Dynamics and mechanisms of intracellular calcium waves elicited by tandem bubble-induced jetting flow
Fenfang Li, Chen Yang, Fang Yuan, Defei Liao, Thomas Li, Farshid Guilak, and Pei Zhong
Ultrasound-induced microbubble oscillation can lead to cell injury or mechanotransduction via calcium signaling processes such as intracellular calcium waves (ICWs). However, the mechanisms by which microbubbles stimulate ICWs remain unknown. Using a microfluidic system with highly controlled bubble−cell interaction, we identified two distinct types of ICWs: a fast response correlating with significant membrane poration, and a slow response triggered by calcium influx through stretch-activated ion channels. The fast ICWs, distinguished from those under physiological conditions, are associated with cell injuries. We further elicited ICWs without cell injury by displacing integrin-binding beads on the cell membrane under mild cavitation conditions. This study provides mechanistic insights into ICWs for guiding ultrasound therapy in tissue modification, drug delivery, and cell mechanotransduction. (See pp. E353–E362.)
Creation of disease-inspired biomaterial environments to mimic pathological events in early calcific aortic valve disease
Ana M. Porras, Jennifer A. Westlund, Austin D. Evans, and Kristyn S. Masters
Due to an insufficient understanding of the mechanisms that drive its progression, there is currently no treatment for calcific aortic valve disease (CAVD) other than valve replacement. In this report, we describe the generation of engineered environments that mimic features found in early CAVD. By combining these engineered models of valve disease with other exogenous cues present during CAVD, we were able to propose a previously unidentified cascade of pathological events and identify specific extracellular components that are critical in regulating this process. These findings demonstrate the utility of engineered in vitro models to perform systematic, controlled studies of events related to disease progression that may lead to future identification of potential targets for therapeutic interventions. (See pp. E363–E371.)
Migration-based selections of antibodies that convert bone marrow into trafficking microglia-like cells that reduce brain amyloid β
Kyung Ho Han, Britni M. Arlian, Matthew S. Macauley, James C. Paulson, and Richard A. Lerner
A migration-based selection system is used to identify antibodies from combinatorial libraries that induce stem cells to both differentiate and selectively traffic to different tissues in adult animals. Significantly, a single agonist antibody induces microglia-like cells, which have the capacity to migrate to the brain and decrease amyloid beta deposition in the brain. (See pp. E372–E381.)
Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release
Thomas Philipp Hoernes, Nina Clementi, Michael Andreas Juen, Xinying Shi, Klaus Faserl, Jessica Willi, Catherina Gasser, Christoph Kreutz, Simpson Joseph, Herbert Lindner, Alexander Hüttenhofer, and Matthias David Erlacher
Translation termination is a crucial process during protein synthesis. Class I release factors (RFs) are in charge of recognizing stop codons and consequently hydrolyzing the peptidyl-tRNA at the ribosomal P site. High-resolution crystal- and cryo-EM structures of RFs bound to the ribosome revealed a network of potential interactions that is formed between the mRNA and RFs; however, it remained enigmatic which interactions are critical for accurate stop codon recognition and peptide release. By using chemically modified stop codon nucleotides, the importance and the contribution of single hydrogen bonds to stop codon recognition was investigated. This approach revealed a detailed picture of chemical groups defining a stop codon and contributing to the discrimination against sense codons during prokaryotic and eukaryotic translation termination. (See pp. E382–E389.)
Computational modeling of three-dimensional ECM-rigidity sensing to guide directed cell migration
Min-Cheol Kim, Yaron R. Silberberg, Rohan Abeyaratne, Roger D. Kamm, and H. Harry Asada
We have investigated how the directed cell migration toward stiffer extracellular matrix (ECM) is guided by filopodial mechanosensing of the surrounding ECM stiffness. As filopodia bind to the surrounding 3D ECM fibers, local force and displacement are created near the filopodium tip in response to the contraction of filopodia. We have looked into the time rate of changes to the force and the relative displacement between each binding site of ECM fiber (or fluorescent microbeads) and that of the filopodium tip. To quantify the local ECM stiffness sensed by the cell, we have aggregated these effects using two approaches of continuum and discrete mechanics and formulated two different equations for the effective rigidities of local ECM perceived by the cell. (See pp. E390–E399.)
Organic matter processing by microbial communities throughout the Atlantic water column as revealed by metaproteomics
Kristin Bergauer, Antonio Fernandez-Guerra, Juan A. L. Garcia, Richard R. Sprenger, Ramunas Stepanauskas, Maria G. Pachiadaki, Ole N. Jensen, and Gerhard J. Herndl
Circumstantial evidence indicates that especially deep-ocean heterotrophic microbes rely on particulate organic matter sinking through the oceanic water column and being solubilized to dissolved organic matter (DOM) prior to utilization rather than on direct uptake of the vast pool of DOM in the deep ocean. Comparative metaproteomics allowed us to elucidate the vertical distribution and abundance of microbially mediated transport processes and thus the uptake of solutes throughout the oceanic water column. Taken together, our data suggest that, while the phylogenetic composition of the microbial community is depth stratified, the composition and substrate specificities of transporters considered in this study are ubiquitous while their relative abundance changes with depth. (See pp. E400–E408.)
Pairwise comparisons across species are problematic when analyzing functional genomic data
Casey W. Dunn, Felipe Zapata, Catriona Munro, Stefan Siebert, and Andreas Hejnol
Comparisons of genome function between species are providing important insight into the evolutionary origins of diversity. Here, we show that comparative functional genomics studies can come to the wrong conclusions if they do not take the relationships of species into account and instead rely on pairwise comparisons between species, as is common practice. We reexamined two previously published studies and found problems with pairwise comparisons that draw both their original conclusions into question. One study found support for the ortholog conjecture, and the other concluded that evolution of gene expression differed in pattern and process between animal phyla vs. within animal phyla. Our results show that, to answer evolutionary questions about genome function, it is critical to consider evolutionary relationships. (See pp. E409–E417.)
Induced GnasR201H expression from the endogenous Gnas locus causes fibrous dysplasia by up-regulating Wnt/β-catenin signaling
Sanjoy Kumar Khan, Prem Swaroop Yadav, Gene Elliott, Dorothy Zhang Hu, Ruoshi Xu, and Yingzi Yang
Understanding molecular and cellular mechanisms of rare genetic diseases provides invaluable insights into the human biology and pathology of both rare and related common diseases. Fibrous dysplasia (FD) is a mosaic disease resulting from postzygotic activating mutations of GNAS. The mouse models we created allowed us to precisely model FD by expressing the FD Gαs mutation under the control of its endogenous genetic locus. We found in our FD mouse models that up-regulated Wnt/β-catenin signaling resulted in impaired differentiation and proliferation of bone marrow stem cells, which in turn caused marrow fibrosis. Our work provides a solid new foundation for therapeutic development of FD and understanding the principles whereby Gαs signaling governs bone formation and maintenance and bone marrow stromal cell differentiation. (See pp. E418–E427.)
Expression of an active Gαs mutant in skeletal stem cells is sufficient and necessary for fibrous dysplasia initiation and maintenance
Xuefeng Zhao, Peng Deng, Ramiro Iglesias-Bartolome, Panomwat Amornphimoltham, Dana J. Steffen, Yunyun Jin, Alfredo A. Molinolo, Luis Fernandez de Castro, Diana Ovejero, Quan Yuan, Qianming Chen, Xianglong Han, Ding Bai, Susan S. Taylor, Yingzi Yang, Michael T. Collins, and J. Silvio Gutkind
We generated a novel conditional inducible mouse model of fibrous dysplasia (FD) by expressing mutant GNAS in skeletal stem cells (SSCs) in a temporally controlled and tissue-specific fashion. Typical FD bone lesions developed rapidly in mutant embryos and postnatal mice. GNAS promoted PKA activation and proliferation of SSCs along the osteogenic lineage but impaired their differentiation to mature osteoblasts and triggered increased osteoclastogenesis and bone resorption. FD lesions reverted on cessation of GNAS expression. Thus, GNAS mutation is sufficient and necessary for FD initiation and maintenance. This model provides a valuable opportunity to identify the molecular mechanism underlying FD progression and accelerate the development of more effective treatment options. (See pp. E428–E437.)
Antagonistic regulation of trafficking to Caenorhabditis elegans sensory cilia by a Retinal Degeneration 3 homolog and retromer
Luis A. Martínez-Velázquez and Niels Ringstad
Sensory neurons concentrate the molecular machinery of sensation into a specialized cellular antenna: the cilium. Proteins destined for the cilium are synthesized in the cell body, and poorly understood mechanisms separate them from other cellular proteins and target them to the cilium. Through genetic studies of this process in sensory neurons of the nematode Caenorhabditis elegans, we show that a sensory neuron-specific factor that mutates in humans to cause photoreceptor degeneration and blindness regulates an early stage in the trafficking of a key molecule for sensation. (See pp. E438–E447.)
Cyclic AMP-dependent plasticity underlies rapid changes in odor coding associated with reward learning
Thierry Louis, Aaron Stahl, Tamara Boto, and Seth M. Tomchik
Cyclic AMP signaling is involved in learning across taxa, but how cAMP modulation drives coherent plasticity across circuits is unclear. Results of this study demonstrate that cAMP-dependent plasticity in the Drosophila brain drives olfactory learning-induced plasticity, which is biased toward appetitive conditioning and modulates motivated behavior. (See pp. E448–E457.)
SRC1 promotes Th17 differentiation by overriding Foxp3 suppression to stimulate RORγt activity in a PKC-θ–dependent manner
Subha Sen, Fei Wang, Jing Zhang, Zhiheng He, Jian Ma, Yousang Gwack, Jianming Xu, and Zuoming Sun
Poor understanding of the mechanisms responsible for the development of IL-17+ and Foxp3+ cells prevents the development of potent clinical treatments to boost protective Th17 immunity and repress the pathogenic Th17 responses that induce autoimmunity. Here we show that SRC1 phosphorylated by TCR signaling kinase PKC-θ functions as a coactivator in vivo to stimulate RORγt activity by disrupting the binding of inhibitory Foxp3 and induce Foxp3 degradation. SRC1 is thus an important checkpoint downstream of TCR signals to promote the dominance of RORγt over Foxp3 to establish an unopposed Th17 differentiation program. Our results thus provide a rationale for the development of SRC1-based treatments to control the scale of Th17 immunity by reciprocal shift of Th17 and T-regulatory cell differentiation. (See pp. E458–E467.)
Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor
Michael Peled, Anna S. Tocheva, Sabina Sandigursky, Shruti Nayak, Elliot A. Philips, Kim E. Nichols, Marianne Strazza, Inbar Azoulay-Alfaguter, Manor Askenazi, Benjamin G. Neel, Adam J. Pelzek, Beatrix Ueberheide, and Adam Mor
Antibodies targeting PD-1 have elicited clinical responses in multiple tumors. Nevertheless, response to anti–PD-1 interventions is limited to a fraction of patients, and a comprehensive understanding of the signaling downstream of PD-1 could provide biomarkers for tumor response. We used affinity purification-mass spectrometry to uncover proteins associated with PD-1 and found that the adaptor protein SAP inhibits PD-1 functions by blocking the enzymatic interaction between the phosphatase SHP2 and the subset of its substrates that also bind to SAP. Signaling downstream of PD-1 in T cell subsets did not correlate with PD-1 expression but was inversely correlated with SAP levels. Thus, SAP is a modulator of PD-1 function and a potential biomarker for reduced responses to PD-1–based interventions. (See pp. E468–E477.)
Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C
Björn Ingelsson, Daniel Söderberg, Tobias Strid, Anita Söderberg, Ann-Charlotte Bergh, Vesa Loitto, Kourosh Lotfi, Mårten Segelmark, Giannis Spyrou, and Anders Rosén
Release of pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) contributes to inflammatory responses and antiviral signaling. Mitochondrial DNA (mtDNA) is a potent DAMP molecule observed in blood circulation of trauma, autoimmune, HIV, and certain cancer patients. Here, we report a previously unrecognized lymphocyte feature that CpG and non-CpG oligodeoxynucleotides of class C promptly induce release of mtDNA as extracellular web-like structures. Lymphocyte mtDNA webs provoked antiviral type I IFN production in peripheral blood mononuclear cells but were devoid of bactericidal proteins. Notably, cells remained viable after the release. Our findings imply an alternative role for lymphocytes in antiviral signaling by utilizing their mtDNA as a rapid signaling molecule to communicate danger. (See pp. E478–E487.)
Distinctive roles of age, sex, and genetics in shaping transcriptional variation of human immune responses to microbial challenges
Barbara Piasecka, Darragh Duffy, Alejandra Urrutia, Hélène Quach, Etienne Patin, Céline Posseme, Jacob Bergstedt, Bruno Charbit, Vincent Rouilly, Cameron R. MacPherson, Milena Hasan, Benoit Albaud, David Gentien, Jacques Fellay, Matthew L. Albert, Lluis Quintana-Murci, and the Milieu Intérieur Consortium
Identifying the drivers of the interindividual diversity of the human immune system is crucial to understand their consequences on immune-mediated diseases. By examining the transcriptional responses of 1,000 individuals to various microbial challenges, we show that age and sex influence the expression of many immune-related genes, but their effects are overall moderate, whereas genetic factors affect a smaller gene set but with a stronger effect. We identify numerous genetic variants that affect transcriptional variation on infection, many of which are associated with autoimmune or inflammatory disorders. These results enable additional exploration of the role of regulatory variants in the pathogenesis of immune-related diseases and improve our understanding of the respective effects of age, sex, and genetics on immune response variation. (See pp. E488–E497.)
Gene regulation and suppression of type I interferon signaling by STAT3 in diffuse large B cell lymphoma
Li Lu, Fen Zhu, Meili Zhang, Yangguang Li, Amanda C. Drennan, Shuichi Kimpara, Ian Rumball, Christopher Selzer, Hunter Cameron, Ashley Kellicut, Amanda Kelm, Fangyu Wang, Thomas A. Waldmann, and Lixin Rui
We demonstrate that STAT3 is a critical transcriptional regulator of the activated B cell–like subtype of diffuse large B cell lymphoma (ABC DLBCL), the most common, aggressive, non-Hodgkin lymphoma. By genome-wide assessment, we have identified target genes of STAT3. Gene regulation by STAT3 in ABC DLBCL accentuates survival signaling pathways while dampening the lethal type I interferon pathway. Knowledge of these STAT3-regulated genes has led to our demonstration that a small-molecule inhibitor in the JAK1-STAT3 signaling pathway synergizes with the type I interferon inducer lenalidomide, suggesting a new therapeutic strategy for ABC DLBCL, a subtype that is particularly difficult to treat and has poor prognosis. (See pp. E498–E505.)
Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite Leishmania
Danyil Grybchuk, Natalia S. Akopyants, Alexei Y. Kostygov, Aleksandras Konovalovas, Lon-Fye Lye, Deborah E. Dobson, Haroun Zangger, Nicolas Fasel, Anzhelika Butenko, Alexander O. Frolov, Jan Votýpka, Claudia M. d’Avila-Levy, Pavel Kulich, Jana Moravcová, Pavel Plevka, Igor B. Rogozin, Saulius Serva, Julius Lukeš, Stephen M. Beverley, and Vyacheslav Yurchenko
Largely overlooked, the viruses of protists have started to attract more attention. Several viruses of the family Totiviridae are currently implicated in the increased pathogenicity of parasitic protozoa such as Leishmania to vertebrate hosts. We conducted a broad survey of RNA viruses within trypanosomatids, one of the iconic groups of protists. These revealed several previously unidentified viral taxa including one designated “Leishbunyaviridae” and a highly divergent virus termed “Leptomonas pyrrhocoris ostravirus 1.” Our studies provide important information on the origins as well as the diversity and distribution of viruses within a group of protists related to the human parasite Leishmania. (See pp. E506–E515.)
Biphasic functions for the GDNF-Ret signaling pathway in chemosensory neuron development and diversification
Christopher R. Donnelly, Amol A. Shah, Charlotte M. Mistretta, Robert M. Bradley, and Brian A. Pierchala
While knowledge of signaling mechanisms orchestrating the development and diversification of peripheral somatosensory neurons is extensive, our understanding of the mechanisms controlling chemosensory neuron specification remains rudimentary. Lingually projecting sensory neurons of the geniculate ganglion are receptive to the five taste qualities, as well as temperature and tactile stimuli, but the mechanisms responsible for the diversification of the unique subpopulations that respond to one, or several, of these stimuli remain unknown. Here we demonstrate that the GDNF-Ret signaling pathway exerts a unique, dual function in peripheral taste system development and postnatal function. Ret acts embryonically to regulate the expression of the chemosensory master regulator Phox2b, thus inducing chemosensory differentiation, while postnatally acting to specify a molecularly unique subpopulation of lingual mechanoreceptors. (See pp. E516–E525.)
KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis
Lihua Zhao, Hanyang Cai, Zhenxia Su, Lulu Wang, Xinyu Huang, Man Zhang, Piaojuan Chen, Xiaozhuan Dai, Heming Zhao, Ravishankar Palanivelu, Xuemei Chen, and Yuan Qin
In flowering plants, the female germ line begins as a single cell known as the megaspore mother cell (MMC) in each ovule. The mechanisms that restrict MMC fate to a single cell remain largely unknown. We show that the Arabidopsis cytochrome P450 gene KLU acts through the chromatin remodeling complex SWR1 to promote WRKY28 expression in ovule primordia. We show that WRKY28 is expressed in a few somatic cells surrounding the MMC and is required to inhibit these cells from acquiring the MMC-like cell fate. Consistent with non–cell-autonomous KLU activity, KLU-expressing cells and WRKY28-expressing cells are neither identical nor adjacently positioned. Our study demonstrates that cell–cell interactions involving only somatic cells in ovule primordia ensure the specification of a single MMC. (See pp. E526–E535.)
Motor dexterity and strength depend upon integrity of the attention-control system
Paul Rinne, Mursyida Hassan, Cristina Fernandes, Erika Han, Emma Hennessy, Adam Waldman, Pankaj Sharma, David Soto, Robert Leech, Paresh A. Malhotra, and Paul Bentley
Simple voluntary movements (e.g., reaching or gripping) deteriorate with distraction, suggesting that the attention-control system—which suppresses distraction—influences motor control. Here, we tested the causal dependency of simple movements on attention control, and its neuroanatomical basis, in healthy elderly and patients with focal brain lesions. Not only did we find that attention control correlates with motor performance, correcting for lesion size, fatigue, etc., but we found a revealing pattern of dissociations: Severe motor impairment could occur with normal attention control whereas impaired attention control never occurred with disproportionately milder motor impairment—suggesting that attention control is required for normal motor performance. One implication is that a component of stroke paralysis arises from poor attentional control, which could itself be a therapeutic target. (See pp. E536–E545.)
Preservation of the genetic diversity of a local common carp in the agricultural heritage rice–fish system
Weizheng Ren, Liangliang Hu, Liang Guo, Jian Zhang, Lu Tang, Entao Zhang, Jiaen Zhang, Shiming Luo, Jianjun Tang, and Xin Chen
This paper contributes to understanding how traditional agriculture can maintain large genetic diversity. We quantify the effects of traditional farmer activities on the genetic diversity of an indigenous common carp in the 1,200-y-old agriculture heritage rice−fish system. We show that small farmer households interdependently incubating fish fry for their rice−fish farming shape the genetic pattern and help to maintain high genetic diversity of this local common carp. We also show how the traditional practice of mixed culturing of diverse color types potentially promotes genetic diversity. We suggest that the locally adapted ways of traditional farmers obtaining and using local genetic resources for their farming play an important role in the biodiversity of farmed crops and animals. It can become a “hotspot” for genetic diversity conservation in agriculture. (See pp. E546–E554.)