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High-resolution computed tomography (HRCT) has become a widely used tool for studying the inner ear morphology of vertebrates. Amphisbaenians are one of the most specialized groups of fossorial reptiles but are poorly understood relative to other squamate reptile. In this paper we survey the anatomy of the inner and middle ear of these fossorial reptiles using HRCT models and we describe qualitatively and quantitatively (using 3D morphometrics) the anatomy of the inner ear. Amphisbaenians are diverse in skull anatomy, especially in the configuration of the snout, which correlates with digging modes. We demonstrate that the ear also exhibits a diversity of configurations, which are independent of phylogenetic relationships. Results from morphological analyses also allow us to describe 11 new potentially informative phylogenetic characters including some that help to diagnose amphisbaenians, such as: 1) the globular vestibule, ii) semicircular canals arranged in a circular trajectory, and iii) an extensive area of interaction between the columella footplate and the lagenar recess. Among extant amphisbaenians, Rhineura floridana has the most unusual inner ear configuration, including a horizontal semicircular canal that is in the same orientation as the inclined snout. The new morphological information helps us to better understand the morphology of headfirst-burrowing fossorial reptiles and contributes new data for resolution of phylogenetic relationships among amphisbaenians., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Clark et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The machairodontine felid Homotherium achieved a global geographic distribution throughout much of the Pleistocene. Accordingly, that large carnivore is important for understanding patterns of community composition. We report on a new record of Homotherium based on a fragmentary premaxilla-maxilla discovered on McFaddin Beach, Texas, along the Gulf of Mexico. Skeletal remains of extinct, Pleistocene vertebrates accumulate on McFaddin Beach. Those fossils appear to originate from submerged deposits on the continental shelf in the Gulf of Mexico, an area that was subaerially exposed in the Late Pleistocene during glacial intervals. Marine erosion and transport altered the externally visible morphology of the current specimen, obscuring and/or damaging taxonomically informative details of the preserved dentition. However, high-resolution X-ray computed tomography revealed diagnostic portions of the unerupted crown of an upper canine within its alveolus. The serrated edges of the canine combined with the position of the incisors demonstrate that the specimen from McFaddin Beach represents a species of Homotherium. That specimen is the latest in a larger sample of Homotherium in Texas that spans most of the Pliocene-Pleistocene. This is the first occurrence of Homotherium from the continental shelf of the Gulf Coast. That landscape may have formed a broad subtropical Gulf Coast corridor that facilitated the dispersal of Neotropical taxa along the coast between Texas and Florida. The associated fauna from McFaddin Beach contains Neotropical mammals common to southern Texas and Florida and indicates that Homotherium was a member of the fauna inhabiting the Gulf Coast corridor during the Late Pleistocene., (© 2024 American Association for Anatomy.)

Amphisbaenians are a specialized fossorial group of reptiles, having developed head-first burrowing, a specialized skull architecture, and an elongated body. This group is generally small-bodied, with some species possessing skulls only a few millimeters long. In this study, we used high-resolution x-ray computed tomography to compare the skulls of 15 specimens from seven of the eight species in the amphisbaenian genus Zygaspis (Zygaspis dolichomenta, Zygaspis ferox, Zygaspis quadrifrons, Zygaspis kafuensis, Zygaspis nigra, Zygaspis vandami, and Zygaspis violacea). Both interspecific and intraspecific variation, including asymmetry, is observed among the cranial bones of the specimens. There are unique morphological features on some cranial bones, including the premaxilla and ectopterygoid of Z. quadrifrons, the pterygoid and vomer of Z. kafuensis, and the extracolumella of Z. nigra. Sexual dimorphism has been previously reported for the species Z. quadrifrons and is observed here as well., (© 2023 American Association for Anatomy.)

Amphisbaenians are a poorly understood clade of fossorial lizards. Because of their derived anatomy and relative scarcity, the systematics of the clade and its placement within squamates has long been controversial. Traditional approaches grouped species into four assemblages according to burrowing behavior and cranial morphology, resulting in the recognition of "shovel-headed," "round-headed," "keel-headed," and "spade-headed" morphotypes. Recent phylogenetic analyses do not support the monophyly of the taxa that share those morphotypes. Detailed analyses of cranial osteology were previously accomplished using high-resolution x-ray computed tomography (HRXCT) for the "shovel-headed" Rhineura hatcherii (Rhineruidae) and the "spade-headed" Diplometopon zarudnyi (Trogonophidae). A detailed description of the "round-headed" Amphisbaena alba was previously completed based upon traditional "dry" skeletal specimens. Seven species of the "round-headed" Blanus (Blanidae) were also analyzed using HRXCT. The goal of that project was a comparative analysis of all extant species of Blanus rather than a detailed, bone-by-bone description of one species, but certainly is useful for comparison with another "round-headed" taxon. The "round-headed" morphotype is by far the most common among amphisbaenians and is much in need of further documentation. We use HRXCT imagery to provide additional data about the disparity in cranial morphology among amphisbaenians. Those data allow us to provide another detailed description of a "round-headed" amphisbaenian, the poorly known southern African species Zygaspis quadrifrons. HRXCT is ideal for this relatively rare and diminutive species. We are able to visualize and describe a detailed reconstruction of the entire skull as well as individual cranial elements. Comparisons with other species that were described in similar detail-D. zarudnyi, Spathorhynchus fossorium, R. hatcherii, and A. alba-and to a lesser degree with Blanus, reveal a complex mosaic of morphological features of the skull in Zygaspis. Preliminary data suggest that intraspecific variation is present within Z. quadrifrons, and interspecific variation among other species of Zygaspis may be sufficient for species-level recognition based on cranial osteology. Our description is, therefore, also intended to serve as a baseline for comparative analysis of other specimens of Z. quadrifrons and of other species within the genus., (© 2023 American Association for Anatomy.)

A snake-like body plan and burrowing lifestyle characterize numerous vertebrate groups as a result of convergent evolution. One such group is the amphisbaenians, a clade of limbless, fossorial lizards that exhibit head-first burrowing behavior. Correlated with this behavior, amphisbaenian skulls are more rigid and coossified than those of nonburrowing lizards. However, due to their lifestyle, there are many gaps in our understanding of amphisbaenian anatomy, including how their cranial osteology varies among individuals of the same species and what that reveals about constraints on the skull morphology of head-first burrowing taxa. We investigated intraspecific variation in the cranial osteology of amphisbaenians using seven individuals of the trogonophid Diplometopon zarudnyi. Variation in both skull and individual skull element morphology was examined qualitatively and quantitatively through three-dimensional (3D) models created from microcomputed tomography data. Qualitative examination revealed differences in the number and position of foramina, the interdigitation between the frontals and parietal, and the extent of coossification among the occipital complex, fused basioccipital and parabasisphenoid ("parabasisphenoid-basioccipital complex"), and elements X. We performed 3D landmark-based geometric morphometrics for the quantitative assessment, revealing shape differences in the skull, premaxilla, maxilla, frontal, and parietal. The observed intraspecific variation may be the result of different stages of ontogenetic development or biomechanical optimization for head-first burrowing. For example, variation in the coossification of the occipital region suggests a potential ontogenetic coossification sequence. Examination of these areas of variation across other head-first burrowing taxa will help determine if the variation is clade-specific or part of a broader macroevolutionary pattern of head-first burrowing., (© 2022 The Authors. Journal of Morphology published by Wiley Periodicals LLC.)

Scolecophidian snakes have long posed challenges for scholars interested in elucidating their anatomy. The importance, and relative paucity, of high-quality anatomical data pertaining to scolecophidians was brought into sharp focus in the late 20 th century as part of a controversy over the phylogeny and ecological origin of snakes. The basal position of scolecophidians in the phylogeny of snakes makes their anatomy, behavior, ecology, and evolution especially important for such considerations. The depauperate fossil record for the group meant that advances in understanding their evolutionary history were necessarily tied to biogeographic distributions and anatomical interpretations of extant taxa. Osteological data, especially data pertaining to the skull and mandible, assumed a dominant role in shaping historical and modern perspectives of the evolution of scolecophidians. Traditional approaches to the exploration of the anatomy of these snakes relied heavily upon serial-sectioned specimens and cleared-and-stained specimens. The application of X-ray computed tomography (CT) to the study of scolecophidians revolutionized our understanding of the osteology of the group, and now, via diffusible iodine-based contrast-enhanced computed tomography (diceCT), is yielding data sets on internal soft anatomical features as well. CT data sets replicate many aspects of traditional anatomical preparations, are readily shared with a global community of scholars, and now are available for unique holotype and other rare specimens. The increasing prevalence and relevance of CT data sets is a strong incentive for the establishment and maintenance of permanent repositories for digital data., (© 2021 American Association for Anatomy.)

Background: Alligator lizards (Gerrhonotinae) are a well-known group of extant North American lizard. Although many fossils were previously referred to Gerrhonotinae, most of those fossils are isolated and fragmentary cranial elements that could not be placed in a precise phylogenetic context, and only a handful of known fossils are articulated skulls. The fossil record has provided limited information on the biogeography and phylogeny of Gerrhonotinae., Results: We redescribe a nearly complete articulated fossil skull from the Pliocene sediments of the Anza-Borrego Desert in southern California, and refer the specimen to the alligator lizard genus Elgaria. The fossil is a representative of a newly described species, Elgaria peludoverde. We created a morphological matrix to assess the phylogeny of alligator lizards and facilitate identifications of fossil gerrhonotines. The matrix contains a considerably expanded taxonomic sample relative to previous morphological studies of gerrhonotines, and we sampled two specimens for many species to partially account for intraspecific variation. Specimen-based phylogenetic analyses of our dataset using Bayesian inference and parsimony inferred that Elgaria peludoverde is part of crown Elgaria. The new species is potentially related to the extant species Elgaria kingii and Elgaria paucicarinata, but that relationship was not strongly supported, probably because of extensive variation among Elgaria. We explored several alternative biogeographic scenarios implied by the geographic and temporal occurrence of the new species and its potential phylogenetic placements., Conclusions: Elgaria peludoverde is the first described extinct species of Elgaria and provides new information on the biogeographic history and diversification of Elgaria. Our research expands the understanding of phylogenetic relationships and biogeography of alligator lizards and strengthens the foundation of future investigations. The osteological data and phylogenetic matrix that we provided will be critical for future efforts to place fossil gerrhonotines. Despite limited intraspecific sampled sizes, we encountered substantial variation among gerrhonotines, demonstrating the value of exploring patterns of variation for morphological phylogenetics and for the phylogenetic placement of fossils. Future osteological investigations on the species we examined and on species we did not examine will continue to augment our knowledge of patterns of variation in alligator lizards and aid in phylogenetics and fossil placement., (© 2021. The Author(s).)

Background: There are limited data on intra- and interspecific osteological variation for many squamate clades. Those data are relevant for phylogenetic analyses that use osteological characters and for apomorphic identifications of fossils. We investigate whether morphological features in the skulls of extant gerrhonotine lizards can be used to distinguish taxa at the species- and genus-level and assess whether newly discovered intra- and interspecific osteological variation alters the utility of previously reported apomorphic features. We examined skulls of species belonging to the gerrhonotine genera Elgaria and Gerrhonotus . These genera contain 17 extant species, but the cranial osteology of only a few species was previously examined. As a result, intra- and interspecific osteological variation of these gerrhonotines is poorly understood., Methods: We employed high-resolution x-ray computed tomography (CT) to scan 25 alcohol-preserved specimens. We provide data on the skulls of all eight species of Elgaria , four for the first time, and five species of Gerrhonotus , three for the first time. We examined 3-D reconstructed skulls of the scanned specimens as well as dry, traditionally prepared skeletons (when they were available)., Results: We found that the purported diagnostic utility of many previously described morphological features is impacted because of substantial morphological variation between and within species. We present an assessment of osteological differences that may be useful to differentiate species of Elgaria and Gerrhonotus , many of which are present on isolated cranial elements commonly recovered as fossils, including the premaxilla, maxilla, parietal, pterygoid, prootic, dentary, and surangular. We demonstrate the importance of documenting patterns of osteological variation using large sample sizes, and the utility of examining disarticulated cranial elements of the squamate skull to identify diagnostic morphology. This study adds to a growing body of literature suggesting that extensive documentation of morphological variation is needed to further our understanding of the phylogenetic and diagnostic utility of morphological features across vertebrate clades. Efforts in that direction likely will benefit from examination of disarticulated skeletal elements., Competing Interests: The authors declare that they have no competing interests., (© 2021 Ledesma et al.)

Multiple tooth rows along the dentary or maxilla are present in many bony and cartilaginous fishes but occur infrequently in amniotes. Although two partially overlapping rows of teeth or a few doubled tooth positions were both previously reported in extant and extinct lizards, multiple rows of teeth across the entire dental or maxillary shelf were not previously documented in any lizard taxon. Here, we report one specimen of the side-blotched lizard Uta stansburiana and two specimens of the rock lizard Petrosaurus mearnsi that have two or more marginal tooth rows that overlap for most of the dental shelf. We also describe several other phrynosomatid lizard specimens with one or two doubled tooth positions per marginal tooth-bearing skeletal element. We examined 520 total specimens of pleurodont iguanian lizards and verified that aberrant tooth row and tooth position morphologies were almost exclusively present in phrynosomatid lizards. We hypothesize that developmental irregularities in the number of odontogenic bands or in the function of zones of inhibition may have resulted in the observed tooth row abnormalities. The expression of multiple tooth rows or doubled tooth positions may be phylogenetically informative morphologies of phrynosomatid lizards. This bears further investigation from developmental, genetic, ecological, and phylogenetic perspectives. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:2014-2025, 2020. © 2019 American Association for Anatomy., (© 2019 American Association for Anatomy.)

Osteoderms constitute a morphological system that plays an important role in squamate systematics. However, their study and visualization have always been difficult due to their isolated occurrence in the skin, among the first organs to be removed during the skeletonization process. High-resolution X-ray computed tomography (HRXCT) offers a nondestructive means of visualizing osteoderms both in their natural relationship to each other and to the underlying cranial bones. Although it is often stated that Varanus komodoensis has a "chain mail" of osteoderms, this morphological system was never described in this taxon. Further, given its size, it might be expected that V. komodoensis would present the extreme of osteoderm development in extant varanids, a group that tends to have weakly developed osteoderms or none at all. Indeed, our HRXCT scan of a 19-year-old captive individual reveals an elaborate mesh of cephalic osteoderms that are incredibly numerous and morphologically diverse. We describe this skeletal system and compare it to the cephalic osteoderms in other varanoids. Anat Rec, 302:1675-1680, 2019. © 2019 American Association for Anatomy., (© 2019 American Association for Anatomy.)

The emergence of new technologies and improved computing power helped to introduce a renewed vitality in morphological research in recent decades. This is especially apparent in the new advances made in understanding the evolutionary morphology of the skeletal system in extinct and extant squamate reptiles. The new data generated as a result of the recent increase in attention are relevant not only for systematic analyses but also are valuable in their own right for contributing to holistic perspectives on organismal evolution, mosaic evolution in the rates of change in different anatomical systems, and broader patterns of macroevolution. A global community of morphological researchers now can share data through online digital collections, but opportunities for continued advance are hindered because we lack even basic data on patterns of variation of the skeletal system for virtually all squamate lineages. Most work on skeletal morphology of squamates is based on a sample size of n = 1; this is an especially noticeable phenomenon for studies relying on X-ray computed tomography technology. We need new collections of skeletal specimens, both material and digital, and new approaches to the study of skeletal morphology. Promising areas for continued research include the recent focus on skeletal elements not traditionally included in morphological studies (especially systematic analyses based upon morphological data) and efforts to elucidate patterns of variation and phylogenetically informative features of disarticulated skeletal elements., (Copyright © 2014 Wiley Periodicals, Inc.)

Background: Previous studies, including those employing diffusion tensor imaging (DTI), have revealed significant disturbances in the white matter of individuals with fetal alcohol spectrum disorders (FASD). Both macrostructural and microstructural abnormalities have been observed across levels of FASD severity. Emerging evidence suggests that these white matter abnormalities are associated with functional deficits. This study used resting-state functional MRI (fMRI) to evaluate the status of network functional connectivity in children with FASD compared with control subjects., Methods: Participants included 24 children with FASD, ages 10 to 17, and 31 matched controls. Neurocognitive tests were administered including Wechsler Intelligence Scales, California Verbal Learning Test (CVLT), and Behavior Rating Inventory of Executive Functioning. High-resolution anatomical MRI data and 6-minute resting-state fMRI data were collected. The resting-state fMRI data were subjected to a graph theory analysis, and 4 global measures of cortical network connectivity were computed: characteristic path length, mean clustering coefficient, local efficiency, and global efficiency., Results: Results revealed significantly altered network connectivity in those with FASD. The characteristic path length was 3.1% higher (p = 0.04, Cohen's d = 0.47), and global efficiency was 1.9% lower (p = 0.04, d = 0.63) in children with FASD compared with controls, suggesting decreased network capacity that may have implications for integrative cognitive functioning. Global efficiency was significantly positively correlated with cortical thickness in frontal (r = 0.38, p = 0.005), temporal (r = 0.28, p = 0.043), and parietal (r = 0.36, p = 0.008) regions. No relationship between facial dysmorphology and functional connectivity was observed. Exploratory correlations suggested that global efficiency and characteristic path length are associated with capacity for immediate verbal memory on the CVLT (r = 0.41, p = 0.05 and r = 0.41, p = 0.01, respectively) among those with FASD., Conclusions: Resting-state functional connectivity measures provide new insight into the integrity of brain networks in clinical populations such as FASD. Results demonstrate that children with FASD have alterations in core components of network function and that these aspects of brain integrity are related to measures of structure and cognitive functioning., (Copyright © 2012 by the Research Society on Alcoholism.)

Diffusion tensor imaging was used to evaluate cerebral white matter in 16 patients (ages 9-18) with myotonic dystrophy type 1 compared to 15 matched controls. Patients with myotonic dystrophy showed abnormalities in mean diffusivity compared to controls in frontal, temporal, parietal, and occipital white matter and in all individual tracts examined. Whole cerebrum mean diffusivity was 8.6 % higher overall in patients with myotonic dystrophy compared to controls. Whole cerebrum fractional anisotropy was also abnormal (10.8 % low overall) in all regions and tracts except corticospinal tracts. Follow-up analysis of parallel and perpendicular diffusivity suggests possible relative preservation of myelin in corticospinal tracts. Correlations between Wechsler working memory performance and mean diffusivity were strong for all regions. Frontal and temporal fractional anisotropy were correlated with working memory as well. Results are consistent with earlier studies demonstrating that significant white matter disturbances are characteristic in young patients with myotonic dystrophy and that these abnormalities are associated with the degree of working memory impairment seen in this disease.

Uropeltids form a diverse clade of highly derived, fossorial snakes that, because of their phylogenetic position among other alethinophidian lineages, may play a key role in understanding the early evolution of cranial morphology in snakes. We include detailed osteological descriptions of crania and mandibles for eight uropeltid species from three nominal genera (Uropeltis, Rhinophis, and Brachyophidium) and emphasize disarticulated elements and the impact of intraspecific variation on previously proposed morphological characters used for phylogenetic analysis. Preliminary analysis of phylogenetic relationships strongly supports a clade composed exclusively of species of Plectrurus, Uropeltis, and Rhinophis. However, monophyly of each of those genera and Melanophidium is not upheld. There is moderate support that Sri Lankan species (e.g., Rhinophis and Uropeltis melanogaster) are monophyletic with respect to Indian uropeltids. Previously proposed characters that are phylogenetically informative include the shape of the nasals, length of the occipital condyle, level of development of the posteroventral process of the dentary, and participation of the parietal in the optic foramen. Additionally, thirty new features that may be systematically informative are identified and described, but were not verified for their utility. Such verification must await availability of additional disarticulated cranial material from a larger sample of taxa. All characters require further testing through increased focus on sources and patterns of intraspecific variation, inclusion of broader taxonomic samples in comparative studies, and exploration of skeletal development, sexual dimorphism, and biogeographic patterns. Additionally, trends in the relative enlargement of the sensory capsules, reduction in cranial ossification and dentition, fusion of elements, and the appearance of novel morphological conditions, such as the structure and location of the suspensorium, may be related to fossoriality and miniaturization in some uropeltid taxa, and may complicate analysis of relationships within Uropeltidae and among alethinophidian snakes.

Background: MRI studies, including recent diffusion tensor imaging (DTI) studies, have shown corpus callosum abnormalities in children prenatally exposed to alcohol, especially in the posterior regions. These abnormalities appear across the range of fetal alcohol spectrum disorders (FASD). Several studies have demonstrated cognitive correlates of callosal abnormalities in FASD including deficits in visual-motor skill, verbal learning, and executive functioning. The goal of this study was to determine whether inter-hemispheric structural connectivity abnormalities in FASD are associated with disrupted inter-hemispheric functional connectivity and disrupted cognition., Methods: Twenty-one children with FASD and 23 matched controls underwent a 6-minute resting-state functional MRI scan as well as anatomical imaging and DTI. Using a semi-automated method, we parsed the corpus callosum and delineated 7 inter-hemispheric white matter tracts with DTI tractography. Cortical regions of interest (ROIs) at the distal ends of these tracts were identified. Right-left correlations in resting fMRI signal were computed for these sets of ROIs, and group comparisons were made. Correlations with facial dysmorphology, cognition, and DTI measures were computed., Results: A significant group difference in inter-hemispheric functional connectivity was seen in a posterior set of ROIs, the para-central region. Children with FASD had functional connectivity that was 12% lower than in controls in this region. Subgroup analyses were not possible owing to small sample size, but the data suggest that there were effects across the FASD spectrum. No significant association with facial dysmorphology was found. Para-central functional connectivity was significantly correlated with DTI mean diffusivity, a measure of microstructural integrity, in posterior callosal tracts in controls but not in FASD. Significant correlations were seen between these structural and functional measures, and Wechsler perceptual reasoning ability., Conclusions: Inter-hemispheric functional connectivity disturbances were observed in children with FASD relative to controls. The disruption was measured in medial parietal regions (para-central) that are connected by posterior callosal fiber projections. We have previously shown microstructural abnormalities in these same posterior callosal regions, and the current study suggests a possible relationship between the two. These measures have clinical relevance as they are associated with cognitive functioning., (Copyright © 2011 by the Research Society on Alcoholism.)

Major depressive disorder (MDD) begins frequently in adolescence and is associated with severe outcomes, but the developmental neurobiology of MDD is not well understood. Research in adults has implicated fronto-limbic neural networks in the pathophysiology of MDD, particularly in relation to the subgenual anterior cingulate cortex (ACC). Developmental changes in brain networks during adolescence highlight the need to examine MDD-related circuitry in teens separately from adults. Using resting state functional magnetic resonance imaging (fMRI), this study examined functional connectivity in adolescents with MDD (n=12) and healthy adolescents (n=14). Seed-based connectivity analysis revealed that adolescents with MDD have decreased functional connectivity in a subgenual ACC-based neural network that includes the supragenual ACC (BA 32), the right medial frontal cortex (BA 10), the left inferior (BA 47) and superior frontal cortex (BA 22), superior temporal gyrus (BA 22), and the insular cortex (BA 13). These preliminary data suggest that MDD in adolescence is associated with abnormal connectivity within neural circuits that mediate emotion processing. Future research in larger, un-medicated samples will be necessary to confirm this finding. We conclude that hypothesis-driven, seed-based analyses of resting state fMRI data hold promise for advancing our current understanding of abnormal development of neural circuitry in adolescents with MDD.

The mechanisms that enable the heart to rapidly increase ATP supply in line with increased demand have not been fully elucidated. Here we used an adenoviral system to express the photoproteins luciferase and aequorin, targeted to the mitochondria or cytosol of adult cardiomyocytes, to investigate the interrelationship between ATP and Ca(2+) in these compartments. In neither compartment were changes in free [ATP] observed upon increased workload (addition of isoproterenol) in myocytes that were already beating. However, when myocytes were stimulated to beat rapidly from rest, in the presence of isoproterenol, a significant but transient drop in mitochondrial [ATP] ([ATP](m)) occurred (on average to 10% of the initial signal). Corresponding changes in cytosolic [ATP] ([ATP](c)) were much smaller (<5%), indicating that [ATP](c) was effectively buffered in this compartment. Although mitochondrial [Ca(2+)] ([Ca(2+)](m)) is an important regulator of respiratory chain activity and ATP production in other cells, the kinetics of mitochondrial Ca(2+) transport are controversial. Parallel experiments in cells expressing mitochondrial aequorin showed that the drop in [ATP](m) occurred over the same time scale as average [Ca(2+)](m) was increasing. Conversely, in the absence or presence of isoproterenol, clear beat-to-beat peaks in [Ca(2+)](m) were observed at 0.9 or 1.3 mum, respectively, concentrations similar to those observed in the cytosol. These results suggest that mitochondrial Ca(2+) transients occur during the contractile cycle and are translated into a time-averaged increase in mitochondrial ATP production that keeps pace with increased cytosolic demand.

Mid-Pleistocene vertebrates in North America are scarce but important for recognizing the ecological effects of climatic change in the absence of humans. We report on a uniquely rich mid-Pleistocene vertebrate sequence from Porcupine Cave, Colorado, which records at least 127 species and the earliest appearances of 30 mammals and birds. By analyzing >20,000 mammal fossils in relation to modern species and independent climatic proxies, we determined how mammal communities reacted to presumed glacial-interglacial transitions between 1,000,000 and 600,000 years ago. We conclude that climatic warming primarily affected mammals of lower trophic and size categories, in contrast to documented human impacts on higher trophic and size categories historically. Despite changes in species composition and minor changes in small-mammal species richness evident at times of climatic change, overall structural stability of mammal communities persisted >600,000 years before human impacts.

To provide empirical evidence of species boundaries and the role of climatic change in affecting evolution, we documented evolution of the sagebrush vole, Lemmiscus curtatus, through hundreds of thousands of years by following populations from the middle Pleistocene to the present. We found that: (i) extant representatives of the species culminate a morphological transition that was initiated within an unusually arid and warm interglacial period, perhaps related to the shift from glacial-interglacial cycles dominated by a 41,000 year periodicity to those dominated by a 100,000 year rhythm; and (ii) sympatry of extant and extinct morphotypes persisted for more than 800,000 years. This exceptionally detailed tracing of extinct populations into extant ones suggests that species such as the one we studied are real entities in space, that their boundaries become fuzzy (although potentially diagnosable) through time and that unusual climatic warming may initiate significant evolutionary change manifested at the morphological level.

Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr(612) and Tyr(941) (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr(896) (a Grb2 binding site); and (iii) Tyr(1229) (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr(1158) and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (>or=20 min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.