Contributors: Malachi Griffith, Christopher A. Miller, Obi L. Griffith, Kilannin Krysiak, Zachary L. Skidmore, Avinash Ramu, Jason R. Walker, Ha X. Dang, Lee Trani, David E. Larson
... Tumors are typically sequenced to depths of 75x–100x (exome) or 30x–50x (whole genome). We demonstrate that current sequencing paradigms are inadequate for tumors that are impure, aneuploid, or clonally heterogeneous. To reassess optimal sequencing strategies, we performed ultra-deep (up to ∼312x) whole genome sequencing and exome capture (up to ∼433x) of a primary acute myeloid leukemia, its subsequent relapse, and a matched normal skin sample. We tested multiple alignment and variant calling algorithms and validated ∼200,000 putative SNVs by sequencing them to depths of ∼1,000x. Additional targeted sequencing provided over 10,000x coverage and ddPCR assays provided up to ∼250,000x sampling of selected sites. We evaluated the effects of different library generation approaches, depth of sequencing, and analysis strategies on the ability to effectively characterize a complex tumor. This dataset, representing the most comprehensively sequenced tumor described to date, will serve as an invaluable community resource (dbGaP: phs000159).
EMG and force production of the flexor hallucis longus muscle in isometric plantarflexion and the push-off phase of walking
Contributors: Annamária Péter, András Hegyi, Lauri Stenroth, Taija Finni, Neil J. Cronin
... Large forces are generated under the big toe in the push-off phase of walking. The largest flexor muscle of the big toe is the flexor hallucis longus (FHL), which likely contributes substantially to these forces. This study examined FHL function at different levels of isometric plantarflexion torque and in the push-off phase at different speeds of walking. FHL and calf muscle activity were measured with surface EMG and plantar pressure was recorded with pressure insoles. FHL activity was compared to the activity of the calf muscles. Force and impulse values were calculated under the big toe, and were compared to the entire pressed area of the insole to determine the relative contribution of big toe flexion forces to the ground reaction force. FHL activity increased with increasing plantarflexion torque level (F=2.8, P=0.024) and with increasing walking speed (F=11.608, P<0.001). No differences were observed in the relative contribution of the force under the big toe to the entire sole between different plantarflexion torque levels (F=0.836, P=0.529). On the contrary, in the push-off phase of walking, peak force under the big toe increased at a higher rate than force under the other areas of the plantar surface (F=3.801, P=0.018), implying a greater relative contribution to total force at faster speeds. Moreover, substantial differences were found between isometric plantarflexion and walking concerning FHL activity relative to that of the calf muscles, highlighting the task-dependant behaviour of FHL.
Contributors: Tuyen Thi Ngoc Tran, Claas Gerding-Reimers, Beate Schölermann, Bettina Stanitzki, Thomas Henkel, Herbert Waldmann, Slava Ziegler
... Natural products represent compound classes with high chemical and structural diversity and various biological activities. Libraries based on natural products are valuable starting point in the search for novel biologically active substances. Here we report on the identification of the natural product podoverine A from the plant Podophyllum versipelle Hance as a novel tubulin-acting agent. A natural product compound collection was subjected to a high-content screen that monitors changes in cytoskeleton and DNA and podoverine A was identified as inhibitor of mitosis. This natural product causes mitotic arrest and inhibits microtubule polymerization in vitro and in cells by targeting the vinca binding site on tubulin.
GR Focus Review - A tectonic model reconciling evidence for the collisions between India, Eurasia and intra-oceanic arcs of the central-eastern Tethys
Contributors: A.D. Gibbons, S. Zahirovic, R.D. Müller, J.M. Whittaker, V. Yatheesh
... Despite several decades of investigations, inferences on the timing and nature of collisions along the Mesozoic–Cenozoic Eurasian margin remain controversial. We assimilate geological and geophysical evidence into a plate tectonic model for the India–Eurasia collision that includes continuously-closing topological plate polygons, constructed from a time-dependent network of evolving plate boundaries, with synthetic plates constructed for now-subducted ocean floor, including back-arc basins that formed on the southern Eurasian margin. Our model is regionally-constrained and self-consistent, incorporating geophysical data from abyssal plains offshore West Australia and East Antarctica, including Jurassic age data from offshore Northwest Australia, limiting much of northern Greater India to a ~1000km-long indenter, originally reaching to the Wallaby–Zenith Fracture Zone. Southern Eurasia and Southeast Asia are riddled with dismembered oceanic arcs indicating long-lived Tethyan intra-oceanic subduction. This intra-oceanic subduction system was well-established from Cretaceous time in the India–Eurasia convergence zone in the NeoTethys, which was consumed during Greater India's northward trajectory towards Eurasia from the Early Cretaceous. Fragments of obducted oceanic crust within the Yarlung-Tsangpo Suture Zone, between India and Eurasia, predominantly date to the Late Jurassic or mid Cretaceous (Barremian–Aptian). The various ophiolites along strike and a hiatus in subduction-related magmatism during the Tithonian–Aptian suggest that there was at least one generation of intra-oceanic arc formation, whose plate boundary configuration remains uncertain. Paleomagnetic and magmatic studies suggest that the intra-oceanic arc was at equatorial latitudes during the Early Cretaceous before subduction resumed further north beneath the Eurasian margin (Lhasa terrane), with another hiatus in subduction-related magmatism along southern Lhasa during ~80–65Ma, possibly as the back-arc spreading centre approached the active Andean-style margin. In our model, Greater India collided with the Tethyan intra-oceanic arc in Paleocene–Eocene time, finally closing the Tethyan seaway from Mid-Late Eocene time, which is consistent with the age of the youngest marine deposits found between India and Eurasia. Geological evidence from the collision zone indicates an age of initial arc–continent collision by ~52Ma, followed by the “soft” (initial) continent–continent collision between India and Eurasia by ~44±2Ma. This timing is supported by marine geophysical data, where the spreading centres in the Indian Ocean record a drastic decline in seafloor spreading rates and changes in spreading directions first at ~52Ma, followed by another reorganisation at ~43Ma. The abandonment of spreading in the Wharton Basin and the onset of extrusion tectonics in Asia by ~36Ma are likely indicators of “hard” (complete) continent collision, and highlight the multi-stage collisional history of this margin. Our continuously evolving network of mid-ocean ridge and subduction zone geometries, and divergence/convergence vectors through time, provide a basis for future refinements to assimilate new data and/or test alternative tectonic scenarios.
Contributors: Gabriel Ouellet Lavallée, Angela Pearson
... Herpes simplex virus 1 (HSV-1) infection induces changes to the host cell nucleus including relocalization of the cellular protein Upstream Binding Factor (UBF) from the nucleolus to viral replication compartments (VRCs). Herein, we tested the hypothesis that UBF is recruited to VRCs to promote viral DNA replication. Surprisingly, infection of UBF-depleted HeLa cells with HSV-1 or HSV-2 produced higher viral titers compared to controls. Reduced expression of UBF also led to a progressive increase in the relative amount of HSV-1 DNA versus controls, and increased levels of HSV-1 ICP27 and TK mRNA and protein, regardless of whether viral DNA replication was inhibited or not. Our results suggest that UBF can inhibit gene expression from viral DNA prior to its replication. A similar but smaller effect on viral titers was observed in human foreskin fibroblasts. This is the first report of UBF having a restrictive effect on replication of a virus.
Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum
Contributors: Wasima Mohamed, Sibnath Ray, Derrick Brazill, Ramamurthy Baskar
... A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA−) did not exhibit tip dominance. A striking phenotype of pkcAˉ was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA− to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules – CadA and CsaA. pkcA− slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA−.
Contributors: Shohei Matsunobu, Yasunori Sasakura
... In most ascidians, the tadpole-like swimming larvae dramatically change their body-plans during metamorphosis and develop into sessile adults. The mechanisms of ascidian metamorphosis have been researched and debated for many years. Until now information on the detailed time course of the initiation and completion of each metamorphic event has not been described. One dramatic and important event in ascidian metamorphosis is tail regression, in which ascidian larvae lose their tails to adjust themselves to sessile life. In the present study, we measured the time associated with tail regression in the ascidian Ciona intestinalis. Larvae are thought to acquire competency for each metamorphic event in certain developmental periods. We show that the timing with which the competence for tail regression is acquired is determined by the time since hatching, and this timing is not affected by the timing of post-hatching events such as adhesion. Because larvae need to adhere to substrates with their papillae to induce tail regression, we measured the duration for which larvae need to remain adhered in order to initiate tail regression and the time needed for the tail to regress. Larvae acquire the ability to adhere to substrates before they acquire tail regression competence. We found that when larvae adhered before they acquired tail regression competence, they were able to remember the experience of adhesion until they acquired the ability to undergo tail regression. The time course of the events associated with tail regression provides a valuable reference, upon which the cellular and molecular mechanisms of ascidian metamorphosis can be elucidated.
Contributors: Ann M. Cavanaugh, Jie Huang, Jau-Nian Chen
... Cardiac neural crest cells are essential for outflow tract remodeling in animals with divided systemic and pulmonary circulatory systems, but their contributions to cardiac development in animals with a single-loop circulatory system are less clear. Here we genetically labeled neural crest cells and examined their contribution to the developing zebrafish heart. We identified two populations of neural crest cells that contribute to distinct compartments of zebrafish cardiovascular system at different developmental stages. A stream of neural crest cells migrating through pharyngeal arches 1 and 2 integrates into the myocardium of the primitive heart tube between 24 and 30h post fertilization and gives rise to cardiomyocytes. A second wave of neural crest cells migrating along aortic arch 6 envelops the endothelium of the ventral aorta and invades the bulbus arteriosus after three days of development. Interestingly, while inhibition of FGF signaling has no effect on the integration of neural crest cells to the primitive heart tube, it prevents these cells from contributing to the outflow tract, demonstrating disparate responses of neural crest cells to FGF signaling. Furthermore, neural crest ablation in zebrafish leads to multiple cardiac defects, including reduced heart rate, defective myocardial maturation and a failure to recruit progenitor cells from the second heart field. These findings add to our understanding of the contribution of neural crest cells to the developing heart and provide insights into the requirement for these cells in cardiac maturation.
Contributors: Åsa Barrefelt, Ying Zhao, Malin K. Larsson, Gabriella Egri, Raoul V. Kuiper, Jörg Hamm, Maryam Saghafian, Kenneth Caidahl, Torkel B. Brismar, Peter Aspelin
... Air-filled polyvinyl alcohol microbubbles (PVA-MBs) were recently introduced as a contrast agent for ultrasound imaging. In the present study, we explore the possibility of extending their application in multimodal imaging by labeling them with a near infrared (NIR) fluorophore, VivoTag-680.
Contributors: O. Kovalchuk Ben-Zaken, I. Nissan, S. Tzaban, A. Taraboulos, E. Zcharia, S. Matzger, I. Shafat, I. Vlodavsky, Y. Tal
... Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrPSc (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrPSc. Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression.