Can I benefit from laboratory automation? A decision aid for the successful introduction of laboratory automation.

The large volumes of samples to be analysed every day would be impossible to manage without laboratory automation. As laboratory procedures have progressed, so have the tasks of laboratory personnel. With this feature article, we would like to provide (bio)chemical practitioners with little or no knowledge of laboratory automation with a guide to help them decide whether to implement laboratory automation and find a suitable system. Especially in small- and medium-sized laboratories, operating a laboratory system means having bioanalytical knowledge, but also being familiar with the technical aspects. However, time, budget and personnel limitations allow little opportunity for personnel to get into the depths of laboratory automation. This includes not only the operation, but also the decision to purchase an automation system. Hasty investments do not only result in slow or non-existent cost recovery, but also occupy valuable laboratory space. We have structured the article as a decision tree, so readers can selectively read chapters that apply to their individual situation. This flexible approach allows each reader to create a personal reading flow tailored to their specific needs. We tried to address a variety of perspectives on the topic, including people who are either supportive or sceptical of laboratory automation, personnel who want or need to automate specific processes, those who are unsure whether to automate and those who are interested in automation but do not know which areas to prioritize. We also help to make a decision whether to reactivate or discard already existing and unused laboratory equipment.

Label-free high-throughput screening via acoustic ejection mass spectrometry put into practice.

Acoustic droplet ejection-open port interface-mass spectrometry (ADE-OPI-MS) is a novel label-free analytical technique, promising to become a versatile readout for high-throughput screening (HTS) applications. The recent introduction of ADE-OPI-MS devices to the laboratory equipment market, paired with their compatibility with laboratory automation platforms, should facilitate the adoption of this technology by a broader community. Towards this goal, instrument robustness in the context of HTS campaigns - where up to millions of samples in complex matrices are tested in a short time frame - represents a major challenge, which explains the absence of detailed literature reports on this subject. Here, we present the results of our first fully automated HTS campaign, based on the ADE-OPI-MS technology, aiming to identify inhibitors of a metabolic enzyme in a >1 million compound library. The report encompasses the assay development and validation steps, as well as the adaptation for HTS requirements, where refinement of the capillary cleaning concept was crucial for final success. Altogether, our study unequivocally demonstrates the applicability of the ADE-OPI-MS technology for HTS-based drug discovery.

Acoustic Ejection Mass Spectrometry: A Fully Automatable Technology for High-Throughput Screening in Drug Discovery.

Acoustic droplet ejection (ADE)-open port interface (OPI)-mass spectrometry (MS) has recently been introduced as a versatile analytical method that combines fast and contactless acoustic sampling with sensitive and accurate electrospray ionization (ESI)-MS-based analyte detection. The potential of the technology to provide label-free measurements in subsecond analytical cycle times makes it an attractive option for high-throughput screening (HTS). Here, we report the first implementation of ADE-OPI-MS in a fully automated HTS environment, based on the example of a biochemical assay aiming at the identification of small-molecule inhibitors of the cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). First, we describe the optimization of the method to enable sensitive and accurate determination of enzyme activity and inhibition in miniaturized 1536-well microtiter plate format. Then we show both results from a validation single-concentration screen using a test set of 5500 compounds, and the subsequent concentration-response testing of selected hits in direct comparison with a previously established matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) readout. Finally, we present the development of an in-line OPI cleaning procedure aiming to match the instrument robustness required for large-scale HTS campaigns. Overall, this work points to critical method development parameters and provides guidance for the establishment of integrated ADE-OPI-MS as HTS-compatible technology for early drug discovery.

Differentiating Primary Pancreatic Lymphoma Versus Primary Splenic Lymphoma: A Case Report.

Background: Both primary pancreatic lymphoma (PPL) and primary splenic lymphoma (PSL) represent rare entities. PPL typically arises in the head of the pancreas but may arise in other locations also. PSL usually presents with nonspecific symptoms, including left upper quadrant pain, weight loss, and fever. This report describes a patient with a large left upper quadrant mass, which initially was believed to be a primary pancreatic mass, but which on final pathology appeared to be consistent with a PSL. Presentation: The patient is a 64-year-old woman who initially presented with symptoms of left upper quadrant abdominal pain and distension; she subsequently was found to have an 18 cm heterogeneous mass arising from the pancreatic tail. She underwent a distal pancreatectomy with splenectomy. Final pathology confirmed a diffuse large B cell lymphoma arising from the splenic parenchyma. Conclusions: Both PPL and PSL are rare causes of left upper quadrant masses. In this case, we describe a large lymphoma that appeared to arise from the tail of the pancreas, but on final pathology was found to be splenic in origin. Differentiating these two clinical entities is important for prognostication and treatment. A multimodal approach with surgical resection followed by chemotherapy is preferred.

MALDI-TOF-Based Affinity Selection Mass Spectrometry for Automated Screening of Protein-Ligand Interactions at High Throughput.

Demonstration of in vitro target engagement for small-molecule ligands by measuring binding to a molecular target is an established approach in early drug discovery and a pivotal step in high-throughput screening (HTS)-based compound triaging. We describe the setup, evaluation, and application of a ligand binding assay platform combining automated affinity selection (AS)-based sample preparation and label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The platform enables mass spectrometry (MS)-based HTS for small-molecule target interactions from single-compound incubation mixtures and is embedded into a regular assay automation environment. Efficient separation of target-ligand complexes is achieved by in-plate size exclusion chromatography (SEC), and small-molecule ligands are subsequently identified by MALDI-TOF analysis. In contrast to alternative HTS-capable binding assay formats, MALDI-TOF AS-MS is capable of identifying orthosteric and allosteric ligands, as shown for the model system protein tyrosine phosphatase 1B (PTP1B), irrespective of protein function. Furthermore, determining relative binding affinities (RBAs) enabled ligand ranking in accordance with functional inhibition and reference data for PTP1B and a number of diverse protein targets. Finally, we present a validation screen of more than 23,000 compounds within 24 h, demonstrating the general applicability of the platform for the HTS-compatible assessment of protein-ligand interactions.

Development of a miniaturized 96-Transwell air-liquid interface human small airway epithelial model.

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor ß1 (TGF-ß1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.

MALDI-TOF Mass Spectrometry-Based High-Throughput Screening for Inhibitors of the Cytosolic DNA Sensor cGAS.

Comprehensive and unbiased detection methods are a prerequisite for high-throughput screening (HTS) campaigns within drug discovery research. Label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an HTS-compatible readout for biochemical test systems to support the drug discovery process. So far, reported HTS applications were based on surface-modified systems or proof-of-concept studies. We present the utilization of a MALDI-TOF-based screening platform to identify inhibitors of human cyclic GMP-AMP synthase (cGAS), a mediator of innate immune response whose aberration has been causally correlated to a number of inflammatory disorders. In this context, the development and validation of a MALDI-TOF-based activity assay is reported to demonstrate fast, robust, and accurate detection of chemical cGAS inhibition by direct quantification of the physiological reaction product cyclic GMP-ATP (cGAMP). Results from a screen of a diverse library of more than 1 million small molecules in 1536-well format against the catalytic cGAS activity are presented with excellent assay performance and data quality. Identified hits were qualified in dose-response experiments and confirmed by RapidFire-MS measurements. Conclusively, the presented data provide the first proof of applicability of direct automated MALDI-TOF MS as a readout strategy for large-scale drug discovery HTS campaigns.

Chemical Derivatization Enables MALDI-TOF-Based High-Throughput Screening for Microbial Trimethylamine (TMA)-Lyase Inhibitors.

Microbial-dependent trimethylamine (TMA) generation from dietary precursors such as choline was recently linked to cardiovascular diseases (CVDs) as well as chronic kidney disease (CKD). Inhibition of TMA-generating enzymes in gut bacteria would be an innovative approach to treat these diseases. The potential to accurately quantify secreted TMA levels highlights the capacity of mass spectrometry (MS) for tracking microbial TMA-lyase activity. However, high-throughput screening (HTS) by conventional MS instrumentation is hampered by limited sample throughput. Recent advancement in liquid handling and instrumentation of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS provides an HTS-compatible MS technology. The deciphering of enzymatic reactions using this label-free readout has been successfully applied but has thus far been limited to peptide/protein-centric activity assays. Here, we demonstrate the versatile applicability of MALDI-TOF by tracking a small molecule within a highly complex sample background. The key to success for this concept was chemical derivatization of the target molecule enabling quantitative assessment of microbial TMA formation. Further, its potential was demonstrated in a side-by-side comparison to RapidFire-MS in a primary screen and subsequent dose-response experiments. Overall, the established assay enables the screening for microbial TMA-lyase inhibitors and serves as a proof of concept for the applicability of MALDI-TOF for demanding assay concepts per se.

Automated MALDI Target Preparation Concept: Providing Ultra-High-Throughput Mass Spectrometry-Based Screening for Drug Discovery.

Label-free, mass spectrometric (MS) deciphering of enzymatic reactions by direct analysis of substrate-to-product conversion provides the next step toward more physiological relevant assays within drug discovery campaigns. Reduced risk of suffering from compound interference combined with diminished necessity for tailored signal mediators emphasizes the valuable role of label-free readouts. However, MS-based detection has not hitherto met high-throughput screening (HTS) requirements because of the lack of HTS-compatible sample introduction. In the present study, we report on a fully automated liquid-handling concept built in-house to concatenate biochemical assays with matrix-assisted laser desorption/ionization time-of-flight closing this technological gap. The integrated reformatting from 384- to 1536-well format enables cycle times of 0.6 s/sample for automated spotting and 0.4 s/sample for MS analysis, matching the requirements of HTS compatibility. In-depth examination of spotting quality, quantification accuracy, and instrument robustness together with the implementation of a protein tyrosine phosphatase 1B (PTP1B) inhibitor screening (4896 compounds) demonstrate the potential of the heavily inquired HTS integration of the label-free MS readout. Overall, the presented data demonstrate that the introduced automation concept makes label-free MS-based readouts accessible for HTS within drug discovery campaigns but also in other research areas requiring ultrafast MS-based detection.

Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

Application of life cycle thinking in multidisciplinary multistakeholder contexts for cross-sectoral planning and implementation of sustainable development projects.

Development planning and implementation is a multifaceted and multiscale task mainly because of the involvement of multiple stakeholders across sectors and disciplines. Even though top-down sectoral planning is commonly practiced, bottom-up cross-sectoral planning involving all relevant stakeholders in a transdisciplinary learning environment has been recognized as a better option, especially if the goal is to drive development projects toward sustainable implementation (Rowe and Fudge 2003; Müller et al. 2005; Global Development Research Center 2008). Even though many planning approaches have this goal, there are limited decision frameworks that are suitable for achieving consensus among stakeholders from multiple disciplines with sectoral objectives and priorities. In most instances, the upstream and downstream effects of development decisions are not thoroughly investigated or communicated with the relevant stakeholders, strongly affecting cross-sectoral integration in the real world (Wiek, Brundiers, et al. 2006). This article presents methodological aspects of developing a stakeholder based life cycle assessment framework (SBLCA) for upstream-downstream decision analysis in a multistakeholder development planning context. The applicability of the framework is demonstrated using simple examples extracted from a pilot case study conducted in Sri Lanka for sustainable posttsunami reconstruction at a village scale. The applicability of SBLCA in specific planning stages, how it promotes transdisciplinary learning and cross-sectoral stakeholder integration in phases of project cycles, and how local stakeholders can practice life cycle thinking in their village development planning and implementation are discussed.