Paget’s disease(PDB)is a late-onset bone remodeling disorder with a broad spectrum of symptoms and complications.One of the most aggressive forms is caused by the P937R mutation in the ZNF687 gene.Although the geneti...Paget’s disease(PDB)is a late-onset bone remodeling disorder with a broad spectrum of symptoms and complications.One of the most aggressive forms is caused by the P937R mutation in the ZNF687 gene.Although the genetic involvement of ZNF687 in PDB has been extensively studied,the molecular mechanisms underlying this association remain unclear.Here,we describe the first Zfp687 knock-in mouse model and demonstrate that the mutation recapitulates the PDB phenotype,resulting in severely altered bone remodeling.Through microcomputed tomography analysis,we observed that 8-month-old mutant mice showed a mainly osteolytic phase,with a significant decrease in the trabecular bone volume affecting the femurs and the vertebrae.Conversely,osteoblast activity was deregulated,producing disorganized bone.Notably,this phenotype became pervasive in 16-month-old mice,where osteoblast function overtook bone resorption,as highlighted by the presence of woven bone in histological analyses,consistent with the PDB phenotype.Furthermore,we detected osteophytes and intervertebral disc degeneration,outlining for the first time the link between osteoarthritis and PDB in a PDB mouse model.RNA sequencing of wild-type and Zfp687 knockout RAW264.7 cells identified a set of genes involved in osteoclastogenesis potentially regulated by Zfp687,e.g.,Tspan7,Cpe,Vegfc,and Ggt1,confirming its role in this process.Strikingly,in this mouse model,the mutation was also associated with a high penetrance of hepatocellular carcinomas.Thus,this study established an essential role of Zfp687 in the regulation of bone remodeling,offering the potential to therapeutically treat PDB,and underlines the oncogenic potential of ZNF687.展开更多
Cup-like nuclear morphological alterations in acute myeloid leukemia(AML)blasts have been widely correlated with Nucleophosmin 1(NPM1)mutations.NPM1-mutated AML has earned recognition as a distinct entity among myeloi...Cup-like nuclear morphological alterations in acute myeloid leukemia(AML)blasts have been widely correlated with Nucleophosmin 1(NPM1)mutations.NPM1-mutated AML has earned recognition as a distinct entity among myeloid tumors,but the absence of a thoroughly established tool for its morphological analysis remains a notable gap.Holographic tomography(HT)can offer a label-free solution for quantitatively assessing the 3D shape of the nucleus based on the volumetric variations of its refractive indices(RIs).However,traditional HT methods analyze adherent cells in a 2D layer,leading to non-isotropic reconstructions due to missing cone artifacts.Here we show for the first time that holo-tomographic flow cytometry(HTFC)achieves quantitative specificity and precise capture of the nucleus volumetric shape in AML cells in suspension.To retrieve nucleus specificity in label-free RI tomograms of flowing AML cells,we conceive and demonstrate in a real-world clinical case a novel strategy for segmenting 3D concave nuclei.This method implies that the correlation between the"phenotype"and"genotype"of nuclei is demonstrated through HTFC by creating a challenging link not yet explored between the aberrant morphological features of AML nuclei and NPM1 mutations.We conduct an ensemble-level statistical characterization of NPM1-wild type and NPM1-mutated blasts to discern their complex morphological and biophysical variances.Our findings suggest that characterizing cup-like nuclei in NPM1-related AML cells by HTFC may enhance the diagnostic approach for these tumors.Furthermore,we integrate virtual reality to provide an immersive fruition of morphological changes in AML cells within a true 3D environment.展开更多
Skeletal muscle tissue engineering(SMTE)has recently emerged to address major clinical challenges such as volumetric muscle loss(VML).Here,we report a rotary wet-spinning(RoWS)biofabrication technique for producing hu...Skeletal muscle tissue engineering(SMTE)has recently emerged to address major clinical challenges such as volumetric muscle loss(VML).Here,we report a rotary wet-spinning(RoWS)biofabrication technique for producing human myo-substitutes with biomimetic architectures and functions.Here,we demonstrate how the proposed technique may be used to establish a welltailored,anisotropic microenvironment that promotes myogenic differentiation of human skeletal muscle–derived pericytes(hPeri).Using high-resolutionmass spectrometry–based proteomics with the integration of literature-derived signaling networks,we uncovered that(i)a 3D biomimetic matrix environment(PEG-fibrinogen)confers a less mitogenic microenvironment compared to standard 2D cultures,favoring the formation of contractile-competent bundles of pericyte-derived myotubes in an anchoring-independent 3D state and(ii)the RoWS method promotes an upregulation of muscle matrix structural protein besides increasing contractile machinery proteins with respect to 3D bulk cultures.Finally,in vivo investigations demonstrate that the 3D-biofabricated myo-substitute is fully compatible with the host ablated muscular tissue,exhibiting myo-substitute engraftment andmuscle regeneration in a mousemodel of VML.Overall,the results show that RoWS offers a superior capability for controlling themyogenic differentiation process on a macroscale and,with future refining,may have the potential to be translated into clinical practice.展开更多
Macroautophagy(referred to as autophagy hereafter)is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles.Previous studies showed tha...Macroautophagy(referred to as autophagy hereafter)is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles.Previous studies showed that autophagy protects against acetaminophen(APAP)-induced injury(AILI)via selective removal of damaged mitochondria and APAP protein adducts.The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes.In the present study,we showed that transcription factor EB(TFEB),a master transcription factor for lysosomal biogenesis,was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers.Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI,respectively.Mechanistically,overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2(NRF2)activation to protect against AILI.We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists.Among these agonists,salinomycin,an anticoccidial and antibacterial agent,activated TFEB and protected against AILI in mice.In conclusion,genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.展开更多
The high mutation rate of SARS-CoV-2 leads to the emergence of multiple variants,some of which are resistant to vaccines and drugs targeting viral elements.Targeting host dependency factors,e.g.cellular proteins requi...The high mutation rate of SARS-CoV-2 leads to the emergence of multiple variants,some of which are resistant to vaccines and drugs targeting viral elements.Targeting host dependency factors,e.g.cellular proteins required for viral replication,would help prevent the development of resistance.However,it remains unclear whether different SARS-CoV-2 variants induce conserved cellular responses and exploit the same core host factors.To this end,we compared three variants of concern and found that the host transcriptional response was conserved,differing only in kinetics and magnitude.Clustered regularly interspaced short palindromic repeats screening identified host genes required for each variant during infection.Most of the genes were shared by multiple variants.We validated our hits with small molecules and repurposed the US Food and Drug Administration-approved drugs.All the drugs were highly active against all the tested variants,including new variants that emerged during the study(Delta and Omicron).Mechanistically,we identified reactive oxygen species production as a key step in early viral replication.Antioxidants such as N-acetyl cysteine(NAC)were effective against all the variants in both human lung cells and a humanized mouse model.Our study supports the use of available antioxidant drugs,such as NAC,as a general and effective anti-COVID-19 approach.展开更多
Nonalcoholic fatty liver disease(NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment.Hyperactivation of mTOR complex1(mTORCl) and subsequent ...Nonalcoholic fatty liver disease(NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment.Hyperactivation of mTOR complex1(mTORCl) and subsequent impairment of the transcription factor EB(TFEB)-mediated autophagy-lysosomal pathway(ALP) are implicated in the development of NAFLD.Accordingly,agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD.The objective of this study was to investigate the effects of nuciferine,a major active component from lotus leaf,on NAFLD and its underlying mechanism of action.Here we show that nuciferine activated ALP and alleviated steatosis,insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner.Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases,thereby suppressing lysosomal localization and activity of mTORC1,which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance.Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORCl-TFEB-ALP axis could represent a novel pharmacological strategy to combat NAFLD.展开更多
基金Italian Association for Cancer Research(AIRC)under IG 2020-ID.25110 projectPOR Campania FESR 2014/2020(Project SATIN)+1 种基金Progetto MISE(F/050011/02/X32)–P.I.Fernando Gianfrancescofunding from the European Calcified Tissue Society(ECTS)and the Italian Society for Osteoporosis,Mineral Metabolism and Skeletal Diseases(SIOMMMS)。
文摘Paget’s disease(PDB)is a late-onset bone remodeling disorder with a broad spectrum of symptoms and complications.One of the most aggressive forms is caused by the P937R mutation in the ZNF687 gene.Although the genetic involvement of ZNF687 in PDB has been extensively studied,the molecular mechanisms underlying this association remain unclear.Here,we describe the first Zfp687 knock-in mouse model and demonstrate that the mutation recapitulates the PDB phenotype,resulting in severely altered bone remodeling.Through microcomputed tomography analysis,we observed that 8-month-old mutant mice showed a mainly osteolytic phase,with a significant decrease in the trabecular bone volume affecting the femurs and the vertebrae.Conversely,osteoblast activity was deregulated,producing disorganized bone.Notably,this phenotype became pervasive in 16-month-old mice,where osteoblast function overtook bone resorption,as highlighted by the presence of woven bone in histological analyses,consistent with the PDB phenotype.Furthermore,we detected osteophytes and intervertebral disc degeneration,outlining for the first time the link between osteoarthritis and PDB in a PDB mouse model.RNA sequencing of wild-type and Zfp687 knockout RAW264.7 cells identified a set of genes involved in osteoclastogenesis potentially regulated by Zfp687,e.g.,Tspan7,Cpe,Vegfc,and Ggt1,confirming its role in this process.Strikingly,in this mouse model,the mutation was also associated with a high penetrance of hepatocellular carcinomas.Thus,this study established an essential role of Zfp687 in the regulation of bone remodeling,offering the potential to therapeutically treat PDB,and underlines the oncogenic potential of ZNF687.
基金supported by the project PRIN 2022 PNRR—flow-cytometry ImaGing by Holographic tomography for predicting TUMor control in Oncology patients treated with Radiotherapy(FIGHT-TUMOR),Prot.P2022ATE2J—funded by the Italian Ministry of University&Research in the framework of Next Generation EUby project“CITOM”—Programma AMICO 2,CNR—UVR—within the PoC 2022—PNRR funded by the Italian Ministry of Business and Made in Italy—UIBM in the framework of Next Generation EUby#NEXTGENERATIONEU(NGEU),Ministry of University and Research(MUR),National Recovery and Resilience Plan(NRRP),project MNESYS(PE0000006)—a multiscale integrated approach to the study of the nervous system in health and disease(DN.155311.10.2022)。
文摘Cup-like nuclear morphological alterations in acute myeloid leukemia(AML)blasts have been widely correlated with Nucleophosmin 1(NPM1)mutations.NPM1-mutated AML has earned recognition as a distinct entity among myeloid tumors,but the absence of a thoroughly established tool for its morphological analysis remains a notable gap.Holographic tomography(HT)can offer a label-free solution for quantitatively assessing the 3D shape of the nucleus based on the volumetric variations of its refractive indices(RIs).However,traditional HT methods analyze adherent cells in a 2D layer,leading to non-isotropic reconstructions due to missing cone artifacts.Here we show for the first time that holo-tomographic flow cytometry(HTFC)achieves quantitative specificity and precise capture of the nucleus volumetric shape in AML cells in suspension.To retrieve nucleus specificity in label-free RI tomograms of flowing AML cells,we conceive and demonstrate in a real-world clinical case a novel strategy for segmenting 3D concave nuclei.This method implies that the correlation between the"phenotype"and"genotype"of nuclei is demonstrated through HTFC by creating a challenging link not yet explored between the aberrant morphological features of AML nuclei and NPM1 mutations.We conduct an ensemble-level statistical characterization of NPM1-wild type and NPM1-mutated blasts to discern their complex morphological and biophysical variances.Our findings suggest that characterizing cup-like nuclei in NPM1-related AML cells by HTFC may enhance the diagnostic approach for these tumors.Furthermore,we integrate virtual reality to provide an immersive fruition of morphological changes in AML cells within a true 3D environment.
基金supported by AFM-Telethon(23551)Fondazione Telethon(TMPGMFU22TT)+2 种基金Muscular Dystrophy Association(MDA 968551)National Science Centre Poland(NCN)within SONATA BIS 12(project no.2022/46/E/ST8/00284)Ministero dell’Istruzione,dell’Universita e della Ricerca(PRIN funding scheme no.201742SBXA_004 and 2022F37JRF).
文摘Skeletal muscle tissue engineering(SMTE)has recently emerged to address major clinical challenges such as volumetric muscle loss(VML).Here,we report a rotary wet-spinning(RoWS)biofabrication technique for producing human myo-substitutes with biomimetic architectures and functions.Here,we demonstrate how the proposed technique may be used to establish a welltailored,anisotropic microenvironment that promotes myogenic differentiation of human skeletal muscle–derived pericytes(hPeri).Using high-resolutionmass spectrometry–based proteomics with the integration of literature-derived signaling networks,we uncovered that(i)a 3D biomimetic matrix environment(PEG-fibrinogen)confers a less mitogenic microenvironment compared to standard 2D cultures,favoring the formation of contractile-competent bundles of pericyte-derived myotubes in an anchoring-independent 3D state and(ii)the RoWS method promotes an upregulation of muscle matrix structural protein besides increasing contractile machinery proteins with respect to 3D bulk cultures.Finally,in vivo investigations demonstrate that the 3D-biofabricated myo-substitute is fully compatible with the host ablated muscular tissue,exhibiting myo-substitute engraftment andmuscle regeneration in a mousemodel of VML.Overall,the results show that RoWS offers a superior capability for controlling themyogenic differentiation process on a macroscale and,with future refining,may have the potential to be translated into clinical practice.
基金We would like to thank Dr.Thomas Ru¨licke at Department of Biomedical Sciences,University of Veterinary Medicine Vienna,Vienna,Austria and Dr.Kurt Zatloukal at The Institute of Pathology,Medical University of Graz,A-8036 Graz,Austria for providing us whole body Sqstm1/p62 knockout mice for the hepatocyte isolation experiment.We also thank Larysa Stroganova at University of Kansas Medical Center for her excellent assistance for the EM studies.This study was supported in part by the National Institute of Health(NIH,USA)funds R01 DK102142,R01 AG072895,R37 AA020518(WXD)and in part by the Intramural Research Program of the National Center for Advancing Translational Sciences,NIH(USA).
文摘Macroautophagy(referred to as autophagy hereafter)is a major intracellular lysosomal degradation pathway that is responsible for the degradation of misfolded/damaged proteins and organelles.Previous studies showed that autophagy protects against acetaminophen(APAP)-induced injury(AILI)via selective removal of damaged mitochondria and APAP protein adducts.The lysosome is a critical organelle sitting at the end stage of autophagy for autophagic degradation via fusion with autophagosomes.In the present study,we showed that transcription factor EB(TFEB),a master transcription factor for lysosomal biogenesis,was impaired by APAP resulting in decreased lysosomal biogenesis in mouse livers.Genetic loss-of and gain-of function of hepatic TFEB exacerbated or protected against AILI,respectively.Mechanistically,overexpression of TFEB increased clearance of APAP protein adducts and mitochondria biogenesis as well as SQSTM1/p62-dependent non-canonical nuclear factor erythroid 2-related factor 2(NRF2)activation to protect against AILI.We also performed an unbiased cell-based imaging high-throughput chemical screening on TFEB and identified a group of TFEB agonists.Among these agonists,salinomycin,an anticoccidial and antibacterial agent,activated TFEB and protected against AILI in mice.In conclusion,genetic and pharmacological activating TFEB may be a promising approach for protecting against AILI.
基金supported by the CaRiPaRo Foundation(NewTarCoV2)the Ministry of Education,University and Research(PRIN-2020KSY3KL)+13 种基金supported by the Telethon Foundation Core Grant,European Research Council(ERC)(CellKarma)Regione Campania(PO-FESR 2014-2020,PO-FESR 2014-2020)Italian Ministry of Health(Piano Operativo Salute Traiettoria 3,‘Genomed’).supported by the Giovanni Armenise-Harvard Foundation,the Telethon Foundation(TCP13013)ERC(ERC Starting Grant,‘MetEpiStem’)supported by ERC(ERC Consolidator 615879)the Bill and Melinda Gates Foundation(OPP1035881 and OPP1097238)the Italian Foundation for Cancer Research(AIRC 21850)the Collaborative Center for XDP at Massachusetts General Hospital(239295)supported by the Italian Foundation for Cancer Research(AIRC-MFAG 25745)University of Padua(STARS Consolidator Grant,‘EMERALD’)supported by the Italian Foundation for Cancer Research(AIRC 2135)Italian Ministry of Health(RCR-201923669115,NET-201602361632)supported by the EVA-GLOBAL project that has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement number 871029EVA-GLOBAL provided access to SARS-CoV-2 Alpha and Delta isolates(human nCoV19 isolate/England/MIG457/2020 and hCoV-19/Netherlands/NH-RIVM-27142/2021_P2).
文摘The high mutation rate of SARS-CoV-2 leads to the emergence of multiple variants,some of which are resistant to vaccines and drugs targeting viral elements.Targeting host dependency factors,e.g.cellular proteins required for viral replication,would help prevent the development of resistance.However,it remains unclear whether different SARS-CoV-2 variants induce conserved cellular responses and exploit the same core host factors.To this end,we compared three variants of concern and found that the host transcriptional response was conserved,differing only in kinetics and magnitude.Clustered regularly interspaced short palindromic repeats screening identified host genes required for each variant during infection.Most of the genes were shared by multiple variants.We validated our hits with small molecules and repurposed the US Food and Drug Administration-approved drugs.All the drugs were highly active against all the tested variants,including new variants that emerged during the study(Delta and Omicron).Mechanistically,we identified reactive oxygen species production as a key step in early viral replication.Antioxidants such as N-acetyl cysteine(NAC)were effective against all the variants in both human lung cells and a humanized mouse model.Our study supports the use of available antioxidant drugs,such as NAC,as a general and effective anti-COVID-19 approach.
基金supported by the National Natural Science Foundation of China (Beijing, Chinagrant Nos. U20A2062, 32022084, and 32002349)+1 种基金Jilin Province Science and Technology Development Project (Changchun, Chinagrant No. 20210508011RQ)
文摘Nonalcoholic fatty liver disease(NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment.Hyperactivation of mTOR complex1(mTORCl) and subsequent impairment of the transcription factor EB(TFEB)-mediated autophagy-lysosomal pathway(ALP) are implicated in the development of NAFLD.Accordingly,agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD.The objective of this study was to investigate the effects of nuciferine,a major active component from lotus leaf,on NAFLD and its underlying mechanism of action.Here we show that nuciferine activated ALP and alleviated steatosis,insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner.Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases,thereby suppressing lysosomal localization and activity of mTORC1,which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance.Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORCl-TFEB-ALP axis could represent a novel pharmacological strategy to combat NAFLD.
