Parallel Samples Research Articles

Automating DUID methods using robotics for rapid high throughput sample preparation – Analysis of Delta-9-Tetrahydrocannabinol and its major metabolite Delta-9-tetrahydrocannabinol acid in whole blood

To fully automate, using liquid-handling robotics, a liquid chromatography tandem mass spectrometry method for the analysis of delta-9-tetrahydrocannabinol (THC) and delta-9-tetrahydrocannabinol acid (THC-A) in whole blood. Uptake in usage of ultra-high performance liquid chromatography (UHPLC) has led to development of methods with very fast (typically < 5 minutes) chromatographic run times used in the forensic toxicology laboratory. Nevertheless, use of whole blood for quantitative analysis continues to present challenges for delivering high throughput methods with rapid analysis times. Whole blood requires the use of solid phase extraction (SPE) for sample pre-treatment and preparation, which is time consuming and labour intensive. Recent developments in SPE technologies have produced 96-well format platforms that are used to extract greater number of samples in parallel and are ideally suitable for automated liquid handlers. With recent sensitivity increases in mass spectrometers, smaller volumes (< 250 μL per aliquot of blood) can be used for extraction in 96-well plates. This facilitates the use of robotic platforms together with rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to give truly high throughput and fast analytical methods, increasing method efficiencies, reducing human intervention and errors. Here we present the development, validation and implementation of a fully automated robotic sample preparation method for whole blood for analysis of THC and THC-A in UK driving under the influence of drugs (DUID) cases. Aliquots (200 μL) of blank blood and casework samples previously dispensed into 96-well plates by a robotic system (BRAND) were prepared by the liquid handling system (Hamilton). Casework samples and blank blood were spiked with internal standard and blank blood samples were further spiked to produce calibrants and quality controls. The plate is then mixed with the addition of a cell lysis solution and the aliquots transferred to a protein/lipid depletion (PPT) plate (Waters). A positive pressure manifold was used to draw the solution through the PPT plate into a collection plate where the precipitate was dried. The precipitate was reconstituted and loaded by the Hamilton onto a C18 microelution SPE plate (Waters). Following extraction, the samples are reconstituted and the collection plate is placed in the autosampler for analysis. The LC-MS/MS method consists of a 2.1 mm × 50 mm, 1.7 μm, C18 column running a 4 minute gradient of water and acetonitrile with 0.1% formic acid. The method was successfully validated, with LLOQs for THC and THC-A of 1 μg/L and 5 μg/L, respectively. Precision (%CV) for THC was < 8% and < 6% for THC-A. Accuracy was within 15% of the spiked concentration for THC and THC-A across all validation batches ( n = 11). The robotic platform was able to undertake sample pre-treatment and extraction in 3.5 hours. A batch of 40 casework samples can be completed in 13 hours including the LC-MS/MS run time. The introduction of this method to our laboratory has resulted in a significant increase in the speed for a sample to be processed and results obtained. We can prepare and extract double the amount of samples in less than half the time taken by our manual method. We have reduced the amount of sample used and decreased our turnaround times all for our customer's benefit. Full automation of the sample preparation of whole blood samples using liquid handling robots can significantly improve laboratory throughput and capacity and efficiently utilise high speed chromatography providing rapid analysis from start to finish.

Upward Flame Spread over Two Parallel Paper Sheets under Reduced Ambient Pressure and Elevated Oxygen Concentration

ABSTRACT The effects of separation distance and environmental parameters (such as ambient pressure and oxygen concentration) on the upward flame spread over two parallel paper sheets were investigated in this work, which would be conducive to enriching the fire dynamic theories and providing a reference for the risk evaluation in building and space fires. It was found that, with the increase of separation distance, the flame spread regimes can be roughly divided into four classes: the flame quenching regime (without inner flame), where the interaction of two parallel papers is mainly dominated by the solid surface radiation; the complete fusion regime, limited by the oxygen diffusion; the partial fusion regime, dominated by the convection; the separated flames regime, dominated by the gas-phase radiation. The flames inside the narrow space are fused under the flame-induced horizontal flow, and extinguished for the oxygen starvation, which reflects the interaction of two parallel samples. Interestingly, although the flames inside and outside the parallel sheets often have different flame lengths and burning intensities, they spread at almost the same rate, which is ascribed to the overlapping of the pyrolysis region on both sides of the thermal thin paper. A critical separation distance was found, where the flame spread rate is largest due to the stoichiometric mixture fraction. The orange-blue layered flames were analyzed, which is related to the boundary layer of the flow field and the mixture of fuel and fresh air. The oxygen concentration has nearly equivalent impacts on the flame-induced buoyancy and horizontal flows, and thus a constant flame fusion angle is maintained. Nevertheless, the enhanced ambient pressure mainly promotes the buoyancy and reduces the flame fusion angle. The flames inside the narrow space are apt to be extinguished under lower ambient pressure with weak buoyancy similar to the reduced gravity and microgravity environments. The transformation of the dominating regimes were observed at a moderate separation distance: the flame quenching regime at pressures below 18 kPa, the complete fusion regime at pressures between 18 ~ 29 kPa, and the partial fusion regime at pressures over 29 kPa.

Critical fields of superconductors with magnetic impurities

The upper critical field ${H}_{c2}$, the field ${H}_{c3}$ for nucleation of the surface superconductivity, and the thermodynamic field ${H}_{c}$ are evaluated within the weak-coupling theory for the isotropic s-wave case with arbitrary transport, and pair-breaking scattering. We find that, for the standard geometry of a half-space sample in a magnetic field parallel to the surface, the ratio $\mathcal{R}={H}_{c3}/{H}_{c2}$ is within the window $1.55\ensuremath{\lesssim}\mathcal{R}\ensuremath{\lesssim}2.34$, regardless of temperature or the scattering type. While the nonmagnetic impurities tend to flatten the $\mathcal{R}(T)$ variation, magnetic scattering merely shifts the maximum of $\mathcal{R}(T)$ to lower temperatures. Surprisingly, while reducing the transition temperature, magnetic scattering has a milder impact on $\mathcal{R}$ than nonmagnetic scattering. The surface superconductivity is quite robust; in fact, the ratio $\mathcal{R}\ensuremath{\approx}1.7$ even in the gapless state. We used Eilenberger's energy functional to evaluate the condensation energy ${F}_{c}$ and the thermodynamic critical field ${H}_{c}$ for any temperature and scattering parameters. By comparing ${H}_{c2}$ and ${H}_{c}$, we find that, unlike transport scattering, the pair-breaking pushes materials toward type-I behavior. We find a peculiar behavior of ${F}_{c}$ as a function of the pair-breaking scattering parameter at the low-$T$ transition from gapped to gapless phases, which has recently been associated with the topological transition in the superconducting density of states.

Failure Mechanism and Acoustic Emission Precursors of Coal Samples considering Bedding Effect under Triaxial Unloading Condition

Bedding increases coal seam anisotropy, which leads to significant differences in the evolution laws of mining stress and strata movement. This work analyzed the dip angles of different layers to analyze the mechanical properties of the coal seam under unloading. The coal sample was subjected to triaxial compression and unloading damage acoustic emission testing. The brittleness characteristics of the coal sample failure in different bedding directions differed significantly. Compared with axial parallel bedding coal samples, axial vertical bedding and inclined stratification reached an ultimate strength. The stress–strain curve decreased sharply and showed visible brittle-drop characteristics. The average strengths of the axially inclined bedding and the parallel layered coal sample decreased compared with the axial vertical bedding coal sample and were 10.20 and 16.12 MPa, respectively, which implies a greater susceptibility to failure during unloading confining pressure tests. Acoustic emission monitoring indicated that the axial vertical bedding and inclined bedding showed sudden destruction of different coal samples, a reduction in axial parallel bedding ductility coal sample characteristics, and stronger unloading damage on the axis parallel to the bedding coal sample. Further, using the acoustic emission ring count rate and the cusp catastrophe theory, the unloading failure prediction of coal samples is carried out. The prediction results are not different from the experimental results, which shows that this method is feasible.

A Kriging-based adaptive parallel sampling approach with threshold value

A Kriging-based adaptive parallel sampling approach with threshold value

Improved alkali-tolerance of FeOx-WO3 catalyst for NO removal via in situ reserving FeOx active species

The improvement of alkali tolerance of de-NOx catalysts remains an immense challenge for selective catalytic reduction (SCR) of NO by NH3. In this work, NOx removal efficiency was nearly retained in the presence of potassium over FeOx-WO3 (FW) catalysts via protecting iron active species using different template agents. Typically, FW using urea (FW-(urea)) as template agent showed superior de-NOx performance after being poisoned by potassium (K/FW-(urea)). Urea could promote the facile binding of potassium with WO3, which acted as alkali-poisoning sites so that iron active species were reserved to a great extent. Additionally, potassium species tended to be aggregated over K/FW-(urea) and thus a lot of iron active sites could be exposed. Besides, K/FW-(urea) catalyst has higher surface acidity, superior redox ability, stronger interaction of WO3 with Fe2O3, and more surface adsorbed oxygen compare with other parallel poisoned samples. This is the reason for the remarkable ability of potassium resistance over K/FW-(urea). The strategy of template regulation could offer a novel route for maintaining considerable NH3-SCR performance in the presence of alkali.

Virtual Blue Noise Lighting

We introduce virtual blue noise lighting, a rendering pipeline for estimating indirect illumination with a blue noise distribution of virtual lights. Our pipeline is designed for virtual lights with non-uniform emission profiles that are more expensive to store, but required for properly and efficiently handling specular transport. Unlike the typical virtual light placement approaches that traverse light paths from the original light sources, we generate them starting from the camera. This avoids two important problems: wasted memory and computation with fully-occluded virtual lights, and excessive virtual light density around high-probability light paths. In addition, we introduce a parallel and adaptive sample elimination strategy to achieve a blue noise distribution of virtual lights with varying density. This addresses the third problem of virtual light placement by ensuring that they are not placed too close to each other, providing better coverage of the (indirectly) visible surfaces and further improving the quality of the final lighting estimation. For computing the virtual light emission profiles, we present a photon splitting technique that allows efficiently using a large number of photons, as it does not require storing them. During lighting estimation, our method allows using both global power-based and local BSDF important sampling techniques, combined via multiple importance sampling. In addition, we present an adaptive path extension method that avoids sampling nearby virtual lights for reducing the estimation error. We show that our method significantly outperforms path tracing and prior work in virtual lights in terms of both performance and image quality, producing a fast but biased estimate of global illumination.

Spacemake: processing and analysis of large-scale spatial transcriptomics data.

BackgroundSpatial sequencing methods increasingly gain popularity within RNA biology studies. State-of-the-art techniques quantify messenger RNA expression levels from tissue sections and at the same time register information about the original locations of the molecules in the tissue. The resulting data sets are processed and analyzed by accompanying software that, however, is incompatible across inputs from different technologies.FindingsHere, we present spacemake, a modular, robust, and scalable spatial transcriptomics pipeline built in Snakemake and Python. Spacemake is designed to handle all major spatial transcriptomics data sets and can be readily configured for other technologies. It can process and analyze several samples in parallel, even if they stem from different experimental methods. Spacemake's unified framework enables reproducible data processing from raw sequencing data to automatically generated downstream analysis reports. Spacemake is built with a modular design and offers additional functionality such as sample merging, saturation analysis, and analysis of long reads as separate modules. Moreover, spacemake employs novoSpaRc to integrate spatial and single-cell transcriptomics data, resulting in increased gene counts for the spatial data set. Spacemake is open source and extendable, and it can be seamlessly integrated with existing computational workflows.

Sequential approximate optimization with adaptive parallel infill strategy assisted by inaccurate Pareto front

Sequential Approximate Optimization (SAO) has been widely used in engineering optimization design problems to improve efficiency. The infilling strategy is one of the critical techniques of the SAO, which is of paramount importance to the surrogate model accuracy and optimization efficiency. In this paper, an adaptive parallel infill strategy for surrogate-based single-objective optimization is proposed within a multi-objective optimization framework to balance exploration and exploitation during the optimization process. Within this method, an inaccurate Pareto Front is adopted to assist the infilling of the sampling points. The proposed SAO method with its adaptive parallel sampling strategy is tested on several numerical test cases and an engineering test case with the optimization results compared to state-of-the-art optimization algorithms. The results show that the proposed SAO with the adaptive parallel sampling strategy possesses excellent performance and better stability.

Advanced unified monophasic lipid extraction protocol with wide coverage on the polarity scale optimized for large-scale untargeted clinical lipidomics analysis of platelets

Lipid extraction is a critical step in sample preparation of lipidomics studies. Biphasic liquid-liquid extraction protocol with methyl tert-butyl ether (MTBE)/methanol (MeOH) as organic solvents are widely adopted by researchers nowadays as an eco-friendly replacement of classic Folch, and Bligh&Dyer protocols. Yet, it has some limitations such as suboptimal performance for the most polar lipids (e.g. acylcarnitines), complicated handling as it requires phase separation, and is therefore non-ideal for large-scale clinical studies. To advance the extraction protocol for large-scale clinical lipidomics, in this study we explored i) 6 different extraction solvent systems, ii) distinct processing procedures (sonication, mechanical cell lysis and bead homogenizer), and iii) also 7 different reconstitution solvents. The extraction systems investigated included biphasic systems MTBE/MeOH/H2O and Hexane/2-propanol (IPA)/1 M acetic acid, and monophasic systems MTBE/MeOH/CHCl3, IPA/H2O (90% IPA), MeOH/MTBE/IPA, and IPA/H2O/MTBE as solvent system for lipid extraction of human platelets. Extraction recovery was investigated by repeated extraction cycles. Subcellular extraction efficiency was assessed by the mitochondria-specific cardiolipins. It turned out that monophasic extraction with MeOH/MTBE/IPA (1.3:1:1, v/v/v), bead homogenizer for cell disruption and MeOH/MTBE (1:1, v/v) as reconstitution solvent provide optimal cellular and subcellular extraction efficiencies for both polar (e.g. acylcarnitines) and apolar lipids (e.g. triglycerides). It is simplified (no phase separation), eco-friendly (reduced solvent consumption and no halogenated ones), fast (5 min for 24 samples in parallel), and can be easily adapted for cells, plasma, and tissue. Therefore, it has great potential for large-scale clinical lipidomics studies.

Spatial barcoding-enabled highly multiplexed immunoassay with digital microfluidics

Digital microfluidics (DMF), facilitating independent manipulation of microliter samples, provides an ideal platform for immunoassay detection; however, suffering limited multiplexity. To address the need, herein we described a digital microfluidics (DMF) platform that realizes spatial barcoding on the Teflon-coated indium tin oxide (ITO) glass side to fulfill highly multiplexed immunoassay (10+) with low-volume samples (∼4 μL) in parallel, representing the highest multiplexing recorded to date for DMF-actuated immunoassay. Planar-based spatial immobilization of multiple capture antibodies was realized on a Teflon-coated ITO glass side, which was then used as the top plate of the DMF device. Droplets containing analytes, secondary antibodies, and fluorescent signaling reporters with low volume, which were electrically manipulated by our DMF control system, were shuttled sequentially along the working electrodes to complete the immuno-reaction. Evaluation of platform performance with recombinant proteins showed excellent sensitivity and reproducibility. To test the feasibility of our platform in analyzing multiplex biomarkers of the immune response, we used lipopolysaccharide-stimulated macrophages as a model system for protein secretion dynamics studies. As a result, temporal profiling of pro-inflammatory cytokine secretion dynamics was obtained. The spatial barcoding strategy presented here is easy-to-operate to enable a more comprehensive evaluation of protein abundance from biological samples, paving the way for new opportunities to realize multiplexity-associated applications with the DMF platform.

Rapid bacterioplankton transcription cascades regulate organic matter utilization during phytoplankton bloom progression in a coastal upwelling system

Coastal upwelling zones are hotspots of oceanic productivity, driven by phytoplankton photosynthesis. Bacteria, in turn, grow on and are the principal remineralizers of dissolved organic matter (DOM) produced in aquatic ecosystems. However, the molecular processes that key bacterial taxa employ to regulate the turnover of phytoplankton-derived DOM are not well understood. We therefore carried out comparative time-series metatranscriptome analyses of bacterioplankton in the Northwest Iberian upwelling system, using parallel sampling of seawater and mesocosms with in situ-like conditions. The mesocosm experiment uncovered a taxon-specific progression of transcriptional responses from bloom development (characterized by a diverse set of taxa in the orders Cellvibrionales, Rhodobacterales, and Pelagibacterales), over early decay (mainly taxa in the Alteromonadales and Flavobacteriales), to senescence phases (Flavobacteriales and Saprospirales taxa). Pronounced order-specific differences in the transcription of glycoside hydrolases, peptidases, and transporters were found, supporting that functional resource partitioning is dynamically structured by temporal changes in available DOM. In addition, comparative analysis of mesocosm and field samples revealed a high degree of metabolic plasticity in the degradation and uptake of carbohydrates and nitrogen-rich compounds, suggesting these gene systems critically contribute to modulating the stoichiometry of the labile DOM pool. Our findings suggest that cascades of transcriptional responses in gene systems for the utilization of organic matter and nutrients largely shape the fate of organic matter on the time scales typical of upwelling-driven phytoplankton blooms.

Exploring Sea Lice Vaccines against Early Stages of Infestation in Atlantic Salmon (Salmo salar).

The sea louse Caligus rogercresseyi genome has opened the opportunity to apply the reverse vaccinology strategy for identifying antigens with potential effects on lice development and its application in sea lice control. This study aimed to explore the efficacy of three sea lice vaccines against the early stage of infestation, assessing the transcriptome modulation of immunized Atlantic salmon. Therein, three experimental groups of Salmo salar (Atlantic salmon) were vaccinated with the recombinant proteins: Peritrophin (prototype A), Cathepsin (prototype B), and the mix of them (prototype C), respectively. Sea lice infestation was evaluated during chalimus I-II, the early-infective stages attached at 7-days post infestation. In parallel, head kidney and skin tissue samples were taken for mRNA Illumina sequencing. Relative expression analyses of genes were conducted to identify immune responses, iron transport, and stress responses associated with the tested vaccines during the early stages of sea lice infection. The vaccine prototypes A, B, and C reduced the parasite burden by 24, 44, and 52% compared with the control group. In addition, the RNA-Seq analysis exhibited a prototype-dependent transcriptome modulation. The high expression differences were observed in genes associated with metal ion binding, molecular processes, and energy production. The findings suggest a balance between the host’s inflammatory response and metabolic process in vaccinated fish, increasing their transcriptional activity, which can alter the early host–parasite interactions. This study uncovers molecular responses produced by three vaccine prototypes at the early stages of infestation, providing new knowledge for sea lice control in the salmon aquaculture.

Optical Dating of Quartz Grains From the Minjiang Fluvial Terraces in the Sonpan Area on the Eastern Margin of the Tibetan Plateau

The fluvial terraces along the banks of the Minjiang River are very important for understanding the tectonic activities of the eastern margin of the Tibetan Plateau and have been widely investigated. However, the reliability of the ages previously reported for the terraces needs further evaluation. In this study, the Minjiang River terraces in the Sonpan area from Hongqiaoguan to the Songpan town were investigated and dated using optically stimulated luminescence (OSL) techniques on quartz grains. Seven strath terraces (T1–T7) were recognized based on the exposures of fluvial sediment and the elevation of bedrock strath, and two of them have been reported by previous studies. The terrace deposits and overlying loess were sampled for OSL dating. The samples exhibited a large scatter in De, even for loess samples, which could be attributed to poor bleaching at deposition. However, the OSL ages obtained using the minimum age model were underestimated, and those obtained using the central age model are considered relatively reliable based on stratigraphic and geomorphological consistency and the comparison of the ages between stratigraphically parallel samples. The results show that the loess samples overlying fluvial terrace sediments were deposited later than terrace formation and their OSL ages cannot represent the terrace formation ages. The T1, T3, T4, T5, and T6 terraces were dated to 13.5 ± 0.6, 29.0 ± 1.7, 48.0 ± 3.3, 44.3 ± 5.2 and 63.8 ± 4.7 ka, respectively. The T4 and T5 terraces may be the same terrace with a weighted mean age of 46.9 ± 2.8 ka. The ages of the T2 and T7 terraces were inferred to be ∼20 and ∼80 ka, respectively, based on the relationship between strath ages and elevations of the other terraces. The mean bedrock river incision rates were calculated to be 1.2 ± 0.1 mm/a for the time period of 64 ka for the T6 terrace—14 ka for the T1 terrace, and 0.15 mm/a for the past 14 ka.

Direct Determination of Free Nicotine Content in Tobacco.

The accurate determination of the free nicotine content in tobacco is of great importance for the quality assessment of tobacco leaves and cigarettes, as well as for the study of tar reduction and cigarette formulation. A method based on solvent extraction and gas chromatography–mass spectrometry (GC–MS) was established for direct determination of free nicotine content in tobacco. Free nicotine extraction from tobacco was optimized, using different solvents and physical techniques (shaking, ultrasonic oscillation, and static extraction). Ultimately, a 24 h static extraction with cyclohexane, followed by GC–MS analysis, gave superior results. The standard addition recovery was 98.0–104.7%, with a limit of detection of 5.3 μg/g and a relative standard deviation between 1.3 and 4.1% (n = 5). Quadratic regression of the standard curve was excellent (R2 ≥ 0.9994). The free nicotine content was determined in 67 tobacco samples, with parallel samples showing relative deviations of 0.1–3.1%. To evaluate the effect of nicotine salts present in tobacco, malic acid, citric acid, acetic acid, and nicotine citrate were spiked into samples prior to extraction and analysis. The results show no interference from bound nicotine compounds on the method. The experimental results show that direct determination of free nicotine in tobacco was achieved using a simple static cyclohexane extraction. Moreover, the high extraction efficiency of free nicotine was also achieved with ease of operation and good repeatability.

Debenzylation of Benzyl-Protected Methylcellulose

Methyl cellulose and its derivatives are widely used in the food industry, cosmetics, and as construction materials. The properties of methyl celluloses (MC) strongly depend on their degrees and positions of substitution. In order to generate MCs with uncommon blocky substitution, we apply fully protected O-benzyl-O-methyl celluloses (BnMC). Such complex polysaccharide derivatives could not be deprotected completely and without shift of the composition by methods usually applied to mono- and oligosaccharides. Therefore, a facile debenzylation method was developed based on photo-initiated free-radical bromination in the presence of hydrobromic acid scavengers followed by alkaline treatment. The reaction proceeds under homogeneous conditions and without the aid of any catalyst. There is no need for expensive equipment, materials, anhydrous reagents, or running the reaction under anhydrous conditions. Reaction parameters were investigated and optimized for successful debenzylation of completely protected BnMC with degrees of methyl substitution (DSMe) around 1.9 (and DSBn around 1.1). Side-product-free and almost complete debenzylation was achieved when 1,2-epoxybutane (0.5 eq./eq. N-bromosuccinimide) and 2,6-di-tert-butylpyridine (0.5 eq./eq. N-bromosuccinimide) were used in the reaction. Furthermore, ATR-IR and 1H NMR spectroscopy confirmed the successful removal of benzyl ether groups. The method was developed to monitor the transglycosylation reaction of the BnMC with permethylated cellulose, for which the deprotection of many small samples in parallel is required. This comprises the determination of the methyl pattern in the glucosyl units by gas-liquid chromatography (GLC), as well as oligosaccharide analysis by liquid chromatography mass spectrometry (LC-MS) after perdeuteromethylation and partial hydrolysis to determine the methyl pattern in the chains. The unavoidable partial chain degradation during debenzylation does not interfere with this analytical application, but, most importantly, the DS and the methyl pattern were almost congruent for the debenzylated product and the original MC, indicating the full success of this approach The presented method provides an unprecedented opportunity for high throughput and parallel debenzylation of complicated glucans, such as BnMC (as a model compound), for analytical purposes. For comparison, debenzylation using Na/NH3 was applied to BnMC and resulted in a completely debenzylated product with a remarkably high recovery yield of 99 mol% and is, thus, the method of choice for synthetic applications, e.g., for the transglycosylation product prepared under the selected conditions in a preparative scale.

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics.

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.

Abstract 3098: The first circulating tumor cell-derived explant (CDX) model of a Merkel cell carcinoma

Abstract NeuroEndocrine Carcinomas (NECs) are rare yet lethal diseases; they are frequently metastatic and have limited treatment options. Whilst progress has been made in understanding the biology of Small Cell Lung Cancer (SCLC), the most studied NEC, research on NECs from other anatomical sites lags behind. In SCLC, circulating tumor cells (CTCs) can be used to generate patient-relevant mouse models; termed CTC-Derived eXplants (CDX) which are faithful in biology and chemotherapy response to donor patients1,2. A pilot prospective study is on-going to assess the potential utility of cell-free DNA (cfDNA) and/or CellSearch (CS) CTCs (EpCAM/cytokeratin positive) as liquid biopsies and feasibility of CDX generation from patients with advanced stage extrapulmonary NECs undergoing palliative chemotherapy. CS CTCs are enumerated before and during treatment and marker-independent RosetteSep-enriched CTCs from parallel blood samples are implanted in immunodeficient mice to attempt CDX generation. Twenty-seven patients were recruited from Jun-19 to Nov-21. CS-CTCs were present in 19/27; 70% pre-treatment samples (mean 57, median 2, range 0-685 CTCs per 7.5mL of blood). CDX generation was successful from a pre-treatment CTC sample from a patient originally diagnosed with a NEC of unknown origin who responded to Platinum/Etoposide. Whole-exome sequencing of this CDX and matched pre-treatment cfDNA from the donor revealed largely overlapping copy number changes and common mutations including an activating PIK3CA mutation. In addition, there was no evidence of TP53 or RB1 loss (hallmarks of SCLC) in neither the CDX nor the donor’s cfDNA. Immunohistochemical (IHC) analysis of this CDX showed a small cell NEC morphology, 90% Ki-67 positive cells, expression of EpCAM, pan-cytokeratins and neuroendocrine (NE) diagnostic markers, concordant with the donor’s biopsy. Further IHC and RNA sequencing revealed expression of the NE transcription factor atonal homolog 1 (ATOH1)2, CK20 and abundant Merkel cell polyomavirus (MCPyV) transcripts. These characteristics prompted reclassification of the original patient diagnosis as Merkel cell carcinoma (MCC). There was only 1 CS-CTC in the parallel pre-treatment blood sample implying that EpCAM-negative CTCs may have given rise to the CDX. The CDX showed in vivo sensitivity to Platinum/Etoposide, mirroring treatment response of the donor. Ex vivo cultures from CDX-derived cells grow as suspension clusters similar to ‘classic’ SCLC and MCPyV-positive MCC cell lines. In summary, this first Merkel Cell Carcinoma CDX recapitulates the biology of the donor’s tumor. As the donor remains alive 25 months from diagnosis of advanced disease, their CDX can serve as an avatar to guide future treatment. 1. Hodgkinson et al., Nature Med 2014, 20: p. 897-903. 2. Simpson et al., Nature Cancer 2020, 1: p. 437-451. Citation Format: Melissa Frizziero, Elaine Kilgour, Kathryn L. Simpson, Dominic Rothwell, Kristopher Frese, Melanie Galvin, Yitao Chen, Alastair Kerr, Sam Humphrey, Mukarram Hossain, Juan W. Valle, Mairéad G. McNamara, Caroline Dive. The first circulating tumor cell-derived explant (CDX) model of a Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3098.

International multi-center evaluation of a rapid antigen test based on gold immunochromatographic assay for detection of severe acute respiratory syndrome coronavirus 2

Objective: The gold immunochromatographic assay for detection of SARS-CoV-2 antigen was evaluated by international multi-center clinical trial. Methods: A total of 1 855 clinical parallel samples with valid test results (for nucleic acid and antigen tests, respectively) were collected from nine countries, including Germany, the United Kingdom, Ukraine, France, India, Thailand, Malaysia, the United States of America and Brazil, with sampling period from January 3, 2021 to September 22, 2021. These samples were detected by SARS-CoV-2 antigen test kit (colloidal gold immunochromatography assay) and nucleic acid detection kit (real-time fluorescent quantitative reverse transcription polymerase chain reaction). Positive coincidence rates [(number of antigen-positive cases/nucleic acid-positive cases)×100%], negative coincidence rates [(number of antigen-negative cases/nucleic acid-negative cases)×100%], total coincidence rates [(number of cases with consistent results for both antigen and nucleic acid detection/number of total cases) ×100%], as well as Kappa values were calculated. The differences of the above indictors among different countries were evaluated by the coefficient of variation. The detection rates of the antigen test for samples with different cycle threshold values (Ct values) for the nucleic acid detection, different characteristics and different mutant strains were analyzed. Results: For all samples, the positive, negative, and total coincidence rate between the antigen test and nucleic acid assay was 90.8% (569/627), 99.7% (1 224/1 228) and 96.7% (1 793/1 855), respectively, and the consistency coefficient Kappa value was 0.924. Among these countries, the coefficient of variation for positive coincidence rates (except for Malaysia with a lot of samples with Ct value>30), negative coincidence rates (except for France without negative samples) and total coincidence rates (except for France) was 6%,<1%, and 6%, respectively. When Ct values were less than 25, the detection rates of antigen test were 83.3%-100% for each countries (the coefficient of variation was 6%); The total detection rate and the coefficient of variation was 93.4% (428/458) and 5%, respectively, for asymptomatic infected persons and cases within 7 days post onset of symptoms; the total detection rate for various SARS-CoV-2 mutant strains was 97.5% (119/122); and it showed negative results for samples from cases infected with other viruses, including influenza A virus subtype H1N1, influenza B virus, respiratory syncytial virus subgroups A and B, coxsackievirus 16, human metapneumovirus, parainfluenza virus types 1 and 4, Epstein-Barr virus and adenovirus. Conclusion: The SARS-CoV-2 antigen test kit showed excellent authenticity, and there were few differences for its indictors among nine countries, therefore it can meet the needs of large-scale early screening of SARS-CoV-2 infection.