Daniela Salvatori - Academia.edu (original) (raw)

Papers by Daniela Salvatori

Cells, Aug 28, 2022

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow ... more The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2 Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

International Journal of Molecular Sciences, Apr 23, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Cell Reports Medicine, 2020

Highlights d SERPINE1-FOSB translocation in hiPSC to model the vascular tumor PHE d CRISPR/Cas9-m... more Highlights d SERPINE1-FOSB translocation in hiPSC to model the vascular tumor PHE d CRISPR/Cas9-mediated gene targeting to engineer hiPSC SERPINE1-FOSB d hiPSC-ECs SERPINE1-FOSB show increased FOSB expression d Functional features of PHE recapitulated by hiPSC-ECs SERPINE1-FOSB

Journal of Allergy and Clinical Immunology, 2019

Phillips have a patent pending for HLA-A*32:01 testing for vancomycin drug reaction and eosinophi... more Phillips have a patent pending for HLA-A*32:01 testing for vancomycin drug reaction and eosinophilia and systemic symptoms. S. Mallal and E. J. Phillips receive royalties from UpToDate and have equity in IIID Pty Ltd, which holds a patent for HLA-B*57:01 testing for abacavir hypersensitivity. E. J. Phillips receives consulting fees from Biocryst. The rest of the authors declare that they have no relevant conflicts of interest.

Protein Engineering, Design and Selection, 2000

Nucleosides, Nucleotides and Nucleic Acids, 2003

N6-Cycloalkyl-2&a... more N6-Cycloalkyl-2',3'-dideoxyadenosine derivatives and (2-chloro)-N6-cycloheptyl-3-deazaadenosine have been synthesized and tested, along with other (deaza)purine (deoxy)nucleosides from our chemical library, as inhibitors of virus replication against Bovine Herpes Virus 1 (BHV-1) and sheep Maedi/Visna Virus (MVV). Most compounds demonstrated good antireplicative activity against MVV, showing also low cell toxicity.

European Journal of Medicinal Chemistry, 2000

Although 2'-deoxy-beta-D-5-azacytidine (Decitabine) and beta-D-5-azacytidine disp... more Although 2'-deoxy-beta-D-5-azacytidine (Decitabine) and beta-D-5-azacytidine display potent antileukemic properties, their therapeutic use is hampered by their sensitivity to nucleophiles and to deamination catalysed by cytidine deaminase. As shown earlier [Shafiee M., Griffon J.-F., Gosselin G., Cambi A., Vincenzetti S., Vita A., Erikson S., Imbach J.-L., Maury G., Biochem. Pharmacol. 56 (1998) 1237-1242], beta-L-enantiomers of cytidine derivatives are resistant to cytidine deaminase. We thus synthesized several 5-azacytosine beta-L-nucleoside analogues to evaluate their enzymatic and biological properties. 2'-Deoxy-beta-L-5-azacytidine (L-Decitabine), beta-L-5-azacytidine, 1-(beta-L-xylo-furanosyl)5-azacytosine, and 1-(2-deoxy-beta-L-threo-pentofuranosyl)5-azacytosine were stereospecifically prepared starting from L-ribose and L-xylose. D- and L-enantiomers of 2'-deoxy-beta-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK) compared to beta-D-deoxycytidine, whereas both enantiomers of beta-5-azacytidine or the L-xylo-analogues were not substrates of the enzyme. As expected, none of the presently reported derivatives of beta-L-5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA). The prepared compounds were tested for their activity against HIV and HBV and they did not show any significant activity or cytotoxicity. In the case of L-Decitabine, this suggests that the enantioselectivities of concerned enzymes other than dCK and CDA might not be favourable.

Comparative Immunology, Microbiology and Infectious Diseases, 2001

A series of beta-D- and beta-L-cytidine analogues were evaluated for their inhibitory effect on t... more A series of beta-D- and beta-L-cytidine analogues were evaluated for their inhibitory effect on the replication of maedi-visna virus (MVV) strains KV1772 and MV1514 cultured on sheep choroid plexus cells and the sheep chondrocyte cell line G81092, respectively. Eleven cytidine analogues were selected for the anti-viral test. Five of them belong to the family of the 2',3'-dideoxycytidine analogues, well known for their activity against human immunodeficiency virus (HIV). The others, all newly synthesized, were potential anti-viral and/or anti-leukemic agents. None of the compounds under study had a toxic effect in both anti-viral assay systems up to a 300 microM concentration. Based on the cytopathic effects (CPE), the virus replication was completely inhibited by the five 2',3'-dideoxycytidine analogues at a concentration of 50 microM, whereas the others six newly synthesized compounds induced titre reductions of 4-5 log units. The effective concentration causing 50% reduction of CPE (EC50) was of 5 microM for the five 2',3'-dideooxycytidine analogues and for beta-L-XyloFc, whereas the value of 50 microM was found for the b-L-XyloC and the four 5-azacytidine compounds tested. All these data reveal a good correlation between inhibition of MVV replication by several nucleoside cytidine analogues and their reported anti-HIV activity.

Education Sciences

Virtual 3D models can be an animal-free alternative to cadaveric dissection to learn spatial anat... more Virtual 3D models can be an animal-free alternative to cadaveric dissection to learn spatial anatomy. The aim of this study was to investigate if the learning outcome differs when studying 3D models with a 360° rotatable interactive monoscopic 3-dimensional (iM3D) or an interactive monoscopic 2-dimensional (iM2D) visualization, and whether the level of visual-spatial ability (VSA) influences learning outcome. A two-center randomized crossover trial was conducted during the Laboratory Animals Science Course (March 2021–March 2022). Participants studied a 3D rat model using iM3D and iM2D. VSA was assessed by a 24-item mental rotation test and learning outcome by two knowledge tests. Data from 69 out of 111 recruited participants were analyzed using linear regression. Participants with low VSA performed significantly worse compared to participants with medium or high VSA when using iM3D, but equally well when using iM2D. When VSA level was disregarded, participants performed equally we...

Scientific Reports

Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby... more Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The s...

Plants

In previous studies, the Argentinean native plants called Zuccagnia punctata (jarilla, pus pus, l... more In previous studies, the Argentinean native plants called Zuccagnia punctata (jarilla, pus pus, lata) and Solanum betaceum (chilto, tree tomato) were reported as new natural sources of antioxidant compounds, mainly chalcones, anthocyanins and rosmarinic acid derivates. The present study deals with the production of antioxidant beverages of Z. punctata (Zp) extract and chilto juice with honey as sweetener. A Zp extract and red chilto juice were obtained according to Food Code and characterized. The beverages were formulated by using maltodextrin (MD) with two dextrose equivalents (DE), 10 and 15, and then spray-dried at an inlet air temperature of 130 °C. The physicochemical, microscopical, phytochemical and functional characteristics of the powders were surveyed. The experiments carried out showed good physical properties for both formulations showing high water solubility with adequate features for handling, transport and storage. The chromatic parameters of both powdered beverages...

Animals

In 2016, the Dutch government declared its commitment to phasing out animal experiments by 2025. ... more In 2016, the Dutch government declared its commitment to phasing out animal experiments by 2025. Although a high number of animal experiments are still performed and the 2025 target will not be met, the commitment remains. Efforts are being made to identify levers that might foster the transition to animal-free science. Education has been found to play a key role in the future of animal-free science and young generations are increasingly seen as key stakeholders. However, their attitudes toward the transition to animal-free innovations have not been investigated. The present article focuses on the values and beliefs held by students, who in 2022, participated in the course ‘Replacing Animal Testing’ (RAT) Challenge, organized by a consortium of Dutch universities. Contextually, students’ motivations to follow the course were investigated. The research was based on a qualitative study, including semi-structured interviews and a literature review. Our analysis of the findings revealed...

Journal of Berry Research, 2021

BACKGROUND: Dark-skin berries constitute a polyphenol-rich source of interest for the development... more BACKGROUND: Dark-skin berries constitute a polyphenol-rich source of interest for the development of functional ingredients. OBJECTIVE: To develop an elderberry powder, addressing technological aspects for maximum bioactive recovery, including physical quality and bioaccesibility of the antioxidant compounds. METHODS: An optimization of the combined process of enzyme-assisted extraction and freeze-drying was undertaken. Polyphenols and anthocyanins were quantified by spectrophotometric and HPLC-DAD analysis along processing stages and an in vitro digestion model was used to study the antioxidant compound activity through gastrointestinal tract and after transepithelial transport across a Caco-2 cell monolayer. Powder physical properties were also evaluated. RESULTS: The best extraction conditions were 45°C and 160 ppm enzyme. 10% maltodextrin was the minimum carrier concentration needed to get a freeze-dried powder with good physical properties and maximum bioactive content. The phe...

Cells, 2019

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient managemen... more Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scena...

Cardiovascular Research, 2019

AimsCardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause o... more AimsCardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause of mortality and morbidity worldwide due in part to the low regenerative capacity of the adult human heart. Human pluripotent stem cell (hPSC)-derived cardiovascular progenitor cells (CPCs) are a potential cell source for cardiac repair. The aim of this study was to examine the impact of extensive remuscularization and coincident revascularization on cardiac remodelling and function in a mouse model of myocardial infarction (MI) by transplanting doxycycline (DOX)-inducible (Tet-On-MYC) hPSC-derived CPCs in vivo and inducing proliferation and cardiovascular differentiation in a drug-regulated manner.Methods and resultsCPCs were injected firstly at a non-cardiac site in Matrigel suspension under the skin of immunocompromised mice to assess their commitment to the cardiovascular lineage and ability to self-renew or differentiate in vivo when instructed by systemically delivered factors inclu...

Stem Cell Reports, 2018

Predicting developmental potency and risk of posttransplantation tumor formation by human pluripo... more Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.

The Journal of Immunology, 2018

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immu... more By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV−/− mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV−/− mice developed higher Ab titers but no autoantibodies. These observations indicate a redunda...

Stem cell reports, Jan 8, 2018

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent ste... more Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, whic...

Neuromuscular Disorders, 2017

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat... more In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1 MommeD1 mice). D4Z4-2.5/Smchd1 MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/ Smchd1 MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.

Stem cell reports, May 9, 2017

The ability to form teratomas in vivo containing multiple somatic cell types is regarded as funct... more The ability to form teratomas in vivo containing multiple somatic cell types is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal dependent, laborious, and only qualitative, the PluriTest and the hPSC ScoreCard assay have been developed as in vitro alternatives. Here we compared normal hPSCs, induced hPSCs (hiPSCs) with reactivated reprogramming transgenes, and human embryonal carcinoma cells (hECs) in these assays. While normal hPSCs gave rise to typical teratomas, the xenografts of the hECs and the hiPSCs with reactivated reprogramming transgenes were largely undifferentiated and malignant. The hPSC ScoreCard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analyzed by the PluriTest, only hECs were identified as abnormal whereas all other cell lines were indistinguishable and resembled normal hPSCs. Our results indicate that pluripotency assa...

Cells, Aug 28, 2022

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow ... more The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2 Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

International Journal of Molecular Sciences, Apr 23, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Cell Reports Medicine, 2020

Highlights d SERPINE1-FOSB translocation in hiPSC to model the vascular tumor PHE d CRISPR/Cas9-m... more Highlights d SERPINE1-FOSB translocation in hiPSC to model the vascular tumor PHE d CRISPR/Cas9-mediated gene targeting to engineer hiPSC SERPINE1-FOSB d hiPSC-ECs SERPINE1-FOSB show increased FOSB expression d Functional features of PHE recapitulated by hiPSC-ECs SERPINE1-FOSB

Journal of Allergy and Clinical Immunology, 2019

Phillips have a patent pending for HLA-A*32:01 testing for vancomycin drug reaction and eosinophi... more Phillips have a patent pending for HLA-A*32:01 testing for vancomycin drug reaction and eosinophilia and systemic symptoms. S. Mallal and E. J. Phillips receive royalties from UpToDate and have equity in IIID Pty Ltd, which holds a patent for HLA-B*57:01 testing for abacavir hypersensitivity. E. J. Phillips receives consulting fees from Biocryst. The rest of the authors declare that they have no relevant conflicts of interest.

Protein Engineering, Design and Selection, 2000

Nucleosides, Nucleotides and Nucleic Acids, 2003

N6-Cycloalkyl-2&a... more N6-Cycloalkyl-2',3'-dideoxyadenosine derivatives and (2-chloro)-N6-cycloheptyl-3-deazaadenosine have been synthesized and tested, along with other (deaza)purine (deoxy)nucleosides from our chemical library, as inhibitors of virus replication against Bovine Herpes Virus 1 (BHV-1) and sheep Maedi/Visna Virus (MVV). Most compounds demonstrated good antireplicative activity against MVV, showing also low cell toxicity.

European Journal of Medicinal Chemistry, 2000

Although 2'-deoxy-beta-D-5-azacytidine (Decitabine) and beta-D-5-azacytidine disp... more Although 2'-deoxy-beta-D-5-azacytidine (Decitabine) and beta-D-5-azacytidine display potent antileukemic properties, their therapeutic use is hampered by their sensitivity to nucleophiles and to deamination catalysed by cytidine deaminase. As shown earlier [Shafiee M., Griffon J.-F., Gosselin G., Cambi A., Vincenzetti S., Vita A., Erikson S., Imbach J.-L., Maury G., Biochem. Pharmacol. 56 (1998) 1237-1242], beta-L-enantiomers of cytidine derivatives are resistant to cytidine deaminase. We thus synthesized several 5-azacytosine beta-L-nucleoside analogues to evaluate their enzymatic and biological properties. 2'-Deoxy-beta-L-5-azacytidine (L-Decitabine), beta-L-5-azacytidine, 1-(beta-L-xylo-furanosyl)5-azacytosine, and 1-(2-deoxy-beta-L-threo-pentofuranosyl)5-azacytosine were stereospecifically prepared starting from L-ribose and L-xylose. D- and L-enantiomers of 2'-deoxy-beta-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK) compared to beta-D-deoxycytidine, whereas both enantiomers of beta-5-azacytidine or the L-xylo-analogues were not substrates of the enzyme. As expected, none of the presently reported derivatives of beta-L-5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA). The prepared compounds were tested for their activity against HIV and HBV and they did not show any significant activity or cytotoxicity. In the case of L-Decitabine, this suggests that the enantioselectivities of concerned enzymes other than dCK and CDA might not be favourable.

Comparative Immunology, Microbiology and Infectious Diseases, 2001

A series of beta-D- and beta-L-cytidine analogues were evaluated for their inhibitory effect on t... more A series of beta-D- and beta-L-cytidine analogues were evaluated for their inhibitory effect on the replication of maedi-visna virus (MVV) strains KV1772 and MV1514 cultured on sheep choroid plexus cells and the sheep chondrocyte cell line G81092, respectively. Eleven cytidine analogues were selected for the anti-viral test. Five of them belong to the family of the 2',3'-dideoxycytidine analogues, well known for their activity against human immunodeficiency virus (HIV). The others, all newly synthesized, were potential anti-viral and/or anti-leukemic agents. None of the compounds under study had a toxic effect in both anti-viral assay systems up to a 300 microM concentration. Based on the cytopathic effects (CPE), the virus replication was completely inhibited by the five 2',3'-dideoxycytidine analogues at a concentration of 50 microM, whereas the others six newly synthesized compounds induced titre reductions of 4-5 log units. The effective concentration causing 50% reduction of CPE (EC50) was of 5 microM for the five 2',3'-dideooxycytidine analogues and for beta-L-XyloFc, whereas the value of 50 microM was found for the b-L-XyloC and the four 5-azacytidine compounds tested. All these data reveal a good correlation between inhibition of MVV replication by several nucleoside cytidine analogues and their reported anti-HIV activity.

Education Sciences

Virtual 3D models can be an animal-free alternative to cadaveric dissection to learn spatial anat... more Virtual 3D models can be an animal-free alternative to cadaveric dissection to learn spatial anatomy. The aim of this study was to investigate if the learning outcome differs when studying 3D models with a 360° rotatable interactive monoscopic 3-dimensional (iM3D) or an interactive monoscopic 2-dimensional (iM2D) visualization, and whether the level of visual-spatial ability (VSA) influences learning outcome. A two-center randomized crossover trial was conducted during the Laboratory Animals Science Course (March 2021–March 2022). Participants studied a 3D rat model using iM3D and iM2D. VSA was assessed by a 24-item mental rotation test and learning outcome by two knowledge tests. Data from 69 out of 111 recruited participants were analyzed using linear regression. Participants with low VSA performed significantly worse compared to participants with medium or high VSA when using iM3D, but equally well when using iM2D. When VSA level was disregarded, participants performed equally we...

Scientific Reports

Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby... more Muscle-specific kinase (MuSK) is crucial for acetylcholine receptor (AChR) clustering and thereby neuromuscular junction (NMJ) function. NMJ dysfunction is a hallmark of several neuromuscular diseases, including MuSK myasthenia gravis. Aiming to restore NMJ function, we generated several agonist monoclonal antibodies targeting the MuSK Ig-like 1 domain. These activated MuSK and induced AChR clustering in cultured myotubes. The most potent agonists partially rescued myasthenic effects of MuSK myasthenia gravis patient IgG autoantibodies in vitro. In an IgG4 passive transfer MuSK myasthenia model in NOD/SCID mice, MuSK agonists caused accelerated weight loss and no rescue of myasthenic features. The MuSK Ig-like 1 domain agonists unexpectedly caused sudden death in a large proportion of male C57BL/6 mice (but not female or NOD/SCID mice), likely caused by a urologic syndrome. In conclusion, these agonists rescued pathogenic effects in myasthenia models in vitro, but not in vivo. The s...

Plants

In previous studies, the Argentinean native plants called Zuccagnia punctata (jarilla, pus pus, l... more In previous studies, the Argentinean native plants called Zuccagnia punctata (jarilla, pus pus, lata) and Solanum betaceum (chilto, tree tomato) were reported as new natural sources of antioxidant compounds, mainly chalcones, anthocyanins and rosmarinic acid derivates. The present study deals with the production of antioxidant beverages of Z. punctata (Zp) extract and chilto juice with honey as sweetener. A Zp extract and red chilto juice were obtained according to Food Code and characterized. The beverages were formulated by using maltodextrin (MD) with two dextrose equivalents (DE), 10 and 15, and then spray-dried at an inlet air temperature of 130 °C. The physicochemical, microscopical, phytochemical and functional characteristics of the powders were surveyed. The experiments carried out showed good physical properties for both formulations showing high water solubility with adequate features for handling, transport and storage. The chromatic parameters of both powdered beverages...

Animals

In 2016, the Dutch government declared its commitment to phasing out animal experiments by 2025. ... more In 2016, the Dutch government declared its commitment to phasing out animal experiments by 2025. Although a high number of animal experiments are still performed and the 2025 target will not be met, the commitment remains. Efforts are being made to identify levers that might foster the transition to animal-free science. Education has been found to play a key role in the future of animal-free science and young generations are increasingly seen as key stakeholders. However, their attitudes toward the transition to animal-free innovations have not been investigated. The present article focuses on the values and beliefs held by students, who in 2022, participated in the course ‘Replacing Animal Testing’ (RAT) Challenge, organized by a consortium of Dutch universities. Contextually, students’ motivations to follow the course were investigated. The research was based on a qualitative study, including semi-structured interviews and a literature review. Our analysis of the findings revealed...

Journal of Berry Research, 2021

BACKGROUND: Dark-skin berries constitute a polyphenol-rich source of interest for the development... more BACKGROUND: Dark-skin berries constitute a polyphenol-rich source of interest for the development of functional ingredients. OBJECTIVE: To develop an elderberry powder, addressing technological aspects for maximum bioactive recovery, including physical quality and bioaccesibility of the antioxidant compounds. METHODS: An optimization of the combined process of enzyme-assisted extraction and freeze-drying was undertaken. Polyphenols and anthocyanins were quantified by spectrophotometric and HPLC-DAD analysis along processing stages and an in vitro digestion model was used to study the antioxidant compound activity through gastrointestinal tract and after transepithelial transport across a Caco-2 cell monolayer. Powder physical properties were also evaluated. RESULTS: The best extraction conditions were 45°C and 160 ppm enzyme. 10% maltodextrin was the minimum carrier concentration needed to get a freeze-dried powder with good physical properties and maximum bioactive content. The phe...

Cells, 2019

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient managemen... more Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scena...

Cardiovascular Research, 2019

AimsCardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause o... more AimsCardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause of mortality and morbidity worldwide due in part to the low regenerative capacity of the adult human heart. Human pluripotent stem cell (hPSC)-derived cardiovascular progenitor cells (CPCs) are a potential cell source for cardiac repair. The aim of this study was to examine the impact of extensive remuscularization and coincident revascularization on cardiac remodelling and function in a mouse model of myocardial infarction (MI) by transplanting doxycycline (DOX)-inducible (Tet-On-MYC) hPSC-derived CPCs in vivo and inducing proliferation and cardiovascular differentiation in a drug-regulated manner.Methods and resultsCPCs were injected firstly at a non-cardiac site in Matrigel suspension under the skin of immunocompromised mice to assess their commitment to the cardiovascular lineage and ability to self-renew or differentiate in vivo when instructed by systemically delivered factors inclu...

Stem Cell Reports, 2018

Predicting developmental potency and risk of posttransplantation tumor formation by human pluripo... more Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.

The Journal of Immunology, 2018

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immu... more By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV−/− mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV−/− mice developed higher Ab titers but no autoantibodies. These observations indicate a redunda...

Stem cell reports, Jan 8, 2018

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent ste... more Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, whic...

Neuromuscular Disorders, 2017

In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat... more In humans, a copy of the DUX4 retrogene is located in each unit of the D4Z4 macrosatellite repeat that normally comprises 8-100 units. The D4Z4 repeat has heterochromatic features and does not express DUX4 in somatic cells. Individuals with facioscapulohumeral muscular dystrophy (FSHD) have a partial failure of somatic DUX4 repression resulting in the presence of DUX4 protein in sporadic muscle nuclei. Somatic DUX4 derepression is caused by contraction of the D4Z4 repeat to 1-10 units (FSHD1) or by heterozygous mutations in genes responsible for maintaining the D4Z4 chromatin structure in a repressive state (FSHD2). One of the FSHD2 genes is the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene. SMCHD1 mutations have also been identified in FSHD1; patients carrying a contracted D4Z4 repeat and a SMCHD1 mutation are more severely affected than relatives with only a contracted repeat or a SMCHD1 mutation. To evaluate the modifier role of SMCHD1, we crossbred mice carrying a contracted D4Z4 repeat (D4Z4-2.5 mice) with mice that are haploinsufficient for Smchd1 (Smchd1 MommeD1 mice). D4Z4-2.5/Smchd1 MommeD1 mice presented with a significantly reduced body weight and developed skin lesions. The same skin lesions, albeit in a milder form, were also observed in D4Z4-2.5 mice, suggesting that reduced Smchd1 levels aggravate disease in the D4Z4-2.5 mouse model. Our study emphasizes the evolutionary conservation of the SMCHD1-dependent epigenetic regulation of the D4Z4 repeat array and further suggests that the D4Z4-2.5/ Smchd1 MommeD1 mouse model may be used to unravel the function of DUX4 in non-muscle tissues like the skin.

Stem cell reports, May 9, 2017

The ability to form teratomas in vivo containing multiple somatic cell types is regarded as funct... more The ability to form teratomas in vivo containing multiple somatic cell types is regarded as functional evidence of pluripotency for human pluripotent stem cells (hPSCs). Since the Teratoma assay is animal dependent, laborious, and only qualitative, the PluriTest and the hPSC ScoreCard assay have been developed as in vitro alternatives. Here we compared normal hPSCs, induced hPSCs (hiPSCs) with reactivated reprogramming transgenes, and human embryonal carcinoma cells (hECs) in these assays. While normal hPSCs gave rise to typical teratomas, the xenografts of the hECs and the hiPSCs with reactivated reprogramming transgenes were largely undifferentiated and malignant. The hPSC ScoreCard assay confirmed the line-specific differentiation propensities in vitro. However, when undifferentiated cells were analyzed by the PluriTest, only hECs were identified as abnormal whereas all other cell lines were indistinguishable and resembled normal hPSCs. Our results indicate that pluripotency assa...