C-ME: a 3D community-based, real-time collaboration tool for scientific research and training

The need for effective collaboration tools is growing as multidisciplinary proteome-wide projects and distributed research teams become more common. The resulting data is often quite disparate, stored in separate locations, and not contextually related. Collaborative Molecular Modeling Environment (C-ME) is an interactive community-based collaboration system that allows researchers to organize information, visualize data on a two-dimensional (2-D) or three-dimensional (3-D) basis, and share and manage that information with collaborators in real time. C-ME stores the information in industry-standard databases that are immediately accessible by appropriate permission within the computer network directory service or anonymously across the internet through the C-ME application or through a web browser. The system addresses two important aspects of collaboration: context and information management. C-ME allows a researcher to use a 3-D atomic structure model or a 2-D image as a contextual basis on which to attach and share annotations to specific atoms or molecules or to specific regions of a 2-D image. These annotations provide additional information about the atomic structure or image data that can then be evaluated, amended or added to by other project members.     

Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals,retinoic acid and Hoxa3

We have investigated the mechanisms involved in generating hindbrain motoneurone subtypes, focusing on somatic motoneurones, which are confined to the caudal hindbrain within rhombomeres 5-8. Following heterotopic transplantation of rhombomeres along the rostrocaudal axis at various developmental stages, we have found that the capacity of rhombomeres to generate somatic motoneurones is labile at the neural plate stage but becomes fixed just after neural tube closure, at stage 10-11. Grafting of somites or retinoic acid-loaded beads beneath the rostral hindbrain induced the formation of somatic motoneurones in rhombomere 4 only, and Hox genes normally expressed more caudally (Hoxa3, Hoxd4) were induced in this region. Targeted overexpression of Hoxa3 in the rostral hindbrain led to the generation of ectopic somatic motoneurones in ventral rhombomeres 1-4, and was accompanied by the repression of the dorsoventral patterning gene Irx3. Taken together, these observations suggest that the somites, retinoic acid and Hox genes play a role in patterning somatic motoneurones in vivo.     

Exercise training biomarkers: influence of short-term diet modification on the blood lactate to rating of perceived exertion (La:RPE) ratio

This study examined the effect of dietary consumption of carbohydrates (CHO) on the blood lactate to rating of perceived exertion (La:RPE) ratio during an intense micro-cycle of exercise training. This ratio is a proposed biomarker of exercise training stress and potential indicator for under- or overtraining in athletes. Sixteen male athletes were randomly assigned into two groups; high CHO (H-CHO; 60% of daily caloric intake) and low CHO (L-CHO; 30% of daily caloric intake). Diets were controlled the day before and for the three days of the micro-cycle. The micro-cycle consisted of three successive days of 60 minutes of intense cycling (～70% of VO2peak). Blood samples were obtained immediately before and after exercise (post) on each day of exercise training (D1, D2, D3) and were analyzed for blood lactate. Rating of perceived exertion (RPE) scores were taken at the end of each exercise session and combined with the post exercise lactate value to form the La:RPE ratio. An analysis of variance (ANOVA) showed a significant difference between the La:RPE ratio for the H-CHO and L-CHO groups at D3 even though the exercise intensity was not significantly different between the groups. Specifically, the ratio was significantly (p < 0.02) lower on D3 in the L-CHO group (～31% lower) than in the H-CHO group. From these findings it is recommended that diet needs to be monitored when using the La:RPE ratio as an exercise training biomarker to determine whether an athlete is truly under-training or overtraining. Athletes or coaches that use the La:RPE ratio as a training biomarker, but do not monitor dietary CHO intake need to interpreted their findings carefully.     

The influence of training on physical and functional growth before, during and after puberty

In boys, the ages at which growth rates for body weight, height, VO2max, maximum O2 pulse and VImax reached their peaks were approximately the same (means and SD: 14.64 +/- 0.98, 14.67 +/- 0.99, 14.71 +/- 1.59, 14.38 +/- 1.36 and 14.64 +/- 1.42 years respectively). There was a positive relationship between the peak velocities of functional capacity indicators (VO2max 0.79 +/- 0.19 1.min-1.year-1, O2 pulse max 4.1 +/- 1.20 ml.year-1, VImax 27.3 +/- 7.15 l.min-1.year-1) and the peak growth velocity of weight and/or height (weight 9.1 +/- 1.92 kg.year-1, height 9.8 +/- 1.92 cm.year-1). A positive relationship between the age at peak velocity of VO2max and O2 pulse max with the age at peak velocity for body weight was also found (r = 0.524 and 0.400 respectively). No relationship was revealed between the age at peak velocity on the one hand and the peak velocities of body weight, height, VO2max, O2 pulse or VImax on the other. Moderate training did not influence acceleration in growth--the age at peak velocity and the peaks of the growth rate did not differ in groups with a different regime of exercise (higher - n = 8, medium - n = 9, lower - n = 12; the peak velocity of VO2max--means and SD--being 0.85 +/- 0.15, 0.76 +/- 0.22 and 0.78 +/- 0.17.min-1.year-1 respectively).     

Calculating the axes of rotation for the subtalar and talocrural joints using 3D bone reconstructions

Orientation of the subtalar joint axis dictates inversion and eversion movements of the foot and has been the focus of evolutionary and clinical studies for a number of years. Previous studies have measured the subtalar joint axis against the axis of the whole foot, the talocrural joint axis and, recently, the principal axes of the talus. The present study introduces a new method for estimating average joint axes from 3D reconstructions of bones and applies the method to the talus to calculate the subtalar and talocrural joint axes. The study also assesses the validity of the principal axes as a reference coordinate system against which to measure the subtalar joint axis. In order to define the angle of the subtalar joint axis relative to that of another axis in the talus, we suggest measuring the subtalar joint axis against the talocrural joint axis. We present corresponding 3D vector angles calculated from a modern human skeletal sample. This method is applicable to virtual 3D models acquired through surface-scanning of disarticulated 'dry' osteological samples, as well as to 3D models created from CT or MRI scans.     

RNA2D3D: a program for generating, viewing, and comparing 3-dimensional models of RNA

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.     

RNA2D3D: A program for Generating,Viewing, and Comparing 3-Dimensional Models of RNA

Abstract

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.     

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.     

Song in the cold is 'hot': memory of and preference for sexual signals perceived under thermal challenge

The environmental conditions under which signals are perceived can affect receiver responses. Many songbird populations produce a song chorus at dawn, when, in cold habitats, they would experience thermal challenge. We recorded temperature and the song activity of Lincoln's sparrows (Melospiza lincolnii) on a high-elevation meadow, and determined that song behaviour is concentrated around the coldest time of the day, at dawn. We hypothesized that this is because male song in the cold is more attractive to females than song in the warm. To test this, we exposed laboratory-housed Lincoln's sparrow females to songs at 1°C and 16°C, which they naturally experience in the wild. Females spent 40 per cent more time close to the speaker during playback at 1°C than at 16°C. When tested at 16°C 1-2 days later, females biased their movement towards the speaker playing songs previously heard at 1°C over 16°C. Thus, female Lincoln's sparrows remembered and affiliated with songs they heard under thermal challenge, indicating that the thermal environment can affect the attractiveness of a sexual signal.     

Microvascular and myocardial contractile responses to ischemia: influence of exercise training.

We hypothesized that exercise training preserves endothelium-dependent relaxation, lessens receptor-mediated constriction of coronary resistance arteries, and reduces myocardial contractile dysfunction in response to ischemia. After 10 wk of treadmill running or cage confinement, regional and global indexes of left ventricular contractile function were not different between trained and sedentary animals in response to three 15-min periods of ischemia (long-term; n = 17), one 5-min bout of ischemia (short-term; n = 18), or no ischemia (sham-operated; n = 24). Subsequently, coronary resistance vessels ( approximately 106 +/- 4 microm ID) were isolated and studied using wire myographs. Maximal ACh-evoked relaxation was approximately 25, 40, and 60% of KCl-induced preconstriction after the long-term, short-term, and sham-operated protocols, respectively, and was similar between groups. Maximal sodium nitroprusside-evoked relaxation also was similar between groups among all protocols, and vasoconstrictor responses to endothelin-1 and U-46619 were not different in trained and sedentary rats after short-term ischemia or sham operation. We did observe that, after long-term ischemia, maximal tension development in response to endothelin-1 and U-46619 was blunted (P < 0.05) in trained animals by approximately 70 and approximately 160%, respectively. These results support our hypothesis that exercise training lessens receptor-mediated vasoconstriction of coronary resistance vessels after ischemia and reperfusion. However, training did not preserve endothelial function of coronary resistance vessels, or myocardial contractile function, after ischemia and reperfusion.     

Exposure to genotoxic agents, host factors, and lifestyle influence the number of centromeric signals in micronuclei: a pooled re-analysis

We pooled data from three biomonitoring studies using the cytokinesis-block micronucleus assay in peripheral blood lymphocytes in combination with fluorescence in situ hybridization. Centromere-positive micronuclei (C+MN) were classified in two groups: those containing one centromere (C1+MN) and those with two or more (Cx+MN). The three studies evaluated untreated cancer patients, welders, and pathologists/anatomists exposed to formaldehyde. The total number of subjects included in the pooled re-analysis was 113. A higher frequency of C+MN was observed in cancer patients and exposed workers, who showed significant differences from controls in all studies. C1+MN were particularly increased in the group of pathologists/anatomists, who showed a 3.29 times higher frequency than controls (95% CI: 2.04-5.30). A borderline increase in Cx+MN was observed in welders when compared to the corresponding control group (FR: 1.31; 95% CI: 0.99-1.74). An evident effect of gender was found, with significantly increased frequencies of all endpoints measuring aneuploidy in females (C+MN, C1+MN, and Cx+MN). Alcohol consumption had a significant effect on total MN frequency and particularly on C+MN and C1+MN. In conclusion, scoring the number of centromeric signals in the micronucleus assay provides additional information about the mechanism of action of various genotoxic agents, and the role of confounding factors may be more specifically accounted for. Indeed, C+MN could be efficiently used in biomonitoring studies as an independent biomarker of exposure and early biological effect. The use of centromeric signals allows the identification of two further endpoints, representing two alternative pathways of chromosome loss, i.e., impaired chromosome migration, leading to increased C1+MN frequency, and centrosome amplification, possibly leading to Cx+MN with two or more centromeric signals.     

Absence of genetic divergence between western corn rootworms (Coleoptera: Chrysomelidae) resistant and susceptible to control by crop rotation

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn, Zea mays L., in North America that has recently invaded Europe. A loss of ovipositional fidelity to cornfields has allowed the species to circumvent crop rotation as a means of control in part of its range in the United States. Analyses of variation at eight microsatellite loci provided no evidence for general genetic differentiation between samples of western corn rootworm collected in soybean, Glycine max (L.) Merr., fields and those collected in cornfields both inside and outside the rotation-resistance problem area. This result suggests that few or no barriers to gene flow exist between rotation-resistant and -susceptible rootworm populations. The implications of this result for the management of western corn rootworm in North America and Europe are discussed.     

Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation.

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.     

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.     

Blood oxidative stress biomarkers: influence of sex,exercise training status,and dietary intake

Background: Sex and lifestyle factors are known to influence the oxidation of protein, lipids, and DNA. Biomarkers such as protein carbonyls (PC), malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) have been commonly used in an attempt to characterize the oxidative status of human subjects.Objective: This study compared resting blood oxidative stress biomarkers, in relation to exercise training status and dietary intake, between men and women.Methods: Exercise-trained and sedentary men and women (with normal menstrual cycles; reporting during the early follicular phase) were recruited from the University of Memphis, Tennessee, campus and surrounding community via recruitment flyers and word of mouth. Participants were categorized by sex and current exercise training status (ie, trained or untrained). Each completed a detailed 5-day food record of all food and drink consumed. Diets were analyzed for kilocalories and macro- and micronutrient (vitamins C, E, A) intake. Venous blood samples were obtained at rest and analyzed for PC, MDA, and 8-OHdG.Results: In the 131 participants (89 men, of whom 74 were exercise trained and 15 untrained, and 42 women, of whom 22 were exercise trained and 20 untrained; mean [SD] age, 24 [4] years), PC did not differ significantly between trained men and women or between untrained men and women. However, trained participants had significantly lower plasma PC (measured in nmol · mg protein^-1) (mean [SEM] 0.0966 [0.0055]) than did untrained participants (0.1036 [0.0098]) (P < 0.05). MDA levels (measured in μmol · L^-1) were significantly lower in trained women (0.4264 [0.0559]) compared with trained men (0.6959 [0.0593]); in trained men and women combined (0.5621 [0.0566]) compared with untrained men and women combined (0.7397 [0.0718]); and in women combined (0.5665 [0.0611]) compared with men combined (0.7338 [0.0789]) (P < 0.05 for all comparisons). No significant differences were noted between any groups for 8-OHdG. Neither PC nor 8-OHdG were correlated to any dietary variable, with the exception of PC and percent of protein in untrained men (r = 0.552; P = 0.033). MDA was positively correlated to protein intake and negatively correlated to percent of carbohydrate and vitamin C intake, primarily in trained men (P ≤ 0.03).Conclusions: In this sample of young healthy adults, oxidative stress was lower in women than in men and in trained compared with untrained individuals, particularly regarding MDA. With the exception of MDA primarily in trained men, dietary intake did not appear to be correlated to biomarkers of oxidative stress.     

Magnitude and direction of DNA bending induced by screw-axis orientation: influence of sequence, mismatches and abasic sites

DNA-bending flexibility is central for its many biological functions. A new bending restraining method for use in molecular mechanics calculations and molecular dynamics simulations was developed. It is based on an average screw rotation axis definition for DNA segments and allows inducing continuous and smooth bending deformations of a DNA oligonucleotide. In addition to controlling the magnitude of induced bending it is also possible to control the bending direction so that the calculation of a complete (2-dimensional) directional DNA-bending map is now possible. The method was applied to several DNA oligonucleotides including A(adenine)-tract containing sequences known to form stable bent structures and to DNA containing mismatches or an abasic site. In case of G:A and C:C mismatches a greater variety of conformations bent in various directions compared to regular B-DNA was found. For comparison, a molecular dynamics implementation of the approach was also applied to calculate the free energy change associated with bending of A-tract containing DNA, including deformations significantly beyond the optimal curvature. Good agreement with available experimental data was obtained offering an atomic level explanation for stable bending of A-tract containing DNA molecules. The DNA-bending persistence length estimated from the explicit solvent simulations is also in good agreement with experiment whereas the adiabatic mapping calculations with a GB solvent model predict a bending rigidity roughly two times larger.     

Rating of perceived exertion as a tool for prescribing and self regulating interval training: a pilot study

The aim of the present study was to analyse the usefulness of the 6-20 rating of perceived exertion (RPE) scale for prescribing and self-regulating high-intensity interval training (HIT) in young individuals. Eight healthy young subjects (age = 27.5±6.7 years) performed maximal graded exercise testing to determine their maximal and reserve heart rate (HR). Subjects then performed two HIT sessions (20 min on a treadmill) prescribed and regulated by their HR (HR: 1 min at 50% alternated with 1 min at 85% of reserve HR) or RPE (RPE: 1 minute at the 9-11 level [very light-fairly light] alternated with 1 minute at the 15-17 level [hard-very hard]) in random order. HR response and walking/running speed during the 20 min of exercise were compared between sessions. No significant difference between sessions was observed in HR during low- (HR: 135±15 bpm; RPE: 138±20 bpm) and high-intensity intervals (HR: 168±15 bpm; RPE: 170±18 bpm). Walking/running speed during low- (HR: 5.7±1.2 km · h⁻¹; RPE: 5.7±1.3 km · h⁻¹) and high-intensity intervals (HR: 7.8±1.9 km · h⁻¹; RPE: 8.2±1.7 km · h⁻¹) was also not different between sessions. No significant differences were observed in HR response and walking/running speed between HIT sessions prescribed and regulated by HR or RPE. This finding suggests that the 6-20 RPE scale may be a useful tool for prescribing and self-regulating HIT in young subjects.     

MuPIT interactive: webserver for mapping variant positions to annotated, interactive 3D structures

Mutation position imaging toolbox (MuPIT) interactive is a browser-based application for single-nucleotide variants (SNVs), which automatically maps the genomic coordinates of SNVs onto the coordinates of available three-dimensional (3D) protein structures. The application is designed for interactive browser-based visualization of the putative functional relevance of SNVs by biologists who are not necessarily experts either in bioinformatics or protein structure. Users may submit batches of several thousand SNVs and review all protein structures that cover the SNVs, including available functional annotations such as binding sites, mutagenesis experiments, and common polymorphisms. Multiple SNVs may be mapped onto each structure, enabling 3D visualization of SNV clusters and their relationship to functionally annotated positions. We illustrate the utility of MuPIT interactive in rationalizing the impact of selected polymorphisms in the PharmGKB database, somatic mutations identified in the Cancer Genome Atlas study of invasive breast carcinomas, and rare variants identified in the exome sequencing project. MuPIT interactive is freely available for non-profit use at http://mupit.icm.jhu.edu.