients into two groups: those with ratios higher than 0.40 and those with ratios lower than 0.40. The cut-off value was used as previously reported by Kim et al.Patients who were alive by December 2020 were censored, and the endpoint of the present study was all-cause mortality.A non-normally distributed variable was reported as a median and interquartile range, while a normally distributed variable was reported as a mean and standard deviation. For categorical variables, the chi-square test was used to compare differences between groups (G1–G4) that were divided by the presence or absence of PEW and the cutoff value of the ECW/TBW ratio at 0.40. For continuous variables, the Kruskal–Wallis test or one-way analysis of variance was used to compare differences. We examined the correlation between simple PEW score and the ECW/TBW ratio using Pearson's correlation coefficient. To examine the differences in the ECW/TBW ratio between the PEW and non-PEW groups, we employed Student's t-test. We used a univariate regression analysis to evaluate at how the ECW/TBW ratio and baseline factors related. Subsequently, using factors that were substantially linked with the ECW/TBW ratio in the univariate regression analysis, we conducted a multivariate regression analysis. We did not include Alb, Cre/BSA, or nPCR in the multivariate linear regression model because they were part of the simple PEW score. We utilized the Kaplan–Meier method to estimate the survival rate and the log-rank test to examine differences in the survival rates. To calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for mortality, we used a univariate Cox analysis. We performed a multivariate Cox analysis with adjustments for sex, age, and covariates, which were significant based on the univariate Cox analysis. Because Alb, Cre, BMI, and nPCR were included in the simple PEW score, these covariates were excluded in the multivariate Cox analysis. We compared the predictabilities for mortality between the baseline risk model, including PEW, and the enriched model with the ECW/TBW ratio, and we calculated the C-index and the net reclassification improvement (NRI).We performed the statistical analyses using SPSS version 28 . Differences with a p-value < 0.05 were considered statistically significant.