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Repeatability of measurements from image analytics is difficult, due to the heterogeneity and complexity of cell samples, exact microscope stage positioning, and slide thickness. We present a method to define and use a reference focal plane that provides repeatable measurements with very high accuracy, by relying on control beads as reference material and a convolutional neural network focused on the control bead images. Previously we defined a reference effective focal plane (REFP) based on the image gradient of bead edges and three specific bead image features. This paper both generalizes and improves on this previous work. First, we refine the definition of the REFP by fitting a cubic spline to describe the relationship between the distance from a bead's center and pixel intensity and by sharing information across experiments, exposures, and fields of view. Second, we remove our reliance on image features that behave differently from one instrument to another. Instead, we apply a convolutional regression neural network (ResNet 18) trained on cropped bead images that is generalizable to multiple microscopes. Our ResNet 18 network predicts the location of the REFP with only a single inferenced image acquisition that can be taken across a wide range of focal planes and exposure times. We illustrate the different strategies and hyperparameter optimization of the ResNet 18 to achieve a high prediction accuracy with an uncertainty for every image tested coming within the microscope repeatability measure of 7.5 µm from the desired focal plane. We demonstrate the generalizability of this methodology by applying it to two different optical systems and show that this level of accuracy can be achieved using only 6 beads per image., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

DNA templates for protein production remain an unexplored source of variability in the performance of cell-free expression (CFE) systems. To characterize this variability, we investigated the effects of two common DNA extraction methodologies, a postprocessing step and manual versus automated preparation on protein production using CFE. We assess the concentration of the DNA template, the quality of the DNA template in terms of physical damage and the quality of the DNA solution in terms of purity resulting from eight DNA preparation workflows. We measure the variance in protein titer and rate of protein production in CFE reactions associated with the biological replicate of the DNA template, the technical replicate DNA solution prepared with the same workflow and the measurement replicate of nominally identical CFE reactions. We offer practical guidance for preparing and characterizing DNA templates to achieve acceptable variability in CFE performance., (Published by Oxford University Press 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants for which human exposure remains ubiquitous. This is of concern since these chemicals can perturb development and cause adverse health effects. For instance, DE-71, a technical mixture of PBDEs, can induce liver toxicity as well as reproductive and developmental toxicity. DE-71 can also disrupt the thyroid hormone (TH) system which may induce developmental neurotoxicity indirectly. However, in developmental toxicity studies, it remains unclear how DE-71 exposure affects the offspring's thyroid hormone system and if this dose-dependently relates to neurodevelopmental effects. To address this, we performed a rat toxicity study by exposing pregnant dams to DE-71 at 0, 40 or 60 mg/kg/day during perinatal development from gestational day 7 to postnatal day 16. We assessed the TH system in both dams and their offspring, as well as potential hearing and neurodevelopmental effects in prepubertal and adult offspring. DE-71 significantly reduced serum T4 and T3 levels in both dams and offspring without a concomitant upregulation of TSH, thus inducing a hypothyroxinemia-like effect. No discernible effects were observed on the offspring's brain function when assessed in motor activity boxes and in the Morris water maze, or on offspring hearing function. Our results, together with a thorough review of the literature, suggest that DE-71 does not elicit a clear dose-dependent relationship between low serum thyroxine (T4) and effects on the rat brain in standard behavioral assays. However, low serum TH levels are in themselves believed to be detrimental to human brain development, thus we propose that we lack assays to identify developmental neurotoxicity caused by chemicals disrupting the TH system through various mechanisms., Competing Interests: The authors have declared that no competing interests exist.

In the United States, footwear examiners make decisions about the sources of crime scene shoe impressions using subjective criteria. This has raised questions about the accuracy, repeatability, reproducibility, and scientific validity of footwear examinations. Currently, most footwear examiners follow a workflow that compares a questioned and test impression with regard to outsole design, size, wear, and randomly acquired characteristics (RACs). We augment this workflow with computer algorithms and statistical analysis so as to improve in the following areas: (1) quantifying the degree of correspondence between the questioned and test impressions with respect to design, size, wear, and RACs, (2) reducing the potential for cognitive bias, and (3) providing an empirical basis for examiner conclusions by developing a reference database of case-relevant pairs of impressions containing known mated and known nonmated impressions. Our end-to-end workflow facilitates all three of these points and is directly relatable to current practice. We demonstrate the workflow, which includes obtaining and interpreting outsole pattern scores, RAC comparison scores, and final scores, on two scenarios-a pristine example (involving very high quality Everspry EverOS scanner impressions) and a mock crime scene example that more closely resembles real casework. These examples not only demonstrate the workflow but also help identify the algorithmic, computational, and statistical challenges involved in improving the system for eventual deployment in casework., (© 2021 American Academy of Forensic Sciences. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)