A Cryptic TOG Domain with a Distinct Architecture Underlies CLASP-Dependent Bipolar Spindle Formation

CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle structure with CLASP mutants displaying a distinctive monopolar spindle phenotype. It has been postulated that cryptic TOG domains underlie CLASP’s ability regulate MT dynamics. Here, we report the crystal structure of the first cryptic TOG domain (TOG2) from human CLASP1, revealing the existence of a bona fide TOG array in the CLASP family. Strikingly, CLASP1 TOG2 exhibits a unique, convex architecture across the tubulin-binding surface that contrasts with the flat tubulin-binding surface of XMAP215 family TOG domains. Mutations in key, conserved TOG2 determinants abrogate the ability of CLASP mutants to rescue bipolar spindle formation in Drosophila cells depleted of endogenous CLASP. These findings highlight the common mechanistic use of TOG domains in XMAP215 and CLASP families to regulate MT dynamics, and suggest that differential TOG domain architecture may confer distinct functions to these critical cytoskeletal regulators

An international network to monitor the structure, composition and dynamics of Amazonian forests (RAINFOR)

The Amazon basin is likely to be increasingly affected by environmental changes: higher temperatures, changes in precipitation, CO2 fertilization and habitat fragmentation. To examine the important ecological and biogeochemical consequences of these changes, we are developing an international network, RAINFOR, which aims to monitor forest biomass and dynamics across Amazonia in a co-ordinated fashion in order to understand their relationship to soil and climate. The network will focus on sample plots established by independent researchers, some providing data extending back several decades. We will also conduct rapid transect studies of poorly monitored regions. Field expeditions analysed local soil and plant properties in the first phase (2001–2002). Initial results suggest that the network has the potential to reveal much information on the continental-scale relations between forest and environment. The network will also serve as a forum for discussion between researchers, with the aim of standardising sampling techniques and methodologies that will enable Amazonian forests to be monitored in a coherent manner in the coming decades

Multi-Country Evaluation of the Sensitivity and Specificity of Two Commercially-Available NS1 ELISA Assays for Dengue Diagnosis

Dengue is the most important mosquito-borne viral disease of humans and an enormous public health burden in affected countries. Early, sensitive and specific diagnosis of dengue is needed for appropriate patient management as well as for early epidemic detection. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offer the possibility of early and rapid diagnosis. Here we evaluated two commercially available ELISA kits for NS1 detection (Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag). Results were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity

TOG–tubulin binding specificity promotes microtubule dynamics and mitotic spindle formation

XMAP215, CLASP, and Crescerin use arrayed tubulin-binding tumor overexpressed gene (TOG) domains to modulate microtubule dynamics. We hypothesized that TOGs have distinct architectures and tubulin-binding properties that underlie each family’s ability to promote microtubule polymerization or pause. As a model, we investigated the pentameric TOG array of a Drosophila melanogaster XMAP215 member, Msps. We found that Msps TOGs have distinct architectures that bind either free or polymerized tubulin, and that a polarized array drives microtubule polymerization. An engineered TOG1-2-5 array fully supported Msps-dependent microtubule polymerase activity. Requisite for this activity was a TOG5-specific N-terminal HEAT repeat that engaged microtubule lattice-incorporated tubulin. TOG5–microtubule binding maintained mitotic spindle formation as deleting or mutating TOG5 compromised spindle architecture and increased the mitotic index. Mad2 knockdown released the spindle assembly checkpoint triggered when TOG5–microtubule binding was compromised, indicating that TOG5 is essential for spindle function. Our results reveal a TOG5-specific role in mitotic fidelity and support our hypothesis that architecturally distinct TOGs arranged in a sequence-specific order underlie TOG array microtubule regulator activity

Galectin-9 plasma levels reflect adverse hematological and immunological features in acute dengue virus infection

Background: Dengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties. Objective: To investigate plasma Gal-9 levels as a biomarker for DENV infection. Study design: We enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness. Results: During the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464. pg/ml; dengue fever patients (DF): 1407. pg/ml; non-dengue febrile illness: 616. pg/ml; healthy: 196. pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P < 0.01). Conclusion: Gal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity

Structures of TOG1 and TOG2 from the human microtubule dynamics regulator CLASP1.

Tubulin-binding TOG domains are found arrayed in a number of proteins that regulate microtubule dynamics. While much is known about the structure and function of TOG domains from the XMAP215 microtubule polymerase family, less in known about the TOG domain array found in animal CLASP family members. The animal CLASP TOG array promotes microtubule pause, potentiates rescue, and limits catastrophe. How structurally distinct the TOG domains of animal CLASP are from one another, from XMAP215 family TOG domains, and whether a specific order of structurally distinct TOG domains in the TOG array is conserved across animal CLASP family members is poorly understood. We present the x-ray crystal structures of Homo sapiens (H.s.) CLASP1 TOG1 and TOG2. The structures of H.s. CLASP1 TOG1 and TOG2 are distinct from each other and from the previously determined structure of Mus musculus (M.m.) CLASP2 TOG3. Comparative analyses of CLASP family TOG domain structures determined to date across species and paralogs supports a conserved CLASP TOG array paradigm in which structurally distinct TOG domains are arrayed in a specific order. H.s. CLASP1 TOG1 bears structural similarity to the free-tubulin binding TOG domains of the XMAP215 family but lacks many of the key tubulin-binding determinants found in XMAP215 family TOG domains. This aligns with studies that report that animal CLASP family TOG1 domains cannot bind free tubulin or microtubules. In contrast, animal CLASP family TOG2 and TOG3 domains have reported microtubule-binding activity but are structurally distinct from the free-tubulin binding TOG domains of the XMAP215 family. H.s. CLASP1 TOG2 has a convex architecture, predicted to engage a hyper-curved tubulin state that may underlie its ability to limit microtubule catastrophe and promote rescue. M.m. CLASP2 TOG3 has unique structural elements in the C-terminal half of its α-solenoid domain that our modeling studies implicate in binding to laterally-associated tubulin subunits in the microtubule lattice in a mode similar to, yet distinct from those predicted for the XMAP215 family TOG4 domain. The potential ability of the animal CLASP family TOG3 domain to engage lateral tubulin subunits may underlie the microtubule rescue activity ascribed to the domain. These findings highlight the structural diversity of TOG domains within the CLASP family TOG array and provide a molecular foundation for understanding CLASP-dependent effects on microtubule dynamics

Reconstructed versus real-time 3-dimensional echocardiography: Comparison with magnetic resonance imaging

Background: The accuracy, reproducibility, and test-retest reliability of 3-dimensional (3D) echocardiography (3DE) with 3D reconstruction (3DR) and real-time (RT) imaging (RT 3DE) exceed that of 2-dimensional echocardiography (2DE). However, image quality with RT 3DE is inferior to 2DE and we sought to determine whether this justified ongoing use of 3DR. Methods: Unselected patients (n = 30, 22 men, age 66 ± 7 years) presenting to the echocardiography laboratory for left ventricular (LV) evaluation were studied with 2DE and RT 3DE; 3DR images were obtained using external localization. The 3D measurements and reconstructions were obtained offline. Magnetic resonance images (MRI) were obtained using true free induction, steady state precession during breath hold and 3D volumes and ejection fraction (EF) were measured using 3D software. A separate cohort of 20 patients (13 men, age 60 ± 12 years) was measured for test-retest variation. Results: All echocardiographic measures underestimated LV volumes and EF compared with MRI, but this was least with RT 3DE. End-diastolic volume by MRI (168 ± 54 mL) was underestimated by RT 3DE (-15 ± 31, P = .02), 3DR (-26 ± 33, P < .01), and 2DE (-57 ± 40, P < .01). Similarly, end-systolic volume by MRI (86 ± 50 mL) was underestimated by RT 3DE (-15 ± 31, P = .02), 3DR (-26 ± 33, P < .01), and 2DE (-57 ± 40, P < .01). However, EF measurements were similar with each method. Test-retest variation was less and interobserver and intraobserver correlations were better with RT 3DE for volumes and EF, compared with 3DR and 2DE. Conclusions: Despite limitations of image quality, RT 3DE is the most feasible and accurate approach for LV volume and EF measurements and follow-up LV assessment in daily practice