Biology subjects

Pasternak, J. A.

Publications and source records attributed to Pasternak, J. A..

4 recordsLinked to original sources

bioRxiv2026

A solid-state heater-imager for quantitative evaluation of colorimetric isothermal nucleic acid amplification on paper

Maintaining precise isothermal conditions in portable nucleic acid amplification tests (NAATs) is critical for reproducible results but remains challenging with conventional single-sided thin-film heaters, which exhibit temperature gradients and strong dependence on ambient conditions. To close this gap, we engineered ThermiQuant VitroMini, a dual-sided heater design that achieves volumetric-level temperature uniformity using thin-film heaters while preserving optical transparency for real-time colorimetric loop-mediated isothermal amplification (LAMP) analysis on microfluidic paper-based analytical devices ({micro}PADs). The device integrates two independently regulated indium tin oxide (ITO) heaters (8 {Omega} each) controlled by independent proportional-integral-derivative (PID) algorithms. Heaters were evaluated under controlled ambient environments of 4 {degrees}C (refrigerated), 23 {degrees}C (room temperature), and 50 {degrees}C (oven). Analytical tests were performed using a colorimetric LAMP assay targeting the SARS-CoV-2 orf7ab gene on {micro}PADs preloaded with dried LAMP reagents, with time-lapse images (30 seconds interval) analyzed via Amplimetrics software. VitroMini maintained 65 {+/-} 0.5 {degrees}C across 4 to 50 {degrees}C ambient conditions and achieved a limit of detection of 34 copies/reaction (4.5 copies/{micro}L) and limit of quantification of 1000 copies/reaction (133 copies/{micro}L), with quantification time (Tq) linearly correlated with log10 DNA concentration. Dual-sided heating eliminated temperature bias, condensation artifacts, and ambient-dependent variability while preserving optical transparency for real-time quantitative LAMP reaction. ThermiQuant VitroMini bridges the gap between benchtop volumetric heaters and portable diagnostic devices, offering a compact, low-power platform for quantitative colorimetric molecular analysis on paper with potential for decentralized and field-deployable applications.

bioengineering↗

bioRxiv2026

ThermiQuant(TM) MegaScan: High-throughput isothermal reactor with quantitative colorimetric readout for paper-based nucleic acid amplification tests

Isothermal nucleic acid amplification tests (NAATs), such as loop-mediated isothermal amplification (LAMP) implemented on microfluidic paper-based analytical devices ({micro}PADs), enable inexpensive and rapid ([≤]60 min) colorimetric molecular diagnostics; however, no existing instrument supports high-throughput (>100 reactions) quantitative analysis of colorimetric isothermal assays on paper substrates under controlled laboratory conditions. To address this gap, we developed ThermiQuant MegaScan, a scanner- and water-bath-based platform that accommodates a 160-reaction {micro}PAD cartridge, maintains uniform incubation at 65 {+/-} 0.5 {degrees}C, and enables real-time imaging every 30 s. We also developed accompanying software, Amplimetrics, for automated {micro}PAD detection and kinetic colorimetric analysis. Using paper-based colorimetric LAMP targeting the SARS-CoV-2 orf7ab region, the assay achieved a limit of detection at 95% probability (LOD95) of 34 copies per reaction (5 copies/{micro}L) and a limit of quantification (LOQ) of 250 copies per reaction (33 copies/{micro}L) using purified synthetic DNA targets, and achieved 72% sensitivity and 100% specificity relative to digital PCR (dPCR) for diluted human nasopharyngeal (NP) swab virus samples. We further evaluated the effects of viral and universal transport media (VTM/UTM) on assay performance and found that linear calibration derived from synthetic targets do not reliably translate to clinical samples in these media. Together, these results establish ThermiQuant MegaScan as a high-throughput laboratory research platform for standardized evaluation, optimization, and benchmarking of paper-based colorimetric nucleic acid amplification assays.

bioengineering↗

bioRxiv2025

Rapid High-Throughput Analysis of Bovine Skeletal Muscle Fiber Morphology via Automated Fluorescent Microscopy and MuscleBos software

Skeletal muscle tissue is comprised of many individual muscle cells (myofibers) that can be classified as different types based on their morphology, histochemistry, enzymatic reactivity, and biochemical characteristics. One of the most common methods of classification of muscle fiber type relies on the local expression of specific myosin heavy chain (MyHC) isoforms. Adult mammalian muscle fibers are generally categorized into four major types including I, IIA, IIX, and IIB. However, the distribution of these muscle fiber types varies across both different species and muscle groups within species, influencing muscle function and physiological responses. In bovine species, skeletal muscle plays a critical role in determining in-vivo metabolic physiological processes and impacting post-harvest meat quality traits. Immunostaining methods using isoform-specific MyHC antibodies have been widely adopted to characterize muscle fiber morphology. However, manual capture and analysis of immunofluorescent images of muscle fiber type staining is time consuming, labor-intensive, and potentially susceptible to investigator bias. To address these limitations, we established and validated a high-throughput method for the analysis of bovine muscle fiber morphology that combines automated fluorescent microscopy with high-content image analysis using a customized version of the MuscleJ plugin for FIJI/ImageJ that we named MuscleBos. This refined method enables rapid quantitative characterization of muscle fiber type profile and fiber type-specific myofiber cross-sectional area in bovine skeletal muscle tissue cross-sections. This methodology should enable valuable deeper insights into future studies of muscle composition in bovine species and its impact on in vivo animal physiology and meat science.

physiology↗

bioRxiv2024

Defining Cellular Diversity at the Swine Maternal-Fetal Interface Using Spatial Transcriptomics and Organoids

The placenta is a dynamic, embryo-derived organ essential for fetal growth and development. While all eutherian mammals have placentas composed of fetal-derived trophoblasts that mediate maternal-fetal exchange, their anatomical and histological structures vary across species due to evolutionary divergence. Despite the cellular heterogeneity of porcine trophoblasts in vivo, understanding the mechanisms driving porcine placental development has been limited by the lack of in vitro models replicating this heterogeneity. In this study, we derived swine trophoblast organoids (sTOs) from full-term porcine placentas, retaining key transcriptional signatures of in vivo trophoblasts. To identify conserved cell populations, we integrated Visium spatial transcriptomics from mid-gestation porcine placentas with single-cell transcriptomics from sTOs. Spatial transcriptomics revealed novel markers of the porcine uterus and placenta, enabling precise separation of histological structures at the maternal-fetal interface. The integration of tissue and sTO transcriptomics showed that sTOs spontaneously differentiate into distinct trophoblast populations, with conserved gene expression and cell communication programs. These findings demonstrate that sTOs recapitulate porcine placental trophoblast populations, offering a powerful model for advancing placentation research. Our work also provides a spatially resolved whole-transcriptome dataset of the porcine maternal-fetal interface, opening new avenues for discoveries in placental development, evolution, and health across mammals.

developmental biology↗