Multivalent Proteins Rapidly and Reversibly Phase-Separate upon Osmotic Cell Volume Change.

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.

BNP-Track: a framework for superresolved tracking.

Superresolution tools, such as PALM and STORM, provide nanoscale localization accuracy by relying on rare photophysical events, limiting these methods to static samples. By contrast, here, we extend superresolution to dynamics without relying on photodynamics by simultaneously determining emitter numbers and their tracks (localization and linking) with the same localization accuracy per frame as widefield superresolution on immobilized emitters under similar imaging conditions (≈50 nm). We demonstrate our Bayesian nonparametric track (BNP-Track) framework on both in cellulo and synthetic data. BNP-Track develops a joint (posterior) distribution that learns and quantifies uncertainty over emitter numbers and their associated tracks propagated from shot noise, camera artifacts, pixelation, background and out-of-focus motion. In doing so, we integrate spatiotemporal information into our distribution, which is otherwise compromised by modularly determining emitter numbers and localizing and linking emitter positions across frames. For this reason, BNP-Track remains accurate in crowding regimens beyond those accessible to other single-particle tracking tools.

Dynamic Recruitment of Single RNAs to Processing Bodies Depends on RNA Functionality.

Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles.

Biological liquid-liquid phase separation has gained considerable attention in recent years as a driving force for the assembly of subcellular compartments termed membraneless organelles. The field has made great strides in elucidating the molecular basis of biomolecular phase separation in various disease, stress response, and developmental contexts. Many important biological consequences of such "condensation" are now emerging from in vivo studies. Here we review recent work from our group and others showing that many proteins undergo rapid, reversible condensation in the cellular response to ubiquitous environmental fluctuations such as osmotic changes. We discuss molecular crowding as an important driver of condensation in these responses and suggest that a significant fraction of the proteome is poised to undergo phase separation under physiological conditions. In addition, we review methods currently emerging to visualize, quantify, and modulate the dynamics of intracellular condensates in live cells. Finally, we propose a metaphor for rapid phase separation based on cloud formation, reasoning that our familiar experiences with the readily reversible condensation of water droplets help understand the principle of phase separation. Overall, we provide an account of how biological phase separation supports the highly intertwined relationship between the composition and dynamic internal organization of cells, thus facilitating extremely rapid reorganization in response to internal and external fluctuations.

Systemic inflammatory syndrome in COVID-19-SISCoV study: systematic review and meta-analysis.

BACKGROUND: There has been a recent upsurge in the cases of Multisystem inflammatory syndrome in children (MIS-C) associated with Coronavirus disease (COVID-19). We performed a systematic review and meta-analysis on the demographic profile, clinical characteristics, complications, management, and prognosis of this emerging novel entity. METHODS: Using a predefined search strategy incorporating MeSH terms and keywords, all known literature databases were searched up till 10th July 2020. The review was done in accordance with PRISMA guidelines and registered in PROSPERO (CRD4202019757). RESULTS: Of the 862 identified publications, 18 studies comprising 833 patients were included for meta-analysis. The socio-demographic profile showed male predilection (p = 0.0085) with no significant racial predisposition. A higher incidence of gastrointestinal symptoms (603/715, 84.3%), myocarditis (191/309, 61.8%), left ventricular dysfunction (190/422, 45.0%), pericardial (135/436, 31.0%) and neurological symptoms (138/602, 22.9%) was reported. Serological evidence of SARS-CoV-2 had higher sensitivity compared to rtPCR (291/800, 36.4% vs 495/752, 65.8%; p < 0.001). Coronary artery anomaly (CAA) was reported in 117/681 in 9 publications (17.2%). A total of 13 (1.6%) fatalities were reported. CONCLUSION: Clinicians need to be vigilant in identifying the constellation of these symptoms in children with clinical or epidemiologic SARS-CoV-2 infection. Early diagnosis and treatment lead to a favorable outcome. IMPACT: Key message This review analyses the demographic profile, clinical spectrum, management strategies, prognosis, and pathophysiology of MIS-C among children with SARS-CoV-2 infection. The stark differences of MIS-C from Kawasaki disease with respect to demographics and clinical spectrum is addressed. Over-reliance on rtPCR for diagnosis can miss the diagnosis of MIS-C. New addition to existing literature The first systematic review and meta-analysis of published literature on MIS-C associated with COVID-19. IMPACT: The article will serve to spread awareness among the clinicians regarding this emerging novel entity, so that diagnosis can be made early and management can be initiated promptly.

High Body Mass Index and Change in Cervical Carotid Artery Position on Serial Scans: A Retrospective Study of an Unexplained Phenomenon.

OBJECTIVE: The purpose of this study was to investigate the relationship between increased body mass index (BMI) and abdominal circumference and prevalence of the change of position of common and internal carotid arteries on serial imaging, termed wandering carotid artery. METHODS: We retrospectively reviewed computed tomography/magnetic resonance imaging neck scans and determined whether the common and internal carotid arteries moved in position on serial scans. We correlated patients' demographic and medical information along with abdominal circumference, BMI, location of the aortic arch, and area of soft tissue surrounding the carotid arteries with the prevalence of a wandering carotid artery. RESULTS: Computed tomography/magnetic resonance imaging neck performed on 56 randomly selected patients between 2017 and 2020 were reviewed. A total of 42.9% of the patients had a wandering common or internal carotid artery. The abdominal circumference and BMI were significantly higher in the patients with wandering carotid arteries compared with the patients without (abdominal circumference = 102.9 ± 14.13 vs 91.61 ± 13.9 cm [ P = 0.01] and BMI = 34.27 ± 8.58 [obese] vs 26.21 ± 4.89 [overweight, P = 0.0001]). After adjusting for age, sex, hypertension, diabetes, atherosclerosis grade, and aortic arch location, the odds of wandering carotid artery was 1.23 (95% confidence interval, 1.1-1.44) times higher for every one-unit increase in BMI. CONCLUSIONS: There is a higher prevalence of wandering common and internal carotid arteries in obese patients with large abdominal circumference irrespective of age, sex, diabetes, hypertension, or carotid atherosclerosis.

From "Cellular" RNA to "Smart" RNA: Multiple Roles of RNA in Genome Stability and Beyond.

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

Cellular Growth Arrest and Persistence from Enzyme Saturation.

Metabolic efficiency depends on the balance between supply and demand of metabolites, which is sensitive to environmental and physiological fluctuations, or noise, causing shortages or surpluses in the metabolic pipeline. How cells can reliably optimize biomass production in the presence of metabolic fluctuations is a fundamental question that has not been fully answered. Here we use mathematical models to predict that enzyme saturation creates distinct regimes of cellular growth, including a phase of growth arrest resulting from toxicity of the metabolic process. Noise can drive entry of single cells into growth arrest while a fast-growing majority sustains the population. We confirmed these predictions by measuring the growth dynamics of Escherichia coli utilizing lactose as a sole carbon source. The predicted heterogeneous growth emerged at high lactose concentrations, and was associated with cell death and production of antibiotic-tolerant persister cells. These results suggest how metabolic networks may balance costs and benefits, with important implications for drug tolerance.

"Are They Listening, and Do They Find It Useful?"-Evaluation of Mid-Rotation Formative Subjective and Objective Feedback to Radiology Trainees.

BACKGROUND: Residents commonly receive only end-of-rotation evaluations and thus are often unaware of their progress during a rotation. In 2021, our neuroradiology section instituted mid-rotation feedback in which rotating residents received formative subjective and objective feedback. The purpose of this study was to describe our feedback method and to evaluate if residents found it helpful. METHODS: Radiology residents rotate 3-4 times on the neuroradiology service for 1-month blocks. At the midpoint of the rotation (2 weeks), 7-10 neuroradiology attendings discussed the rotating residents' subjective performance. One attending was tasked with facilitating this discussion and taking notes. Objective metrics were obtained from our dictation software. Compiled feedback was relayed to residents via email. A 16-question anonymous survey was sent to 39 radiology residents (R1-R4) to evaluate the perceived value of mid-rotation feedback. Odds ratios and 95% confidence intervals were computed using logistic regression. RESULTS: Sixty-nine percent (27/39) of residents responded to the survey; 92.6% (25/27) of residents reported receiving mid-rotation feedback in ≥50% of neuroradiology rotations; 92.3% (24/26) of residents found the subjective feedback helpful; 88.4% (23/26) of residents reported modifying their performance as suggested (100% R1-R2 vs 70% R3-R4; OR: 15.4 CI:1.26, >30.0);59.1% (13/22) of residents found the objective metrics helpful (75% R1-R2 vs 40% R3-R4; OR: 3.92 CI:0.74, 24.39) and 68.2% (15/22) stated they modified their performance based on these metrics (83.3% R1-R2 vs 50.0% R3-R4; OR:4.2 CI:0.73, 30.55); and 84.6% (22/26) of residents stated that mid-rotation subjective feedback and 45.5% (10/22) stated that mid-rotation objective feedback should be implemented in other sections. CONCLUSIONS: Majority of residents found mid-rotation feedback to be helpful in informing them about their progress and areas for improvement in the neuroradiology rotation, more so for subjective feedback than objective feedback. The majority of residents stated all rotations should provide mid-rotation subjective feedback.

Intrinsically disordered sequences can tune fungal growth and the cell cycle for specific temperatures.

Temperature can impact every reaction essential to a cell. For organisms that cannot regulate their own temperature, adapting to temperatures that fluctuate unpredictably and on variable timescales is a major challenge. Extremes in the magnitude and frequency of temperature changes are increasing across the planet, raising questions as to how the biosphere will respond. To examine mechanisms of adaptation to temperature, we collected wild isolates from different climates of the fungus Ashbya gossypii, which has a compact genome of only ∼4,600 genes. We found control of the nuclear division cycle and polarized morphogenesis, both critical processes for fungal growth, were temperature sensitive and varied among the isolates. The phenotypes were associated with naturally varying sequences within the glutamine-rich region (QRR) IDR of an RNA-binding protein called Whi3. This protein regulates both nuclear division and polarized growth via its ability to form biomolecular condensates. In cells and in cell-free reconstitution assays, we found that temperature tunes the properties of Whi3-based condensates. Exchanging Whi3 sequences between isolates was sufficient to rescue temperature-sensitive phenotypes, and specifically, a heptad repeat sequence within the QRR confers temperature-sensitive behavior. Together, these data demonstrate that sequence variation in the size and composition of an IDR can promote cell adaptation to growth at specific temperature ranges. These data demonstrate the power of IDRs as tuning knobs for rapid adaptation to environmental fluctuations.

Fluorogenic RNA-based biomaterials for imaging and tracking the cargo of extracellular vesicles.

Extracellular vesicles (EVs), or exosomes, play important roles in physiological and pathological cellular communication and have gained substantial traction as biological drug carriers. EVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. To fully capitalize on the potential of EVs as drug carriers, it is important to study and understand the intricacies of EV function and EV RNA-based communication. Here we developed a genetically encodable RNA-based biomaterial, termed EXO-Probe, for tracking EV RNAs. The EXO-Probe comprises an EV-loading RNA sequence (EXO-Code), fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of EV RNA and colocalization with markers of multivesicular bodies; imaging RNA within EVs, and non-destructive quantification of EVs. Overall, the new RNA-based biomaterial provides a useful and versatile means to interrogate the role of EVs in cellular communication via RNA trafficking to EVs and to study cellular sorting decisions. The system will also help lay the foundation to further improve the therapeutic efficacy of EVs as drug carriers.

Introduction to Techniques Used to Study Mosquito Neuroanatomy and Neural Circuitry.

Mosquitoes transmit deadly pathogens from person to person as they obtain the blood meal that is essential for their life cycle. Female mosquitoes of many species are unable to reproduce without consuming protein that they obtain from blood. This developmental stage makes them highly efficient disease vectors of deadly pathogens. They can transmit pathogens between members of the same species and different species that can provide a route for evolving zoonotic viruses to jump from animals to humans. One possible way to develop novel strategies to combat pathogen transmission by mosquitoes is to study the sensory systems that drive mosquito reproductive behaviors, in particular the neural architecture and circuits of mosquito sensory afferent neurons, the central circuits that process sensory information, and the downstream circuits that drive reproductive behaviors. The study of mosquito neuroanatomy and circuitry also benefits basic neuroscience, allowing for comparative neuroanatomy in insect species, which has great value in the current model species-heavy landscape of neuroscience. Here, we introduce two important techniques that are used to study neuroanatomy and neural circuitry-namely, immunofluorescent labeling and neural tracing. We describe how to apply these approaches to study mosquito neuroanatomy and describe considerations for researchers using the techniques.

"The Up and Down Approach" to Localize and Exclude Sites of Intraspinal CSF Leaks.

Myelography is a commonly performed procedure to locate cerebrospinal fluid (CSF) leaks in patients with spontaneous intracranial hypotension. Often, the site of leak within the spinal canal cannot be located creating a diagnostic dilemma for clinicians. This technical report describes a novel method to locate and exclude intraspinal CSF leaks in patients with multiple potential sites of CSF leak using a lumbar and cervical approach to inject intrathecal contrast and subsequently performing CT myelography.

Head and neck vessel magnetic resonance angiography appearance and artifacts after therapeutic intravenous ferumoxytol infusion.

Intravenous ferumoxytol infusions are an effective treatment option for iron deficiency anemia. Ferumoxytol contains a superparamagnetic iron oxide core which causes artifacts on multiple MRI brain sequences. However, in our experience, there is not much information on the appearance of intracranial and neck vessels on MR angiography (MRA) after recent therapeutic i.v. administration of ferumoxytol. MRA is an integral part of the work-up for multiple diseases processes including for acute stroke and for detection of aneurysm(s), vasculopathy/vasculitis, vascular malformations, among others and are often performed without the acquisition of MRI brain. Without proper knowledge of the appearance of vessels after administration of i.v. feruomoxytol, radiologists may misinterpret the findings leading to unnecessary further investigation or errant diagnosis. We present the case of a patient who underwent MRI brain and MRA head and neck imaging after recent therapeutic i.v. infusion of ferumoxytol and discuss relevant imaging findings and imaging artifact caused by this medication.

Utilizing functional cell-free extracts to dissect ribonucleoprotein complex biology at single-molecule resolution.

Cellular machineries that drive and regulate gene expression often rely on the coordinated assembly and interaction of a multitude of proteins and RNA together called ribonucleoprotein complexes (RNPs). As such, it is challenging to fully reconstitute these cellular machines recombinantly and gain mechanistic understanding of how they operate and are regulated within the complex environment that is the cell. One strategy for overcoming this challenge is to perform single molecule fluorescence microscopy studies within crude or recombinantly supplemented cell extracts. This strategy enables elucidation of the interaction and kinetic behavior of specific fluorescently labeled biomolecules within RNPs under conditions that approximate native cellular environments. In this review, we describe single molecule fluorescence microcopy approaches that dissect RNP-driven processes within cellular extracts, highlighting general strategies used in these methods. We further survey biological advances in the areas of pre-mRNA splicing and transcription regulation that have been facilitated through this approach. Finally, we conclude with a summary of practical considerations for the implementation of the featured approaches to facilitate their broader future implementation in dissecting the mechanisms of RNP-driven cellular processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Biomolecular condensates in fungi are tuned to function at specific temperatures.

Temperature can impact every reaction and molecular interaction essential to a cell. For organisms that cannot regulate their own temperature, a major challenge is how to adapt to temperatures that fluctuate unpredictability and on variable timescales. Biomolecular condensation offers a possible mechanism for encoding temperature-responsiveness and robustness into cell biochemistry and organization. To explore this idea, we examined temperature adaptation in a filamentous-growing fungus called Ashbya gossypii that engages biomolecular condensates containing the RNA-binding protein Whi3 to regulate mitosis and morphogenesis. We collected wild isolates of Ashbya that originate in different climates and found that mitotic asynchrony and polarized growth, which are known to be controlled by the condensation of Whi3, are temperature sensitive. Sequence analysis in the wild strains revealed changes to specific domains within Whi3 known to be important in condensate formation. Using an in vitro condensate reconstitution assay we found that temperature impacts the relative abundance of protein to RNA within condensates and that this directly impacts the material properties of the droplets. Finally, we found that exchanging Whi3 genes between warm and cold isolates was sufficient to rescue some, but not all, condensate-related phenotypes. Together these data demonstrate that material properties of Whi3 condensates are temperature sensitive, that these properties are important for function, and that sequence optimizes properties for a given climate.

BNP-Track: A framework for superresolved tracking.

Assessing dynamic processes at single molecule scales is key toward capturing life at the level of its molecular actors. Widefield superresolution methods, such as STORM, PALM, and PAINT, provide nanoscale localization accuracy, even when distances between fluorescently labeled single molecules ("emitters") fall below light's diffraction limit. However, as these superresolution methods rely on rare photophysical events to distinguish emitters from both each other and background, they are largely limited to static samples. In contrast, here we leverage spatiotemporal correlations of dynamic widefield imaging data to extend superresolution to simultaneous multiple emitter tracking without relying on photodynamics even as emitter distances from one another fall below the diffraction limit. We simultaneously determine emitter numbers and their tracks (localization and linking) with the same localization accuracy per frame as widefield superresolution does for immobilized emitters under similar imaging conditions (≈50nm). We demonstrate our results for both in cellulo data and, for benchmarking purposes, on synthetic data. To this end, we avoid the existing tracking paradigm relying on completely or partially separating the tasks of emitter number determination, localization of each emitter, and linking emitter positions across frames. Instead, we develop a fully joint posterior distribution over the quantities of interest, including emitter tracks and their total, otherwise unknown, number within the Bayesian nonparametric paradigm. Our posterior quantifies the full uncertainty over emitter numbers and their associated tracks propagated from origins including shot noise and camera artefacts, pixelation, stochastic background, and out-of-focus motion. Finally, it remains accurate in more crowded regimes where alternative tracking tools cannot be applied.

"We are everyone's ASHAs but who's there for us?" a qualitative exploration of perceptions of work stress and coping among rural frontline workers in Madhya Pradesh, India.

OBJECTIVE: More than a million female village-level lay providers called 'Accredited Social Health Activists (ASHAs)', who deliver primary care, face high levels of stress due to work demands and low compensation, within the context of poverty and gender inequality. Evidence on ASHAs has focused on workplace challenges from a system perspective, without sufficient probing into individual-level stress. This study aims to gain perspectives into the experiences of work stress, the related health symptoms, and the responses to stress among ASHAs in India. METHODS: Focus group discussions (FGDs) conducted with ASHAs in Sehore district, Madhya Pradesh, were audio-recorded and transcribed. Grounded theory was used to generate themes under the various domains of ASHAs' work and domestic life. We identified pathways between the conditions that trigger stressful events, experiences of these events, resulting perceptions, effects on health and wellbeing, and approaches used by ASHAs to respond to stress. RESULTS: Six FGDs with 59 ASHAs generated the following themes: (a) Facility: Workload, undue pressures, unstructured work; ASHAs' relationships with seniors (e.g., feelings of being disrespected, blamed, or targeted), and low access to physical and administrative resources; (b) Home: Feelings of guilt for putting less time for family/child care; disrespect by the elderly for a poorly incentivised job; (c) Community: Low acceptance by the villagers; caste- and gender-bias; difficult community-level relationships (emotional labour, fear/stigma towards her services); (d) Somatic and psychological symptoms: headache, exhaustion, depressive symptoms (to cite a few); and (e) Responses to stress: Motivation (support from peers, family, a sense of identity/pride, incentives), Individual strengths (e.g., social responsibility), and spiritual recourse mechanisms. CONCLUSIONS: This study will inform the development of a strengths-based coaching intervention to address work stress among ASHAs. The findings are relevant to building the evidence on alleviation of work stress among female frontline cadres in low-resource settings globally.

Unusual progression of osmotic demyelination after liver transplantation on MRI brain.

Osmotic demyelination syndrome, comprised of central pontine and extrapontine myelinolysis, is an important and potentially fatal complication primarily related to rapid overcorrection of serum sodium leading to devastating neurological symptoms. While traditionally presenting in the pons, we report the case of a 43-year-old female patient who recently underwent a liver transplant and developed extrapontine myelinolysis and subsequently central pontine myelinolysis resulting in irreversible spastic quadriparesis. This rare case highlights the variability of presentation of osmotic demyelination syndrome on imaging.

Membrane surfaces regulate assembly of ribonucleoprotein condensates.

Biomolecular condensates organize biochemistry, yet little is known about how cells control the position and scale of these structures. In cells, condensates often appear as relatively small assemblies that do not coarsen into a single droplet despite their propensity to fuse. Here, we report that ribonucleoprotein condensates of the glutamine-rich protein Whi3 interact with the endoplasmic reticulum, which prompted us to examine how membrane association controls condensate size. Reconstitution revealed that membrane recruitment promotes Whi3 condensation under physiological conditions. These assemblies rapidly arrest, resembling size distributions seen in cells. The temporal ordering of molecular interactions and the slow diffusion of membrane-bound complexes can limit condensate size. Our experiments reveal a trade-off between locally enhanced protein concentration at membranes, which favours condensation, and an accompanying reduction in diffusion, which restricts coarsening. Given that many condensates bind endomembranes, we predict that the biophysical properties of lipid bilayers are key for controlling condensate sizes throughout the cell.