bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting global conservation funding to limit immediate biodiversity declines
Authors: Waldron Anthony , Mooers Arne O. , Miller Daniel C. , Nibbelink Nate , Redding David , Kuhn Tyler S. , Roberts J. Timmons , Gittleman John L.
Published: July 2013

Inadequate funding levels are a major impediment to effective global biodiversity conservation and are likely associated with recent failures to meet United Nations biodiversity targets. Some countries are more severely underfunded than others and therefore represent urgent financial priorities. However, attempts to identify these highly underfunded countries have been hampered for decades by poor and incomplete data on actual spending, coupled with uncertainty and lack of consensus over the relative size of spending gaps. Here, we assemble a global database of annual conservation spending. We then develop a statistical model that explains 86% of variation in conservation expenditures, and use this to identify countries where funding is robustly below expected levels. The 40 most severely underfunded countries contain 32% of all threatened mammalian diversity and include neighbors in some of the world’s most biodiversity-rich areas (Sundaland, Wallacea, and Near Oceania). However, very modest increases in international assistance would achieve a large improvement in the relative adequacy of global conservation finance. Our results could therefore be quickly applied to limit immediate biodiversity losses at relatively little cost.
Identification of a μ-δ opioid receptor heteromer-biased agonist with antinociceptive activity
Authors: Gomes Ivone , Fujita Wakako , Gupta Achla , Saldanha S. Adrian , Negri Ana , Pinello Christine E. , Eberhart Christina , Roberts Edward , Filizola Marta , Hodder Peter , Devi Lakshmi A.
Published: July 2013

G protein-coupled receptors play a pivotal role in many physiological signaling pathways. Mounting evidence suggests that G protein-coupled receptors, including opioid receptors, form dimers, and dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological role of dimerization in vivo has not been well-explored because of the lack of tools to study these dimers in endogenous systems. To address this problem, we previously generated antibodies to μ-δ opioid receptor (μOR-δOR) dimers and used them to study the pharmacology and signaling by this heteromer. We also showed that the heteromer exhibits restricted distribution in the brain and that its abundance is increased in response to chronic morphine administration. Thus, the μOR-δOR heteromer represents a potentially unique target for the development of therapeutics to treat pain. Here, we report the identification of compounds targeting μOR-δOR heteromers through high-throughput screening of a small-molecule library. These compounds exhibit activity in μOR-δOR cells but not μOR or δOR cells alone. Among them, CYM51010 was found to be a μOR-δOR–biased ligand, because its activity is blocked by the μOR-δOR heteromer antibody. Notably, systemic administration of CYM51010 induced antinociceptive activity similar to morphine, and chronic administration of CYM51010 resulted in lesser antinociceptive tolerance compared with morphine. Taken together, these results suggest that CYM51010, a μOR-δOR–biased ligand, could serve as a scaffold for the development of a unique type (heteromer-biased) of drug that is more potent and without the severe side effects associated with conventional clinical opioids.
Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate
Authors: Sokolova Ekaterina , Spruijt Evan , Hansen Maike M. K. , Dubuc Emilien , Groen Joost , Chokkalingam Venkatachalam , Piruska Aigars , Heus Hans A. , Huck Wilhelm T. S.
Published: July 2013

Liquid–liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in
Escherichia coli
cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation.
Structural and functional analysis of the yeast
N
-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism
Authors: Nasuno Ryo , Hirano Yoshinori , Itoh Takafumi , Hakoshima Toshio , Hibi Takao , Takagi Hiroshi
Published: July 2013

Mpr1 (sig
m
a1278b gene for
p
roline-analog
r
esistance 1), which was originally isolated as
N
-acetyltransferase detoxifying the proline analog
l
-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the
l
-proline and
l
-arginine metabolism by acetylating
l
-Δ
1
-pyrroline-5-carboxylate, leading to the
l
-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related
N
-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate
cis
-4-hydroxy-
l
-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent
k
cat
value. The replacement of Asn135 led to a remarkable increase in the apparent
K
m
value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the
l
-Δ
1
-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.
Anti-VEGF– and anti-VEGF receptor–induced vascular alteration in mouse healthy tissues
Authors: Yang Yunlong , Zhang Yin , Cao Ziquan , Ji Hong , Yang Xiaojuan , Iwamoto Hideki , Wahlberg Eric , Länne Toste , Sun Baocun , Cao Yihai
Published: July 2013

Systemic therapy with anti-VEGF drugs such as bevacizumab is widely used for treatment of human patients with various solid tumors. However, systemic impacts of such drugs in host healthy vasculatures remain poorly understood. Here, we show that, in mice, systemic delivery of an anti-VEGF or an anti–VEGF receptor (VEGFR)-2 neutralizing antibody caused global vascular regression. Among all examined tissues, vasculatures in endocrine glands, intestinal villi, and uterus are the most affected in response to VEGF or VEGFR-2 blockades. Thyroid vascular fenestrations were virtually completely blocked by VEGF blockade, leading to marked accumulation of intraendothelial caveolae vesicles. VEGF blockade markedly increased thyroid endothelial cell apoptosis, and withdrawal of anti-VEGF resulted in full recovery of vascular density and architecture after 14 d. Prolonged anti-VEGF treatment resulted in a significant decrease of the circulating level of the predominant thyroid hormone free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid functions. Conversely, VEGFR-1–specific blockade produced virtually no obvious phenotypes. These findings provide structural and functional bases of anti-VEGF–specific drug-induced side effects in relation to vascular changes in healthy tissues. Understanding anti-VEGF drug-induced vascular alterations in healthy tissues is crucial to minimize and even to avoid adverse effects produced by currently used anti-VEGF–specific drugs.
Bomb-curve radiocarbon measurement of recent biologic tissues and applications to wildlife forensics and stable isotope (paleo)ecology
Authors: Uno Kevin T. , Quade Jay , Fisher Daniel C. , Wittemyer George , Douglas-Hamilton Iain , Andanje Samuel , Omondi Patrick , Litoroh Moses , Cerling Thure E.
Published: July 2013

Above-ground thermonuclear weapons testing from 1952 through 1962 nearly doubled the concentration of radiocarbon (
14
C) in the atmosphere. As a result, organic material formed during or after this period may be radiocarbon-dated using the abrupt rise and steady fall of the atmospheric
14
C concentration known as the bomb-curve. We test the accuracy of accelerator mass spectrometry radiocarbon dating of 29 herbivore and plant tissues collected on known dates between 1905 and 2008 in East Africa. Herbivore samples include teeth, tusks, soft tissue, hair, and horn. Tissues formed after 1955 are dated to within 0.3–1.3 y of formation, depending on the tissue type, whereas tissues older than
ca.
1955 have high age uncertainties (>17 y) due to the Suess effect.
14
C dating of tissues has applications to stable isotope (paleo)ecology and wildlife forensics. We use data from 41 additional samples to determine growth rates of tusks, molars, and hair, which improve interpretations of serial stable isotope data for (paleo)ecological studies.
14
C dating can also be used to calculate the time interval represented in periodic histological structures in dental tissues (i.e., perikymata), which in turn may be used as chronometers in fossil teeth. Bomb-curve
14
C dating of confiscated animal tissues (e.g., ivory statues) can be used to determine whether trade of the item is legal, because many Convention of International Trade of Endangered Species restrictions are based on the age of the tissue, and thus can serve as a powerful forensic tool to combat illegal trade in animal parts.
Crystal structure and versatile functional roles of the COP9 signalosome subunit 1
Authors: Lee Jung-Hoon , Yi Lina , Li Jixi , Schweitzer Katrin , Borgmann Marc , Naumann Michael , Wu Hao
Published: July 2013

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) plays key roles in many biological processes, such as repression of photomorphogenesis in plants and protein subcellular localization, DNA-damage response, and NF-κB activation in mammals. It is an evolutionarily conserved eight-protein complex with subunits CSN1 to CSN8 named following the descending order of molecular weights. Here, we report the crystal structure of the largest CSN subunit, CSN1 from
Arabidopsis thaliana
(atCSN1), which belongs to the Proteasome, COP9 signalosome, Initiation factor 3 (PCI) domain containing CSN subunit family, at 2.7 Å resolution. In contrast to previous predictions and distinct from the PCI-containing 26S proteasome regulatory particle subunit Rpn6 structure, the atCSN1 structure reveals an overall globular fold, with four domains consisting of helical repeat-I, linker helix, helical repeat-II, and the C-terminal PCI domain. Our small-angle X-ray scattering envelope of the CSN1–CSN7 complex agrees with the EM structure of the CSN alone (apo-CSN) and suggests that the PCI end of each molecule may mediate the interaction. Fitting of the CSN1 structure into the CSN–Skp1-Cul1-Fbox (SCF) EM structure shows that the PCI domain of CSN1 situates at the hub of the CSN for interaction with several other subunits whereas the linker helix and helical repeat-II of CSN1 contacts SCF using a conserved surface patch. Furthermore, we show that, in human, the C-terminal tail of CSN1, a segment not included in our crystal structure, interacts with IκBα in the NF-κB pathway. Therefore, the CSN complex uses multiple mechanisms to hinder NF-κB activation, a principle likely to hold true for its regulation of many other targets and pathways.
Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane
Authors: Mayers Jonathan R. , Wang Lei , Pramanik Jhuma , Johnson Adam , Sarkeshik Ali , Wang Yueju , Saengsawang Witchuda , Yates John R. , Audhya Anjon
Published: July 2013

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using
Caenorhabditis elegans
, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.
Formation of hexagonal and cubic ice during low-temperature growth
Authors: Thürmer Konrad , Nie Shu
Published: July 2013

From our daily life we are familiar with hexagonal ice, but at very low temperature ice can exist in a different structure––that of cubic ice. Seeking to unravel the enigmatic relationship between these two low-pressure phases, we examined their formation on a Pt(111) substrate at low temperatures with scanning tunneling microscopy and atomic force microscopy. After completion of the one-molecule-thick wetting layer, 3D clusters of hexagonal ice grow via layer nucleation. The coalescence of these clusters creates a rich scenario of domain-boundary and screw-dislocation formation. We discovered that during subsequent growth, domain boundaries are replaced by growth spirals around screw dislocations, and that the nature of these spirals determines whether ice adopts the cubic or the hexagonal structure. Initially, most of these spirals are single, i.e., they host a screw dislocation with a Burgers vector connecting neighboring molecular planes, and produce cubic ice. Films thicker than ∼20 nm, however, are dominated by double spirals. Their abundance is surprising because they require a Burgers vector spanning two molecular-layer spacings, distorting the crystal lattice to a larger extent. We propose that these double spirals grow at the expense of the initially more common single spirals for an energetic reason: they produce hexagonal ice.
EGF shifts human airway basal cell fate toward a smoking-associated airway epithelial phenotype
Authors: Shaykhiev Renat , Zuo Wu-Lin , Chao IonWa , Fukui Tomoya , Witover Bradley , Brekman Angelika , Crystal Ronald G.
Published: July 2013

The airway epithelium of smokers acquires pathological phenotypes, including basal cell (BC) and/or goblet cell hyperplasia, squamous metaplasia, structural and functional abnormalities of ciliated cells, decreased number of secretoglobin (SCGB1A1)-expressing secretory cells, and a disordered junctional barrier. In this study, we hypothesized that smoking alters airway epithelial structure through modification of BC function via an EGF receptor (EGFR)-mediated mechanism. Analysis of the airway epithelium revealed that EGFR is enriched in airway BCs, whereas its ligand EGF is induced by smoking in ciliated cells. Exposure of BCs to EGF shifted the BC differentiation program toward the squamous and epithelial–mesenchymal transition-like phenotypes with down-regulation of genes related to ciliogenesis, secretory differentiation, and markedly reduced junctional barrier integrity, mimicking the abnormalities present in the airways of smokers in vivo. These data suggest that activation of EGFR in airway BCs by smoking-induced EGF represents a unique mechanism whereby smoking can alter airway epithelial differentiation and barrier function.
Stoichiometry of SecYEG in the active translocase of
Escherichia coli
varies with precursor species
Authors: Mao Chunfeng , Cheadle Carl E. , Hardy Simon J. S. , Lilly Angela A. , Suo Yuying , Sanganna Gari Raghavendar Reddy , King Gavin M. , Randall Linda L.
Published: July 2013

We have established a reconstitution system for the translocon SecYEG in proteoliposomes in which 55% of the accessible translocons are active. This level corresponds to the fraction of translocons that are active in vitro when assessed in their native environment of cytoplasmic membrane vesicles. Assays using these robust reconstituted proteoliposomes and cytoplasmic membrane vesicles have revealed that the number of SecYEG units involved in an active translocase depends on the precursor undergoing transfer. The active translocase for the precursor of periplasmic galactose-binding protein contains twice the number of heterotrimeric units of SecYEG as does that for the precursor of outer membrane protein A.
Mn(II,III) oxidation and MnO
2
mineralization by an expressed bacterial multicopper oxidase
Authors: Butterfield Cristina N. , Soldatova Alexandra V. , Lee Sung-Woo , Spiro Thomas G. , Tebo Bradley M.
Published: July 2013

Reactive Mn(IV) oxide minerals are ubiquitous in the environment and control the bioavailability and distribution of many toxic and essential elements and organic compounds. Their formation is thought to be dependent on microbial enzymes, because spontaneous Mn(II) to Mn(IV) oxidation is slow. Several species of marine
Bacillus
spores oxidize Mn(II) on their exosporium, the outermost layer of the spore, encrusting them with Mn(IV) oxides. Molecular studies have identified the
mnx
(Mn oxidation) genes, including
mnxG
, encoding a putative multicopper oxidase (MCO), as responsible for this two-electron oxidation, a surprising finding because MCOs only catalyze single-electron transfer reactions. Characterization of the enzymatic mechanism has been hindered by the lack of purified protein. By purifying active protein from the
mnxDEFG
expression construct, we found that the resulting enzyme is a blue (absorption maximum 590 nm) complex containing MnxE, MnxF, and MnxG proteins. Further, by analyzing the Mn(II)- and (III)-oxidizing activity in the presence of a Mn(III) chelator, pyrophosphate, we found that the complex facilitates both electron transfers from Mn(II) to Mn(III) and from Mn(III) to Mn(IV). X-ray absorption spectroscopy of the Mn mineral product confirmed its similarity to Mn(IV) oxides generated by whole spores. Our results demonstrate that Mn oxidation from soluble Mn(II) to Mn(IV) oxides is a two-step reaction catalyzed by an MCO-containing complex. With the purification of active Mn oxidase, we will be able to uncover its mechanism, broadening our understanding of Mn mineral formation and the bioinorganic capabilities of MCOs.
P
II
signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate
Authors: Radchenko Martha V. , Thornton Jeremy , Merrick Mike
Published: July 2013

P
II
proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. In all these organisms, P
II
proteins coordinate many facets of nitrogen metabolism by interacting with and regulating the activities of enzymes, transcription factors, and membrane transport proteins. The primary mode of signal perception by P
II
proteins derives from their ability to bind the effector molecules 2-oxoglutarate (2-OG) and ATP or ADP. The role of 2-OG as an indicator of cellular nitrogen status is well understood, but the function of ATP/ADP binding has remained unresolved. We have now shown that the
Escherichia coli
P
II
protein, GlnK, has an ATPase activity that is inhibited by 2-OG. Hence, when a drop in the cellular 2-OG pool signals nitrogen sufficiency, 2-OG depletion of GlnK causes bound ATP to be hydrolyzed to ADP, leading to a conformational change in the protein. We propose that the role of ATP/ADP binding in
E. coli
GlnK is to effect a 2-OG-dependent molecular switch that drives a conformational change in the T loops of the P
II
protein. We have further shown that two other P
II
proteins,
Azospirillum brasilense
GlnZ and
Arabidopsis thaliana
P
II
, have a similar ATPase activity, and we therefore suggest that this switch mechanism is likely to be a general property of most members of the P
II
protein family.
Highly penetrative, drug-loaded nanocarriers improve treatment of glioblastoma
Authors: Zhou Jiangbing , Patel Toral R. , Sirianni Rachael W. , Strohbehn Garth , Zheng Ming-Qiang , Duong Nha , Schafbauer Thomas , Huttner Anita J. , Huang Yiyun , Carson Richard E. , Zhang Ying , Sullivan David J. , Piepmeier Joseph M. , Saltzman W. Mark
Published: July 2013

Current therapy for glioblastoma multiforme is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two major advances in technology: (i) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery and (ii) repurposed compounds, previously used in Food and Drug Administration-approved products, which were identified through library screening to target BCSCs. Using fluorescence imaging and positron emission tomography, we demonstrate that brain-penetrating nanoparticles can be delivered to large intracranial volumes in both rats and pigs. We identified several agents (from Food and Drug Administration-approved products) that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by convection-enhanced delivery, one of these agents, dithiazanine iodide, significantly increased survival in rats bearing BCSC-derived xenografts. This unique approach to controlled delivery in the brain should have a significant impact on treatment of glioblastoma multiforme and suggests previously undescribed routes for drug and gene delivery to treat other diseases of the central nervous system.
Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance
Authors: Okamoto Masanori , Peterson Francis C. , Defries Andrew , Park Sang-Youl , Endo Akira , Nambara Eiji , Volkman Brian F. , Cutler Sean R.
Published: July 2013

Abscisic acid (ABA) is an essential molecule in plant abiotic stress responses. It binds to soluble pyrabactin resistance1/PYR1-like/regulatory component of ABA receptor receptors and stabilizes them in a conformation that inhibits clade A type II C protein phosphatases; this leads to downstream SnRK2 kinase activation and numerous cellular outputs. We previously described the synthetic naphthalene sulfonamide ABA agonist pyrabactin, which activates seed ABA responses but fails to trigger substantial responses in vegetative tissues in
Arabidopsis thaliana
. Here we describe quinabactin, a sulfonamide ABA agonist that preferentially activates dimeric ABA receptors and possesses ABA-like potency in vivo. In
Arabidopsis
, the transcriptional responses induced by quinabactin are highly correlated with those induced by ABA treatments. Quinabactin treatments elicit guard cell closure, suppress water loss, and promote drought tolerance in adult
Arabidopsis
and soybean plants. The effects of quinabactin are sufficiently similar to those of ABA that it is able to rescue multiple phenotypes observed in the ABA-deficient mutant
aba2
. Genetic analyses show that quinabactin’s effects in vegetative tissues are primarily mediated by dimeric ABA receptors. A PYL2-quinabactin-HAB1 X-ray crystal structure solved at 1.98-Å resolution shows that quinabactin forms a hydrogen bond with the receptor/PP2C “lock” hydrogen bond network, a structural feature absent in pyrabactin-receptor/PP2C complexes. Our results demonstrate that ABA receptors can be chemically controlled to enable plant protection against water stress and define the dimeric receptors as key targets for chemical modulation of vegetative ABA responses.
Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts
Authors: Mehrshahi Payam , Stefano Giovanni , Andaloro Joshua Michael , Brandizzi Federica , Froehlich John E. , DellaPenna Dean
Published: July 2013

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.
Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells
Authors: Stadler Sonja C. , Vincent C. Theresa , Fedorov Victor D. , Patsialou Antonia , Cherrington Brian D. , Wakshlag Joseph J. , Mohanan Sunish , Zee Barry M. , Zhang Xuesen , Garcia Benjamin A. , Condeelis John S. , Brown Anthony M. C. , Coonrod Scott A. , Allis C. David
Published: July 2013

Peptidylarginine deiminase 4 (PAD4) is a Ca
2+
-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3β (GSK3β), both in vitro and in vivo. Among other functions, GSK3β is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3β protein levels, increased TGF-β signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3β is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3β is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.
Improved El Niño forecasting by cooperativity detection
Authors: Ludescher Josef , Gozolchiani Avi , Bogachev Mikhail I. , Bunde Armin , Havlin Shlomo , Schellnhuber Hans Joachim
Published: July 2013

Although anomalous episodic warming of the eastern equatorial Pacific, dubbed El Niño by Peruvian fishermen, has major (and occasionally devastating) impacts around the globe, robust forecasting is still limited to about 6 mo ahead. A significant extension of the prewarning time would be instrumental for avoiding some of the worst damages such as harvest failures in developing countries. Here we introduce a unique avenue toward El Niño prediction based on network methods, inspecting emerging teleconnections. Our approach starts from the evidence that a large-scale cooperative mode—linking the El Niño basin (equatorial Pacific corridor) and the rest of the ocean—builds up in the calendar year before the warming event. On this basis, we can develop an efficient 12-mo forecasting scheme, i.e., achieve some doubling of the early-warning period. Our method is based on high-quality observational data available since 1950 and yields hit rates above 0.5, whereas false-alarm rates are below 0.1.
Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process
Authors: Wu Danielle , Schaffler Mitchell B. , Weinbaum Sheldon , Spray David C.
Published: July 2013

Osteocytes are bone cells that form cellular networks that sense mechanical loads distributed throughout the bone tissue. Interstitial fluid flow in the lacunar canalicular system produces focal strains at localized attachment sites around the osteocyte cell process. These regions of periodic attachment between the osteocyte cell membrane and its canalicular wall are sites where pN-level fluid-flow induced forces are generated in vivo. In this study, we show that focally applied forces of this magnitude using a newly developed Stokesian fluid stimulus probe initiate rapid and transient intercellular electrical signals in vitro. Our experiments demonstrate both direct gap junction coupling and extracellular purinergic P2 receptor signaling between MLO-Y4 cells in a connected bone cell network. Intercellular signaling was initiated by pN-level forces applied at integrin attachment sites along both appositional and distal unapposed cell processes, but not initiated at their cell bodies with equivalent forces. Electrical coupling was evident in 58% of all cell pairs tested with appositional connections; coupling strength increased with the increasing number of junctional connections. Apyrase, a nucleotide-degrading enzyme, suppressed and abolished force-induced effector responses, indicating a contribution from ATP released by the stimulated cell. This work extends the understanding of how osteocytes modulate their microenvironment in response to mechanical signals and highlights mechanisms of intercellular relay of mechanoresponsive signals in the bone network.
Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca
2+
release and contractility
Authors: Nelson Benjamin R. , Wu Fenfen , Liu Yun , Anderson Douglas M. , McAnally John , Lin Weichun , Cannon Stephen C. , Bassel-Duby Rhonda , Olson Eric N.
Published: July 2013

Excitation–contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca
2+
transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca
2+
release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (
Stac3
) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca
2+
release from the SR of cultured myotubes from
Stac3
mutant mice could be restored by application of 4-chloro-
m
-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca
2+
store, and ryanodine receptors are functional in
Stac3
mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.
Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression
Authors: Li Huang , Wang Zhijia , Zhou Xuexia , Cheng Yuanming , Xie Zhiqin , Manley James L. , Feng Ying
Published: July 2013

Significance

Splicing of mRNA precursors occurs in two sequential transesterification steps. We characterize a highly unusual inhibition of splicing in which the reaction is blocked between the two steps. We demonstrate that RNA secondary structure and an exonic splicing silencer element (ESS) can independently mediate second-step splicing repression in vitro and cause exon exclusion in vivo. Importantly, we provide evidence that far upstream element-binding protein 1, a single-stranded DNA- and RNA-binding protein initially identified as a regulator of
MYC
transcription and recently implicated in several cancers, binds the ESS and functions as a splicing regulator in vitro and in vivo.
Nutrient enrichment, biodiversity loss, and consequent declines in ecosystem productivity
Authors: Isbell Forest , Reich Peter B. , Tilman David , Hobbie Sarah E. , Polasky Stephen , Binder Seth
Published: July 2013

Anthropogenic drivers of environmental change often have multiple effects, including changes in biodiversity, species composition, and ecosystem functioning. It remains unknown whether such shifts in biodiversity and species composition may, themselves, be major contributors to the total, long-term impacts of anthropogenic drivers on ecosystem functioning. Moreover, although numerous experiments have shown that random losses of species impact the functioning of ecosystems, human-caused losses of biodiversity are rarely random. Here we use results from long-term grassland field experiments to test for direct effects of chronic nutrient enrichment on ecosystem productivity, and for indirect effects of enrichment on productivity mediated by resultant species losses. We found that ecosystem productivity decreased through time most in plots that lost the most species. Chronic nitrogen addition also led to the nonrandom loss of initially dominant native perennial C
4
grasses. This loss of dominant plant species was associated with twice as great a loss of productivity per lost species than occurred with random species loss in a nearby biodiversity experiment. Thus, although chronic nitrogen enrichment initially increased productivity, it also led to loss of plant species, including initially dominant species, which then caused substantial diminishing returns from nitrogen fertilization. In contrast, elevated CO
2
did not decrease grassland plant diversity, and it consistently promoted productivity over time. Our results support the hypothesis that the long-term impacts of anthropogenic drivers of environmental change on ecosystem functioning can strongly depend on how such drivers gradually decrease biodiversity and restructure communities.
The Effect of Visual Degradation on Anticipatory and Compensatory Steering Control
Authors: Frissen Ilja , Mars Franck
Published: July 2013

It has long been held that steering a vehicle is subserved by two distinct visual processes, a compensatory one for maintaining lane position and an anticipatory one for previewing the curvature of the upcoming road. In this study, we investigated the robustness of these two steering control processes by systematically degrading their visual inputs. Performance was measured at the level of vehicle position and at the level of the actions on the steering wheel. The results show that the compensatory process is more robust to visual degradation than the anticipatory process. The results are also consistent with the idea that steering is under the supervision of a combination of compensatory and anticipatory mechanisms, although they suggest that the quality of the sensory information will determine how information is combined.
Lysosome-mediated processing of chromatin in senescence
Authors: Ivanov Andre , Pawlikowski Jeff , Manoharan Indrani , van Tuyn John , Nelson David M. , Rai Taranjit Singh , Shah Parisha P. , Hewitt Graeme , Korolchuk Viktor I. , Passos Joao F. , Wu Hong , Berger Shelley L. , Adams Peter D.
Published: July 2013

Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly γ-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.
The Bishop-Phelps-Bollobás Theorem for bilinear forms
Authors: Acosta María , Becerra-Guerrero Julio , García Domingo , Maestre Manuel
Published: July 2013

In this paper we provide versions of the Bishop-Phelps-Bollobás Theorem for bilinear forms. Indeed we prove the first positive result of this kind by assuming uniform convexity on the Banach spaces. A characterization of the Banach space

Y
Y

satisfying a version of the Bishop-Phelps-Bollobás Theorem for bilinear forms on

ℓ
1

×
Y

\ell _1 \times Y

is also obtained. As a consequence of this characterization, we obtain positive results for finite-dimensional normed spaces, uniformly smooth spaces, the space

C

(
K
)

\mathcal {C}(K)

of continuous functions on a compact Hausdorff topological space

K
K

and the space

K
(
H
)

K(H)

of compact operators on a Hilbert space

H
H

. On the other hand, the Bishop-Phelps-Bollobás Theorem for bilinear forms on

ℓ
1

×

L
1

(
μ
)

\ell _1 \times L_1 (\mu )

fails for any infinite-dimensional

L
1

(
μ
)

L_1 (\mu )

, a result that was known only when

L
1

(
μ
)
=

ℓ
1

L_1 (\mu ) = \ell _1

.
Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin
Authors: Howell Bonnie , Larsson Niklas , Gullberg Martin , Cassimeris Lynne
Published: July 2013

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.
Unusual Centrosome Cycle inDictyostelium: Correlation of Dynamic Behavior and Structural Changes
Authors: Ueda Masahiro , Schliwa Manfred , Euteneuer Ursula
Published: July 2013

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a γ-tubulin–green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyosteliumcentrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G1/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of γ-tubulin–green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.
Birefringence Imaging Directly Reveals Architectural Dynamics of Filamentous Actin in Living Growth Cones
Authors: Katoh Kaoru , Hammar Katherine , Smith Peter J. S. , Oldenbourg Rudolf
Published: July 2013

We have investigated the dynamic behavior of cytoskeletal fine structure in the lamellipodium of nerve growth cones using a new type of polarized light microscope (the Pol-Scope). Pol-Scope images display with exquisite resolution and definition birefringent fine structures, such as filaments and membranes, without having to treat the cell with exogenous dyes or fluorescent labels. Furthermore, the measured birefringence of protein fibers in the thin lamellipodial region can be interpreted in terms of the number of filaments in the bundles. We confirmed that birefringent fibers are actin-based using conventional fluorescence-labeling methods. By recording movies of time-lapsed Pol-Scope images, we analyzed the creation and dynamic composition of radial fibers, filopodia, and intrapodia in advancing growth cones. The strictly quantitative information available in time-lapsed Pol-Scope images confirms previously deduced behavior and provides new insight into the architectural dynamics of filamentous actin.
Disruption of a Dynamin Homologue Affects Endocytosis, Organelle Morphology, and Cytokinesis inDictyostelium discoideum
Authors: Wienke Dirk C. , Knetsch Menno L.W. , Neuhaus Eva M. , Reedy Mary C. , Manstein Dietmar J.
Published: July 2013

The identification and functional characterization ofDictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.