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A combined nanotherapeutic approach targeting farnesoid X receptor,ferroptosis,and fibrosis for nonalcoholic steatohepatitis treatment

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Acta Pharmaceutica Sinica B 2024年第5期14卷 2228-2246页

作者： Jiangtao Fu Pingping Zhang Zhiguo Sun Guodong Lu Qi Cao Yiting Chen Wenbin Wu Jiabao Zhang Chunlin Zhuang Chunquan Sheng Jiajun Xu Ying Lu Pei Wang The center for Basic Research and Innovation of Medicine and Pharmacy(MOE) School of PharmacyNaval Medical UniversityShanghai 200433China National Demonstration center for experimental Pharmaceutical Education Naval Medical University/Second Military Medical UniversityShanghai 200433China department of Diving and Hyperbaric Medicine Naval Special Medical CenterNaval Medical UniversityShanghai 200433China

Obeticholic acid(OCA),a farnesoid X receptor(FXR)agonist with favorable effects on fatty and glucose metabolism,has been considered the leading candidate drug for nonalcoholic steatohepatitis(NASH)***,its limited effectiveness in resolving liver fibrosis and lipotoxicity-induced cell death remains a major ***,a newly recognized form of cell death characterized by uncontrolled lipid peroxidation,is involved in the progression of *** oxide(NO)is a versatile biological molecule that can degrade extracellular *** this study,we developed a PEGylated thiolated hollow mesoporous silica nanoparticles(MSN)loaded with OCA,as well as a ferroptosis inhibitor liproxsatin-1 and a NO donor S-nitrosothiol(ONL@MSN).Biochemical analyses,histology,multiplexed flow cytometry,bulk-tissue RNA sequencing,and fecal 16S ribosomal RNA sequencing were utilized to evaluate the effects of the combined nanoparticle(ONL@MSN)in a mouse NASH *** with the OCA-loaded nanoparticles(O@MSN),ONL@MSN not only protected against hepatic steatosis but also greatly ameliorated fibrosis and ***@MSN also displayed enhanced therapeutic actions on the maintenance of intrahepatic macrophages/monocytes homeostasis,inhibition of immune response/lipid peroxidation,and correction of microbiota *** findings present a promising synergistic nanotherapeutic strategy for the treatment of NASH by simultaneously targeting FXR,ferroptosis,and fibrosis.

关键词： Nonalcoholic steatohepatitis Nanotherapy Farnesoid X receptor Ferroptosis Fibrosis Obeticholic acid S-Nitrosothiol Liproxstatin-1

Artificial cavernosa-like tissue based on multibubble Matrigel and a human corpus cavernous fibroblast scaffold

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Asian Journal of Andrology 2024年第3期26卷 260-267页

作者： Yu-Zhuo Chen Yi-Hong Zhou Min-Bo Yan Ming Xiao Biao Liu Ying-Hao Yin Xiao-Li Tan Yong-Quan Huang Yu-Hong Lin Ting Xie Jia-Li Tian Qi Wang Jian-Ying Li Zi-Zhou Meng Zheng Li Emily Xing Yu-Xin Tang Ya-Wei Li Zhong-Zhen Su Liang-Yu Zhao department of Ultrasound The Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000China department of Interventional Medicine Guangdong Provincial Key Laboratory of Biomedical ImagingThe Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000China department of Urology The Fifth Affiliated HospitalSun Yat-sen UniversityZhuhai 519000China department of Andrology the Center for Men’s HealthUrologic Medical CenterShanghai Key Laboratory of ReproductiveMedicineShanghai General HospitalShanghai Jiao Tong University School of MedicineShanghai 200080China Cedars-Sinai Medical center Los AngelesCA 90024USA

Ex vivo tissue culture of the human corpus cavernosum(cc)can be used to explore the tissue structural changes and complex signaling *** present,artificial Cc-like tissues based on acellular or three-dimensional(3D)-printed scaffolds are used to solve the scarcity of primary penis tissue ***,inconvenience and high costs limit the wide application of such ***,we describe a simple,fast,and economical method of constructing artificial CC-like *** Cc fibroblasts(FBs),endothelial cells(ECs),and smooth muscle cells(SMCs)were expanded in vitro and mixed with Matrigel in specific proportions.A large number of bubbles were formed in the mixture by vortexing combined with pipette blowing,creating a porous,spongy,and spatial *** Cc FBs produced a variety of signaling factors,showed multidirectional differentiation potential,and grew in a 3D grid in Matrigel,which is necessary for CC-like tissue to maintain a porous structure as a cell *** the C-like tissue,ECs covered the surface of the lumen,and SMCs were located inside the trabeculae,similar to the structure of the primary *** cell components remained stable for 3 days in vitro,but the EC content decreased on the 7th ***/integrated(WNT)signaling activation led to lumen atrophy and increased tissue fibrosis in CC-like tissue,inducing the same changes in characteristics as in the primary *** study describes a preparation method for human artificial cC-like tissue that may provide an improved experimental platform for exploring the function and structure of the cC and conducting drug screening for erectile dysfunction therapy.

关键词： artificial tissue corpus cavernosum ex vivo fibroblasts

Elucidating and manipulating pressure-induced water intrusion–extrusion in tunable hydrophobic Co/Zn bimetallic ZIFs:Roles of pore size and hydrogen bond

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Nano Research 2024年第1期17卷 344-353页

作者： Diyi Fang Chuanlei Liu Yu Chen Qilong Peng Kongguo Wu Yuxiang Chen Hao Jiang Yuan Wu Benxian Shen Qiumin Wu Di Wu Hui Sun International Joint Research center of Green Energy Chemical Engineering School of Chemical EngineeringEast China University of Science and TechnologyShanghai 200237China Alexandra Navrotsky Institute for experimental Thermodynamics The Gene and Linda Voiland School of Chemical Engineering and BioengineeringMaterials Science and Engineeringand Department of ChemistryWashington State UniversityWashington 99163USA

Porous heterogeneous lyophobic systems(HLSs)find potential applications in energy restoring,dissipating,and ***,the development of controllable HLSs still lacks rational structure design of nanoporous materials matching the molecular sizes of adopted *** that,thoroughly understanding the underlying transportation mechanism in the confined nano channels is greatly *** this work,a series of Co/Zn bimetallic zeolitic imidazolate frameworks(ZIFs)with tunable structures were synthesized via regulating the Co to Zn ratios and employed to investigate the intrusion–extrusion of liquid water in confined *** characterizations confirm the heterometallic coordination in the Co/Zn-doped *** intrusion–extrusion experiments unlock the relationship between the intrusion pressure and the nanopore size and realize the evolution of the HLSs between molecular spring and *** addition,cycling tests indicate the reversible structure change of Co/Zn ZIFs encountering pressure-induced water *** combination with molecular dynamics simulations,we present that the water multimers intrude into nanopores of ZIFs in chain-like forms along with dissociation of hydrogen bonds(HBs).Water molecules in the pre-intrusion state exhibit reduced HBs in response to the increase of pressure and linear structure with 1.6–3.0 HBs on *** transition to the post-intrusion situation,the associative configuration of water tends to exhibit the tetrahedral ***,we highlight the roles of pore size and HB in synergically dominating the pressure-induced intrusion–extrusion of liquid water in hydrophobic ***,the present work can also guide the development of functional guest–host systems based on porous architectures.

关键词： heterometallic zeolitic imidazolate frameworks nanopore intrusion hydrogen bonds

Recombinant adeno-associated virus 8-mediated inhibition of microRNA let-7a ameliorates sclerosing cholangitis in a clinically relevant mouse model

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World Journal of Gastroenterology 2024年第5期30卷 471-484页

作者： Hui Hua Qian-Qian Zhao Miriam Nkesichi Kalagbor Guo-Zhi Yu Man Liu Zheng-Rui Bian Bei-Bei Zhang Qian Yu Yin-Hai Xu Ren-Xian Tang Kui-Yang Zheng Chao Yan Jiangsu Key Laboratory of Immunity and Metabolism Department of Pathogenic Biology and ImmunologyNational Demonstration Center for Experimental Basic Medical Science EducationLaboratory of Infection and ImmunityXuzhou Medical UniversityXuzhou 221004Jiangsu ProvinceChina department of Laboratory Medicine The Affiliated Hospital of Xuzhou Medical UniversityXuzhou 221002Jiangsu ProvinceChina

BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment *** adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been *** To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing *** A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC *** sacrifice,the liver and the serum were collected from each *** hepatobiliary injuries,hepatic inflammation and fibrosis were *** targets of let-7a-5p and downstream molecule NF-κB were detected using Western *** rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 *** reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary ***,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic *** Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.

关键词： Primary sclerosing cholangitis Recombinant adeno-associated virus 8 Let-7a-5p Therapeutic effects Inflammation

Targeted anti-tumor synergistic effects of Myc decoy oligodeoxynucleotides-loaded selenium nanostructure combined with chemoradiotherapy on LNCaP prostate cancer cells

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Oncology Research 2024年第1期32卷 101-125页

作者： ROGHAYEH GHORBANI MAHMOUD GHARBAVI ALI SHARAFI ELHAM RISMANI HAMED REZAEEJAM YOUSEF MORTAZAVI BEHROOZ JOHARI department of Medical Biotechnology School of MedicineZanjan University of Medical SciencesZanjanIran Nanotechnology Research center Ahvaz Jundishapur University of Medical SciencesAhvazIran Zanjan Pharmaceutical Biotechnology Research center Zanjan University of Medical SciencesZanjanIran department of Pharmaceutical Biotechnology School of PharmacyZanjan University of Medical SciencesZanjanIran Molecular Medicine department Biotechnology Research CenterPasteur Institute of IranPasteur AvenueTehranIran department of radiology Technology School of Allied Medical SciencesZanjan University of Medical SciencesZanjanIran

In the present study,we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNCaP prostate cancer *** decoy ODNs were designed based on the promoter of Bcl-2 gene and analyzed by molecular docking and molecular dynamics *** were loaded on the synthesized Se@BSA@Chi-MTX *** physicochemical characteristics of nanostructures were determined by FTIR,DLS,UV-vis,TEM,EDX,in vitro release,and hemolysis ***,the cytotoxicity properties of them with and without X-irradiation were investigated by uptake,MTT,cell cycle,apoptosis,and scratch assays on the LNCaP cell *** results of DLS and TEM showed negative charge(−9 mV)and nanometer size(40 nm)for Se@BSA@Chi-DEC-MTX NPs,*** results of FTIR,UV-vis,and EDX showed the proper interaction of different parts and the correct synthesis of *** results of hemolysis showed the hemocompatibility of this nanoparticle in concentrations less than 6 mg/*** ODNs release from the nanostructures showed a pH-dependent manner,and the release rate was 15%higher in acidic *** targeted Se@BSA@Chi-labeled ODN-MTX NPs were efficiently taken up by LNCaP cells by targeting the prostate-specific membrane antigen(PSMA).The significant synergistic effects of nanostructure(containing MTX drug)treatment along with X-irradiation showed cell growth inhibition,apoptosis induction(~57%),cell cycle arrest(G2/M phase),and migration inhibition(up to 90%)compared to the *** results suggested that the Se@BSA@Chi-DEC-MTX NPs can potentially suppress the cell growth of LNCaP *** nanostructure system can be a promising approach for targeted drug delivery and chemoradiotherapy in prostate cancer treatment.

关键词： Chemoradiotherapy Combination therapy Decoy oligodeoxynucleotides Myc transcription factor Selenium nanoparticle Prostate cancer

Deep learning framework for comprehensive molecular and prognostic stratifications of triple-negative breast cancer

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Fundamental Research 2024年第3期4卷 678-689页

作者： Shen Zhao Chao-Yang Yan Hong Lv Jing-Cheng Yang Chao You Zi-Ang Li Ding Ma Yi Xiao Jia Hu Wen-Tao Yang Yi-Zhou Jiang Jun Xu Zhi-Ming Shao Key Laboratory of Breast Cancer in Shanghai Department of Breast SurgeryFudan University Shanghai Cancer CenterShanghai 200032China Institute for AI in Medicine School of Artificial IntelligenceNanjing University of Information Science and TechnologyNanjing 210044China department of Pathology Fudan University Shanghai Cancer CenterShanghai 200032China Greater Bay Area Institute of Precision Medicine Guangzhou 511466China department of radiology Fudan University Shanghai Cancer CenterShanghai 200032China

Triple-negative breast cancer(TNBC)is the most challenging breast cancer *** stratification and target therapy bring clinical benefit for TNBC patients,but it is difficult to implement comprehensive molecular testing in clinical ***,using our multi-omics TNBC cohort(N=425),a deep learning-based framework was devised and validated for comprehensive predictions of molecular features,subtypes and prognosis from pathological whole slide *** framework first incorporated a neural network to decompose the tissue on WSIs,followed by a second one which was trained based on certain tissue types for predicting different ***-omics molecular features were analyzed including somatic mutations,copy number alterations,germline mutations,biological pathway activities,metabolomics features and immunotherapy *** was shown that the molecular features with therapeutic implications can be predicted including the somatic PIK3CA mutation,germline BRCA2 mutation and PD-L1 protein expression(area under the curve[AUC]:0.78,0.79 and 0.74 respectively).The molecular subtypes of TNBC can be identified(AUC:0.84,0.85,0.93 and 0.73 for the basal-like immune-suppressed,immunomodulatory,luminal androgen receptor,and mesenchymal-like subtypes respectively)and their distinctive morphological patterns were revealed,which provided novel insights into the heterogeneity of TNBC.A neural network integrating image features and clinical covariates stratified patients into groups with different survival outcomes(log-rank P<0.001).Our prediction framework and neural network models were externally validated on the TNBC cases from TCGA(N=143)and appeared robust to the changes in patient *** potential clinical translation,we built a novel online platform,where we modularized and deployed our framework along with the validated *** can realize real-time one-stop prediction for new *** summary,using only pathological WSIs,our proposed framework

关键词： Triple-negative breast cancer Deep learning Digital pathology Patient stratification Online platform

Metabolic reprogramming by Syntenin-1 directs RA FLS and endothelial cell-mediated inflammation and angiogenesis

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Cellular & Molecular Immunology 2024年第1期21卷 33-46页

作者： Anja Meyer Stephanie R.Zack Wes Nijim Adel Burgos Vishwa Patel Brian Zanotti Michael V.Volin M.Asif Amin Myles J.Lewis Costantino Pitzalis Shiva Arami Joseph A.Karam Nadera J.Sweiss Shiva Shahrara Jesse Brown VA Medical center ChicagoILUSA department of Medicine Division of RheumatologyThe University of Illinois at ChicagoChicagoILUSA department of Microbiology and Immunology Midwestern UniversityDowners GroveILUSA Division of Rheumatology and Clinical Autoimmunity center of Excellence University of MichiganAnn ArborMIUSA Centre for experimental Medicine and Rheumatology William Harvey Research InstituteBarts and The London School of Medicine and DentistryQueen Mary University of LondonLondonUnited Kingdom Centre for experimental Medicine&Rheumatology William Harvey Research InstituteQueen Mary University of London and Barts NIHR BRC&NHS TrustLondonUK department of Biomedical Sciences Humanitas Universityand Humanitas Research HospitalMilanItaly department of Orthopedic Surgery the University of Illinois at ChicagoChicagoILUSA

A novel rheumatoid arthritis(RA)synovial fluid protein,Syntenin-1,and its receptor,Syndecan-1(SDC-1),are colocalized on RA synovial tissue endothelial cells and fibroblast-like synoviocytes(FLS).Syntenin-1 exacerbates the inflammatory landscape of endothelial cells and RA FLS by upregulating transcription of IRF1/5/7/9,IL-1β,IL-6,and CCL2 through SDC-1 ligation and HIF1α,or mTOR ***,Syntenin-1 orchestrates RA FLS and endothelial cell invasion via SDC-1 and/or mTOR *** Syntenin-1 reprogrammed endothelial cells,the dynamic expression of metabolic intermediates coincides with escalated glycolysis along with unchanged oxidative factors,AMPK,PGC-1α,citrate,and inactive oxidative ***,RA FLS rewired by Syntenin-1 displayed a modest glycolytic-ATP accompanied by a robust mitochondrial-ATP *** enriched mitochondrial-ATP detected in Syntenin-1 reprogrammed RA FLS was coupled with mitochondrial fusion and fission recapitulated by escalated Mitofusin-2 and DRP1 *** found that VEGFR1/2 and Notch1 networks are responsible for the crosstalk between Syntenin-1 rewired endothelial cells and RA FLS,which are also represented in RA *** to RA explants,morphological and transcriptome studies authenticated the importance of VEGFR1/2,Notch1,RAPTOR,and HIF1αpathways in Syntenin-1 arthritic mice and their obstruction in SDC-1 deficient ***,dysregulation of SDC-1,mTOR,and HIF1αnegated Syntenin-1 inflammatory phenotype in RA explants,while inhibition of HIF1αimpaired synovial angiogenic imprint amplified by *** conclusion,since the current therapies are ineffective on Syntenin-1 and SDC-1 expression in RA synovial tissue and blood,targeting this pathway and its interconnected metabolic intermediates may provide a novel therapeutic strategy.

关键词： Syntenin-1 Syndecan-1 RA FLS RA explants immunometabolism

Adenosine triphosphate mediates the pain tolerance effect of manual acupuncture at Zusanli(ST36) in mice

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Journal of Traditional Chinese Medicine 2024年第4期44卷 660-669页

作者： LI Zhongzheng ZHAO Yadan Ma Weigang Zhang Yonglong XU Zhifang XI Qiang LI Yanqi QIN Siru ZHANG Zichen WANG Songtao ZHAO Xue LIU Yangyang GUO Yi GUO Yongming Research center of experimental Acupuncture Science Tianjin University of Traditional Chinese Medicine National Clinical Research center for Chinese Medicine Acupuncture and Moxibustion department of Acupuncture and Moxibustion Qingdao Hiser Hospital Affiliated of Qingdao University(Qingdao Traditional Chinese Medicine Hospital) School of Traditional Chinese Medicine Tianjin University of Traditional Chinese Medicine School of Acupuncture and Moxibustion and Tuina Tianjin University of Traditional Chinese Medicine

OBJECTIVE: To investigate the mechanisms behind the effects of acupuncture in Traditional Chinese Medicine,we delved into the adenosine triphosphate/peripheral purinergic P2X receptor 3(ATP/P2X3) receptor signaling system as an indicator of the body's energy state,commonly referred to as "Qi".METHODS: The tail-flick test was utilized to explore the impact of acupuncture on pain tolerance threshold(PTT)in mice, while also assessing adenosine(ADO) levels and adenylate energy charge(EC) at Zusanli(ST36). The study further investigated the dose-dependent effects of acupuncture on PTT and ADO levels at Zusanli(ST36).To shed light on the underlying mechanisms of acupuncture's effects, the study examined the impact of ATP, a P2X3 receptor antagonist, and adenosine disodium on PTT following acupuncture administration. RESULTS: Acupuncture at Zusanli(ST36) led to significant improvements in PTT in mice, with the most effective interventions being twirling for 2 min and needle retention for 28 min. These interventions also resulted in significant increases in ATP levels. The effects of acupuncture were further augmented by administration of different doses of ATP at Zusanli(ST36), and pretreatment with a P2X3 receptor antagonist decreased PTT. Adenylate EC peaked at 30 min after intraperitoneal injection of ATP, and pretreatment with various doses of i.p. ATP 30 min prior to acupuncture increased PTT in a dose-dependent manner. Additionally, pretreatment with an i.p. or intramuscular injection of adenosine disodium enhanced the effects of acupuncture. CONCLUSION: This research provides compelling evidence that ATP is involved in the regulation of PTT through acupuncture, revealing new avenues for achieving enhanced clinical outcomes.

关键词： acupuncture adenosine triphosphate energy state point ST36 (Zusanli) receptors, purinergic P2X3

Spleen volume is associated with overt hepatic encephalopathy after transjugular intrahepatic portosystemic shunt in patients with portal hypertension

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World Journal of Gastrointestinal Surgery 2024年第7期16卷 2054-2064页

作者： Chun-Juan Zhao Chao Ren Zhen Yuan Guo-Hui Bai Jin-Yu Li Long Gao Jin-Hui Li Ze-Qi Duan Dui-Ping Feng Hui Zhang College of Medical imaging Shanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina department of radiology The First Hospital of Shanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina department of Occupational Health School of Public HealthShanxi Key Laboratory of Environmental Health Impairment and PreventionNHC Key Laboratory of PneumoconiosisMOE Key Laboratory of Coal Environmental Pathogenicity and PreventionShanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina department of Oncological and Vascular Intervention The First Hospital of Shanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina Shanxi Provincial Clinical Research center for Interventional Medicine The First Hospital of Shanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina First Clinical Medical College Shanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina Shanxi Key Laboratory of Intelligent imaging and Nanomedicine The First Hospital of Shanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina Intelligent imaging Big Data and Functional Nano-imaging Engineering Research center of Shanxi Province The First Hospital of Shanxi Medical UniversityTaiyuan 030001Shanxi ProvinceChina

BACKGROUND Portal shunt and immune status related to the spleen are related to the occurrence of hepatic encephalopathy(HE).It is unknown whether spleen volume before transjugular intrahepatic portosystemic shunt(TIPS)is related to postoperative *** To investigate the relationship between spleen volume and the occurrence of *** This study included 135 patients with liver cirrhosis who underwent TIPS,and liver and spleen volumes were elevated upon computed tomography *** Kaplan-Meier curve was used to compare the difference in the incidence rate of HE among patients with different spleen *** and multivariate Cox regression analyses were performed to identify the factors affecting overt HE(OHE).Restricted cubic spline was used to examine the shapes of the dose-response association between spleen volumes and OHE *** The results showed that 37(27.2%)of 135 patients experienced OHE during a 1-year follow-up *** with preoperative spleen volume(901.30±471.90 cm3),there was a significant decrease in spleen volume after TIPS(697.60±281.0 cm^(3))in OHE *** the severity of OHE increased,the spleen volume significantly decreased(P<0.05).Compared with patients with a spleen volume≥782.4 cm^(3),those with a spleen volume<782.4 cm^(3) had a higher incidence of HE(P<0.05).Cox regression analysis showed that spleen volume was an independent risk factor for post-TIPS OHE(hazard ratio=0.494,P<0.05).Restricted cubic spline model showed that with an increasing spleen volume,OHE risk showed an initial increase and then decrease(P<0.05).CONCLUSION Spleen volume is related to the occurrence of OHE after *** spleen volume is an independent risk factor for post-TIPS OHE.

关键词： Hepatic encephalopathy Transjugular intrahepatic portosystemic shunt Spleen volume Portal hypertension Cirrhosis