92 results on '"Dhaenens M"'
Search Results
2. Laser microdissection for the assessment of the clonal relationship between chronic lymphocytic leukemia/small lymphocytic lymphoma and proliferating B cells within lymph node pseudofollicles
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Vandewoestyne, M L, Pede, V C, Lambein, K Y, Dhaenens, M F, Offner, F C, Praet, M M, Philippé, J J, Kipps, T J, and Deforce, D L
- Published
- 2011
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3. Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.
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Scheerlinck, E., Dhaenens, M., Van Soom, A., Peelman, L., De Sutter, P., Van Steendam, K., and Deforce, D.
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PROTEOMICS , *DENATURATION of proteins , *ALKYLATION , *CHEMICAL reduction , *LYSIS , *LIQUID chromatography-mass spectrometry - Abstract
Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography–tandem mass spectrometry (LC–MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS E ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC–MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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4. Neutrophil Elastase in the capacity of the “H2A-specific protease”.
- Author
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Dhaenens, M., Glibert, P., Lambrecht, S., Vossaert, L., Van Steendam, K., Elewaut, D., and Deforce, D.
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LEUCOCYTE elastase , *PROTEOLYTIC enzymes , *HISTONES , *CHROMATIN , *MONOCYTES , *MASS spectrometry , *EMBRYONIC stem cells - Abstract
Highlights: [•] The H2A specific protease (H2Asp) clips histone H2A at V114 but was never identified since its discovery over 35 years ago. [•] We developed an AQUA approach for high throughput quantitation of V114 clipping. [•] We show that H2Asp actually is Neutrophil Elastase (NE). [•] NE associates strongly with nucleosomes in differential gradient experiments. [•] We emphasize the potential implications of H2AV114 clipping in vitro and in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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5. Dendritic cells from spondylarthritis-prone HLA-B27-transgenic rats display altered cytoskeletal dynamics, class II major histocompatibility complex expression, and viability.
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Dhaenens M, Fert I, Glatigny S, Haerinck S, Poulain C, Donnadieu E, Hacquard-Bouder C, André C, Elewaut D, Deforce D, and Breban M
- Abstract
OBJECTIVE: Spondylarthritis (SpA) is characterized by spinal and peripheral joint inflammation, frequently combined with extraarticular manifestations. Despite the well-established association of SpA with the class I major histocompatibility complex (MHC) allele HLA-B27, there are still different, parallel hypotheses on the relationship between HLA-B27 and disease mechanisms. The present study was undertaken to investigate several characteristics of mature dendritic cells (DCs), which are believed to be essential for triggering disease in a model of SpA in HLA-B27-transgenic rats. METHODS: We combined different whole-proteome approaches (2-dimensional polyacrylamide gel electrophoresis and iTRAQ) to define the most aberrant molecular processes occurring in spleen DCs. Videomicroscopy and flow cytometry were used to confirm both cytoskeletal and class II MHC expression deficiencies. RESULTS: Our proteome studies provided evidence of up-regulation of proteins involved in class I MHC loading, and unfolded protein response, along with a striking down-regulation of several cytoskeleton-reorganizing proteins. The latter result was corroborated by findings of deficient motility, altered morphology, and decreased immunologic synapse formation. Furthermore, class II MHC surface expression was reduced in DCs from B27-transgenic rats, and this could be linked to differences in class II MHC-induced apoptotic sensitivity. Finally, we found reduced viability of the CD103+CD4- DC subpopulation, which likely exerts tolerogenic function. CONCLUSION: Taken together, our findings have different important implications regarding the physiology of B27-transgenic rat DCs, which have a putative role in spontaneous disease in these rats. In particular, the reduced motility and viability of putatively tolerogenic CD4+ DCs could play an important role in initiating the inflammatory process, resulting in SpA. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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6. 390 EXPLORATION OF THE PROTEOME OF HUMAN ARTICULAR CHONDROCYTES BY A SHOTGUN PROTEOMICS APPROACH
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Lambrecht, S., Dhaenens, M., Verbruggen, G., Elewaut, D., and Deforce, D.
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- 2007
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7. T-2 toxin induced Salmonella Typhimurium intoxication results in decreased Salmonella numbers in the cecum contents of pigs, despite marked effects on Salmonella-host cell interactions
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Verbrugghe Elin, Vandenbroucke Virginie, Dhaenens Maarten, Shearer Neil, Goossens Joline, De Saeger Sarah, Eeckhout Mia, D'Herde Katharina, Thompson Arthur, Deforce Dieter, Boyen Filip, Leyman Bregje, Van Parys Alexander, De Backer Patrick, Haesebrouck Freddy, Croubels Siska, and Pasmans Frank
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Veterinary medicine ,SF600-1100 - Abstract
Abstract The mycotoxin T-2 toxin and Salmonella Typhimurium infections pose a significant threat to human and animal health. Interactions between both agents may result in a different outcome of the infection. Therefore, the aim of the presented study was to investigate the effects of low and relevant concentrations of T-2 toxin on the course of a Salmonella Typhimurium infection in pigs. We showed that the presence of 15 and 83 μg T-2 toxin per kg feed significantly decreased the amount of Salmonella Typhimurium bacteria present in the cecum contents, and a tendency to a reduced colonization of the jejunum, ileum, cecum, colon and colon contents was noticed. In vitro, proteomic analysis of porcine enterocytes revealed that a very low concentration of T-2 toxin (5 ng/mL) affects the protein expression of mitochondrial, endoplasmatic reticulum and cytoskeleton associated proteins, proteins involved in protein synthesis and folding, RNA synthesis, mitogen-activated protein kinase signaling and regulatory processes. Similarly low concentrations (1-100 ng/mL) promoted the susceptibility of porcine macrophages and intestinal epithelial cells to Salmonella Typhimurium invasion, in a SPI-1 independent manner. Furthermore, T-2 toxin (1-5 ng/mL) promoted the translocation of Salmonella Typhimurium over an intestinal porcine epithelial cell monolayer. Although these findings may seem in favour of Salmonella Typhimurium, microarray analysis showed that T-2 toxin (5 ng/mL) causes an intoxication of Salmonella Typhimurium, represented by a reduced motility and a downregulation of metabolic and Salmonella Pathogenicity Island 1 genes. This study demonstrates marked interactions of T-2 toxin with Salmonella Typhimurium pathogenesis, resulting in bacterial intoxication.
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- 2012
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8. M066 COV2MS: A tool for simultaneous longitudinal epidemiological monitoring of a variety of pathogens.
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Van Puyvelde, B., Van Uytfanghe, K., and Dhaenens, M.
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PATHOGENIC microorganisms - Published
- 2022
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9. La methode mathematique de Ruch et Ugi. Application au dedoublement partiel selon horeau
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Vigneron, J.P., Dhaenens, M., and Horeau, A.
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- 1977
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10. Configurations absolues des methylbenzhydryl, isopropylbenzhydryl et isopropylbenzylcarbinols
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Vigneron, J.P., Dhaenens, M., and Horeau, A.
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- 1977
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11. Preparation des methyl-4 heptanols-3 erythro et threo optiquement purs.
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Vigneron, J.P., Méric, R., and Dhaenens, M.
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- 1980
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12. Nouvelle methode pour porter au maximum la purete optique d'un produit partiellement dedouble sans l'aide d'aucune substance chirale
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Vigneron, J.P., Dhaenens, M., and Horeau, A.
- Published
- 1973
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13. Mise en évidence d'une anomalie de régulation du cytosquelette dans les cellules dendritiques (DC) de rats transgéniques pour le HLA-B27 et la beta-2 microglobuline humaine (rats-B27)
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Hacquard-Bouder, C., Poulain, C., Fert, I., Dhaenens, M., Haerinck, S., André, C., Elewaut, D., Deforce, D., and Breban, M.
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- 2007
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14. Effects of hypothermia and hypoxia on cytochrome P450-mediated drug metabolism in neonatal Göttingen minipigs.
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Stroe MS, De Clerck L, Dhaenens M, Dennis RS, Deforce D, Carpentier S, Annaert P, Leys K, Smits A, Allegaert K, Van Ginneken C, and Van Cruchten S
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- Animals, Swine, Female, Male, Swine, Miniature, Cytochrome P-450 Enzyme System metabolism, Cytochrome P-450 Enzyme System genetics, Animals, Newborn, Hypothermia, Induced, Hypoxia metabolism, Microsomes, Liver metabolism, Liver metabolism
- Abstract
Asphyxiated neonates often undergo therapeutic hypothermia (TH) to reduce morbidity and mortality. As perinatal asphyxia and TH impact neonatal physiology, this could also influence enzyme functionality. Therefore, this study aimed to unravel the impact of age, hypothermia and hypoxia on porcine hepatic cytochrome P450 (CYP) gene expression, protein abundance and activity. Hepatic CYP expression, protein abundance and activity were assessed in naive adult and neonatal Göttingen minipigs, alongside those from an (non-survival) in vivo study, where four conditions-control (C), therapeutic hypothermia (TH), hypoxia (H), hypoxia and TH (H + TH)-were examined. Naive neonatal Göttingen minipigs exhibited 75% lower general CYP activity and different gene expression patterns than adults. In vitro hypothermia (33°C) decreased general CYP activity in adult liver microsomes by 36%. Gene expression was not different between TH and C while hypoxia up-regulated several genes (i.e., CYP3A29 [expression ratio; E
R = 5.1472] and CYP2C33 [ER = 3.2292] in the H group and CYP2C33 [ER = 2.4914] and CYP2C42 [ER = 4.0197] in the H + TH group). The medical treatment and the interventions over 24 h, along with hypoxia and TH, affected the protein abundance. These data on CYP expression, abundance and activity in young animals can be valuable in building physiologically-based pharmacokinetic models for neonatal drug dose predictions., (© 2024 The Author(s). Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)- Published
- 2024
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15. The interplay of dietary mycotoxins and oncogenic viruses toward human carcinogenesis: a scoping review.
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Mouchtaris Michailidis T, De Saeger S, Khoueiry R, Odongo GA, Bader Y, Dhaenens M, Herceg Z, and De Boevre M
- Abstract
Background: Mycotoxins, fungal metabolites prevalent in many foods, are recognized for their role in carcinogenesis, especially when interacting with oncogenic viruses., Objectives: This scoping review synthesizes current evidence on the human cancer risk associated with mycotoxin exposure and oncogenic virus infections., Methods: Searches were conducted on PubMed, Embase, and Web of Science. Studies were selected based on the PECOS framework. Data extraction involved narrative and qualitative presentation of findings, with meta-analysis where feasible. Risk of bias and outcome quality were assessed using the OHAT tool and GRADE approach., Results: From 25 included studies, 18 focused on aflatoxins and hepatitis viruses in hepatocellular carcinoma (HCC). Four studies examined aflatoxin B1 (AFB1) and human papilloma virus (HPV) in cervical cancer, while three investigated AFB1 with Epstein-Barr virus (EBV) in lymphomagenesis. The review highlights a significant synergistic effect between AFB1 and hepatitis B and C viruses in HCC development. Significant interactions between AFB1 and HPV, as well as AFB1 and EBV, were observed, but further research is needed., Conclusions: The synergistic impact of mycotoxins and oncogenic viruses is a critical public health concern. Future research, especially prospective cohort studies and investigations into molecular mechanisms, is essential to address this complex issue.
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- 2024
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16. Mass Spectrometry-based Profiling of Single-cell Histone Post-translational Modifications to Dissect Chromatin Heterogeneity.
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Cutler R, Corveleyn L, Ctortecka C, Cantlon J, Jacome Vaca SA, Deforce D, Vijg J, Dhaenens M, Papanastasiou M, Carr SA, and Sidoli S
- Abstract
Single-cell proteomics confidently quantifies cellular heterogeneity, yet precise quantification of post-translational modifications, such as those deposited on histone proteins, has remained elusive. Here, we developed a robust mass spectrometry-based method for the unbiased analysis of single-cell histone post-translational modifications (schPTM). schPTM identifies both single and combinatorial histone post-translational modifications (68 peptidoforms in total), which includes nearly all frequently studied histone post-translational modifications with comparable reproducibility to traditional bulk experiments. As a proof of concept, we treated cells with sodium butyrate, a histone deacetylase inhibitor, and demonstrated that our method can i) distinguish between treated and non-treated cells, ii) identify sub-populations of cells with heterogeneous response to the treatment, and iii) reveal differential co-regulation of histone post-translational modifications in the context of drug treatment. The schPTM method enables comprehensive investigation of chromatin heterogeneity at single-cell resolution and provides further understanding of the histone code.
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- 2024
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17. A Targeted LC-MRM 3 Proteomic Approach for the Diagnosis of SARS-CoV-2 Infection in Nasopharyngeal Swabs.
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Drouin N, Elfrink HL, Boers SA, van Hugten S, Wessels E, de Vries JJC, Groeneveld GH, Miggiels P, Van Puyvelde B, Dhaenens M, Budding AE, Ran L, Masius R, Takats Z, Boogaerds A, Bulters M, Muurlink W, Oostvogel P, Harms AC, van der Lubben M, and Hankemeier T
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- Humans, Chromatography, Liquid methods, Coronavirus Nucleocapsid Proteins metabolism, Sensitivity and Specificity, Mass Spectrometry methods, Phosphoproteins, COVID-19 diagnosis, COVID-19 virology, SARS-CoV-2 isolation & purification, Proteomics methods, Nasopharynx virology
- Abstract
Since its first appearance, severe acute respiratory syndrome coronavirus 2 quickly spread around the world and the lack of adequate PCR testing capacities, especially during the early pandemic, led the scientific community to explore new approaches such as mass spectrometry (MS). We developed a proteomics workflow to target several tryptic peptides of the nucleocapsid protein. A highly selective multiple reaction monitoring-cubed (MRM
3 ) strategy provided a sensitivity increase in comparison to conventional MRM acquisition. Our MRM3 approach was first tested on an Amsterdam public health cohort (alpha-variant, 760 participants) detecting viral nucleocapsid protein peptides from nasopharyngeal swabs samples presenting a cycle threshold value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immunopurification. A second iteration of the MS-diagnostic test, able to analyze more than 400 samples per day, was clinically validated on a Leiden-Rijswijk public health cohort (delta-variant, 2536 participants) achieving 99.9% specificity and 93.1% sensitivity for patients with cycle threshold values up to 35. In this manuscript, we also developed and brought the first proof of the concept of viral variant monitoring in a complex matrix using targeted MS., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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18. Acoustic ejection mass spectrometry empowers ultra-fast protein biomarker quantification.
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Van Puyvelde B, Hunter CL, Zhgamadze M, Savant S, Wang YO, Hoedt E, Raedschelders K, Pope M, Huynh CA, Ramanujan VK, Tourtellotte W, Razavi M, Anderson NL, Martens G, Deforce D, Fu Q, Dhaenens M, and Van Eyk JE
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- Humans, Peptides, Coronavirus Nucleocapsid Proteins analysis, Phosphoproteins, Biomarkers blood, COVID-19 diagnosis, COVID-19 virology, COVID-19 blood, SARS-CoV-2 immunology, Mass Spectrometry methods
- Abstract
The global scientific response to COVID 19 highlighted the urgent need for increased throughput and capacity in bioanalytical laboratories, especially for the precise quantification of proteins that pertain to health and disease. Acoustic ejection mass spectrometry (AEMS) represents a much-needed paradigm shift for ultra-fast biomarker screening. Here, a quantitative AEMS assays is presented, employing peptide immunocapture to enrich (i) 10 acute phase response (APR) protein markers from plasma, and (ii) SARS-CoV-2 NCAP peptides from nasopharyngeal swabs. The APR proteins were quantified in 267 plasma samples, in triplicate in 4.8 h, with %CV from 4.2% to 10.5%. SARS-CoV-2 peptides were quantified in triplicate from 145 viral swabs in 10 min. This assay represents a 15-fold speed improvement over LC-MS, with instrument stability demonstrated across 10,000 peptide measurements. The combination of speed from AEMS and selectivity from peptide immunocapture enables ultra-high throughput, reproducible quantitative biomarker screening in very large cohorts., (© 2024. The Author(s).)
- Published
- 2024
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19. First insights into human mobility in Neolithic Belgium using strontium isotopic analysis and proteomics: A case study of Grotte de La Faucille (Sclayn, province of Namur).
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van Hattum I, Costas-Rodríguez M, Hobin K, Vanhaecke F, Vandendriessche H, Collet H, Cattelain P, Toussaint M, Goffette Q, Dhaenens M, Palmer JLA, Daled S, Crombé P, and De Groote I
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- Male, Adult, Humans, Belgium, Isotopes analysis, Strontium analysis, Proteomics, Strontium Isotopes analysis
- Abstract
Objectives: So far, no
87 Sr/86 Sr mobility studies have been done for Neolithic remains from Belgium and information on the Sr isotopic variability in the region is scarce. This study aims to explore mobility in a Final Neolithic population from the funerary cave 'Grotte de La Faucille', contribute to the understanding of the isotopic composition of bioavailable Sr in Belgium, assess evidence for male mobility using proteomic analysis, and explore possible places of origin for nonlocal individuals., Materials and Methods: The87 Sr/86 Sr isotope ratio of dental enamel from six adults and six juveniles was determined. Liquid chromatography mass spectrometry-based protein analysis was employed to identify individuals of male biological sex.87 Sr/86 Sr of micromammal teeth, snail shells, and modern plants from three geological areas in Belgium were measured to establish isotopic signatures for bioavailable strontium. Nonlocality was assessed by comparing human87 Sr/86 Sr isotope ratios to the87 Sr/86 Sr range for bioavailable Sr., Results: Four individuals yielded87 Sr/86 Sr isotope ratios consistent with a nonlocal origin. No statistical differences were found between adults and juveniles. Three males were detected in the sample set, of which two show nonlocal87 Sr/86 Sr values., Discussion: This study provides evidence for mobility in Final Neolithic Belgium. The four nonlocal87 Sr/86 Sr signatures correspond with the87 Sr/86 Sr of bio-available Sr in Dutch South Limburg, the Black Forest in Southwest Germany, and regions of France, such as parts of the Paris Basin and the Vosges. The results support the ruling hypothesis of connections with Northern France, brought to light by archeological research., (© 2023 Wiley Periodicals LLC.)- Published
- 2023
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20. Cathepsin-L Secreted by High-Quality Bovine Embryos Exerts an Embryotrophic Effect In Vitro.
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Raes A, Wydooghe E, Pavani KC, Bogado Pascottini O, Van Steendam K, Dhaenens M, Boel A, Heras S, Heindryckx B, Peelman L, Deforce D, Van Nieuwerburgh F, Opsomer G, Van Soom A, and Smits K
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- Cattle, Animals, Humans, Zygote, Blastocyst metabolism, Cathepsins metabolism, Culture Media pharmacology, Culture Media metabolism, Fertilization in Vitro, Embryo Culture Techniques, Embryonic Development
- Abstract
While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups improves blastocyst yield and quality. Paracrine embryotrophins could be responsible for this improved embryo development, but their identity remains largely unknown. We hypothesize that supplementation of embryotrophic proteins to a culture medium could be the key to improve individual embryo production. In this study, proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as being secreted by both excellent- and good-quality embryos, while being absent in the medium conditioned by poor-quality embryos. The embryotrophic role of cathepsin-L was explored in vitro, whereby bovine zygotes were cultured individually for 8 days with or without cathepsin-L. Preliminary dose-response experiments pointed out 100 ng/mL as the optimal concentration of cathepsin-L in embryo culture medium. Supplementation of cathepsin-L to individual culture systems significantly improved blastocyst development and quality in terms of blastocoel formation at day 7, and the hatching ratio and apoptotic cell ratio at day 8, compared to the control. Taken together, cathepsin-L acts as an important embryotrophin by increasing embryo quality, and regulating blastulation and hatching in bovine in vitro embryo production.
- Published
- 2023
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21. Proteomic Comparison of Three Wild-Type Pseudorabies Virus Strains and the Attenuated Bartha Strain Reveals Reduced Incorporation of Several Tegument Proteins in Bartha Virions.
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Delva JL, Daled S, Van Waesberghe C, Almey R, Jansens RJJ, Deforce D, Dhaenens M, and Favoreel HW
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- Animals, Capsid metabolism, Proteomics, Swine, Viral Proteins immunology, Vaccines, Attenuated immunology, Herpesvirus 1, Suid metabolism, Pseudorabies prevention & control, Swine Diseases prevention & control, Swine Diseases virology, Viral Vaccines immunology
- Abstract
Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily and the causative agent of Aujeszky's disease in pigs. Driven by the large economic losses associated with PRV infection, several vaccines and vaccine programs have been developed. To this day, the attenuated Bartha strain, generated by serial passaging, represents the golden standard for PRV vaccination. However, a proteomic comparison of the Bartha virion to wild-type (WT) PRV virions is lacking. Here, we present a comprehensive mass spectrometry-based proteome comparison of the attenuated Bartha strain and three commonly used WT PRV strains: Becker, Kaplan, and NIA3. We report the detection of 40 structural and 14 presumed nonstructural proteins through a combination of data-dependent and data-independent acquisition. Interstrain comparisons revealed that packaging of the capsid and most envelope proteins is largely comparable in-between all four strains, except for the envelope protein pUL56, which is less abundant in Bartha virions. However, distinct differences were noted for several tegument proteins. Most strikingly, we noted a severely reduced incorporation of the tegument proteins IE180, VP11/12, pUS3, VP22, pUL41, pUS1, and pUL40 in Bartha virions. Moreover, and likely as a consequence, we also observed that Bartha virions are on average smaller and more icosahedral compared to WT virions. Finally, we detected at least 28 host proteins that were previously described in PRV virions and noticed considerable strain-specific differences with regard to host proteins, arguing that the potential role of packaged host proteins in PRV replication and spread should be further explored. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha-an attenuated strain created by serial passaging-represents an exceptional success story in alphaherpesvirus vaccination. Here, we used mass spectrometry to analyze the Bartha virion composition in comparison to three established WT PRV strains. Many viral tegument proteins that are considered nonessential for viral morphogenesis were drastically less abundant in Bartha virions compared to WT virions. Interestingly, many of the proteins that are less incorporated in Bartha participate in immune evasion strategies of alphaherpesviruses. In addition, we observed a reduced size and more icosahedral morphology of the Bartha virions compared to WT PRV. Given that the Bartha vaccine strain elicits potent immune responses, our findings here suggest that differences in protein packaging may contribute to its immunogenicity. Further exploration of these observations could aid the development of efficacious vaccines against other alphaherpesvirus vaccines such as HSV-1/2 or EHV-1.
- Published
- 2022
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22. Cov 2 MS: An Automated and Quantitative Matrix-Independent Assay for Mass Spectrometric Measurement of SARS-CoV-2 Nucleocapsid Protein.
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Van Puyvelde B, Van Uytfanghe K, Van Oudenhove L, Gabriels R, Van Royen T, Matthys A, Razavi M, Yip R, Pearson T, Drouin N, Claereboudt J, Foley D, Wardle R, Wyndham K, Hankemeier T, Jones D, Saelens X, Martens G, Stove CP, Deforce D, Martens L, Vissers JPC, Anderson NL, and Dhaenens M
- Subjects
- Humans, COVID-19 Testing, Clinical Laboratory Techniques methods, Mass Spectrometry methods, Peptides, Sensitivity and Specificity, SARS-CoV-2, COVID-19
- Abstract
The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov
2 MS assay is compatible with most matrices including nasopharyngeal swabs, saliva, and plasma and has increased sensitivity into the attomole range, a 1000-fold improvement compared to direct detection in a matrix. A strong positive correlation was observed with qPCR detection beyond a quantification cycle of 30-31, the level where no live virus can be cultured. The automatable sample preparation and reduced LC dependency allow analysis of up to 500 samples per day per instrument. Importantly, peptide enrichment allows detection of the N protein in pooled samples without sensitivity loss. Easily multiplexed, we detect variants and propose targets for Influenza A and B detection. Thus, the Cov2 MS assay can be adapted to test for many different pathogens in pooled samples, providing longitudinal epidemiological monitoring of large numbers of pathogens within a population as an early warning system.- Published
- 2022
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23. Micro-Topographies Induce Epigenetic Reprogramming and Quiescence in Human Mesenchymal Stem Cells.
- Author
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Vermeulen S, Van Puyvelde B, Bengtsson Del Barrio L, Almey R, van der Veer BK, Deforce D, Dhaenens M, and de Boer J
- Abstract
Biomaterials can control cell and nuclear morphology. Since the shape of the nucleus influences chromatin architecture, gene expression and cell identity, surface topography can control cell phenotype. This study provides fundamental insights into how surface topography influences nuclear morphology, histone modifications, and expression of histone-associated proteins through advanced histone mass spectrometry and microarray analysis. The authors find that nuclear confinement is associated with a loss of histone acetylation and nucleoli abundance, while pathway analysis reveals a substantial reduction in gene expression associated with chromosome organization. In light of previous observations where the authors found a decrease in proliferation and metabolism induced by micro-topographies, they connect these findings with a quiescent phenotype in mesenchymal stem cells, as further shown by a reduction of ribosomal proteins and the maintenance of multipotency on micro-topographies after long-term culture conditions. Also, this influence of micro-topographies on nuclear morphology and proliferation is reversible, as shown by a return of proliferation when re-cultured on a flat surface. The findings provide novel insights into how biophysical signaling influences the epigenetic landscape and subsequent cellular phenotype., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)
- Published
- 2022
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24. An interactive mass spectrometry atlas of histone posttranslational modifications in T-cell acute leukemia.
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Provez L, Van Puyvelde B, Corveleyn L, Demeulemeester N, Verhelst S, Lintermans B, Daled S, Roels J, Clement L, Martens L, Deforce D, Van Vlierberghe P, and Dhaenens M
- Subjects
- Humans, Epigenesis, Genetic, Mass Spectrometry methods, Peptides chemistry, Protein Processing, Post-Translational, T-Lymphocytes chemistry, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma, Histones metabolism, Leukemia
- Abstract
The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development., (© 2022. The Author(s).)
- Published
- 2022
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25. Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.
- Author
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Zijlmans DW, Talon I, Verhelst S, Bendall A, Van Nerum K, Javali A, Malcolm AA, van Knippenberg SSFA, Biggins L, To SK, Janiszewski A, Admiraal D, Knops R, Corthout N, Balaton BP, Georgolopoulos G, Panda A, Bhanu NV, Collier AJ, Fabian C, Allsop RN, Chappell J, Pham TXA, Oberhuemer M, Ertekin C, Vanheer L, Athanasouli P, Lluis F, Deforce D, Jansen JH, Garcia BA, Vermeulen M, Rivron N, Dhaenens M, Marks H, Rugg-Gunn PJ, and Pasque V
- Subjects
- Cell Differentiation genetics, Chromatin genetics, Histones genetics, Humans, Trophoblasts metabolism, Pluripotent Stem Cells, Polycomb Repressive Complex 2 genetics, Polycomb Repressive Complex 2 metabolism
- Abstract
Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates., (© 2022. The Author(s).)
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- 2022
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26. A comprehensive LFQ benchmark dataset on modern day acquisition strategies in proteomics.
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Van Puyvelde B, Daled S, Willems S, Gabriels R, Gonzalez de Peredo A, Chaoui K, Mouton-Barbosa E, Bouyssié D, Boonen K, Hughes CJ, Gethings LA, Perez-Riverol Y, Bloomfield N, Tate S, Schiltz O, Martens L, Deforce D, and Dhaenens M
- Subjects
- Animals, Chromatography, Liquid methods, Humans, Mass Spectrometry methods, Proteome, Benchmarking, Proteomics
- Abstract
In the last decade, a revolution in liquid chromatography-mass spectrometry (LC-MS) based proteomics was unfolded with the introduction of dozens of novel instruments that incorporate additional data dimensions through innovative acquisition methodologies, in turn inspiring specialized data analysis pipelines. Simultaneously, a growing number of proteomics datasets have been made publicly available through data repositories such as ProteomeXchange, Zenodo and Skyline Panorama. However, developing algorithms to mine this data and assessing the performance on different platforms is currently hampered by the lack of a single benchmark experimental design. Therefore, we acquired a hybrid proteome mixture on different instrument platforms and in all currently available families of data acquisition. Here, we present a comprehensive Data-Dependent and Data-Independent Acquisition (DDA/DIA) dataset acquired using several of the most commonly used current day instrumental platforms. The dataset consists of over 700 LC-MS runs, including adequate replicates allowing robust statistics and covering over nearly 10 different data formats, including scanning quadrupole and ion mobility enabled acquisitions. Datasets are available via ProteomeXchange (PXD028735)., (© 2022. The Author(s).)
- Published
- 2022
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27. A large scale mass spectrometry-based histone screening for assessing epigenetic developmental toxicity.
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Verhelst S, Van Puyvelde B, Willems S, Daled S, Cornelis S, Corveleyn L, Willems E, Deforce D, De Clerck L, and Dhaenens M
- Subjects
- Humans, Proof of Concept Study, Histone Code, Mass Spectrometry, Protein Processing, Post-Translational, Teratogens analysis, Toxicity Tests methods
- Abstract
Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose-response study was performed on a proof-of-concept library of ten compounds (1) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), (2) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), (3) classified as non-embryotoxic by ECVAM (Penicillin G), and (4) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine)., (© 2022. The Author(s).)
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- 2022
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28. Add mass spectrometry to the pandemic toolbox.
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Van Puyvelde B and Dhaenens M
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- Humans, Mass Spectrometry, Proteomics, SARS-CoV-2, COVID-19, Pandemics
- Abstract
A new protocol step improves robustness and ease-of-use for mass spectrometry in the clinic, opening the door to mass deployment to monitor infectious agents., Competing Interests: BV, MD No competing interests declared, (© 2021, Van Puyvelde and Dhaenens.)
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- 2021
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29. An experimental design to extract more information from MS-based histone studies.
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De Clerck L, Willems S, Daled S, Van Puyvelde B, Verhelst S, Corveleyn L, Deforce D, and Dhaenens M
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- Histone Code, Protein Processing, Post-Translational, Proteomics, Histones metabolism, Research Design
- Abstract
Histone-based chromatin organization paved the way for eukaryotic genome complexity. Because of their key role in information management, the histone posttranslational modifications (hPTM), which mediate their function, have evolved into an alphabet that has more letters than there are amino acids, together making up the "histone code". The resulting combinatorial complexity is manifold higher than what is usually encountered in proteomics. Consequently, a considerably bigger part of the acquired MSMS spectra remains unannotated to date. Adapted search parameters can dig deeper into the dark histone ion space, but the lack of false discovery rate (FDR) control and the high level of ambiguity when searching combinatorial PTMs makes it very hard to assess whether the newly assigned ions are informative. Therefore, we propose an easily adoptable time-lapse enzymatic deacetylation (HDAC1) of a commercial histone extract as a quantify-first strategy that allows isolating ion populations of interest, when studying e.g. acetylation on histones, that currently remain in the dark. By adapting search parameters to study potential issues in sample preparation, data acquisition and data analysis, we stepwise managed to double the portion of annotated precursors of interest from 10.5% to 21.6%. This strategy is intended to make up for the lack of validated FDR control and has led to several adaptations of our current workflow that will reduce the portion of the dark histone ion space in the future. Finally, this strategy can be applied with any enzyme targeting a modification of interest.
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- 2021
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30. Ferroptosis Induction in Multiple Myeloma Cells Triggers DNA Methylation and Histone Modification Changes Associated with Cellular Senescence.
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Logie E, Van Puyvelde B, Cuypers B, Schepers A, Berghmans H, Verdonck J, Laukens K, Godderis L, Dhaenens M, Deforce D, and Vanden Berghe W
- Subjects
- Histone Code, Humans, Multiple Myeloma pathology, Cellular Senescence, DNA Methylation, DNA, Neoplasm metabolism, Ferroptosis, Histones metabolism, Multiple Myeloma metabolism, Neoplasm Proteins metabolism
- Abstract
Disease relapse and therapy resistance remain key challenges in treating multiple myeloma. Underlying (epi-)mutational events can promote myelomagenesis and contribute to multi-drug and apoptosis resistance. Therefore, compounds inducing ferroptosis, a form of iron and lipid peroxidation-regulated cell death, are appealing alternative treatment strategies for multiple myeloma and other malignancies. Both ferroptosis and the epigenetic machinery are heavily influenced by oxidative stress and iron metabolism changes. Yet, only a limited number of epigenetic enzymes and modifications have been identified as ferroptosis regulators. In this study, we found that MM1 multiple myeloma cells are sensitive to ferroptosis induction and epigenetic reprogramming by RSL3, irrespective of their glucocorticoid-sensitivity status. LC-MS/MS analysis revealed the formation of non-heme iron-histone complexes and altered expression of histone modifications associated with DNA repair and cellular senescence. In line with this observation, EPIC BeadChip measurements of significant DNA methylation changes in ferroptotic myeloma cells demonstrated an enrichment of CpG probes located in genes associated with cell cycle progression and senescence, such as Nuclear Receptor Subfamily 4 Group A member 2 (NR4A2). Overall, our data show that ferroptotic cell death is associated with an epigenomic stress response that might advance the therapeutic applicability of ferroptotic compounds.
- Published
- 2021
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31. Histone clipping: the punctuation in the histone code.
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Dhaenens M
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- Protein Processing, Post-Translational, Histone Code, Histones genetics, Histones metabolism
- Abstract
Histone clipping was first discovered in the 1960s and still is a lingering mystery. Considering the essential roles of histones in regulating eukaryotic transcription through the histone code, clipping is a post-translational modification that appeals to the imagination. In this issue of EMBO Reports, Marruecos and colleagues investigate histone H4 clipping during intestinal development (Marruecos et al, 2021), and are providing crucial clues to finally elucidate the intricacies of this elusive modification., (© 2021 The Author.)
- Published
- 2021
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32. Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.
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Van Puyvelde B, Van Uytfanghe K, Tytgat O, Van Oudenhove L, Gabriels R, Bouwmeester R, Daled S, Van Den Bossche T, Ramasamy P, Verhelst S, De Clerck L, Corveleyn L, Willems S, Debunne N, Wynendaele E, De Spiegeleer B, Judak P, Roels K, De Wilde L, Van Eenoo P, Reyns T, Cherlet M, Dumont E, Debyser G, t'Kindt R, Sandra K, Gupta S, Drouin N, Harms A, Hankemeier T, Jones DJL, Gupta P, Lane D, Lane CS, El Ouadi S, Vincendet JB, Morrice N, Oehrle S, Tanna N, Silvester S, Hannam S, Sigloch FC, Bhangu-Uhlmann A, Claereboudt J, Anderson NL, Razavi M, Degroeve S, Cuypers L, Stove C, Lagrou K, Martens GA, Deforce D, Martens L, Vissers JPC, and Dhaenens M
- Abstract
Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550., Competing Interests: The authors declare the following competing financial interest(s): L. Van Oudenhove, J. Claereboudt, S. Oehrle, N. Tanna, and J. P. C. Vissers are employed by Waters Corporation. C. S. Lane, S. El Ouadi, J.-B. Vincendet, and N. Morrice are employed by Sciex. F. Sigloch and A. Bhangu-Uhlmann are employed by Polyquant GmbH. M. Razavi and L. Anderson are employed by SISCAPA., (© 2021 The Authors. Published by American Chemical Society.)
- Published
- 2021
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33. Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions.
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Daled S, Willems S, Van Puyvelde B, Corveleyn L, Verhelst S, De Clerck L, Deforce D, and Dhaenens M
- Abstract
Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX
® , Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423.- Published
- 2021
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34. Hepatic Cytochrome P450 Abundance and Activity in the Developing and Adult Göttingen Minipig: Pivotal Data for PBPK Modeling.
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Buyssens L, De Clerck L, Schelstraete W, Dhaenens M, Deforce D, Ayuso M, Van Ginneken C, and Van Cruchten S
- Abstract
The Göttingen Minipig is gaining ground as nonrodent species in safety testing of drugs for pediatric indications. Due to developmental changes in pharmacokinetics and pharmacodynamics, physiologically based pharmacokinetic (PBPK) models are built to better predict drug exposure in children and to aid species selection for nonclinical safety studies. These PBPK models require high quality physiological and ADME data such as protein abundance of drug metabolizing enzymes. These data are available for man and rat, but scarce for the Göttingen Minipig. The aim of this study was to assess hepatic cytochrome P450 (CYP) protein abundance in the developing Göttingen Minipig by using mass spectrometry. In addition, sex-related differences in CYP protein abundance and correlation of CYP enzyme activity with CYP protein abundance were assessed. The following age groups were included: gestational day (GD) 84-86 ( n = 8), GD 108 ( n = 6), postnatal day (PND) 1 ( n = 8), PND 3 ( n = 8), PND 7 ( n = 8), PND 28 ( n = 8) and adult ( n = 8). Liver microsomes were extracted and protein abundance was compared to that in adult animals. Next, the CYP protein abundance was correlated to CYP enzyme activity in the same biological samples. In general, CYP protein abundance gradually increased during development. However, we observed a stable protein expression over time for CYP4A24 and CYP20A1 and for CYP51A1, a high protein expression during the fetal stages was followed by a decrease during the first month of life and an increase toward adulthood. Sex-related differences were observed for CYP4V2_2a and CYP20A1 at PND 1 with highest expression in females for both isoforms. In the adult samples, sex-related differences were detected for CYP1A1, CYP1A2, CYP2A19, CYP2E1_2, CYP3A22, CYP4V2_2a and CYP4V2_2b with higher values in female compared to male Göttingen Minipigs. The correlation analysis between CYP protein abundance and CYP enzyme activity showed that CYP3A22 protein abundance correlated clearly with the metabolism of midazolam at PND 7. These data are remarkably comparable to human data and provide a valuable step forward in the construction of a neonatal and juvenile Göttingen Minipig PBPK model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Buyssens, De Clerck, Schelstraete, Dhaenens, Deforce, Ayuso, Van Ginneken and Van Cruchten.)
- Published
- 2021
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35. The CD147 Protein Complex Is Involved in Entry of Chikungunya Virus and Related Alphaviruses in Human Cells.
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De Caluwé L, Coppens S, Vereecken K, Daled S, Dhaenens M, Van Ostade X, Deforce D, Ariën KK, and Bartholomeeusen K
- Abstract
Chikungunya virus (CHIKV) is an arbovirus with a global spread and significant public health impact. It is a positive stranded RNA alphavirus belonging to the Togaviridae family. However, many questions about the replication cycle of CHIKV remain unanswered. The entry process of CHIKV is not completely understood nor are the associated virus-receptor interactions fully identified. Here, we designed an affinity purification mass spectrometry coupled approach that allowed the identification of factors that facilitate entry of CHIKV in human cells. The identified entry factors were further validated using CRISPR/Cas9. In HEK293T cells we identified the CD147 protein complex as an entry factor for CHIKV. We further showed the involvement of the CD147 protein complex in the replication cycle of related alphaviruses. Interestingly, CD147 contains similar protein domains as the previously identified alphavirus entry factor MXRA8., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 De Caluwé, Coppens, Vereecken, Daled, Dhaenens, Van Ostade, Deforce, Ariën and Bartholomeeusen.)
- Published
- 2021
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36. Comprehensive histone epigenetics: A mass spectrometry based screening assay to measure epigenetic toxicity.
- Author
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Verhelst S, De Clerck L, Willems S, Van Puyvelde B, Daled S, Deforce D, and Dhaenens M
- Abstract
Evidence of the involvement of epigenetics in pathologies such as cancer, diabetes, and neurodegeneration has increased global interest in epigenetic modifications. For nearly thirty years, it has been known that cancer cells exhibit abnormal DNA methylation patterns. In contrast, the large-scale analysis of histone post-translational modifications (hPTMs) has lagged behind because classically, histone modification analysis has relied on site specific antibody-based techniques. Mass spectrometry (MS) is a technique that holds the promise to picture the histone code comprehensively in a single experiment. Therefore, we developed an MS-based method that is capable of tracking all possible hPTMs in an untargeted approach. In this way, trends in single and combinatorial hPTMs can be reported and enable prediction of the epigenetic toxicity of compounds. Moreover, this method is based on the use of human cells to provide preliminary data, thereby omitting the need to sacrifice laboratory animals. Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort. Still, this novel toxicoepigenetic assay and the data it generates holds great potential for, among others, pharmaceutical industry, food science, clinical diagnostics and, environmental toxicity screening. •There is a growing interest in epigenetic modifications, and more specifically in histone post-translational modifications (hPTMs).•We describe an MS-based workflow that is capable of tracking all possible hPTMs in an untargeted approach that makes use of human cells.•Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)
- Published
- 2020
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37. An Alphaherpesvirus Exploits Antimicrobial β-Defensins To Initiate Respiratory Tract Infection.
- Author
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Van Cleemput J, Poelaert KCK, Laval K, Vanderheijden N, Dhaenens M, Daled S, Boyen F, Pasmans F, and Nauwynck HJ
- Subjects
- Animals, Anti-Infective Agents adverse effects, Cell Line, Epithelial Cells virology, Herpesviridae Infections virology, Herpesvirus 1, Equid, Horse Diseases virology, Horses, Host-Pathogen Interactions physiology, Immune Evasion, Rabbits, Respiratory Tract Infections drug therapy, Viral Envelope Proteins, beta-Defensins adverse effects, Alphaherpesvirinae physiology, Anti-Infective Agents pharmacology, Respiratory Tract Infections immunology, Respiratory Tract Infections virology, beta-Defensins pharmacology
- Abstract
β-Defensins protect the respiratory tract against the myriad of microbial pathogens entering the airways with each breath. However, this potentially hostile environment is known to serve as a portal of entry for herpesviruses. The lack of suitable respiratory model systems has precluded understanding of how herpesvirus virions overcome the abundant mucosal β-defensins during host invasion. We demonstrate how a central alphaherpesvirus, equine herpesvirus type 1 (EHV1), actually exploits β-defensins to invade its host and initiate viral spread. The equine β-defensins (eBDs) eBD1, -2, and -3 were produced and secreted along the upper respiratory tract. Despite the marked antimicrobial action of eBD2 and -3 against many bacterial and viral pathogens, EHV1 virions were resistant to eBDs through the action of the viral glycoprotein M envelope protein. Pretreatment of EHV1 virions with eBD2 and -3 increased the subsequent infection of rabbit kidney (RK13) cells, which was dependent on viral N-linked glycans. eBD2 and -3 also caused the aggregation of EHV1 virions on the cell surface of RK13 cells. Pretreatment of primary equine respiratory epithelial cells (EREC) with eBD1, -2, and -3 resulted in increased EHV1 virion binding to and infection of these cells. EHV1-infected EREC, in turn, showed an increased production of eBD2 and -3 compared to that seen in mock- and influenza virus-infected EREC. In addition, these eBDs attracted leukocytes, which are essential for EHV1 dissemination and which serve as latent infection reservoirs. These novel mechanisms provide new insights into herpesvirus respiratory tract infection and pathogenesis. IMPORTANCE How herpesviruses circumvent mucosal defenses to promote infection of new hosts through the respiratory tract remains unknown due to a lack of host-specific model systems. We used the alphaherpesvirus equine herpesvirus type 1 (EHV1) and equine respiratory tissues to decipher this key event in general alphaherpesvirus pathogenesis. In contrast to several respiratory viruses and bacteria, EHV1 resisted potent antimicrobial equine β-defensins (eBDs) eBD2 and eBD3 by the action of glycoprotein M. Instead, eBD2 and -3 facilitated EHV1 particle aggregation and infection of rabbit kidney (RK13) cells. In addition, virion binding to and subsequent infection of respiratory epithelial cells were increased upon preincubation of these cells with eBD1, -2, and -3. Infected cells synthesized eBD2 and -3, promoting further host cell invasion by EHV1. Finally, eBD1, -2, and -3 recruited leukocytes, which are well-known EHV1 dissemination and latency vessels. The exploitation of host innate defenses by herpesviruses during the early phase of host colonization indicates that highly specialized strategies have developed during host-pathogen coevolution., (Copyright © 2020 Van Cleemput et al.)
- Published
- 2020
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38. Removing the Hidden Data Dependency of DIA with Predicted Spectral Libraries.
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Van Puyvelde B, Willems S, Gabriels R, Daled S, De Clerck L, Vande Casteele S, Staes A, Impens F, Deforce D, Martens L, Degroeve S, and Dhaenens M
- Subjects
- Computational Biology methods, Databases, Protein, HeLa Cells, Humans, Peptide Library, Software, Chromatography, Liquid methods, Data Mining methods, Mass Spectrometry methods, Peptides analysis, Proteome analysis, Proteomics methods
- Abstract
Data-independent acquisition (DIA) generates comprehensive yet complex mass spectrometric data, which imposes the use of data-dependent acquisition (DDA) libraries for deep peptide-centric detection. Here, it is shown that DIA can be redeemed from this dependency by combining predicted fragment intensities and retention times with narrow window DIA. This eliminates variation in library building and omits stochastic sampling, finally making the DIA workflow fully deterministic. Especially for clinical proteomics, this has the potential to facilitate inter-laboratory comparison., (© 2020 The Authors. Proteomics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2020
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39. Deletion of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 in Mouse T Cells Protects Against Development of Autoimmune Arthritis but Leads to Spontaneous Osteoporosis.
- Author
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Gilis E, Gaublomme D, Staal J, Venken K, Dhaenens M, Lambrecht S, Coudenys J, Decruy T, Schryvers N, Driege Y, Dumas E, Demeyer A, De Muynck A, van Hengel J, Van Hoorebeke L, Deforce D, Beyaert R, and Elewaut D
- Subjects
- Animals, Arthritis, Experimental immunology, Arthritis, Rheumatoid immunology, Lymphocyte Activation genetics, Mice, Osteoporosis immunology, Signal Transduction, T-Lymphocytes, Regulatory immunology, Arthritis, Experimental genetics, Arthritis, Rheumatoid genetics, Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein genetics, Osteoporosis genetics, Sequence Deletion immunology
- Abstract
Objective: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) plays a crucial role in innate and adaptive immune signaling by modulating the threshold for activation of immune cells, including Treg cells. Therefore, MALT-1 is regarded to be an interesting therapeutic target in several immune-mediated diseases. The goal of this study was to examine the role of MALT-1 in experimental animal models of rheumatoid arthritis (RA)., Methods: MALT-1 activation was assessed by measuring cleavage of the deubiquitinase CYLD in lymphocytes from mice with collagen-induced arthritis (CIA). Furthermore, the impact of MALT-1 deficiency on arthritis was evaluated in Malt1
KO mice with CIA or with collagen antibody-induced arthritis (CAIA). T cell-specific MALT-1 deficiency was measured in mice with deletion of T cell-specific MALT-1 (Malt1Tcell KO ), and the time-dependent effects of MALT-1 deficiency were assessed in mice with deletion of tamoxifen-inducible T cell-specific MALT-1 (Malt1iTcell KO ). Bone density was determined in MALT-1-deficient mice using micro-computed tomography and femur-bending tests. Reconstitution of Treg cells was performed using adoptive transfer experiments., Results: MALT-1 activation was observed in the lymphocytes of mice with CIA. T cell-specific MALT-1 deletion in the induction phase of arthritis (incidence of arthritis, 25% in control mice versus 0% in Malt1iTcell KO mice; P < 0.05), but not in the effector phase of arthritis, completely protected mice against the development of CIA. Consistent with this finding, MALT-1 deficiency had no impact on CAIA, an effector phase model of RA. Finally, mice with MALT-1 deficiency showed a spontaneous decrease in bone density (mean ± SEM trabecular thickness, 46.3 ± 0.7 μm in control mice versus 40 ± 1.1 μm in Malt1KO mice; P < 0.001), which was linked to the loss of Treg cells in these mice., Conclusion: Overall, these data in murine models of RA highlight MALT-1 as a master regulator of T cell activation, which is relevant to the pathogenesis of autoimmune arthritis. Furthermore, these findings show that MALT-1 deficiency can lead to spontaneous osteoporosis, which is associated with impaired Treg cell numbers., (© 2019, American College of Rheumatology.)- Published
- 2019
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40. Untargeted histone profiling during naive conversion uncovers conserved modification markers between mouse and human.
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De Clerck L, Taelman J, Popovic M, Willems S, Van der Jeught M, Heindryckx B, De Sutter P, Marks H, Deforce D, and Dhaenens M
- Subjects
- Animals, Cell Differentiation physiology, Cells, Cultured, Epigenome physiology, Humans, Mice, Pluripotent Stem Cells metabolism, Signal Transduction physiology, Biomarkers metabolism, Histones metabolism, Human Embryonic Stem Cells metabolism
- Abstract
Recent progress has enabled the conversion of primed human embryonic stem cells (hESCs) to the naive state of pluripotency, resembling the well-characterized naive mouse ESCs (mESCs). However, a thorough histone epigenetic characterization of this conversion process is currently lacking, while its likeness to the mouse model has not been clearly established. Here, we profile the histone epigenome of hESCs during conversion in a time-resolved experimental design, using an untargeted mass spectrometry-based approach. In total, 23 histone post-translational modifications (hPTMs) changed significantly over time. H3K27Me3 was the most prominently increasing marker hPTM in naive hESCs. This is in line with previous reports in mouse, prompting us to compare all the shared hPTM fold changes between mouse and human, revealing a set of conserved hPTM markers for the naive state. Principally, we present the first roadmap of the changing human histone epigenome during the conversion of hESCs from the primed to the naive state. This further revealed similarities with mouse, which hint at a conserved mammalian epigenetic signature of the ground state of pluripotency.
- Published
- 2019
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41. hSWATH: Unlocking SWATH's Full Potential for an Untargeted Histone Perspective.
- Author
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De Clerck L, Willems S, Noberini R, Restellini C, Van Puyvelde B, Daled S, Bonaldi T, Deforce D, and Dhaenens M
- Subjects
- Acetylation drug effects, Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Line, Tumor, Female, Histone Deacetylase Inhibitors pharmacology, Humans, Peptide Library, Reproducibility of Results, Chromatography, Liquid methods, Histones metabolism, Peptides metabolism, Protein Processing, Post-Translational, Proteomics methods, Tandem Mass Spectrometry methods
- Abstract
Mass spectrometry (MS) has become the technique of choice for large-scale analysis of histone post-translational modifications (hPTMs) and their combinatorial patterns, especially in untargeted settings where novel discovery-driven hypotheses are being generated. However, MS-based histone analysis requires a distinct sample preparation, acquisition, and data analysis workflow when compared to traditional MS-based approaches. To this end, sequential window acquisition of all theoretical fragment ion spectra (SWATH) has great potential, as it allows for untargeted accurate identification and quantification of hPTMs. Here, we present a complete SWATH workflow specifically adapted for the untargeted study of histones (hSWATH). We assess its validity on a technical dataset of time-lapse deacetylation of a commercial histone extract using HDAC1, which contains a ground truth, i.e., acetylated substrate peptides reduce in intensity. We successfully apply this workflow in a biological setting and subsequently investigate the differential response to HDAC inhibition in different breast cancer cell lines.
- Published
- 2019
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42. Introducing the YPIC challenge.
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Dhaenens M
- Published
- 2019
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43. Aims & scope.
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Dhaenens M
- Published
- 2019
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44. Editorial: The next generation in (EuPA Open) Proteomics.
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Dhaenens M
- Published
- 2019
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45. Host intestinal biomarker identification in a gut leakage model in broilers.
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De Meyer F, Eeckhaut V, Ducatelle R, Dhaenens M, Daled S, Dedeurwaerder A, De Gussem M, Haesebrouck F, Deforce D, and Van Immerseel F
- Subjects
- Animals, Biomarkers, Inflammation metabolism, Inflammation physiopathology, Intestinal Diseases metabolism, Intestinal Diseases physiopathology, Poultry Diseases metabolism, Proteomics, Chickens, Inflammation veterinary, Intestinal Diseases veterinary, Intestines physiopathology, Poultry Diseases physiopathology
- Abstract
Intestinal health problems are a major issue in the poultry industry. Quantifiable easy-to-measure biomarkers for intestinal health would be of great value to monitor subclinical intestinal entities that cause performance problems and to evaluate control methods for intestinal health. The aim of the study was to identify host protein biomarkers for intestinal inflammation and intestinal barrier damage. Proteomic analysis was conducted on ileal and colonic content samples of broilers under an experimental gut damage and inflammation model. Effects of the challenge treatment resulted in a worse gut condition based on macroscopic gut appearance (p < 0.0001). Also microscopic changes such as shortening of the villi and increased crypt depth (p < 0.0001) as well as higher infiltration of T-lymphocytes (p < 0.0001) were seen in the duodenal tissue of challenged animals. Several candidate proteins associated with inflammation, serum leakage and/or tissue damage were identified with an increased abundance in intestinal content of challenged animals (p < 0.05). Conversely, brush border enzymes were less abundant in intestinal content of challenged animals (p < 0.05). These candidate biomarkers have potential to be used in the field for detection of gut barrier failure in broilers.
- Published
- 2019
- Full Text
- View/download PDF
46. The role of small proteins in Burkholderia cenocepacia J2315 biofilm formation, persistence and intracellular growth.
- Author
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Van Acker H, Crabbé A, Jurėnas D, Ostyn L, Sass A, Daled S, Dhaenens M, Deforce D, Teirlinck E, De Keersmaecker H, Braeckmans K, Van Melderen L, and Coenye T
- Abstract
Burkholderia cenocepacia infections are difficult to treat due to resistance, biofilm formation and persistence. B. cenocepacia strain J2315 has a large multi-replicon genome (8.06 Mb) and the function of a large fraction of (conserved) hypothetical genes remains elusive. The goal of the present study is to elucidate the role of small proteins in B. cenocepacia , focusing on genes smaller than 300 base pairs of which the function is unknown. Almost 10% (572) of the B. cenocepacia J2315 genes are smaller than 300 base pairs and more than half of these are annotated as coding for hypothetical proteins. For 234 of them no similarity could be found with non-hypothetical genes in other bacteria using BLAST. Using available RNA sequencing data obtained from biofilms, a list of 27 highly expressed B. cenocepacia J2315 genes coding for small proteins was compiled. For nine of them expression in biofilms was also confirmed using LC-MS based proteomics and/or expression was confirmed using eGFP translational fusions. Overexpression of two of these genes negatively impacted growth, whereas for four others overexpression led to an increase in biofilm biomass. Overexpression did not have an influence on the MIC for tobramycin, ciprofloxacin or meropenem but for five small protein encoding genes, overexpression had an effect on the number of persister cells in biofilms. While there were no significant differences in adherence to and invasion of A549 epithelial cells between the overexpression mutants and the WT, significant differences were observed in intracellular growth/survival. Finally, the small protein BCAM0271 was identified as an antitoxin belonging to a toxin-antitoxin module. The toxin was found to encode a tRNA acetylase that inhibits translation. In conclusion, our results confirm that small proteins are present in the genome of B. cenocepacia J2315 and indicate that they are involved in various biological processes, including biofilm formation, persistence and intracellular growth., (© 2019 The Authors.)
- Published
- 2019
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- View/download PDF
47. Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics.
- Author
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Crabbé A, Ostyn L, Staelens S, Rigauts C, Risseeuw M, Dhaenens M, Daled S, Van Acker H, Deforce D, Van Calenbergh S, and Coenye T
- Subjects
- Anti-Bacterial Agents pharmacology, Biofilms drug effects, Cells, Cultured, Epithelial Cells drug effects, Epithelial Cells metabolism, Epithelial Cells microbiology, Humans, Lung drug effects, Lung microbiology, Microbial Sensitivity Tests, Pseudomonas Infections metabolism, Pseudomonas Infections microbiology, Culture Media, Conditioned pharmacology, Lung metabolism, Metabolome, Proton-Motive Force drug effects, Pseudomonas Infections drug therapy, Pseudomonas aeruginosa drug effects, Tobramycin pharmacology
- Abstract
Antibiotic susceptibility of bacterial pathogens is typically evaluated using in vitro assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy in vitro and in vivo, with some antibiotics being effective in vitro but not in vivo or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen Pseudomonas aeruginosa, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an in vivo-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against P. aeruginosa, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics in vitro and in vivo may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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48. Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency.
- Author
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van Mierlo G, Dirks RAM, De Clerck L, Brinkman AB, Huth M, Kloet SL, Saksouk N, Kroeze LI, Willems S, Farlik M, Bock C, Jansen JH, Deforce D, Vermeulen M, Déjardin J, Dhaenens M, and Marks H
- Subjects
- Animals, Cell Differentiation, Chromatin genetics, Histones genetics, Histones metabolism, Mice, Mouse Embryonic Stem Cells metabolism, Pluripotent Stem Cells metabolism, Polycomb Repressive Complex 2 genetics, Protein Processing, Post-Translational, Chromatin metabolism, DNA Methylation, Epigenesis, Genetic, Mouse Embryonic Stem Cells cytology, Pluripotent Stem Cells cytology, Polycomb Repressive Complex 2 metabolism, Proteome analysis
- Abstract
The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 are highly abundant in 2i/LIF ESCs, and H3K27me3 is distributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability or transcriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
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49. Plasma Functionalization of Polycaprolactone Nanofibers Changes Protein Interactions with Cells, Resulting in Increased Cell Viability.
- Author
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Asadian M, Dhaenens M, Onyshchenko I, De Waele S, Declercq H, Cools P, Devreese B, Deforce D, Morent R, and De Geyter N
- Subjects
- Cell Line, Cell Survival, Humans, Wettability, Cell Proliferation, Materials Testing, Nanofibers chemistry, Polyesters chemistry
- Abstract
The surface properties of electrospun scaffolds can greatly influence protein adsorption and, thus, strongly dictate cell-material interactions. In this study, we aim to investigate possible correlations between the surface properties of argon, nitrogen, and ammonia and helium plasma-functionalized polycaprolactone (PCL) nanofibers (NFs) and their cellular interactions by examining the protein corona patterns of the plasma-treated NFs as well as the cell membrane proteins involved in cell proliferation. As a result of the performed plasma treatments, PCL NFs morphology was preserved, while wettability was improved profoundly after all treatments because of the incorporation of polar surface groups. Depending on the discharge gas, different types of groups are incorporated, which influenced the resultant cell-material interactions. Argon plasma-functionalized PCL NFs, only enriched by oxygen-containing functional groups, were found to show the best cell-material interactions, followed by N
2 and He/NH3 plasma-treated samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry clearly indicated an increased protein retention compared with non-treated PCL NFs. The nine proteins retained best on plasma-treated NF are important mediators of extracellular matrix interaction, illustrating the importance thereof for cell proliferation and the viability of cells. Finally, 92 proteins that can be used to differentiate how the different plasma treatments are clustered and subjected to a gene ontology study, illustrating the importance of keratinization and extracellular matrix organization.- Published
- 2018
- Full Text
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50. Estimating the Reliability of Low-Abundant Signals and Limited Replicate Measurements through MS2 Peak Area in SWATH.
- Author
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Limonier F, Willems S, Waeterloos G, Sneyers M, Dhaenens M, and Deforce D
- Subjects
- Humans, Reproducibility of Results, Mass Spectrometry methods, Peptide Fragments analysis, Proteins analysis, Proteomics methods, Software
- Abstract
Sequential windows acquisition of all theoretical fragment ions mass spectrometry (SWATH-MS) provides large-scale protein quantification with high accuracy and selectivity. Nevertheless, reliable quantification of low-abundant signals in complex samples remains challenging, as recently illustrated in a multicenter benchmark study of different label-free software tools. Here, the SWATH Replicates Analysis 2.0 template from Sciex is used to highlight that the relationship between the MS2 peak area and the variability can be described by a function. This functional relationship appears to be largely insensitive to variation in samples or acquisition conditions, suggesting a device-intrinsic property. By using a power regression, it is shown that the MS2 peak area can be used to predict the quantification repeatability without relying on replicate injections, thus contributing to high-throughput confident quantification of low-abundant signals with SWATH-MS., (© 2018 The Authors. Proteomics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2018
- Full Text
- View/download PDF
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