Meningococcus Hijacks a ß2-adrenoceptor/ß-Arrestin pathway to cross brain microvasculature endothelium.

Following pilus-mediated adhesion to human brain endothelial cells, meningococcus (N. meningitidis), the bacterium causing cerebrospinal meningitis, initiates signaling cascades, which eventually result in the opening of intercellular junctions, allowing meningeal colonization. The signaling receptor activated by the pathogen remained unknown. We report that N. meningitidis specifically stimulates a biased ß2-adrenoceptor/ß-arrestin signaling pathway in endothelial cells, which ultimately traps ß-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by ß-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas ß-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of ß-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier. The identification of the mechanism used for hijacking host cell signaling machineries opens perspectives for treatment and prevention of meningococcal infection.

Voxelated soft matter via multimaterial multinozzle 3D printing.

There is growing interest in voxelated matter that is designed and fabricated voxel by voxel1-4. Currently, inkjet-based three-dimensional (3D) printing is the only widely adopted method that is capable of creating 3D voxelated materials with high precision1-4, but the physics of droplet formation requires the use of low-viscosity inks to ensure successful printing5. By contrast, direct ink writing, an extrusion-based 3D printing method, is capable of patterning a much broader range of materials6-13. However, it is difficult to generate multimaterial voxelated matter by extruding monolithic cylindrical filaments in a layer-by-layer manner. Here we report the design and fabrication of voxelated soft matter using multimaterial multinozzle 3D (MM3D) printing, in which the composition, function and structure of the materials are programmed at the voxel scale. Our MM3D printheads exploit the diode-like behaviour that arises when multiple viscoelastic materials converge at a junction to enable seamless, high-frequency switching between up to eight different materials to create voxels with a volume approaching that of the nozzle diameter cubed. As exemplars, we fabricate a Miura origami pattern14 and a millipede-like soft robot that locomotes by co-printing multiple epoxy and silicone elastomer inks of stiffness varying by several orders of magnitude. Our method substantially broadens the palette of voxelated materials that can be designed and manufactured in complex motifs.

Maintained mitochondrial integrity without oxygen in the anoxia tolerant crucian carp.

Very few vertebrates survive without oxygen (anoxia) for more than a few minutes. The crucian carp (Carassius carassius) survive months of anoxia at low temperatures and we hypothesised that they maintain mitochondrial membrane potential and function. Isolated crucian carp cardiomyocytes indeed maintained mitochondrial membrane potential after blocking complex-IV of the electron transport system with cyanide, while those of anoxia-intolerant trout depolarized. When complexes I-III were inhibited, crucian carp mitochondria depolarized, indicating that these complexes need to function during anoxia. Mitochondrial membrane potential depended on reversal of ATP synthase in chemical anoxia, as blocking with cyanide combined with oligomycin to inhibit ATP-synthase lead to depolarization. ATP-synthase activity was reduced in the heart after one week of anoxia in crucian carp, together with a downregulation of ATP-synthase subunit gene expression. However, the morphology of cardiac mitochondria were not affected by one-week anoxia, even with a large increase in mitofusin-2 expression. Cardiac citrate synthase activity was not affected by anoxia, while cytochrome-C oxidase activity was increased. We show how mitochondria respond to anoxia. A mechanistic understanding of how mitochondrial function can be maintained in anoxia may provide new perspectives to reduce mitochondrial damage in anoxia-sensitive organisms.

A Visual, In-Expensive, and Wireless Capillary Rheometer for Characterizing Wholly-Cellular Bioinks.

Rheological measurements with in situ visualization can elucidate the microstructural origin of complex flow behaviors of an ink. However, existing commercial rheometers suffer from high costs, the need for dedicated facilities for microfabrication, a lack of design flexibility, and cabling that complicates operation in sterile or enclosed environments. To address these limitations, a low-cost ($300) visual, in-expensive and wireless rheometer (VIEWR) using 3D-printed and off-the-shelf components is presented. VIEWR measurements are validated by steady-state and transient flow responses for different complex fluids, and microstructural flow profiles and evolution of yield-planes are revealed via particle image velocimetry. Using the VIEWR, a wholly-cellular bioink system comprised of compacted cell aggregates is characterized, and complex yield-stress and viscoelastic responses are captured via concomitantly visualizing the spatiotemporal evolution of aggregate morphology. A symmetric hyperbolic extensional-flow geometry is further constructed inside a capillary tube using digital light processing. Such geometries allow for measuring the extensional viscosity at varying deformation rates and further visualizing the alignment and stretching of aggregates under external flow. Synchronized but asymmetric evolution of aggregate orientation and strain through the neck is visualized. Using varying geometries, the jamming and viscoelastic deformation of aggregates are shown to contribute to the extensional viscosity of the wholly-cellular bioinks.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Control of CCR5 Cell-Surface Targeting by the PRAF2 Gatekeeper.

The cell-surface targeting of neo-synthesized G protein-coupled receptors (GPCRs) involves the recruitment of receptors into COPII vesicles budding at endoplasmic reticulum exit sites (ERESs). This process is regulated for some GPCRs by escort proteins, which facilitate their export, or by gatekeepers that retain the receptors in the ER. PRAF2, an ER-resident four trans- membrane domain protein with cytoplasmic extremities, operates as a gatekeeper for the GB1 protomer of the heterodimeric GABAB receptor, interacting with a tandem di-leucine/RXR retention motif in the carboxyterminal tail of GB1. PRAF2 was also reported to interact in a two-hybrid screen with a peptide corresponding to the carboxyterminal tail of the chemokine receptor CCR5 despite the absence of RXR motifs in its sequence. Using a bioluminescence resonance energy transfer (BRET)-based subcellular localization system, we found that PRAF2 inhibits, in a concentration-dependent manner, the plasma membrane export of CCR5. BRET-based proximity assays and Co-IP experiments demonstrated that PRAF2/CCR5 interaction does not require the presence of a receptor carboxyterminal tail and involves instead the transmembrane domains of both proteins. The mutation of the potential di-leucine/RXR motif contained in the third intracellular loop of CCR5 does not affect PRAF2-mediated retention. It instead impairs the cell-surface export of CCR5 by inhibiting CCR5's interaction with its private escort protein, CD4. PRAF2 and CD4 thus display opposite roles on the cell-surface export of CCR5, with PRAF2 inhibiting and CD4 promoting this process, likely operating at the level of CCR5 recruitment into COPII vesicles, which leave the ER.

Flow-enhanced vascularization and maturation of kidney organoids in vitro.

Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

Blood unit segments accurately represent the biophysical properties of red blood cells in blood bags but not hemolysis.

BACKGROUND: The biophysical properties of red blood cells (RBCs) provide potential biomarkers for the quality of donated blood. Blood unit segments provide a simple and nondestructive way to sample RBCs in clinical studies of transfusion efficacy, but it is not known whether RBCs sampled from segments accurately represent the biophysical properties of RBCs in blood bags. STUDY DESIGN AND METHODS: RBCs were sampled from blood bags and segments every two weeks during 8 weeks of storage at 4°C. RBC deformability was measured by deformability-based sorting using the microfluidic ratchet device in order to derive a rigidity score. Standard hematological parameters, including mean corpuscular volume (MCV), red cell distribution width (RDW), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and hemolysis were measured at the same time points. RESULTS: Deformability of RBCs stored in blood bags was retained over 4 weeks storage, but a progressive loss of deformability was observed at weeks 6 and 8. This trend was mirrored in blood unit segments with a strong correlation to the blood bag data. Strong correlations were also observed between blood bag and segment for MCV, MCHC, and MCH but not for hemolysis. CONCLUSION: RBCs sampled from blood unit segments accurately represent the biophysical properties of RBCs in blood bags but not hemolysis. Blood unit segments provide a simple and nondestructive sample for measuring RBC biophysical properties in clinical studies.

Society for Maternal-Fetal Medicine Special Statement: Best-practice recommendations for ultrasound network connectivity.

Prenatal ultrasound is an indispensable tool used by obstetrical care providers to assist in the everyday care of their pregnant patients. Alongside advancements in imaging, the electronic systems that support this technology have become more advanced. However, it is currently difficult for these individual systems to communicate with each other "out of the box." There is also minimal standardization of the type and format of data transmitted within these systems. Clinicians and system vendors must work collaboratively to create clinical and technical standards to serve as the foundation for increased interoperability among the various systems within each institutional network. Therefore, the Society for Maternal-Fetal Medicine Clinical Informatics Committee established an Ultrasound Electronic Health Record Subcommittee to facilitate collaboration between clinicians, including maternal-fetal medicine subspecialists, and ultrasound network component vendors. Based on the work of this subcommittee, the purpose of this document is to provide: (1) a basic understanding of ultrasound network architecture and capabilities, and (2) best-practice recommendations for electronic health record order design, obstetrical clinical data standards, and billing and coding practices.

Sensory attenuation from action observation.

A central claim of many embodied approaches to cognition is that understanding others' actions is achieved by covertly simulating the observed actions and their consequences in one's own motor system. If such a simulation occurs, it may be accomplished through forward models, a component of the motor system already known to perform simulations of actions and their consequences in order to support sensory-monitoring of one's own actions. Forward-model simulations cause an attenuation of sensory intensity, so if the simulations hypothesized by embodied cognition are indeed provided by forward models, then action observation should trigger this sensory attenuation. To test this hypothesis, the experiments reported here measured the perceived intensity of a touch sensation on the finger when participants observed an active touch (a finger reaching to touch a ball) vs. a passive touch (a ball rolling to touch an unmoving finger). The touch sensation was perceived as less intense during observation of active touch in comparison with observation of passive touch, providing evidence that forward models are indeed engaged during action observation. The strength of this sensory attenuation is compared and contrasted with a well-established sensory-amplification effect caused by visual attention. This sensory-amplification effect has not generally been considered in studies related to sensory attenuation in action observation, which may explain conflicting results reported in the field.

Systematic review and meta-analysis of anal motor and rectal sensory dysfunction in male and female patients undergoing anorectal manometry for symptoms of faecal incontinence.

AIM: Manometry is the best established technique to assess anorectal function in faecal incontinence. By systematic review, pooled prevalences of anal hypotonia/hypocontractility and rectal hypersensitivity/hyposensitivity in male and female patients were determined in controlled studies using anorectal manometry. METHODS: Searches of MEDLINE and Embase were completed. Screening, data extraction and bias assessment were performed by two reviewers. Meta-analysis was performed based on a random effects model with heterogeneity evaluated by I2 . RESULTS: Of 2116 identified records, only 13 studies (2981 faecal incontinence patients; 1028 controls) met the inclusion criteria. Anal tone was evaluated in 10 studies and contractility in 11; rectal sensitivity in five. Only three studies had low risk of bias. Pooled prevalence of anal hypotonia was 44% (95% CI 32-56, I2 = 96.35%) in women and 27% (95% CI 14-40, I2 = 94.12%) in men. The pooled prevalence of anal hypocontractility was 69% (95% CI 57-81; I2 = 98.17%) in women and 36% (95% CI 18-53; I2 = 96.77%) in men. Pooled prevalence of rectal hypersensitivity was 10% (95% CI 4-15; I2 = 80.09%) in women and 4% (95% CI 1-7; I2 = 51.25%) in men, whereas hyposensitivity had a pooled prevalence of 7% (95% CI 5-9; I2 = 0.00%) in women compared to 19% (95% CI 15-23; I2 = 0.00%) in men. CONCLUSIONS: The number of appropriately controlled studies of anorectal manometry is small with fewer still at low risk of bias. Results were subject to gender differences, wide confidence intervals and high heterogeneity indicating the need for international collective effort to harmonize practice and reporting to improve certainty of diagnosis.

T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments.

It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. Here we studied a mouse model of human T-cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single-cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting that a stochastic mechanism underlies these processes. Yet, although T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells while perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, to combat the invasion by and survival of chemo-resistant T-ALL cells.

Reconstructing the heart using iPSCs: Engineering strategies and applications.

Induced pluripotent stem cells (iPSCs) have emerged as a key component of cardiac tissue engineering, enabling studies of cardiovascular disease mechanisms, drug responses, and developmental processes in human 3D tissue models assembled from isogenic cells. Since the very first engineered heart tissues were introduced more than two decades ago, a wide array of iPSC-derived cardiac spheroids, organoids, and heart-on-a-chip models have been developed incorporating the latest available technologies and materials. In this review, we will first outline the fundamental biological building blocks required to form a functional unit of cardiac muscle, including iPSC-derived cells differentiated by soluble factors (e.g., small molecules), extracellular matrix scaffolds, and exogenous biophysical maturation cues. We will then summarize the different fabrication approaches and strategies employed to reconstruct the heart in vitro at varying scales and geometries. Finally, we will discuss how these platforms, with continued improvements in scalability and tissue maturity, can contribute to both basic cardiovascular research and clinical applications in the future.

Temporal binding and agency under startle.

Forward models are a component of the motor system that predicts the sensory consequences of our actions. These models play several key roles in motor control and are hypothesized to underlie (among other things) the two phenomena under investigation in this experiment: The feeling of agency that we have over self-initiated actions (as opposed to reflexes), and "temporal binding", in which self-caused sensations are judged to have occurred earlier in time than they actually did. This experiment probes the connection between forward models and both of these phenomena using the "Startle" paradigm. In the Startle paradigm, a startlingly loud sound causes people to initiate a prepared action at a very short latency. It is hypothesized that the latency of a startle-initiated action is so short that normal cortical operations (including forward models) are circumvented. This experiment replicates the temporal-binding effect and simultaneously measures participants' sense of agency over their actions. The results show that both the temporal-binding effect and the sense of agency we have over our own actions is disrupted under the startle paradigm in line with the theory that these phenomena both rely on forward models. Furthermore, this experiment provides evidence in support of the claim that a startle-induced action is qualitatively different from other actions.

Beta-arrestins operate an on/off control switch for focal adhesion kinase activity.

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-ß-arrestin complex to the plasma membrane, ß-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient ß-arrestin and by a specific inhibitor of ß-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of ß-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

TRPV1 Activation Promotes ß-arrestin2 Interaction with the Ribosomal Biogenesis Machinery in the Nucleolus:Implications for p53 Regulation and Neurite Outgrowth.

Transient receptor potential vanilloids (TRPV1) are non-selective cation channels that sense and transduce inflammatory pain signals. We previously reported that activation of TRPV1 induced the translocation of ß-arrestin2 (ARRB2) from the cytoplasm to the nucleus, raising questions about the functional role of ARRB2 in the nucleus. Here, we determined the ARRB2 nuclear signalosome by conducting a quantitative proteomic analysis of the nucleus-sequestered L395Q ARRB2 mutant, compared to the cytosolic wild-type ARRB2 (WT ARRB2), in a heterologous expression system. We identified clusters of proteins that localize to the nucleolus and are involved in ribosomal biogenesis. Accordingly, L395Q ARRB2 or WT ARRB2 after capsaicin treatment were found to co-localize and interact with the nucleolar marker nucleophosmin (NPM1), treacle protein (TCOF1) and RNA polymerase I (POL I). We further investigated the role of nuclear ARRB2 signaling in regulating neuroplasticity. Using neuroblastoma (neuro2a) cells and dorsal root ganglia (DRG) neurons, we found that L395Q ARRB2 mutant increased POL I activity, inhibited the tumor suppressorp53 (p53) level and caused a decrease in the outgrowth of neurites. Together, our results suggest that the activation of TRPV1 promotes the ARRB2-mediated regulation of ribosomal biogenesis in the nucleolus. The ARRB2-TCOF1-p53 checkpoint signaling pathway might be involved in regulating neurite outgrowth associated with pathological pain conditions.

Incorporating Network Connectivity into Stream Classification Frameworks.

Stream classification frameworks are important tools for conserving aquatic resources. Yet despite their utility, most classification frameworks have not incorporated network connectivity. We developed and compared three biologically informed stream classification frameworks considering the effects of variables indexing local habitat and/or connectivity on stream fish communities. The first framework classified streams according to local environmental variables largely following the precedent set by previous stream classifications. The second framework classified streams according solely to network connectivity variables, while the third framework considered both local and connectivity variables. Using fish community data from 291 wadeable streams in South Carolina, USA, we used conditional inference tree analyses to identify either seven or eight discrete types of wadeable streams within each framework. Classifications were evaluated on their ability to describe community composition at a subset of sites not used in model training, and canonical correspondence analysis suggested that each framework performed similarly in describing overall community variation, with about 19% of variation explained. After accounting for the effects of biogeography and land use in our analytical approach, each classification explained a substantially higher amount of community variation with 46% of variation explained by our connectivity-informed classification and 42% explained by our locally informed classification. Classifications differed in their ability to describe elements of community structure; a classification incorporating connectivity predicted species richness better than the one that did not. This study ultimately addresses an important knowledge gap in the classification literature while providing broader implications for the conservation of aquatic organisms and their habitats.

Integrating Regional Frameworks and Local Variability for Riverine Bioassessment.

Regional frameworks enable bioassessment methods to detect anthropogenic effects on ecosystems amid natural variability. Conventional approaches to regionalization have used coarse geographical frameworks to separate sites similar in their ecological (ecoregion) or faunal (basin) characteristics. Expectations for individual streams are then adjusted for within-region variability in local environmental characteristics. Integrating regional frameworks and local variability may improve the sensitivity and performance of bioassessments. In this study, we used a biologically-informed stream classification to develop an integrated regional framework for bioassessment considering the effects of ecoregion, basin, and local environmental variables on wadeable stream fish communities of South Carolina, USA. Our integrated framework was compared against conventional regional frameworks indexing ecoregions or basins alone. Frameworks were evaluated by their ability to (1) efficiently partition community variation and (2) allow for the detection of anthropogenic effects on fish communities. We found an integrated framework better described natural variability in stream fish communities. In addition, we found highly regional relationships between fish metrics and anthropogenic disturbance among frameworks, suggesting appropriate bioassessment metrics will differ across regions in our study area. Differences in community response to disturbance among frameworks emphasize the importance of testing metrics for their hypothesized sensitivity before using them in bioassessment. This study ultimately supports the integration of regional frameworks across spatial scales to classify streams for bioassessment, and provides an analytical framework from which to evaluate biotic variation and metric utility in the context of bioassessment.

Interaural recalibration of phonetic categories.

Recalibration is a learning process in which perceptual boundaries between speech-sounds adjust through exposure to a supplementary source of information. Using a dichotic-listening methodology, the experiments reported here establish interaural recalibration-in which an ambiguous speech sound in one ear is recalibrated on the basis of a clear sound presented to the other ear. This demonstrates a previously unknown form of recalibration and shows that location-specific recalibration occurs even when people are unaware of location differences between the sounds involved.

Planning for historic urban environments under austerity conditions: Insights from post-crash Ireland.

In this paper, we examine how conservation-planning and local regeneration in historic urban cores have been re-shaped under austerity conditions and how local planners and local government more generally have negotiated or navigated this emerging austerity terrain. We seek to contribute to wider debates on 'austerity urbanism', by examining the impacts of austerity on local planning and how planning officials have attempted to moderate austerity largely imposed by central government (entrenched roll-back neoliberalism) but often through the further roll-out of neoliberalism in local growth strategies. Drawing on the experience of three Irish urban centres, we examine efforts to 'sell' the historic city. Both nationally and within the three case study areas, a common overarching theme was evident in the initial post-crisis response to urban development: an emphasis on utilising heritage as a potential economic regeneration pathway. However, while drawing on intangible heritage and heritage narratives for place-branding, the actual protection of tangible built heritage assets was undermined through a greater emphasis on 'flexible' planning responses to managing heritage, which seek to minimise barriers to development.