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Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm's anterior-posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.
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Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.
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Transferencia Resonante de Energía de Fluorescencia , Proteínas , Transferencia Resonante de Energía de Fluorescencia/métodos , Reproducibilidad de los Resultados , Proteínas/química , Conformación Molecular , LaboratoriosRESUMEN
Animals interact with each other in species-specific reproducible patterns. These patterns of organization are captured by social network analysis, and social interaction networks (SINs) have been described for a wide variety of species including fish, insects, birds, and mammals. The aim of this study is to understand the evolution of social organization in Drosophila Using a comparative ecological, phylogenetic, and behavioral approach, the different properties of SINs formed by 20 drosophilids were compared. We investigate whether drosophilid network structures arise from common ancestry, a response to the species' past climate, other social behaviors, or a combination of these factors. This study shows that differences in past climate predicted the species' current SIN properties. The drosophilid phylogeny offered no value to predicting species' differences in SINs through phylogenetic signal tests. This suggests that group-level social behaviors in drosophilid species are shaped by divergent climates. However, we find that the social distance at which flies interact correlated with the drosophilid phylogeny, indicating that behavioral elements of SINs have remained largely unchanged in their evolutionary history. We find a significant correlation of leg length to social distance, outlining the interdependence of anatomy and complex social structures. Although SINs display a complex evolutionary relationship across drosophilids, this study suggests that the ecology, and not common ancestry, contributes to diversity in social structure in Drosophila.
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Evolución Biológica , Drosophila , Ambiente , Conducta Social , Animales , Drosophila/clasificación , Drosophila/genética , Drosophila/fisiología , Femenino , Masculino , FilogeniaRESUMEN
To render the sodium ion battery (SIB) competitive among other technologies, the processes behind sodium storage in hard carbon anodes must be understood. For this purpose, electrochemical impedance spectroscopy (EIS) is usually undervalued, since fitting the spectra with equivalent circuit models requires an a priori knowledge about the system at hand. The analysis of the distribution of relaxation times (DRT) is an alternative, which refrains from fitting arbitrarily nested equivalent circuits. In this paper, the sodiation and desodiation of a hard carbon anode is studied by EIS at different states of charge (SOC). By reconstructing the DRT function, highly resolved information on the number and relative contribution of individual electrochemical processes is derived. During the sloping part of the sodiation curve, mass transport is found to be the most dominant source of resistance but rapidly diminishes when the plateau phase is reached. An equivalent circuit model qualitatively reproducing the experimental data of the sloping region was built upon the DRT results, which is particularly useful for future EIS studies on hard carbon SIB anodes. More importantly, this work contributes to establish EIS as a practical tool to directly study electrode processes without the bias of a previously assumed model.
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An algorithm for the simulation and evaluation of cyclic voltammetry (CV) at macroporous electrodes such as felts, foams, and layered structures is presented. By considering 1D, 2D, and 3D arrays of electrode sheets, cylindrical microelectrodes, hollow-cylindrical microelectrodes, and hollow-spherical microelectrodes the internal diffusion domains of the macroporous structures are approximated. A universal algorithm providing the time-dependent surface concentrations of the electrochemically active species, required for simulating cyclic voltammetry responses of the individual planar, cylindrical, and spherical microelectrodes, is presented as well. An essential ingredient of the algorithm, which is based on Laplace integral transformation techniques, is the use of a modified Talbot contour for the inverse Laplace transformation. It is demonstrated that first-order homogeneous chemical kinetics preceding and/or following the electrochemical reaction and electrochemically active species with non-equal diffusion coefficients can be included in all diffusion models as well. The proposed theory is supported by experimental data acquired for a reference reaction, the oxidation of [Fe(CN)6 ]4- at platinum electrodes as well as for a technically relevant reaction, the oxidation of VO2+ at carbon felt electrodes. Based on our calculation strategy, we provide a powerful open source tool for simulating and evaluating CV data implemented into a Python graphical user interface (GUI).
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Staphylococcus aureus is part of the human nasal and skin microbiomes along with other bacterial commensals and opportunistic pathogens. Nutrients are scarce in these habitats, demanding effective nutrient acquisition and competition strategies. How S. aureus copes with phosphate limitation is still unknown. Wall teichoic acid (WTA), a polyol-phosphate polymer, could serve as a phosphate source, but whether S. aureus can utilize it during phosphate starvation remains unknown. S. aureus secretes a glycerophosphodiesterase, GlpQ, that cleaves a broad variety of glycerol-3-phosphate (GroP) headgroups of deacylated phospholipids, providing this bacterium with GroP as a carbon and phosphate source. Here we demonstrate that GlpQ can also use glycerophosphoglycerol derived from GroP WTA from coagulase-negative Staphylococcus lugdunensis, Staphylococcus capitis, and Staphylococcus epidermidis, which share the nasal and skin habitats with S. aureus Therefore, S. aureus GlpQ is the first reported WTA-hydrolyzing enzyme, or teichoicase, from Staphylococcus Activity assays revealed that unmodified WTA is the preferred GlpQ substrate, and the results from MS analysis suggested that GlpQ uses an exolytic cleavage mechanism. Importantly, GlpQ did not hydrolyze the ribitol-5-phosphate WTA polymers of S. aureus, underscoring its role in interspecies competition rather than in S. aureus cell wall homeostasis or WTA recycling. glpQ expression was strongly up-regulated under phosphate limitation, and GlpQ allowed S. aureus to grow in the presence of GroP WTA as the sole phosphate source. Our study reveals a novel and unprecedented strategy of S. aureus for acquiring phosphate from bacterial competitors under the phosphate-limiting conditions in the nasal or skin environments.
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Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Fosfatos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/metabolismo , Glicosilación , Espectrometría de Masas , Especificidad por SustratoRESUMEN
In order to quantitatively investigate the kinetic performance and the pore size distribution of carbon felt electrodes for the application in vanadium redox flow batteries, the theory of cyclic voltammetry (CV) is derived for a random network of cylindrical microelectrodes on the base of convolutive modeling. In this context we present an algorithm based on the use of a modified Talbot contour for inverse Laplace transformation, providing the mass transfer functions required for the calculation of the CV responses in external cylindrical finite diffusion space. First-order homogenous chemical kinetics preceding and/or following the electrochemical reactions are implemented in this algorithm as well. The VO2+ oxidation is investigated as model reaction at pristine and electrochemically aged commercial carbon felt electrodes. A fit of simulated data to experimental data clearly shows that an electrochemical aging predominantly affects the kinetics of the electron transfer reaction and that internal electrode surfaces and pore size distributions remain constant. The estimated pore size distributions are in excellent agreement with porosimetry measurements, validating our theory and providing a new strategy to determine electrode porosities and electrode kinetics simultaneously via CV.
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The facultative pathogen Staphylococcus aureus colonizes the human anterior nares and causes infections of various organ systems. Which carbon, energy, and phosphate sources can be utilized by S. aureus in nutrient-poor habitats has remained largely unknown. We describe that S. aureus secretes a glycerophosphodiesterase (glycerophosphodiester phosphodiesterase, EC 3.1.4.46), GlpQ, degrading the glycerophosphodiester (GPD) head groups of phospholipids such as human phosphatidylcholine (GroPC). Deletion of glpQ completely abolished the GroPC-degrading activity in S. aureus culture supernatants. GroPC has been detected in human tissues and body fluids probably as a result of phospholipid remodelling and degradation. Notably, GroPC promoted S. aureus growth under carbon- and phosphate-limiting conditions in a GlpQ-dependent manner indicating that GlpQ permits S. aureus to utilize GPD-derived glycerol-3-phosphate as a carbon and phosphate sources. Thus, S. aureus can use a broader spectrum of nutrients than previously thought which underscores its capacity to adapt to the highly variable and nutrient-poor surroundings.
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Hidrolasas Diéster Fosfóricas/metabolismo , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glicerofosfatos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Staphylococcus aureus/enzimología , Staphylococcus aureus/genéticaRESUMEN
Organisms rarely act in isolation. Their decisions and movements are often heavily influenced by direct and indirect interactions with conspecifics. For example, we each represent a single node within a social network of family and friends, and an even larger network of strangers. This group membership can affect our opinions and actions. Similarly, when in a crowd, we often coordinate our movements with others like fish in a school, or birds in a flock. Contributions of the group to individual behaviors are observed across a wide variety of taxa but their biological mechanisms remain largely unknown. With the advent of powerful computational tools as well as the unparalleled genetic accessibility and surprisingly rich social life of Drosophila melanogaster, researchers now have a unique opportunity to investigate molecular and neuronal determinants of group behavior. Conserved mechanisms and/or selective pressures in D. melanogaster can likely inform a much wider phylogenetic scale. Here, we highlight two examples to illustrate how quantitative and genetic tools can be combined to uncover mechanisms of two group behaviors in D. melanogaster: social network formation and collective behavior. Lastly, we discuss future challenges towards a full understanding how coordinated brain activity across many individuals gives rise to the behavioral patterns of animal societies.
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Conducta Animal , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Animales , Genes de Insecto , Fenómenos Genéticos , Genética , Fenómenos Fisiológicos del Sistema Nervioso , Vías Nerviosas , Neurociencias , Conducta SocialRESUMEN
Social behaviour emerges from the local environment but is constrained by the animal's life history and its evolutionary lineage. In this perspective, we consider the genus Drosophila and provide an overview of how these constraints can shape how individuals interact. Our focus is restricted to visual and chemical signals and how their use varies across species during courtship - currently the only social behaviour well-studied across many Drosophila species. We broadly categorize species into four climatic groups - cosmopolitan, tropical, temperate and arid - which serve as discussion points as we review comparative behavioural and physiological studies and relate them to the abiotic conditions of a species environment. We discuss how the physiological and behavioural differences among many fly species may reflect life history differences as much as, or even more than, differences in phylogeny. This perspective serves not only to summarize what has been studied across drosophilids, but also to identify questions and outline gaps in the literature worth pursuing for progressing the understanding of behavioural evolution in Drosophila.
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Drosophila/fisiología , Conducta Sexual Animal , Comunicación Animal , Animales , Cortejo , Drosophila/genética , Femenino , Masculino , Feromonas/metabolismo , Filogenia , Especificidad de la Especie , Percepción VisualRESUMEN
We report a new limitation on the ability of physical systems to perform computation-one that is based on generalizing the notion of memory, or storage space, available to the system to perform the computation. Roughly, we define memory as the maximal amount of information that the evolving system can carry from one instant to the next. We show that memory is a limiting factor in computation even in lieu of any time limitations on the evolving system-such as when considering its equilibrium regime. We call this limitation the space-bounded Church-Turing thesis (SBCT). The SBCT is supported by a simulation assertion (SA), which states that predicting the long-term behavior of bounded-memory systems is computationally tractable. In particular, one corollary of SA is an explicit bound on the computational hardness of the long-term behavior of a discrete-time finite-dimensional dynamical system that is affected by noise. We prove such a bound explicitly.
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Flies display transient social interactions in groups. However, whether fly-fly interactions are stochastic or structured remains unknown. We hypothesized that groups of flies exhibit patterns of social dynamics that would manifest as nonrandom social interaction networks. To test this, we applied a machine vision system to track the position and orientation of flies in an arena and designed a classifier to detect interactions between pairs of flies. We show that the vinegar fly, Drosophila melanogaster, forms nonrandom social interaction networks, distinct from virtual network controls (constructed from the intersections of individual locomotor trajectories). In addition, the formation of interaction networks depends on chemosensory cues. Gustatory mutants form networks that cannot be distinguished from their virtual network controls. Olfactory mutants form networks that are greatly disrupted compared with control flies. Different wild-type strains form social interaction networks with quantitatively different properties, suggesting that the genes that influence this network phenotype vary across and within wild-type populations. We have established a paradigm for studying social behaviors at a group level in Drosophila and expect that a genetic dissection of this phenomenon will identify conserved molecular mechanisms of social organization in other species.
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Conducta Animal/fisiología , Drosophila melanogaster/fisiología , Conducta Social , Animales , Animales Modificados Genéticamente , Células Quimiorreceptoras/fisiología , Drosophila melanogaster/genética , Femenino , Masculino , Modelos Biológicos , Mutación , Fenotipo , Olfato/genética , Olfato/fisiologíaRESUMEN
Brain single photon emission CT (SPECT) scans indirectly show functional activity via measurement of regional cerebral blood flow. In conventional SPECT scans, the typical tomographic slices are produced. In three-dimensional thresholded SPECT scans, pixels representing activity below a certain threshold are discarded. A retrospective analysis of 427 patients shows that three-dimensional thresholded SPECT scans yield a sensitivity for predicting clinical attention deficit hyperactivity disorder of 54% [95% confidence interval (CI), 46%-61%; specificity, 76%; 95% CI, 71%-81%] compared with 4% sensitivity [95% CI, 2%-8%; specificity, 97%; 95% CI, 94%-98%] for conventional SPECT scans. For 170 of the patients originating from a general psychiatry practice, conventional SPECT showed 10% sensitivity (95% CI, 4%-23%) and 98% specificity (95% CI, 93%-99%), whereas three-dimensional thresholded SPECT showed 83% sensitivity (95% CI, 68%-91%) and 77% specificity (95% CI, 69%-83%). These findings indicate that a much stronger signal is obtained when the three-dimensional thresholded SPECT scan is performed rather than the conventional SPECT scan in detecting attention deficit hyperactivity disorder and suggest similar results may be obtained for other psychiatric disorders.
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Trastorno por Déficit de Atención con Hiperactividad/diagnóstico , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Imagenología Tridimensional , Tomografía Computarizada de Emisión de Fotón Único , Adolescente , Adulto , Trastorno por Déficit de Atención con Hiperactividad/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Mapeo Encefálico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica , Psicotrópicos/uso terapéutico , Estudios Retrospectivos , Adulto JovenRESUMEN
Cryo-electron tomography (cryo-ET) has begun to provide intricate views of cellular architecture at unprecedented resolutions. Considerable efforts are being made to further optimize and automate the cryo-ET workflow, from sample preparation to data acquisition and analysis, to enable visual proteomics inside of cells. Here, we will discuss the latest advances in cryo-ET that go hand in hand with their application to the actin cytoskeleton. The development of deep learning tools for automated annotation of tomographic reconstructions and the serial lift-out sample preparation procedure will soon make it possible to perform high-resolution structural biology in a whole new range of samples, from multicellular organisms to organoids and tissues.
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Citoesqueleto de Actina , Tomografía con Microscopio Electrónico , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Humanos , Animales , Microscopía por Crioelectrón , Análisis de la Célula IndividualRESUMEN
In many cases, the hybridization of two or more excitation modes in solids has led to new and useful dispersion relations of waves. Well-studied examples are phonon polaritons, plasmon polaritons, particle-plasmon polaritons, cavity polaritons, and magnetic resonances at optical frequencies. In all of these cases, the lowest propagating mode couples to a finite-frequency localized resonance. Herein, the unusual metamaterial phonon dispersion relations arising from the hybridization of an ordinary acoustical phonon mode with a back-folded soft or easy phonon mode of a monomode elastic metamaterial are discussed. Conceptually, the single easy mode can have strictly zero wave velocity. In reality, its wave velocity is very much smaller than that of all other modes. Considering polymeric three-dimensional printed elastic monomode metamaterials at ultrasound frequencies, it is shown theoretically and experimentally that the resulting pronounced avoided crossing, with a frequency splitting comparable to the mid-frequency, leads to backward-wave behavior for the lowest band over a broad frequency range, conceptually at zero loss.
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Social networks are a mathematical representation of interactions among individuals which are prevalent across various animal species. Studies of human populations have shown the breadth of what can spread throughout a social network: obesity, smoking cessation, happiness, drug use and divorce. 'Betweenness centrality' is a key property of social networks that indicates an individual's importance in facilitating communication and cohesion within the network. Heritability of betweenness centrality has been suggested in several species, however the genetic regulation of this property remains enigmatic. Here, we demonstrate that the gene CG14109, referred to as degrees of kevin bacon (dokb), influences betweenness centrality in Drosophila melanogaster. We identify strain-specific alleles of dokb with distinct amino acid sequences and when the dokb allele is exchanged between strains, flies exhibit the betweenness centrality pattern dictated by the donor allele. By inserting a GAL4 reporter into the dokb locus, we confirm that dokb is expressed in the central nervous system. These findings define a novel genetic entry point to study social network structure and thereby establish gene-to-social structure relationships. While dokb sequence homology is exclusive to Diptera, we anticipate that dokb-associated molecular pathways could unveil convergent neural mechanisms of social behaviour that apply in diverse animal species.
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Proteínas de Drosophila , Drosophila melanogaster , Animales , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Alelos , Masculino , Femenino , Conducta Animal , Conducta Social , Red SocialRESUMEN
In classical Cauchy elasticity, 3D materials exhibit six eigenmodes of deformation. Following the 1995 work of Milton and Cherkaev, extremal elastic materials can be classified by the number of eigenmodes, N, out of these six that are "easy". Using Greek number words, this leads to hexamode (N = 6), pentamode (N = 5), tetramode (N = 4), trimode (N = 3), dimode (N = 2), and monomode (N = 1) materials. While hexamode materials are unstable in all regards, the possibility of pentamode metamaterials ("meta-fluids") has attracted considerable attention throughout the last decade. Here, inspired by the 2021 theoretical work of Wei, Liu, and Hu, microstructured 3D polymer-based tetramode metamaterials are designed and characterized by numerical band-structure calculations, fabricated by laser printing, characterized by ultrasound experiments, and compared to the theoretical ideal. An application in terms of a compact and broadband polarizer for acoustical phonons at ultrasound frequencies is demonstrated.
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Actin contributes to an exceptionally wide range of cellular processes through the assembly and disassembly of highly dynamic and ordered structures. Visualizing these structures in cells can help us understand how the molecular players of the actin machinery work together to produce force-generating systems. In recent years, cryo-electron tomography (cryo-ET) has become the method of choice for structural analysis of the cell interior at the molecular scale. Here we review advances in cryo-ET workflows that have enabled this transformation, especially the automation of sample preparation procedures, data collection, and processing. We discuss new structural analyses of dynamic actin assemblies in cryo-preserved cells, which have provided mechanistic insights into actin assembly and function at the nanoscale. Finally, we highlight the latest visual proteomics studies of actin filaments and their interactors reaching sub-nanometer resolutions in cells.
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Actinas , Tomografía con Microscopio Electrónico , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodosRESUMEN
The present study aimed to examine the relationship between perfectionism, OCD symptom dimensions, harm avoidance, and incompleteness at varying levels (i.e., higher-order/subscale) in college students. College students (n = 548) completed measures of perfectionism, obsessive-compulsive symptoms, harm avoidance and incompleteness. The results revealed that all five higher-order dimensions of perfectionism were related to incompleteness but only three were related to harm avoidance. Parallel mediation revealed an indirect relationship between concern for mistakes and doubts about actions and checking and cleaning behaviors, respectively, through incompleteness. An indirect relationship between concern for mistakes and doubts about actions with ordering, respectively, through both incompleteness and harm avoidance was found. However, incompleteness played a stronger role than harm avoidance in this relationship. These results suggest that incompleteness might be a stronger motivator than harm avoidance for perfectionistic individuals. Therefore, interventions targeting incompleteness should be implemented to potentially circumvent the development or worsening of obsessive-compulsive symptoms.
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Actin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin filaments, which individually generate piconewton forces, can produce forces reaching tens of nanonewtons. Here we use in situ cryo-electron tomography to unveil how the nanoscale architecture of macrophage podosomes enables basal membrane protrusion. We show that the sum of the actin polymerization forces at the membrane is not sufficient to explain podosome protrusive forces. Quantitative analysis of podosome organization demonstrates that the core is composed of a dense network of bent actin filaments storing elastic energy. Theoretical modelling of the network as a spring-loaded elastic material reveals that it exerts forces of a few tens of nanonewtons, in a range similar to that evaluated experimentally. Thus, taking into account not only the interface with the membrane but also the bulk of the network, is crucial to understand force generation by actin machineries. Our integrative approach sheds light on the elastic behavior of dense actin networks and opens new avenues to understand force production inside cells.