Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

<p>Abstract</p> <p>Background</p> <p>The availability of low cost sequencing has spurred its application to discovery and typing of variation, including variation induced by mutagenesis. Mutation discovery is challenging as it requires a substantial amount of sequencing and analysis to detect very rare changes and distinguish them from noise. Also challenging are the cases when the organism of interest has not been sequenced or is highly divergent from the reference.</p> <p>Results</p> <p>We describe the development of a simple method for reduced representation sequencing. Input DNA was digested with a single restriction enzyme and ligated to Y adapters modified to contain a sequence barcode and to provide a compatible overhang for ligation. We demonstrated the efficiency of this method at SNP discovery using rice and arabidopsis. To test its suitability for the discovery of very rare SNP, one control and three mutagenized rice individuals (1, 5 and 10 mM sodium azide) were used to prepare genomic libraries for Illumina sequencers by ligating barcoded adapters to <it>NlaIII </it>restriction sites. For genome-dependent discovery 15-30 million of 80 base reads per individual were aligned to the reference sequence achieving individual sequencing coverage from 7 to 15×. We identified high-confidence base changes by comparing sequences across individuals and identified instances consistent with mutations, i.e. changes that were found in a single treated individual and were solely GC to AT transitions. For genome-independent discovery 70-mers were extracted from the sequence of the control individual and single-copy sequence was identified by comparing the 70-mers across samples to evaluate copy number and variation. This <it>de novo </it>"genome" was used to align the reads and identify mutations as above. Covering approximately 1/5 of the 380 Mb genome of rice we detected mutation densities ranging from 0.6 to 4 per Mb of diploid DNA depending on the mutagenic treatment.</p> <p>Conclusions</p> <p>The combination of a simple and cost-effective library construction method, with Illumina sequencing, and the use of a bioinformatic pipeline allows practical SNP discovery regardless of whether a genomic reference is available.</p

The Perception Of Walking Speed In A Virtual Environment

Studies of locomotion in virtual environments assume that correct geometric principles define the relationship between walking speed and environmental flow. However, we have observed that geometrically correct optic flow appears to be too slow during simulated locomotion on a treadmill. Experiment 1 documents the effect in a head-mounted display. Experiment 2 shows that the effect is eliminated when the gaze is directed downward or to the side, or when the walking speed is slow. Experiment 3 shows that the effect is unchanged by stride length. Experiment 4 verifies that the effect is not attributable to image jitter. The change in perceived speed from straight ahead to side or down gaze coincides with a shift from expanding optic flow to lamellar flow. Therefore, we hypothesize that lamellar flow is necessary for accurate speed perception, and that a limited field of view eliminates this cue during straight-ahead gaze

Effect of metoprolol on myocardial function and energetics in patients with nonischemic dilated cardiomyopathy: A randomized, double-blind, placebo-controlled study

AbstractObjectives. This study examined the of metoprolol on left ventricular performance, efficiency, neurohormonal activation and myocardial respiratory quotient in patients with dilated cardiomyopathy.Background.The mechanism by which beta-adrenergie blockade improves ejection fraction in patients with dilated cardiomyopathy remains an enigma. Thus, we undertook an extensive hemodynamic evaluation of this mechanism. In addition, because animal models have shown that catecholamine exposure may increase relative fatty acid utilization, we hypothesized that antagonism of sympathetic stimulation may result in increased carbohydrate utilization.Methods. This was a randomized, double-blind, prospective trial in which 24 men with nonischemic dilated cardiomyopathy underwent cardiac catheterization before and after 3 months of therapy with metoprolol (n = 15) or placebo (n = 9) in addition to standard therapy. Pressure-volume relations were examined using a micromanometer catheter and digital ventriculography.Results. At baseline, the placebo-treated patients had somewhat more advanced left ventricular dysfunction. Ejection fraction and left ventricular performance improved only in the metoprolol-treated patients. Stroke and minute work increased without an increase in myocardial oxgen consumption, suggesting increased myocardial efficiency. Further increases in ejection fraction were seen between 3 and 6 months in the metoprolol group. The placebo group had a significant increase in ejection fraction only after crossover to metoprolol. A significant relation the change in coronary sinus norepinephrine and myocardial respiratory quotient was seen, suggesting a possible effect of adrenergic deactivation on substrate utilization.Conclusions. These data demonstrate that in patients with cardiomyopthy, metoprolol treatment improves myocardial performance and energetics, and favorably alters substrate utilization. Beta-adrenergic blocking agents, such as metoprolol, are hemodynamically and energetically beneficial in the treatment of myocardial failure

Differences in cerebral response to esophageal acid stimuli and psychological anticipation in GERD subtypes - An fMRI study

<p>Abstract</p> <p>Background</p> <p>To evaluate whether there are differences in the cerebral response to intraesophageal acid and psychological anticipation stimuli among subtypes of gastroesophageal reflux disease (GERD).</p> <p>Methods</p> <p>Thirty nine patients with GERD and 11 healthy controls were enrolled in this study after gastroscopy and 24 hr pH monitoring. GERD subjects were divided into four subgroups: RE (reflux esophagitis), NERD+ (non-erosive reflux disease with excessive acid reflux), NERD-SI+ (normal acid exposure and positive symptom index) and NERD-SI+ (normal acid exposure and negative symptom index, but responded to proton pump inhibitor trial). Cerebral responses to intraesophageal acid and psychological anticipation were evaluated with fMRI.</p> <p>Results</p> <p>During intraesophageal acid stimulation, the prefrontal cortex (PFC) region was significantly activated in all subgroups of GERD; the insular cortex (IC) region was also activated in RE, NERD+ and NERD-SI- groups; the anterior cingulated cortex (ACC) region was activated only in RE and NERD-SI- groups. The RE subgroup had the shortest peak time in the PFC region after acid was infused, and presented the greatest change in fMRI signals in the PFC and ACC region (<it>P </it>= 0.008 and <it>P </it>= 0.001, respectively). During psychological anticipation, the PFC was significantly activated in both the control and GERD groups. Activation of the IC region was found in the RE, NERD-SI+ and NERD-SI- subgroups. The ACC was activated only in the NERD-SI+ and NERD-SI- subgroups. In the PFC region, the NERD-SI- subgroup had the shortest onset time (<it>P </it>= 0.008) and peak time (<it>P </it>< 0.001). Compared with actual acid infusion, ACC in RE and IC in NERD+ were deactivated while additional areas including the IC and ACC were activated in the NERD-SI+ group; and in NERD-SI- group, onset-time and peak time in the PFC and IC areas were obviously shorter in induced anticipation than in actual acid infusion.</p> <p>Conclusions</p> <p>The four subgroups of GERD patients and controls showed distinctly different activation patterns and we therefore conclude GERD patients have different patterns of visceral perception and psychological anticipation. Psychological factors play a more important role in NERD-SI+ and NERD-SI- groups than in RE and NERD+ groups.</p

Radiation Protection Studies for LCLS Tune Up Dump

The Linac Coherent Light Source (LCLS) at the Stanford Linear Accelerator Center is a pioneer fourth generation hard x-ray free electron laser that shall start to deliver laser pulses in 2009. Among other components of LCLS that present radiation protection concerns, the tune up dump (tdund) is of special interest because it also constitutes an issue for machine protection, as it is placed close to radiation sensitive components, like electronic devices and permanent magnets in the undulators. This paper first introduces the stopper of tdund looking at the heat load, and then it describes the shielding around the dump necessary to maintain the prompt and residual dose within design values. Next, preliminary comparisons of the magnetization loss in a dedicated on-site magnet irradiation experiment with FLUKA simulations serve to characterize the magnetic response to radiation of magnets like those of LCLS. The previous knowledge, together with the limit for the allowed demagnetization, are used to estimate the lifetime of the undulator. Further simulations provide guidelines on which lifetime can be expected for an electronic device placed at a given distance of tdund

Evaluation of Petrifilm™ Select E. coli Count Plate medium to discriminate antimicrobial resistant Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Screening and enumeration of antimicrobial resistant <it>Escherichia coli </it>directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select <it>E. coli </it>Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant <it>E. coli</it>.</p> <p>Method</p> <p>A range of <it>E. coli </it>isolates were tested by agar dilution method comparing the Minimal Inhibitory Concentration (MIC) for eight antimicrobials obtained by Mueller-Hinton II agar, MacConkey agar and SEC plates. Kappa statistics was used to assess the levels of agreement when classifying strains as resistant, intermediate or susceptible.</p> <p>Results</p> <p>SEC plate showed that 74% of all strains agreed within ± 1 log₂dilution when comparing MICs with Mueller-Hinton II media. High agreement levels were found for gentamicin, ampicillin, chloramphenicol and cefotaxime, resulting in a kappa value of 0.9 and 100% agreement within ± 1 log₂dilution. Significant variances were observed for oxytetracycline and sulphamethoxazole. Further tests showed that the observed discrepancy in classification of susceptibility to oxytetracycline by the two media could be overcome when a plate-dependent breakpoint of 64 mg/L was used for SEC plates. For sulphamethoxazole, SEC plates provided unacceptably high MICs.</p> <p>Conclusion</p> <p>SEC plates showed good agreement with Mueller-Hinton II agar in MIC studies and can be used to screen and discriminate resistant <it>E. coli </it>for ampicillin, cephalothin, streptomycin, chloramphenicol, cefotaxime and gentamicin using CLSI standardized breakpoints, but not for sulphamethoxazole. SEC plates can also be used to discriminate oxytetracycline-resistant <it>E. coli </it>if a plate-dependent breakpoint value of 64 mg/L is used.</p

Evaluation of gliovascular functions of AQP4 readthrough isoforms

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared All

High Resolution Intravital Imaging of Subcellular Structures of Mouse Abdominal Organs Using a Microstage Device

Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated ‘microstage’ that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox