"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.

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.

"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.

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.

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.

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.

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.

"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.

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.

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.

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.

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.

A qualitative exploration of perceived needs and barriers of individuals with schizophrenia, caregivers and clinicians in using mental health applications in Madhya Pradesh, India.

Introduction: About 3.5 million people are living with schizophrenia in India, with most failing to receive minimally adequate care. Digital mental health applications could potentially decrease this treatment gap; however, these applications should be tailored to meet the needs and overcoming barriers of its end-users to ensure their adoption and sustained usage. Few studies in India have explored the perspectives of target stakeholders to understand how digital tools could be viable for supporting care. Therefore, this study explores the perceived needs and barriers of patients with schizophrenia, caregivers and clinicians in using digital mental health applications. Methods: Focus group discussions (FGDs) were conducted with patients having schizophrenia attending outpatient clinics at a government tertiary hospital, and their caregivers, and mental health clinicians in Bhopal, Madhya Pradesh, India. FGDs were audio-recorded and coded. Framework analysis was employed to guide the analysis, involving deductive and inductive generation of themes, data triangulation and comparison of perspectives between participant groups. Results: Six FGDs were conducted with individuals with schizophrenia (n â€‹= â€‹11), their caregivers (n â€‹= â€‹14), and mental health clinicians (n â€‹= â€‹19). Four a priori themes were established: a) Prior experiences with health applications; b) Content of a mental health application; c) Involvement of caregivers in mental health application usage and d) Supporting doctors' work through mental health applications. Additionally, two themes were generated inductively: a) Qualities of a mental health application and b) Data privacy and confidentiality. Conclusion: Exploration of stakeholder perspectives on the content, features, and uses of mental health applications is crucial to yield initial insights about the use of these digital programs in India. This study generated a multitude of suggestions on app functionality and components, which can guide ongoing efforts to develop and deliver digital mental health applications for patients living with schizophrenia in low-resource settings, with limited access to mental health services.

Radiology Performed Fluoroscopy-Guided Lumbar Punctures Decrease Volume of Diagnostic Study Interpretation - Impact on Resident Training and Potential Solutions.

OBJECTIVES: Lumbar punctures performed in radiology departments have significantly increased over the last few decades and are typically performed in academic centers by radiology trainees using fluoroscopy guidance. Performing fluoroscopy-guided lumbar punctures (FGLPs) can often constitute a large portion of a trainee's workday and the impact of performing FGLPs on the trainee's clinical productivity (i.e. dictating reports on neuroradiology cross-sectional imaging) has not been studied. The purpose of the study was to evaluate the relationship between the number of FGLPs performed and cross-sectional neuroimaging studies dictated by residents during their neuroradiology rotation (NR). MATERIAL AND METHODS: The number of FGLPs and myelograms performed and neuroimaging studies dictated by radiology residents on our neuroradiology service from July 2008 to December 2017 were retrospectively reviewed. The relationship between the number of FGLPs performed and neuroimaging studies (CT and MRI) dictated per day by residents was examined. RESULTS: Radiology residents (n = 84) performed 3437 FGLPs and myelograms and interpreted 33402 cross-sectional studies. Poisson regression demonstrated an exponential decrease in number of studies dictated daily with a rising number of FGLPs performed (P = 0.0001) and the following formula was derived: Number of expected studies dictated per day assuming no FGLPs × e-0.25 x number of FGLPs = adjusted expected studies dictated for the day. CONCLUSION: We quantified the impact performing FGLPs can have on the number of neuroimaging reports residents dictate on the NR. We described solutions to potentially decrease unnecessary FGLP referrals including establishing departmental guidelines for FGLP referrals and encouraging bedside lumbar punctures attempts before referral. We also emphasized equally distributing the FGLPs among trainees to mitigate procedural burden.

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.

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.

A qualitative assessment of barriers and facilitators to implementing recommended infant nutrition practices in Mumbai, India.

BACKGROUND: Childhood malnutrition has been a longstanding crisis in Mumbai, India. Despite national IYCF (Infant Young Child Feeding) guidelines to promote best practices for infant/toddler feeding, nearly one-third of children under age five are stunted or underweight. To improve child nutrition, interventions should address the cultural, social, and environmental influences on infant feeding practices. This study is an in-depth qualitative assessment of family barriers and facilitators to implementing recommended nutrition practices in two Mumbai slum communities, within the context of an existing nutrition education-based intervention by a local non-governmental non-profit organization. METHODS: The population was purposively sampled to represent a variety of household demographics. Data were collected through 33 in-depth semi-structured interviews with caregivers (mothers and paternal grandmothers) of children age 0-2 years. Transcripts were translated and transcribed, and analyzed using qualitative analysis procedures and software. RESULTS: A complex set of barriers and facilitators influence mothers'/caregivers' infant-toddler feeding practices. Most infants were fed complementary foods and non-nutritious processed snacks, counter to IYCF recommendations. Key barriers included: lack of nutrition knowledge and experience, receiving conflicting messages from different sources, limited social support, and poor self-efficacy for maternal decision-making. Key facilitators included: professional nutrition guidance, personal self-efficacy and empowerment, and family support. Interventions to improve child nutrition should address mothers'/caregivers' key barriers and facilitators to recommended infant-toddler feeding practices. CONCLUSIONS: Nutrition interventions should prioritize standard messaging across healthcare providers, engage all family members, target prevention of early introduction of sugary and non-nutritious processed foods, and strengthen maternal self-efficacy for following IYCF recommended guidelines.

Infrahyoid wandering carotid arteries.

Knowledge of retropharyngeal carotid arteries in patients is important in order to avoid potential catastrophic injuries. Previous studies demonstrated that carotid arteries can change in position at or near the level of the hyoid bone on serial scans. We report the presence of wandering carotid arteries at the level of the cricoid cartilage and superiorly in the neck of a 74-year-old female patient over multiple months. The potential for carotid arteries to randomly change positions in the suprahyoid and infrahyoid neck should be known by clinicians to avoid misdiagnosing occult neck masses and to avoid potential arterial injury during neck surgery. Although the exact etiology for wandering carotid arteries remains uncertain, we propose that reduced axial tension on carotid arteries and increased body mass Index may play a causative role.

Following the messenger: Recent innovations in live cell single molecule fluorescence imaging.

Messenger RNAs (mRNAs) convey genetic information from the DNA genome to proteins and thus lie at the heart of gene expression and regulation of all cellular activities. Live cell single molecule tracking tools enable the investigation of mRNA trafficking, translation and degradation within the complex environment of the cell and in real time. Over the last 5 years, nearly all tools within the mRNA tracking toolbox have been improved to achieve high-quality multi-color tracking in live cells. For example, the bacteriophage-derived MS2-MCP system has been improved to facilitate cloning and achieve better signal-to-noise ratio, while the newer PP7-PCP system now allows for orthogonal tracking of a second mRNA or mRNA region. The coming of age of epitope-tagging technologies, such as the SunTag, MoonTag and Frankenbody, enables monitoring the translation of single mRNA molecules. Furthermore, the portfolio of fluorogenic RNA aptamers has been expanded to improve cellular stability and achieve a higher fluorescence "turn-on" signal upon fluorogen binding. Finally, microinjection-based tools have been shown to be able to track multiple RNAs with only small fluorescent appendages and to track mRNAs together with their interacting partners. We systematically review and compare the advantages, disadvantages and demonstrated applications in discovering new RNA biology of this refined, expanding toolbox. Finally, we discuss developments expected in the near future based on the limitations of the current methods. This article is categorized under: RNA Export and Localization > RNA Localization RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

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.