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Standard single‐cell (sc) proteomics of disease states inferred from multicellular organs or organoids cannot currently be related to single‐cell physiology. Here, a scPatch‐Clamp/Proteomics platform is developed on single neurons generated from hiPSCs bearing an Alzheimer's disease (AD) genetic mutation and compares them to isogenic wild‐type controls. This approach provides both current and voltage electrophysiological data plus detailed proteomics information on single‐cells. With this new method, the authors are able to observe hyperelectrical activity in the AD hiPSC‐neurons, similar to that observed in the human AD brain, and correlate it to ≈1400 proteins detected at the single neuron level. Using linear regression and mediation analyses to explore the relationship between the abundance of individual proteins and the neuron's mutational and electrophysiological status, this approach yields new information on therapeutic targets in excitatory neurons not attainable by traditional methods. This combined patch‐proteomics technique creates a new proteogenetic‐therapeutic strategy to correlate genotypic alterations to physiology with protein expression in single‐cells. [ABSTRACT FROM AUTHOR]

This paper describes a new methodology for characterising the plastic zone ahead of a fatigue crack. This methodology is applied to a set of experimental data obtained by synchrotron X‐ray diffraction on a bainitic steel compact tension specimen. The methodology is based on generating the equivalent Von Mises strain field from the X‐ray experimental elastic strain maps. Based on the material response, a threshold is then applied on the equivalent strain maps to identify the size and shape of the plastic zone. The experimental plastic zone lies between the plane strain and plane stress Westergaard's bounds but closer to the plane strain theoretical prediction confirming that the volume analyzed is predominantly subjected to plane strain conditions. However, the observed plastic zone has a somewhat flatter shape, extending further from the crack plane but less extended in the crack growing direction. Highlights: This paper describes a new methodology to infer the plastic zone in the bulk of materials.Methodology was tested successfully on a CT specimen made of bainitic steel.Results are consistent with data collected at the midplane through the thickness. [ABSTRACT FROM AUTHOR]

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA‐binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP‐bound mRNAs. We show ER stress‐induced activation of Inositol requiring enzyme‐1 (IRE1), an ER‐resident stress‐sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress‐induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis. Synopsis: Targeting IRE1 function and substrate(s) provides a novel therapeutic approach to atherosclerosis. We found a key role for IRE1‐mediated FMRP phosphorylation that suppresses the expression of cholesterol transporters and efferocytosis receptors in macrophages and promotes atherosclerosis progression. FMRP is a novel IRE1 kinase substrate.Lipid‐induced, IRE1‐mediated FMRP phosphorylation leads to post‐transcriptional suppression of cholesterol transporters and efferocytosis regulators.Ablation of IRE1 kinase activity or suppression of FMRP expression enhances efferocytosis and cholesterol transport in vitro and in vivo.IRE1 kinase inhibition by a small molecule inhibitor or genetic deletion of FMRP in macrophages alleviates atherosclerotic plaque formation. [ABSTRACT FROM AUTHOR]

Aurora kinase A (AURKA) is a conserved kinase that plays crucial roles in numerous cellular processes. Although AURKA overexpression is frequent in human cancers, its pleiotropic functions and multifaceted regulation present challenges in its therapeutic targeting. Key to overcoming these challenges is to identify and characterize the full range of AURKA interactors, which are often weak and transient. Previous proteomic studies were limited in monitoring dynamic and non‐mitotic AURKA interactions. Here, we generate the proximity interactome of AURKA in asynchronous cells, which consists of 440 proteins involving multiple biological processes and cellular compartments. Importantly, AURKA has extensive proximate and physical interactions to centriolar satellites, key regulators of the primary cilium. Loss‐of‐function experiments identify satellites as negative regulators of AURKA activity, abundance, and localization in quiescent cells. Notably, loss of satellites activates AURKA at the basal body, decreases centrosomal IFT88 levels, and causes ciliogenesis defects. Collectively, our results provide a resource for dissecting spatiotemporal regulation of AURKA and uncover its proteostatic regulation by satellites as a new mechanism for its ciliary functions. SYNOPSIS: This study presents the in vivo proximity interactome of AURKA in asynchronous cells, which reveals a new regulatory and functional relationship between AURKA and centriolar satellites. AURKA proximity interactome is composed of 440 proteins involving diverse biological processes and cellular compartments.AURKA has extensive interactions with centriolar satellite proteins.Depletion of centriolar satellites increases cellular and centrosomal abundance and activation of AURKA.AURKA inhibition rescues the ciliogenesis defects of cells depleted for centriolar satellites. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are cell‐derived membrane vesicles that are released into the extracellular space. EVs encapsulate key proteins and mediate intercellular signalling pathways. Recently, primary cilia have been shown to release EVs under fluid‐shear flow, but many proteins encapsulated in these vesicles have never been identified. Primary cilia are ubiquitous mechanosensory organelles that protrude from the apical surface of almost all human cells. Primary cilia also serve as compartments for signalling pathways, and their defects have been associated with a wide range of human genetic diseases called ciliopathies. To better understand the mechanism of ciliopathies, it is imperative to know the distinctive protein profiles of the differently sourced EVs (cilia vs cytosol). Here, we isolated EVs from ciliated wild‐type (WT) and non‐ciliated IFT88 knockout (KO) mouse endothelial cells using fluid‐shear flow followed by a conventional method of EV isolation. EVs isolated from WT and KO exhibited distinctive sizes. Differences in EV protein contents were studied using liquid chromatography with tandem mass spectrometry (LC‐MS‐MS) and proteomic comparative analysis, which allowed us to classify proteins between ciliary EVs and cytosolic EVs derived from WT and KO, respectively. A total of 79 proteins were exclusively expressed in WT EVs, 145 solely in KO EVs, and 524 in both EVs. Our bioinformatics analyses revealed 29% distinct protein classes and 75% distinct signalling pathways between WT and KO EVs. Based on our statistical analyses and in vitro studies, we identified NADPH‐cytochrome P450 reductase (POR), and CD166 antigen (CD166) as potential biomarkers for ciliary and cytosolic EVs, respectively. Our protein‐protein interaction network analysis revealed that POR, but not CD166, interacted with either established or strong ciliopathy gene candidates. This report shows the unique differences between EVs secreted from cilia and the cytosol. These results will be important in advancing our understanding of human genetic diseases. [ABSTRACT FROM AUTHOR]

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody‐based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high‐affinity and specific CD22 ligands onto NK‐92MI and cytokine‐induced natural killer cells to achieve tumor‐specific CD22 targeting. These CD22‐ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand‐modified NK‐92MI cells with the E‐selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual‐functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively. [ABSTRACT FROM AUTHOR]

Primary cilia are shown to have membrane swelling, also known as ciliary bulbs. However, the role of these structures and their physiological relevance remains unknown. Here, it is reported that a ciliary bulb has extracellular vesicle (EV)‐like characteristics. The ciliary extracellular‐like vesicle (cELV) has a unique dynamic movement and can be released by mechanical fluid force. To better identify the cELV, differential multidimensional proteomic analyses are performed on the cELV. A database of 172 cELV proteins is generated, and all that examined are confirmed to be in the cELV. Repressing the expression of these proteins in vitro and in vivo inhibits cELV formation. In addition to the randomized heart looping, hydrocephalus, and cystic kidney in fish, compensated heart contractility is observed in both fish and mouse models. Specifically, low circulation of cELV results in hypotension with compensated heart function, left ventricular hypertrophy, cardiac fibrosis, and arrhythmogenic characteristics, which result in a high mortality rate in mice. Furthermore, the overall ejection fraction, stroke volume, and cardiac output are significantly decreased in mice lacking cELV. It is thus proposed that the cELV as a nanocompartment within a primary cilium plays an important role in cardiovascular functions. [ABSTRACT FROM AUTHOR]

Aim: To investigate the interdependence between risk factors associated with long-term intellectual development in individuals with tuberous sclerosis complex (TSC).Method: The Tuberous Sclerosis 2000 Study is a prospective longitudinal study of individuals with TSC. In phase 1 of the study, baseline measures of intellectual ability, epilepsy, cortical tuber load, and mutation were obtained for 125 children (63 females, 62 males; median age=39mo). In phase 2, at an average of 8 years later, intellectual abilities were estimated for 88 participants with TSC and 35 unaffected siblings. Structural equation modelling was used to determine the risk pathways from genetic mutation through to IQ at phase 2.Results: Intellectual disability was present in 57% of individuals with TSC. Individuals without intellectual disability had significantly lower mean IQ compared to unaffected siblings, supporting specific genetic factors associated with intellectual impairment. Individuals with TSC who had a slower gain in IQ from infancy to middle childhood were younger at seizure onset and had increased infant seizure severity. Structural equation modelling indicated indirect pathways from genetic mutation, to tuber count, to seizure severity in infancy, through to IQ in middle childhood and adolescence.Interpretation: Early-onset and severe epilepsy in the first 2 years of life are associated with increased risk of long-term intellectual disability in individuals with TSC, emphasizing the importance of early and effective treatment or prevention of epilepsy.What This Paper Adds: Intellectual disability was present in 57% of individuals with tuberous sclerosis complex (TSC). Those with TSC without intellectual disability had significantly lower mean IQ compared to unaffected siblings. Earlier onset and greater severity of seizures in the first 2 years were observed in individuals with a slower gain in intellectual ability. Risk pathways through seizures in the first 2 years predict long-term cognitive outcomes in individuals with TSC. [ABSTRACT FROM AUTHOR]

Progress in proteomics research has led to a demand for powerful analytical tools with high separation efficiency and sensitivity for confident identification and quantification of proteins, posttranslational modifications, and protein complexes expressed in cells and tissues. This demand has significantly increased interest in capillary electrophoresis‐mass spectrometry (CE‐MS) in the past few years. This review provides highlights of recent advances in CE‐MS for proteomics research, including a short introduction to top‐down mass spectrometry and native mass spectrometry (native MS), as well as a detailed overview of CE methods. Both the potential and limitations of these methods for the analysis of proteins and peptides in synthetic and biological samples and the challenges of CE methods are discussed, along with perspectives about the future direction of CE‐MS. @ 2019 Wiley Periodicals, Inc. Mass Spec Rev 00:1–16, 2019. [ABSTRACT FROM AUTHOR]

Additive manufacturing of metallic components is regarded as one of the more exciting developments in engineering. The combined attractions of near net shape, tailored composition, and geometry optimisation have led to much interest in the various processes used and a drive to improve the mechanical properties to match those of wrought parts. In this paper, we reflect on the apparent lack of ambition in optimising the structural integrity of parts made using these new manufacturing processes. The current research focus seems to be either on largely irrelevant static properties, or on quantifying the fatigue response in a way that would be familiar to engineers in the 19th Century. Given the work on the role of microstructure and fatigue, which dates back to Ewing and Humphrey in 1903 reaching its zenith in the 1980s and 90s with Keith Miller in the vanguard, and recent developments in both imaging technologies and sophisticated numerical modelling, all the elements are in place for a much more rigorous, and ultimately more fruitful, approach to understand the structural integrity of additive manufactured components. [ABSTRACT FROM AUTHOR]

The Paip2 protein is a factor regulating mRNA translation and stability in the cytoplasm. It has also been found in the nuclei of several cell types in Drosophila. Here, we aim to elucidate the functions of Paip2 in the cell nucleus. We find that nuclear Paip2 is a component of an ~300‐kDa protein complex. Paip2 interacts with mRNA capping factor and factors of RNA polymerase II (Pol II) transcription initiation and early elongation. Paip2 functionally cooperates with the Cbp80 subunit of the cap‐binding complex, with both proteins ensuring proper Pol II C‐terminal domain (CTD) Ser5 phosphorylation at the promoter. Thus, Paip2 is a novel player at the stage of mRNA capping and early Pol II elongation. [ABSTRACT FROM AUTHOR]

A new combined experimental and modelling approach has been developed in order to understand the physical mechanisms that lead to crack nucleation in a polycrystalline aluminium alloy AA2024 undergoing cyclic loading. Four‐point bending low‐cycle fatigue tests were performed inside the chamber of a scanning electron microscope on specimens with a through‐thickness central hole, introduced to localize stresses and strains in a small region on the top surface of the sample. Fatigue crack initiation and small crack growth mechanisms were analyzed through high‐resolution scanning electron microscope images, local orientation measurements using electron‐back‐scattered‐diffraction, and local strain measurements using digital image correlation. A crystal plasticity finite element model was developed to simulate the cyclic deformation behaviour of AA2024. Two‐dimensional Voronoi‐based microstructures were generated, and the material parameters for the constitutive equations (including both isotropic and kinematic hardening) were identified using monotonic and fully reversed cyclic tests. A commonly used fatigue crack initiation criterion found in the literature, the maximum accumulated plastic slip, was evaluated in the crystal plasticity finite element model but could not predict the formation of cracks away from the edge of the hole in the deformed specimens. A new criterion combining 2 parameters: The maximum accumulated slip over each individual (critical) slip system and the maximum accumulated slip over all slip systems were formulated to reproduce the experimental locations of crack nucleation in the microstructure. [ABSTRACT FROM AUTHOR]

Summary: Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by mutations in TSC1 or TSC2. Epilepsy occurs in 80%‐90% of affected individuals during their lifetime, and up to one‐third of children with TSC will develop epileptic (infantile) spasms, for which vigabatrin has been shown to be particularly effective. Epilepsy severity and epileptic spasms are consistent markers of risk for the development of intellectual impairment in TSC. Although previous studies demonstrate a bimodal distribution of intellectual ability in TSC, recent findings suggest a unimodal distribution, which may reflect a change in IQ distribution over time. We compared 3 large historical UK cohorts of TSC (n = 331) that show varied distributions of intellectual ability, first ruling out differences in study methodology. Later‐born individuals had a higher frequency of reported spasms and higher likelihood of vigabatrin administration, but were less likely to have profound intellectual impairment, compared to the earlier‐born individuals. Our findings suggest that epileptic spasms went undetected in the older patients and therefore were not treated, leading to a higher occurrence of profound impairment, whereas the later born cohort had better access to treatment. These findings support the importance of early identification and treatment of seizures in TSC. [ABSTRACT FROM AUTHOR]

Pachytene pi RNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from pi RNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived pi RNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved pi RNA pathway protein Maelstrom ( MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene pi RNA primary transcripts. We provide evidence of functional significance of these complexes in Mael 129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael 129-null mutant testes possess low levels of pi RNAs derived from MAEL-associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor pi RNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene pi RNAs and MIWI. [ABSTRACT FROM AUTHOR]

MBF and SBF transcription factors regulate a large family of coordinately expressed G1/S genes required for early cell-cycle functions including DNA replication and repair. SBF is inactivated upon S-phase entry by Clb/CDK whereas MBF targets are repressed by the co-repressor, Nrm1. Using genome-wide expression analysis of cells treated with methyl methane sulfonate (MMS), hydroxyurea (HU) or camptothecin (CPT), we show that genotoxic stress during S phase specifically induces MBF-regulated genes. This occurs via direct phosphorylation of Nrm1 by Rad53, the effector checkpoint kinase, which prevents its binding to MBF target promoters. We conclude that MBF-regulated genes are distinguished from SBF-regulated genes by their sensitivity to activation by the S-phase checkpoint, thereby, providing an effective mechanism for enhancing DNA replication and repair and promoting genome stability. [ABSTRACT FROM AUTHOR]

The cognitive impairment in patients with Alzheimer’s disease is closely associated with synaptic loss in the neocortex and limbic system. Although the neurotoxic effects of aggregated amyloid-β oligomers in Alzheimer’s disease have been studied extensively in experimental models, less is known about the characteristics of these aggregates across the spectrum of Alzheimer’s disease. In this study, postmortem frontal cortex samples from controls and patients with Alzheimer’s disease were fractionated and analyzed for levels of oligomers and synaptic proteins. We found that the levels of oligomers correlated with the severity of cognitive impairment (blessed information-memory-concentration score and mini-mental state examination) and with the loss of synaptic markers. Reduced levels of the synaptic vesicle protein, vesicle-associated membrane protein-2, and the postsynaptic protein, postsynaptic density-95, correlated with the levels of oligomers in the various fractions analyzed. The strongest associations were found with amyloid-β dimers and pentamers. Co-immunoprecipitation and double-labeling experiments supported the possibility that amyloid-β and postsynaptic density-95 interact at synaptic sites. Similarly, in transgenic mice expressing high levels of neuronal amyloid precursor protein, amyloid-β co-immunoprecipitated with postsynaptic density-95. This was accompanied by a decrease in the levels of the postsynaptic proteins Shank1 and Shank3 in patients with Alzheimer’s disease and in the brains of amyloid precursor protein transgenic mice. In conclusion, this study suggests that the presence of a subpopulation of amyloid-β oligomers in the brains of patients with Alzheimer’s disease might be related to alterations in selected synaptic proteins and cognitive impairment. Structured digital abstract • : Shank3 (uniprotkb: ), SAPAP1 (uniprotkb: ) and Shank1 (uni-protkb: ) colocalize ( ) by cosedimentation in solution ( ) • : A-beta (uniprotkb: ) and A-beta (uniprotkb: ) physically interact ( ) by cosedimentation in solution ( ) • : A-beta (uniprotkb: ) physically interacts ( ) with PSD95 (uniprotkb: ) by anti bait coimmunoprecipitation ( ) • , , : A-beta (uniprotkb: ) physically interacts ( ) with Psd95 (uniprotkb: ) by anti bait coimmunoprecipitation ( ) • : Psd95 (uniprotkb: ) and Map2 (uniprotkb: ) colocalize ( ) by fluorescence microscopy ( ) • : SHANK3 (uniprotkb: ), SHANK1 (uniprotkb: ) and SAPAP1 (uniprotkb: ) colocalize ( ) by cosedimentation in solution ( ) • : A-beta (uniprotkb: ) and A-beta (uniprotkb: ) physically interact ( ) by cosedimentation in solution ( ) • : A-beta (uniprotkb: ) and Psd95 (uniprotkb: ) colocalize ( ) by cosedimentation in solution ( ) • : A-beta (uniprotkb: ) and Psd95 (uniprotkb: ) colocalize ( ) by cosedimentation in solution ( ) [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-mediated ABA signalling. [ABSTRACT FROM AUTHOR]

Pontin (Pont) and Reptin (Rept) are paralogous ATPases that are evolutionarily conserved from yeast to human. They are recruited in multiprotein complexes that function in various aspects of DNA metabolism. They are essential for viability and have antagonistic roles in tissue growth, cell signalling and regulation of the tumour metastasis suppressor gene, KAI1, indicating that the balance of Pont and Rept regulates epigenetic programmes critical for development and cancer progression. Here, we describe Pont and Rept as antagonistic mediators of Drosophila Hox gene transcription, functioning with Polycomb group (PcG) and Trithorax group proteins to maintain correct patterns of expression. We show that Rept is a component of the PRC1 PcG complex, whereas Pont purifies with the Brahma complex. Furthermore, the enzymatic functions of Rept and Pont are indispensable for maintaining Hox gene expression states, highlighting the importance of these two antagonistic factors in transcriptional output. [ABSTRACT FROM AUTHOR]