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Laura-Oana Albulescu; Chunfang Xie; Stuart Ainsworth; Jaffer Alsolaiss; Edouard Crittenden; Charlotte A. Dawson; Rowan Softley; Keirah E. Bartlett; Robert A. Harrison; Jeroen Kool; Nicholas R. Casewell. Author Correction: A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite. Nature Communications 2021, 12, 1 -1.
AMA StyleLaura-Oana Albulescu, Chunfang Xie, Stuart Ainsworth, Jaffer Alsolaiss, Edouard Crittenden, Charlotte A. Dawson, Rowan Softley, Keirah E. Bartlett, Robert A. Harrison, Jeroen Kool, Nicholas R. Casewell. Author Correction: A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite. Nature Communications. 2021; 12 (1):1-1.
Chicago/Turabian StyleLaura-Oana Albulescu; Chunfang Xie; Stuart Ainsworth; Jaffer Alsolaiss; Edouard Crittenden; Charlotte A. Dawson; Rowan Softley; Keirah E. Bartlett; Robert A. Harrison; Jeroen Kool; Nicholas R. Casewell. 2021. "Author Correction: A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite." Nature Communications 12, no. 1: 1-1.
Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA2s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA2 proteins. We, therefore, conclude that PLA2s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.
Taline Kazandjian; Arif Arrahman; Kristina Still; Govert Somsen; Freek Vonk; Nicholas Casewell; Mark Wilkinson; Jeroen Kool. Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib. Toxins 2021, 13, 302 .
AMA StyleTaline Kazandjian, Arif Arrahman, Kristina Still, Govert Somsen, Freek Vonk, Nicholas Casewell, Mark Wilkinson, Jeroen Kool. Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib. Toxins. 2021; 13 (5):302.
Chicago/Turabian StyleTaline Kazandjian; Arif Arrahman; Kristina Still; Govert Somsen; Freek Vonk; Nicholas Casewell; Mark Wilkinson; Jeroen Kool. 2021. "Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib." Toxins 13, no. 5: 302.
This study presents, for the first time, the successful application of analyzing a whole gas chromatography (GC) chromatogram by nuclear magnetic resonance (NMR) spectroscopy using a continuous repeatable and stable (n = 280) high-resolution (HR) GC fractionation platform with a 96-well plate. Typically with GC– or liquid chromatography–mass spectrometry analysis, (isomer) standards and/or additional NMR analysis are needed to confirm the identification and/or structure of the analyte of interest. In the case of complex substances (e.g., UVCBs), isomer standards are often unavailable and NMR spectra too complex to achieve this. This proof of concept study shows that a HR GC fractionation collection platform was successfully applied to separate, purify, and enrich isomers in complex substances from a whole GC chromatogram, which would facilitate NMR analysis. As a model substance, a chlorinated paraffin (CP) mixture (>8,000 isomers) was chosen. NMR spectra were obtained from all 96 collected fractions, which provides important information for unravelling their full structure. As a proof of concept, a spectral interpretation of a few NMR spectra was made to assign sub-structures. More research is ongoing for the full characterization of CP isomers using multivariate statistical analysis. For the first time, up to only a few CP isomers per fraction were isolated from a highly complex mixture. These may be further purified and certified as standards, which are urgently needed, and can also be used for persistency, bioaccumulation, or toxicity studies.
Louise M. van Mourik; Elwin Janssen; Robin Breeuwer; Willem Jonker; Jacco Koekkoek; Arif Arrahman; Jeroen Kool; Pim E. G. Leonards. Combining High-Resolution Gas Chromatographic Continuous Fraction Collection with Nuclear Magnetic Resonance Spectroscopy: Possibilities of Analyzing a Whole GC Chromatogram. Analytical Chemistry 2021, 93, 6158 -6168.
AMA StyleLouise M. van Mourik, Elwin Janssen, Robin Breeuwer, Willem Jonker, Jacco Koekkoek, Arif Arrahman, Jeroen Kool, Pim E. G. Leonards. Combining High-Resolution Gas Chromatographic Continuous Fraction Collection with Nuclear Magnetic Resonance Spectroscopy: Possibilities of Analyzing a Whole GC Chromatogram. Analytical Chemistry. 2021; 93 (15):6158-6168.
Chicago/Turabian StyleLouise M. van Mourik; Elwin Janssen; Robin Breeuwer; Willem Jonker; Jacco Koekkoek; Arif Arrahman; Jeroen Kool; Pim E. G. Leonards. 2021. "Combining High-Resolution Gas Chromatographic Continuous Fraction Collection with Nuclear Magnetic Resonance Spectroscopy: Possibilities of Analyzing a Whole GC Chromatogram." Analytical Chemistry 93, no. 15: 6158-6168.
Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of different pathologies, including haemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, haemolytic activity can greatly debilitate victims and contributes to systemic haemotoxicity. Current assays designed for studying haemolytic activity are not suitable for rapid screening of a large number of toxic compounds. Consequently, in this study, a high-throughput haemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving haemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for haemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The haemolytic venom toxins identified were mainly phospholipases A2s and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralisation of venom cytotoxins.
Chunfang Xie; Matyas A. Bittenbinder; Julien Slagboom; Arif Arrahman; Sven Bruijns; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Juan J. García-Vallejo; Jeroen Kool. Erythrocyte haemotoxicity profiling of snake venom toxins after nanofractionation. Journal of Chromatography B 2021, 1176, 122586 .
AMA StyleChunfang Xie, Matyas A. Bittenbinder, Julien Slagboom, Arif Arrahman, Sven Bruijns, Govert W. Somsen, Freek J. Vonk, Nicholas R. Casewell, Juan J. García-Vallejo, Jeroen Kool. Erythrocyte haemotoxicity profiling of snake venom toxins after nanofractionation. Journal of Chromatography B. 2021; 1176 ():122586.
Chicago/Turabian StyleChunfang Xie; Matyas A. Bittenbinder; Julien Slagboom; Arif Arrahman; Sven Bruijns; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Juan J. García-Vallejo; Jeroen Kool. 2021. "Erythrocyte haemotoxicity profiling of snake venom toxins after nanofractionation." Journal of Chromatography B 1176, no. : 122586.
Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite.
Laura-Oana Albulescu; Chunfang Xie; Stuart Ainsworth; Jaffer Alsolaiss; Edouard Crittenden; Charlotte A. Dawson; Rowan Softley; Keirah E. Bartlett; Robert A. Harrison; Jeroen Kool; Nicholas R. Casewell. A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite. Nature Communications 2020, 11, 1 -14.
AMA StyleLaura-Oana Albulescu, Chunfang Xie, Stuart Ainsworth, Jaffer Alsolaiss, Edouard Crittenden, Charlotte A. Dawson, Rowan Softley, Keirah E. Bartlett, Robert A. Harrison, Jeroen Kool, Nicholas R. Casewell. A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite. Nature Communications. 2020; 11 (1):1-14.
Chicago/Turabian StyleLaura-Oana Albulescu; Chunfang Xie; Stuart Ainsworth; Jaffer Alsolaiss; Edouard Crittenden; Charlotte A. Dawson; Rowan Softley; Keirah E. Bartlett; Robert A. Harrison; Jeroen Kool; Nicholas R. Casewell. 2020. "A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite." Nature Communications 11, no. 1: 1-14.
Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for Gs, Gq, and Gi signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds.
Teemu Suutari; Sabrina N. Rahman; Henry F. Vischer; Dick Van Iperen; Arto Merivaara; Marjo Yliperttula; Rob Leurs; Jeroen Kool; Tapani Viitala. Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways. Analytical Chemistry 2020, 92, 14509 -14516.
AMA StyleTeemu Suutari, Sabrina N. Rahman, Henry F. Vischer, Dick Van Iperen, Arto Merivaara, Marjo Yliperttula, Rob Leurs, Jeroen Kool, Tapani Viitala. Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways. Analytical Chemistry. 2020; 92 (21):14509-14516.
Chicago/Turabian StyleTeemu Suutari; Sabrina N. Rahman; Henry F. Vischer; Dick Van Iperen; Arto Merivaara; Marjo Yliperttula; Rob Leurs; Jeroen Kool; Tapani Viitala. 2020. "Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways." Analytical Chemistry 92, no. 21: 14509-14516.
Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A2 inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper, Bothrops jararaca, Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A2, while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments.
Chunfang Xie; Julien Slagboom; Laura-Oana Albulescu; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Jeroen Kool. Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms. Acta Pharmaceutica Sinica B 2020, 10, 1835 -1845.
AMA StyleChunfang Xie, Julien Slagboom, Laura-Oana Albulescu, Govert W. Somsen, Freek J. Vonk, Nicholas R. Casewell, Jeroen Kool. Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms. Acta Pharmaceutica Sinica B. 2020; 10 (10):1835-1845.
Chicago/Turabian StyleChunfang Xie; Julien Slagboom; Laura-Oana Albulescu; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Jeroen Kool. 2020. "Neutralising effects of small molecule toxin inhibitors on nanofractionated coagulopathic Crotalinae snake venoms." Acta Pharmaceutica Sinica B 10, no. 10: 1835-1845.
Animal-derived antivenoms are the only specific therapies currently available for the treatment of snake envenoming, but these products have a number of limitations associated with their efficacy, safety and affordability for use in tropical snakebite victims. Small molecule drugs and drug candidates are regarded as promising alternatives for filling the critical therapeutic gap between snake envenoming and effective treatment. In this study, by using an advanced analytical technique that combines chromatography, mass spectrometry and bioassaying, we investigated the effect of several small molecule inhibitors that target phospholipase A2 (varespladib) and snake venom metalloproteinase (marimastat, dimercaprol and DMPS) toxin families on inhibiting the activities of coagulopathic toxins found in Viperinae snake venoms. The venoms of Echis carinatus, Echis ocellatus, Daboia russelii and Bitis arietans, which are known for their potent haemotoxicities, were fractionated in high resolution onto 384-well plates using liquid chromatography followed by coagulopathic bioassaying of the obtained fractions. Bioassay activities were correlated to parallel recorded mass spectrometric and proteomics data to assign the venom toxins responsible for coagulopathic activity and assess which of these toxins could be neutralized by the inhibitors under investigation. Our results showed that the phospholipase A2-inhibitor varespladib neutralized the vast majority of anticoagulation activities found across all of the tested snake venoms. Of the snake venom metalloproteinase inhibitors, marimastat demonstrated impressive neutralization of the procoagulation activities detected in all of the tested venoms, whereas dimercaprol and DMPS could only partially neutralize these activities at the doses tested. Our results provide additional support for the concept that combinations of small molecules, particularly the combination of varespladib with marimastat, serve as a drug-repurposing opportunity to develop new broad-spectrum inhibitor-based therapies for snakebite envenoming.
Chunfang Xie; Laura-Oana Albulescu; Mátyás A. Bittenbinder; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Jeroen Kool. Neutralizing Effects of Small Molecule Inhibitors and Metal Chelators on Coagulopathic Viperinae Snake Venom Toxins. Biomedicines 2020, 8, 297 .
AMA StyleChunfang Xie, Laura-Oana Albulescu, Mátyás A. Bittenbinder, Govert W. Somsen, Freek J. Vonk, Nicholas R. Casewell, Jeroen Kool. Neutralizing Effects of Small Molecule Inhibitors and Metal Chelators on Coagulopathic Viperinae Snake Venom Toxins. Biomedicines. 2020; 8 (9):297.
Chicago/Turabian StyleChunfang Xie; Laura-Oana Albulescu; Mátyás A. Bittenbinder; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Jeroen Kool. 2020. "Neutralizing Effects of Small Molecule Inhibitors and Metal Chelators on Coagulopathic Viperinae Snake Venom Toxins." Biomedicines 8, no. 9: 297.
Phospholipase A2 (PLA2) enzymes are important toxins found in many snake venoms, and they can exhibit a variety of toxic activities including causing hemolysis and/or anticoagulation. In this study, the inhibiting effects of the small molecule PLA2 inhibitor varespladib on snake venom PLA2s was investigated by nanofractionation analytics, which combined chromatography, mass spectrometry (MS), and bioassays. The venoms of the medically important snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus, Echis ocellatus, and Oxyuranus scutellatus were separated by liquid chromatography (LC) followed by nanofractionation and interrogation of the fractions by a coagulation assay and a PLA2 assay. Next, we assessed the ability of varespladib to inhibit the activity of enzymatic PLA2s and the coagulopathic toxicities induced by fractionated snake venom toxins, and identified these bioactive venom toxins and those inhibited by varespladib by using parallel recorded LC-MS data and proteomics analysis. We demonstrated here that varespladib was not only capable of inhibiting the PLA2 activities of hemotoxic snake venoms, but can also effectively neutralize the coagulopathic toxicities (most profoundly anticoagulation) induced by venom toxins. While varespladib effectively inhibited PLA2 toxins responsible for anticoagulant effects, we also found some evidence that this inhibitory molecule can partially abrogate procoagulant venom effects caused by different toxin families. These findings further emphasize the potential clinical utility of varespladib in mitigating the toxic effects of certain snakebites.
Chunfang Xie; Laura-Oana Albulescu; Kristina B. M. Still; Julien Slagboom; Yumei Zhao; Zhengjin Jiang; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Jeroen Kool. Varespladib Inhibits the Phospholipase A2 and Coagulopathic Activities of Venom Components from Hemotoxic Snakes. Biomedicines 2020, 8, 165 .
AMA StyleChunfang Xie, Laura-Oana Albulescu, Kristina B. M. Still, Julien Slagboom, Yumei Zhao, Zhengjin Jiang, Govert W. Somsen, Freek J. Vonk, Nicholas R. Casewell, Jeroen Kool. Varespladib Inhibits the Phospholipase A2 and Coagulopathic Activities of Venom Components from Hemotoxic Snakes. Biomedicines. 2020; 8 (6):165.
Chicago/Turabian StyleChunfang Xie; Laura-Oana Albulescu; Kristina B. M. Still; Julien Slagboom; Yumei Zhao; Zhengjin Jiang; Govert W. Somsen; Freek J. Vonk; Nicholas R. Casewell; Jeroen Kool. 2020. "Varespladib Inhibits the Phospholipase A2 and Coagulopathic Activities of Venom Components from Hemotoxic Snakes." Biomedicines 8, no. 6: 165.
Animal-derived antivenoms are the only specific therapies currently available for the treatment of snake envenoming, but these products have a number of limitations associated with their efficacy, safety and affordability for use in tropical snakebite victims. Small molecule drugs and drug candidates are regarded as promising alternatives for filling the critical therapeutic gap between snake envenoming and effective treatment. In this study, by using an advanced analytical technique that combines chromatography, mass spectrometry and bioassaying, we investigated the effect of several small molecule inhibitors that target phospholipase A2 (varespladib) and snake venom metalloproteinase (marimastat, dimercaprol and DMPS) toxin families on inhibiting the activities of coagulopathic toxins found in Viperinae snake venoms. The venoms of Echis carinatus, Echis ocellatus, Daboia russelii and Bitis arietans, which are known for their potent coagulopathic toxicities, were fractionated in high resolution onto 384-well plates using liquid chromatography followed by coagulopathic bioassaying of the obtained fractions. Bioassay activities were correlated to parallel recorded mass spectrometric and proteomics data to assign the venom toxins responsible for coagulopathic activity and assess which of these toxins could be neutralized by the inhibitors under investigation. Our results showed that the phospholipase A2-inhibitor varespladib neutralized the vast majority of anticoagulation activities found across all of the tested snake venoms. Of the snake venom metalloproteinase inhibitors, marimastat demonstrated impressive neutralization of the procoagulation activities detected in all of the tested venoms, whereas dimercaprol and DMPS could only partially neutralize these activities at the doses tested. Our results provide additional support for the concept that combination of small molecules, particularly the combination of varespladib with marimastat, serve as a drug-repurposing opportunity to develop new broad-spectrum inhibitor-based therapies for snakebite envenoming.
Chunfang Xie; Laura Albulescu; Matyas A Bittenbinder; Govert Somsen; Freek Vonk; Nicholas R Casewell; Jeroen Kool. Neutralizing effects of small molecule inhibitors and metal chelators on coagulopathic Viperinae snake venom toxins. 2020, 1 .
AMA StyleChunfang Xie, Laura Albulescu, Matyas A Bittenbinder, Govert Somsen, Freek Vonk, Nicholas R Casewell, Jeroen Kool. Neutralizing effects of small molecule inhibitors and metal chelators on coagulopathic Viperinae snake venom toxins. . 2020; ():1.
Chicago/Turabian StyleChunfang Xie; Laura Albulescu; Matyas A Bittenbinder; Govert Somsen; Freek Vonk; Nicholas R Casewell; Jeroen Kool. 2020. "Neutralizing effects of small molecule inhibitors and metal chelators on coagulopathic Viperinae snake venom toxins." , no. : 1.
Many organisms, ranging from plants to mammals, contain phospholipase A2 enzymes (PLA2s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA2s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA2 activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA2s. The methodology was demonstrated for assessment of PLA2 activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii.
Kristina B.M. Still; Julien Slagboom; Sarah Kidwai; Chunfang Xie; Yumei Zhao; Bastiaan Eisses; Zhengjin Jiang; Freek J. Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. Development of high-throughput screening assays for profiling snake venom phospholipase A2 activity after chromatographic fractionation. Toxicon 2020, 184, 28 -38.
AMA StyleKristina B.M. Still, Julien Slagboom, Sarah Kidwai, Chunfang Xie, Yumei Zhao, Bastiaan Eisses, Zhengjin Jiang, Freek J. Vonk, Govert W. Somsen, Nicholas R. Casewell, Jeroen Kool. Development of high-throughput screening assays for profiling snake venom phospholipase A2 activity after chromatographic fractionation. Toxicon. 2020; 184 ():28-38.
Chicago/Turabian StyleKristina B.M. Still; Julien Slagboom; Sarah Kidwai; Chunfang Xie; Yumei Zhao; Bastiaan Eisses; Zhengjin Jiang; Freek J. Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. 2020. "Development of high-throughput screening assays for profiling snake venom phospholipase A2 activity after chromatographic fractionation." Toxicon 184, no. : 28-38.
Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and fiscal snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using multiple in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings strongly support the translation of combinations of safe and affordable enzyme inhibitors as novel broad-spectrum therapeutics for snakebite.
Laura-Oana Albulescu; Chunfang Xie; Stuart Ainsworth; Jaffer Alsolaiss; Edouard Crittenden; Charlotte A. Dawson; Rowan Softley; Keirah E. Bartlett; Robert A. Harrison; Jeroen Kool; Nicholas R. Casewell. A therapeutic combination of two small molecule toxin inhibitors provides pancontinental preclinical efficacy against viper snakebite. 2020, 1 .
AMA StyleLaura-Oana Albulescu, Chunfang Xie, Stuart Ainsworth, Jaffer Alsolaiss, Edouard Crittenden, Charlotte A. Dawson, Rowan Softley, Keirah E. Bartlett, Robert A. Harrison, Jeroen Kool, Nicholas R. Casewell. A therapeutic combination of two small molecule toxin inhibitors provides pancontinental preclinical efficacy against viper snakebite. . 2020; ():1.
Chicago/Turabian StyleLaura-Oana Albulescu; Chunfang Xie; Stuart Ainsworth; Jaffer Alsolaiss; Edouard Crittenden; Charlotte A. Dawson; Rowan Softley; Keirah E. Bartlett; Robert A. Harrison; Jeroen Kool; Nicholas R. Casewell. 2020. "A therapeutic combination of two small molecule toxin inhibitors provides pancontinental preclinical efficacy against viper snakebite." , no. : 1.
Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors. Snakebite is a neglected tropical disease that results in more than 100,000 deaths every year. Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to suffering life-threating hemorrhage. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood coagulation cascade and based on the initial results and the medical relevance of the species, seven venoms were selected for in-depth analysis of the responsible toxins using advanced identification techniques. Our findings reveal a number of anticoagulant toxins that have not yet been reported before as such. The methodology described herein not only enables the identification of both known and unknown toxins that cause impairment of the blood coagulation, but offers a throughput platform to effectively screen for inhibitory molecules relevant for the development of next generation snakebite treatments.
Julien Slagboom; Marija Mladić; Chunfang Xie; Taline D. Kazandjian; Freek Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches. PLOS Neglected Tropical Diseases 2020, 14, e0007802 .
AMA StyleJulien Slagboom, Marija Mladić, Chunfang Xie, Taline D. Kazandjian, Freek Vonk, Govert W. Somsen, Nicholas R. Casewell, Jeroen Kool. High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches. PLOS Neglected Tropical Diseases. 2020; 14 (4):e0007802.
Chicago/Turabian StyleJulien Slagboom; Marija Mladić; Chunfang Xie; Taline D. Kazandjian; Freek Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. 2020. "High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches." PLOS Neglected Tropical Diseases 14, no. 4: e0007802.
Snakebites cause upwards of 1.8 million envenomings, 138,000 deaths and 500,000 cases of long term morbidity each year. Viper snake venoms (family Viperidae) generally contain a high proportion of proteases which can cause devastating effects such as hemorrhage, coagulopathy, edema, necrosis, and severe pain, in envenomed victims. In this study, analytical techniques were combined with enzymatic assays to develop a novel method for the detection of snake venom protease activity by using rhodamine-110-peptide substrate. In the so called at-line nanofractionation set up, crude venoms were first separated with reversed phase liquid chromatography, after which fractions were collected onto 384-well plates. Protease activity assays were then performed in the 384-well plates and bioassay chromatograms were constructed revealing protease activity. Parallel obtained UV absorbance, MS and proteomics data from a previous study facilitated toxin identification. The application of the rhodamine-110-peptide substrate assay showed significantly greater sensitivity compared to prior assays using casein-FITC as the substrate. Moreover, cross referencing UV and MS data and resulted in the detection of a number of tentative proteases suspected to exhibit protease activity, including snake venom serine proteases from Calloselasma rhodostoma and Daboia russelli venom and a snake venom metalloproteinase from the venom of Echis ocellatus. Our data demonstrate that his methodology can be a useful tool for selectively identifying snake venom proteases, and can be applied to provide a better understanding of protease-induced pathologies and the development of novel therapeutics for treating snakebite.
Coleen Neumann; Julien Slagboom; Govert W. Somsen; Freek Vonk; Nicholas R. Casewell; Carmen L. Cardoso; Jeroen Kool. Development of a generic high-throughput screening assay for profiling snake venom protease activity after high-resolution chromatographic fractionation. Toxicon 2020, 178, 61 -68.
AMA StyleColeen Neumann, Julien Slagboom, Govert W. Somsen, Freek Vonk, Nicholas R. Casewell, Carmen L. Cardoso, Jeroen Kool. Development of a generic high-throughput screening assay for profiling snake venom protease activity after high-resolution chromatographic fractionation. Toxicon. 2020; 178 ():61-68.
Chicago/Turabian StyleColeen Neumann; Julien Slagboom; Govert W. Somsen; Freek Vonk; Nicholas R. Casewell; Carmen L. Cardoso; Jeroen Kool. 2020. "Development of a generic high-throughput screening assay for profiling snake venom protease activity after high-resolution chromatographic fractionation." Toxicon 178, no. : 61-68.
Many organisms, ranging from plants to mammals, contain phospholipase A2enzymes (PLA2s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA2s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA2activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA2s. The methodology was demonstrated for assessment of PLA2activity profiles of venoms from the snake speciesBothrops asper,Echis carinatus,Echis coloratus, Echis ocellatus,Oxyuranus scutellatusandDaboia russelii russelii.
Kristina B.M. Still; Julien R Slagboom; Sarah Kidwai; Chunfang Xie; Bastiaan Eisses; Freek J. Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. Development of high-throughput screening assays for profiling snake venom Phospholipase A2activity after high-resolution chromatographic fractionation. 2020, 1 .
AMA StyleKristina B.M. Still, Julien R Slagboom, Sarah Kidwai, Chunfang Xie, Bastiaan Eisses, Freek J. Vonk, Govert W. Somsen, Nicholas R. Casewell, Jeroen Kool. Development of high-throughput screening assays for profiling snake venom Phospholipase A2activity after high-resolution chromatographic fractionation. . 2020; ():1.
Chicago/Turabian StyleKristina B.M. Still; Julien R Slagboom; Sarah Kidwai; Chunfang Xie; Bastiaan Eisses; Freek J. Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. 2020. "Development of high-throughput screening assays for profiling snake venom Phospholipase A2activity after high-resolution chromatographic fractionation." , no. : 1.
Venomous snakebite is one of the world’s most lethal neglected tropical diseases. Animal-derived antivenoms are the only standardized specific therapies currently available for treating snakebite envenoming, but due to venom variation, often this treatment is not effective in counteracting all clinical symptoms caused by the multitude of injected toxins. In this study, the coagulopathic toxicities of venoms from the medically relevant snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus and Echis ocellatus were assessed. The venoms were separated by liquid chromatography (LC) followed by nanofractionation and parallel mass spectrometry (MS). A recently developed high-throughput coagulation assay was employed to assess both the pro- and anticoagulant activity of separated venom toxins. The neutralization capacity of antivenoms on separated venom components was assessed and the coagulopathic venom peptides and enzymes that were either neutralized or remained active in the presence of antivenom were identified by correlating bioassay results with the MS data and with off-line generated proteomics data. The results showed that most snake venoms analyzed contained both procoagulants and anticoagulants. Most anticoagulants were identified as phospholipases A2s (PLA2s) and most procoagulants correlated with snake venom metalloproteinases (SVMPs) and serine proteases (SVSPs). This information can be used to better understand antivenom neutralization and can aid in the development of next-generation antivenom treatments.
Chunfang Xie; Julien Slagboom; Laura-Oana Albulescu; Ben Bruyneel; Kristina B. M. Still; Freek J. Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. Antivenom Neutralization of Coagulopathic Snake Venom Toxins Assessed by Bioactivity Profiling Using Nanofractionation Analytics. Toxins 2020, 12, 53 .
AMA StyleChunfang Xie, Julien Slagboom, Laura-Oana Albulescu, Ben Bruyneel, Kristina B. M. Still, Freek J. Vonk, Govert W. Somsen, Nicholas R. Casewell, Jeroen Kool. Antivenom Neutralization of Coagulopathic Snake Venom Toxins Assessed by Bioactivity Profiling Using Nanofractionation Analytics. Toxins. 2020; 12 (1):53.
Chicago/Turabian StyleChunfang Xie; Julien Slagboom; Laura-Oana Albulescu; Ben Bruyneel; Kristina B. M. Still; Freek J. Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. 2020. "Antivenom Neutralization of Coagulopathic Snake Venom Toxins Assessed by Bioactivity Profiling Using Nanofractionation Analytics." Toxins 12, no. 1: 53.
This chapter introduces bioactivity and bioaffinity terms in relation to mixture profiling and gives the significance of bioactivity and/or bioaffinity profiling of biologically active mixtures in general, and for bioactive mixtures in drug discovery research in particular. Further, the chapter gives an overview of the common and less common analytical approaches for bioactivity profiling of bioactive mixtures. Special focus is put on bioassay-guided fractionation as the standard technique employed (in identification and purification of bioactive molecules from a bioactive mixture), and on state-of-the-art post-column bioactivity profiling approaches, also providing examples and limitations of these analytical methods. On-column and pre-column bioactivity profiling analytics is also discussed. Examples of bioactive molecules identified and purified from different natural products are given with emphasis on molecules isolated from animal venoms. Finally, this chapter briefly discusses the importance of bioactivity profiling of metabolic mixtures in drug discovery.
Marija Mladic; Wilfried M. A. Niessen; Govert W. Somsen; Jeroen Kool. Analytics for Bioactivity Profiling of Complex Mixtures with a Focus on Venoms. Methods in Molecular Biology 2019, 2068, 27 -49.
AMA StyleMarija Mladic, Wilfried M. A. Niessen, Govert W. Somsen, Jeroen Kool. Analytics for Bioactivity Profiling of Complex Mixtures with a Focus on Venoms. Methods in Molecular Biology. 2019; 2068 ():27-49.
Chicago/Turabian StyleMarija Mladic; Wilfried M. A. Niessen; Govert W. Somsen; Jeroen Kool. 2019. "Analytics for Bioactivity Profiling of Complex Mixtures with a Focus on Venoms." Methods in Molecular Biology 2068, no. : 27-49.
Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates before direct analysis using a plasma coagulation assay. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.Author summarySnakebite is a neglected tropical disease that results in more than 100,000 deaths every year. Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to suffering life-threating hemorrhage. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood coagulation cascade and based on the initial results and the medical relevance of the species, seven venoms were selected for in-depth analysis of the responsible toxins using advanced identification techniques. Our findings reveal a number of anticoagulant toxins that have not yet been reported before as such. The methodology described herein not only enables the identification of both known and unknown toxins that cause impairment of the blood coagulation, but offers a throughput platform to effectively screen for inhibitory molecules relevant for the development of next generation snakebite treatments.
Julien Slagboom; Marija Mladić; Chunfang Xie; Freek Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches. 2019, 780155 .
AMA StyleJulien Slagboom, Marija Mladić, Chunfang Xie, Freek Vonk, Govert W. Somsen, Nicholas R. Casewell, Jeroen Kool. High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches. . 2019; ():780155.
Chicago/Turabian StyleJulien Slagboom; Marija Mladić; Chunfang Xie; Freek Vonk; Govert W. Somsen; Nicholas R. Casewell; Jeroen Kool. 2019. "High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches." , no. : 780155.
High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases.
Barbara M. Zietek; Kristina B. M. Still; Kevin Jaschusch; Ben Bruyneel; Freek Ariese; Tinco J. F. Brouwer; Matthijs Luger; Rob J. Limburg; Joost C. Rosier; Dick J. V. Iperen; Nicholas R. Casewell; Govert W. Somsen; Jeroen Kool. Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation. Analytical Chemistry 2019, 91, 10458 -10466.
AMA StyleBarbara M. Zietek, Kristina B. M. Still, Kevin Jaschusch, Ben Bruyneel, Freek Ariese, Tinco J. F. Brouwer, Matthijs Luger, Rob J. Limburg, Joost C. Rosier, Dick J. V. Iperen, Nicholas R. Casewell, Govert W. Somsen, Jeroen Kool. Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation. Analytical Chemistry. 2019; 91 (16):10458-10466.
Chicago/Turabian StyleBarbara M. Zietek; Kristina B. M. Still; Kevin Jaschusch; Ben Bruyneel; Freek Ariese; Tinco J. F. Brouwer; Matthijs Luger; Rob J. Limburg; Joost C. Rosier; Dick J. V. Iperen; Nicholas R. Casewell; Govert W. Somsen; Jeroen Kool. 2019. "Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation." Analytical Chemistry 91, no. 16: 10458-10466.
We describe the development of a high-resolution, noncontact fraction collector for liquid chromatography (LC) separations, allowing high-resolution fractionation in high-density well plates. The device is based on a low-dead-volume solenoid valve operated at 1–30 Hz for accurate collection of fractions of equal volume. The solenoid valve was implemented in a modified autosampler resulting in the so-called FractioMate fractionator. The influence of the solenoid supply voltage on solvent release was determined and the effect of the frequency, flow rate, and mobile phase composition was studied. For this purpose, droplet release was visually assessed for a wide range of frequencies and flow rates, followed by quantitative evaluation of a selection of promising settings for highly accurate, repeatable, and stable fraction collection. The potential of the new fraction collector for LC-based bioactivity screening was demonstrated by fractionating the LC eluent of a mixture of estrogenic and androgenic compounds, and a surface water sample (blank and spiked with bioactives) combining mass spectrometric detection and two reporter gene assays for bioactivity detection of the fractions. Additionally, a mixture of two compounds was repeatedly LC separated and fractionated to assess the feasibility of the system for analyte isolation followed by nuclear magnetic resonance analysis.
Willem Jonker; Koen De Vries; Niels Althuisius; Dick Van Iperen; Elwin Janssen; Rob Ten Broek; Corine Houtman; Nick Zwart; Timo Hamers; Marja H. Lamoree; Bert Ooms; Johannes Hidding; Govert W. Somsen; Jeroen Kool. Compound Identification Using Liquid Chromatography and High-Resolution Noncontact Fraction Collection with a Solenoid Valve. SLAS TECHNOLOGY: Translating Life Sciences Innovation 2019, 24, 543 -555.
AMA StyleWillem Jonker, Koen De Vries, Niels Althuisius, Dick Van Iperen, Elwin Janssen, Rob Ten Broek, Corine Houtman, Nick Zwart, Timo Hamers, Marja H. Lamoree, Bert Ooms, Johannes Hidding, Govert W. Somsen, Jeroen Kool. Compound Identification Using Liquid Chromatography and High-Resolution Noncontact Fraction Collection with a Solenoid Valve. SLAS TECHNOLOGY: Translating Life Sciences Innovation. 2019; 24 (6):543-555.
Chicago/Turabian StyleWillem Jonker; Koen De Vries; Niels Althuisius; Dick Van Iperen; Elwin Janssen; Rob Ten Broek; Corine Houtman; Nick Zwart; Timo Hamers; Marja H. Lamoree; Bert Ooms; Johannes Hidding; Govert W. Somsen; Jeroen Kool. 2019. "Compound Identification Using Liquid Chromatography and High-Resolution Noncontact Fraction Collection with a Solenoid Valve." SLAS TECHNOLOGY: Translating Life Sciences Innovation 24, no. 6: 543-555.