Genetic Variation and Population Structure in Native Americans

We examined genetic diversity and population structure in the American landmass using 678 autosomal microsatellite markers genotyped in 422 individuals representing 24 Native American populations sampled from North, Central, and South America. These data were analyzed jointly with similar data available in 54 other indigenous populations worldwide, including an additional five Native American groups. The Native American populations have lower genetic diversity and greater differentiation than populations from other continental regions. We observe gradients both of decreasing genetic diversity as a function of geographic distance from the Bering Strait and of decreasing genetic similarity to Siberians-signals of the southward dispersal of human populations from the northwestern tip of the Americas. We also observe evidence of: (1) a higher level of diversity and lower level of population structure in western South America compared to eastern South America, (2) a relative lack of differentiation between Mesoamerican and Andean populations, (3) a scenario in which coastal routes were easier for migrating peoples to traverse in comparison with inland routes, and (4) a partial agreement on a local scale between genetic similarity and the linguistic classification of populations. These findings offer new insights into the process of population dispersal and differentiation during the peopling of the Americas.

Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction

Tissue engineering represents an attractive approach for the treatment of congestive heart failure. The influence of the differentiation of myogenic graft for functional recovery is not defined. We engineered a biodegradable skeletal muscle graft (ESMG) tissue and investigated its functional effect after implantation on the epicardium of an infarcted heart segment. ESMGs were synthesized by mixing collagen (2 mg/mL), Matrigel (2 mg/mL), and rat skeletal muscle cells (10(6)). Qualitative and quantitative aspects of ESMGs were optimized. Two weeks following coronary ligation, the animals were randomized in three groups: ESMG glued to the epicardial surface with fibrin (ESMG, n = 7), fibrin alone (fibrin, n = 5), or sham operation (sham, n = 4). Echocardiography, histology, and immunostaining were performed 4 weeks later. A cohesive three-dimensional tissular structure formed in vitro within 1 week. Myoblasts differentiated into randomly oriented myotubes. Four weeks postimplantation, ESMGs were vascularized and invaded by granulation tissue. Mean fractional shortening (FS) was, however, significantly increased in the ESMG group as compared with preimplantation values (42 +/- 6 vs. 33 +/- 5%, P < 0.05) and reached the values of controlled noninfarcted animals (control, n = 5; 45 +/- 3%; not significant). Pre- and postimplantation FS did not change over these 4 weeks in the sham group and the fibrin-treated animals. This study showed that it is possible to improve systolic heart function following myocardial infarction through implantation of differentiated muscle fibers seeded on a gel-type scaffold despite a low rate of survival.

Vertebroplasty: experimental characterization of polymethylmethacrylate bone cement spreading as a function of viscosity, bone porosity, and flow rate

STUDY DESIGN: This is an experimental study on an artificial vertebra model and human cadaveric spine. OBJECTIVE: Characterization of polymethylmethacrylate (PMMA) bone cement distribution in the vertebral body as a function of cement viscosity, bone porosity, and injection speed. Identification of relevant parameters for improved cement flow predictability and leak prevention in vertebroplasty. SUMMARY OF BACKGROUND DATA: Vertebroplasty is an efficient procedure to treat vertebral fractures and stabilize osteoporotic bone in the spine. Severe complications result from bone cement leakage into the spinal canal or the vascular system. Cement viscosity has been identified as an important parameter for leak prevention but the influence of bone structure and injection speed remain obscure. METHODS: An artificial vertebra model based on open porous aluminum foam was used to simulate bone of known porosity. Fifty-six vertebroplasties with 4 different starting viscosity levels and 2 different injection speeds were performed on artificial vertebrae of 3 different porosities. A validation on a human cadaveric spine was executed. The experiments were radiographically monitored and the shape of the cement clouds quantitatively described with the 2 indicators circularity and mean cement spreading distance. RESULTS: An increase in circularity and a decrease in mean cement spreading distance was observed with increasing viscosity, with the most striking change occurring between 50 and 100 Pas. Larger pores resulted in significantly reduced circularity and increased mean cement spreading distance whereas the effect of injection speed on the 2 indicators was not significant. CONCLUSION: Viscosity is the key factor for reducing the risk of PMMA cement leakage and it should be adapted to the degree of osteoporosis encountered in each patient. It may be advisable to opt for a higher starting viscosity but to inject the material at a faster rate.

Investigating within-canopy variation of functional traits and cellular structure of sugar maple (Acer saccharum) leaves

Patterns of increasing leaf mass per area (LMA), area-based leaf nitrogen (Narea), and carbon isotope composition (δ13C) with increasing height in the canopy have been attributed to light gradients or hydraulic limitation in tall trees. Theoretical optimal distributions of LMA and Narea that scale with light maximize canopy photosynthesis; however, sub-optimal distributions are often observed due to hydraulic constraints on leaf development. Using observational, experimental, and modeling approaches, we investigated the response of leaf functional traits (LMA, density, thickness, and leaf nitrogen), leaf carbon isotope composition (δ13C), and cellular structure to light availability, height, and leaf water potential (Ψl) in an Acer saccharum forest to tease apart the influence of light and hydraulic limitations. LMA, leaf and palisade layer thickness, and leaf density were greater at greater light availability but similar heights, highlighting the strong control of light on leaf morphology and cellular structure. Experimental shading decreased both LMA and area-based leaf nitrogen (Narea) and revealed that LMA and Narea were more strongly correlated with height earlier in the growing season and with light later in the growing season. The supply of CO2 to leaves at higher heights appeared to be constrained by stomatal sensitivity to vapor pressure deficit (VPD) or midday leaf water potential, as indicated by increasing δ13C and VPD and decreasing midday Ψl with height. Model simulations showed that daily canopy photosynthesis was biased during the early growing season when seasonality was not accounted for, and was biased throughout the growing season when vertical gradients in LMA and Narea were not accounted for. Overall, our results suggest that leaves acclimate to light soon after leaf expansion, through an accumulation of leaf carbon, thickening of palisade layers and increased LMA, and reduction in stomatal sensitivity to Ψl or VPD. This period of light acclimation in leaves appears to optimize leaf function over time, despite height-related constraints early in the growing season. Our results imply that vertical gradients in leaf functional traits and leaf acclimation to light should be incorporated in canopy function models in order to refine estimates of canopy photosynthesis.

Structure and superorganization of acetylcholine receptor-rapsyn complexes

The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor-rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters.

Combined effect of heterogeneity, anisotropy and saturation on steady state flow and transport: Structure recognition and numerical simulation

1 Natural soil profiles may be interpreted as an arrangement of parts which are characterized by properties like hydraulic conductivity and water retention function. These parts form a complicated structure. Characterizing the soil structure is fundamental in subsurface hydrology because it has a crucial influence on flow and transport and defines the patterns of many ecological processes. We applied an image analysis method for recognition and classification of visual soil attributes in order to model flow and transport through a man-made soil profile. Modeled and measured saturation-dependent effective parameters were compared. We found that characterizing and describing conductivity patterns in soils with sharp conductivity contrasts is feasible. Differently, solving flow and transport on the basis of these conductivity maps is difficult and, in general, requires special care for representation of small-scale processes.

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

Developmental Changes in the Structure and Composition of the Postsynaptic Density

The development of the brain and its underlying circuitry is dependent on the formation of trillions of chemical synapses, which are highly specialized contacts that regulate the flow of information from one neuron to the next. It is through these synaptic connections that neurons wire together into networks capable of performing specific tasks, and activity-dependent changes in their structural and physiological state is one way that the brain is thought to adapt and store information. At the ultrastructural level, developmental and activity-dependent changes in the size and shape of dendritic spines have been well documented, and it is widely believed that structural changes in spines are a hallmark sign of synapse maturation and alteration of synaptic physiology. While changes in spine structure have been studied extensively, changes in one of its most prominent components, the postsynaptic density (PSD), have largely evaded observation. The PSD is a protein-rich organelle on the cytoplasmic side of the postsynaptic membrane, where it sits in direct opposition to the presynaptic terminal. The PSD functions both to cluster neurotransmitter receptors at the cell surface as well as organize the intracellular signaling molecules responsible for transducing extracellular signals to the postsynaptic cell. Much is known about the chemical composition of the PSD, but the structural arrangement of its molecular components is not well documented. Adding to the difficulty of understanding such a complex mass of protein machinery is the fact that its protein composition is known to change in response to synaptic activity, meaning that its structure is plastic and no two PSDs are identical. Here, immuno-gold labeling and electron tomography of PSDs isolated throughout development was used to track changes in both the structure and molecular composition of the PSD. State-of-the-art cryo-electron tomography was used to study the fine structure of the PSD during development, and provides an unprecedented glimpse into its molecular architecture in an un-fixed, unstained and hydrated state. Through this analysis, large structural and compositional changes are apparent and suggest a model by which the PSD is first assembled as a mesh-like lattice of proteins that function as support for the later recruitment of various PSD components. Spatial analysis of the recruitment of proteins into the PSD demonstrated that its assembly has an underlying order.

A study of xlcaax-1: Patterns of localization, possible function and usefulness as a developmental marker

Morphogenesis is the process by which the 3-dimensional structure of the developing embryo takes shape. We are studying xlcaax-1, a gene whose product can be used as a molecular marker for several morphogenetic events. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole mount immunocytochemistry and immunoperoxidase staining of tissue sections showed that during development the xlcaax-1 protein accumulation was coincident with the differentiation of the epidermis, pronephros and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein was localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein served as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. The xlcaax-1 protein was most abundant in kidney and immunogold EM analysis showed that the xlcaax-1 protein was highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. The xlcaax-1 protein was also localized in other ion transporting epithelia. The localization pattern and preliminary functional assays of xlcaax-1 suggest that the protein may function in association with an ion transport channel or pump.^ Cell migration and cell-cell interactions play important roles in numerous processes during morphogenesis. One of these is the formation of the pronephric (wolffian) duct (PD), which connects the pronephros to the cloaca. It is currently accepted that in most amphibians the pronephric duct is formed by active migration of the pronephric duct rudiment (PDR) cells along a pre-determined pathway. However, there is evidence that in Xenopus, the PD may be formed entirely by in situ segregation of cells out of the lateral mesoderm. In this study, we showed, using PDR ablation and X. laevis - X. borealis chimeras, that PD elongation in Xenopus required both active cell migration and an induced recruitment of cells from the posterior lateral plate mesoderm. We also showed that PDR cell migration was limited to only a few stages during development and that this temporal control is due, at least in part, to changes in the competence of the PD pathway to support cell migration. In addition, our data suggested that an alkaline phosphatase (APase) adhesion gradient may be involved in determining this competence. ^

Regulation and function of {\it Xparaxis\/} during the development of paraxial mesoderm in {\it Xenopus laevis\/}

Genes of the basic helix-loop-helix transcription factor family have been implicated in many different developmental processes from neurogenesis to myogenesis. The recently cloned bHLH transcription factor, paraxis, has been found to be expressed in the paraxial mesoderm of the mouse suggesting a role for paraxis in the development of this mesodermal subtype which gives rise to the axial muscle, skeleton, and dermis of the embryo. In order to perform in vivo gain of function assays and obtain a better understanding of the possible roles of paraxis in mesodermal and somitic development, we have successfully identified homologues of paraxis in the frog, Xenopus laevis, where the process of mesodermal induction and development is best understood. The two homologues, Xparaxis-a and Xparaxis-b, are conserved with respect to their murine homologue in structure and expression within the embryo. Xparaxis genes are expressed immediately after gastrulation in the paraxial mesoderm of Xenopus embryos and are down regulated in the myotome of the mature somite with continued expression in the undifferentiated dermatome. Overexpression of Xparaxis-b in Xenopus embryos caused defects in the organization and morphology of the somites. This effect was not dependent on DNA binding of Xparaxis but is likely due to its dimerization with other bHLH factors. Co-injections with XE12 did not diminish the effects indicating that the defects were not the result of limiting amounts of XE12. We also demonstrated that Xparaxis does not cause obvious defects in the cell adhesions and movements required for proper mesoderm patterning during gastrulation. The paraxis proteins also lacked the ability to activate transcription as GAL4 fusion proteins in a GAL4 reporter assay, indicating that the genes may function more as modulators of the activity of dimerization partners than as positively acting cell determination factors. In agreement with this, Xparaxis is regulated in response to other pathways of bHLH gene action, in that XE12 can activate Xparaxis-b, in vivo. In addition we show regulation of Xparaxis in response to mMyoD induced myogenesis pathways, again suggesting Xparaxis plays an important role in the patterning and organization of the paraxial mesoderm. ^

Human TNF receptor p75/80 gene structure and promoter characterization

Tumor necrosis factor receptor p75/80 ((TNF-R p75/80) is a 75 kDa type 1 transmembrane protein expressed predominately on cells of hematopoietic lineage. TNF-R p75/80 belongs to the TNF receptor superfamily characterized by cysteine-rich extracellular regions composed of three to six disulfide-linked domains. In the present report, we have characterized, for the first time, the complete gene structure for human TNF-R p75/80 which spans approximately 43 kbp. The gene consists of 10 exons (ranging from 34 bp to 2.5 kbp) and 9 introns (343 bp to 19 kbp). Consensus elements for transcription factors involved in T cell development and activation were noted in the 5$\sp\prime$ flanking region including TCF-1, Ikaros, AP-1, CK-2, IL-6RE, ISRE, GAS, NF-$\kappa$B and SP1, as well as an unusually high GC content and CpG frequency that appears characteristic of some TNF-R family members. The unusual (GATA)$\sb{\rm n}$ and (GAA)(GGA) repeats found within intron 1 may prove useful for further genome analysis within the 1p36 chromosomal locus. The human TNF-R p75/80 gene structure will permit further assessment of its involvement in normal hematopoietic cell development and function, autoimmune disease, and non-random translocations in hematopoietic malignancies. The region 1.8 kb 5$\sp\prime$ of the ATG was able to drive luciferase expression when transfected into cell lines expressing TNF-R p75/80. Further characterization of the 5$\sp\prime$-regulatory region will aid in determining factors and signal transduction pathways involved in regulating TNF-R p75/80 expression. ^

The structural substrate of brain function: analysis of brain structural networks based on diffusion-weighted imaging

The brain is a complex neural network with a hierarchical organization and the mapping of its elements and connections is an important step towards the understanding of its function. Recent developments in diffusion-weighted imaging have provided the opportunity to reconstruct the whole-brain structural network in-vivo at a large scale level and to study the brain structural substrate in a framework that is close to the current understanding of brain function. However, methods to construct the connectome are still under development and they should be carefully evaluated. To this end, the first two studies included in my thesis aimed at improving the analytical tools specific to the methodology of brain structural networks. The first of these papers assessed the repeatability of the most common global and local network metrics used in literature to characterize the connectome, while in the second paper the validity of further metrics based on the concept of communicability was evaluated. Communicability is a broader measure of connectivity which accounts also for parallel and indirect connections. These additional paths may be important for reorganizational mechanisms in the presence of lesions as well as to enhance integration in the network. These studies showed good to excellent repeatability of global network metrics when the same methodological pipeline was applied, but more variability was detected when considering local network metrics or when using different thresholding strategies. In addition, communicability metrics have been found to add some insight into the integration properties of the network by detecting subsets of nodes that were highly interconnected or vulnerable to lesions. The other two studies used methods based on diffusion-weighted imaging to obtain knowledge concerning the relationship between functional and structural connectivity and about the etiology of schizophrenia. The third study integrated functional oscillations measured using electroencephalography (EEG) and functional magnetic resonance imaging (fMRI) as well as diffusion-weighted imaging data. The multimodal approach that was applied revealed a positive relationship between individual fluctuations of the EEG alpha-frequency and diffusion properties of specific connections of two resting-state networks. Finally, in the fourth study diffusion-weighted imaging was used to probe for a relationship between the underlying white matter tissue structure and season of birth in schizophrenia patients. The results are in line with the neurodevelopmental hypothesis of early pathological mechanisms as the origin of schizophrenia. The different analytical approaches selected in these studies also provide arguments for discussion of the current limitations in the analysis of brain structural networks. To sum up, the first studies presented in this thesis illustrated the potential of brain structural network analysis to provide useful information on features of brain functional segregation and integration using reliable network metrics. In the other two studies alternative approaches were presented. The common discussion of the four studies enabled us to highlight the benefits and possibilities for the analysis of the connectome as well as some current limitations.

Copeptin Is Associated with Kidney Length, Renal Function, and Prevalence of Simple Cysts in a Population-Based Study.

Arginine vasopressin (AVP) has a key role in osmoregulation by facilitating water transport in the collecting duct. Recent evidence suggests that AVP may have additional effects on renal function and favor cyst growth in polycystic kidney disease. Whether AVP also affects kidney structure in the general population is unknown. We analyzed the association of copeptin, an established surrogate for AVP, with parameters of renal function and morphology in a multicentric population-based cohort. Participants from families of European ancestry were randomly selected in three Swiss cities. We used linear multilevel regression analysis to explore the association of copeptin with renal function parameters as well as kidney length and the presence of simple renal cysts assessed by ultrasound examination. Copeptin levels were log-transformed. The 529 women and 481 men had median copeptin levels of 3.0 and 5.2 pmol/L, respectively (P<0.001). In multivariable analyses, the copeptin level was associated inversely with eGFR (β=-2.1; 95% confidence interval [95% CI], -3.3 to -0.8; P=0.002) and kidney length (β=-1.2; 95% CI, -1.9 to -0.4; P=0.003) but positively with 24-hour urinary albumin excretion (β=0.11; 95% CI, 0.01 to 0.20; P=0.03) and urine osmolality (β=0.08; 95% CI, 0.05 to 0.10; P<0.001). A positive association was found between the copeptin level and the presence of renal cysts (odds ratio, 1.6; 95% CI, 1.1 to 2.4; P=0.02). These results suggest that AVP has a pleiotropic role in renal function and may favor the development of simple renal cysts.

Are shrubs really a sign of declining ecosystem function? Disentangling the myths and truths of woody encroachment in Australia

Since European settlement, there has been a dramatic increase in the density, cover and distribution of woody plants in former grassland and open woodland. There is a widespread belief that shrub encroachment is synonymous with declines in ecosystem functions, and often it is associated with landscape degradation or desertification. Indeed, this decline in ecosystem functioning is considered to be driven largely by the presence of the shrubs themselves. This prevailing paradigm has been the basis for an extensive program of shrub removal, based on the view that it is necessary to reinstate the original open woodland or grassland structure from which shrublands are thought to have been derived. We review existing scientific evidence, particularly focussed on eastern Australia, to question the notion that shrub encroachment leads to declines in ecosystem functions. We then summarise this scientific evidence into two conceptual models aimed at optimising landscape management to maximise the services provided by shrub-encroached areas. The first model seeks to reconcile the apparent conflicts between the patch- and landscape-level effects of shrubs. The second model identifies the ecosystem services derived from different stages of shrub encroachment. We also examined six ecosystem services provided by shrublands (biodiversity, soil C, hydrology, nutrient provision, grass growth and soil fertility) by using published and unpublished data. We demonstrated the following: (1) shrub effects on ecosystems are strongly scale-, species- and environment-dependent and, therefore, no standardised management should be applied to every case; (2) overgrazing dampens the generally positive effect of shrubs, leading to the misleading relationship between encroachment and degradation; (3) woody encroachment per se does not hinder any of the functions or services described above, rather it enhances many of them; (4) no single shrub-encroachment state (including grasslands without shrubs) will maximise all services; rather, the provision of ecosystem goods and services by shrublands requires a mixture of different states; and (5) there has been little rigorous assessment of the long-term effectiveness of removal and no evidence that this improves land condition in most cases. Our review provides the basis for an improved, scientifically based understanding and management of shrublands, so as to balance the competing goals of providing functional habitats, maintaining soil processes and sustaining pastoral livelihoods.