The origin of squamates revealed by a Middle Triassic lizard from the Italian Alps.

Modern squamates (lizards, snakes and amphisbaenians) are the world's most diverse group of tetrapods along with birds 1 and have a long evolutionary history, with the oldest known fossils dating from the Middle Jurassic period-168 million years ago2-4. The evolutionary origin of squamates is contentious because of several issues: (1) a fossil gap of approximately 70 million years exists between the oldest known fossils and their estimated origin5-7; (2) limited sampling of squamates in reptile phylogenies; and (3) conflicts between morphological and molecular hypotheses regarding the origin of crown squamates6,8,9. Here we shed light on these problems by using high-resolution microfocus X-ray computed tomography data from the articulated fossil reptile Megachirella wachtleri (Middle Triassic period, Italian Alps 10 ). We also present a phylogenetic dataset, combining fossils and extant taxa, and morphological and molecular data. We analysed this dataset under different optimality criteria to assess diapsid reptile relationships and the origins of squamates. Our results re-shape the diapsid phylogeny and present evidence that M. wachtleri is the oldest known stem squamate. Megachirella is 75 million years older than the previously known oldest squamate fossils, partially filling the fossil gap in the origin of lizards, and indicates a more gradual acquisition of squamatan features in diapsid evolution than previously thought. For the first time, to our knowledge, morphological and molecular data are in agreement regarding early squamate evolution, with geckoes-and not iguanians-as the earliest crown clade squamates. Divergence time estimates using relaxed combined morphological and molecular clocks show that lepidosaurs and most other diapsids originated before the Permian/Triassic extinction event, indicating that the Triassic was a period of radiation, not origin, for several diapsid lineages.

Virtual histology of archaeological human deciduous prenatal enamel through synchrotron X-ray computed microtomography images.

Virtual histology is increasingly utilized to reconstruct the cell mechanisms underlying dental morphology for fragile fossils when physical thin sections are not permitted. Yet, the comparability of data derived from virtual and physical thin sections is rarely tested. Here, the results from archaeological human deciduous incisor physical sections are compared with virtual ones obtained by phase-contrast synchrotron radiation computed microtomography (SRµCT) of intact specimens using a multi-scale approach. Moreover, virtual prenatal daily enamel secretion rates are compared with those calculated from physical thin sections of the same tooth class from the same archaeological skeletal series. Results showed overall good visibility of the enamel microstructures in the virtual sections which are comparable to that of physical ones. The highest spatial resolution SRµCT setting (effective pixel size = 0.9 µm) produced daily secretion rates that matched those calculated from physical sections. Rates obtained using the lowest spatial resolution setup (effective pixel size = 2.0 µm) were higher than those obtained from physical sections. The results demonstrate that virtual histology can be applied to the investigated samples to obtain reliable and quantitative measurements of prenatal daily enamel secretion rates.

Growth of Neanderthal infants from Krapina (120-130 ka), Croatia.

Modern humans have a slow and extended period of childhood growth, but to what extent this ontogenetic pathway was present in Neanderthals is debated. Dental development, linked to the duration of somatic growth across modern primates, is the main source for information about growth and development in a variety of fossil primates, including humans. Studies of Neanderthal permanent teeth report a pace of development either similar to recent humans or relatively accelerated. Neanderthal milk teeth, which form and emerge before permanent teeth, provide an opportunity to determine which pattern was present at birth. Here we present a comparative study of the prenatal and early postnatal growth of five milk teeth from three Neanderthals (120 000-130 000 years ago) using virtual histology. Results reveal regions of their milk teeth formed quickly before birth and over a relatively short period of time after birth. Tooth emergence commenced towards the earliest end of the eruption schedules displayed by extant human children. Advanced dental development is consistent with expectations for Neanderthal infant feeding.

Synchrotron radiation X-ray microtomography for the visualization of intra-cochlear anatomy in human temporal bones implanted with a perimodiolar cochlear implant electrode array.

Recently, synchrotron radiation computed microtomography (SRµCT) has emerged as a promising tool for non-destructive, in situ visualization of cochlear implant electrode arrays inserted into a human cochlea. Histological techniques have been the `gold standard' technique for accurate localization of cochlear implant electrodes but are suboptimal for precise three-dimensional measurements. Here, an SRµCT experimental setup is proposed that offers the benefit of a high spatial and contrast resolution (isotropic voxel size = 4.95 µm and propagation-based phase-contrast imaging), while visualizing the soft-tissue structures and electrode array of the cochlear implant simultaneously. In this work, perimodiolar electrode arrays have been tested, which incorporate thick and closely spaced platinum-iridium contacts and wiring. These data can assist cochlear implant and hearing research, can be used to verify electrode segmentation techniques for clinical computed tomography or could be utilized to evaluate cochlear implant electrode array designs.

Analysis of Intracellular Magnesium and Mineral Depositions during Osteogenic Commitment of 3D Cultured Saos2 Cells.

In this study, we explore the behaviour of intracellular magnesium during bone phenotype modulation in a 3D cell model built to mimic osteogenesis. In addition, we measured the amount of magnesium in the mineral depositions generated during osteogenic induction. A two-fold increase of intracellular magnesium content was found, both at three and seven days from the induction of differentiation. By X-ray microscopy, we characterized the morphology and chemical composition of the mineral depositions secreted by 3D cultured differentiated cells finding a marked co-localization of Mg with P at seven days of differentiation. This is the first experimental evidence on the presence of Mg in the mineral depositions generated during biomineralization, suggesting that Mg incorporation occurs during the bone forming process. In conclusion, this study on the one hand attests to an evident involvement of Mg in the process of cell differentiation, and, on the other hand, indicates that its multifaceted role needs further investigation.

A compact and flexible induction furnace for in situ X-ray microradiograhy and computed microtomography at Elettra: design, characterization and first tests.

A compact and versatile induction furnace for in situ high-resolution synchrotron and laboratory hard X-ray microradiography and computed microtomography is described. The furnace can operate from 773 to 1723 K. Its programmable controller enables the user to specify multiple heating and cooling ramp rates as well as variable dwell times at fixed temperatures allowing precise control of heating and cooling rates to within 5 K. The instrument can work under a controlled atmosphere. Thanks to the circular geometry of the induction coils, the heat is homogeneously distributed in the internal volume of the graphite cell (ca. 150 mm3) where the sample holder is located. The thermal gradient within the furnace is less than 5 K over a height of ca. 5 mm. This new furnace design is well suited to the study of melting and solidification processes in geomaterials, ceramics and several metallic alloys, allowing fast heating (tested up to 6.5 K s-1) and quenching (up to 21 K s-1) in order to freeze the sample microstructure and chemistry under high-temperature conditions. The sample can be held at high temperatures for several hours, which is essential to follow phenomena with relatively slow dynamics, such as crystallization processes in geomaterials. The utility of the furnace is demonstrated through a few examples of experimental applications performed at the Elettra synchrotron laboratory (Trieste, Italy).

Enhanced contrast in X-ray microtomographic images of the membranous labyrinth using different X-ray sources and scanning modes.

The vestibular system, located in the inner ear, plays a crucial role in balance and gaze stabilisation by sensing head movements. The interconnected tubes with membranous walls of the vestibular system are located in the skull bone (the 'membranous labyrinth'). Unfortunately, these membranes are very hard to visualise using three-dimensional (3D) X-ray imaging techniques. This difficulty arises due to the embedment of the membranes in the dense skull bone, the thinness of the membranes, and the small difference in X-ray absorption between the membranes and the surrounding fluid. In this study, we compared the visualisation of very small specimens (lizard heads with vestibular systems smaller than 3 mm) by X-ray computed micro-tomography (µCT) based on synchrotron radiation and conventional sources. A visualisation protocol using conventional X-ray µCT would be very useful thanks to the ease of access and lower cost. Careful optimisation of the acquisition parameters enables detection of the membranes by using µCT scanners based on conventional microfocus sources, but in some cases a low contrast-to-noise ratio (CNR) prevents fast and reliable segmentation of the membranes. Synchrotron radiation µCT proved to be preferable for the visualisation of the small samples with very thin membranes, because of their high demands for spatial and contrast resolution. The best contrast was obtained by using synchrotron radiation µCT working in phase-contrast mode, leading to up to twice as high CNRs than the best conventional µCT results. The CNR of the synchrotron radiation µCT scans was sufficiently high enough to enable the construction of a 3D model by the means of semi-automatic segmentation of the membranous labyrinth. Membrane thickness was found to range between 2.7 and 36.3 µm. Hence, the minimal membrane thickness was found to be much smaller than described previously in the literature (between 10 and 50 µm).

Inner tooth morphology of Homo erectus from Zhoukoudian. New evidence from an old collection housed at Uppsala University, Sweden.

Locality 1, in the Lower Cave of the Zhoukoudian cave complex, China, is one of the most important Middle Pleistocene paleoanthropological and archaeological sites worldwide, with the remains of c. 45 Homo erectus individuals, 98 mammalian taxa, and thousands of lithic tools recovered. Most of the material collected before World War II was lost. However, besides two postcranial elements rediscovered in China in 1951, four human permanent teeth from the 'Dragon Bone Hill,' collected by O. Zdansky between 1921 and 1923, were at the time brought to the Paleontological Institute of Uppsala University, Sweden, where they are still stored. This small sample consists of an upper canine (PMU 25719), an upper third molar (PMU M3550), a lower third premolar crown (PMU M3549), and a lower fourth premolar (PMU M3887). Some researchers have noted the existence of morpho-dimensional differences between the Zhoukoudian and the H. erectus dental assemblage from Sangiran, Java. However, compared to its chrono-geographical distribution, the Early to Middle Pleistocene dental material currently forming the Chinese-Indonesian H. erectus hypodigm is quantitatively meager and still poorly characterized for the extent of its endostructural variation. We used micro-focus X-ray tomography techniques of virtual imaging coupled with geometric morphometrics for comparatively investigating the endostructural conformation (tissue proportions, enamel thickness distribution, enamel-dentine junction morphology, pulp cavity shape) of the four specimens stored in Uppsala, all previously reported for their outer features. The results suggest the existence of time-related differences between continental and insular Southeast Asian dental assemblages, the Middle Pleistocene Chinese teeth apparently retaining an inner signature closer to the likely primitive condition represented by the Early Pleistocene remains from Java, while the Indonesian stock evolved toward tooth structural simplification.

Dynamic intensity normalization using eigen flat fields in X-ray imaging.

In X-ray imaging, it is common practice to normalize the acquired projection data with averaged flat fields taken prior to the scan. Unfortunately, due to source instabilities, vibrating beamline components such as the monochromator, time varying detector properties, or other confounding factors, flat fields are often far from stationary, resulting in significant systematic errors in intensity normalization. In this work, a simple and efficient method is proposed to account for dynamically varying flat fields. Through principal component analysis of a set of flat fields, eigen flat fields are computed. A linear combination of the most important eigen flat fields is then used to individually normalize each X-ray projection. Experiments show that the proposed dynamic flat field correction leads to a substantial reduction of systematic errors in projection intensity normalization compared to conventional flat field correction.

Rethinking the area of protection "natural resources" in life cycle assessment.

Life cycle impact assessment (LCIA) in classical life cycle assessment (LCA) aims at analyzing potential impacts of products and services typically on three so-called areas of protection (AoPs): Natural Environment, Human Health, and Natural Resources. This paper proposes an elaboration of the AoP Natural Resources. It starts with analyzing different perspectives on Natural Resources as they are somehow sandwiched in between the Natural Environment (their cradle) and the human-industrial environment (their application). Reflecting different viewpoints, five perspectives are developed with the suggestion to select three in function of classical LCA. They result in three safeguard subjects: the Asset of Natural Resources, their Provisioning Capacity, and their role in Global Functions. Whereas the Provisioning Capacity is fully in function of humans, the global functions go beyond provisioning as they include nonprovisioning functions for humans and regulating and maintenance services for the globe as a whole, following the ecosystem services framework. A fourth and fifth safeguard subject has been identified: recognizing the role Natural Resources for human welfare, either specifically as building block in supply chains of products and services as such, either with or without their functions beyond provisioning. But as these are far broader as they in principle should include characterization of mechanisms within the human industrial society, they are considered as subjects for an integrated sustainability assessment (LCSA: life cycle sustainability assessment), that is, incorporating social, economic and environmental issues.

The late Early Pleistocene human dental remains from Uadi Aalad and Mulhuli-Amo (Buia), Eritrean Danakil: macromorphology and microstructure.

Fieldwork performed during the last 15 years in various Early Pleistocene East African sites has significantly enlarged the fossil record of Homo erectus sensu lato (s.l.). Additional evidence comes from the Danakil Depression of Eritrea, where over 200 late Early to early Middle Pleistocene sites have been identified within a ∼1000 m-thick sedimentary succession outcropping in the Dandiero Rift Basin, near Buia. Along with an adult cranium (UA 31), which displays a blend of H. erectus-like and derived morpho-architectural features and three pelvic remains, two isolated permanent incisors (UA 222 and UA 369) have also been recovered from the 1 Ma (millions of years ago) Homo-bearing outcrop of Uadi Aalad. Since 2010, our surveys have expanded to the nearby (4.7 km) site of Mulhuli-Amo (MA). This is a fossiliferous area that has been preliminarily surveyed because of its exceptional concentration of Acheulean stone tools. So far, the site has yielded 10 human remains, including the unworn crown of a lower permanent molar (MA 93). Using diverse analytical tools (including high resolution µCT and µMRI), we analysed the external and internal macromorphology and microstructure of the three specimens, and whenever possible compared the results with similar evidence from early Homo, H. erectus s.l., H. antecessor, H. heidelbergensis (from North Africa), Neanderthals and modern humans. We also assessed the UA 369 lower incisor from Uadi Aalad for root completion timing and showed that it compares well with data for root apex closure in modern human populations.

The Middle Pleistocene human metatarsal from Sedia del Diavolo (Rome, Italy).

The peopling of Europe during the Middle Pleistocene is a debated topic among paleoanthropologists. Some authors suggest the coexistence of multiple human lineages in this period, while others propose a single evolving lineage from Homo heidelbergensis to Homo neanderthalensis. The recent reassessment of the stratigraphy at the Sedia del Diavolo (SdD) site (Latium, Italy), now dated to the beginning of marine isotope stage (MIS) 8, calls for a revision of the human fossils from the site. In this paper, we present the morphometric, biomechanical and palaeopathological study of the second right metatarsal SdD2, to both re-evaluate its taxonomical affinities and possibly determine the levels of physical activity experienced by the individual during lifetime. Results demonstrate the persistence of archaic features in SdD2 suggesting new insights into the technology and hunting strategies adopted by Homo between MIS 9 and MIS 8.

Bone marrow oxytocin mediates the anabolic action of estrogen on the skeleton.

Estrogen uses two mechanisms to exert its effect on the skeleton: it inhibits bone resorption by osteoclasts and, at higher doses, can stimulate bone formation. Although the antiresorptive action of estrogen arises from the inhibition of the MAPK JNK, the mechanism of its effect on the osteoblast remains unclear. Here, we report that the anabolic action of estrogen in mice occurs, at least in part, through oxytocin (OT) produced by osteoblasts in bone marrow. We show that the absence of OT receptors (OTRs) in OTR(-/-) osteoblasts or attenuation of OTR expression in silenced cells inhibits estrogen-induced osteoblast differentiation, transcription factor up-regulation, and/or OT production in vitro. In vivo, OTR(-/-) mice, known to have a bone formation defect, fail to display increases in trabecular bone volume, cortical thickness, and bone formation in response to estrogen. Furthermore, osteoblast-specific Col2.3-Cre(+)/OTR(fl/fl) mice, but not TRAP-Cre(+)/OTR(fl/fl) mice, mimic the OTR(-/-) phenotype and also fail to respond to estrogen. These data attribute the phenotype of OTR deficiency to an osteoblastic rather than an osteoclastic defect. Physiologically, feed-forward OT release in bone marrow by a rising estrogen concentration may facilitate rapid skeletal recovery during the latter phases of lactation.

PITRE: software for phase-sensitive X-ray image processing and tomography reconstruction.

Synchrotron-radiation computed tomography has been applied in many research fields. Here, PITRE (Phase-sensitive X-ray Image processing and Tomography REconstruction) and PITRE_BM (PITRE Batch Manager) are presented. PITRE supports phase retrieval for propagation-based phase-contrast imaging/tomography (PPCI/PPCT), extracts apparent absorption, refractive and scattering information of diffraction enhanced imaging (DEI), and allows parallel-beam tomography reconstruction for conventional absorption CT data and for PPCT phase retrieved and DEI-CT extracted information. PITRE_BM is a batch processing manager for PITRE: it executes a series of tasks, created via PITRE, without manual intervention. Both PITRE and PITRE_BM are coded in Interactive Data Language (IDL), and have a user-friendly graphical user interface. They are freeware and can run on Microsoft Windows systems via IDL Virtual Machine, which can be downloaded for free and does not require a license. The data-processing principle and some examples of application will be presented.

Brief communication: two human fossil deciduous molars from the Sangiran dome (Java, Indonesia): outer and inner morphology.

Currently, the human deciduous dental record from the Pleistocene deposits of the Sangiran Dome, Java, consists of only eight specimens. Here we report two deciduous crowns collected near the village of Pucung. While their precise geo-chronological context remains unknown, a provenance from the Early-Middle Pleistocene Kabuh Formation, or from the Early Pleistocene "Grenzbank Zone," is very likely. These isolated specimens consist of an upper first molar (PCG.1) and a lower second molar (PCG.2). Taxonomic discrimination of the Indonesian tooth record is difficult because of the convergence in crown size and appearance between Pongo and Homo. Accordingly, as PCG.2 still bears a concretion masking most of its features, we coupled the outer analysis of the two specimens with an investigation of their inner morphology. In addition to external characteristics, virtual imaging and quantitative assessment of inner morphology and tissue proportions support an attribution to the taxon Homo, and we preliminary allocate both specimens toH. erectus.

Oxytocin is an anabolic bone hormone.

We report that oxytocin (OT), a primitive neurohypophyseal hormone, hitherto thought solely to modulate lactation and social bonding, is a direct regulator of bone mass. Deletion of OT or the OT receptor (Oxtr) in male or female mice causes osteoporosis resulting from reduced bone formation. Consistent with low bone formation, OT stimulates the differentiation of osteoblasts to a mineralizing phenotype by causing the up-regulation of BMP-2, which in turn controls Schnurri-2 and 3, Osterix, and ATF-4 expression. In contrast, OT has dual effects on the osteoclast. It stimulates osteoclast formation both directly, by activating NF-kappaB and MAP kinase signaling, and indirectly through the up-regulation of RANK-L. On the other hand, OT inhibits bone resorption by mature osteoclasts by triggering cytosolic Ca(2+) release and NO synthesis. Together, the complementary genetic and pharmacologic approaches reveal OT as a novel anabolic regulator of bone mass, with potential implications for osteoporosis therapy.

PyPore3D: An Open Source Software Tool for Imaging Data Processing and Analysis of Porous and Multiphase Media.

In this work, we propose the software library PyPore3D, an open source solution for data processing of large 3D/4D tomographic data sets. PyPore3D is based on the Pore3D core library, developed thanks to the collaboration between Elettra Sincrotrone (Trieste) and the University of Trieste (Italy). The Pore3D core library is built with a distinction between the User Interface and the backend filtering, segmentation, morphological processing, skeletonisation and analysis functions. The current Pore3D version relies on the closed source IDL framework to call the backend functions and enables simple scripting procedures for streamlined data processing. PyPore3D addresses this limitation by proposing a full open source solution which provides Python wrappers to the the Pore3D C library functions. The PyPore3D library allows the users to fully use the Pore3D Core Library as an open source solution under Python and Jupyter Notebooks PyPore3D is both getting rid of all the intrinsic limitations of licensed platforms (e.g., closed source and export restrictions) and adding, when needed, the flexibility of being able to integrate scientific libraries available for Python (SciPy, TensorFlow, etc.).

3D-localisation of cochlear implant electrode contacts in relation to anatomical structures from in vivo cone-beam computed tomography.

Positioning of the cochlear implant (CI) electrode in relation to the anatomical structures is a key factor for the hearing outcome and the preservation of residual hearing after cochlear implantation. Determining the exact electrode's location is therefore expected to play an important role in optimisation of the electrode design, the surgical techniques and the post-operative device fitting. The aim of this study is the development and validation of a robust and efficient computerised algorithm for three-dimensional (3D) localisation of the CI-electrode contacts with respect to the relevant cochlear structures, such as the basilar membrane and the modiolus, from modern clinical in vivo cone-beam computed tomography (CBCT). In the presented algorithm, the pre- and post-implantation CBCT are spatially aligned. To localise the anatomical structures, a cochlear microanatomical template derived from lab-based X-ray computed microtomography (µCT) measurements is warped to match the patient-specific cochlear shape acquired from pre-implantation CBCT. The electrode-contact locations, determined from the post-operative CBCT, are superimposed onto the cochlear fine-structure of the microanatomical template to localise the array. The accuracy of this method was validated in a temporal bone study by comparing the distance of the electrode contacts from the modiolar wall, as derived by the algorithm from CBCTs, with the distance determined from synchrotron-radiation (SR) µCT on the same specimens. Due to the achievable spatial resolution, good tissue contrast and limited presence of metallic artifacts, the SRµCT technique is considered to be a golden standard in the proposed approach. In contrast to other approaches, this validation method allowed for the evaluation of the final electrode-to-modiolus distance (EMD) error, and covers the error in co-alignment of the images, in the determination of the electrode contact location and in the localisation of the cochlear structures. The absolute mean error on the EMD parameter was determined at 0.11 mm (max = 0.29 mm, SD = 0.07 mm) across five samples, slightly lower than the voxel size of the CBCT-scans. In a retrospective study, the algorithm was applied to identify scalar translocations of the electrode from clinical in vivo CBCT datasets of 23 CI-recipients, which showed perfect (100%) agreement with the blinded opinion of two experienced neuroradiologists.

A novel endocast technique providing a 3D quantitative analysis of the gastrovascular system in Rhizostoma pulmo: An unexpected through-gut in cnidaria.

The investigation of jellyfish gastrovascular systems mainly focused on stain injections and dissections, negatively affected by thickness and opacity of the mesoglea. Therefore, descriptions are incomplete and data about tridimensional structures are scarce. In this work, morphological and functional anatomy of the gastrovascular system of Rhizostoma pulmo (Macri 1778) was investigated in detail with innovative techniques: resin endocasts and 3D X-ray computed microtomography. The gastrovascular system consists of a series of branching canals ending with numerous openings within the frilled margins of the oral arms. Canals presented a peculiar double hemi-canal structure with a medial adhesion area which separates centrifugal and centripetal flows. The inward flow involves only the "mouth" openings on the internal wing of the oral arm and relative hemi-canals, while the outward flow involves only the two outermost wings' hemi-canals and relative "anal" openings on the external oral arm. The openings differentiation recalls the functional characteristics of a through-gut apparatus. We cannot define the gastrovascular system in Rhizostoma pulmo as a traditional through-gut, rather an example of adaptive convergence, that partially invalidates the paradigm of a single oral opening with both the uptake and excrete function.

Tracing the mobility of a Late Epigravettian (~ 13 ka) male infant from Grotte di Pradis (Northeastern Italian Prealps) at high-temporal resolution.

We present the results of a multi-disciplinary investigation on a deciduous human tooth (Pradis 1), recently recovered from the Epigravettian layers of the Grotte di Pradis archaeological site (Northeastern Italian Prealps). Pradis 1 is an exfoliated deciduous molar (Rdm2), lost during life by an 11-12-year-old child. A direct radiocarbon date provided an age of 13,088-12,897 cal BP (95% probability, IntCal20). Amelogenin peptides extracted from tooth enamel and analysed through LC-MS/MS indicate that Pradis 1 likely belonged to a male. Time-resolved 87Sr/86Sr analyses by laser ablation mass spectrometry (LA-MC-ICPMS), combined with dental histology, were able to resolve his movements during the first year of life (i.e. the enamel mineralization interval). Specifically, the Sr isotope ratio of the tooth enamel differs from the local baseline value, suggesting that the child likely spent his first year of life far from Grotte di Pradis. Sr isotopes are also suggestive of a cyclical/seasonal mobility pattern exploited by the Epigravettian human group. The exploitation of Grotte di Pradis on a seasonal, i.e. summer, basis is also indicated by the faunal spectra. Indeed, the nearly 100% occurrence of marmot remains in the entire archaeozoological collection indicates the use of Pradis as a specialized marmot hunting or butchering site. This work represents the first direct assessment of sub-annual movements observed in an Epigravettian hunter-gatherer group from Northern Italy.