Biallelic loss-of-function variants in ATM (Ataxia Telangiectasia Mutated) cause Ataxia Telangiectasia (AT), a rare disorder associated with cerebellar degeneration and ataxia, cancer predisposition, infertility, grow...Biallelic loss-of-function variants in ATM (Ataxia Telangiectasia Mutated) cause Ataxia Telangiectasia (AT), a rare disorder associated with cerebellar degeneration and ataxia, cancer predisposition, infertility, growth retardation, etc. ATM is a phosphoinositide 3-kinase-related kinase (PIKK) with a role in DNA repair and maintenance of genome stability. Studying a multisystem genetic disease like AT requires animal models to ascertain its pathogenesis at the level of tissues, organs and the organism. Due to its small size, cheap maintenance, large progeny, rapid development and initial transparency, zebrafish (Danio rerio) is an increasingly popular vertebrate model organism, suitable for genetic modifications and large-scale in vivo therapeutic screens as embryos are chemically permeable to small compounds. Currently, no zebrafish model for AT exists.1 We generated atm knock-outs through CRIPSR-Cas9 mutagenesis. We show that atm conserved its function as a tumour suppressor gene and is involved in gametogenesis and fertility. Therefore, this mutant is of great value for further studies investigating the role of atm in reproduction and tumorigenesis.展开更多
Hereditary tyrosinemia type 1(HT1)is a life-threatening disease caused by the patient's inability to break down tyrosine due to loss-of-function mutations in the fumarylacetoacetate hydrolase(FAH)enzyme(Fig.S1).Cu...Hereditary tyrosinemia type 1(HT1)is a life-threatening disease caused by the patient's inability to break down tyrosine due to loss-of-function mutations in the fumarylacetoacetate hydrolase(FAH)enzyme(Fig.S1).Currently,the only available life-saving treatment is nitisinone(NTBC).However,nitisinone therapy comes with debilitating side effects and requires a strict drug regime combined with a tyrosine-and phenylalanine-restricted diet.Consequently,therapy adherence is often experienced as an additional burden.In this study,transcriptional profiling was conducted parallel to high-resolution metabolomics on,respectively,liver tissue and serum samples of Fah-deficient mice.The experimental workflow of our study is shown in Figure 1A.Canonical pathway analyses showed manifest activation of the NRF2-stress response,associated with hepatocellular damage and mainly driven by a markedly increased oxidative burden.Other altered canonical pathways were primarily a downstream consequence of the latter.Furthermore,we observed an overall aminoacidemia and changes related to aminoacyl-tRNA biosynthesis,which postulates that short-term nitisinone discontinuation can already put the patient at risk for hepatocellular carcinoma development.Finally,we did not only identify changes in numerous pathways,directly and indirectly,related to the metabolism of tyrosine and glutathione but also the presence of“side-chain”metabolites,such as y-glutamyl-and N-acetyl-coupled amino acids,indicating that non-directly related pathways like phase II biotransformation reactions are also affected.展开更多
In mammals,plant lectinshave been shown to possess immunomodulatory properties,acting in both the innate and adaptive immune system to modulate the production of mediators of the immune response,ultimately improving h...In mammals,plant lectinshave been shown to possess immunomodulatory properties,acting in both the innate and adaptive immune system to modulate the production of mediators of the immune response,ultimately improving host defences.At present,knowledge of immunomodulatory effects of plant lectins in insects is scarce.Treatment of insect cells with the Orysa sativa lectin,Orysata,was previously reported to induce cell aggregation,mimicking the immune process of encapsulation.In this project we investigated the potential immunomodulatory effects of this mannose-binding lectin using Drosophila melanogaster S2 cells.Identification of the Orysata binding partners on the surface of S2 cells through a pull-down assay andproteomic analysis revealed 221 putative interactors,several of which were immunity-related proteins.Subsequent qPCR analysis revealed the upregulation of Toll-and immune deficiency(IMD)-regulated antimicrobial peptides(Drs,Mtk,AttA,and Dpt)and signal transducers(Rel and Hid)belonging to the IMD pathway.In addition,the iron-binding protein Transferrin 3 was identified as a putative interactor for Orysata,and treatment of S2 cells with Orysata was shown to reduce the intracellular iron concentration.All together,we believe these results offer a new perspective on the effects by which plant lectins influence insect cells and contribute to the study of their immunomodulatory properties.展开更多
Fluorescence microscopy is the method of choice for studying intracellular dynamics.However,its success depends on the.availability of specific and stable markers.A prominent example of markers that are rapidly gainin...Fluorescence microscopy is the method of choice for studying intracellular dynamics.However,its success depends on the.availability of specific and stable markers.A prominent example of markers that are rapidly gaining interest are nanobodies(Nbs.-15 kDa),which can be functionalized with bright and photostable organic fluorophores.Due to their relatively small size and high specificity,Nbs offer great potential for high-quality long-term subcellular imaging,but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells.We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability.Being a laser-based technology,it is readily compatible with light microscopy and the typical cell recipients used for that.Spurred by these promising initial results,we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells.We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm,actin-bundling protein Fascin,and the histone H2A/H2B heterodimers.With an efficiency of more than 80%labeled cells and minimal toxicity(-2%),photoporation proved to be an excellent intracellular delivery method for Nbs.Time-lapse microscopy revealed that cell division rate and migration remained unaffected,confirming excellent cell viability and functionality.We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells,laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.展开更多
Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe.In order to germinate,it must perceive various structurally divergent host-exuded signals,including isothiocyanates(IT...Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe.In order to germinate,it must perceive various structurally divergent host-exuded signals,including isothiocyanates(ITCs)and strigolactones(SLs).However,the receptors involved are still uncharacterized.Here,we identify five putative SL receptors in P.ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination.We demonstrate the high plasticity of PrKAI2d3,which allows it to interact with different chemicals,including ITCs.The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants.We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs.Additionally,we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P.ramosa germination with bioactivity comparable to that of ITCs.This study demonstrates that P.ramosa has extended its signal perception system during evolution,a fact that should be considered for the development of specific and efficient biocontrol methods.展开更多
基金supported by‘Kom op Tegen Kanker–Emmanuel Van der Schueren’(No.365M02318)by‘UGent-Bijzonder OnderzoeksFonds’(No.BOF15 GOA/011).
文摘Biallelic loss-of-function variants in ATM (Ataxia Telangiectasia Mutated) cause Ataxia Telangiectasia (AT), a rare disorder associated with cerebellar degeneration and ataxia, cancer predisposition, infertility, growth retardation, etc. ATM is a phosphoinositide 3-kinase-related kinase (PIKK) with a role in DNA repair and maintenance of genome stability. Studying a multisystem genetic disease like AT requires animal models to ascertain its pathogenesis at the level of tissues, organs and the organism. Due to its small size, cheap maintenance, large progeny, rapid development and initial transparency, zebrafish (Danio rerio) is an increasingly popular vertebrate model organism, suitable for genetic modifications and large-scale in vivo therapeutic screens as embryos are chemically permeable to small compounds. Currently, no zebrafish model for AT exists.1 We generated atm knock-outs through CRIPSR-Cas9 mutagenesis. We show that atm conserved its function as a tumour suppressor gene and is involved in gametogenesis and fertility. Therefore, this mutant is of great value for further studies investigating the role of atm in reproduction and tumorigenesis.
基金funded by the Research Foundation-Flanders(FWO)(No.1518619N and G041521N)the Research Council(OZR)of the Vrije Universiteit Brussel(VUB),the Hercules Foundation,Wetenschappelijk Fonds Willy Gepts(WFWG)from the UZ Brussel and a Medical Grant from Swedish Orphan Biovitrum(SOBI)(No.AIIFUND37).
文摘Hereditary tyrosinemia type 1(HT1)is a life-threatening disease caused by the patient's inability to break down tyrosine due to loss-of-function mutations in the fumarylacetoacetate hydrolase(FAH)enzyme(Fig.S1).Currently,the only available life-saving treatment is nitisinone(NTBC).However,nitisinone therapy comes with debilitating side effects and requires a strict drug regime combined with a tyrosine-and phenylalanine-restricted diet.Consequently,therapy adherence is often experienced as an additional burden.In this study,transcriptional profiling was conducted parallel to high-resolution metabolomics on,respectively,liver tissue and serum samples of Fah-deficient mice.The experimental workflow of our study is shown in Figure 1A.Canonical pathway analyses showed manifest activation of the NRF2-stress response,associated with hepatocellular damage and mainly driven by a markedly increased oxidative burden.Other altered canonical pathways were primarily a downstream consequence of the latter.Furthermore,we observed an overall aminoacidemia and changes related to aminoacyl-tRNA biosynthesis,which postulates that short-term nitisinone discontinuation can already put the patient at risk for hepatocellular carcinoma development.Finally,we did not only identify changes in numerous pathways,directly and indirectly,related to the metabolism of tyrosine and glutathione but also the presence of“side-chain”metabolites,such as y-glutamyl-and N-acetyl-coupled amino acids,indicating that non-directly related pathways like phase II biotransformation reactions are also affected.
基金Pengyu Chen is the recipient of a doctoral grant provided by the Chinese Scholarship Council(CSC,China)This research was also funded by the Research Foundation-Flanders(FWO-Vlaanderen,Belgium)and the Special Research Fund from the Ghent University(Belgium).
文摘In mammals,plant lectinshave been shown to possess immunomodulatory properties,acting in both the innate and adaptive immune system to modulate the production of mediators of the immune response,ultimately improving host defences.At present,knowledge of immunomodulatory effects of plant lectins in insects is scarce.Treatment of insect cells with the Orysa sativa lectin,Orysata,was previously reported to induce cell aggregation,mimicking the immune process of encapsulation.In this project we investigated the potential immunomodulatory effects of this mannose-binding lectin using Drosophila melanogaster S2 cells.Identification of the Orysata binding partners on the surface of S2 cells through a pull-down assay andproteomic analysis revealed 221 putative interactors,several of which were immunity-related proteins.Subsequent qPCR analysis revealed the upregulation of Toll-and immune deficiency(IMD)-regulated antimicrobial peptides(Drs,Mtk,AttA,and Dpt)and signal transducers(Rel and Hid)belonging to the IMD pathway.In addition,the iron-binding protein Transferrin 3 was identified as a putative interactor for Orysata,and treatment of S2 cells with Orysata was shown to reduce the intracellular iron concentration.All together,we believe these results offer a new perspective on the effects by which plant lectins influence insect cells and contribute to the study of their immunomodulatory properties.
基金K.B.acknowledges financial support from the European Research Council(ERC)under the European Union's Horizon 2020 research and innovation program(No.648124)from the Ghent University Special Research Fund(No.01B04912)+3 种基金with gratitude.J.L.gratefully acknowledges the financial support from the China Scholarship Council(CSC)(No.201506750012)the Ghent University Special Research Fund(No.01SC1416)T.H.and J.G.acknowledges financial support from the Fonds Wetenschappelijk Onderzoek(No.G.0559.16N)Ghent University(BOF-GOA)(No.BOF13/GOA010)。
文摘Fluorescence microscopy is the method of choice for studying intracellular dynamics.However,its success depends on the.availability of specific and stable markers.A prominent example of markers that are rapidly gaining interest are nanobodies(Nbs.-15 kDa),which can be functionalized with bright and photostable organic fluorophores.Due to their relatively small size and high specificity,Nbs offer great potential for high-quality long-term subcellular imaging,but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells.We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability.Being a laser-based technology,it is readily compatible with light microscopy and the typical cell recipients used for that.Spurred by these promising initial results,we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells.We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm,actin-bundling protein Fascin,and the histone H2A/H2B heterodimers.With an efficiency of more than 80%labeled cells and minimal toxicity(-2%),photoporation proved to be an excellent intracellular delivery method for Nbs.Time-lapse microscopy revealed that cell division rate and migration remained unaffected,confirming excellent cell viability and functionality.We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells,laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.
基金supported by the Institut Jean-Pierre Bourgin’s Plant Observatory technological platforms and has benefited from the facilities and expertise of the I2BC proteomic platform(Proteomic-Gif,SICaPS)supported by Infrastructures en Biologie Santéet Agronomie,Ile de France Region,Plan Cancer,CNRS,and Paris-Sud University+3 种基金The CHARM3AT Labex program(ANR-11-LABX-39)is also acknowledged for its supportthe recipient of an AgreenSkills award from the European Union in the framework of the Marie-Curie FP7 COFUND People Program and a fellowship from Saclay Plant Sciences(ANR-17-EUR-0007)A.J.is indebted to the Research Foundation Flanders for a Structural Basic Research fellowship(project 1S15817N)a travel grant in the framework of a Tournesol fellowship(project VS04418N).
文摘Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe.In order to germinate,it must perceive various structurally divergent host-exuded signals,including isothiocyanates(ITCs)and strigolactones(SLs).However,the receptors involved are still uncharacterized.Here,we identify five putative SL receptors in P.ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination.We demonstrate the high plasticity of PrKAI2d3,which allows it to interact with different chemicals,including ITCs.The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants.We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs.Additionally,we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P.ramosa germination with bioactivity comparable to that of ITCs.This study demonstrates that P.ramosa has extended its signal perception system during evolution,a fact that should be considered for the development of specific and efficient biocontrol methods.