BIOM-13. DEVELOPMENT AND VALIDATION OF AN LC-MS/MS ASSAY TO MEASURE GLUTATHIONE, GLUTATHIONE DISULFIDE, CYSTEINE, AND CYSTINE IN HUMAN BRAIN AND HUMAN GLIOBLASTOMA

BACKGROUND Oxidative stress is implicated in many pathological conditions. Herein, we report on our development and validation of a sensitive and rapid LC-MS/MS method for the determination of oxidative stress biomarkers glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys) and cystine (CySS) in human brain and glioblastoma tissue. METHODS Freshly-acquired human glioblastoma tissue was homogenized with N-ethylmaleimide solution to prevent thiol oxidation. Analytes were then extracted from homogenate samples by protein precipitation with 2% sulfosalicylic acid (SSA). Stable isotope-labeled analytes were used as internal standards. Independent calibration curves for thiols and disulfides were prepared in analytical solutions. Three levels of quality controls were prepared in human brain homogenate. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS Linear regression model was used to cover a concentration ranging 0.4-100 µmol/L for GSSG/CySS and 1-400 µmol/L for GSH/Cys. Chromatographic separation was optimized on Intrada Amino Acid column with total run time of 5 min using gradient elution. For all analytes the maximum coefficient of variation for intra- and inter-day precision was 11.4% and the accuracy was within 80.9-113.7% in analytical solution and matrix. The analytes were stable in brain homogenate for 1 hour and 3 hours at room temperature (RT) and 4 °C, respectively. Stability at -80°C was demonstrated for at least 35 days in human brain homogenate. Stability of stock and working solutions was demonstrated for at least 4 hours (RT) and 25 days (-20°C). CONCLUSIONS A bioanalytical method to quantify GSH, GSSG, Cys, and CySS is successfully developed and validated. The method is currently applied to measure thiols and related disulfides in human glioblastoma tissue undergoing 5-aminolevulinic acid sonodynamic therapy (NCT04559685).

Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

Numerical calibration of railway bridge based on measurement data

<p>Bridges rarely behave precisely according to design assumptions. In most cases, they have some hidden reserves and behave preferable under traffic loads. To take these benefits into consideration numerical models can be calibrated based on measured structural response. The case study presented herein shows the calibration process for a railway truss bridge in Austria and the comparison of calibration results obtained by two individual teams. Each team did an individual and independent calibration based on different finite element models based on measured train passages. Both calibrations improved the precision of the calculated model response compared to the initial model, but also showed that the calibration parameters must be chosen with care to ensure plausibility of the results.</p>

Base-substitution mutation rate across the nuclear genome of Alpheus snapping shrimp and the timing of isolation by the Isthmus of Panama

The formation of the Isthmus of Panama and final closure of the Central American Seaway (CAS) provides an independent calibration point for examining the rate of DNA substitutions. This vicariant event has been widely used to estimate the substitution rate across mitochondrial genomes and to date evolutionary events in other taxonomic groups. Nuclear sequence data is increasingly being used to complement mitochondrial datasets for phylogenetic and evolutionary investigations; these studies would benefit from information regarding the rate and pattern of DNA substitutions derived from the nuclear genome. To estimate this genomewide neutral mutation rate (μ), genotype-by-sequencing (GBS) datasets were generated for three transisthmian species pairs in Alpheus snapping shrimp. Using a Bayesian coalescent approach (G-PhoCS) applied to 44,960 GBS loci, we estimated μ to be 2.64E-9 substitutions/site/year, when calibrated with the closure of the CAS at 3 Ma. This estimate is remarkably similar to experimentally derived mutation rates in model arthropod systems, strengthening the argument for a recent closure of the CAS. To our knowledge this is the first use of transisthmian species pairs to calibrate the rate of molecular evolution from GBS data.

Spine Cop: Posture Correction Monitor and Assistant

Back and spine-related issues are frequent maladies that most people have or will experience during their lifetime. A common and sensible observation that can be made is regarding the posture of an individual. We present a new approach that combines accelerometer, gyroscope, and magnetometer sensor data in combination with permanent magnets assembled as a wearable device capable of real-time spine posture monitoring. An independent calibration of the device is required for each user. The sensor data is processed by a probabilistic classification algorithm that compares the real-time data with the calibration result, verifying whether the data point lies within regions of confidence defined by a computed threshold. An incorrect posture classification is considered if both accelerometer and magnetometer classify the posture as incorrect. A pilot trial was performed in a single adult test subject. The combination of the magnets and magnetometer greatly improved the posture classification accuracy (89%) over the accuracy obtained when only accelerometer data were used (47%). The validation of this method was based on image analysis.

The ESA Permanent Facility for Altimetry Calibration: Monitoring Performance of Radar Altimeters for Sentinel-3A, Sentinel-3B and Jason-3 Using Transponder and Sea-Surface Calibrations with FRM Standards

This work presents the latest calibration results for the Copernicus Sentinel-3A and -3B and the Jason-3 radar altimeters as determined by the Permanent Facility for Altimetry Calibration (PFAC) in west Crete, Greece. Radar altimeters are used to provide operational measurements for sea surface height, significant wave height and wind speed over oceans. To maintain Fiducial Reference Measurement (FRM) status, the stability and quality of altimetry products need to be continuously monitored throughout the operational phase of each altimeter. External and independent calibration and validation facilities provide an objective assessment of the altimeter’s performance by comparing satellite observations with ground-truth and in-situ measurements and infrastructures. Three independent methods are employed in the PFAC: Range calibration using a transponder, sea-surface calibration relying upon sea-surface Cal/Val sites, and crossover analysis. Procedures to determine FRM uncertainties for Cal/Val results have been demonstrated for each calibration. Biases for Sentinel-3A Passes No. 14, 278 and 335, Sentinel-3B Passes No. 14, 71 and 335, as well as for Jason-3 Passes No. 18 and No. 109 are given. Diverse calibration results by various techniques, infrastructure and settings are presented. Finally, upgrades to the PFAC in support of the Copernicus Sentinel-6 ‘Michael Freilich’, due to launch in November 2020, are summarized.

A fast calibration approach for onboard LiDAR-camera systems

Outdoor surveillance and security robots have a wide range of industrial, military, and civilian applications. In order to achieve autonomous navigation, the LiDAR-camera system is widely applied by outdoor surveillance and security robots. The calibration of the LiDAR-camera system is essential and important for robots to correctly acquire the scene information. This article proposes a fast calibration approach that is different from traditional calibration algorithms. The proposed approach combines two independent calibration processes, which are the calibration of LiDAR and the camera to robot platform, so as to address the relationship between LiDAR sensor and camera sensor. A novel approach to calibrate LiDAR to robot platform is applied to improve accuracy and robustness. A series of indoor experiments are carried out and the results show that the proposed approach is effective and efficient. At last, it is applied to our own outdoor security robot platform to detect both positive and negative obstacles in a field environment, in which two Velodyne-HDL-32 LiDARs and a color camera are employed. The real application illustrates the robustness performance of the proposed approach.

Performance Comparison of Three Density-Independent Calibration Functions for Microwave Moisture Sensing in Unshelled Peanuts during Drying

HighlightsReal-time, free-space transmission measurements of relative complex permittivity in unshelled peanuts during dryingDynamic application: temperature, density and moisture content changing during dryingThree density-independent calibration functions evaluated for accuracy in real-time moisture content determinationReal-time moisture content determination with standard error of performance (SEP) = 0.55% moisture content for all calibration functionsCalibration function most commonly used with microwave moisture sensor was determined to be most accurate; SEP = 0.448% moisture contentAbstract. A microwave moisture sensor, developed within USDA ARS, has been used to determine moisture content in unshelled peanuts during drying. Relative complex permittivities of the peanuts obtained from free-space transmission measurements at 5.8 GHz are used for the moisture determination. Due to variations in density caused by drying, it is advantageous to estimate moisture content independent of bulk density. Therefore, moisture content was estimated with three density-independent calibration functions to assess which one provided optimal accuracy. One of the functions is based on the measured attenuation and phase shift, and the other two are permittivity based (one of which is commonly used with the microwave moisture sensor). The sensor was calibrated for peanut pod moisture content determination over a temperature range of 10°C to 40°C and a moisture content range of 6.5% to 19% wet basis (w.b.). Statistical analysis showed high coefficients of determination (r2), = 0.97 for the calibration with each function. Peanut pod moisture content was determined with the sensor in real-time as peanuts dried, and estimated moisture content was compared to the reference oven drying method. While the standard error of performance (SEP) for the three functions was = 0.55% moisture content, the calibration function most commonly used with the microwave sensor was observed to be the most accurate (SEP = 0.448% moisture content). Microwave sensing is a viable solution for nondestructive, real-time determination of moisture content in peanuts in dynamic situations such as drying. Keywords: Complex permittivity, Dielectric properties, Free-space measurements, Microwave sensing, Moisture content, Peanut drying.