Optimization of PCR conditions to amplify microsatellite loci in the bunchgrass lizard (Sceloporus slevini) genomic DNA

<p>Abstract</p> <p>Background</p> <p>Microsatellites, also called Simple Sequence Repeats (SSRs), repetitions of nucleotide motifs of 1-5 bases, are currently the markers of choice due to their abundant distribution in the genomes, and suitability for high-throughput analysis. A total of five different primer pairs were optimized for polymerase chain reaction (PCR) to amplify microsatellite loci in total genomic DNA of bunchgrass lizards (<it>Sceloporus slevini</it>) collected from three sites in southeastern Arizona; the Sonoita Plain, Chiricahua Mountains and Huachuca Mountains.</p> <p>Findings</p> <p>The primers used for current investigation were originally designed for the Eastern Fence Lizard (<it>Sceloporus undulatus</it>). Five primer pairs were selected based on annealing temperatures for optimizing the PCR conditions to amplify with bunchgrass lizards. Different concentrations of DNA and annealing temperature were optimized. While keeping other reagents constant, a DNA concentration, 37.5 ng in the final reaction volume and PCR conditions of an initial denaturation of 94°C for five minutes, an annealing temperature of 55°C and final extension of 72°C for four minutes gave the best amplification for all the primer pairs.</p> <p>Conclusions</p> <p>Modifying the standard protocol for annealing temperatures and final extension time increases the success of cross amplification of specific microsatellite loci in the bunchgrass lizard. A loading volume of 5 ul DNA at a concentration of 10 ng/ul and a 2% agarose for gel electrophoresis were observed the best for cross amplification of selected five primer pairs on bunch grass lizard.</p> <p>Trial Registration</p> <p>The research was conducted with Arizona Game and Fish Department scientific collecting permits SP565256, SP657407 & SP749119 to Dr. Christian A d'Orgeix.</p

ERK Is Involved in the Reorganization of Somatosensory Cortical Maps in Adult Rats Submitted to Hindlimb Unloading

Sensorimotor restriction by a 14-day period of hindlimb unloading (HU) in the adult rat induces a reorganization of topographic maps and receptive fields. However, the underlying mechanisms are still unclear. Interest was turned towards a possible implication of intracellular MAPK signaling pathway since Extracellular-signal-Regulated Kinase 1/2 (ERK1/2) is known to play a significant role in the control of synaptic plasticity. In order to better understand the mechanisms underlying cortical plasticity in adult rats submitted to a sensorimotor restriction, we analyzed the time-course of ERK1/2 activation by immunoblot and of cortical reorganization by electrophysiological recordings, on rats submitted to hindlimb unloading over four weeks. Immunohistochemistry analysis provided evidence that ERK1/2 phosphorylation was increased in layer III neurons of the somatosensory cortex. This increase was transient, and parallel to the changes in hindpaw cortical map area (layer IV). By contrast, receptive fields were progressively enlarged from 7 to 28 days of hindlimb unloading. To determine whether ERK1/2 was involved in cortical remapping, we administered a specific ERK1/2 inhibitor (PD-98059) through osmotic mini-pump in rats hindlimb unloaded for 14 days. Results demonstrate that focal inhibition of ERK1/2 pathway prevents cortical reorganization, but had no effect on receptive fields. These results suggest that ERK1/2 plays a role in the induction of cortical plasticity during hindlimb unloading

Motor-Cortical Interaction in Gilles de la Tourette Syndrome

BACKGROUND: In Gilles de la Tourette syndrome (GTS) increased activation of the primary motor cortex (M1) before and during movement execution followed by increased inhibition after movement termination was reported. The present study aimed at investigating, whether this activation pattern is due to altered functional interaction between motor cortical areas. METHODOLOGY/PRINCIPAL FINDINGS: 10 GTS-patients and 10 control subjects performed a self-paced finger movement task while neuromagnetic brain activity was recorded using Magnetoencephalography (MEG). Cerebro-cerebral coherence as a measure of functional interaction was calculated. During movement preparation and execution coherence between contralateral M1 and supplementary motor area (SMA) was significantly increased at beta-frequency in GTS-patients. After movement termination no significant differences between groups were evident. CONCLUSIONS/SIGNIFICANCE: The present data suggest that increased M1 activation in GTS-patients might be due to increased functional interaction between SMA and M1 most likely reflecting a pathophysiological marker of GTS. The data extend previous findings of motor-cortical alterations in GTS by showing that local activation changes are associated with alterations of functional networks between premotor and primary motor areas. Interestingly enough, alterations were evident during preparation and execution of voluntary movements, which implies a general theme of increased motor-cortical interaction in GTS

Beneficial Effects of Estrogen in a Mouse Model of Cerebrovascular Insufficiency

BACKGROUND: The M(5) muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M(5) muscarinic acetylcholine knockout mice (M5R(-/-) mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain. METHODOLOGY/PRINCIPAL FINDINGS: We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R(-/-) mice. Here we show that female M5R(-/-) mice did not show the reduction in vascular area observed in male M5R(-/-) mice. However, ovariectomized female M5R(-/-) mice displayed phenotypic changes similar to male M5R(-/-) mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17beta-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERalpha, ERbeta, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R(-/-) mice treated with E2. Treatment with E2 also improved the performance of male M5R(-/-) mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R(-/-) mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that M5R(-/-) mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction

Atmospheric electrification in dusty, reactive gases in the solar system and beyond

Detailed observations of the solar system planets reveal a wide variety of local atmospheric conditions. Astronomical observations have revealed a variety of extrasolar planets none of which resembles any of the solar system planets in full. Instead, the most massive amongst the extrasolar planets, the gas giants, appear very similar to the class of (young) Brown Dwarfs which are amongst the oldest objects in the universe. Despite of this diversity, solar system planets, extrasolar planets and Brown Dwarfs have broadly similar global temperatures between 300K and 2500K. In consequence, clouds of different chemical species form in their atmospheres. While the details of these clouds differ, the fundamental physical processes are the same. Further to this, all these objects were observed to produce radio and X-ray emission. While both kinds of radiation are well studied on Earth and to a lesser extent on the solar system planets, the occurrence of emission that potentially originate from accelerated electrons on Brown Dwarfs, extrasolar planets and protoplanetary disks is not well understood yet. This paper offers an interdisciplinary view on electrification processes and their feedback on their hosting environment in meteorology, volcanology, planetology and research on extrasolar planets and planet formation