Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.     

Spatial interactions between sympatric carnivores: asymmetric avoidance of an intraguild predator

Interactions between intraguild species that act as both competitors and predator–prey can be especially complex. We studied patterns of space use by the black-footed ferret (Mustela nigripes), a prairie dog (Cynomys spp.) specialist, and the American badger (Taxidea taxus), a larger generalist carnivore that competes for prairie dogs and is known to kill ferrets. We expected that ferrets would spatially avoid badgers because of the risk of predation, that these patterns of avoidance might differ between sexes and age classes, and that the availability of food and space might influence these relationships. We used location data from 60 ferrets and 15 badgers to model the influence of extrinsic factors (prairie dog density and colony size) and intrinsic factors (sex, age) on patterns of space use by ferrets in relation to space use by different sex and age categories of badgers. We documented asymmetric patterns of avoidance of badgers by ferrets based on the sex of both species. Female ferrets avoided adult female badgers, but not male badgers, and male ferrets exhibited less avoidance than female ferrets. Additionally, avoidance decreased with increasing densities of prairie dogs. We suggest that intersexual differences in space use by badgers create varying distributions of predation risk that are perceived by the smaller carnivore (ferrets) and that females respond more sensitively than males to that risk. This work advances understanding about how competing species coexist and suggests that including information on both intrinsic and extrinsic factors might improve our understanding of behavioral interactions between sympatric species.     

Psychophysical Investigations into the Role of Low-Threshold C Fibres in Non-Painful Affective Processing and Pain Modulation

We recently showed that C low-threshold mechanoreceptors (CLTMRs) contribute to touch-evoked pain (allodynia) during experimental muscle pain. Conversely, in absence of ongoing pain, the activation of CLTMRs has been shown to correlate with a diffuse sensation of pleasant touch. In this study, we evaluated (1) the primary afferent fibre types contributing to positive (pleasant) and negative (unpleasant) affective touch and (2) the effects of tactile stimuli on tonic muscle pain by varying affective attributes and frequency parameters. Psychophysical observations were made in 10 healthy participants. Two types of test stimuli were applied: stroking stimulus using velvet or sandpaper at speeds of 0.1, 1.0 and 10.0 cm/s; focal vibrotactile stimulus at low (20 Hz) or high (200 Hz) frequency. These stimuli were applied in the normal condition (i.e. no experimental pain) and following the induction of muscle pain by infusing hypertonic saline (5%) into the tibialis anterior muscle. These observations were repeated following the conduction block of myelinated fibres by compression of sciatic nerve. In absence of muscle pain, all participants reliably linked velvet-stroking to pleasantness and sandpaper-stroking to unpleasantness (no pain). Likewise, low-frequency vibration was linked to pleasantness and high-frequency vibration to unpleasantness. During muscle pain, the application of previously pleasant stimuli resulted in overall pain relief, whereas the application of previously unpleasant stimuli resulted in overall pain intensification. These effects were significant, reproducible and persisted following the blockade of myelinated fibres. Taken together, these findings suggest the role of low-threshold C fibres in affective and pain processing. Furthermore, these observations suggest that temporal coding need not be limited to discriminative aspects of tactile processing, but may contribute to affective attributes, which in turn predispose individual responses towards excitatory or inhibitory modulation of pain.     

Technical Note: Calcium and carbon stable isotope ratios as paleodietary indicators

Calcium stable isotope ratios are hypothesized to vary as a function of trophic level. This premise raises the possibility of using calcium stable isotope ratios to study the dietary behaviors of fossil taxa and to test competing hypotheses on the adaptive origins of euprimates. To explore this concept, we measured the stable isotope composition of contemporary mammals in northern Borneo and northwestern Costa Rica, two communities with functional or phylogenetic relevance to primate origins. We found that bone collagen δ¹³C and δ¹⁵N values could differentiate trophic levels in each assemblage, a result that justifies the use of these systems to test the predicted inverse relationship between bioapatite δ¹³C and δ⁴⁴Ca values. As expected, taxonomic carnivores (felids) showed a combination of high δ¹³C and low δ⁴⁴Ca values; however, the δ⁴⁴Ca values of other faunivores were indistinguishable from those of primary consumers. We suggest that the trophic insensitivity of most bioapatite δ⁴⁴Ca values is attributable to the negligible calcium content of arthropod prey. Although the present results are inconclusive, the tandem analysis of δ⁴⁴Ca and δ¹³C values in fossils continues to hold promise for informing paleodietary studies and we highlight this potential by drawing attention to the stable isotope composition of the Early Eocene primate Cantius. Am J Phys Anthropol 154:633–643, 2014. © 2014 Wiley Periodicals, Inc.     

Sex ratios and Ophryocystis elektroscirrha infection levels of Danaus plexippus during spring migration through Oklahoma,USA

Monarch butterflies, Danaus plexippus L. (Lepidoptera: Nymphalidae), have a multiple brood migration in the spring as they move between their overwintering grounds and summer breeding grounds. In Oklahoma, USA, monarchs produce at least one generation in the spring, which develops and continues the northward migration, leaving Oklahoma without a breeding population during the hot summer months. Female monarchs leave the overwintering grounds prior to males, but it is not clear whether females re‐colonize areas along the migration route prior to, or at the same time as males. Male‐to‐female ratios are 1:1 at emergence, but studies have identified a male‐biased sex ratio in the field. Both males and females are susceptible to infection by the obligate protozoan parasite, Ophryocystis elektroscirrha McLaughlin & Myers (OE), which reduces flight abilities and life spans of infected individuals. We examine sex ratios during the spring migration through Oklahoma and whether sex ratios or OE infection estimates vary with capture technique (active or passive). Our data suggest populations are male‐biased during the 1st week of spring migration in Oklahoma, but shift to female‐biased by the 3rd week in both cool and warm springs. Therefore, males may leave southern areas prior to females or migrate longer distances per day. Active sampling (i.e., netting) did not bias sex compared to passive sampling (i.e., sticky traps). Significantly fewer OE‐carrying monarchs (with two or more spores) were captured via netting than by sticky traps which may be caused by sticky trap glue affecting tape sampling effectiveness, but there was no difference in the number of heavily infected individuals (more than 100 spores). Therefore, data from netted monarchs may underestimate OE infection rates within populations.     

Estimating base cation weathering rates in Canadian forest soils using a simple texture-based model

Mineral weathering is the primary source of long-term buffering capacity in soils and is important for forest nutrient sustainability. Regional assessments of weathering rates in Canada and the U.S. have employed an empirical clay-based Soil Texture Approximation (STA) owing to limited data availability, although the STA is rarely calibrated before application to a study area. Soil weathering rates estimated with the STA at 75 sites in Canadian forests (6–367 eq ha^?1 year^?1) were on average seven times lower than estimates obtained using the PROFILE model and when mineralogy was not available, the Analysis to Mineralogy model and parameter estimation (143–2,119 eq ha^?1 year^?1). Comparison with a catchment mass balance at a subset of sites in Ontario (n = 19) demonstrated the reliability of PROFILE weathering estimates. A revised (generalized) STA model for total base cation weathering was developed at the 75 study sites to incorporate soil silt content (%) and loss-on-ignition (LOI, %) (BC _w = (1.73 + 0.03 · silt ? 0.06 · LOI) · 1,000 · depth). The model performance (R _adj ²  = 73%) and relative bias (?1%) suggested that the revised STA may have broad application to forest soils in Canada but may not necessarily be suited to all soil texture classes.     

Selection for methotrexate resistance in mammalian cells bearing a <Emphasis Type="Italic">Drosophila</Emphasis> dihydrofolate reductase transgene

Antifolates, such as methotrexate (MTX), are the treatment of choice for numerous cancers. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms. In Drosophila, however, a resistant phenotype has only been observed to occur by mutation resulting in a MTX-resistant DHFR. It is unclear if differences in gene structure and/or genome organization between Drosophila and mammals contribute to the observed differences in acquired drug resistance. To investigate if gene structure is involved, Drosophila Dhfr cDNA was transfected into a line of CHO cells that do not express endogenous DHFR. These transgenic cells, together with wild-type CHO cells, were selected for 19 months for resistance to increasing concentrations of MTX, from 50- to 200-fold over the initial concentration. Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance.     

Molecular docking and 3D-QSAR studies of HIV-1 protease inhibitors

HIV-1 protease is an obligatory enzyme in the replication process of the HIV virus. The abundance of structural information on HIV-1PR has made the enzyme an attractive target for computer-aided drug design strategies. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV-1PR inhibitors with better efficacy profiles and reduced toxicity. In the present investigation, molecular modeling studies were performed on a series of 54 cyclic urea analogs with symmetric P2/P2′ substituents. The binding modes of these inhibitors were determined by docking. The docking results also provided a reliable conformational superimposition scheme for the 3D-QSAR studies. To gain insight into the steric, electrostatic, hydrophobic and hydrogen-bonding properties of these molecules and their influence on the inhibitory activity, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed. Two different alignment schemes viz. receptor-based and atom-fit alignment, were used in this study to build the QSAR models. The derived 3D-QSAR models were found to be robust with statistically significant r ² and r ² _pred values and have led to the identification of regions important for steric, hydrophobic and electronic interactions. The predictive ability of the models was assessed on a set of molecules that were not included in the training set. Superimposition of the 3D-contour maps generated from these models onto the active site of enzyme provided additional insight into the structural requirements of these inhibitors. The CoMFA and CoMSIA models were used to design some new inhibitors with improved binding affinity. Pharmacokinetic and toxicity predictions were also carried out for these molecules to gauge their ADME and safety profile. The computational results may open up new avenues for synthesis of potent HIV-1 protease inhibitors.