Stable carbon and nitrogen isotope discrimination in soft tissues of the leatherback turtle (Dermochelys coriacea): Insights for trophic studies of marine turtles

The trophic ecology of marine vertebrates has been increasingly studied via stable isotope analysis of body tissues. However, the theoretical basis for using stable isotopes to elucidate consumer–prey relationships remains poorly validated for most taxa despite numerous studies using this technique in natural systems. In this study, we measured stable carbon and stable nitrogen diet-tissue discrimination (Δ_dt) in whole blood, red blood cells, blood plasma solutes, and skin of leatherback sea turtles (Dermochelys coriacea; N = 7) maintained in captivity for up to 424 days and fed an isotopically consistent control diet with a mean C:N ratio of 2.94:1.00 and an energetic content of 20.16 ± 0.39 kJ g^− 1 Dry Mass. We used a random-effect repeated measure model to evaluate isotopic consistency among tissue samples collected on days 276, 348, and 424. Both δ¹³C and δ¹⁵N remained consistent among sampling events in all tissues (all 95% posterior intervals for the slopes of a linear model included zero), indicating that all tissues had fully integrated diet-derived stable isotope compositions. Mean tissue-specific δ¹³C ranged from − 18.30 ± 0.16‰ (plasma solutes) to − 15.54 ± 0.14‰ (skin), whereas mean δ¹⁵N was from 10.06 ± 0.22‰ (whole blood) to 11.46 ± 0.10‰ (plasma solutes). The computed Δ_dt factors for carbon ranged from − 0.58‰ (plasma solutes) to + 2.25‰ (skin), whereas Δ_dt for nitrogen was from + 1.49 (red blood cells) to + 2.85 (plasma solutes). As the only discrimination factors available for leatherback turtles, our data will be useful for future interpretations of field-derived stable isotope data for this species. The inherent variability in Δ_dt values among individuals was low, which supports the value of these data for dietary reconstructions. However, it is important to note that tissue-specific discrimination factors for leatherbacks contrast with the widely accepted values for endothermic species (0–1‰ for C, 3–5‰ for N), and are also different from values established for hard-shelled turtles. This underscores the need for species- and tissue-specific discrimination factors before interpreting trophic studies of wild animals, including marine turtles.     

Acupuncture for smokers: lack of long-term therapeutic effect in a controlled study.

Two types of acupuncture therapy, one aimed specifically at smoking withdrawal and the other aimed at enhancing relaxation, were compared with self-monitoring in 75 healthy men that wished to stop smoking. During the 2 weeks following treatment there was no significant difference in the adjusted mean daily smoking rates of the subjects receiving acupuncture therapy of the two types, but their combined rate was significantly lower than the rate of the subjects in the self-monitoring group. However, at 1, 3, and 6 months following treatment there were no longer statistically significant differences between the three treatment groups in the adjusted mean smoking rates. At no time were there significant differences between the three treatment groups in the proportion of subjects that stopped smoking during the study. Although acupuncture appears to have become a popular treatment for cigarette smokers, its effectiveness remains to be proven in the treatment of tobacco addiction.     

Methods to collect, preserve, and prepare elasmobranch tissues for stable isotope analysis

Stable isotope analysis has the potential to expand our understanding of elasmobranch ecology. However, elasmobranchs share unique traits (i.e., retention of urea, lack of adipose tissue, cartilaginous skeletons) that require modified preparation techniques. Alternative tissue collection and preservation methods would allow sampling from ichthyology collections and at remote locations. We compared different collection, preservation, and preparation techniques to identify treatments that yielded robust isotopic data. Blood components collected in tubes coated with lithium heparin (an anti-coagulant) were not isotopically distinct from blood collected in no-additive tubes. Compared to frozen muscle, ethanol-treated muscle had altered ??¹³C values, but similar ??¹⁵N values. Finally, we removed lipids and urea with petroleum ether and deionized water, respectively. Although untreated and treated muscle had similar amino acid compositions, treated muscle preferentially lost ¹⁴N and had greater C:N ratios. These results indicate that urea affects isotope ratios and that water treatment removes urea without altering muscle protein composition. Although not exhaustive, our study begins to address the need for elasmobranch-specific methods.     

Irradiation-induced hypoxia in bones and soft tissues: an experimental study

Bone marrow and subcutaneous tissue pO2 and pCO2 were measured by means of implanted tissue tonometers in irradiated and nonirradiated rabbit hind limbs. The x-ray dose was 500, 1000, 1500, 2000, and 3000 rads. Tissue gas tensions were measured 1 day and 5 and 11 weeks after radiation. The pCO2 changes in both tissues were slight but not statistically significant. The subcutaneous tissue pO2 decreased during the acute phase of irradiation injury, and the effect of irradiation was dose-dependent. Later on, irradiation had no significant effects on the subcutaneous pO2, although light microscopy of the affected tissues showed fibrosis and blood vessel changes. The response of the subcutaneous pO2 to systemic hyperoxia also increased in the chronic phase of irradiation injury as a sign of improved microcirculation. The bone marrow showed a high radiosensitivity. Irradiation caused a rapid dose-dependent decrease of the marrow pO2, and the marrow pO2 decreased with time during the chronic phase of irradiation injury. The marrow pO2 responded slowly and marginally to an increment of arterial pO2 during breathing 100% oxygen as further evidence of impaired vascular pattern. The results showed that irradiation causes only a transient impairment of tissue perfusion in the skin. However, irradiation-damaged marrow was characterized by progressive tissue hypoxia.     

AMP-deaminase in elasmobranch fish: a comparative histochemical and enzymatic study

AMP-deaminase activity was measured in white muscle from a wide range of fish, including one cyclostome, 13 chondrosteans, and one teleost to elucidate the pattern of the AMP-deaminase activity in white muscle of fish. Compared to a mammalian (rat) muscle extract, low enzyme activities are found in the cyclostome and two elasmobranchs from two families (Scyliorhinidae, Hexanchidae). In contrast, higher AMP-deaminase activities, similar to mammals, are expressed in Squalidae, all families of skates, Chimaeridae and in the teleostean fish. We then compared AMP-deaminase activities in red and white muscles from two representative elasmobranch fish, the dogfish (Scyliorhinus canicula) and the thornback ray (Raja clavata). The fibre type composition and distribution of the locomotory musculature were determined in these two elasmobranchs to establish a relationship between the morphology, the type of fibres of the locomotion-implicated muscles and the AMP-deaminase activity. Experimental data are discussed with respect to the layout of fibres in the myotome. In both species, three fibre types were identified. In the two fish myotomes, most of the axial muscles are white fibres while red fibres constitute a thin sheet. Some differences were observed between the two species in the distribution of intermediate fibres: in dogfish, these are located between the red and white fibres; in thornback ray, some are dispersed within the white fibre region, while others form an intermediary layer like in dogfish. These results suggest that in the course of evolution, an amplification of the AMP-deaminase activity in muscle was coupled with increase of complexity of the muscular structure.     

New method for comparing DNA primary sequences based on a discrimination measure

We introduce a new approach to compare DNA primary sequences. The core of our method is a new measure of pairwise distances among sequences. Using the primitive discrimination substrings of sequence S and Q, a discrimination measure DM(S, Q) is defined for the similarity analysis of them. The proposed method does not require multiple alignments and is fully automatic. To illustrate its utility, we construct phylogenetic trees on two independent data sets. The results indicate that the method is efficient and powerful.     

Carbon and nitrogen dynamics in a maritime Antarctic stream

• 1 The carbon and nitrogen dynamics in a maritime Antarctic lake outflow stream were investigated. The stream and the algal communities could be split into two zones: a semi-aquatic margin consisting of a perennial cyanobacteria/diatom mat and a flowing channel with a similar perennial mat that was overgrown by annual filamentous chlorophytes during the course of the summer.
• 2 Neither algal community was limited by nutrient availability. Major nutrients were always available in the stream water. There were slight differences in the atomic ratios of the mats, the N:P ratios in the channel mat being lower than those in the marginal mat. However, both these and the total dissolved N:P ratio in the stream water were all close to those that indicate a balanced supply.
• 3 There was no net carbon or nitrogen accumulation by the marginal mat suggesting that uptake processes were balanced by loss processes.
• 4 Maximum rates of carbon fixation (0.1–0.5mgCg^?1 dry weight h^?1) were similar to those of other perennial Antarctic algal mats. Productivity appeared to be limited by physical factors, but the effects of irradiance and temperature could not be separated.
• 5 There were no heterocystous cyanobacteria in the mat communities and rates of atmospheric nitrogen fixation were very low (0–10ngNmg^?1 mat Nh^?1). Fixation accounted for only 0.3% of the nitrogen accumulation of the channel mats, but was higher in the marginal mat where uptake of other sources of nitrogen was also low.
• 6 Nitrogen accumulation by the channel mat averaged 0.34gNm^?2 day^?1. Only 0.05gNm^?2 day^?1 was accounted for by the uptake of dissolved inorganic nitrogen (nitrate plus ammonium). The major (80%) source of nitrogen appeared to be dissolved organic nitrogen. Recycling of nitrogen within the stream ecosystem may also be important.

     

Prospects for measuring the rheologic characteristics of human soft tissues based on the recording of their transverse rigidity

It is shown by an example of a forearm that to determine objective characteristics of rheological properties of soft human tissues by measuring their transverse stiffness (K) one should first investigate the relation between K and the diameter of the used indentor (d). The nonlinearity of the dependence K(d) for indentor diameters from 2 to 8 mm is demonstrated and it is assumed that this nonlinearity is due to heterogeneity of tissue properties in the directions normal to the axis of force action.     

Carbon and nitrogen limitations on soybean seedling development

Carbon and nitrogen limitations on symbiotically grown soybean seedlings (Glycine max [L.] Merr.) were assessed by providing 0.0, 1.0, or 8.0 millimolar NH₄NO₃ and 320 or 1,000 microliters CO₂/liter for 22 days after planting. Maximum development of the Rhizobium-soybean symbiosis, as determined by acetylene reduction, was measured in the presence of 1.0 millimolar NH₄NO₃ under both levels of CO₂. Raising NH₄NO₃ from 0.0 to 8.0 millimolar under 320 microliters CO₂/liter increased plant dry weight by 251% and Kjeldahl N content by 287% at 22 days after planting. Increasing NH₄NO₃ from 1.0 to 8.0 millimolar under 320 microliters CO₂/liter increased total dry weight and Kjeldahl N by 100 and 168%, respectively, on day 22. Raising CO₂ from 320 to 1,000 microliters CO₂/liter during the same period had no significant effect on Kjeldahl N content of plants grown with 0.0 or 1.0 millimolar NH₄NO₃. The maximum CO₂ treatment effects were observed in plants supplied with 8.0 millimolar NH₄NO₃, where dry weight and Kjeldahl N content were increased 64% and 20%, respectively. An increase in shoot CO₂-exchange rate associated with the CO₂-enrichment treatment was reflected in a significant increase in leaf dry weight and starch content for plants grown with 1,000 microliters CO₂/liter under all combined N treatments. These data show directly that seedling growth in symbiotically grown soybeans was limited primarily by N availability. The failure of the CO₂-enrichment treatment to increase total plant N significantly in Rhizobium-dependent plants indicates that root nodule development and functioning in such plants was not limited by photosynthate production.     

Carbon and nitrogen stocks in physical fractions of a subtropical Acrisol as influenced by long-term no-till cropping systems and N fertilisation

The long-term soil management effects on C and N stocks of soil physical fractions are still poorly understood for South American subtropical soils. This study aimed (i) to evaluate the influence of cereal- and legume-based cropping systems and N fertilisation on C and N stocks of the sand-, silt- and clay-size fractions of a no-tilled subtropical Acrisol in southern Brazil, (ii) to compute the Carbon Management Index (CMI) for those cropping systems using physical fractionation data, and (iii) to investigate the possible existence of finite capacity of those soil physical fractions to store C and N. Soil samples of a long-term experiment were collected from the 0–2.5 and 2.5–7.5 cm layers of three no-till cropping systems [fallow bare soil, oat/maize (O/M) and pigeon pea+maize (P+M)] under two N fertilisation levels (0 and 180 kg N ha^–1). However, for fallow bare soil, only the non-fertilised sub-plot was sampled. An adjacent native grassland soil was sampled as a reference. The C and N stocks of the three soil physical fractions were higher in the legume-based cropping system (P+M) than in O/M and bare soil, because of the higher residue input in the former. The P+M cropping system restored the C and N stocks in sand- and silt-size fractions to the same levels found in grassland soil. Higher C and N stocks in all physical fractions were also obtained with N fertilisation. The C and N stocks and the C:N ratio were most affected by cropping systems in the sand- and least in the clay-size fraction. Particulate organic matter was found in the silt-size fraction, showing this fraction is not only constituted by mineral-associated organic mater, as commonly believed. Taking grassland soil as reference (CMI = 100), the CMI ranged from 46, in O/M no N, to 517, in P+M with N, pointing to a better soil management in the latter. The clay-size fraction tended to show a finite capacity to store C and N (48.8 g C kg^–1 and 4.9 g N kg^–1 of clay), which was not verified in sand- and silt-size fractions. The adoption of no-tillage and legume-based cropping systems with high residue input are adequate soil management strategies to improve soil quality and make the agricultural production systems more sustainable in subtropical regions.     

Environmental factors affecting basic nitrogen metabolism and seasonal levels of various nitrogen fractions in tissues of bilberry, Vaccinium myrtillus

Bilberry tissues accumulated nitrogen for the winter in the form of reduced low-molecular weight amino compounds. The storage organ was principally the underground stem and the oldest parts of the aerial shoot. Most of the nitrogen was stored in arginine and ammonium compounds, and less in glutamine and other amino acids. Proteins did not accumulate during the winter. The soluble nitrogenous compounds were discharged from storage in May, when nitrogen was translocated from the lower parts of the stem to the growing leaves and buds. Amino acid compositions and concentrations in winter were almost identical under the snow and in snowless areas, only the concentration of glutamine being lower and that of glutathione higher in the snowless area. The level of total protein, particularly in the leaves and buds was much higher in a nitrogen-polluted industrial area than in unpolluted urban forests. The same difference was observed in total amino compounds, but among individual substances it only appeared in ammonium compounds. Certain species differences in the amino acid pool were recorded between V. myrtillus and V. vitis-idaea.     

The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks Comment on: stable isotope dynamics in elasmobranch fishes

Stable isotopes of nitrogen and carbon (δ¹⁵N and δ¹³C) provide an important tool to examine diet, trophic position and movement/migration of both aquatic and terrestrial animals. Over the past 10 years, there have been repeated calls to tighten up basic assumptions when applying stable isotopes, one of the most important being the application of accurate, species-specific diet-tissue discrimination factors (DTDFs). Taxa- or species-specific DTDFs are required for (i) predicting dietary sources to a consumer using stable isotope mixing models and (ii) for estimating trophic position relative to primary consumers or known base species. Logan & Lutcavage (2010) recently presented data on stable isotope dynamics in elasmobranch fishes and concluded that DTDFs for teleost fish were suitable for elasmobranch fish, endorsing the generally applied value of 3.4‰. When considering (i) a recent study which found that DTDFs were lower for large sharks than teleost fish (Hussey et al., 2010) and (ii) that the Logan and Lutcavage study did not experimentally address the issue of DTDFs, we would argue that this conclusion is misleading. We demonstrate this point by estimating the proportion of prey items of a captive shark with a known diet history by modelling the δ¹⁵N values of the shark and its prey. The often repeated implication of inaccurate DTDFs is clear, with model results highly variable depending on the selected DTDF. In addition, model results for the standard teleost DTDF of 3.4‰ provided erroneous estimates of prey consumption. The suggestion that DTDFs for teleost fish are suitable for elasmobranchs may mislead investigators to choose DTDFs which are likely not applicable to their study species. Caution is therefore warranted in advocating this approach. Continued experimental work to examine stable isotopes in sharks is required and recommendations are made.     

Finite element implementation of a generalized Fung-elastic constitutive model for planar soft tissues

Numerical simulations of the anisotropic mechanical properties of soft tissues and tissue-derived biomaterials using accurate constitutive models remain an important and challenging research area in biomechanics. While most constitutive modeling efforts have focused on the characterization of experimental data, only limited studies are available on the feasibility of utilizing those models in complex computational applications. An example is the widely utilized exponential constitutive model proposed by Fung. Although present in the biomechanics literature for several decades, implementation of this model into finite element (FE) simulations has been limited. A major reason for limited numerical implementations are problems associated with inherent numerical instability and convergence. To address this issue, we developed and applied two restrictions for a generalized Fung-elastic constitutive model necessary to achieve numerical stability. These are (1) convexity of the strain energy function, and (2) the condition number of material stiffness matrix set lower than a prescribed value. These constraints were implemented in the nonlinear regression used for constitutive model parameter estimation to the experimental biaxial mechanical data. We then implemented the generalized Fung-elastic model into a commercial FE code (ABAQUS, Pawtucket, RI, USA). Single element and multi-element planar biaxial test simulations were conducted to verify the accuracy and robustness of the implementation. Results indicated that numerical convergence and accurate FE implementation were consistently obtained. The present study thus presents an integrated framework for accurate and robust implementation of pseudo-elastic constitutive models for planar soft tissues. Moreover, since our approach is formulated within a general FE code, it can be straightforwardly adopted across multiple software platforms.     

Dietary myo-inositol hexaphosphate prevents dystrophic calcifications in soft tissues: a pilot study in Wistar rats

Myo-inositol hexaphosphate (InsP6) is an abundant component of plant seeds. It is also found in significant levels in blood and mammalian tissues, but they are totally dependent on their dietary intake. In the present paper, we describe studies on the effect of InsP6 on a model of dystrophic calcification, which was chemically induced by subcutaneous injection of a 0.1% KMnO4 solution. Male Wistar rats were randomly divided into four groups for treatment over 31 days. A: animals consuming a purified diet in which InsP6 was absent but to which 1% of InsP6 (as sodium salt) was added. In this group, the InsP6 plasma levels (0.393 +/- 0.013 microM) were similar to those observed in rats consuming a standard diet. B: animals consuming only the purified diet in which InsP6 was absent. In this case the InsP6 plasma levels decreased (0.026 +/- 0.006 microM); C: animals consuming the same purified diet as group B but received daily subcutaneous injections of 50 microg kg(-1) etidronate during the last 14 days. In this case the InsP6 plasma levels were also very low (0.025 +/- 0.007 microM); D: animals consuming the same diet as group B but a 6% of carob germ (InsP6 rich product) was added. The InsP6 plasma levels (0.363 +/- 0.035 microM) were also similar to those observed in rats consuming a standard diet. After 21 days plaque formation was induced. Calcification plaques were allowed to proceed for 10 days, after which the plaque material present was excised, dried and weighed. It was found that the presence of myo-inositol hexaphosphate (phytate) in plasma at normal concentrations (0.3-0.4 microM) clearly inhibited the development of dystrophic calcifications in soft tissues. These results demonstrates that myo-inositol hexaphosphate acts as an inhibitor of calcium salt crystallization.     

Mechanical characterization of anisotropic planar biological soft tissues using large indentation: a computational feasibility study

Traditionally, the complex mechanical behavior of planar soft biological tissues is characterized by (multi)axial tensile testing. While uniaxial tests do not provide sufficient information for a full characterization of the material anisotropy, biaxial tensile tests are difficult to perform and tethering effects limit the analyses to a small central portion of the test sample. In both cases, determination of local mechanical properties is not trivial. Local mechanical characterization may be performed by indentation testing. Conventional indentation tests, however, often assume linear elastic and isotropic material properties, and therefore these tests are of limited use in characterizing the nonlinear, anisotropic material behavior typical for planar soft biological tissues. In this study, a spherical indentation experiment assuming large deformations is proposed. A finite element model of the aortic valve leaflet demonstrates that combining force and deformation gradient data, one single indentation test provides sufficient information to characterize the local material behavior. Parameter estimation is used to fit the computational model to simulated experimental data. The aortic valve leaflet is chosen as a typical example. However, the proposed method is expected to apply for the mechanical characterization of planar soft biological materials in general.     

Carbon and nitrogen source effects on basidiomycetes exopolysaccharide production

The capability to synthesize the extracellular polysaccharide (EPS) is widespread among eight mushroom species which accumulated 0.6–2.2 g/1 of EPS in submerged cultivation. Glucose, maltose, and mannitol were the most appropriate carbon sources for biomass and EPS production. Organic nitrogen sources appeared to be the most suitable nitrogen sources for biomass and EPS accumulation. The cultivation process in shake flasks was successfully reproduced in a laboratory fermentor with enhanced EPS production. The highest yield of EPS (3.8–4.0 g/1) was achieved in cultivation of Agaricus nevoi and Inonotus levis.     

Identifying a minimal rheological configuration: a tool for effective and efficient constitutive modeling of soft tissues

We describe a modeling methodology intended as a preliminary step in the identification of appropriate constitutive frameworks for the time-dependent response of biological tissues. The modeling approach comprises a customizable rheological network of viscous and elastic elements governed by user-defined 1D constitutive relationships. The model parameters are identified by iterative nonlinear optimization, minimizing the error between experimental and model-predicted structural (load-displacement) tissue response under a specific mode of deformation. We demonstrate the use of this methodology by determining the minimal rheological arrangement, constitutive relationships, and model parameters for the structural response of various soft tissues, including ex vivo perfused porcine liver in indentation, ex vivo porcine brain cortical tissue in indentation, and ex vivo human cervical tissue in unconfined compression. Our results indicate that the identified rheological configurations provide good agreement with experimental data, including multiple constant strain rate load/unload tests and stress relaxation tests. Our experience suggests that the described modeling framework is an efficient tool for exploring a wide array of constitutive relationships and rheological arrangements, which can subsequently serve as a basis for 3D constitutive model development and finite-element implementations. The proposed approach can also be employed as a self-contained tool to obtain simplified 1D phenomenological models of the structural response of biological tissue to single-axis manipulations for applications in haptic technologies.