ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure.

Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

Compositional Control of Phase-Separated Cellular Bodies.

Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-separated liquids organized by multivalent interactions among proteins and RNA molecules. Although many components of various cellular bodies are known, general principles that define body composition are lacking. We modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold molecules form phase-separated liquids that concentrate low valency client proteins. Clients partition differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. Composition can switch rapidly through changes in scaffold concentration or valency. Natural PML NBs and P bodies show analogous partitioning behavior, suggesting how their compositions could be controlled by levels of PML SUMOylation or cellular mRNA concentration, respectively. The data suggest a conceptual framework for considering the composition and control thereof of cellular bodies assembled through heterotypic multivalent interactions.

A global timing mechanism regulates cell-type-specific wiring programmes.

The assembly of neural circuits is dependent on precise spatiotemporal expression of cell recognition molecules1-5. Factors controlling cell type specificity have been identified6-8, but how timing is determined remains unknown. Here we describe induction of a cascade of transcription factors by a steroid hormone (ecdysone) in all fly visual system neurons spanning target recognition and synaptogenesis. We demonstrate through single-cell sequencing that the ecdysone pathway regulates the expression of a common set of targets required for synaptic maturation and cell-type-specific targets enriched for cell-surface proteins regulating wiring specificity. Transcription factors in the cascade regulate the expression of the same wiring genes in complex ways, including activation in one cell type and repression in another. We show that disruption of the ecdysone pathway generates specific defects in dendritic and axonal processes and synaptic connectivity, with the order of transcription factor expression correlating with sequential steps in wiring. We also identify shared targets of a cell-type-specific transcription factor and the ecdysone pathway that regulate specificity. We propose that neurons integrate a global temporal transcriptional module with cell-type-specific transcription factors to generate different cell-type-specific patterns of cell recognition molecules regulating wiring.

The Stress Granule Transcriptome Reveals Principles of mRNA Accumulation in Stress Granules.

Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here, we describe the stress granule transcriptome of yeast and mammalian cells through RNA-sequencing (RNA-seq) analysis of purified stress granule cores and single-molecule fluorescence in situ hybridization (smFISH) validation. While essentially every mRNA, and some noncoding RNAs (ncRNAs), can be targeted to stress granules, the targeting efficiency varies from <1% to >95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-seq analysis by smFISH reveals that only 10% of bulk mRNA molecules accumulate in mammalian stress granules and that only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest that stress granules may not represent a specific biological program of messenger ribonucleoprotein (mRNP) assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes.

Isolation of mammalian stress granule cores for RNA-Seq analysis.

Stress granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian stress granules contain stable structures, referred to as "cores" that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from stress granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of stress granules and may assist in the discovery of the assembly mechanisms and functions of stress granules in a variety of biological contexts.

Isolation of yeast and mammalian stress granule cores.

Stress granules are dynamic, conserved RNA-protein (RNP) assemblies that form when translation is limiting; and are related to pathological aggregates in degenerative disease. Mammalian stress granules are comprised of two structures - an unstable shell and more stable cores. Herein we describe methodology for isolation of stress granule cores from both yeast and mammalian cells. The protocol consists of first enriching for stress granule cores using centrifugation and then further purifying stress granule cores using immunoprecipitation. The stress granule core isolation protocol provides a starting point for assisting future endeavors aimed at discovering conserved RNA regulatory mechanisms and potential links between RNP aggregation and degenerative disease.

Designing a Naturally Inspired Conductive Copolymer to Engineer Wearable Bioadhesives for Sensing Applications.

The design of adhesive and conductive soft hydrogels using biopolymers with tunable mechanical properties has received significant interest in the field of wearable sensors for detecting human motions. These hydrogels are primarily fabricated through the modification of biopolymers to introduce cross-linking sites, the conjugation of adhesive components, and the incorporation of conductive materials into the hydrogel network. The development of a multifunctional copolymer that integrates adhesive and conductive properties within a single polymer chain with suitable cross-linking sites eliminates the need for biopolymer modification and the addition of extra conductive and adhesive components. In this study, we synthesized a copolymer based on poly([2-(methacryloyloxy)ethyl] trimethylammonium chloride-co-dopamine methacrylamide) (p(METAC-DMA)) using a controlled radical polymerization, allowing for the efficient conjugation of both adhesive and conductive units within a single polymer chain. Subsequently, our multifunctional hydrogel named Gel-MD was fabricated by mixing the p(METAC-DMA) copolymer with non-modified gelatin in which cross-linking took place in an oxidative environment. We confirmed the biocompatibility of the Gel-MD hydrogel through in vitro studies using NIH 3T3 cells as well as in vivo subcutaneous implantation in rats. Furthermore, the Gel-MD hydrogel was effective and sensitive in detecting various human motions, making it a promising wearable sensor for health monitoring and diagnosis.

A Highly Stretchable, Conductive, and Transparent Bioadhesive Hydrogel as a Flexible Sensor for Enhanced Real-Time Human Health Monitoring.

Real-time continuous monitoring of non-cognitive markers is crucial for the early detection and management of chronic conditions. Current diagnostic methods are often invasive and not suitable for at-home monitoring. An elastic, adhesive, and biodegradable hydrogel-based wearable sensor with superior accuracy and durability for monitoring real-time human health is developed. Employing a supramolecular engineering strategy, a pseudo-slide-ring hydrogel is synthesized by combining polyacrylamide (pAAm), ß-cyclodextrin (ß-CD), and poly 2-(acryloyloxy)ethyltrimethylammonium chloride (AETAc) bio ionic liquid (Bio-IL). This novel approach decouples conflicting mechano-chemical effects arising from different molecular building blocks and provides a balance of mechanical toughness (1.1 × 106 Jm-3), flexibility, conductivity (≈0.29 S m-1), and tissue adhesion (≈27 kPa), along with rapid self-healing and remarkable stretchability (≈3000%). Unlike traditional hydrogels, the one-pot synthesis avoids chemical crosslinkers and metallic nanofillers, reducing cytotoxicity. While the pAAm provides mechanical strength, the formation of the pseudo-slide-ring structure ensures high stretchability and flexibility. Combining pAAm with ß-CD and pAETAc enhances biocompatibility and biodegradability, as confirmed by in vitro and in vivo studies. The hydrogel also offers transparency, passive-cooling, ultraviolet (UV)-shielding, and 3D printability, enhancing its practicality for everyday use. The engineered sensor demonstratesimproved efficiency, stability, and sensitivity in motion/haptic sensing, advancing real-time human healthcare monitoring.

Spatial profiling of the interplay between cell type- and vision-dependent transcriptomic programs in the visual cortex.

How early sensory experience during "critical periods" of postnatal life affects the organization of the mammalian neocortex at the resolution of neuronal cell types is poorly understood. We previously reported that the functional and molecular profiles of layer 2/3 (L2/3) cell types in the primary visual cortex (V1) are vision-dependent (Tan et al., Neuron, 108(4), 2020; Cheng et al., Cell, 185(2), 2022). Here, we characterize the spatial organization of L2/3 cell types with and without visual experience. Spatial transcriptomic profiling based on 500 genes recapitulates the zonation of L2/3 cell types along the pial-ventricular axis in V1. By applying multi-tasking theory (Adler et al., Cell Systems, 8, 2019), we suggest that the spatial zonation of L2/3 cell types is linked to the continuous nature of their gene expression profiles, which can be represented as a 2D manifold bounded by three archetypal cell types ("A", "B", and "C"). By comparing normally reared and dark reared L2/3 cells, we show that visual deprivation-induced transcriptomic changes comprise two independent gene programs. The first, induced specifically in the visual cortex, includes immediate-early genes and genes associated with metabolic processes. It manifests as a change in cell state that is orthogonal to cell type-specific gene expression programs. By contrast, the second program impacts L2/3 cell type identity, regulating a subset of cell type-specific genes and shifting the distribution of cells within the L2/3 manifold, with a depression of the B-type and C-type and a gain of the A-type. Through an integrated analysis of spatial transcriptomic measurements with single-nucleus RNA-seq data from our previous study, we describe how vision patterns L2/3 cortical cell types during the postnatal critical period. Significance statement: Layer 2/3 (L2/3) glutamatergic neurons are important sites of experience-dependent plasticity and learning in the mammalian cortex. Their properties vary continuously with cortical depth and depend upon experience. Here, by applying spatial transcriptomics and different computational approaches in the mouse primary visual cortex, we show that vision regulates orthogonal gene expression programs underlying cell states and cell types. Visual deprivation not only induces an activity-dependent cell state, but also alters the composition of L2/3 cell types, which are appropriately described as a transcriptomic continuum. Our results provide insights into how experience shapes transcriptomes that may, in turn, sculpt brain wiring, function, and behavior.

Human Skeletal Muscle Myoblast Culture in Aligned Bacterial Nanocellulose and Commercial Matrices.

Bacterial nanocellulose (BNC) is a durable, flexible, and dynamic biomaterial capable of serving a wide variety of fields, sectors, and applications within biotechnology, healthcare, electronics, agriculture, fashion, and others. BNC is produced spontaneously in carbohydrate-rich bacterial culture media, forming a cellulosic pellicle via a nanonetwork of fibrils extruded from certain genera. Herein, we demonstrate engineering BNC-based scaffolds with tunable physical and mechanical properties through postprocessing. Human skeletal muscle myoblasts (HSMMs) were cultured on these scaffolds, and in vitro electrical stimulation was applied to promote cellular function for tissue engineering applications. We compared physiologic maturation markers of human skeletal muscle myoblast development using a 2.5-dimensional culture paradigm in fabricated BNC scaffolds, compared to two-dimensional (2D) controls. We demonstrate that the culture of human skeletal muscle myoblasts on BNC scaffolds developed under electrical stimulation produced highly aligned, physiologic morphology of human skeletal muscle myofibers compared to unstimulated BNC and standard 2D culture. Furthermore, we compared an array of metrics to assess the BNC scaffold in a rigorous head-to-head study with commercially available, clinically approved matrices, Kerecis Omega3 Wound Matrix (Marigen) and Phoenix as well as a gelatin methacryloyl (GelMA) hydrogel. The BNC scaffold outcompeted industry standard matrices as well as a 20% GelMA hydrogel in durability and sustained the support of human skeletal muscle myoblasts in vitro. This work offers a robust demonstration of BNC scaffold cytocompatibility with human skeletal muscle cells and sets the basis for future work in healthcare, bioengineering, and medical implant technological development.

IgA Levels among Type 2 Diabetic and Non-Diabetic Patients with Periodontitis: A Prospective Clinical Study.

OBJECTIVES: To estimate gingival crevicular immunoglobulin A(IgA) using enzyme-linked immunosorbent assay (ELISA) among type II diabetic patients with periodontitis. MATERIALS AND METHODS: A non-randomized study was done of 40 periodontitis subjects with a mean age of 50 years and were recruited into two groups, Group A (Type II controlled diabetics with HbA1c < 7%) and Group B (non-diabetics with HbA1c between 4 and 6%). Both the groups underwent nonsurgical periodontal therapy (NSPT). The clinical parameters were recorded at baseline, 1, and 3 months. GCF sample was collected for the estimation of crevicular IgA at baseline and at 3 months. STATISTICAL ANALYSIS: Results were analyzed using parametric tests paired t-test and Student's t-test for every assessment point. The level of significance was set at p < 0.05. RESULTS: Difference in IgA levels and clinical parameters was seen between diabetic and non-diabetic groups, which was statistically significant. CONCLUSION: Changes in crevicular IgA levels in patients with diabetic periodontitis can be used as a novel biomarker in assessing the inflammatory status.

Ascertaining Framingham heart failure phenotype from inpatient electronic health record data using natural language processing: a multicentre Atherosclerosis Risk in Communities (ARIC) validation study.

OBJECTIVES: Using free-text clinical notes and reports from hospitalised patients, determine the performance of natural language processing (NLP) ascertainment of Framingham heart failure (HF) criteria and phenotype. STUDY DESIGN: A retrospective observational study design of patients hospitalised in 2015 from four hospitals participating in the Atherosclerosis Risk in Communities (ARIC) study was used to determine NLP performance in the ascertainment of Framingham HF criteria and phenotype. SETTING: Four ARIC study hospitals, each representing an ARIC study region in the USA. PARTICIPANTS: A stratified random sample of hospitalisations identified using a broad range of International Classification of Disease, ninth revision, diagnostic codes indicative of an HF event and occurring during 2015 was drawn for this study. A randomly selected set of 394 hospitalisations was used as the derivation dataset and 406 hospitalisations was used as the validation dataset. INTERVENTION: Use of NLP on free-text clinical notes and reports to ascertain Framingham HF criteria and phenotype. PRIMARY AND SECONDARY OUTCOME MEASURES: NLP performance as measured by sensitivity, specificity, positive-predictive value (PPV) and agreement in ascertainment of Framingham HF criteria and phenotype. Manual medical record review by trained ARIC abstractors was used as the reference standard. RESULTS: Overall, performance of NLP ascertainment of Framingham HF phenotype in the validation dataset was good, with 78.8%, 81.7%, 84.4% and 80.0% for sensitivity, specificity, PPV and agreement, respectively. CONCLUSIONS: By decreasing the need for manual chart review, our results on the use of NLP to ascertain Framingham HF phenotype from free-text electronic health record data suggest that validated NLP technology holds the potential for significantly improving the feasibility and efficiency of conducting large-scale epidemiologic surveillance of HF prevalence and incidence.

Brain Signals to Rescue Aphasia, Apraxia and Dysarthria Speech Recognition.

In this paper, we propose a deep learning-based algorithm to improve the performance of automatic speech recognition (ASR) systems for aphasia, apraxia, and dysarthria speech by utilizing electroencephalography (EEG) features recorded synchronously with aphasia, apraxia, and dysarthria speech. We demonstrate a significant decoding performance improvement by more than 50% during test time for isolated speech recognition task and we also provide preliminary results indicating performance improvement for the more challenging continuous speech recognition task by utilizing EEG features. The results presented in this paper show the first step towards demonstrating the possibility of utilizing non-invasive neural signals to design a real-time robust speech prosthetic for stroke survivors recovering from aphasia, apraxia, and dysarthria. Our aphasia, apraxia, and dysarthria speech-EEG data set will be released to the public to help further advance this interesting and crucial research.

Augmented reality in patient education and health literacy: a scoping review protocol.

BACKGROUND: Health literacy enables the patients in understanding the basic healthcare information and taking informed health decisions; thus, it is a desirable goal of any healthcare system. It increases patients' adherence to treatment, improves the quality of care and eases the overall burden on the healthcare system. In recent years, technological solutions are being increasingly used in educating patients and achieving better health literacy. Augmented reality (AR) provides powerful, contextual and situated learning experiences and supplements the real world with virtual objects. AR could potentially be an effective learning methodology for the patients, thus, warranting a comprehensive overview of the current state of AR in patient education and health literacy. METHODS: The proposed scoping review will be based on the framework developed by Arksey and O'Malley, including the refinements suggested by Levac et al. A systematic search for references in the published literature will be conducted in nine research databases-Institute of Electrical and Electronics Engineers (IEEE), Cumulative Index to Nursing and Allied Health Literature (CINAHL), PubMed, PsycInfo, Embase, Web of Science, Scopus, Association for Computing Machinery (ACM) and Association for Information Systems eLibrary (AISeL). The unpublished studies from ProQuest Dissertations and Theses, Conference Proceedings Citation Index and grey literature references obtained from a web search will also be included. Databases will be searched from inception to 14 January 2020. Two independent reviewers will screen the studies from the search results in two successive stages of title/abstract screening followed by full-text screening. Data variables will be extracted from the selected studies to characterise study design, type of AR technology employed and the relational factors affecting patient education. Lastly, key stakeholders will be consulted to gather their insights about the study findings. ETHICS AND DISSEMINATION: The results will be disseminated through stakeholder meetings and conference presentations. The data used are from publicly available secondary sources, so this study does not require ethical review.

Generation of a glycosylated asparagine residue through chemoselective acylation of a glycosylhydrazide.

Herein, we report the first selective anomeric N-acylation of a glycosylhydrazide. We show that this transformation can be harnessed to generate amino acid building blocks including FmocAsn(GlcNAc)OH (1), a residue that has been previously shown to be a competent reagent in the solid-phase peptide synthesis of N-linked glycopeptides.

Distinct stages in stress granule assembly and disassembly.

Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.

Global analysis of yeast mRNPs.

Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNA-protein complexes (mRNPs) is essential to understanding these processes. In a global survey of Saccharomyces cerevisiae mRNA-binding proteins, we identified 120 proteins that cross-link to mRNA, including 66 new mRNA-binding proteins. These include kinases, RNA-modification enzymes, metabolic enzymes and tRNA- and rRNA-metabolism factors. These proteins show dynamic subcellular localization during stress, including assembly into stress granules and processing bodies (P bodies). Cross-linking and immunoprecipitation (CLIP) analyses of the P-body components Pat1, Lsm1, Dhh1 and Sbp1 identified sites of interaction on specific mRNAs, revealing positional binding preferences and co-assembly preferences. When taken together, this work defines the major yeast mRNP proteins, reveals widespread changes in their subcellular location during stress and begins to define assembly rules for P-body mRNPs.