Computational Approaches and Observer Variation in the 3D Musculoskeletal Modeling of the Heads of Anolis.

High-resolution imaging, 3D modeling, and quantitative analyses are equipping evolutionary biologists with new approaches to understanding the variation and evolution of the musculoskeletal system. However, challenges with interpreting DiceCT data and higher order use of modeled muscles have not yet been fully explored, and the error in and accuracy of some digital methods remain unclear. West Indian Anolis lizards are a model clade for exploring patterns in functional adaptation, ecomorphology, and sexual size dimorphism in vertebrates. These lizards possess numerous jaw muscles with potentially different anatomies that sculpt the adductor chamber of the skull. Here we test approaches to quantifying the musculoskeletal shape of the heads of two species of Anolis: A. pulchellus and A. sagrei. We employ comparative approaches such as DiceCT segmentation of jaw muscles, 3D surface attachment mapping, and 3D landmarking with the aim of exploring muscle volumes, 3D muscle fiber architecture, and sexual dimorphism of the skull. We then compare sources of measurement error in these 3D analyses while also presenting new 3D musculoskeletal data from the Anolis feeding apparatus. These findings demonstrate the accessibility and repeatability of these emerging techniques as well as provide details regarding the musculoskeletal anatomy of the heads of A. pulchellus and A. sagrei which show potential for further research of comparative biomechanics and evolution in the clade.

3D Muscle Architecture of the Pectoral Muscles of European Starling (Sturnus vulgaris).

Avian flight is achieved through a number of modifications to the body, including the pectoral girdle, yet little is known about the architecture of the pectoral musculature. Muscle architecture is a critical variable in determining the biomechanical function of the vertebrate musculoskeletal system; however, accurate three-dimensional (3D) understanding of muscle architecture has been historically difficult to acquire. Here, we present a musculoskeletal model of a European starling (Sturnus vulgaris) pectoral girdle generated from iodine contrast-enhanced micro-computed-tomography (CT) data and 3D fiber tracking analysis. We used a template-based fiber-tracking algorithm to reconstruct muscle fibers in 3D based on grayscale differences in CT images, which allowed us to estimate fascicle lengths, pennation angles, muscle volumes, and physiological cross-sectional area. Our modeled muscles were qualitatively accurate; however, quantitative muscle architecture data differed between digital and traditional gross-dissection methods reflecting the complex organization of the tissue and differing natures of data collection. We found that model quality is affected by the resolution of CT image data and the fiber-tracking program's input parameters. Nonetheless, digital fiber tracking offers numerous advantages over gross-dissection methods, most importantly, the ability to visualize and quantify entire muscles in three-dimensions, yielding a much more accurate estimation of whole muscle architecture.

Membrane muscle function in the compliant wings of bats.

Unlike flapping birds and insects, bats possess membrane wings that are more similar to many gliding mammals. The vast majority of the wing is composed of a thin compliant skin membrane stretched between the limbs, hand, and body. Membrane wings are of particular interest because they may offer many advantages to micro air vehicles. One critical feature of membrane wings is that they camber passively in response to aerodynamic load, potentially allowing for simplified wing control. However, for maximum membrane wing performance, tuning of the membrane structure to aerodynamic conditions is necessary. Bats possess an array of muscles, the plagiopatagiales proprii, embedded within the wing membrane that could serve to tune membrane stiffness, or may have alternative functions. We recorded the electromyogram from the plagiopatagiales proprii muscles of Artibeus jamaicensis, the Jamaican fruit bat, in flight at two different speeds and found that these muscles were active during downstroke. For both low- and high-speed flight, muscle activity increased between late upstroke and early downstroke and decreased at late downstroke. Thus, the array of plagiopatagiales may provide a mechanism for bats to increase wing stiffness and thereby reduce passive membrane deformation. These muscles also activate in synchrony, presumably as a means to maximize force generation, because each muscle is small and, by estimation, weak. Small differences in activation timing were observed when comparing low- and high-speed flight, which may indicate that bats modulate membrane stiffness differently depending on flight speed.

The impact of extended care on the long-term maintenance of weight loss: a systematic review and meta-analysis.

Behavioural weight management interventions consistently produce 8-10% reductions in body weight, yet most participants regain weight after treatment ends. One strategy for extending the effects of behavioural interventions has been the provision of extended care. The current study is a systematic review and meta-analysis of the literature on the effect of extended care on maintenance of weight loss. Through database searches (using PubMED, PsychInfo and Cochrane Reviews) and manual searches through reference lists of related publications, 463 studies were identified. Of these, 11 were included in the meta-analysis and an additional two were retained for qualitative analysis. The average effect of extended care on weight loss maintenance was g=0.385 (95% confidence interval: 0.281, 0.489; P<0.0001). This effect would lead to the maintenance of an additional 3.2 kg weight loss over 17.6 months post-intervention in participants provided extended care compared with control. There was no significant heterogeneity between studies, Q=5.63, P=0.845, and there was minimal evidence for publication bias. These findings suggest that extended care is a viable and efficacious solution to addressing long-term maintenance of lost weight. Given the chronic disease nature of obesity, extended care may be necessary for long-term health benefits.

Erythropoietin elevates VO2,max but not voluntary wheel running in mice.

Voluntary activity is a complex trait, comprising both behavioral (motivation, reward) and anatomical/physiological (ability) elements. In the present study, oxygen transport was investigated as a possible limitation to further increases in running by four replicate lines of mice that have been selectively bred for high voluntary wheel running and have reached an apparent selection limit. To increase oxygen transport capacity, erythrocyte density was elevated by the administration of an erythropoietin (EPO) analogue. Mice were given two EPO injections, two days apart, at one of two dose levels (100 or 300 microg kg(-1)). Hemoglobin concentration ([Hb]), maximal aerobic capacity during forced treadmill exercise (VO2,max) and voluntary wheel running were measured. [Hb] did not differ between high runner (HR) and non-selected control (C) lines without EPO treatment. Both doses of EPO significantly (P<0.0001) increased [Hb] as compared with sham-injected animals, with no difference in [Hb] between the 100 microg kg(-1) and 300 microg kg(-1) dose levels (overall mean of 4.5 g dl(-1) increase). EPO treatment significantly increased VO2,max by approximately 5% in both the HR and C lines, with no dosexline type interaction. However, wheel running (revolutions per day) did not increase with EPO treatment in either the HR or C lines, and in fact significantly decreased at the higher dose in both line types. These results suggest that neither [Hb] per se nor VO2,max is limiting voluntary wheel running in the HR lines. Moreover, we hypothesize that the decrease in wheel running at the higher dose of EPO may reflect direct action on the reward pathway of the brain.

The morphological basis of hallucal orientation in extant birds.

The perching foot of living birds is commonly characterized by a reversed or opposable digit I (hallux). Primitively, the hallux of nonavian theropod dinosaurs was unreversed and lay parallel to digits II-IV. Among basal birds, a unique digital innovation evolved in which the hallux opposes digits II-IV. This digital configuration is critical for grasping and perching. I studied skeletons of modern birds with a range of hallucal designs, from unreversed (anteromedially directed) to fully reversed (posteriorly directed). Two primary correlates of hallucal orientation were revealed. First, the fossa into which metatarsal I articulates is oriented slightly more posteriorly on the tarsometatarsus, rotating the digit as a unit. Second, metatarsal I exhibits a distinctive torsion of its distal shaft relative to its proximal articulation with the tarsometatarsus, reorienting the distal condyles and phalanges of digit I. Herein, I present a method that facilitates the re-evaluation of hallucal orientation in fossil avians based on morphology alone. This method also avoids potential misinterpretations of hallucal orientation in fossil birds that could result from preserved appearance alone.

Novel suicide ligands of tubulin arrest cancer cells in S-phase.

It is presently accepted that the mechanism of action for all anti-tumor tubulin ligands involves the perturbation of microtubule dynamics during the G2/M phase of cell division and subsequent entry into apoptosis [1]. In this report, we challenge the established dogma by describing a unique mechanism of action caused by a novel series of tubulin ligands, halogenated derivatives of acetamido benzoyl ethyl ester. We have developed a suicide ligand for tubulin, which covalently attaches to the target and shows potent cancericidal activity in tissue culture assays and in animal tumor models. These compounds target early S-phase at the G1/S transition rather than the G2/M phase and mitotic arrest. Bcl-2 phosphorylation, a marker of mitotic microtubule inhibition by other tubulin ligands was dramatically altered, phosphorylation was rapid and biphasic rather than a slow linear event. The halogenated ethyl ester series of derivatives thus constitute a unique set of tubulin ligands which induce a novel mechanism of apoptosis.

Conservation of intergenic spacer length in ribosomal DNA of the tailed frog, Ascaphus truei.

We have examined the organization of cloned rDNA [encoding ribosomal RNA (rRNA)] repeat units from the tailed frog, Ascaphus truei, and have compared rDNA spacer lengths in the genomes of eleven individuals from two widely-separated populations. This comparison has shown that the A. truei spacer is always very short (about 1.5 kb) and that it is remarkably constant in length. In none of the individuals tested were more than two spacer-length classes found and the maximum difference in spacer length found in comparisons both within single animals and across both populations was about 120 bp. We point out those structural features that may contribute to the unusual stability of this spacer and the consequent absence of the extensive length heterogeneities found amongst rDNA repeat units in most genomes.

Short interspersed repeats from Xenopus that contain multiple octamer motifs are related to known transposable elements.

We have identified in an intron of an X. laevis alpha-tubulin gene a member of a novel family of short (226-431 bp) interspersed repetitive elements. We have isolated other members of this family, which we term Ocr, from ovary cDNA and genome libraries and have identified another two in the published sequences of an H1B histone gene cluster and an actin gene intron. The termini of the Ocr elements are formed by a 19 bp inverted repeat that has clear sequence homologies to those of certain large transposable elements, such as 1723 (Xenopus) and Ac (maize). However, the Ocr elements do not appear to be deletion derivatives of larger transposons. The internal regions of the Ocr elements contain multiple copies of the octamer motif (ATTTGCAT) arranged as divergently-orientated dyads. We have shown by a gel mobility shift assay that these octamer dyads specifically bind what is presumably an OTF-type activator protein in oocyte nuclear extracts. We speculate that short interspersed repetitive families of this type may be generated by a mechanism of replicative transposition that uses a DNA intermediate and involves the interaction of DNA-binding proteins also utilised in other cellular processes.

Premature termination of transcription can be induced on an injected alpha-tubulin gene in Xenopus oocytes.

The Xenopus laevis alpha-tubulin gene X alpha T14, which is highly expressed during oogenesis, exhibits accurate and efficient transcription initiation when microinjected into X. laevis oocytes. However, we found previously in nuclease protection assays of transcripts from injected X alpha T14 that many protected fragments that were shorter than expected could be produced. We show here by exonuclease VII mapping, Northern (RNA) blotting, and gel fractionation of RNA that these fragments were caused by truncated transcripts that share the same initiation sites as mature transcripts but whose 3' ends are located in the 5' leader just 45 to 72 nucleotides downstream. We present evidence from the analysis of in vitro pulse-labeled RNA that these truncated transcripts are formed by premature transcription termination rather than by RNA processing. At low template levels, very little premature termination occurred, but as more DNA was injected, the proportion of transcripts that were prematurely terminated increased steadily, even at template levels at which the initiation machinery was unsaturated. At high template levels, most transcripts were prematurely terminated. These results suggest that some sort of saturable antitermination function operates in oocytes in a manner that is dependent on the number of appropriate templates available rather than on the number of polymerases that initiate transcription. They also suggest that measures of initiation frequency may not always be a reliable means of assessing the amount of transcription of injected genes in oocytes.

An oocyte-expressed alpha-tubulin gene in Xenopus laevis; sequences required for the initiation of transcription.

We have studied the expression of X alpha T14, a member of the alpha-tubulin multigene family in Xenopus laevis. Small amounts of X alpha T14 RNA are detectable in a range of cell types, but much higher levels are present in ovary and tissue culture cells. In oocytes X alpha T14 transcripts accumulate during early vitellogenesis but their level declines in more advanced stages. Faithful and efficient initiation of transcription occurred on cloned X alpha T14 injected into oocytes even at low template levels. We have examined the amount of transcript produced by various deletion mutants relative to a co-injected control gene. The presence of 200bp of DNA 5' and 53bp of DNA 3' to the initiation site sufficed for high levels of promoter activity, although maximum activity required 560 bp of 5' flanking DNA. The DNA between -200 and -60 was necessary for transcription in oocytes and contains several sequence motifs implicated in transcriptional regulation including three CCAAT boxes and a sequence resembling a heat shock element. An 8 bp deletion that removed the latter element from 5kb of 5'-flanking DNA reduced promoter activity by 60%.

Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts.

1. The activities of ecto- and cytosolic 5'-nucleotidase (EC 3.1.3.5), adenosine kinase (EC 2.7.1.20), adenosine deaminase (EC 3.5.4.4) and AMP deaminase (EC 3.5.4.6) were compared in ventricular myocardium from man, rats, rabbits, guinea pigs, pigeons and turtles. The most striking variation was in the activity of the ecto-5'-nucleotidase, which was 20 times less active in rabbit heart and 300 times less active in pigeon heart than in rat heart. The cytochemical distribution of ecto-5'-nucleotidase was also highly variable between species. 2. Adenosine formation was quantified in pigeon and rat ventricular myocardium in the presence of inhibitors of adenosine kinase and adenosine deaminase. 3. Both adenosine formation rates and the proportion of ATP catabolized to adenosine were greatest during the first 2 min of total ischaemia at 37 degrees C. Adenosine formation rates were 410 +/- 40 nmol/min per g wet wt. in pigeon hearts and 470 +/- 60 nmol/min per g wet wt. in rat hearts. Formation of adenosine accounted for 46% of ATP plus ADP broken down in pigeon hearts and 88% in rat hearts. 4. The data show that, in both pigeon and rat hearts, adenosine is the major catabolite of ATP in the early stages of normothermic myocardial ischaemia. The activity of ecto-5'-nucleotidase in pigeon ventricle (16 +/- 4 nmol/min per g wet wt.) was insufficient to account for adenosine formation, indicating the existence of an alternative catabolic pathway.

Metabolic damage to human saphenous vein during preparation for coronary artery bypass grafting.

Segments of saphenous vein from patients undergoing coronary artery by-pass graft surgery were frozen in liquid nitrogen immediately on dissection (control), after stripping of the adventitia and side branch ligation (manipulation), after distention with blood (distention), or at completion of the last proximal anastomosis (prepared vein). Vein was stored during the operation in patient's heparinised arterial blood at room temperature. Frozen vein was extracted with perchloric acid. ATP, ADP, and AMP, adenosine, inosine and hypoxanthine concentrations were measured by high pressure liquid chromatography. Prepared vein had ca 50% lower ATP concentrations and ATP/ADP ratio than control vein, higher concentrations of inosine and hypoxanthine and lower concentrations of AMP and adenosine. ATP concentration and ATP/ADP ratio did not correlate with the time elapsed between dissection and freezing of the prepared vein. The characteristic changes seen in prepared vein were not seen when control vein was simply stored in arterial blood at 23 degrees C, in normal saline at 23 degrees C or 4 degrees C, in Krebs-Ringer bicarbonate buffer at 37 degrees C or at St Thomas's Hospital cardioplegic solution at 4 degrees C. Distention with unlimited pressure did not distension at less than 300 mmHg gave rise to the same changes in ATP concentration and ATP/ADP ratio as in the prepared vein. These results show that vein suffered metabolic changes during preparation for bypass grafting and suggest that uncontrolled distention may contribute to these changes. Such biochemical measurements provide a quantitative estimate of tissue damage and allow objective comparison of different preparative techniques.

Catecholamine-induced necrosis and hypertrophy. Hemodynamic factors.

Catecholamine-induced myocardial hypertrophy and necrosis in rats have been measured and compared following treatment with different catecholamines. Significant degrees of both hypertrophy (whether measured as biventricular weight or biventricular/body weight ratio) and necrosis (measured by enzyme histochemical techniques on a standardized series of cryostat sections through the apex of each heart) occurred following 10 days' treatment with daily isoproterenol (0.5 or 5 mg/kg s.c.) or dobutamine (5 mg/kg s.c.) (N = 6-12). These agents given to conscious restrained animals lowered blood pressure and increased heart rate for 3, 6, or 1 hr, respectively. Neither hypertrophy nor necrosis occurred after norepinephrine (1 mg/kg) or dopamine (5 mg/kg); both these agents acutely increased blood pressure for about 30 min. Hemodynamic factors may therefore contribute to catecholamine-induced necrosis, which may in turn contribute to the associated hypertrophy.