A study of lead uptake and distribution in horns from lead-dosed goats using synchrotron radiation-induced micro X-ray fluorescence elemental imaging.

OBJECTIVE: The principal goal of this study was to investigate the uptake and distribution of lead (Pb) in the horns of Pb-dosed goats, and to explore possible links to their historical Pb dosing records. Horn is a keratinized material that grows in discrete increments with the potential to preserve the historical record of past environmental exposures. While previous studies have leveraged this potential to examine environmental and biological phenomena in horns, Pb uptake has never been explored. METHODS: Horns were collected post-mortem from three goats that had been previously used to produce blood lead reference materials for the New York State proficiency testing program. The animals were periodically dosed with lead acetate, administered orally in a capsule, over a 5 to 8-year period. Horn cross sections were taken from each animal and analyzed using synchrotron radiation-induced micro X-ray fluorescence spectrometry (SR-µXRF) at the Cornell High Energy Synchrotron Source (CHESS). RESULTS: Elemental distribution maps were obtained by SR-µXRF for Pb, Ca, S, Se, and three other elements (Br, Zn and Cu), with values reported quantitatively as a mass fraction (µg/g for trace elements and mg/g for Ca and S). Accumulations of Pb were clearly visible as a series of narrow "rings" in each of the horn samples analyzed. The elements Ca, S, Br, Zn, and Cu were also detected as discrete rings within each cross-section, with Br strongly correlated with S in the samples examined. A marginal increase in Se may coincide with Pb accumulation in horn cross-sections. Annual mineralization estimates based on the relative distribution of Ca and S were used to establish a tentative timeline for horn growth, with each timeline linked to the pattern of Pb accumulation in the corresponding horn cross-section sample. CONCLUSIONS: Following ingestion, absorbed Pb is eventually deposited into caprine horns, resulting in discrete accumulations or "rings." Elemental mapping by SR-µXRF clearly show Ca-rich layers that vary with annual periodicity, consistent with previous reports of horn mineralization. Localized enrichment of Cu, Zn, Br and S appear to coincide with the keratinized regions related to the annual growth ring pattern in horns. Spatial analysis of horns for Pb accumulation may be useful as a qualitative marker of time-resolved exposures that may reflect specific periods of acute Pb absorption.

Microstructure and mechanical properties of different keratinous horns.

Animal horns play an important role during intraspecific combat. This work investigates the microstructure and mechanical properties of horns from four representative ruminant species: the bighorn sheep (Ovis canadensis), domestic sheep (Ovis aries), mountain goat (Oreamnos americanus) and pronghorn (Antilocapra americana), aiming to understand the relation between evolved microstructures and mechanical properties. Microstructural similarity is found where disc-shaped keratin cells attach edge-to-edge along the growth direction of the horn core (longitudinal direction) forming a lamella; multiple lamellae are layered face to face along the impact direction (radial direction, perpendicular to horn core growth direction), forming a wavy pattern surrounding a common feature, the tubules. Differences among species include the number and shape of the tubules, the orientation of aligned lamellae and the shape of keratin cells. Water absorption tests reveal that the pronghorn horn has the largest water-absorbing ability due to the presence of nanopores in the keratin cells. The loading direction (compressive and tensile) and level of hydration vary among the horns from different species. The differences in mechanical properties among species may relate to their different fighting behaviours: high stiffness and strength in mountain goat to support the forces during stabbing; high tensile strength in pronghorn for interlocked pulling; impact energy absorption properties in domestic and bighorn sheep to protect the skull during butting. These design rules based on evolutionary modifications among species can be applied in synthetic materials to meet different mechanical requirements.

Peptidome characterization of the antipyretic fraction of Bubali Cornu aqueous extract by nano liquid chromatography with orbitrap mass spectrometry detection.

In the present study, the antipyretic activity of Bubali Cornu (water buffalo horn) fraction and its metabolomics were investigated. The fraction decreased rat rectal temperature, and 13 endogenous metabolites were identified as potential biomarkers. Selected metabolites were involved in arachidonic acid metabolism and glycerophospholipid metabolism etc. Following treatment with the fraction, four metabolites, pyroglutamic acid, palmitelaidic acid, leukotriene A4, and prostaglandin A2 were reversed. In addition, the levels of interleukin-1ß, tumor necrosis factor-α, prostaglandin E2 , and cyclic adenosine monophosphate in plasma were also reversed after treatment as determined by enzyme linked immunosorbent assay. Furthermore, nano-flow liquid chromatography with orbitrap mass spectrometry detection was used to analyze the peptides in the fraction. In total, 824 peptide sequences mainly from keratins were determined, with Keratin 14, Keratin 34, and Keratin 86 representing the three main types of keratin hydrolysis in water buffalo horn based on peptide heat maps. Of the identified peptides, 81.2% were hydrophilic and the molecular weight of 70.3% of identified peptides was lower than 2000 Da. According to the metabolomics- and peptidomics-based approach used in the present study, it is feasible to identify and analyze the active peptide matrix from animal-horn-derived traditional Chinese medicines.

Molecular identification of antelope horn by melting curve analysis.

Antelope horn is a valuable Chinese traditional medicine and widely used in clinic. However, with the deterioration of antelope's living environment and a lot of killing, the saiga population begins falling and in some places plummet. Since the increasing demand of this expensive and good bioactive medicine, the horn of artiodactyla animals is often used as the antelope horn. The adulterated or impostor not only cause damage to clinical medicine but also affect the antelope resources protection and sustainable development. Here, in order to establish a melting curve analysis (MCA) method to distinguish the antelope horn from other animal horns and identify the decoction pieces and Chinese patent medicine in a fast and easy way, animal horns and its decoction pieces, Chinese patent medicines were collected from the market and the DNA of all the collected samples were extracted. The melting curve of two universal fragments (COI and Cyt b) was scanned and Cyt b was selected as feasibility fragment for identifying authentic antelope horn from eight adulterant animal horns. After optimizing the condition for MCA, inspecting the precision and the replication of the method, a reference melting curve modern was established and we performed MCA on the antelope horns, fakes, and adulterants on a 1:1 mix, decoction pieces, and Chinese patent medicine. Thus, this study provides fast and easy methods so that MCA can detect the truth, fakes, and adulterations of antelope horns.

Microstructure and mechanical properties of horns derived from three domestic bovines.

The microstructure and mechanical properties of horns derived from three domestic bovines (buffalo, cattle and sheep) were examined. The effects of water content, sampling position and orientation of three bovid horns on mechanical properties were systematically investigated by uniaxial tension and micron indentation tests. Meanwhile, the material composition and metal element contents were determined by Raman spectroscopy and elemental analysis respectively, and the microstructures of the horns were measured by scanning electron microscopy (SEM). Results show that the mechanical properties of horns have negative correlation with water contents and depend on sampling position and orientation. The spatial variations of the mechanical properties in horns are attributed to the different keratinization degrees in the proximal, middle and distal parts. And the mechanical properties of horns in the longitudinal direction are better than those in transverse. Among the three kinds of horns, the mechanical properties of buffalo horn are the best, followed by cattle horn, and those in sheep horn are the worst. This is due to the differences in material composition, metal element, and the microstructures of the horns. But the mechanical properties of buffalo horns are not dependent on the source of the buffalo. Therefore, regular engineered buffalo keratinous materials with standard mechanical properties can be obtained from different buffalo horns by using proper preparation methods.

Beetle horns and horned beetles: emerging models in developmental evolution and ecology.

Many important questions in developmental biology increasingly interface with related questions in other biological disciplines such as evolutionary biology and ecology. In this article, we review and summarize recent progress in the development of horned beetles and beetle horns as study systems amenable to the integration of a wide range of approaches, from gene function analysis in the laboratory to population ecological and behavioral studies in the field. Specifically, we focus on three key questions at the current interface of developmental biology, evolutionary biology and ecology: (1) the developmental mechanisms underlying the origin and diversification of novel, complex traits, (2) the relationship between phenotypic diversification and the diversification of genes and transcriptomes, and (3) the role of behavior as a leader or follower in developmental evolution. For each question we discuss how work on horned beetles is contributing to our current understanding of key issues, as well as highlight challenges and opportunities for future studies.

Identification of novel exons and transcripts by comprehensive RNA-Seq of horn cancer transcriptome in Bos indicus.

Horn cancer (HC) is a widely prevalent cancer amongst Bos indicus cattle and frequently associated with economic losses due to reduction in draught and milk production capacity. To identify novel transcripts and characterize HC transcriptome, we employed high throughput RNA-Seq analysis of HC and horn normal (HN) tissue. Total of 584,450 and 920,450 high quality reads were analyzed for HN and HC tissue, respectively, resulting in the identification of 16,803 unique genes comprising of 14,754 annotated genes and 2049 novel transcripts. We found 251 significant differentially expressed genes in HC transcriptome, which includes 52 putative novel transcripts. We validated presence of 23 out of 28 novel transcripts by RT-PCR and confirmed differential expression of 12 novel transcripts through RT-qPCR on 4 samples each of HC and HN. Functional analysis revealed that HC is characterized by deregulation of genes involved in epithelial cell differentiation and complement cascade. Our study will serve as a step further in detailed characterization of HC transcriptome and provide firm base to explore and mitigate HC at finer resolution.

A study to compare circulating flunixin, meloxicam and gabapentin concentrations with prostaglandin E2 levels in calves undergoing dehorning.

The purpose of this study was to investigate the pharmacokinetics of intravenous flunixin (2.2 mg/kg b.w.), oral meloxicam (1mg/kg b.w.), oral gabapentin (15 mg/kg b.w.) alone or co-administrated with meloxicam as well as the effects of these compounds on prostaglandin E2 (PGE2) synthesis in calves subjected to surgical dehorning. Plasma samples collected up to 24h after drug administration were analyzed by liquid chromatography/mass spectrometry, whereas blood PGE2 levels were measured by immunoenzymatic assay. In plasma, the terminal half-live of flunixin, meloxicam and gabapentin were 6.0 h (range, 3.4-11.0 h), 16.7h (range, 13.7-21.3h) and 15.3h (range, 11-32.9h), respectively. The co-administration of single doses of gabapentin and meloxicam did not seem to affect the pharmacokinetic profile of the two drugs except for gabapentin that reached significantly (P<0.05) higher maximum serum concentration (Cmax) when co-administered with meloxicam, than when administered alone. At 5, 360 and 720 min after dehorning, a significant (P<0.01) decrease in PGE2 concentration was observed in flunixin-treated animals compared with control calves. Moreover, circulating log PGE2 concentrations were inversely proportional to log flunixin concentrations (R(2)=0.75; P<0.0001). None of the other drugs significantly affected blood PGE2 levels. Further assessment of oral meloxicam and gabapentin in established pain models is required to formulate science based analgesic recommendations to enhance animal well-being after dehorning.

Insulin signaling as a mechanism underlying developmental plasticity: the role of FOXO in a nutritional polyphenism.

We investigated whether insulin signaling, known to mediate physiological plasticity in response to changes in nutrition, also facilitates discrete phenotypic responses such as polyphenisms. We test the hypothesis that the gene FOXO--which regulates growth arrest under nutrient stress--mediates a nutritional polyphenism in the horned beetle, Onthophagus nigriventris. Male beetles in the genus Onthophagus vary their mating strategy with body size: large males express horns and fight for access to females while small males invest heavily in genitalia and sneak copulations with females. Given that body size and larval nutrition are linked, we predicted that 1) FOXO expression would differentially scale with body size (nutritional status) between males and females, and 2) manipulation of FOXO expression would affect the nutritional polyphenism in horns and genitalia. First, we found that FOXO expression varied with body size in a tissue- and sex-specific manner, being more highly expressed in the abdominal tissue of large (horned) males, in particular in regions associated with genitalia development. Second, we found that knockdown of FOXO through RNA-interference resulted in the growth of relatively larger copulatory organs compared to control-injected individuals and significant, albeit modest, increases in relative horn length. Our results support the hypothesis that FOXO expression in the abdominal tissue limits genitalia growth, and provides limited support for the hypothesis that FOXO regulates relative horn length through direct suppression of horn growth. Both results support the idea that tissue-specific FOXO expression may play a general role in regulating scaling relationships in nutritional polyphenisms by signaling traits to be relatively smaller.

Application of 12S rRNA gene for the identification of animal-derived drugs.

PURPOSE. Animal-derived drugs are the major source of biological products and traditional medicine, but they are often difficult to identify, causing confusion in the clinical application. Among these medicinal animals, a number of animal species are endangered, leading to the destruction of biodiversity. The identification of animal-derived drugs and their alternatives would be a first step toward biodiversity conservation and safe medication. Until now, no effective method for identifying animal-derived drugs has been demonstrated; DNA-based species identification presents a brand-new technique. METHODS. We designed primers to amplify a 523-bp fragment of 12S rRNA and generated sequences for 13 individuals within six medicinal animal species. We examined the efficiency of species recognition based on this sequence, and we also tested the taxonomic affiliations against the GenBank database. RESULTS. All the tested drugs were identified successfully, and a visible gap was found between the inter-specific and intra-specific variation. We further demonstrated the importance of data exploration in DNA-based species identification practice by examining the sequence characteristics of relative genera in GenBank. This region of the 12S rRNA gene had a 100% success rate of species recognition within the six medicinal animal species. CONCLUSIONS. We propose that the 12S rRNA locus might be universal for identifying animal-derived drugs and their adulterants. The development of 12S rRNA for indentifying animal-derived drugs that share a common gene target would contribute significantly to the clinical application of animal-derived drugs and the conservation of medicinal animal species. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Finite element analysis of ramming in Ovis canadensis.

The energy produced during the ramming of bighorn sheep (Ovis canadensis) would be expected to result in undesirable stresses in their frontal skull, which in turn would cause brain injury; yet, this animal seems to suffer no ill effects. In general, horn is made of an α-keratin sheath covering a bone. Despite volumes of data on the ramming behavior of Ovis canadensis, the extent to which structural components of horn and horn-associated structure or tissue absorb the impact energy generated by the ramming event is still unknown. This study investigates the hypothesis that there is a mechanical relationship present among the ramming event, the structural constituents of the horn, and the horn-associated structure. The three-dimensional complex structure of the bighorn sheep horn was successfully constructed and modeled using a computed tomography (CT) scan and finite element (FE) method, respectively. Three different three-dimensional quasi-static models, including a horn model with trabecular bone, a horn model with compact bone that instead of trabecular bone, and a horn model with trabecular bone as well as frontal sinuses, were studied. FE simulations were used to compare distributions of principal stress in the horn and the frontal sinuses and the strain energy under quasi-static loading conditions. It was noticed that strain energy due to elastic deformation of the complex structure of horn modeled with trabecular bone and with trabecular bone and frontal sinus was different. In addition, trabecular bone in the horn distributes the stresses over a larger volume, suggesting a mechanical link between the structural constituents and the ramming event. This phenomenon was elucidated through the principal stress distribution in the structure. This study will help designers in choosing appropriate material combinations for the successful design of protective structures against a similar impact.

Keratinolytic activity from new recombinant fusant AYA2000, derived from endophytic Micromonospora strains.

Two different endophytic strains, ESRAA1997 and ALAA2000, were isolated from the Egyptian herbal plant Anastatica hierochuntica. The 2 strains produced alkaline serine protease and were identified based on their phenotypic and chemotypic characteristics as different strains of Micromonospora spp. Both strains grew and produced keratinase, using different keratinous waste substances as the sole source of carbon and nitrogen. In our study, the activity and properties of keratinase enzymes of the wild strains ESRAA1997 and ALAA2000 were altered by genetic recombination through protoplast fusion between them, leading to a potent keratinolytic fusant Micromonospora strain AYA2000 with improved properties (activity, stability, specificity, and tolerance to inhibitors). Using a mixture of yeast extract, peptone, and malt extract as a supplement to the bovine hair medium increased keratinase production by 48%, and addition of 1% glucose suppressed enzyme production by Micromonospora strain AYA2000. The enzyme was purified by ammonium sulphate precipitation and DEAE-cellulose chromatography followed by gel filtration. The molecular weight, estimated using SDS-PAGE, was 39 kDa. The enzyme exhibited remarkable activity towards all keratinous wastes used and could also adapt to a broad range of pH and temperatures, with optima at pH 11 and 60 °C. The enzyme was not influenced by chelating reagents, metal ions, or alcohols. These properties make AYA2000 keratinase an ideal candidate for biotechnological application.

EST and microarray analysis of horn development in Onthophagus beetles.

BACKGROUND: The origin of novel traits and their subsequent diversification represent central themes in evo-devo and evolutionary ecology. Here we explore the genetic and genomic basis of a class of traits that is both novel and highly diverse, in a group of organisms that is ecologically complex and experimentally tractable: horned beetles. RESULTS: We developed two high quality, normalized cDNA libraries for larval and pupal Onthophagus taurus and sequenced 3,488 ESTs that assembled into 451 contigs and 2,330 singletons. We present the annotation and a comparative analysis of the conservation of the sequences. Microarrays developed from the combined libraries were then used to contrast the transcriptome of developing primordia of head horns, prothoracic horns, and legs. Our experiments identify a first comprehensive list of candidate genes for the evolution and diversification of beetle horns. We find that developing horns and legs show many similarities as well as important differences in their transcription profiles, suggesting that the origin of horns was mediated partly, but not entirely, by the recruitment of genes involved in the formation of more traditional appendages such as legs. Furthermore, we find that horns developing from the head and prothorax differ in their transcription profiles to a degree that suggests that head and prothoracic horns are not serial homologs, but instead may have evolved independently from each other. CONCLUSION: We have laid the foundation for a systematic analysis of the genetic basis of horned beetle development and diversification with the potential to contribute significantly to several major frontiers in evolutionary developmental biology.

Differential recruitment of limb patterning genes during development and diversification of beetle horns.

The origins of novel complex phenotypes represent one of the most fundamental, yet largely unresolved, issues in evolutionary biology. Here we explore the developmental genetic regulation of beetle horns, a class of traits that lacks obvious homology to traits in other insects. Furthermore, beetle horns are remarkably diverse in their expression, including sexual dimorphisms, male dimorphisms, and interspecific differences in location of horn expression. At the same time, beetle horns share aspects of their development with that of more traditional appendages. We used larval RNA interference-mediated gene function analysis of 3 cardinal insect appendage patterning genes, dachshund, homothorax, and Distal-less, to investigate their role in development and diversification of beetle horns within and between species. Transcript depletion of all 3 patterning genes generated phenotypic effects very similar to those documented in previous studies that focused on general insect development. In addition, we found that Distal-less and homothorax, but not dachshund, regulate horn expression in a species-, sex-, body region-, and body size-dependent manner. Our results demonstrate differential co-option of appendage patterning genes during the evolution and radiation of beetle horns. Furthermore, our results illustrate that regulatory genes whose functions are otherwise highly conserved nevertheless retain the capacity to acquire additional functions, and that little phylogenetic distance appears necessary for the evolution of sex- and species-specific differences in these functions.

On the origin and evolutionary diversification of beetle horns.

Many scarab beetles produce rigid projections from the body called horns. The exaggerated sizes of these structures and the staggering diversity of their forms have impressed biologists for centuries. Recent comparative studies using DNA sequence-based phylogenies have begun to reconstruct the historical patterns of beetle horn evolution. At the same time, developmental genetic experiments have begun to elucidate how beetle horns grow and how horn growth is modulated in response to environmental variables, such as nutrition. We bring together these two perspectives to show that they converge on very similar conclusions regarding beetle evolution. Horns do not appear to be difficult structures to gain or lose, and they can diverge both dramatically and rapidly in form. Although much of this work is still preliminary, we use available information to propose a conceptual developmental model for the major trajectories of beetle horn evolution. We illustrate putative mechanisms underlying the evolutionary origin of horns and the evolution of horn location, shape, allometry, and dimorphism.

Activity levels of 137Cs and 40K in the skin and the cutaneous organs of a cow.

We have performed an extensive study to determine the activity distributions of radiocesium (137Cs) and of the radioisotope 40K of potassium in the whole body of a cow. ICRP assumes that cesium and potassium are distributed homogeneously throughout the whole body of an organism. The current investigation measured concentrations of 137Cs and 40K in components of the skin, horns, and hooves of a cow. Activity levels of 137Cs were caused by the long-term ingestion following the Chernobyl fallout, whereas the naturally occurring potassium (40K) is an essential part of cow's normal diet. The cow was born at the time of the fallout following the Chernobyl accident and had ingested heavily contaminated forage during its entire lifetime. Activities of 137Cs and 40K were determined simultaneously by gamma spectrometry. All activities are related to the day of slaughter and include also corrections for self attenuation of photons caused by the different densities of the samples. Radionuclide concentrations in samples were corrected for moisture losses incurred during freezing and sample preparation. Surface contamination on the skin was estimated by rinsing it in heated water followed by removal of the epidermis and hair. In comparison with the activities of the components of the skin we observed a considerable amount of external contamination on the surface of the skin. But with respect to whole body countings of the animal this amount of external contamination appears to be negligible. It was found that activity ratios of 137Cs to 40K are greater than 1 in all measured components. The average activity concentrations of 137Cs and 40K in the common integument are 23.3 Bq kg(-1) and 13.3 Bq kg(-1), respectively. The highest activity concentrations of 137Cs and 40K were measured in clean hair and hypodermis. Despite being its largest organ, the cow's skin contains probably less than 1% of the animal's whole body 137Cs activity.

The mineralization of crystalline inorganic components in Japanese serow horn.

The Japanese serow (Capricornis crispus) is protected as a special natural monument in Japan. The ring count of the soft X-ray photographs of Japanese serow horn was found to be a useful criteria to determine the ages exactly. The mineralization process in Japanese serow horn was examined microscopic, ICP and X-ray diffraction methods. The incremental lines appeared as light and dark layers in the section stained for fuchsin and methylen blue. Mineral depositions were observed among the keratin fibers, no matrix vesicle in the electron dense regions. X-ray diffraction pattern of crystalline inorganic components in Japanese serow horn was determined as beta-tricalcium phosphate (TCP), hydroxyapatite (HA) and unknown phase. ICP measurement was also carried out. The horn contained trace elements of K besides Na, Ca, Fe and P. The Ca/P molar was found to be 2.9. The ratio was much higher than the theoretical value of HA. Presumably, keratin was the seed which might be related to mineralization and higher Ca activity was detected in the initial phase of epitaxial growth. Analytical results of the measurement of trace elements in Japanese serow horn by using ICP method seemed to be correlated with the evaluation of environmental conditions. The present study indicated that the mineralization of Japanese serow horn directly related with deposition Ca-deficient HA among the keratin fibers.