Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A\u27s involvement in mutation of endogenous or exogenous DNA

Structural basis for mutation-induced destabilization of profilin 1 in ALS

Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis

Anaplasma phagocytophilum: REPERCUSSÃO DA INFECÇÃO DA FAUNA SILVESTRE EM CÃES E GATOS DE UMA ZONA ENDÉMICA

A população silvestre pode albergar uma elevada densidade de ixodídeos associados à transmissão de agentes patogénicos. Este facto é particularmente importante quando se assiste ao aumento descontrolado das populações silvestres, como se verifica, actualmente, com os javalis em Portugal. O presente estudo, procurou detectar evidências de infecção activa por Anaplasma phagocytophilum, agente zoonótico cuja transmissão poderá estar potenciada com o aumento da população de javalis no Parque Natural da Serra da Arrábida (PNSA). Foram estudados 21 javalis de três populações distintas, 35 cães e um gato com sinais clínicos, laboratoriais e/ou epidemiológicos compatíveis com doença associada a ixodídeos, atendidos no Hospital Veterinário da Arrábida (HVA) e 80 ixodídeos, capturados na vegetação (N=61) ou a parasitar animais (N=19) em áreas do PNSA. A pesquisa de ADN bacteriano foi realizada por PCR convencional genérico para Anaplasma/Ehrlichia e por PCR em tempo real específico para A. phagocytophilum. Embora em nenhuma das amostras tenha sido possível identificar reacções positivas as restrições temporais e espaciais deste estudo exploratório reforçam a importância de se realizar uma vigilância epidemiológica mais abrangente de agentes patogénicos associados a ixodídeos no PNSA

Hamiltonian Noether theorem for gauge systems and two time physics

The Noether theorem for Hamiltonian constrained systems is revisited. In particular, our review presents a novel method to show that the gauge transformations are generated by the conserved quantities associated with the first class constraints. We apply our results to the relativistic point particle, to the Friedberg et al. model and, with special emphasis, to two time physics.Comment: 20 pages, Latex, references added, the "massless" sense of (87) is clarifie

Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 A. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A-ssDNA complex defines the 5\u27-3\u27 directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics

Characterization and Mitigation of Insufficiencies In Automated Driving Systems

Automated Driving (AD) systems have the potential to increase safety, comfort and energy efficiency. Recently, major automotive companies have started testing and validating AD systems (ADS) on public roads. Nevertheless, the commercial deployment and wide adoption of ADS have been moderate, partially due to system functional insufficiencies (FI) that undermine passenger safety and lead to hazardous situations on the road. In contrast to system faults that are analyzed by the automotive functional safety standard ISO 26262, FIs are defined in ISO 21448 Safety Of The Intended Functionality (SOTIF). FIs are insufficiencies in sensors, actuators and algorithm implementations, including neural networks and probabilistic calculations. Examples of FIs in ADS include inaccurate ego-vehicle localization on the road, incorrect prediction of a cyclist maneuver, unreliable detection of a pedestrian in rainy weather using cameras and image processing algorithms, etc. The main goal of the study is to formulate a generic architectural design pattern, which is compatible with existing methods and ADS, to improve FI mitigation and enable faster commercial deployment of ADS. First, the authors studied the 2021 autonomous vehicles disengagement reports published by the California Department of Motor Vehicles (DMV). The data clearly show that disengagements are five times more often caused by FIs rather than by system faults. They then made a comprehensive list of insufficiencies and their characteristics by analyzing over 10 hours of publicly available road test videos. In particular, the authors identified insufficiency types in four major categories: world model, motion plan, traffic rule, and operational design domain. The insufficiency characterization helps making the SOTIF analyses of triggering conditions more systematic and comprehensive. To handle faults, modern ADS already integrate multiple AD channels, where each channel is composed of sensors and processors running AD software. The characterization study triggered a hypothesis that these heterogeneous channels can also complement each other’s capabilities to mitigate insufficiencies in vehicle operation. To verify the hypothesis, the authors built an open-loop automated driving simulation environment based on the LG SVL simulator. Three realistic AD channels (Baidu Apollo, Autoware.Auto, and comma.ai openpilot) were tested in the same driving scenario. The experiments suggest that even advanced AD channels have insufficiencies that can be mitigated by switching control to another (possibly less advanced) AD channel at the right moment. Based on the FI characterization, simulation experiments and literature survey, the authors define a novel generic architectural design pattern Daruma to dynamically select the channel that is least likely to have a FI at the moment. The key component of the pattern does cross-channel analysis, in which planned trajectories and world models from different AD channels are mutually evaluated. The output of the cross-channel analysis is combined with more traditional fault detections in a safety fusion component. The safety fusion then feeds an aggregated per-channel safety score to the high-level arbiter, which eventually selects the AD channel to control the vehicle. The formulated architectural pattern can help manufactures of autonomous vehicles in mitigating FIs. Limitations of the study suggest interesting future work, including algorithmic research on cross-channel analysis and safety fusion, as well as evaluation of the cross-channel analysis in simulations and road tests

Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of The Royal Society for personal use, not for redistribution. The definitive version was published in Journal of The Royal Society Interface 6 (2010): 549-560, doi:10.1098/rsif.2009.0299.Many cephalopods exhibit remarkable dermal iridescence, a component of their complex, dynamic camouflage and communication. In the species Euprymna scolopes, the light-organ iridescence is static and is due to reflectin protein-based platelets assembled into lamellar thin-film reflectors called iridosomes, contained within iridescent cells called iridocytes. Squid in the family Loliginidae appear to be unique in that the dermis possesses a dynamic iridescent component, with reflective, colored structures that are assembled and disassembled under the control of the muscarinic cholinergic system and the associated neurotransmitter acetylcholine (Mathger et al. 2004). Here we present the sequences and characterization of three new members of the reflectin family associated with the dynamically changeable iridescence in Loligo and not found in static Euprymna iridophores. In addition, we show that application of genistein, a protein tyrosine kinase inhibitor, suppresses acetylcholine- and calcium-induced iridescence in Loligo. We further demonstrate that two of these novel reflectins are extensively phosphorylated in concert with the activation of iridescence by exogenous acetylcholine. This phosphorylation and the correlated iridescence can be blocked with genistein. Our results suggest that tyrosine phosphorylation of reflectin proteins is involved in the regulation of dynamic iridescence in Loligo.We gratefully acknowledge support from Anteon contract F33615-03-D-5408 to the Marine Biological Laboratory, Woods Hole, MA and grant # W911NF-06-1-0285 from the Army Research Office to D.E.M