Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation
Contributors: Xiaofei Cheng, Ping Deng, Hongguang Cui, Aiming Wang
... Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells.
The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left–right asymmetry in zebrafish
Contributors: Jason J. Gokey, Agnik Dasgupta, Jeffrey D. Amack
... Asymmetric fluid flows generated by motile cilia in a transient ‘organ of asymmetry’ are involved in establishing the left–right (LR) body axis during embryonic development. The vacuolar-type H+-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer’s vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures—neuromasts and olfactory placodes—suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry.
Contributors: Mathilde Flamand, Bastien Herlin, Smaranda Leu-Semenescu, Valérie Attali, Claire Launois, Isabelle Arnulf
... Choking during sleep may be caused by various respiratory and non-respiratory problems.
Contributors: Hisako Takigawa-Imamura, Ritsuko Morita, Takafumi Iwaki, Takashi Tsuji, Kenichi Yoshikawa
... In the early stage of tooth germ development, the bud of the dental epithelium is invaginated by the underlying mesenchyme, resulting in the formation of a cap-like folded shape. This bud-to-cap transition plays a critical role in determining the steric design of the tooth. The epithelial-mesenchymal interaction within a tooth germ is essential for mediating the bud-to-cap transition. Here, we present a theoretical model to describe the autonomous process of the morphological transition, in which we introduce mechanical interactions among cells. Based on our observations, we assumed that peripheral cells of the dental epithelium bound tightly to each other to form an elastic sheet, and mesenchymal cells that covered the tooth germ would restrict its growth. By considering the time-dependent growth of cells, we were able to numerically show that the epithelium within the tooth germ buckled spontaneously, which is reminiscent of the cap-stage form. The difference in growth rates between the peripheral and interior parts of the dental epithelium, together with the steric size of the tooth germ, were determining factors for the number of invaginations. Our theoretical results provide a new hypothesis to explain the histological features of the tooth germ.
Letter to the Editor - Preoperative Echocardiography First Diagnosed and Intraoperative Echocardiography Altered the Surgical Plan in Intravenous Leiomyomatosis
Contributors: Ying-Hsuan Tai, Su-Man Lin, Chiao-Po Hsu, Wen-Chung Yu, Chun-Sung Sung
Absence of Dystrophin Related Protein-2 disrupts Cajal bands in a patient with Charcot–Marie–Tooth disease
Contributors: Kathryn M. Brennan, Yunhong Bai, Chiara Pisciotta, Suola Wang, Shawna M.E. Feely, Mark Hoegger, Laurie Gutmann, Steven A. Moore, Michael Gonzalez, Diane L. Sherman
... Using exome sequencing in an individual with Charcot–Marie–Tooth disease (CMT) we have identified a mutation in the X-linked dystrophin-related protein 2 (DRP2) gene. A 60-year-old gentleman presented to our clinic and underwent clinical, electrophysiological and skin biopsy studies. The patient had clinical features of a length dependent sensorimotor neuropathy with an age of onset of 50 years. Neurophysiology revealed prolonged latencies with intermediate conduction velocities but no conduction block or temporal dispersion. A panel of 23 disease causing genes was sequenced and ultimately was uninformative. Whole exome sequencing revealed a stop mutation in DRP2, c.805C>T (Q269*). DRP2 interacts with periaxin and dystroglycan to form the periaxin–DRP2–dystroglycan complex which plays a role in the maintenance of the well-characterized Cajal bands of myelinating Schwann cells. Skin biopsies from our patient revealed a lack of DRP2 in myelinated dermal nerves by immunofluorescence. Furthermore electron microscopy failed to identify Cajal bands in the patient's dermal myelinated axons in keeping with ultrastructural pathology seen in the Drp2 knockout mouse. Both the electrophysiologic and dermal nerve twig pathology support the interpretation that this patient's DRP2 mutation causes characteristic morphological abnormalities recapitulating the Drp2 knockout model and potentially represents a novel genetic cause of CMT.
Contributors: Alexander Dunkel
... Assessing information on aspects of identification, perception, emotion, and social interaction with respect to the environment is of particular importance to the fields of natural resource management. Our ability to visualize this type of information has rapidly improved with the proliferation of social media sites throughout the Internet in recent years. While many methods to extract information on human behavior from crowdsourced geodata already exist, this paper focuses on visualizing landscape perception for application to the fields of landscape and urban planning. Visualization of peoples’ perceptual responses to landscape is demonstrated with crowdsourced photo geodata from Flickr, a popular photo sharing community. A basic, general method to map, visualize, and evaluate perception and perceptual values is proposed. The approach utilizes common tools for spatial knowledge discovery and builds on existing research, but is specifically designed for implementation within the context of landscape perception analysis and particularly suited as a base for further evaluation in multiple scenarios. To demonstrate the process in application, three novel types of visualizations are presented: the mapping of sightlines in Yosemite Valley, the assessment of landscape change in the area surrounding the High Line in Manhattan, and individual location analysis for Coit Tower in San Francisco. The results suggest that analyzing crowdsourced data may contribute to a more balanced assessment of the perceived landscape, which provides a basis for a better integration of public values into planning processes.
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.