Evaluation of a Vivo-Morpholino Delivery Method to the Brain and the Affect on Physical Activity

Evaluation of a Vivo-Morpholino Delivery Method to the Brain and the Affect on Physical Activity *David P. Ferguson MS, Emily E. Schmitt MS, J. Timothy Lightfoot PhD FACSM Biology of Physical Activity Lab, Texas A&M University, College Station, TX, 77843 *To be judged in the doctoral category Physical inactivity has been shown to be correlated to various disease and conditions. Therefore, there is interest in the genetic mechanisms that control daily physical activity. Vivo-morpholinos are a new molecular biology tool that allows for the transient silencing of specific genes in an animal model, thereby allowing for a systematic method to turn off potential candidate genes involved in the regulation of physical activity. Vivo-morpholinos have not been shown to be effective at silencing genes in the brain due to the fact that the vivo-morpholino cannot cross the blood brain barrier. To counteract this, a tail vein injection (55 ul total volume; 11mg/kg vivo-morpholino; 6.5ug/kg RMP7) was given on three consecutive days containing the bradykinin analog RMP7 and a vivo-morpholino targeting Vmat2 to male C57/LJ mice (n=6). RMP7 has been shown to increase blood brain barrier permeability while Vmat2 is a dopamine transporter and is thought to be involved in the regulation of voluntary physical activity. Control animals received either RMP7 plus saline (n=6) or RMP7 plus a vivo-morpholino “scramble” control (n=6). Physical activity was measured by wheel running. Results showed there was not a significant (p=0.24) knockdown in Vmat2 in the brain with RMP7 administration as compared to control animals. Interestingly there was a significant (p=0.001) knockdown in daily physical activity in the Vmat2 treated animals compared to the control group. RMP7 may still be a viable option for vivo-morpholino delivery in the brain; however an increased dosage may be required

Transiently silencing genes associated with voluntary physical activity using intravenous injection of Vivo‐morpholinos

Physical inactivity has been associated with several diseases and conditions with multiple candidate genes proposed to regulate voluntary physical activity. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. The novel molecular biology tool, Vivo‐morpholinos, is a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo‐morpholinos in a mouse model for voluntary physical activity with several sub‐objectives. We observed that Vivomorpholinos achieved between 60 – 97% knockdown of Drd1‐, Vmat2‐, and Glut4‐protein in skeletal muscle, the delivery moiety of Vivo‐morpholinos (scramble) did not influence physical activity and that a cocktail of multiple Vivo‐morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo‐morpholinos injected intravenously alone did not significantly knockdown Vmat2‐protein expression in the brain (p=0.28). However, the use of a bradykinin analog to increase blood‐brain‐barrier permeability in conjunction with the Vivomorpholinos significantly (p=0.0001) decreased Vmat2‐protein in the brain with a corresponding later over‐expression of Vmat2 coincident with a significant (p=0.0016) increase in physical activity. We conclude that with appropriate research design, Vivo‐morpholinos can be a valuable tool in determining causal gene‐phenotype relationships in whole animal models

The Correlation of Physical Activity and Body Composition in Inbred Mice

The Correlation of Physical Activity and Body Composition in Inbred Mice. Analisa .M Jimenez*; David .P Ferguson MS RCEP; J. Timothy Lightfoot, PhD FACSM RCEPCES I would like to participate in the undergraduate category. Biology of Physical Activity Lab, Texas A&M University, College Station TX, 77843-4243 Obesity is a growing epidemic related to physical inactivity, cardiovascular disease, and Type II diabetes. In smaller mouse cohorts, there has been no association reported between physical activity and body composition indices. The purpose of this experiment was to determine if there was a correlation between the physical activity level and body composition in a large cohort of inbred mouse strains. Using a GE Lunar Piximus (Madison, WI), body composition of 420 mice across 17 strains was analyzed for association with percent body fat, lean mass, and fat mass. Activity data using a running wheel had previously been measured in each of these mice. Pearson correlations were determined using the indices of body composition and physical activity level with alpha \u3c0.05 set a priori. There was a significant (p=0.013) correlation (r2 = 0.87) between mouse strain and fat mass (p \u3c 0.05). Additionally, there trends for lean mass (p=0.053) and total body weight (p=0.056) to be correlated to mouse strain. There was not a significance association between the body composition characteristics (fat mass (p=0.013), lean mass (p=0.053), and percent body fat (p=0.068)) and physical activity in the mice. From these results, we have supported previous literature showing a genetic predisposition for fat mass within each mouse strain. Further, the lack of significant relationship between body composition and daily activity is not surprising given previous published data in smaller mouse cohorts. Our data support previous suggestions that body composition and the inherent drive to be active are independent of each other

The Dust Content of Galaxy Clusters

We report on the detection of reddening toward z ~ 0.2 galaxy clusters. This is measured by correlating the Sloan Digital Sky Survey cluster and quasar catalogs and by comparing the photometric and spectroscopic properties of quasars behind the clusters to those in the field. We find mean E(B-V) values of a few times 10^-3 mag for sight lines passing ~Mpc from the clusters' center. The reddening curve is typical of dust but cannot be used to distinguish between different dust types. The radial dependence of the extinction is shallow near the cluster center suggesting that most of the detected dust lies at the outskirts of the clusters. Gravitational magnification of background z ~ 1.7 sources seen on Mpc (projected) scales around the clusters is found to be of order a few per cent, in qualitative agreement with theoretical predictions. Contamination by different spectral properties of the lensed quasar population is unlikely but cannot be excluded.Comment: 4 pages, 3 figure

Acute post-disaster medical needs of patients with diabetes: emergency department use in New York City by diabetic adults after Hurricane Sandy.

OBJECTIVE: To evaluate the acute impact of disasters on diabetic patients, we performed a geospatial analysis of emergency department (ED) use by New York City diabetic adults in the week after Hurricane Sandy. RESEARCH DESIGN AND METHODS: Using an all-payer claims database, we retrospectively analyzed the demographics, insurance status, and medical comorbidities of post-disaster ED patients with diabetes who lived in the most geographically vulnerable areas. We compared the patterns of ED use among diabetic adults in the first week after Hurricane Sandy\u27s landfall to utilization before the disaster in 2012. RESULTS: In the highest level evacuation zone in New York City, postdisaster increases in ED visits for a primary or secondary diagnosis of diabetes were attributable to a significantly higher proportion of Medicare patients. Emergency visits for a primary diagnosis of diabetes had an increased frequency of certain comorbidities, including hypertension, recent procedure, and chronic skin ulcers. Patients with a history of diabetes visited EDs in increased numbers after Hurricane Sandy for a primary diagnosis of myocardial infarction, prescription refills, drug dependence, dialysis, among other conditions. CONCLUSIONS: We found that diabetic adults aged 65 years and older are especially at risk for requiring postdisaster emergency care compared to other vulnerable populations. Our findings also suggest that there is a need to support diabetic adults particularly in the week after a disaster by ensuring access to medications, aftercare for patients who had a recent procedure, and optimize their cardiovascular health to reduce the risk of heart attacks

Avirulent Strains of Toxoplasma Gondii Infect Macrophages by Active Invasion from the Phagosome

Unlike most intracellular pathogens that gain access into host cells through endocytic pathways, Toxoplasma gondii initiates infection at the cell surface by active penetration through a moving junction and subsequent formation of a parasitophorous vacuole. Here, we describe a noncanonical pathway for T. gondii infection of macrophages, in which parasites are initially internalized through phagocytosis, and then actively invade from within a phagosomal compartment to form a parasitophorous vacuole. This phagosome to vacuole invasion (PTVI) pathway may represent an intermediary link between the endocytic and the penetrative routes for host cell entry by intracellular pathogens. The PTVI pathway is preferentially used by avirulent strains of T. gondii and confers an infectious advantage over virulent strains for macrophage tropism

Absence of microemboli on transcranial Doppler identifies low-risk patients with asymptomatic carotid stenosis who do not warrant endarterectomy or stenting

Background and Purpose - Carotid endarterectomy clearly benefits patients with symptomatic severe stenosis (SCS), but the risk of stroke is so low for asymptomatic patients (ACS) that the number needed to treat is very high. We studied transcranial Doppler (TCD) embolus detection as a method for identifying patients at higher risk who would have a lower number needed to treat. Methods - Patients with carotid stenosis of ≥60% by Doppler ultrasound who had never been symptomatic (81%) or had been asymptomatic for at least 18 months (19%) were studied with TCD embolus detection for up to 1 hour on 2 occasions a week apart; patients were followed for 2 years. Results - 319 patients were studied, age (standard deviation) 69.68 (9.12) years; 32 (10%) had microemboli at baseline (TCD+). Events were more likely to occur in the first year. Patients with microemboli were much more likely to have microemboli 1 year later (34.4 versus 1.4%; P\u3c0.0001) and were more likely to have a stroke during the first year of follow-up (15.6%, 95% CI, 4.1 to 79; versus 1%, 95% CI, 1.01 to 1.36; P\u3c0.0001). Conclusions - Our findings indicate that TCD- ACS will not benefit from endarterectomy or stenting unless it can be done with a risk \u3c1%; TCD+ may benefit as much as SCS if their surgical risk is not higher. These findings suggest that ACS should be managed medically with delay of surgery or stenting until the occurrence of symptoms or emboli. © 2005 American Heart Association, Inc

Stellar Models of Multiple Populations in Globular Clusters. I. The Main Sequence of NGC 6752

We present stellar atmosphere and evolution models of main sequence stars in two stellar populations of the Galactic globular cluster NGC 6752. These populations represent the two extremes of light-element abundance variations in the cluster. NGC 6752 is a benchmark cluster in the study of multiple stellar populations because of the rich array of spectroscopic abundances and panchromatic Hubble Space Telescope photometry. The spectroscopic abundances are used to compute stellar atmosphere and evolution models. The synthetic spectra for the two populations show significant differences in the ultraviolet and, for the coolest temperatures, in the near-infrared. The stellar evolution models exhibit insignificant differences in the H-R diagram except on the lower main sequence. The appearance of multiple sequences in the colour-magnitude diagrams (CMDs) of NGC 6752 is almost exclusively due to spectral effects caused by the abundance variations. The models reproduce the observed splitting and/or broadening of sequences in a range of CMDs. The ultraviolet CMDs are sensitive to variations in carbon, nitrogen, and oxygen but the models are not reliable enough to directly estimate abundance variations from photometry. On the other hand, the widening of the lower main sequence in the near-infrared CMD, driven by oxygen-variation via the water molecule, is well-described by the models and can be used to estimate the range of oxygen present in a cluster from photometry. We confirm that it is possible to use multiband photometry to estimate helium variations among the different populations, with the caveat that the estimated amount of helium-enhancement is model-dependent.Comment: Accepted for publication in MNRAS; 19 pages, 13 figure

Niche-Associated Activation of Rac Promotes the Asymmetric Division of <i>Drosophila</i> Female Germline Stem Cells

Background: Drosophila female germline stem cells (GSCs) reside adjacent to a cellular niche that secretes Bone Morphogenetic Protein (BMP) ligands and anchors the GSCs through adherens junctions. The GSCs divide asymmetrically such that one daughter remains in the niche as a GSC, while the other is born away from the niche and differentiates. However, given that the BMP signal can be diffusible, it remains unclear how a local extracellular asymmetry is sufficient to result in a robust pattern of asymmetric division. Methods and Findings: Here we show that GSCs are polarized with respect to the cellular niche. We first use a modified biosensor to demonstrate that the small GTPase Rac is asymmetrically activated within the GSC at the niche-GSC interface. Experiments using loss-of-function and gain-of-function mutations in Rac indicate that asymmetric Rac activity both localizes the microtubule binding protein Apc2 to orient one GSC centrosome at the niche-GSC interface during interphase and activates the Jun N-terminal kinase pathway to increase the ability of the GSC to respond to BMP ligands. Other processes act in concert with each function of Rac. Specifically, we demonstrate that the GSC cell cycle arrests at prometaphase if centrosomes are misoriented. Conclusions: Thus, the GSCs, an adult stem cell present in a cellular niche, have a niche-associated polarity that couples control of the division plane with increased response to an extracellular maintenance signal. Other processes work in parallel with the Rac-mediated polarity to ensure a robust pattern of asymmetric division. We suggest that all adult stem cells likely employ multiple, independently acting mechanisms to ensure asymmetric division to maintain tissue homeostasis.</p

Carbonate determination in soils by mid-IR spectroscopy with regional and continental scale models

A Partial Least Squares (PLS) carbonate (CO3) prediction model was developed for soils throughout the contiguous United States using mid-infrared (MIR) spectroscopy. Excellent performance was achieved over an extensive geographic and chemical diversity of soils. A single model for all soil types performed very well with a root mean square error of prediction (RMSEP) of 12.6 g kg-1 and was further improved if Histosols were excluded (RMSEP 11.1 g kg-1). Exclusion of Histosols was particularly beneficial for accurate prediction of CO3 values when the national model was applied to an independent regional dataset. Little advantage was found in further narrowing the taxonomic breadth of the calibration dataset, but higher precision was obtained by running models for a restricted range of CO3. A model calibrated using only on the independent regional dataset, was unable to accurately predict CO3 content for the more chemically diverse national dataset. Ten absorbance peaks enabling CO3 prediction by mid-infrared (MIR) spectroscopy were identified and evaluated for individual and combined predictive power. A single-band model derived from an absorbance peak centered at 1796 cm-yielded the lowest RMSEP of 13.5 g kg-1 for carbonate prediction compared to other single-band models. This predictive power is attributed to the strength and sharpness of the peak, and an apparent minimal overlap with confounding co-occurring spectral features of other soil components. Drawing from the 10 identified bands, multiple combinations of 3 or 4 peaks were able to predict CO3 content as well as the full-spectrum national models. Soil CO3 is an excellent example of a soil parameter that can be predicted with great effectiveness and generality, and MIR models could replace direct laboratory measurement as a lower cost, high quality alternative