A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

Mechanical response and conformational amplification in α-helical coiled coils

α-Helical coiled coils (CCs) are ubiquitous tertiary structural domains that are often found in mechanoproteins. CCs have mechanical rigidity and are often involved in force transmission between protein domains. Although crystal structures of CCs are available, information about their conformational flexibility is limited. The role of hydrophobic interactions in determining the CC conformation is not clear. In this work we examined the mechanical responses of typical CCs and constructed a coarse-grained mechanical model to describe the conformation of the protein. The model treats α-helices as elastic rods. Hydrophobic bonds arranged in a repeated pattern determine the CC structure. The model is compared with molecular-dynamics simulations of CCs under force. We also estimate the effective bending and twisting persistence length of the CC. The model allows us to examine unconventional responses of the CC, including significant conformational amplification upon binding of a small molecule. We find that the CC does not behave as a simple elastic rod and shows complex nonlinear responses. These results are significant for understanding the role of CC structures in chemoreceptors, motor proteins, and mechanotransduction in general.     

From falling to flying: the path to powered flight of a robotic samara nano air vehicle

This paper details the development of a nano-scale (>15 cm) robotic samara, or winged seed. The design of prototypes inspired by naturally occurring geometries is presented along with a detailed experimental process which elucidates similarities between mechanical and robotic samara flight dynamics. The helical trajectories of a samara in flight were observed to differ in-flight path and descent velocity. The body roll and pitch angular rates for the differing trajectories were observed to be coupled to variations in wing pitch, and thus provide a means of control. Inspired by the flight modalities of the bio-inspired samaras, a robotic device has been created that mimics the autorotative capability of the samara, whilst providing the ability to hover, climb and translate. A high-speed camera-based motion capture system is used to observe the flight dynamics of the mechanical and robotic samara. Similarities in the flight dynamics are compared and discussed as it relates to the design of the robotic samara.     

Community-based knowledge transfer and exchange: Helping community-based organizations link research to action

Background

Community-based organizations (CBOs) are important stakeholders in health systems and are increasingly called upon to use research evidence to inform their advocacy, program planning, and service delivery efforts. CBOs increasingly turn to community-based research (CBR) given its participatory focus and emphasis on linking research to action. In order to further facilitate the use of research evidence by CBOs, we have developed a strategy for community-based knowledge transfer and exchange (KTE) that helps CBOs more effectively link research evidence to action. We developed the strategy by: outlining the primary characteristics of CBOs and why they are important stakeholders in health systems; describing the concepts and methods for CBR and for KTE; comparing the efforts of CBR to link research evidence to action to those discussed in the KTE literature; and using the comparison to develop a framework for community-based KTE that builds on both the strengths of CBR and existing KTE frameworks.

Discussion

We find that CBR is particularly effective at fostering a climate for using research evidence and producing research evidence relevant to CBOs through community participation. However, CBOs are not always as engaged in activities to link research evidence to action on a larger scale or to evaluate these efforts. Therefore, our strategy for community-based KTE focuses on: an expanded model of 'linkage and exchange' (i.e., producers and users of researchers engaging in a process of asking and answering questions together); a greater emphasis on both producing and disseminating systematic reviews that address topics of interest to CBOs; developing a large-scale evidence service consisting of both 'push' efforts and efforts to facilitate 'pull' that highlight actionable messages from community relevant systematic reviews in a user-friendly way; and rigorous evaluations of efforts for linking research evidence to action.

Summary

Through this type of strategy, use of research evidence for CBO advocacy, program planning, and service delivery efforts can be better facilitated and continually refined through ongoing evaluations of its impact.

     

Designing and engineering evolutionary robust genetic circuits

Background  

One problem with engineered genetic circuits in synthetic microbes is their stability over evolutionary time in the absence of selective pressure. Since design of a selective environment for maintaining function of a circuit will be unique to every circuit, general design principles are needed for engineering evolutionary robust circuits that permit the long-term study or applied use of synthetic circuits.     

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community-wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single-locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.     

Tree species effects on ecosystem water-use efficiency in a high-elevation, subalpine forest

Ecosystem water-use efficiency (eWUE; the ratio of net ecosystem productivity to evapotranspiration rate) is a complex landscape-scale parameter controlled by both physical and biological processes occurring in soil and plants. Leaf WUE (lWUE; the ratio of leaf CO₂ assimilation rate to transpiration rate) is controlled at short time scales principally by leaf stomatal dynamics and this control varies among plant species. Little is known about how leaf-scale variation in lWUE influences landscape-scale variation in eWUE. We analyzed approximately seven thousand 30-min averaged eddy covariance observations distributed across 9 years in order to assess eWUE in two neighboring forest communities. Mean eWUE was 19% lower for the community in which Engelmann spruce and subalpine fir were dominant, compared to the community in which lodgepole pine was dominant. Of that 19% difference, 8% was attributed to residual bias in the analysis that favored periods with slightly drier winds for the spruce-fir community. In an effort to explain the remaining 11% difference, we assessed patterns in lWUE using C isotope ratios. When we focused on bulk tissue from older needles we detected significant differences in lWUE among tree species and between upper and lower canopy needles. However, when these differences were scaled to reflect vertical and horizontal leaf area distributions within the two communities, they provided no power to explain differences in eWUE that we observed in the eddy covariance data. When we focused only on bulk needle tissue of current-year needles for 3 of the 9 years, we also observed differences in lWUE among species and in needles from upper and lower parts of the canopy. When these differences in lWUE were scaled to reflect leaf area distributions within the two communities, we were able to explain 6.3% of the differences in eWUE in 1 year (2006), but there was no power to explain differences in the other 2 years (2003 and 2007). When we examined sugars extracted from needles at 3 different times during the growing season of 2007, we could explain 3.8–6.0% of the differences in eWUE between the two communities, but the difference in eWUE obtained from the eddy covariance record, and averaged over the growing season for this single year, was 32%. Thus, overall, after accounting for species effects on lWUE, we could explain little of the difference in eWUE between the two forest communities observed in the eddy covariance record. It is likely that water and C fluxes from soil, understory plants, and non-needle tissues, account for most of the differences observed in the eddy covariance data. For those cases where we could explain some of the difference in eWUE on the basis of species effects, we partitioned the scaled patterns in lWUE into two components: a component that is independent of canopy leaf area distribution, and therefore only dependent on species-specific differences in needle physiology; and a component that is independent of species differences in needle physiology, and only dependent on species-specific influences on canopy leaf area distribution. Only the component that is dependent on species influences on canopy leaf area distribution, and independent of inherent species differences in needle physiology, had potential to explain differences in eWUE between the two communities. Thus, when tree species effects are important, canopy structure, rather than species-specific needle physiology, has more potential to explain patterns in eWUE.     

Prediction of wheat response to an application of phosphorus under field conditions using diffusive gradients in thin-films (DGT) and extraction methods

The ability of the Diffusive Gradients in Thin Films (DGT) technique and two other established testing methods (Colwell, resin) to predict wheat responsiveness to applied P from 35 field trials across southern Australia was investigated. Regression analysis of relative early dry matter production and grain yield responses demonstrated that the DGT method predicted plant responsiveness to applied P more accurately than Colwell P and resin P at sites where maximum yields were reached with P rates used (20 out of 35). The measured concentration in soils at the DGT surface, C_DGT, explained 74% of the variation in response for both early dry matter and grain, compared to 7% for early dry matter and 35% for grain using the resin P method. No significant relationships could be obtained for Colwell P although modifying the Colwell test data using Phosphorus Buffering Index resulted in a correct response prediction for 11 of the 20 field sites compared to 18 for DGT and 14 for resin P. These observations suggest that the DGT technique can assess plant available P in soils with significantly greater accuracy than traditional soil P testing methods. The critical P threshold, expressed as C_DGT, was 255 μg L^?1 for early dry matter and 66 μg L^?1 for grain.     

Naegleria gruberi De Novo Basal Body Assembly Occurs via Stepwise Incorporation of Conserved Proteins

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, γ-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally γ-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.The beautiful and enigmatic pinwheel structures of centrioles and basal bodies have captured the imaginations of cell biologists for over a century. These small (∼1-μm) organelles are composed largely of a cylinder of nine microtubule triplets (11). The surrounding amorphous material harbors the microtubule-organizing activities of the centrosome, placing centrioles at the hub of the microtubule cytoskeleton. Metazoan centrosomes define mitotic spindle poles, and their centrioles are called basal bodies when used to form cilia (29). Moreover, in 1900 Meeves showed in a series of classical experiments that centrioles and basal bodies are interconvertible structures (34). Centrioles must replicate exactly once per cell cycle, as duplication errors can lead to problems with chromosome segregation and cell morphology (17).Virtually all animal cells have a pair of centrosomal centrioles that duplicate via “templated” assembly, with the new centriole developing perpendicular and attached to a preexisting centriole (4). Centrioles can also be formed “de novo” in cytosol devoid of preexisting centrioles and basal bodies (20). In addition to many in vivo examples (20), terminally differentiated fibroblasts held in S phase can assemble centrioles de novo after removal of preexisting centrioles by laser microsurgery (15).The amoeboflagellate Naegleria gruberi grows as an amoeba that completely lacks a cytoplasmic microtubule cytoskeleton. However, when exposed to stressors such as temperature, osmotic, or pH changes, Naegleria rapidly differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton from scratch, including two basal bodies and flagella (8). This differentiation occurs synchronously, with approximately 90% of cells growing visible flagella in a 15-min window (T₅₀ = 65 min after initiation of differentiation). As part of this differentiation, Naegleria has been shown to assemble the pinwheel structure of the basal bodies de novo, about 10 min before flagella are seen (11).Two centrosomal proteins that have been studied during Naegleria differentiation are centrin and γ-tubulin. Centrin is a calcium-binding phosphoprotein that is an integral component of the wall and lumen of basal bodies and of the pericentriolar lattice in many organisms (4, 19). During differentiation, Naegleria induces synthesis of centrin protein, which then localizes specifically to basal body structures throughout differentiation (18). γ-Tubulin is a general microtubule nucleation factor that localizes to microtubule-organizing centers (MTOCs) of many types. Surprisingly, Naegleria''s γ-tubulin homolog has been reported to localize to basal body precursor complexes and then move to the other end of the cell before disappearing completely (32).A third protein that has come under recent scrutiny for its role in centriole duplication is SAS-6, a functionally conserved coiled-coil protein required for the formation of diverse basal body precursor structures (7, 21,–23, 31). In Caenorhabditis elegans and Drosophila melanogaster, SAS-6 is recruited at S phase to form the “central tube,” a cylindrical basal body precursor that lacks microtubules (22, 23). SAS-6 is also required for the formation of the flat ring or cartwheel with nine radiating spokes, which is the first structure to be formed in the Chlamydomonas basal body (21).To determine if Naegleria is likely to have typical basal body components, we identified conserved basal body genes in the Naegleria genome. We also made antibodies to and localized Naegleria''s homologs of SAS-6 and γ-tubulin. Finally, we have determined the order of expression and incorporation of these proteins, as well as centrin, during Naegleria de novo basal body assembly.     

The length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis

Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 70 x 200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) defective interfering particle, DI-T. By imaging the uptake of individual particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained.