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Biophysics
Nrp2 is sufficient to instruct circuit formation of mitral-cells to mediate odour-induced attractive social responses
Jul 21, 2017   Nature Communications
Inokuchi K, Imamura F, Takeuchi H, Kim R, Okuno H, Nishizumi H, Bito H, Kikusui T, Sakano H
Nrp2 is sufficient to instruct circuit formation of mitral-cells to mediate odour-induced attractive social responses
Jul 21, 2017
Nature Communications
Odour information induces various innate responses that are critical to the survival of the individual and for the species. An axon guidance molecule, Neuropilin 2 (Nrp2), is known to mediate targeting of olfactory sensory neurons (primary neurons), to the posteroventral main olfactory bulb (PV MOB) in mice. Here we report that Nrp2-positive (Nrp2+) mitral cells (MCs, second-order neurons) play crucial roles in transmitting attractive social signals from the PV MOB to the anterior part of medial amygdala (MeA). Semaphorin 3F, a repulsive ligand to Nrp2, regulates both migration of Nrp2+ MCs to the PV MOB and their axonal projection to the anterior MeA. In the MC-specific Nrp2 knockout mice, circuit formation of Nrp2+ MCs and odour-induced attractive social responses are impaired. In utero, electroporation demonstrates that activation of the Nrp2 gene in MCs is sufficient to instruct their circuit formation from the PV MOB to the anterior MeA.
Estimating relative mitochondrial DNA copy number using high throughput sequencing data
Jul 23, 2017   Genomics
Zhang P, Lehmann BD, Samuels DC, Zhao S, Zhao YY, Shyr Y, Guo Y
Estimating relative mitochondrial DNA copy number using high throughput sequencing data
Jul 23, 2017
Genomics
We hypothesize that the relative mitochondria copy number (MTCN) can be estimated by comparing the abundance of mitochondrial DNA to nuclear DNA reads using high throughput sequencing data. To test this hypothesis, we examined relative MTCN across 13 breast cancer cell lines using the RT-PCR based NovaQUANT Human Mitochondrial to Nuclear DNA Ratio Kit as the gold standard. Six distinct computational approaches were used to estimate the relative MTCN in order to compare to the RT-PCR measurements. The results demonstrate that relative MTCN correlates well with the RT-PCR measurements using exome sequencing data, but not RNA-seq data. Through analysis of copy number variants (CNVs) in The Cancer Genome Atlas, we show that the two nuclear genes used in the NovaQUANT assay to represent the nuclear genome often experience CNVs in tumor cells, questioning the accuracy of this gold-standard method when it is applied to tumor cells. Copyright © 2017. Published by Elsevier Inc.
Channel opening and gating mechanism in AMPA-subtype glutamate receptors
Jul 24, 2017   Nature Add nature.com free-link Cancel
Twomey EC, Yelshanskaya MV, Grassucci RA, Frank J, Sobolevsky AI
Channel opening and gating mechanism in AMPA-subtype glutamate receptors
Jul 24, 2017
Nature
AMPA-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the neurotransmitter glutamate, AMPA receptors are critical for synaptic strength and dysregulation of AMPA receptor-mediated signalling is linked to numerous neurological diseases. Here, we use cryo-electron microscopy to solve the structures of AMPA receptor-auxiliary subunit complexes in the apo, antagonist and agonist-bound states and elucidate the iris-like mechanism of ion channel opening. The ion channel selectivity filter is formed by the extended portions of the re-entrant M2 loops, while the helical portions of M2 contribute to extensive hydrophobic interfaces between AMPA receptor subunits in the ion channel. We show how the permeation pathway changes upon channel opening and identify conformational changes throughout the entire AMPA receptor that accompany activation and desensitization. Our findings provide a framework for understanding gating across the family of ionotropic glutamate receptors and the role of AMPA receptors in excitatory neurotransmission.
Identifying Risk for Acute Kidney Injury in Infants and Children Following Cardiac Arrest
Jul 24, 2017   Pediatric Critical Care Medicine : A Journal Of The Society Of Critical Care Medicine And The World Federation Of Pediatric Intensive And Critical Care Societies
Neumayr TM, Gill J, Fitzgerald JC, Gazit AZ, Pineda JA, Berg RA, Dean JM, Moler FW, Doctor A
Identifying Risk for Acute Kidney Injury in Infants and Children Following Cardiac Arrest
Jul 24, 2017
Pediatric Critical Care Medicine : A Journal Of The Society Of Critical Care Medicine And The World Federation Of Pediatric Intensive And Critical Care Societies
Our goal was to identify risk factors for acute kidney injury in children surviving cardiac arrest. Retrospective analysis of a public access dataset. Fifteen children's hospitals associated with the Pediatric Emergency Care Applied Research Network. Two hundred ninety-six subjects between 1 day and 18 years old who experienced in-hospital or out-of-hospital cardiac arrest between July 1, 2003, and December 31, 2004. None. Our primary outcome was development of acute kidney injury as defined by the Acute Kidney Injury Network criteria. An ordinal probit model was developed. We found six critical explanatory variables, including total number of epinephrine doses, postcardiac arrest blood pressure, arrest location, presence of a chronic lung condition, pH, and presence of an abnormal baseline creatinine. Total number of epinephrine doses received as well as rate of epinephrine dosing impacted acute kidney injury risk and severity of acute kidney injury. This study is the first to identify risk factors for acute kidney injury in children after cardiac arrest. Our findings regarding the impact of epinephrine dosing are of particular interest and suggest potential for epinephrine toxicity with regard to acute kidney injury. The ability to identify and potentially modify risk factors for acute kidney injury after cardiac arrest may lead to improved morbidity and mortality in this population.
Can time-resolved NIRS provide the sensitivity to detect brain activity during motor imagery consistently?
Jul 24, 2017   Biomedical Optics Express
Abdalmalak A, Milej D, Diop M, Shokouhi M, Naci L, Owen AM, St Lawrence K
Can time-resolved NIRS provide the sensitivity to detect brain activity during motor imagery consistently?
Jul 24, 2017
Biomedical Optics Express
Previous functional magnetic resonance imaging (fMRI) studies have shown that a subgroup of patients diagnosed as being in a vegetative state are aware and able to communicate by performing a motor imagery task in response to commands. Due to the fMRI's cost and accessibility, there is a need for exploring different imaging modalities that can be used at the bedside. A promising technique is functional near infrared spectroscopy (fNIRS) that has been successfully applied to measure brain oxygenation in humans. Due to the limited depth sensitivity of continuous-wave NIRS, time-resolved (TR) detection has been proposed as a way of enhancing the sensitivity to the brain, since late arriving photons have a higher probability of reaching the brain. The goal of this study was to assess the feasibility and sensitivity of TR fNIRS in detecting brain activity during motor imagery. Fifteen healthy subjects were recruited in this study, and the fNIRS results were validated using fMRI. The change in the statistical moments of the distribution of times of flight (number of photons, mean time of flight and variance) were calculated for each channel to determine the presence of brain activity. The results indicate up to an 86% agreement between fMRI and TR-fNIRS and the sensitivity ranging from 64 to 93% with the highest value determined for the mean time of flight. These promising results highlight the potential of TR-fNIRS as a portable brain computer interface for patients with disorder of consciousness.
BdCIPK31, a Calcineurin B-Like Protein-Interacting Protein Kinase, Regulates Plant Response to Drought and Salt Stress
Jul 24, 2017   Frontiers In Plant Science
Luo Q, Wei Q, Wang R, Zhang Y, Zhang F, He Y, Zhou S, Feng J, Yang G, He G
BdCIPK31, a Calcineurin B-Like Protein-Interacting Protein Kinase, Regulates Plant Response to Drought and Salt Stress
Jul 24, 2017
Frontiers In Plant Science
Calcineurin B-like protein interacting protein kinases (CIPKs) are vital elements in plant abiotic stress signaling pathways. However, the functional mechanism of CIPKs has not been understood clearly, especially in Brachypodium distachyon, a new monocot model plant. In this study, BdCIPK31, a CIPK gene from B. distachyon was characterized. BdCIPK31 was downregulated by polyethylene glycol, NaCl, H2O2, and abscisic acid (ABA) treatments. Transgenic tobacco plants overexpressing BdCIPK31 presented improved drought and salt tolerance, and displayed hypersensitive response to exogenous ABA. Further investigations revealed that BdCIPK31 functioned positively in ABA-mediated stomatal closure, and transgenic tobacco exhibited reduced water loss under dehydration conditions compared with the controls. BdCIPK31 also affected Na+/K+ homeostasis and root K+ loss, which contributed to maintain intracellular ion homeostasis under salt conditions. Moreover, the reactive oxygen species scavenging system and osmolyte accumulation were enhanced by BdCIPK31 overexpression, which were conducive for alleviating oxidative and osmotic damages. Additionally, overexpression of BdCIPK31 could elevate several stress-associated gene expressions under stress conditions. In conclusion, BdCIPK31 functions positively to drought and salt stress through ABA signaling pathway. Overexpressing BdCIPK31 functions in stomatal closure, ion homeostasis, ROS scavenging, osmolyte biosynthesis, and transcriptional regulation of stress-related genes.
Soluble epoxide hydrolase activation by S-nitrosation contributes to cardiac ischemia-reperfusion injury
Jul 24, 2017   Journal Of Molecular And Cellular Cardiology
Ding Y, Li Y, Zhang X, He J, Lu D, Fang X, Wang Y, Wang J, Zhang Y, Qiao X, Gan LM, Chen C, Zhu Y
Soluble epoxide hydrolase activation by S-nitrosation contributes to cardiac ischemia-reperfusion injury
Jul 24, 2017
Journal Of Molecular And Cellular Cardiology
Cardiac ischemia-reperfusion (I/R) injury always accompanies recanalization treatment for myocardial infarction. Here we found soluble epoxide hydrolase (sEH), which metabolizes cardioprotective epoxyeicosatrienoic acids into less effective diols, was rapidly activated during myocardial reperfusion in both mouse and rat models in expression-independent manner. Similar activation was mimicked by nitric oxide (NO) donor dose-dependently in vitro, along with an obvious induction of sEH S-nitrosation, a short-term post-translational modification, which diminished in sEH Cys-141-Ala mutant. In vivo, I/R induced sEH S-nitrosation could be reversed by NO synthase inhibitor L-NAME, with protective effect on cardiac dysfunction, which however vanished in sEH-/- mice. Further, a protective effect against I/R injury in the initial phase of reperfusion was observed in eNOS-/- mice, indicating inhibition of NO as a sEH-based cardioprotective in early time of I/R injury. Besides, sEH inhibitor directly targeting on activated sEH during cardiac reperfusion significant reduced infarct size after I/R in vivo. In summary, our findings show the critical role of sEH S-nitrosation in cardiac I/R injury and inhibiting sEH S-nitrosation may be a new therapeutic strategy clinically. Copyright © 2017. Published by Elsevier Ltd.
High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain
Jul 24, 2017   Cell Stem Cell
Zhou T, Tan L, Cederquist GY, Fan Y, Hartley BJ, Mukherjee S, Tomishima M, Brennand KJ, Zhang Q, Schwartz RE, Evans T, Studer L, Chen S
High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates that Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain
Jul 24, 2017
Cell Stem Cell
Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients. Copyright © 2017 Elsevier Inc. All rights reserved.
Crystal Structure of the C-terminal Domain of Human eIF2D and its Implications on Eukaryotic Translation Initiation
Jul 24, 2017   Journal Of Molecular Biology
Vaidya AT, Lomakin IB, Joseph NN, Dmitriev SE, Steitz TA
Crystal Structure of the C-terminal Domain of Human eIF2D and its Implications on Eukaryotic Translation Initiation
Jul 24, 2017
Journal Of Molecular Biology
Protein synthesis is a key process in all living organisms. In eukaryotes, Initiation Factor 2 (eIF2) plays an important role in translation initiation as it selects and delivers the initiator tRNA to the small ribosomal subunit. Under stress conditions, phosphorylation of the α-subunit of eIF2 downregulates cellular protein synthesis. However, translation of certain mRNAs continues via the eIF2D dependent non-canonical initiation pathway. The molecular mechanism of this process remains elusive. In addition, eIF2D plays a role in translation re-initiation and ribosome recycling. Currently, there has been no structural information of eIF2D. We have now determined the crystal structure of the C-terminal domains of eIF2D at 1.4Å resolution. One domain has the fold similar to that of eIF1, which is crucial for the scanning and initiation codon selection. The second domain has a known SWIB/MDM2 fold, which was not observed before in other translation initiation factors. Our structure reveals atomic details of inter-domain interactions in the C-terminal part of eIF2D and sheds light on the possible role of these domains in eIF2D during translation. Copyright © 2017. Published by Elsevier Ltd.
Conformational Changes of CFTR upon Phosphorylation and ATP Binding
Jul 24, 2017   Cell
Zhang Z, Liu F, Chen J
Conformational Changes of CFTR upon Phosphorylation and ATP Binding
Jul 24, 2017
Cell
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from an ATP-binding cassette transporter. CFTR channel gating is strictly coupled to phosphorylation and ATP hydrolysis. Previously, we reported essentially identical structures of zebrafish and human CFTR in the dephosphorylated, ATP-free form. Here, we present the structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to 3.4 Å resolution. Comparison of the two conformations shows major structural rearrangements leading to channel opening. The phosphorylated regulatory domain is disengaged from its inhibitory position; the nucleotide-binding domains (NBDs) form a "head-to-tail" dimer upon binding ATP; and the cytoplasmic pathway, found closed off in other ATP-binding cassette transporters, is cracked open, consistent with CFTR's unique channel function. Unexpectedly, the extracellular mouth of the ion pore remains closed, indicating that local movements of the transmembrane helices can control ion access to the pore even in the NBD-dimerized conformation. Copyright © 2017 Elsevier Inc. All rights reserved.
Behaviorally Selective Engagement of Short-Latency Effector Pathways by Motor Cortex
Jul 24, 2017   Neuron
Miri A, Warriner CL, Seely JS, Elsayed GF, Cunningham JP, Churchland MM, Jessell TM
Behaviorally Selective Engagement of Short-Latency Effector Pathways by Motor Cortex
Jul 24, 2017
Neuron
Blocking motor cortical output with lesions or pharmacological inactivation has identified movements that require motor cortex. Yet, when and how motor cortex influences muscle activity during movement execution remains unresolved. We addressed this ambiguity using measurement and perturbation of motor cortical activity together with electromyography in mice during two forelimb movements that differ in their requirement for cortical involvement. Rapid optogenetic silencing and electrical stimulation indicated that short-latency pathways linking motor cortex with spinal motor neurons are selectively activated during one behavior. Analysis of motor cortical activity revealed a dramatic change between behaviors in the coordination of firing patterns across neurons that could account for this differential influence. Thus, our results suggest that changes in motor cortical output patterns enable a behaviorally selective engagement of short-latency effector pathways. The model of motor cortical influence implied by our findings helps reconcile previous observations on the function of motor cortex. Copyright © 2017 Elsevier Inc. All rights reserved.
Modeling RNA Secondary Structure with Sequence Comparison and Experimental Mapping Data
Jul 24, 2017   Biophysical Journal
Tan Z, Sharma G, Mathews DH
Modeling RNA Secondary Structure with Sequence Comparison and Experimental Mapping Data
Jul 24, 2017
Biophysical Journal
Secondary structure prediction is an important problem in RNA bioinformatics because knowledge of structure is critical to understanding the functions of RNA sequences. Significant improvements in prediction accuracy have recently been demonstrated though the incorporation of experimentally obtained structural information, for instance using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) mapping. However, such mapping data is currently available only for a limited number of RNA sequences. In this article, we present a method for extending the benefit of experimental mapping data in secondary structure prediction to homologous sequences. Specifically, we propose a method for integrating experimental mapping data into a comparative sequence analysis algorithm for secondary structure prediction of multiple homologs, whereby the mapping data benefits not only the prediction for the specific sequence that was mapped but also other homologs. The proposed method is realized by modifying the TurboFold II algorithm for prediction of RNA secondary structures to utilize basepairing probabilities guided by SHAPE experimental data when such data are available. The SHAPE-mapping-guided basepairing probabilities are obtained using the RSample method. Results demonstrate that the SHAPE mapping data for a sequence improves structure prediction accuracy of other homologous sequences beyond the accuracy obtained by sequence comparison alone (TurboFold II). The updated version of TurboFold II is freely available as part of the RNAstructure software package. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Analysis of RNA-protein interactions in vertebrate embryos using UV crosslinking approaches
Jul 23, 2017   Methods (San Diego, Calif.)
Despic V, Dejung M, Butter F, Neugebauer KM
Analysis of RNA-protein interactions in vertebrate embryos using UV crosslinking approaches
Jul 23, 2017
Methods (San Diego, Calif.)
A decade ago, we believed that at least 300 RNA binding proteins (RBPs) were encoded in our genomes based on annotations of known or predicted RNA binding domains. Deciphering the roles of those RBPs in regulated gene expression was a vast frontier awaiting exploration. Since then, the field has developed a number of key tools that navigate the landscape of cellular RNA. These rely principally on UV crosslinking to create covalent bonds between RBPs and target RNAs in vivo, revealing not only target identities but also local binding sites upon RNA-Seq. More recently, a reverse protocol - mRNA interactome capture - has enabled the identification of the proteins that interact with mRNA. Astonishingly, the number of RBPs has grown to more than 1,000, and we must now understand what they do. Here, we discuss the application of these methods to model organisms, focusing on the zebrafish Danio rerio, which provide unique biological contexts for the analysis of RBPs and their functions. Copyright © 2017. Published by Elsevier Inc.
Cryo-EM structure and biochemical analysis reveal the basis of the functional difference between human PI3KC3-C1 and -C2
Jul 21, 2017   Cell Research
Ma M, Liu JJ, Li Y, Huang Y, Ta N, Chen Y, Fu H, Ye MD, Ding Y, Huang W, Wang J, Dong MQ, Yu L, Wang HW
Cryo-EM structure and biochemical analysis reveal the basis of the functional difference between human PI3KC3-C1 and -C2
Jul 21, 2017
Cell Research
Phosphatidylinositol 3-phosphate (PI3P) plays essential roles in vesicular trafficking, organelle biogenesis and autophagy. Two class III phosphatidylinositol 3-kinase (PI3KC3) complexes have been identified in mammals, the ATG14L complex (PI3KC3-C1) and the UVRAG complex (PI3KC3-C2). PI3KC3-C1 is crucial for autophagosome biogenesis, and PI3KC3-C2 is involved in various membrane trafficking events. Here we report the cryo-EM structures of human PI3KC3-C1 and PI3KC3-C2 at sub-nanometer resolution. The two structures share a common L-shaped overall architecture with distinct features. EM examination revealed that PI3KC3-C1 "stands up" on lipid monolayers, with the ATG14L BATs domain and the VPS34 C-terminal domain (CTD) directly contacting the membrane. Biochemical dissection indicated that the ATG14L BATs domain is responsible for membrane anchoring, whereas the CTD of VPS34 determines the orientation. Furthermore, PI3KC3-C2 binds much more weakly than PI3KC3-C1 to both PI-containing liposomes and purified endoplasmic reticulum (ER) vesicles, a property that is specifically determined by the ATG14L BATs domain. The in vivo ER localization analysis indicated that the BATs domain was required for ER localization of PI3KC3. We propose that the different lipid binding capacity is the key factor that differentiates the functions of PI3KC3-C1 and PI3KC3-C2 in autophagy.Cell Research advance online publication 21 July 2017; doi:10.1038/cr.2017.94.
Crystal structure of a low molecular weight activator Blm-pep with yeast 20S proteasome - insights into the enzyme activation mechanism
Jul 22, 2017   Scientific Reports
Witkowska J, Giżyńska M, Grudnik P, Golik P, Karpowicz P, Giełdoń A, Dubin G, Jankowska E
Crystal structure of a low molecular weight activator Blm-pep with yeast 20S proteasome - insights into the enzyme activation mechanism
Jul 22, 2017
Scientific Reports
Proteasomes are responsible for protein turnover in eukaryotic cells, degrading short-lived species but also removing improperly folded or oxidatively damaged ones. Dysfunction of a proteasome results in gradual accumulation of misfolded/damaged proteins, leading to their aggregation. It has been postulated that proteasome activators may facilitate removal of such aggregation-prone proteins and thus prevent development of neurodegenerative disorders. However, the discovery of pharmacologically relevant compounds is hindered by insufficient structural understanding of the activation process. In this study we provide a model peptidic activator of human proteasome and analyze the structure-activity relationship within this novel scaffold. The binding mode of the activator at the relevant pocket within the proteasome has been determined by X-ray crystallography. This crystal structure provides an important basis for rational design of pharmacological compounds. Moreover, by providing a novel insight into the proteasome gating mechanism, our results allow the commonly accepted model of proteasome regulation to be revisited.
Cellular interfaces with hydrogen-bonded organic semiconductor hierarchical nanocrystals
Jul 22, 2017   Nature Communications
Sytnyk M, Jakešová M, Litviňuková M, Mashkov O, Kriegner D, Stangl J, Nebesářová J, Fecher FW, Schöfberger W, Sariciftci NS, Schindl R, Heiss W, Głowacki ED
Cellular interfaces with hydrogen-bonded organic semiconductor hierarchical nanocrystals
Jul 22, 2017
Nature Communications
Successful formation of electronic interfaces between living cells and semiconductors hinges on being able to obtain an extremely close and high surface-area contact, which preserves both cell viability and semiconductor performance. To accomplish this, we introduce organic semiconductor assemblies consisting of a hierarchical arrangement of nanocrystals. These are synthesised via a colloidal chemical route that transforms the nontoxic commercial pigment quinacridone into various biomimetic three-dimensional arrangements of nanocrystals. Through a tuning of parameters such as precursor concentration, ligands and additives, we obtain complex size and shape control at room temperature. We elaborate hedgehog-shaped crystals comprising nanoscale needles or daggers that form intimate interfaces with the cell membrane, minimising the cleft with single cells without apparent detriment to viability. Excitation of such interfaces with light leads to effective cellular photostimulation. We find reversible light-induced conductance changes in ion-selective or temperature-gated channels.Nanomaterials that form a bioelectronic interface with cells are fascinating tools for controlling cellular behavior. Here, the authors photostimulate single cells with spiky assemblies of semiconducting quinacridone nanocrystals, whose nanoscale needles maximize electronic contact with the cells.
CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells
Jul 22, 2017   Scientific Reports
Ziegler S, Weiss E, Schmitt AL, Schlegel J, Burgert A, Terpitz U, Sauer M, Moretta L, Sivori S, Leonhardt I, Kurzai O, Einsele H, Loeffler J
CD56 Is a Pathogen Recognition Receptor on Human Natural Killer Cells
Jul 22, 2017
Scientific Reports
Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response.
Structural and Functional Insights into Human Re-initiation Complexes
Jul 22, 2017   Molecular Cell
Weisser M, Schäfer T, Leibundgut M, Böhringer D, Aylett CHS, Ban N
Structural and Functional Insights into Human Re-initiation Complexes
Jul 22, 2017
Molecular Cell
After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA. Copyright © 2017 Elsevier Inc. All rights reserved.
CRISPulator: a discrete simulation tool for pooled genetic screens
Jul 22, 2017   BMC Bioinformatics
Nagy T, Kampmann M
CRISPulator: a discrete simulation tool for pooled genetic screens
Jul 22, 2017
BMC Bioinformatics
The rapid adoption of CRISPR technology has enabled biomedical researchers to conduct CRISPR-based genetic screens in a pooled format. The quality of results from such screens is heavily dependent on the selection of optimal screen design parameters, which also affects cost and scalability. However, the cost and effort of implementing pooled screens prohibits experimental testing of a large number of parameters. We present CRISPulator, a Monte Carlo method-based computational tool that simulates the impact of screen parameters on the robustness of screen results, thereby enabling users to build intuition and insights that will inform their experimental strategy. CRISPulator enables the simulation of screens relying on either CRISPR interference (CRISPRi) or CRISPR nuclease (CRISPRn). Pooled screens based on cell growth/survival, as well as fluorescence-activated cell sorting according to fluorescent reporter phenotypes are supported. CRISPulator is freely available online ( http://crispulator.ucsf.edu ). CRISPulator facilitates the design of pooled genetic screens by enabling the exploration of a large space of experimental parameters in silico, rather than through costly experimental trial and error. We illustrate its power by deriving non-obvious rules for optimal screen design.
Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination
Jul 21, 2017   DNA Repair
Orta ML, Pastor N, Burgos-Morón E, Domínguez I, Calderón-Montaño JM, Huertas Castaño C, López-Lázaro M, Helleday T, Mateos S
Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination
Jul 21, 2017
DNA Repair
Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It's well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR. Copyright © 2017 Elsevier B.V. All rights reserved.
Increased ParB level affects expression of stress response, adaptation and virulence operons and potentiates repression of promoters adjacent to the high affinity binding sites parS3 and parS4 in Pseudomonas aeruginosa
Jul 21, 2017   PloS One
Kawalek A, Glabski K, Bartosik AA, Fogtman A, Jagura-Burdzy G
Increased ParB level affects expression of stress response, adaptation and virulence operons and potentiates repression of promoters adjacent to the high affinity binding sites parS3 and parS4 in Pseudomonas aeruginosa
Jul 21, 2017
PloS One
Similarly to its homologs in other bacteria, Pseudomonas aeruginosa partitioning protein ParB facilitates segregation of newly replicated chromosomes. Lack of ParB is not lethal but results in increased frequency of anucleate cells production, longer division time, cell elongation, altered colony morphology and defective swarming and swimming motility. Unlike in other bacteria, inactivation of parB leads to major changes of the transcriptome, suggesting that, directly or indirectly, ParB plays a role in regulation of gene expression in this organism. ParB overproduction affects growth rate, cell division and motility in a similar way as ParB deficiency. To identify primary ParB targets, here we analysed the impact of a slight increase in ParB level on P. aeruginosa transcriptome. ParB excess, which does not cause changes in growth rate and chromosome segregation, significantly alters the expression of 176 loci. Most notably, the mRNA level of genes adjacent to high affinity ParB binding sites parS1-4 close to oriC is reduced. Conversely, in cells lacking either parB or functional parS sequences the orfs adjacent to parS3 and parS4 are upregulated, indicating that direct ParB- parS3/parS4 interactions repress the transcription in this region. In addition, increased ParB level brings about repression or activation of numerous genes including several transcriptional regulators involved in SOS response, virulence and adaptation. Overall, our data support the role of partitioning protein ParB as a transcriptional regulator in Pseudomonas aeruginosa.
Formation of singlet oxygen by decomposition of protein hydroperoxide in photosystem II
Jul 21, 2017   PloS One
Pathak V, Prasad A, Pospíšil P
Formation of singlet oxygen by decomposition of protein hydroperoxide in photosystem II
Jul 21, 2017
PloS One
Singlet oxygen (1O2) is formed by triplet-triplet energy transfer from triplet chlorophyll to O2 via Type II photosensitization reaction in photosystem II (PSII). Formation of triplet chlorophyll is associated with the change in spin state of the excited electron and recombination of triplet radical pair in the PSII antenna complex and reaction center, respectively. Here, we have provided evidence for the formation of 1O2 by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex. Protein hydroperoxide is formed by protein oxidation initiated by highly oxidizing chlorophyll cation radical and hydroxyl radical formed by Type I photosensitization reaction. Under highly oxidizing conditions, protein hydroperoxide is oxidized to protein peroxyl radical which either cyclizes to dioxetane or recombines with another protein peroxyl radical to tetroxide. These highly unstable intermediates decompose to triplet carbonyls which transfer energy to O2 forming 1O2. Data presented in this study show for the first time that 1O2 is formed by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex.
Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa
Jul 21, 2017   PLoS Pathogens
Wang D, Chen W, Huang S, He Y, Liu X, Hu Q, Wei T, Shang H, Gan J, Chen H
Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa
Jul 21, 2017
PLoS Pathogens
Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/β-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved β-sheets pocket. The dimerization of the N-terminal H1 and H1' α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents.
13 C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids
Jul 21, 2017   The FEBS Journal
Zhang M, Yu XW, Xu Y, Jouhten P, Swapna GVT, Glaser RW, Hunt JF, Montelione GT, Maaheimo H, Szyperski T
13 C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids
Jul 21, 2017
The FEBS Journal
Carbon metabolism of Crabtree negative yeast Pichia pastoris was profiled using 13 C NMR to delineate regulation during exponential growth and to study the import of two precursors for branched-chain amino acid biosynthesis, α-ketoisovalerate and α-ketobutyrate. Cells were grown in aerobic batch cultures containing (i) only glucose, (ii) glucose along with the precursors, or (iii) glucose and Val. The study provided the following new insights. First, 13 C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree negative Pichia stipitis and Crabtree positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between Pichia stipitis and Saccharomyces cerevisiae. Second, excess α-ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α-ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α-ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2 H,13 C,15 N-labeled proteins containing protonated Ile, Val and Leu methyl groups in P. pastoris for NMR-based structural biology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Community detection in sequence similarity networks based on attribute clustering
Jul 24, 2017   PloS One
Chowdhary J, Löffler FE, Smith JC
Community detection in sequence similarity networks based on attribute clustering
Jul 24, 2017
PloS One
Networks are powerful tools for the presentation and analysis of interactions in multi-component systems. A commonly studied mesoscopic feature of networks is their community structure, which arises from grouping together similar nodes into one community and dissimilar nodes into separate communities. Here, the community structure of protein sequence similarity networks is determined with a new method: Attribute Clustering Dependent Communities (ACDC). Sequence similarity has hitherto typically been quantified by the alignment score or its expectation value. However, pair alignments with the same score or expectation value cannot thus be differentiated. To overcome this deficiency, the method constructs, for pair alignments, an extended alignment metric, the link attribute vector, which includes the score and other alignment characteristics. Rescaling components of the attribute vectors qualitatively identifies a systematic variation of sequence similarity within protein superfamilies. The problem of community detection is then mapped to clustering the link attribute vectors, selection of an optimal subset of links and community structure refinement based on the partition density of the network. ACDC-predicted communities are found to be in good agreement with gold standard sequence databases for which the "ground truth" community structures (or families) are known. ACDC is therefore a community detection method for sequence similarity networks based entirely on pair similarity information. A serial implementation of ACDC is available from https://cmb.ornl.gov/resources/developments.

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