PurposeIntraventricular blood flow dynamics are associated with cardiac function. Accurate, noninvasive, and easy assessments of hemodynamic quantities (such as velocity, vortex, and pressure)... Show morePurposeIntraventricular blood flow dynamics are associated with cardiac function. Accurate, noninvasive, and easy assessments of hemodynamic quantities (such as velocity, vortex, and pressure) could be an important addition to the clinical diagnosis and treatment of heart diseases. However, the complex time-varying flow brings many challenges to the existing noninvasive image-based hemodynamic assessments. The development of reliable techniques and analysis tools is essential for the application of hemodynamic biomarkers in clinical practice.MethodsIn this study, a time-resolved particle tracking method, Shake-the-Box, was applied to reconstruct the flow in a realistic left ventricle (LV) silicone model with biological valves. Based on the obtained velocity, 4D pressure field was calculated using a Poisson equation-based pressure solver. Furthermore, flow analysis by proper orthogonal decomposition (POD) of the 4D velocity field has been performed.ResultsAs a result of the Shake-the-Box algorithm, we have extracted: (i) particle positions, (ii) particle tracks, and finally, (iii) 4D velocity fields. From the latter, the temporal evolution of the 3D pressure field during the full cardiac cycle was obtained. The obtained maximal pressure difference extracted along the base-to-apex was about 2.7 mmHg, which is in good agreement with those reported in vivo. The POD analysis results showed a clear picture of different scale of vortices in the pulsatile LV flow, together with their time-varying information and corresponding kinetic energy content. To reconstruct 95% of the kinetic energy of the LV flow, only the first six POD modes would be required, leading to significant data reduction.ConclusionsThis work demonstrated Shake-the-Box is a promising technique to accurately reconstruct the left ventricle flow field in vitro. The good spatial and temporal resolutions of the velocity measurements enabled a 4D reconstruction of the pressure field in the left ventricle. The application of POD analysis showed its potential in reducing the complexity of the high-resolution left ventricle flow measurements. For future work, image analysis, multi-modality flow assessments, and the development of new flow-derived biomarkers can benefit from fast and data-reducing POD analysis. Show less
Madern, J.M.; Voorneveld, J.; Rack, J.G.M.; Kistemaker, H.A.V.; Ahel, I.; Marel, G.A. van der; ... ; Filippov, D.V. 2023
Adenosine diphosphate (ADP) ribosylation is an importantpost-translationalmodification (PTM) that plays a role in a wide variety of cellularprocesses. To study the enzymes responsible for the... Show moreAdenosine diphosphate (ADP) ribosylation is an importantpost-translationalmodification (PTM) that plays a role in a wide variety of cellularprocesses. To study the enzymes responsible for the establishment,recognition, and removal of this PTM, stable analogues are invaluabletools. We describe the design and synthesis of a 4-thioribosyl APRrpeptide that has been assembled by solid phase synthesis. The key4-thioribosyl serine building block was obtained in a stereoselectiveglycosylation reaction using an alkynylbenzoate 4-thioribosyl donor. Show less
The modification of substrates with ADP-ribose (ADPr) is im-portant in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it... Show moreThe modification of substrates with ADP-ribose (ADPr) is im-portant in, for example, antiviral immunity and cancer. Recently, several reagents were developed to detect ADP-ribosylation; however, it is unknown whether they recognise ADPr, specific amino acid-ADPr linkages, or ADPr with the surrounding protein backbone. We first optimised methods to prepare extracts con-taining ADPr-proteins and observe that depending on the amino acid modified, the modification is heatlabile. We tested the re-activity of available reagents with diverse ADP-ribosylated pro-tein and RNA substrates and observed that not all reagents are equally suited for all substrates. Next, we determined cross -reactivity with adenylylated RNA, AMPylated proteins, and me-tabolites, including NADH, which are detected by some reagents. Lastly, we analysed ADP-ribosylation using confocal microscopy, where depending on the fixation method, either mitochondrion, nucleus, or nucleolus is stained. This study allows future work dissecting the function of ADP-ribosylation in cells, both on protein and on RNA substrates, as we optimised sample prepa-ration methods and have defined the reagents suitable for specific methods and substrates. Show less
Voorneveld, J.; Kloet, M.S.; Wijngaarden, S.; Kim, R.Q.; Moutsiopoulou, A.; Verdegaal, M.; ... ; Noort, G.J.V. van 2022
We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method... Show moreWe describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an alpha-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5 '- hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes. Show less
Voorneveld, J.; Kloet, M.S.; Wijngaarden, S.; Kim, R.Q.; Moutsiopoulou, A.; Verdegaal, M.; ... ; Noort, G.J.V. van 2022
We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method... Show moreWe describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an alpha-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5 '- hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes. Show less
This thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route... Show moreThis thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route towards ADP-ribosylated nucleic acids. Furthermore, two photoaffinity probes for PARP1 have been developed and assessed in living cells and two activity based probes have been synthesized, designed for CD38. Show less
Mutluer, F.O.; Velde, N. van der; Voorneveld, J.; Bosch, J.G.; Roos-Hesselink, J.W.; Geest, R.J. van der; ... ; Bosch, A. van den 2021
Background The aim of this systematic review was to evaluate current inter-modality agreement of noninvasive clinical intraventricular flow (IVF) assessment with 3 emerging imaging modalities:... Show moreBackground The aim of this systematic review was to evaluate current inter-modality agreement of noninvasive clinical intraventricular flow (IVF) assessment with 3 emerging imaging modalities: echocardiographic particle image velocimetry (EPIV), vector flow mapping (VFM), and 4-dimensional flow cardiovascular magnetic resonance imaging (4D flow CMR). Methods We performed a systematic literature review in the databases EMBASE, Medline OVID and Cochrane Central for identification of studies evaluating left ventricular (LV) flow patterns using one of these flow visualization modalities. Of the 2224 initially retrieved records, 10 EPIV, 23 VFM, and 25 4D flow CMR studies were included in the final analysis. Results Vortex parameters were more extensively studied with EPIV, while LV energetics and LV transport mechanics were mainly studied with 4D flow CMR, and LV energy loss and vortex circulation were implemented by VFM studies. Pooled normative values are provided for these parameters. The meta- analysis for the values of two vortex morphology parameters, vortex length and vortex depth, failed to reveal a significant change between heart failure patients and healthy controls. Conclusion Agreement between the different modalities studying intraventricular flow is low and different methods of measurement and reporting were used among studies. A multimodality framework with a standardized set of flow parameters is necessary for implementation of noninvasive flow visualization in daily clinical practice. The full potential of noninvasive flow visualization in addition to diagnostics could also include guiding medical or interventional treatment. Show less
Purpose To assess errors associated with EPI-accelerated intracardiac 4D flow MRI (4DEPI) with EPI factor 5, compared with non-EPI gradient echo (4DGRE). Methods Three 3T MRI experiments were... Show morePurpose To assess errors associated with EPI-accelerated intracardiac 4D flow MRI (4DEPI) with EPI factor 5, compared with non-EPI gradient echo (4DGRE). Methods Three 3T MRI experiments were performed comparing 4DEPI to 4DGRE: steady flow through straight tubes, pulsatile flow in a left-ventricle phantom, and intracardiac flow in 10 healthy volunteers. For each experiment, 4DEPI was repeated with readout and blip phase-encoding gradient in different orientations, parallel or perpendicular to the flow direction. In vitro flow rates were compared with timed volumetric collection. In the left-ventricle phantom and in vivo, voxel-based speed and spatio-temporal median speed were compared between sequences, as well as mitral and aortic transvalvular net forward volume. Results In steady-flow phantoms, the flow rate error was largest (12%) for high velocity (>2 m/s) with 4DEPI readout gradient parallel to the flow. Voxel-based speed and median speed in the left-ventricle phantom were <= 5.5% different between sequences. In vivo, mean net forward volume inconsistency was largest (6.4 +/- 8.5%) for 4DEPI with nonblip phase-encoding gradient parallel to the main flow. The difference in median speed for 4DEPI versus 4DGRE was largest (9%) when the 4DEPI readout gradient was parallel to the flow. Conclusions Velocity and flow rate are inaccurate for 4DEPI with EPI factor 5 when flow is parallel to the readout or blip phase-encoding gradient. However, mean differences in flow rate, voxel-based speed, and spatio-temporal median speed were acceptable (<= 10%) when comparing 4DEPI to 4DGRE for intracardiac flow in healthy volunteers. Show less
Liu, Q.; Knobloch, G.; Voorneveld, J.; Meeuwenoord, N.J.; Overkleeft, H.S.; Marel, G.A. van der; ... ; Filippov, D.V. 2021
ADP-ribosylation is a pivotal post-translational modification that mediates various important cellular processes producing negatively charged biopolymer, poly (ADP-ribose), the functions of which... Show moreADP-ribosylation is a pivotal post-translational modification that mediates various important cellular processes producing negatively charged biopolymer, poly (ADP-ribose), the functions of which need further elucidation. Toward this end, the availability of well-defined ADP-ribose (ADPr) oligomers in sufficient quantities is a necessity. In this work, we demonstrate the chemical synthesis of linear ADPr oligomers of defined, increasing length using a modified solid phase synthesis method. An advanced phosphoramidite building block temporarily protected with the base sensitive Fm-group was designed and implemented in the repeating pyrophosphate formation via a P(v)-P(iii) coupling procedure on Tentagel solid support. Linear ADPr oligomers up to a pentamer were successfully synthesized and their affinity for the poly-(ADP-ribose)-binding macrodomain of the human oncogenic helicase and chromatin remodeling enzyme ALC1 was determined. Our data reveal a length-dependent binding manner of the nucleic acid, with larger ADPr oligomers exhibiting higher binding enthalpies for ALC1, illustrating how the activity of this molecular machine is gated by PAR. Show less
Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked... Show morePoly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry. Show less
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr... Show moreADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase. Show less
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr... Show moreADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase. Show less
Voorneveld, J.; Florea, B.I.; Mendowicz, R.J.; Veer, M.S. van der; Gagestein, B.; Stelt, M. van der; ... ; Filippov, D.V. 2020
The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes poly adenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair... Show moreThe poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes poly adenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to DNA damage sites. Inhibition of PARP activity by olaparib can cause cell death which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work we present the design, synthesis and biological evaluation of photo-activatable affinity probes inspired by the olaparib molecule which are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS-PAGE, western blotting and label-free LC-MS/MS quantification analysis show that the probes target the PARP-1 protein and are selectively outcompeted by olaparib suggesting binding in the same enzymatic pocket. Show less
Mechelen, J. van; Voorneveld, J.; Overkleeft, H.S.; Filippov, D.V.; Marel, G.A. van der; Codée, J.D.C. 2020
2,4-Diamino-2,4,6-trideoxyglucose (bacillosamine) is a monosaccharide found in many pathogenic bacteria, variation in the functionalities appended to the amino groups occurs depending on the... Show more2,4-Diamino-2,4,6-trideoxyglucose (bacillosamine) is a monosaccharide found in many pathogenic bacteria, variation in the functionalities appended to the amino groups occurs depending on the species the sugar is derived from. We here report the first synthesis of bacillosamine synthons that allow for the incorporation of two different functionalities at the C-2-N-acetyl and C-4-amines. We have developed chemistry to assemble a set of conjugation ready Neisseria meningitidis C-2-N-acetyl bacillosamine saccharides, carrying either an acetyl or (R)- or (S)-glyceroyl at the C-4 amine. The glyceroyl bacillosamines have been further extended at the C-3-OH with an α-d-galactopyranose to provide structures that occur as post-translational modifications of N. meningitidis PilE proteins, which make up the bacterial pili. Show less
ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the... Show moreADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2'-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates. Show less
