Imaging reticulospinal neurons in the lamprey brainstem using calcium indicator

Abstract only availableImaging reticulospinal neurons in the lamprey brainstem using calcium indicator In the lamprey, a lower vertebrate, reticulospinal (RS) neurons in the brain are the output elements of the command system that activate spinal pattern generators and initiate swimming. In order to better understand the locomotor command system in the lamprey, it is necessary to determine the locations of neurons in the network, as well as their connectivity and patterns of activity. Calcium indicator dyes are an important technique for labeling and monitoring neuron activity. During impulse, calcium enters neurons and binds to the dye, increasing the fluorescence of the dye and creating an optical image that can be recorded and analyzed. In the present study, Calcium Green dextran amine was applied to the transected spinal cord at 20% body length (BL). After retrograde transport of the dye and labeling of RS neurons, the brain and spinal cord were removed and placed on a slide for viewing under a microscope equipped for fluorescence. Electrical stimulation of the spinal cord activated labeled RS neurons in the brain, resulting in a fluorescence increase that was recorded by an S-VHS video camera. The next step will be to image RS neuron activity during actual swimming movements. For this purpose, RS neurons will be labeled in a semi-intact preparation in which the brain and upper spinal cord are exposed and the lower half of the body is free to produce swimming movements. As a control experiment, the spinal cord was transected and Calcium Green applied at 60% BL. Semi-intact preparations were observed to produce swimming movements. Imaging of the isolated brain and rostral spinal cord showed RS neuron labeling and fluorescent changes similar to when tracer was applied at 20% BL. These results lay the groundwork for imaging brain neuron activity during actual swimming behavior.Life Sciences Undergraduate Research Opportunity Progra

Assessing biocompatibility of porcine tissue for hernia repair using flow cytometry [abstract]

Abstract only availableThe current standard for abdominal hernia repair uses synthetic meshes, which often break down, leading to complications and a high rate of recurrence. A biologic prosthetic, derived from animal tissue, would provide a more natural substrate for tissue remodeling and an improved host response. The success of a biologic material for tissue repair first depends on preventing an immune reaction post implantation. Engineering an appropriate construct requires removal of cells from the donor tissue, stabilization of the resulting collagen matrix with cross-linkers, and sterilization prior to implantation. The central tendon of the porcine diaphragm is a novel material for use as a biologic implant. This study is a preliminary investigation of the biocompatibility of porcine diaphragm as a hernia mesh material. Diaphragm tissue was de-cellularized by one of two methods and cross-linked with one of two chemical agents. Combinations of these two treatments were cultured with mouse fibroblast cells. Viability was assessed with flow cytometry, using propridium iodine to stain non-vital cells. Cell viability on treated tissue was compared to untreated tissue and control cell sets. Results indicate superior biocompatibility of one preparation, with viability high enough to warrant further studies. The next step will be implantation of the material into an animal model. With successful preparation, biological constructs can improve the host tissue response and reduce the need for revision surgery, thereby replacing conventional synthetic meshes for hernia repair.College of Engineering Undergraduate Research Optio

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Imaging reticulospinal neurons in the lamprey brainstem using calcium indicator [abstract]

Abstract only availableIn the lamprey, a lower vertebrate, reticulospinal (RS) neurons in the brain are the output elements of the command system that activate spinal pattern generators and initiate swimming. In order to better understand the locomotor command system in the lamprey, it is necessary to determine the locations of neurons in the network, as well as their connectivity and patterns of activity. Calcium indicator dyes are an important technique for labeling and monitoring neuron activity. During impulse, calcium enters neurons and binds to the dye, increasing the fluorescence of the dye and creating an optical image that can be recorded and analyzed. In the present study, Calcium Green dextran amine was applied to the transected spinal cord at 20% body length (BL). After retrograde transport of the dye and labeling of RS neurons, the brain and spinal cord were removed and placed on a slide for viewing under a microscope equipped for fluorescence. Electrical stimulation of the spinal cord activated labeled RS neurons in the brain, resulting in a fluorescence increase that was recorded by an S-VHS video camera. The next step will be to image RS neuron activity during actual swimming movements. For this purpose, RS neurons will be labeled in a semi-intact preparation in which the brain and upper spinal cord are exposed and the lower half of the body is free to produce swimming movements. As a control experiment, the spinal cord was transected and Calcium Green applied at 60% BL. Semi-intact preparations were observed to produce swimming movements. Imaging of the isolated brain and rostral spinal cord showed RS neuron labeling and fluorescent changes similar to when tracer was applied at 20% BL. These results lay the groundwork for imaging brain neuron activity during actual swimming behavior.Life Sciences Undergraduate Research Opportunity Progra