Three-dimensional soft tissue preservation revealed in the skin of a non-avian dinosaur

The most commonly preserved soft tissues associated with ornithischian dinosaurs are skin remains. The apparent resistance of hadrosaur skin to decay, and its abundance in the fossil record relative to that of other tetrapods, has been attributed to factors such as thickness and composition. Here we report additional intrinsic factors within hadrosaur skin: 3D-preserved eumelanin-bearing bodies, dermal cells and blood vessel fragments in an organic matrix composed of protein fossilization products. The skin is much thinner than that of living mammals of similar size. It is likely that the preservation of hadrosaur skin is related to the arrangement of the layers composing it.     

Decaying in different clays: implications for soft‐tissue preservation

Lagerstätten, places where soft‐bodied organisms became mineralized, provide a substantial bulk of palaeobiological information, but the detailed mechanisms of how soft‐tissue preservation takes place remain debatable. An experimental taphonomy approach, which allows for direct study of decay and mineralization, offers a means to study the preservational potential of different soft‐bodied organisms under controlled conditions. Here we compare the preservational capacity of two types of clay (kaolinite and montmorillonite) through a long‐term (24 month) experiment involving the burial and decay of small crustaceans. Our experimental design is innovative in that it models catastrophic sedimentation in fine‐grained colloidal suspension, which is believed to form Lagerstätten deposits. We demonstrated better preservation of buried organisms in clays compared to water, and in kaolinite compared to montmorillonite. As aluminium cations were present in high concentrations in kaolinite sediment but not in montmorillonite, the better preservation in kaolinite is attributed to the tanning properties of aluminium, which catalyses cross‐linking in proteins, protecting them from bacterial degradation. Anaerobic environments and acidification also slow down decay, but they are less effective than tanning. Kaolinite and montmorillonite replaced the crustacean integuments differently: in the remains buried in kaolinite, Al and Si were detected in equal proportions, while in those buried in montmorillonite, the Si content appeared to be much higher even in comparison with the initial sample of the clay. These variations probably arose from the different dynamics of acidic hydrolysis in the two clays associated with anaerobic decomposition of organic matter. Our results show that the preservation mechanism includes multi‐component interactions between the solution, mineral, sediment and organic remains; taken separately, any single component explains little. The specific conditions that occur within the colloidal clay sediments can facilitate conservation and start fast mineralization according to chemical properties and elemental content.     

Disarticulation patterns in Ordovician crinoids: Implications for the evolutionary history of connective tissue in the Crinoidea

Taphonomic information is examined to evaluate the early history of connective tissues in the Crinoidea. The pattern of stalk segmentation of Middle and Late Ordovician crinoids is consistent with the two-ligament (intercolumnal and through-going ligaments) pattern present in living isocrinid crinoids and interpreted for fossil isocrinids, holocrinids, and Lower Mississippian crinoids. A single rhombiferan was also examined; its taphonomic pattern is also indicative of this style of tissue organization. Furthermore, the taphonomy of all Middle and Late Ordovician crinoids may reflect that they lacked discretely organized muscles between arm brachials, which is consistent with the hypothesis that muscles evolved as a connective tissue between plates only once within the Crinoidea, during the Early Devonian. These data indicate that the two-ligament organization of the stalk is a primitive feature among the Crinoidea and perhaps even among stalked echinoderms. Therefore, the autotomy function of this column-tissue organization among living crinoids is an exaptation. On the other hand, discretely organized muscles as connective tissue in crinoid arms is a derived trait that first appeared during the middle Paleozoic; this adaptation proved very successful for the advanced cladid crinoids.     

Connective tissue growth factor is necessary for retinal capillary basal lamina thickening in diabetic mice.

Experimental prevention of basal lamina (BL) thickening of retinal capillaries ameliorates early vascular changes caused by diabetes. Connective tissue growth factor (CTGF) is upregulated early in diabetes in the human retina and is a potent inducer of expression of BL components. We hypothesize that CTGF is causally involved in diabetes-induced BL thickening of retinal capillaries. To test this hypothesis, we compared the effects of streptozotocin (STZ)-induced diabetes on retinal capillary BL thickness between wild-type mice (CTGF+/+) and mice lacking one functional CTGF allele (CTGF+/-). Differences in BL thickness were calculated by quantitative analysis of electron microscopic images of transversally sectioned capillaries in and around the inner nuclear layer of the retina. We show that BL thickening was significant in diabetic CTGF+/+ mice compared with control CTGF+/+ mice, whereas diabetes did not significantly induce BL thickening in CTGF+/- mice. We conclude that CTGF expression is necessary for diabetes-induced BL thickening and suggest that reduction of CTGF levels may be protective against the development of diabetic retinopathy.     

Unveiling biases in soft‐tissue phosphatization: extensive preservation of musculature in the Cretaceous (Cenomanian) polychaete Rollinschaeta myoplena (Annelida: Amphinomidae)

The process of soft‐tissue phosphatization (the replication of labile tissues by calcium phosphate) is responsible for many instances of high‐resolution soft tissue preservation, often revealing anatomical insights into the animals that so preserved. However, while much work has gone into exploring key issues such as biases and micro‐controls, phosphatization remains poorly understood as a taphonomic process. Here, using camera lucida, plain‐light microscopy and SEM imagery, we address this issue by describing the taphonomy and fidelity of the musculature of Rollinschaeta myoplena Parry et al., a phosphatized annelid from the Cretaceous Konservat‐Lagerstätten of Hakel and Hjoula, Lebanon, with an unprecedented quantity of three‐dimensional soft‐tissue preservation. Analysis highlights two strong, previously recognized biases affecting the process of phosphatization: (1) a taxonomic bias restricted to R. myoplena that triggers unusually extensive phosphatization; and (2) a tissue bias whereby longitudinal and parapodial musculature show markedly higher fidelity in comparison to the musculature of the intestine and body wall circular muscles. Potential explanations for these biases include internal phosphate‐enrichment by relative muscle density, the relative rate of decay and the physiology of musculature. Incongruence between experimental decay series for polychaetes and the prevalence of labile tissue preservation over recalcitrant tissues in R. myoplena exposes the limits of decay experiments for understanding exceptional preservation.     

Chemical preservation of tail feathers from Anchiornis huxleyi,a theropod dinosaur from the Tiaojishan Formation (Upper Jurassic,China)

A panel of geochemical techniques is used here to investigate the taphonomy of fossil feathers preserved in association with the skeleton of the Jurassic theropod Anchiornis huxleyi. Extant feathers were analysed in parallel to test whether the soft tissues morphologically preserved in the fossil also exhibit a high degree of chemical preservation. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) indicate that clays and iron oxide pseudomorphs occur in the surrounding sediment and also reveal the preservation of melanosome-like microbodies in the fossil. Carbon gradient along a depth profile and co-occurrence of carbon and sulphur are shown in the fossil by elastic backscattering (EBS) and particle-induced x-ray emission (PIXE), which are promising techniques for the elemental analysis of fossil soft tissues. The molecular composition of modern and fossil soft tissues was assessed from micro-attenuated total reflectance fourier transform infrared spectroscopy (micro-ATR FTIR), solid-state ¹³C nuclear magnetic resonance (CP-MAS ¹³C NMR) and pyrolysis gas chromatography mass spectrometry in the presence of TMAH (TMAH-Py-GC-MS). Results indicate that the proteinaceous material that comprises the modern feathers is not present in the fossil feathers. The fossil feathers and the embedding sediment exhibit a highly aliphatic character. However, substantial differences exist between these samples, revealing that the organic matter of the fossil feathers is, at least partially, derived from original constituents of the feathers. Our results suggest that, despite the morphological preservation of Anchiornis feathers, original proteins, that is keratin, were probably not preserved in the 160-myr-old feathers.     

Species composition of dabbling duck assemblages: ecomorphological patterns compared with null models

Ecomorphological patterns of breeding dabbling duck (Anas spp.) assemblages were studied in six regions in northern Europe. Observed spacings among species in terms of bill lamellar density and body length were compared with expected spacings based on null models incorporating different levels of constraints (regional species pools, species relative abundances, lake size and habitat requirements of species). Deviations of observed spacings from expected ones were compared with prey abundance and prey size diversity in the lakes. Observed spacings in terms of body length, but not in terms of bill lamellar density, were greater than expected on the basis of null models. The most abundant species were generally relatively more different than less abundant species in terms of body length but not in terms of bill lamellar density. Deviations between observed and expected spacings in terms of body length were more like those predicted by the competition hypothesis in lakes with low food abundance than in lakes with high food abundance. Patterns in bill lamellar spacings were not related to food abundance nor to food size diversity. In general, patterns in body length spacings were consistent with the competition hypothesis whether the null model used in comparisons was constrained or not.     

An easy technique for the clearing of histochemically stained plant tissue

The sites of GUS-activity in strongly pigmented whole-mount preparations or thick handmade sections of plant material can easily be visualized by clearing it with chlorallactophenol (CLP), without affecting the ClBr-indigo precipitate.     

Compensatory growth in the gibel carp following feed deprivation: temporal patterns in growth, nutrient deposition, feed intake and body composition

To investigate the nature of compenstory growth in fish, an 8 week study at 28°C was performed on juvenile gibel carp Carassius auratus gibelio weighing 6·6 g. Fish were starved for 0 (control), 1 (S1) or 2 (S2) weeks and then re-fed to satiation for 5 weeks. Weekly changes in weight gain, feed intake and body composition were monitored during re-feeding. No significant difference was found in final body weight between the three groups, indicating complete compensation in the deprived fish. The deprived groups caught up in body weight with that of the control after 2 weeks of re-feeding. Body fat: lean body mass ratio was restored to the control level within 1 week of re-feeding. In the re-feeding period, weekly gains in body weight, protein, lipid, ash and energy in the S1 group were significantly higher than in the controls for 1 week. For the S2 group, weekly gains in body weight, lipid, ash and energy were higher than in the controls for 2 weeks, and gain in protein was higher than in the controls for 3 weeks, though gain in body energy became elevated again during the last 2 weeks of the experiment. Feed intake remained higher than the control level for 3 weeks in the S1 group and 4 weeks in the S2 group. Growth efficiency was not significantly different among the three groups in any of the weeks during re-feeding. Compensatory responses in growth and especially feed intake tended to last longer than the recovery of body composition.     

Three-dimensional structure of basal body triplet revealed by electron cryo-tomography

Basal bodies and centrioles play central roles in microtubule (MT)‐organizing centres within many eukaryotes. They share a barrel‐shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo‐tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo‐atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non‐tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ‐tubulin and other components participate in the assembly of the basal body.     

On the preservation of carapaces of some limnic ostracods: An exercise in actuopalaeontology

The way in which the ostracod carapace decomposes through biological and physico-chemical processes is illustrated with Recent and Neogene non-marine ostracods (candonines, herpetocyprines and cytherids) from lacustrine and groundwater habitats. The degree of carapace preservation for Recent Candoninae, in the Mondsee (Austria) is shown to be dependent on the sedimentary microenvironment. The carapaces of herpetocyprines, because of their special morphological structure, decay very easily. This could explain the rare occurrrence of this ostracod group in Palaeogene deposits in Europe.     

Taphonomy of Clypeaster fragments: preservation and taphofacies

The preservation of echinoid fragments of the common echinoid genus Clypeaster recovered from bulk sediment samples is used to explore the possibilities of recognizing taphonomic patterns and establishing taphofacies within a shallow water carbonate environment (Northern Bay of Safaga, Red Sea, Egypt). This is accomplished by the comparison and quantification of the taphonomic signatures of numerous fragments of roughly similar size. The main taphonomic processes observed are abrasion and destruction of plate surfaces, including tubercles and plate edges. Encrustation, especially by foraminifera, can be common and intense. Intraplate fracturing and surface features, such as scratch marks, were also quantified. Four different taphofacies with different characteristic signatures were separated using a cluster analysis of cumulative taphonomic grades. The distribution of taphofacies clearly reflects the topographic complexity of the study area and can be explained by ambient environmental parameters that reflect the general picture of wave and current intensities and sedimentation patterns. The potential use of taphofacies analysis as an independent measure of environmental heterogeneity in palaeoecological studies is confirmed in that taphofacies analysis can reveal parameters not immediately obvious when investigating the textural and biotic characteristics of a sediment.     

Impact of tissue preservation on collagen fiber architecture

Microarchitectural features of collagen-rich extracellular matrices provide the mechanical foundation for tissue function and exhibit topographical cues that influence cellular behavior including proliferation, migration and protein expression. Preservation of tissue microarchitecture is required for accurate evaluation of tissue characteristics and pathology. It is unclear whether common tissue preservation methods possess equal ability to preserve microarchitecture. We investigated collagen microarchitecture in samples that had been flash frozen, fixed in formalin or preserved in RNAlater®, and which contained both collagen-rich and collagen-sparse regions. Fibrillar collagen organization was characterized using picrosirius red staining and second harmonic generation (SHG) microscopy. Maintenance of collagen fiber characteristics compared to the gold standard of flash freezing depended on both the method of preservation and the local collagen content of the tissue. Both formalin fixation and RNAlater® preserved collagen fiber characteristics similar to flash freezing in collagen-rich areas of the tissue, but not in collagen-sparse regions. Analysis using picrosirius red staining indicated preservation-dependent changes in overall tissue architecture and suprafibrillar organization. Together with considerations of cost, ease of use, storage conditions and ability to use the preserved tissue for RNA or protein analysis, our quantitative characterization of the effects of preservation method on collagen microarchitecture may help investigators select the most appropriate preservation approach for their needs.     

An essential quality control mechanism at the eukaryotic basal body prior to intraflagellar transport

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain-containing protein that is specifically located at the mature basal body transitional fibres. RNA interference-mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum-specific TBCC-like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the 'quality' of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.     

Experimental analysis of soft‐tissue fossilization: opening the black box

Taphonomic experiments provide important insights into fossils that preserve the remains of decay‐prone soft tissues, tissues that are usually degraded and lost prior to fossilization. These fossils are among the most scientifically valuable evidence of ancient life on Earth, giving us a view into the past that is much less biased and incomplete than the picture provided by skeletal remains alone. Although the value of taphonomic experiments is beyond doubt, a lack of clarity regarding their purpose and limitations, and ambiguity in the use of terminology, are hampering progress. Here we distinguish between processes that promote information retention and those that promote information loss, in order to clarify the distinction between fossilization and preservation. Recognizing distinct processes of decay, mineralization and maturation, the sequence in which they act, and the potential for interactions, has important consequences for analysis of fossils, and for the design of taphonomic experiments. The purpose of well‐designed taphonomic experiments is generally to understand decay, maturation and preservation individually, thus limiting the number of variables involved. Much work remains to be done, but these methodologically reductionist foundations will allow researchers to build towards more complex taphonomic experiments and a more holistic understanding and analysis of the interactions between decay, maturation and preservation in the fossilization of non‐biomineralized remains. Our focus must remain on the key issue of understanding what exceptionally preserved fossils reveal about the history of biodiversity and evolution, rather than on debating the scope and value of an experimental approach.     

Lower critical temperature and cold-induced thermogenesis of lean and overweight humans are inversely related to body mass and basal metabolic rate

It is colloquially stated that body size plays a role in the human response to cold, but the magnitude and details of this interaction are unclear. To explore the inherent influence of body size on cold-exposed metabolism, we investigated the relation between body composition and resting metabolic rate in humans at thermoneutrality and during cooling within the nonshivering thermogenesis range. Body composition and resting energy expenditure were measured in 20 lean and 20 overweight men at thermoneutrality and during individualized cold exposure. Metabolic rates as a function of ambient temperature were investigated considering the variability in body mass and composition. We observed an inverse relationship between body size and the lower critical temperature (LCT), i.e. the threshold where thermoneutrality ends and cold activates thermogenesis. LCT was higher in lean than overweight subjects (22.1 ± 0.6 vs 19.5 ± 0.5 °C, p < 0.001). Below LCT, minimum conductance was identical between lean and overweight (100 ± 4 vs 97 ± 3 kcal/°C/day respectively, p = 0.45). Overweight individuals had higher basal metabolic rate (BMR) explained mostly by the higher lean mass, and lower cold-induced thermogenesis (CIT) per degree of cold exposure. Below thermoneutrality, energy expenditure did not scale to lean body mass. Overweight subjects had lower heat loss per body surface area (44.7 ± 1.3 vs 54.7 ± 2.3 kcal/°C/m²/day, p < 0.001). We conclude that larger body sizes possessed reduced LCT as explained by higher BMR related to more lean mass rather than a change in whole-body conductance. Thus, larger individuals with higher lean mass need to be exposed to colder temperatures to activate CIT, not because of increased insulation, but because of a higher basal heat generation. Our study suggests that the distinct effects of body size and composition on energy expenditure should be taken in account when exploring the metabolism of humans exposed to cold.     

Aerial photogrammetry and tag-derived tissue density reveal patterns of lipid-store body condition of humpback whales on their feeding grounds

Monitoring the body condition of free-ranging marine mammals at different life-history stages is essential to understand their ecology as they must accumulate sufficient energy reserves for survival and reproduction. However, assessing body condition in free-ranging marine mammals is challenging. We cross-validated two independent approaches to estimate the body condition of humpback whales (Megaptera novaeangliae) at two feeding grounds in Canada and Norway: animal-borne tags (n = 59) and aerial photogrammetry (n = 55). Whales that had a large length-standardized projected area in overhead images (i.e. whales looked fatter) had lower estimated tissue body density (TBD) (greater lipid stores) from tag data. Linking both measurements in a Bayesian hierarchical model to estimate the true underlying (hidden) tissue body density (uTBD), we found uTBD was lower (−3.5 kg m⁻³) in pregnant females compared to adult males and resting females, while in lactating females it was higher (+6.0 kg m⁻³). Whales were more negatively buoyant (+5.0 kg m⁻³) in Norway than Canada during the early feeding season, possibly owing to a longer migration from breeding areas. While uTBD decreased over the feeding season across life-history traits, whale tissues remained negatively buoyant (1035.3 ± 3.8 kg m⁻³) in the late feeding season. This study adds confidence to the effectiveness of these independent methods to estimate the body condition of free-ranging whales.     

Hierarchies in the energy budget: Thyroid hormones and the evolution of human life history patterns

The evolution of human life history characteristics required dramatic shifts in energy allocation mechanisms compared with our primate ancestors. Thyroid hormones, such as thyroxine (T4) and triiodothyronine (T3), are sensitive to energy balance, and are significant determinants for both tissue-specific and whole-body metabolic rate. Thus, thyroid hormones are in part responsible for setting the body's overall energy budget and likely played an important role in the evolution of human life history patterns. We propose that the dynamics of mammalian T3 production, uptake, and action have evolved so that energy allocation prioritizes the high demands of brain development and functioning, often at the expense of growth and reproduction. This paper explores the role of thyroid hormone dynamics in the evolution of human encephalization, prolonged childhood and adolescence, long lifespans, reproduction, and human aging.     

American and korean ginseng tissue cultures: Growth,chemical analysis,and plantlet production

Summary Roots, stems, or leaves of American (Panax quinquefolium) and Korean (Panax ginsing) ginseng were grown as callus or supension tissue cultures. Tissue cultures ofP. ginseng would occasionally form plantlets. The fundamental chemical composition, inorganic analysis, and saponin (panaquilin) content of American and Korean ginseng plants and tissue cultures were determined. The crude saponin content is very similar to, but approximately one-half (1.3%, fresh weight) of that present in ginseng roots. Two-dimensional thin layer chromatographic analysis revealed minor differences in the panaquilins present in American and Korean ginseng tissue cultures. The sapogenin, panaxadiol, was isolated from Korean ginseng callus.     

Transgenic and knockout rodents: Novel insights into mechanisms of body weight regulation

Transgenic animals that over- or underexpress a protein of interest have been used to study obesity development, prevention, and susceptibility to diet-induced obesity such as a high-fat diet. Several transgenic models are resistant to diet-induced obesity including those that overexpress the insulin-sensitive glucose transporter, GLUT4, in adipose tissue only. In this animal there is increased adipose tissue mass but the animal maintains its insulin sensitivity. The overexpression of lipoprotein lipase (LPL) in skeletal muscle and the elimination of a protein kinase A subunit both resulted in lean and obesity resistant animals. By directing the production of the diphtheria toxin A chain to adipose tissue only the resulting animals not only had less adipose tissue mass but were resistant to MSG-induced obesity. Conversely, transgenic models with decreased brown adipose tissue or its function have all resulted in obese animals, highlighting the importance of thermoregulation in body weight maintenance. The use of transgenic technology in the field of obesity has emphasized the regional differences among fat pads as well as the dissimilarity between genders in fuel metabolism. Several transgenic models have separated obesity from insulin resistance allowing the importance of each state to be studied individually. Results using transgenic animals have re-emphasized that obesity is a polygenic disease.