A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments

Microfluidics microFACS for Life Detection

A prototype micro-scale Fluorescent Activated Cell Sorter (microFACS) for life detection has been built and is undergoing testing. A functional miniature microfluidics instrument with the ability to remotely distinguish live or dead bacterial cells from abiotic particulates in ice or permafrost of icy bodies of the solar system would be of fundamental value to NASA. The use of molecular probes to obtain the bio-signature of living or dead cells could answer the most fundamental question of Astrobiology: Does life exist beyond Earth? The live-dead fluorescent stains to be used in the microFACS instrument function only with biological cell walls. The detection of the cell membranes of living or dead bacteria (unlike PAH's and many other Biomarkers) would provide convincing evidence of present or past life. This miniature device rapidly examine large numbers of particulates from a polar ice or permafrost sample and distinguish living from dead bacteria cells and biological cells from mineral grains and abiotic particulates and sort the cells and particulates based on a staining system. Any sample found to exhibit fluorescence consistent with living cells could then be used in conjunction with a chiral labeled release experiment or video microscopy system to seek addition evidence for cellular metabolism or motility. Results of preliminary testing and calibration of the microFACS prototype instrument system with pure cultures and enrichment assemblages of microbial extremophiles will be reported

IMMUNODEFICIENT R2G2 MOUSE STRAIN YIELDS SPLEENS WITH UNUSUAL CYTOARCHITECTURE AND SYMPATHETIC INNERVATION

The nervous system and immune system contact one another through two-way communication in order to establish and preserve homeostasis. The sympathetic neurotransmitter norepinephrine has an impact on how the immune system responds by affecting regional blood flow and activation of adrenergic receptors on leukocytes. Former studies showed that immune cells are capable of releasing nerve growth factor allowing for the establishment and continuation of sympathetic nerves in targeted tissues. From this gathered information, it was hypothesized that sympathetic nerves would prove to be less frequent in spleens from the immunodeficient R2G2 mouse strain (Envigo) when compared to 129P3/J (129) and C57BL/6 (C57) strains. R2G2 mice are an immunodeficient strain that lacks functional T, B, and natural killer cells. Ten to eleven week aged-matched male mice were measured by body weight, spleen weight, and temperature. Spleens were cut and fixed for histological investigation. Sympathetic nerves were labeled by immunostaining tyrosine hydroxylase (TH). Hematoxylin & eosin (H&E) was used to stain spleen sections in order to evaluate cytoarchitecture. Von Willebrand factor (VWF) was used to immunostain for megakaryocytes. R2G2 mice showed slightly higher temperatures and body weights but yielded a significantly smaller spleen weight (R2G2, 38.20 ± 1.48; 129, 65.08 ± 11.71; C57, 81.33 ± 8.38; P\u3c 0.0001, ANOVA). TH stain revealed sympathetic innervation in all strains but location and morphology differed in R2G2 mice compared to controls. Control spleens had nerves which entered white pulp regions of the spleen and were closely related to leukocytes. Fiber profiles in the controls were filamentous with small acute bends. R2G2 differed by having (TH+) nerve fibers more associated with arteries and less localized in the surrounding parenchyma. The fibers were abnormally swollen and held a more granular shape instead of a filamentous shape. The H&E stain showed clear red and white pulp zones in the control spleens with 129 showing more distinct germinal centers than C57. R2G2 H&E sections showed cytoarchitecture with indistinct pulp areas. VWF staining revealed R2G2 mice had an abundant amount of megakaryocytes versus control mice megakaryocyte counts (R2G2, 11.28 ± 3.87 per 20X field; 129, 1.73 ± 0.70; C57, 1.42 ± 0.13; P\u3c 0.0001, ANOVA) and extramedullary hematopoiesis was highly prominent. This evidence supports that leukocytes secrete neurotrophic factors or are vital to establishing normal growth of TH+ nerves toward the white pulp. Leukocytes may not be required for sympathetic innervation of blood vessels in the spleen, however, lack of leukocytes shows TH+ nerve fibers with abnormal morphology in severely immune threatened mice

Loss of Sympathetic Nerves in Spleens From Patients With End Stage Sepsis

The spleen is an important site for central regulation of immune function by noradrenergic sympathetic nerves, but little is known about this major region of neuroimmune communication in humans. Experimental studies using animal models have established that sympathetic innervation of the spleen is essential for cholinergic anti-inflammatory responses evoked by vagal nerve stimulation, and clinical studies are evaluating this approach for treating inflammatory diseases. Most data on sympathetic nerves in spleen derive from rodent studies, and this work has established that remodeling of sympathetic innervation can occur during inflammation. However, little is known about the effects of sepsis on spleen innervation. Our primary goals were to (i) localize noradrenergic nerves in human spleen by immunohistochemistry for tyrosine hydroxylase (TH), a specific noradrenergic marker, (ii) determine if nerves occur in close apposition to leukocytes, and (iii) determine if splenic sympathetic innervation is altered in patients who died from end stage sepsis. Staining for vesicular acetylcholine transporter (VAChT) was done to screen for cholinergic nerves. Archived paraffin tissue blocks were used. Control samples were obtained from trauma patients or patients who died after hemorrhagic stroke. TH + nerves were associated with arteries and arterioles in all control spleens, occurring in bundles or as nerve fibers. Individual TH + nerve fibers entered the perivascular region where some appeared in close apposition to leukocytes. In marked contrast, spleens from half of the septic patients lacked TH + nerves fibers and the average abundance of TH + nerves for the septic group was only 16% of that for the control group (control: 0.272 ± 0.060% area, n = 6; sepsis: 0.043 ± 0.026% area, n = 8; P \u3c 0.005). All spleens lacked cholinergic innervation. Our results provide definitive evidence for the distribution of noradrenergic nerves in normal human spleen and the first evidence for direct sympathetic innervation of leukocytes in human spleen. We also provide the first evidence for marked loss of noradrenergic nerves in patients who died from sepsis. Such nerve loss could impair neuroimmunomodulation and may not be limited to the spleen

Deletion of Neurturin Impairs Development of Cholinergic Nerves and Heart Rate Control in Postnatal Mouse Hearts

The neurotrophic factor neurturin is required for normal cholinergic innervation of adult mouse heart and bradycardic responses to vagal stimulation. Our goals were to determine effects of neurturin deletion on development of cardiac chronotropic and dromotropic functions, vagal baroreflex response, and cholinergic nerve density in nodal regions of postnatal mice. Experiments were performed on postnatal C57BL/6 wild-type (WT) and neurturin knockout (KO) mice. Serial electrocardiograms were recorded noninvasively from conscious pups using an ECGenie apparatus. Mice were treated with atenolol to evaluate and block sympathetic effects on heart rate (HR) and phenylephrine (PE) to stimulate the baroreflex. Immunohistochemistry was used to label cholinergic nerves in paraffin sections. WT and KO mice showed similar age-dependent increases in HR and decreases in PR interval between postnatal days (P) 2.5 and 21. Treatment with atenolol reduced HR significantly in WT and KO pups at P7.5. PE caused a reflex bradycardia that was significantly smaller in KO pups. Cholinergic nerve density was significantly less in nodal regions of P7.5 KO mice. We conclude that cholinergic nerves have minimal influence on developmental changes in HR and PR, QRS, and QTc intervals in mouse pups. However, cholinergic nerves mediate reflex bradycardia by 1 week postnatally. Deletion of neurturin impairs cholinergic innervation of the heart and the vagal efferent component of the baroreflex early during postnatal development

The Effects of Nicotine in the Neonatal Quinpirole Rodent Model of Psychosis: Neural Plasticity Mechanisms and Nicotinic Receptor Changes

Neonatal quinpirole (NQ) treatment to rats increases dopamine D2 receptor sensitivity persistent throughout the animal’s lifetime. In Experiment 1, we analyzed the role of α7 and α4β2 nicotinic receptors (nAChRs) in nicotine behavioral sensitization and on the brain-derived neurotrophic factor (BDNF) response to nicotine in NQ- and neonatally saline (NS)-treated rats. In Experiment 2, we analyzed changes in α7 and α4β2 nAChR density in the nucleus accumbens (NAcc) and dorsal striatum in NQ and NS animals sensitized to nicotine. Male and female Sprague-Dawley rats were neonatally treated with quinpirole (1 mg/kg) or saline from postnatal days (P)1–21. Animals were given ip injections of either saline or nicotine (0.5 mg/kg free base) every second day from P33 to P49 and tested on behavioral sensitization. Before each injection, animals were ip administered the α7 nAChR antagonist methyllycaconitine (MLA; 2 or 4 mg/kg) or the α4β2 nAChR antagonist dihydro beta erythroidine (DhβE; 1 or 3 mg/kg). Results revealed NQ enhanced nicotine sensitization that was blocked by DhβE. MLA blocked the enhanced nicotine sensitization in NQ animals, but did not block nicotine sensitization. NQ enhanced the NAcc BDNF response to nicotine which was blocked by both antagonists. In Experiment 2, NQ enhanced nicotine sensitization and enhanced α4β2, but not 7, nAChR upregulation in the NAcc. These results suggest a relationship between accumbal BDNF and α4β2 nAChRs and their role in the behavioral response to nicotine in the NQ model which has relevance to schizophrenia, a behavioral disorder with high rates of tobacco smoking

Ursinus College Alumni Journal, March 1967

The paradox of urbia • The joyful acceptance of technopolis • Toward a new kind of freedom • Bill Daniels leads a creative life of action in urbia • Who is educated to plan the urban environment? • From the President • Concerning Freeland Seminary: An alumna writes a history of Collegeville • Discovery in Europe, student style • Personal calls will climax \u2767 Centennial Fund • Candidates for office • Springtime is alumni time • Student magazine focuses on political affairs • A theory of non-interference • From the Perkiomen to the Potomac • In the land of the four 70s • Sporting scene: Wrestling; Indoor track; Cross country; Basketball; Women\u27s hockey; Other women\u27s sports; Scheduling correction • Campus clippings: Financial support; Student lounge a success; Selma sheriff speaks; The amazing Evening School; Questionnaires returned; Book on urbia still available; Honor graduate; Sign to show the way • Class notebook • Physicians Alumni Club of Ursinus College organizes • Weddings • Births • In memoriamhttps://digitalcommons.ursinus.edu/alumnijournal/1088/thumbnail.jp

A Single Cell Transcriptomics Map of Paracrine Networks in the Intrinsic Cardiac Nervous System

We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion, the right atrial ganglionic plexus (RAGP) that is a critical mediator of sinoatrial node (SAN) activity. This 3D representation of RAGP used neuronal tracing to extensively map the spatial distribution of the subset of neurons that project to the SAN. RNA-seq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to the central nervous system and a surprising finding that cholinergic and catecholaminergic markers are coexpressed, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. High-throughput qPCR of hundreds of laser capture microdissected single neurons confirmed these findings and revealed a high dimensionality of neuromodulatory factors that contribute to dynamic control of the heart. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease