Relationships between dental microwear and diet in Carnivora (Mammalia) — Implications for the reconstruction of the diet of extinct taxa

Food consumption causes distinct microwear patterns on teeth, especially in mammals that actively masticate food. Here we perform a microwear analysis to assess the relationships between diet and microwear features of diverse Carnivora. Our database includes approximately 230 individuals of 17 extant species having different diets. We analyse both slicing and grinding facets of M1 and m1. The proposed method is reproducible and allows the differentiation, especially on slicing facets, of microwear poles that are significantly distinct from one another. In carnivorans, the microwear features mainly result from their foraging behavior and the proportion of certain food items consumed. We applied our method to extinct taxa such as the amphicyonid Amphicyon major. The results on the m1 slicing facet indicate dietary similarities between this large Miocene predator and the extant red fox; results from the m1 grinding facet do not have equivalent in extant taxa, however.     

Evaluation of plasma enzyme activities using gas chromatography–mass spectrometry based steroid signatures

The simultaneous quantification of 65 plasma steroids, including 22 androgens, 15 estrogens, 15 corticoids and 13 progestins, was developed using gas chromatography-mass spectrometry (GC–MS). The extraction efficiency of the catechol estrogens was improved by the addition of l-ascorbic acid in several steps. All steroids, as their trimethylsilyl derivatives, were well separated with good peak shapes within a 50 min run. The devised method provided good linearity (correlation coefficient, r² > 0.993), while the limit of quantification ranged from 0.2 to 2.0 ng mL^?1. The precision (% CV) and accuracy (% bias) were 2.0–12.4% and 93.5–109.2%, respectively. The metabolic changes were evaluated by applying this method to plasma samples obtained from 26 healthy male subjects grouped according to the pre- and post-administration of dutasteride, which inhibits 5α-reductase isoenzyme types 1 and 2. The levels of three plasma steroids, such as dihydrotestosterone, 5α-androstanedione and allotetrahydrocortisol, were decreased significantly after drug administration, while the levels of testosterone and 5β-androstane-3β,17α-diol were increased. In addition, the ratios of the steroid precursors and their metabolites, which represent the activities of the related enzymes, were z-score transformed for visualization in heat maps generated using supervised hierarchical clustering analysis. These results validated the data transformation because 5α-reductase is an indicator for the biological actions of dutasteride. GC–MS base quantitative visualization might be found in the integration with the mining biomarkers in drug evaluations and hormone-dependent diseases.     

Can diet composition estimates using stable isotope analysis of feathers predict growth and condition in nestling mountain bluebirds (Sialia currucoides)?

Insectivorous birds breeding in seasonal environments provision their dependent young during periods when prey diversity and abundance vary. Consequently, the composition and nutritional value of diets parents feed to their offspring may differ within and among broods, potentially affecting the condition of nestlings. In a population of mountain bluebirds (Sialia currucoides), we used two methods to estimate diet composition for individual nestlings: direct observation of provisioning using video recordings at 5 and 9 days post‐hatch, and stable isotopes of the δ¹³C and δ¹⁵N in nestling feathers and prey followed by analysis with mixing models. We determined the macronutrient content (% fat and lean mass) and estimated the metabolized energy from each type of prey. We evaluated whether different methods of estimating diet composition would produce similar results, and whether the types of prey nestlings ate at one or both ages affected their morphology, growth rates, or blood ketone concentration. We found that bluebirds fed their young 5 main types of prey: beetles, cicadas, grasshoppers, insect larvae, and spiders. Both observational and mixing model estimates of diet composition indicated that larvae are traded off with grasshoppers and that fewer larvae are provided to nestlings as the season progresses. In evaluating how diet influences individual growth and condition, estimates from direct observations had greater explanatory power than those from mixing models, indicating that diets rich in the most energy‐dense prey (greatest fat content; cicadas and larvae) were associated with larger size and higher body condition, and faster rate of mass gain and growth of tarsus. Lower value prey had more limited, specific effects on nestlings, but may still be important dietary components. While isotopic methods produced estimates of diet composition that were generally informative, when applied to explain the growth and condition of nestlings they proved less useful.     

The use of strontium and barium analyses for the reconstruction of the diet of the early medieval coastal population of Gdańsk (Poland): A preliminary study

Barium and strontium analyses yield an important perspective on temporal shifts in diet in relation to social and environmental circumstances. This research focuses on reconstructing dietary strategies of individuals in the early medieval (12-13th century) population of Gdańsk on the coast of the Baltic Sea. To describe these strategies where seafood rich in minerals was included in the diet, levels of strontium, barium, calcium and phosphorus were measured in first permanent molars of adult men and women whose remains were excavated from the churchyard in the city centre. Faunal remains from the excavation were analysed as an environmental background with respect to the content of the above-mentioned elements. The impact of diagenesis on the odontological material under study was also determined by an analysis of the soil derived from the grave and non-grave surroundings. For verification of diagenetic processes, the calcium/phosphorus index was used. Strontium, calcium, phosphorus and barium levels were determined with the spectrophotometric method using the latest generation plasma spectrophotometer Elan 6100 ICP-MS. From the results of the analysis of taphonomic parameters such as the soil pH, potential ion exchange in the grave surroundings and the Ca/P ratio, it can be inferred that diagenetic factors had little impact on the studied material. From this pilot study we can conclude that in the early Middle Ages in the Baltic Sea basin, seafood was included in the diet from early childhood and at the same time the diet contained calcium-rich milk products (also rich in minerals). The lack of sex differences may indicate the absence of a sex-specific nutritional strategy in childhood and early adolescence.     

Detection of 5-hydroxymethylcytosine in a combined glycosylation restriction analysis (CGRA) using restriction enzyme Taq(α)I

5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA base in mammalian cells that is believed to be another important epigenetic modification. Here we report the use of a methylation-insensitive restriction enzyme Taq^αI coupled with selective chemical labeling of 5-hmC in a combined glycosylation restriction analysis (CGRA) to detect 5-hmC in TCGA sequences. This method, differentiates fully versus hemi-hydroxymethylated cytosine in the CpG dinucleotide, adds a new tool to facilitate biological studies of 5-hmC.     

Chemical shift assignments for F-plasmid TraI (381–569)

TraI, the F plasmid-encoded nickase, is a 1,756 amino acid protein essential for conjugative transfer of F plasmid DNA from one bacterium to another. While crystal structures of N- and C-terminal domains of F TraI have been determined, central domains of the protein are structurally unexplored. These middle domains (between residues 306 and 1,500) are known to both bind single-stranded DNA (ssDNA) and unwind DNA through a highly processive helicase activity. Of this central region, the more C-terminal portion (~900–1500) appears related to helicase RecD of the E. coli RecBCD complex. The more N-terminal portion (306–900), however, shows limited sequence similarity to other proteins. In an attempt to define the structure of well-folded domains of this middle region and discern their function, we have isolated stable regions of TraI following limited proteolysis. One of these regions, TraI (381–569), was identified and a genetic construct encoding it was engineered. The protein was expressed, purified, and the sequence-specific chemical shifts for it were assigned.     

Tissue-specific response of δ15N in adult Pacific herring (Clupea pallasi) following an isotopic shift in diet

The objective of this study was to measure the tissue-specific response of isotope δ¹⁵N to changes in isotopic signature of diet in an adult Pacific herring, Clupea pallasi, and to examine the importance of growth and metabolism in this shift. This was accomplished by placing wild adult Pacific herring in captivity and monitoring isotopic shift in tissues with a corresponding isotopic shift in diet, and the application of a metabolism/growth mixing model. Tissues examined were blood, eye, heart, liver, and white muscle. One group of herring was given a δ¹⁵N diet depleted by approximately 5.4‰, and another given a ¹⁵N-enriched diet labeled with 98 atom% l-phenylalanine. This study showed that (i) isotopic response of individual tissues following an isotopic shift in diet varied in both rate of change and fractionation level, (ii) most of this isotopic shift is due to growth, and (iii) white muscle and liver tissue appeared the most responsive to isotopic shift in diet, reaching isotopic equilibrium with diet in a matter of months (not years). For trophic studies using δ¹⁵N, these results indicate that field measurement of Pacific herring should be done after much of summer growth has occurred.     

Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume–rhizobia symbiosis

Root nodule (RN) symbiosis is a mutualistic interaction observed between nitrogen-fixing soil bacteria and nodulating plants, which are scattered in only four orders of angiosperms called nitrogen-fixing clade. Most of legumes engage in RN symbiosis with rhizobia. Molecular genetic analyses with legumes and non-leguminous nodulating plants revealed that RN symbiosis utilizes early signalling components that are required for symbiosis with arbuscular mycorrhizal (AM) fungi. However detailed evolutionary processes are still largely unknown. Comparative analyses with non-nodulating species phylogenetically related to legumes could be better strategies to study the evolution of RN symbiosis in legumes. Polygala paniculata is a non-leguminous species that belongs to a family different from legumes but that is classified into the same order, Fabales. It has appropriate characteristics for cultivation in laboratories: small body size, high fertility and short lifecycles. Therefore, we further assessed whether this species is suitable as a model species for comparative studies with legumes. We first validated that the plant we obtained in Palau was truly P. paniculata by molecular phylogenetic analysis using rbcL sequences. The estimated genome size of this species was less than those of two model legumes, Lotus japonicus and Medicago truncatula. We determined conditions for cultivation in vitro and for hairy root formation from P. paniculata seedlings. It would facilitate to investigate gene functions in this species. The ability of P. paniculata to interact with AM fungi was confirmed by inoculation with Rhizophagus irregularis, suggesting the presence of early signalling factors that might be involved in RN symbiosis. Unexpectedly, branching of root hairs was observed when inoculated with Mesorhizobium loti broad host range strain NZP2037, indicating that P. paniculata has the biological potential to respond to rhizobia. We propose that P. paniculata is used as a model plant for the evolutionary study of RN symbiosis.

     

Ontogenetic diet shift in Commerson’s dolphin (Cephalorhynchus commersonii commersonii) off Tierra del Fuego

The Commerson’s dolphin (Cephalorhynchus c. commersonii, Lacépède, 1804) is the most common endemic dolphin of the coastal waters of Tierra del Fuego incidentally caught in artisanal coastal gill nets. A better understanding of its trophic ecology is essential to facilitate the effective management and conservation of its population. The aim of this study was to analyze possible shifts in diet among age and sex classes by analyzing bone collagen δ¹³C and δ¹⁵N values collected from 220 individuals along the coast of Tierra del Fuego. Additional analysis of potential prey allowed us to use a stable isotope mixing model to quantify resource use. Isotopic comparisons suggest that all age classes share the same foraging areas. We found, however, a significant dietary shift: juveniles consume a higher mean (±SD) proportion (60 ± 7 %) of pelagic Sprattus fuegensis than adults (36 ± 10 %), and the consumption of benthopelagic species such as Illex argentinus and Loligo gahi is higher in adults (33 ± 12 %) than juveniles (20 ± 7 %). This may result from an improvement in foraging skills and expansion of habitats in adulthood. While males move greater distances in search of mates and resources, a lack of isotopic differences between the sexes suggests little evidence for sex-related resource partitioning. A significant decrease in δ¹⁵N values between calves and juveniles indicates a weaning period between 0.5 and 1 year as was shown in previous captive studies on this species. Our results also suggest that artisanal fisheries, despite being a major source of mortality, do not affect the long-term food habits of Commerson’s dolphin since they showed a weak trophic overlap.     

Multicenter evaluation of an enzyme immunoassay (Platelia® Aspergillus) for the detection of Aspergillus antigen in serum

Invasive aspergillosis is a serious problem for immunocompromised patients, especially if neutropenic. The diagnosis of this infection is complicated, since clinical symptoms are often similar to those of other fungal diseases. The chance of detecting the presence of a specific antigen in the serum could confirm the suspected clinical diagnosis and. perhaps, be useful for the follow-up of the patient. The Medical Mycology Committee of the Associazione Microbiologi Clinici Italiani (AMCLI) decided to evaluate in a multicenter prospective study (from I November 1998 to 28 February 1999) the performance of the Platelia Aspergillus Kit (Bio-Rad) for the detection of Aspergillus galactomannan in human serum. The enrolled patients included various groups of immunosuppressed patients (mostly neutropenic). Blood samples were drawn at the time of enrollment. This decision was based upon a clinical diagnosis of probable aspergillosis (antibiotic non-responsive fever for at least 96 hours, cough, hemophthosis and positive chest X-ray). Additional blood samples were drawn on days 3, 6, 9, 12, 15 and 21. Culture and histopathologic examinations were performed according to the individual laboratory workflow. For each patient the laboratory filled a form with all the available clinical information, to create a database on which to evaluate the results of the test. During the study, 187 patients with various kinds of immunosuppression were enrolled. A total of 256 sera were tested: for 117 patients (62.6%) only the basal sample was tested, whereas for the 70 symptomatic patients (37.4%) multiple specimens (range: 1-6) were tested. The results allowed the laboratories to exclude (68.6%) or confirm (31.5%: confirmed and/or probable) the clinical diagnosis of invasive aspergillosis; 4 cases remained undetermined. Based on the results of this study, it seems that the use of this test should be limited to those patients with clinical symptoms of aspergillosis.     

RAPD technique for taxonomic studies of Pellia epiphylla‐complex (Hepaticae, Metzgeriales)

The random amplified polymorphic DNA (RAPD) technique was used to study the genomic relationships of three liverworts from the genus Pellia: P. epiphylla, P. borealis and P. neesiana. Altogether 150 characters (150 DNA fragments obtained using PCR) were scored. These characters were used to create a matrix of pairwise distances between all the pairs of taxa. Both distance (UPGMA, Fitch–Margoliash and Neighbor–Joining) and binary character‐state (Wagner and Camin‐Sokal parsimony and compatibility) methods were applied for trees' construction. Our results strongly support distinction of the recently discovered sibling species of P. epiphylla – species N and P. epiphylla – species S, which have an allopatric distribution in Poland (N – North, S – South Poland). Moreover, our data also supports the hypothesis of a hybrid origin (alloploid) of the polyploid P. borealis from P.epiphylla−N×P.epiphylla−S. P. neesiana was excluded as a donor of either of the genomes of P. borealis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification of lipase from Geotrichum candidum: conditions optimized for enzyme production using Box–Behnken design

The fungus Geotrichum candidum was selected from isolates of oil-mill waste as a potent lipase producer. Factors affecting lipase production by the fungus G. candidum in yeast-extract-peptone medium have been optimized by using a Box–Behnken design with seven variables to identify the significant correlation between effects of these variables in the production of the enzyme lipase. The experimental values were found to be in accordance with the predicted values, the correlation coefficient is 0.9957. It was observed that the variables days (6), pH (7.0), temperature (30 °C), carbon (1.25%), nitrogen (2.0%), Tween (1.0%) and salt concentrations (0.5 mM) were the optimum conditions for maximum lipase production (87.7 LU/ml). The enzyme was purified to homogeneity with an apparent molecular mass of 32 kDa by SDS-PAGE. The optimum pH at 40 °C was 7.0 and the optimum temperature at pH 7.0 was 40 °C. The enzyme was stable within a pH range of 6.5 to 8.5 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, and EDTA. However, the presence of Ca²⁺ and Ba²⁺ ions enhanced the activity of the enzyme.     

Derivation of 13C chemical shift surfaces for the anomeric carbons of oligosaccharides and glycopeptides using ab initio methodology

The dependence between the anomeric carbon chemical shift and the glycosidic bond , dihedral angles in oligosaccharide and glycopeptide model compounds was studied by Gauge-Including Atomic Orbital (GIAO) ab initio calculations. Complete chemical shift surfaces versus and for d-Glcp-d-Glcp disaccharides with (11), (12), (13), and (14) linkages in both - and -configurations were computed using a 3-21G basis set, and scaled to reference results from calculations at the 6-311G^** level of theory. Similar surfaces were obtained for GlcNAcThr and GlcNAcSer model glycopeptides in - and -configurations, using in this case different conformations for the peptide moiety. The results obtained for both families of model compounds are discussed. We also present the determination of empirical formulas of the form ¹³C=f(,) obtained by fitting the raw ab initio data to trigonometric series expansions suitable for use in molecular mechanics and dynamics simulations. Our investigations are consistent with experimental observations and earlier calculations performed on smaller glycosidic bond models, and show the applicability of chemical shift surfaces in the study of the conformational behavior of oligosaccharides and glycopeptides.     

Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme–substrate docking studies

Ethyl (S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced (S)-ECHB exclusively, whereas YOR120W and YOL151W made (R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced (S)-ECHB exclusively.     

Cytochrome P-450 for 11beta- and 18-hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two?

The 11beta- and 18-hydroxylase activities of a highly purified cytochrome P-450 from bovine adrenocortical mitochondria have been examined in detail with a view to determining whether the two activities are shown by a single protein or by two distinct proteins. The purified enzyme shows a single N-terminal residue (glutamic acid) and its amino acid composition is reported. Both enzyme activities decay considerably during storage at 4 degrees C for 11 days and the rates of decay are similar for the two activities. Metyrapone inhibits both activities competitively (Ki = 1.50 and 1.43 micrometer for 11beta- and 18-hydroxylase, respectively). Carbon monoxide inhibits both activities and the ratio CO:O2 for 50% inhibition is similar for the two activities (K = 1.69 and 1.53). A variety of nonspecific inhibitors produce approximately the same inhibition of both activities. Finally, antiserum produced by rabbits to the purified enzyme on double diffusion in agarose gels gives a single band with the purified enzyme. Increasing concentrations of antiserum added to the assay system produce increasing and proportionate inhibition of both activities. The evidence strongly supports earlier suggestions that the two hydroxylase activities occur in a single protein.     

Identifying carbon sources and trophic position of coral reef fishes using diet and stable isotope (δ15N and δ13C) analyses in two contrasted bays in Moorea, French Polynesia

Stable isotope ratios (δ¹⁵N and δ¹³C) and diet of three fish species, Stegastes nigricans, Chaetodon citrinellus and Epinephelus merra, were analyzed on the fringing coral reefs of two bays that are differentially exposed to river runoff on Moorea Island, French Polynesia. S. nigricans and C. citrinellus relied mostly on turf algae and presented similar trophic levels and δ¹⁵N values, whereas E. merra fed on large invertebrates (crabs and shrimps) and had higher trophic levels and δ¹⁵N values. Discrepancies existed between stomach content and stable isotope analyses for the relative importance of food items. Bayesian mixing models indicated that sedimented organic matter was also an important additional food for S. nigricans and C. citrinellus, and fishes for E. merra. The main sources of organic matter involved in the food webs ending with these species were algal turfs and surface sediments, while water particulate organic matter was barely used. Significant spatial differences in C and N isotopic ratios for sources and fishes were found within and between bays. Lower ¹³C and higher ¹⁵N values were observed for various compartments of the studied trophic network at the end of each bay than at the entrance. Differences were observed between bays, with organic sources and consumers being, on average, slightly more ¹³C-depleted and ¹⁵N-enriched in Cook’s Bay than in Opunohu Bay, linked with a higher mean annual flow of the river at Cook’s Bay. Our results suggest that rivers bring continental material into these two bays, which is partly incorporated into the food webs of fringing coral reefs at least close to river mouths. Thus, continental inputs can influence the transfer of organic matter within coral reef food webs depending on the diet of organisms.     

CAD–CAM prosthetically guided bone regeneration using preformed titanium mesh for the reconstruction of atrophic maxillary arches

The protocol presented here is intended to minimise the intervention in bone reconstruction surgery when severe atrophy or deformity is present in the maxillary arches. A patient underwent augmentation of an atrophic maxillary arch using titanium mesh and particulate autogenous plus bovine demineralised bone. After computed tomography data elaboration, computer-aided design and computer-aided machining were used to plan the augmentation of bone volume to improve the implant position needed to support the final dental prosthesis. The augmented maxilla was rapidly prototyped in plastic, and the titanium mesh was tested on this model before the surgical intervention. Then, the preformed titanium mesh was implanted in the maxillary arch with bone grafting. The bone was augmented relative to the position of the implants for the definitive fixed implant-supported rehabilitation. The protocol presented here is a viable, reproducible way to determine the correct bone augmentation for the final implant-supported prosthetic rehabilitation.     

ASP – a simulation-based power calculator for genetic linkage studies of qualitative traits, using sib-pairs

An easy-to-use, simulation-based power calculator (ASP) for linkage analysis using sib-pair designs (concordant or discordant) has been developed and made publicly available via the Internet. The program employs a diallelic model for the trait locus, at which parental/offspring genotypes are simulated, assuming Hardy-Weinberg equilibrium in the parental generation. Genotypes at a linked multiallelic marker locus are simulated conditional upon the inheritance pattern at the trait locus, allowing for recombination. Marker genotypes are tested for non-Mendelian identity-by-descent sharing, using both an unrestricted and a restricted likelihood ratio test, the latter representing an extension of the "mean test" from fully to partially informative families. The power of user-defined datasets is estimated by the number of simulations giving significant results at varying type I error levels.     

Development of an enzyme activity screening system for β-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting

Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying β-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.