Validating the Perceived Active School Travel Enablers and Barriers - Child (PASTEB-C) questionnaire.

OBJECTIVES: Presently, child-specific tools and instruments related to active school travel (AST) are lacking. This methodological shortcoming often contributes to suboptimal AST behaviour evaluations and intervention programming. The aim of this paper was to develop and validate a theoretically informed child-specific scale regarding multiple perceived barriers and enablers known to impact children's participation in AST. STUDY DESIGN: Mixed methods. METHODS: A mixed-methods and multistudy scale development approach featuring the application of social-ecological theory, a validation pilot study (n = 80), and test-retest study (n = 96) was conducted in collaboration with children in Ontario, Canada. In tandem with completing cognitive interviews and online surveys, multiple analyses, including a qualitative thematic analysis, along with weighted Cohen's kappa, Cronbach's alpha, and confirmatory factor analysis were undertaken. RESULTS: Qualitative analyses of the developed tool addressed face validity concerns related to the response options and definitions of terms used. Following the reliability analyses of 40 items, two confirmatory factor analyses were run to assess the construct validation of perceived AST barriers and enablers, and resulted in the development of the 24-item Perceived Active School Travel Enablers and Barriers - Child (PASTEB-C) questionnaire. CONCLUSION: The developed PASTEB-C questionnaire may be used to inform the programming and development of AST interventions, as well as conduct child-specific AST research.

Substantial variability in morphological scaling among bumblebee colonies.

Differences in organ scaling among individuals may play an important role in determining behavioural variation. In social insects, there are well-documented intraspecific differences in colony behaviour, but the extent that organ scaling differs within and between colonies remains unclear. Using 12 different colonies of the bumblebee Bombus terrestris, we aim to address this knowledge gap by measuring the scaling relationships between three different organs (compound eyes, wings and antennae) and body size in workers. Though colonies were exposed to different rearing temperatures, this environmental variability did not explain the differences of the scaling relationships. Two colonies had differences in wing versus antenna slopes, three colonies showed differences in wing versus eye slopes and a single colony has differences between eye versus antenna slopes. There are also differences in antennae scaling slopes between three different colonies, and we present evidence for putative trade-offs in morphological investment. We discuss the utility of having variable scaling among colonies and the implication for understanding variability in colony fitness and behaviour.

Establishing the tolerability to broiler chickens and laying hens of nonanoic acid at practical levels of use as a feed flavouring.

1. The following experiments were conducted to evaluate the effects of nonanoic acid (NA) in broilers and laying hens, at practical levels as a flavouring in complete feed.2. In the first experiment, 1100, one-day-old Ross 308 chicks, half male and female, were randomly assigned to 50 floor pens containing 22 chicks each. Chicks were fed one of five treatment diets containing either 0 (control), 100, 300, 500 or 1,000 mg NA/kg complete feed for 42 days.3. The NA treatment had no effect on ADFI, but there was a linear relationship with ADG and FCR. No differences were observed in blood parameters or tissue pathology among treatment groups.4. In a second study, 150 Hyline hens aged 24 weeks old were randomly assigned to 50 pens containing three birds each. Laying hens were fed one of five treatment diets containing 0 (control), 100, 300, 500 or 1,000 mg NA/kg complete feed for 56 days.5. Treatment with NA has no effect on live weight, ADFI or egg production in laying hens, and there were no observed changes in tissue pathology.6. The results supported the toleration of NA in broilers or layers at dietary levels of up to 1,000 mg/kg.

Wound dressing following primary total hip arthroplasty: a prospective randomised controlled trial.

OBJECTIVE: This study compared three methods of surgical wound dressing in patients undergoing primary total hip arthroplasty to determine their effect on wound leakage. METHOD: Total hip arthroplasties were randomised to 3 groups: 2-octyl cyanoacrylate (Dermabond-Ethicon Inc, G) with Opsite (Smith & Nephew; O) [G+O], 2-octyl cyanoacrylate with Tegaderm (3M; T) [G+T], and Opsite without 2-octyl cyanoacrylate [O]. Postoperative wound leakage was assessed and graded daily until discharge, the frequency of the dressing changes was also recorded. Patients were clinically reviewed at three months to assess cosmesis of their surgical scar and wound complications. RESULTS: In all 211 total hip arthoplasties were included. A greater proportion of patients' dressings remained dry on day 1 postoperatively in the two groups using 2-octyl cyanoacrylate (G+O and G+T) compared to the no glue group (O; p=0.0001). The G+T group had a significantly lower proportion of patients with increased leakage of wounds on days 2 and 3 postoperatively compared with both G+O and O groups (p=0.0043). The overall rate of dressing change for G+O was 8%, G+T 5%, and O 13%. Overall wound cosmesis was similar in all groups (p=0.690). CONCLUSION: The reduction in frequency of dressing changes coupled with low levels of wound leakage observed using the combination of the glue and nonabsorbent dressings (O+T), makes this combination of wound closing products ideal for facilitating enhanced recovery and early discharge programmes in elective hip arthroplasty.

C5 nerve root palsy following decompression of the cervical spine: a systematic evaluation of the literature.

C5 nerve root palsy is a rare and potentially debilitating complication of cervical spine surgery. Currently, however, there are no guidelines to help surgeons to prevent or treat this complication. We carried out a systematic review of the literature to identify the causes of this complication and options for its prevention and treatment. Searches of PubMed, Embase and Medline yielded 60 articles for inclusion, most of which addressed C5 palsy as a complication of surgery. Although many possible causes were given, most authors supported posterior migration of the spinal cord with tethering of the nerve root as being the most likely. Early detection and prevention of a C5 nerve root palsy using neurophysiological monitoring and variations in surgical technique show promise by allowing surgeons to minimise or prevent the incidence of C5 palsy. Conservative treatment is the current treatment of choice; most patients make a full recovery within two years.

How dim is dim? Precision of the celestial compass in moonlight and sunlight.

Prominent in the sky, but not visible to humans, is a pattern of polarized skylight formed around both the Sun and the Moon. Dung beetles are, at present, the only animal group known to use the much dimmer polarization pattern formed around the Moon as a compass cue for maintaining travel direction. However, the Moon is not visible every night and the intensity of the celestial polarization pattern gradually declines as the Moon wanes. Therefore, for nocturnal orientation on all moonlit nights, the absolute sensitivity of the dung beetle's polarization detector may limit the precision of this behaviour. To test this, we studied the straight-line foraging behaviour of the nocturnal ball-rolling dung beetle Scarabaeus satyrus to establish when the Moon is too dim--and the polarization pattern too weak--to provide a reliable cue for orientation. Our results show that celestial orientation is as accurate during crescent Moon as it is during full Moon. Moreover, this orientation accuracy is equal to that measured for diurnal species that orient under the 100 million times brighter polarization pattern formed around the Sun. This indicates that, in nocturnal species, the sensitivity of the optical polarization compass can be greatly increased without any loss of precision.

Biological monitoring for trimethylbenzene exposure: a human volunteer study and a practical example in the workplace.

This paper presents data from both a human volunteer study looking at exposure to 1,3,5-trimethylbenzene (TMB) and an occupational hygiene study of a printing firm using screen wash containing technical grade TMB. The biomarkers measured were TMB in blood and breath, and urinary dimethylbenzoic acids (DMBAs). The volunteer (N = 4) study showed that TMB was rapidly absorbed into the bloodstream reaching a mean level of 0.85 micromol l(-1) during a 4 h exposure to 25 p.p.m. TMB. There was little decline 1 h post-exposure possibly indicating storage of TMB in adipose tissue. Breath TMB levels peaked within an hour of exposure commencing and averaged 137 nmol l(-1) during exposure. Elimination of TMB in breath was biphasic with an initial half-life of 60 min. Peak excretion of urinary DMBA occurred 4-8 h after the end of exposure and averaged 40 mmol mol(-1) creatinine. Elimination of DMBA in urine was biphasic with half-lives of 13 and 60 h indicating that accumulation of body burden throughout the working week is likely if exposure is repeated. The occupational hygiene study demonstrated an excellent correlation between personal air TMB levels and post-shift urinary DMBA levels (r = 0.997) collected on the third working day. The regression equation from this study indicates that 8 h exposure to 25 p.p.m. TMB would result in a urinary DMBA level of 206 mmol mol(-1) creatinine. All workers showed pre-shift levels of DMBA from exposure to TMB on previous days. Both urinary DMBA and breath TMB levels can be used as biomarkers of TMB exposure. Urine samples should be taken post-shift towards the end of the working week as significant body burden accumulation throughout the working week can be expected. Breath sampling is more suited to task or single-shift monitoring.

Fmoc solid phase synthesis of polyamides containing pyrrole and imidazole amino acids.

[structure: see text]. Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available peptide synthesizers.

Recruitment and retention of elderly patients in clinical trials: issues and strategies.

Clinicians and researchers alike are shifting their focus to elderly patients in order to target the most effective treatments for a variety of psychiatric conditions. Clinical trials with elderly patients are the necessary because they consume the largest number of prescription medications. There are special challenges and considerations in designing and conducting clinical studies. The authors review the various phases of such research, including recruitment of appropriate patients and retention of those enrolled, and they make suggestions, using examples from already completed research studies, illustrating the methods found to be most successful.

Alternative heterocycles for DNA recognition: an N-methylpyrazole/N-methylpyrrole pair specifies for A.T/T.A base pairs.

Side-by-side pairs of three five-membered rings, N-methylpyrrole (Py), N-methylimidazole (Im), and N-methylhydroxy-pyrrole (Hp), have been demonstrated to distinguish each of the four Watson Crick base pairs in the minor groove of DNA. However, not all DNA sequences targeted by these pairing rules achieve affinities and specificities comparable to DNA binding proteins. We have initiated a search for new heterocycles which can expand the sequence repetoire currently available. Two heterocyclic aromatic amino acids. N-methylpyrazole (Pz) and 4-methylthiazole (Th), were incorporated into a single position of an eight-ring polyamide of sequence ImImXPy-gamma-lmPyPyPy-beta-Dp to examine the modulation of affinity and specificity for DNA binding by a Pz/Py pair and or a Th/Py pair. The X/Py pairings Pz/Py and Th/Py were evaluated by quantitative DNase I footprint titrations on a DNA fragment with the four sites 5'-TGGNCA-3' (N=T, A, G, C). The Pz/Py pair binds T.A and A.T with similar affinity to a Py/Py pair but with improved specificity. disfavoring both G.C and C.G by about 100-fold. The Th/Py pair binds poorly to all four Watson Crick base pairs. These results demonstrate that in some instances new heterocyclic aromatic amino acid pairs can be incorporated into imidazole-pyrrole polyamides to mimic the DNA specificity of Py/Py pairs which may be relevant as biological criteria in animal studies become important.

Kinetic consequences of covalent linkage of DNA binding polyamides.

Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subunits can bind in the minor groove of DNA at predetermined sequences with subnanomolar affinity and high specificity. Covalent linkage of polymer subunits using a gamma-aminobutyric acid linker has been shown to increase both the affinity and specificity of polyamides. Using a fluorescence detected stopped-flow assay, we have studied the differences in association and dissociation kinetics of a series of polyamides representing unlinked, hairpin and cyclic analogues of the four ring polyamide ImPyPyPy-beta-Dp. Whereas the large differences seen in the equilibrium association constants between the unlinked and covalently linked polyamides are primarily due to higher association rate constants, discrimination between matched and mismatched sites by each polyamide can be ascribed in large part to differences in their dissociation rate constants. The consequences of this kinetic behavior for future design are discussed.

Recognition of the DNA minor groove by pyrrole-imidazole polyamides: comparison of desmethyl- and N-methylpyrrole.

Polyamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), and N-methyl-3-hydroxypyrrole (Hp) are synthetic ligands that recognize predetermined DNA sequences with affinities and specificities comparable to many DNA-binding proteins. As derivatives of the natural products distamycin and netropsin, Py/Im/Hp polyamides have retained the N-methyl substituent, although structural studies of polyamide:DNA complexes have not revealed an obvious function for the N-methyl. In order to assess the role of the N-methyl moiety in polyamide:DNA recognition, a new monomer, desmethylpyrrole (Ds), where the N-methyl moiety has been replaced with hydrogen, was incorporated into an eight-ring hairpin polyamide by solid-phase synthesis. MPE footprinting, affinity cleavage, and quantitative DNase I footprinting revealed that replacement of each Py residue with Ds resulted in identical binding site size and orientation and similar binding affinity for the six-base-pair (bp) target DNA sequence. Remarkably, the Ds-containing polyamide exhibited an 8-fold loss in specificity for the match site versus a mismatched DNA site, relative to the all-Py parent. Polyamides with Ds exhibit increased water solubility, which may alter the cell membrane permeability properties of the polyamide. The addition of Ds to the repertoire of available monomers may prove useful as polyamides are applied to gene regulation in vivo. However, the benefits of Ds incorporation must be balanced with a potential loss in specificity.

Asymmetric DNA binding by a homodimeric bHLH protein.

Protein-DNA interactions that lie outside of the core recognition sequence for the Drosophila bHLH transcription factor Deadpan (Dpn) were investigated using minor groove binding pyrrole-imidazole polyamides. Electrophoretic mobility shift assays and DNase I footprinting demonstrate that hairpin polyamides bound immediately upstream, but not immediately downstream of the Dpn homodimer selectively inhibit protein-DNA complex formation. Mutation of the Dpn consensus binding site from the asymmetric sequence 5'-CACGCG-3' to the palindromic sequence 5'-CACGTG-3' abolishes asymmetric inhibition. A Dpn mutant containing the unnatural amino acid norleucine in place of lysine at position 80 in the bHLH loop region is not inhibited by the polyamide, suggesting that the epsilon amino group at this position is responsible for DNA contacts outside the major groove. We conclude that the nonpalindromic Dpn recognition site imparts binding asymmetry by providing unique contacts to the basic region of each monomer in the bHLH homodimer.

Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration.

The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents.

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove.

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.

Anti-repression of RNA polymerase II transcription by pyrrole-imidazole polyamides.

Pyrrole-imidazole polyamides are ligands that bind in the minor groove of DNA with high affinity and sequence selectivity. Molecules of this class have been shown to disrupt specific transcription factor-DNA interactions and to inhibit basal and activated transcription from various RNA polymerase II and III promoters. A set of eight-ring hairpin-motif pyrrole-imidazole polyamides has been designed to bind within the binding site for the human cytomegalovirus (CMV) UL122 immediate early protein 2 (IE86). IE86 represses transcription of the CMV major immediate early promoter (MIEP) through its cognate cis recognition sequence (crs) located between the TATA box and the transcription initiation site. The designed polyamides bind to their target DNA sequence with nanomolar affinities and with a high degree of sequence selectivity. The polyamides effectively block binding of IE86 protein to the crs in DNase I footprinting experiments. A mismatch polyamide, containing a single imidazole to pyrrole substitution, and also a polyamide binding to a site located 14 base pairs upstream of the repressor binding site, do not prevent IE86 binding to the crs. IE86-mediated transcriptional repression in vitro is relieved by a match polyamide but not by a mismatch polyamide. Transcription from a DNA template harboring a mutation in the crs is not affected either by IE86 protein or by the match polyamides. These results demonstrate that this new class of small molecules, the pyrrole-imidazole polyamides, are not only effective inhibitors of basal and activated transcription, but also can be used to activate transcription by blocking the DNA-binding activity of a repressor protein.

The tax protein-DNA interaction is essential for HTLV-I transactivation in vitro.

The human T-cell leukemia virus type-1 (HTLV-I)-encoded Tax protein enhances viral gene transcription through interaction with three repeated DNA elements located in the viral promoter. These elements, called viral CREs, are composed of an off-consensus eight base-pair cyclic AMP response element (CRE), immediately flanked by sequences that are rich in guanine and cytosine residues. Recent biochemical experiments have demonstrated that in the presence of the cellular protein CREB, Tax directly binds the viral CRE G+C-rich sequences via interaction with the minor groove. To determine the functional significance of the Tax-DNA interaction, we synthesized minor groove-binding pyrrole-imidazole polyamides which bind specifically to the G+C-rich sequences in the viral CREs. At concentrations where the polyamides specifically protect the G+C-rich sequences from MPE:Fe cleavage, the polyamides block the Tax-DNA interaction. At precisely these same concentrations, the polyamides specifically inhibit Tax transactivation in vitro, without altering CREB-activated transcription or basal transcription from the same promoter. Together, these data provide strong evidence that Tax-viral CRE interaction is essential for Tax function in vitro, and suggest that targeted disruption of the Tax-DNA minor groove interaction with polyamides may provide a novel approach for inhibiting viral replication in vivo.

Inhibition of Ets-1 DNA binding and ternary complex formation between Ets-1, NF-kappaB, and DNA by a designed DNA-binding ligand.

Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for TBP, Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor Ets-1 is specifically inhibited by a polyamide bound in the minor groove. Ets-1 is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-kappaB, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1. NF-kappaB.DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for NF-kappaB also inhibits NF-kappaB binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA.

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.