Cryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo... Show moreCryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo-EM techniques to gain insight into the structural changes of the human pathogen, Vibrio cholerae, as it transitions between different environments. A combination of established and novel techniques is used to prepare the individual cells for cryogenic electron tomography (cryo-ET). For example, I designed a manual plunge freezing apparatus to prepare cryo-EM samples off site and subsequently image them with cryo-ET. Furthermore, I used light microscopy and serial block face scanning EM imaging to visualize changes to the cells’ morphology and structure when transitioning from the environment, into the natural host, the zebrafish (Danio rerio), and back into the environment. In addition, this thesis demonstrates how ultraviolet-C radiation of cryo-EM samples of V. cholerae and the ICP1 bacteriophage can be used to inactivate the pathogen while retaining their ultrastructural details. Lastly, this thesis outlines current and novel methods for processing of larger, more complex samples for cryo-ET. These techniques, together with new models of host-pathogen interactions, offer new tools for exploring microbial interactions with their environments. Show less
Background: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit... Show moreBackground: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit components, buffers and even plastics has resulted in suboptimal testing procedures worldwide. Alternative workflows have been implemented to overcome these difficulties. Recently a liquid based sample prep has been launched as solution to overcome limitations in relation to nucleic acid extraction.Objective: Multicenter evaluation of the QlAprep& Viral RNA UM kit (QIA P&A) for rapid sample preparation and real-time PCR detection of SARS-CoV-2 in comparison to standardized laboratory testing methods.Study design: Selected samples of the routine diagnostic workflow at Clinical Microbiology Laboratories of four Dutch hospitals have been subjected to the rapid QIA P&A protocol and the results have been compared to routine diagnostic data.Results: Combining results of manual and automated procedures, a total of 377 clinical samples of which 202 had been tested positive with a wide range of C-T values, showed almost complete concordance in the QIA P&A assay for samples up to C-T values of 33 with one exception of C-T 31. Prospectively 60 samples were tested and also showed 100 % concordance with 5 positives. The method has been automated by two centres.Conclusions: Despite an input of only 8 mu L of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic. Show less
The human metabolome provides a direct physiological read-out of an individual's actual health state and includes biomarkers that may predict disease or response to a treatment. The discovery and... Show moreThe human metabolome provides a direct physiological read-out of an individual's actual health state and includes biomarkers that may predict disease or response to a treatment. The discovery and validation of these metabolomic biomarkers requires large-scale cohort studies, typically involving thousands of samples. This analytical challenge drives novel technological developments to enable faster, cheaper, and more comprehensive metabolomic analysis: more for less.This review summarises recent (2012–2018) developments towards this goal in all aspects of the analytical workflow, in relation to NMR but primarily to mass spectrometry (MS). Recent trends include miniaturisation and automation of extraction techniques, online coupling to fast analysis methods including direct infusion ion mobility MS, integrated microfluidic devices, and sharing and standardizing metabolomics software and data.The technological advances in metabolomics support its widespread application, integration with other -omics fields, and ultimately disease prediction and precision medicine. Show less
Martin Lorenzo, M.; Balluff, B.; Sanz-Maroto, A.; Zeijl, R.J.M. van; Vivanco, F.; Alvarez-Llamas, G.; McDonnell, L.A. 2014
Biotechnology increasingly delivers highly promising protein-based biopharmaceutical candidates to the drug development funnel. For successful biopharmaceutical drug development, reliable... Show moreBiotechnology increasingly delivers highly promising protein-based biopharmaceutical candidates to the drug development funnel. For successful biopharmaceutical drug development, reliable bioanalytical methods enabling quantification of drugs in biological fluids (plasma, urine, tissue, etc.) are required to generate toxicokinetic (TK), pharmacokinetic (PK), and bioavailability data. A clear observable trend is that liquid chromatography coupled to (tandem) mass spectrometry (LC–MS(/MS)) is more and more replacing ligand binding assays (LBA) for the bioanalytical determination of protein-based biopharmaceuticals in biological matrices, mainly due to improved selectivity and linear dynamic ranges. Practically all MS-based quantification methods for protein-based biopharmaceuticals traditionally rely on (targeted) proteomic techniques and include “seven critical factors”: (1) internal standardization, (2) protein purification, (3) enzymatic digestion, (4) selection of signature peptide(s), (5) peptide purification, (6) liquid chromatographic separation and (7) mass spectrometric detection. For this purpose, the variety of applied strategies for all “seven critical factors” in current literature on MS-based protein quantification have been critically reviewed and evaluated. Special attention is paid to the quantification of therapeutic monoclonal antibodies (mAbs) in serum and plasma since this is a very promising and rapidly expanding group of biopharmaceuticals. Additionally, the review aims to predict the impact of strategies moving away from traditional protein cleavage isotope dilution mass spectrometry (PC-IDMS) toward approaches that are more dedicated to bioanalysis. Show less
