Rh Glycoprotein as an Ammonia Transport Molecule

Fish use their gills to excrete ammonia in order to eliminate nitrogenous waste. We hypothesize that this mechanism is accomplished by one or more transport proteins in the Rh glycoprotein (RhxG) family. Longhorn sculpin (Myoxocephalus octodecemspinosus) cDNA was amplified using polymerase chain reaction (PCR) and then the PCR products were visualized on an agarose gel. The cDNA from the gel bands was then sequenced and the gene sequence fragments were assembled and completed by rapid amplification of the cDNA ends (RACE). By this process we have obtained large portions of the gene sequences of the four known paralogues located in the sculpin gill (RhA, RhB, RhC1, and RhC2). Also, in vivo ammonia-loading experiments were done to determine the effect of increased internal ammonia on protein and mRNA expression. Treatment groups were exposed to a single ammonium bicarbonate, distilled water, or ammonium chloride (5 mM kg-1) infusion; then gill tissue was collected 4 hr postinfusion and analyzed using quantitative PCR to test changes in mRNA levels and dot blots for changes in RhxG protein levels. Preliminary QPCR data showed a trend of increase in response to ammonia loading. A second infusion test, with a chronic (8 hr) double load of ammonium bicarbonate, was completed with QPCR and dot blot analysis done on the gill tissue. Ambient water samples were also collected to determine in vivo ammonia efflux. In conclusion, from this data we have found protein expression changes in response to increased internal ammonia

Regulation of NMDA Receptor channel block and desensitization by intracellular calcium

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels found at nearly all vertebrate excitatory synapses that contribute to a multitude of nervous system functions. Unique biophysical properties, including high Ca2+ permeability, voltage-dependent Mg2+ block, and slow gating kinetics, allow NMDARs to control the magnitude and timing of Ca2+ influx following synaptic events. Ca2+ influx through NMDARs drives an array of signaling pathways that regulate critical neuronal functions such as synaptic plasticity and cell survival. Abnormal NMDAR activity is involved in a remarkable range of nervous system disorders including schizophrenia, major depressive disorder, stroke, neuropathic pain, and neurodegenerative diseases. Specifically, NMDAR overactivation can lead to accumulation of toxic levels of Ca2+¬¬¬ that initate cell death signaling pathways. Because of the core involvement of NMDARs in normal brain physiology as well as brain pathologies, NMDARs are attractive targets for neurotherapeutic drugs. The NMDAR channel blocker memantine, a clinically approved treatment for Alzheimer’s disease, displays a combination of clinical utility and tolerability unique amongst NMDAR antagonists. We recently discovered that memantine enhances NMDAR desensitization by stabilizing a Ca2+-dependent desensitized receptor state. Stabilization of a Ca2+-dependent state by memantine offers a rational mechanism by which memantine can target specific NMDAR subpopulations involved in disease: preferential inhibition of NMDARs in neurons experiencing long durations of high Ca2+ influx. Therefore, we systematically investigated the relation between channel blocker potency, intracellular Ca2+ concentration ([Ca2+]i, and NMDAR desensitization. We found that while potency of memantine depended on [Ca2+]i, the potency of another clinically useful channel blocker, ketamine, was [Ca2+]i-independent. Utilizing this discrepancy, we compared the memantine and ketamine binding sites and identified a residue in the NMDAR transmembrane domain that strongly contributes to NMDAR desensitization and memantine potency. Lastly, we characterized novel NMDAR channel blockers and discovered that potency of a memantine derivative was also dependent on [Ca2+]i. The data presented in this dissertation provide key insight into how [Ca2+]i affects channel blocker activity and NMDAR desensitization, and ultimately improve our understanding of the structural and functional mechanisms underlying the effects of channel blocking drugs on NMDAR function

Adaptive foveated single-pixel imaging with dynamic super-sampling

As an alternative to conventional multi-pixel cameras, single-pixel cameras enable images to be recorded using a single detector that measures the correlations between the scene and a set of patterns. However, to fully sample a scene in this way requires at least the same number of correlation measurements as there are pixels in the reconstructed image. Therefore single-pixel imaging systems typically exhibit low frame-rates. To mitigate this, a range of compressive sensing techniques have been developed which rely on a priori knowledge of the scene to reconstruct images from an under-sampled set of measurements. In this work we take a different approach and adopt a strategy inspired by the foveated vision systems found in the animal kingdom - a framework that exploits the spatio-temporal redundancy present in many dynamic scenes. In our single-pixel imaging system a high-resolution foveal region follows motion within the scene, but unlike a simple zoom, every frame delivers new spatial information from across the entire field-of-view. Using this approach we demonstrate a four-fold reduction in the time taken to record the detail of rapidly evolving features, whilst simultaneously accumulating detail of more slowly evolving regions over several consecutive frames. This tiered super-sampling technique enables the reconstruction of video streams in which both the resolution and the effective exposure-time spatially vary and adapt dynamically in response to the evolution of the scene. The methods described here can complement existing compressive sensing approaches and may be applied to enhance a variety of computational imagers that rely on sequential correlation measurements.Comment: 13 pages, 5 figure

Geometric Entropy for Lead vs Top-Rope Rock Climbing

International Journal of Exercise Science 9(2): 168-174, 2016. The complexity of movement of a rock climber’s center of mass during an ascent has been described as geometric entropy (GE). It has been proposed that lower geometric entropy could represent more fluid and economical movement during climbing. The purpose of the present study was to measure GE during rock climbing ascents under a lead condition (LD), where the climber connects a safety rope to several intermediate anchors during the ascent and under a top-rope condition (TR), where the safety rope is always anchored above the climber. Six experienced rock climbers volunteered to participate in the study. Each participant ascended a route on natural rock outdoors under three conditions. The first ascent was performed in a top-rope condition as an accommodation trial. The two remaining ascents were performed as LD and top-rope (TR2) in random order. Each LD and TR2 ascent was recorded via digital video at 30 Hz. A single point at the back center of each climber’s waist harness was manually digitized from the video images at 6 Hz and interpreted as the climber’s center of mass (CM). The displacement of CM was expressed as the line of motion (LM). Geometric Entropy (GE) was calculated as GE = ln((2∙LM)/CH)), where CH was the value of the convex hull about the LM. A within subjects, repeated measures ANOVA with Bonferroni post hoc testing was utilized to test for differences among ascent conditions with significance set at P \u3c0.05. Mean (±s.d) values for LM and GE were 81.5±11.3 m vs 77.6±7.3 m and 1.021±0.133 vs 0.924±0.062 for LD and TR2 respectively. There were no significant differences for LM and GE between ascent conditions. It was concluded that LM and GE do not vary between LD and TR ascent conditions

The Effects of Surgical Antiseptics and Time Delays on RNA Isolated From Human and Rodent Peripheral Nerves

Peripheral Nerve Injury (PNI) is common following blunt or penetrating trauma with an estimated prevalence of 2% among the trauma population. The resulting economic and societal impacts are significant. Nerve regeneration is a key biological process in those recovering from neural trauma. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) and RNA sequencing (RNA seq) are investigative methods that are often deployed by researchers to characterize the cellular and molecular mechanisms that underpin this process. However, the ethical and practical challenges associated with studying human nerve injury have meant that studies of nerve injury have largely been limited to rodent models of renervation. In some circumstances it is possible to liberate human nerve tissue for study, for example during reconstructive nerve repair. This complex surgical environment affords numerous challenges for optimizing the yield of RNA in sufficient quantity and quality for downstream RT-qPCR and/or RNA seq applications. This study characterized the effect of: (1) Time delays between surgical liberation and cryopreservation and (2) contact with antiseptic surgical reagents, on the quantity and quality of RNA isolated from human and rodent nerve samples. It was found that time delays of greater than 3 min between surgical liberation and cryopreservation of human nerve samples significantly decreased RNA concentrations to be sub-optimal for downstream RT-qPCR/RNA seq applications (<5 ng/μl). Minimizing the exposure of human nerve samples to antiseptic surgical reagents significantly increased yield of RNA isolated from samples. The detrimental effect of antiseptic reagents on RNA yield was further confirmed in a rodent model where RNA yield was 8.3-fold lower compared to non-exposed samples. In summary, this study has shown that changes to the surgical tissue collection protocol can have significant effects on the yield of RNA isolated from nerve samples. This will enable the optimisation of protocols in future studies, facilitating characterisation of the cellular and molecular mechanisms that underpin the regenerative capacity of the human peripheral nervous system

Identification of single nucleotide polymorphisms from the transcriptome of an organism with a whole genome duplication

BACKGROUND: The common ancestor of salmonid fishes, including rainbow trout (Oncorhynchus mykiss), experienced a whole genome duplication between 20 and 100 million years ago, and many of the duplicated genes have been retained in the trout genome. This retention complicates efforts to detect allelic variation in salmonid fishes. Specifically, single nucleotide polymorphism (SNP) detection is problematic because nucleotide variation can be found between the duplicate copies (paralogs) of a gene as well as between alleles. RESULTS: We present a method of differentiating between allelic and paralogous (gene copy) sequence variants, allowing identification of SNPs in organisms with multiple copies of a gene or set of genes. The basic strategy is to: 1) identify windows of unique cDNA sequences with homology to each other, 2) compare these unique cDNAs if they are not shared between individuals (i.e. the cDNA is homozygous in one individual and homozygous for another cDNA in the other individual), and 3) give a “SNP score” value between zero and one to each candidate sequence variant based on six criteria. Using this strategy we were able to detect about seven thousand potential SNPs from the transcriptomes of several clonal lines of rainbow trout. When directly compared to a pre-validated set of SNPs in polyploid wheat, we were also able to estimate the false-positive rate of this strategy as 0 to 28% depending on parameters used. CONCLUSIONS: This strategy has an advantage over traditional techniques of SNP identification because another dimension of sequencing information is utilized. This method is especially well suited for identifying SNPs in polyploids, both outbred and inbred, but would tend to be conservative for diploid organisms

Social competence in pediatric brain tumor survivors: application of a model from social neuroscience and developmental psychology.

Pediatric brain tumor (BT) survivors are at risk for psychosocial late effects across many domains of functioning, including neurocognitive and social. The literature on the social competence of pediatric BT survivors is still developing and future research is needed that integrates developmental and cognitive neuroscience research methodologies to identify predictors of survivor social adjustment and interventions to ameliorate problems. This review discusses the current literature on survivor social functioning through a model of social competence in childhood brain disorder and suggests future directions based on this model. Interventions pursuing change in survivor social adjustment should consider targeting social ecological factors