Fast and sensitive GCaMP calcium indicators for imaging neural populations.

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

Transcriptomic mapping uncovers Purkinje neuron plasticity driving learning.

Cellular diversification is critical for specialized functions of the brain including learning and memory1. Single-cell RNA sequencing facilitates transcriptomic profiling of distinct major types of neuron2-4, but the divergence of transcriptomic profiles within a neuronal population and their link to function remain poorly understood. Here we isolate nuclei tagged5 in specific cell types followed by single-nucleus RNA sequencing to profile Purkinje neurons and map their responses to motor activity and learning. We find that two major subpopulations of Purkinje neurons, identified by expression of the genes Aldoc and Plcb4, bear distinct transcriptomic features. Plcb4+, but not Aldoc+, Purkinje neurons exhibit robust plasticity of gene expression in mice subjected to sensorimotor and learning experience. In vivo calcium imaging and optogenetic perturbation reveal that Plcb4+ Purkinje neurons have a crucial role in associative learning. Integrating single-nucleus RNA sequencing datasets with weighted gene co-expression network analysis uncovers a learning gene module that includes components of FGFR2 signalling in Plcb4+ Purkinje neurons. Knockout of Fgfr2 in Plcb4+ Purkinje neurons in mice using CRISPR disrupts motor learning. Our findings define how diversification of Purkinje neurons is linked to their responses in motor learning and provide a foundation for understanding their differential vulnerability to neurological disorders.

SLEAP: A deep learning system for multi-animal pose tracking.

The desire to understand how the brain generates and patterns behavior has driven rapid methodological innovation in tools to quantify natural animal behavior. While advances in deep learning and computer vision have enabled markerless pose estimation in individual animals, extending these to multiple animals presents unique challenges for studies of social behaviors or animals in their natural environments. Here we present Social LEAP Estimates Animal Poses (SLEAP), a machine learning system for multi-animal pose tracking. This system enables versatile workflows for data labeling, model training and inference on previously unseen data. SLEAP features an accessible graphical user interface, a standardized data model, a reproducible configuration system, over 30 model architectures, two approaches to part grouping and two approaches to identity tracking. We applied SLEAP to seven datasets across flies, bees, mice and gerbils to systematically evaluate each approach and architecture, and we compare it with other existing approaches. SLEAP achieves greater accuracy and speeds of more than 800 frames per second, with latencies of less than 3.5 ms at full 1,024 × 1,024 image resolution. This makes SLEAP usable for real-time applications, which we demonstrate by controlling the behavior of one animal on the basis of the tracking and detection of social interactions with another animal.

The final piece of the Triangle of U: Evolution of the tetraploid Brassica carinata genome.

Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species.

Study protocol: infectious diseases consortium (I3D) for study on integrated and innovative approaches for management of respiratory infections: respiratory infections research and outcome study (RESPIRO).

BACKGROUND: Community-acquired respiratory infections are a leading cause of illness and death globally. The aetiologies of community-acquired pneumonia remain poorly defined. The RESPIRO study is an ongoing prospective observational cohort study aimed at developing pragmatic logistical and analytic platforms to accurately identify the causes of moderate-to-severe community-acquired pneumonia in adults and understand the factors influencing disease caused by individual pathogens. The study is currently underway in Singapore and has plans for expansion into the broader region. METHODS: RESPIRO is being conducted at three major tertiary hospitals in Singapore. Adults hospitalised with acute community-acquired pneumonia or lower respiratory tract infections, based on established clinical, laboratory and radiological criteria, will be recruited. Over the course of the illness, clinical data and biological samples will be collected longitudinally and stored in a biorepository for future analysis. DISCUSSION: The RESPIRO study is designed to be hypothesis generating, complementary to and easily integrated with other research projects and clinical trials. The detailed clinical database and biorepository will yield insights into the epidemiology and outcomes of community-acquired lower respiratory tract infections in Singapore and the surrounding region and offers the opportunity to deeply characterise the microbiology and immunopathology of community-acquired pneumonia.

A systems framework for remedying dysfunction in US democracy.

Democracy often fails to meet its ideals, and these failures may be made worse by electoral institutions. Unwanted outcomes include elite polarization, unresponsive representatives, and the ability of a faction of voters to gain power at the expense of the majority. Various reforms have been proposed to address these problems, but their effectiveness is difficult to predict against a backdrop of complex interactions. Here we outline a path for systems-level modeling to help understand and optimize repairs to US democracy. Following the tradition of engineering and biology, models of systems include mechanisms with dynamical properties that include nonlinearities and amplification (voting rules), positive feedback mechanisms (single-party control, gerrymandering), negative feedback (checks and balances), integration over time (lifetime judicial appointments), and low dimensionality (polarization). To illustrate a systems-level approach, we analyze three emergent phenomena: low dimensionality, elite polarization, and antimajoritarianism in legislatures. In each case, long-standing rules now contribute to undesirable outcomes as a consequence of changes in the political environment. Theoretical understanding at a general level will also help evaluate whether a proposed reform's benefits will materialize and be lasting, especially as conditions change again. In this way, rigorous modeling may not only shape new lines of research but aid in the design of effective and lasting reform.

Urban people's preferences for life-sustaining treatment or artificial nutrition and hydration in advance decisions.

BACKGROUND: The Patient Right to Autonomy Act (PRAA), implemented in Taiwan in 2019, enables the creation of advance decisions (AD) through advance care planning (ACP). This legal framework allows for the withholding and withdrawal of life-sustaining treatment (LST) or artificial nutrition and hydration (ANH) in situations like irreversible coma, vegetative state, severe dementia, or unbearable pain. This study aims to investigate preferences for LST or ANH across various clinical conditions, variations in participant preferences, and factors influencing these preferences among urban residents. METHODS: Employing a survey of legally structured AD documents and convenience sampling for data collection, individuals were enlisted from Taipei City Hospital, serving as the primary trial and demonstration facility for ACP in Taiwan since the commencement of the PRAA in its inaugural year. The study examined ADs and ACP consultation records, documenting gender, age, welfare entitlement, disease conditions, family caregiving experience, location of ACP consultation, participation of second-degree relatives, and the intention to participate in ACP. RESULTS: Data from 2337 participants were extracted from electronic records. There was high consistency in the willingness to refuse LST and ANH, with significant differences noted between terminal diseases and extremely severe dementia. Additionally, ANH was widely accepted as a time-limited treatment, and there was a prevalent trend of authorizing a health care agent (HCA) to make decisions on behalf of participants. Gender differences were observed, with females more inclined to decline LST and ANH, while males tended towards accepting full or time-limited treatment. Age also played a role, with younger participants more open to treatment and authorizing HCA, and older participants more prone to refusal. CONCLUSION: Diverse preferences in LST and ANH were shaped by the public's current understanding of different clinical states, gender, age, and cultural factors. Our study reveals nuanced end-of-life preferences, evolving ADs, and socio-demographic influences. Further research could explore evolving preferences over time and healthcare professionals' perspectives on LST and ANH decisions for neurological patients..

Demographics, employment, and proximity to covid-19 as predictors of preparedness among Taiwanese health social workers.

COVID-19 struck the world violently and cause negative psychological consequences on health professionals. The preparedness of social workers for the pandemic is critical while facing these challenges and pressures. The study aimed to explore what are the roles of demographic, employment, and proximity to Covid-19 in predicting preparedness for the next wave of COVID among social workers in Taiwan. A total of 158 participants were conveniently sampled and multiple regression, univariate analysis, and two-way ANOVA were conducted. The results demonstrated that the demographic and employment variables significantly predicted preparedness, and there were significant differences among demographics on preparedness and an interaction effect between seniority and age. Consequently, middle-aged social workers with junior seniority years may have more difficulties in their preparation for the current situation. The implication of our findings is also discussed.

Combination immunotherapy including OncoVEXmGMCSF creates a favorable tumor immune micro-environment in transgenic BRAF murine melanoma.

Talimogene Laherparepvec (OncoVEXmGMCSF), an oncolytic virus, immune checkpoint inhibitor anti-programmed cell death protein 1 (anti-PD1), and BRAF inhibition (BRAFi), are all clinically approved for treatment of melanoma patients and are effective through diverse mechanisms of action. Individually, these therapies also have an effect on the tumor immune microenvironment (TIME). Evaluating the combination effect of these three therapies on the TIME can help determine when combination therapy is most appropriate for further study. In this study, we use a transgenic murine melanoma model (Tyr::CreER; BRAFCA/+; PTENflox/flox), to evaluate the TIME in response to combinations of BRAFi, anti-PD1, and OncoVEXmGMCSF. We find that mice treated with the triple combination BRAFi + anti-PD1 + OncoVEXmGMCSF have decreased tumor growth compared to BRAFi alone and prolonged survival compared to control. Flow cytometry shows an increase in percent CD8 + /CD3 + cytotoxic T Lymphocytes (CTLs) and a decrease in percent FOXP3 + /CD4 + T regulatory cells (Tregs) in tumors treated with OncoVEXmGMCSF compared to mice not treated with OncoVEXmGMCSF. Immunogenomic analysis at 30d post-treatment shows an increase in Th1 and interferon-related genes in mice receiving OncoVEXmGMCSF + BRAFi. In summary, treatment with combination BRAFi + anti-PD1 + OncoVEXmGMCSF is more effective than any single treatment in controlling tumor growth, and groups receiving OncoVEXmGMCSF had more tumoral infiltration of CTLs and less intratumoral Tregs in the TIME. This study provides rational basis to combine targeted agents, oncolytic viral therapy, and checkpoint inhibitors in the treatment of melanoma.

Fast animal pose estimation using deep neural networks.

The need for automated and efficient systems for tracking full animal pose has increased with the complexity of behavioral data and analyses. Here we introduce LEAP (LEAP estimates animal pose), a deep-learning-based method for predicting the positions of animal body parts. This framework consists of a graphical interface for labeling of body parts and training the network. LEAP offers fast prediction on new data, and training with as few as 100 frames results in 95% of peak performance. We validated LEAP using videos of freely behaving fruit flies and tracked 32 distinct points to describe the pose of the head, body, wings and legs, with an error rate of <3% of body length. We recapitulated reported findings on insect gait dynamics and demonstrated LEAP's applicability for unsupervised behavioral classification. Finally, we extended the method to more challenging imaging situations and videos of freely moving mice.

Publisher Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

In the version of this paper originally published, important figure labels in Fig. 3d were not visible. An image layer present in the authors' original figure that included two small dashed outlines and text labels indicating ROI 1 and ROI 2, as well as a scale bar and the name of the cell label, was erroneously altered during image processing. The figure has been corrected in the HTML and PDF versions of the paper.

Author Correction: Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

The version of this paper originally published cited a preprint version of ref. 12 instead of the published version (Proc. Natl. Acad. Sci. USA 115, 5594-5599; 2018), which was available before this Nature Methods paper went to press. The reference information has been updated in the PDF and HTML versions of the article.

Prompt successful response to a COVID-19 outbreak: Performance of community-based rapid screening station.

An outbreak occurred in Wanhua District of Taipei City. It was traced to a cluster infection originating from a teahouse. To prevent further large-scaled community spread, the Taipei City Government established the first community rapid test screening station. This report describes the station's strategy and performance and key factors that contributed to its operation. The project involves collaboration among various departments of Taipei City Government, including the health, environmental, police, transportation, and fire departments. The station provides rapid screening, polymerase chain reaction (PCR) testing, and immediate isolation and follow-up medical services upon the detection of a positive case. These services are accessible to local residents and are intended to ease hospitals' burdens. In 36 days, a total of 8532 people were tested, and 419 confirmed cases were identified. Over the same period, the weekly number of positive cases in Wanhua District decreased from 356 to 40, and the PCR positive rate decreased from 21.7% to 1.2%. The policy of establishing rapid screening station, contact tracing and mask wearing policy are key strategies for interrupting chains of transmission of COVID-19. This intervention has become a model for preventing the spread of the epidemic and establishing community rapid screening stations in Taiwan.

Virtual Reality-Based Education for Patients Undergoing Radiation Therapy.

We built a virtual reality (VR) application that runs on a commercially available standalone VR headset that allows patients to view a virtual simulation of themselves receiving radiotherapy. The purpose of this study was to determine if this experience can improve patient understanding of radiotherapy and/or reduce patient anxiety. We created software that reads data from our clinical treatment planning system and renders the plan on a life-size "virtual linear accelerator." The patient's CT simulation data is converted into a 3D translucent virtual human shown lying on the treatment table while visible yellow radiation beams are delivered to the target volumes in the patient. We conducted a prospective study to determine if showing patients their radiotherapy plan in VR improves patient education and/or reduces anxiety about treatment. A total of 43 patients were enrolled. The most common plans were 3D breast tangents and intensity-modulated radiotherapy prostate plans. Patients were administered pre- and post-experience questionnaires. Thirty-two patients (74%) indicated that they "strongly agree" that the VR session gave them a better understanding of how radiotherapy will be used to treat their cancer. Of the 21 patients who expressed any anxiety about radiotherapy beforehand, 12 (57%) said that the VR session helped decrease their anxiety about undergoing radiotherapy. In our single-institution, single-arm prospective patient study, we found that the majority of patients reported that the personalized VR experience was educational and can reduce anxiety. VR technology has potential to be a powerful adjunctive educational tool for cancer patients about to undergo radiotherapy.

Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR.

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

The p75NTR Influences Cerebellar Circuit Development and Adult Behavior via Regulation of Cell Cycle Duration of Granule Cell Progenitors.

Development of brain circuitry requires precise regulation and timing of proliferation and differentiation of neural progenitor cells. The p75 neurotrophin receptor (p75NTR) is highly expressed in the proliferating granule cell precursors (GCPs) during development of the cerebellum. In a previous paper, we showed that proNT3 promoted GCP cell cycle exit via p75NTR. Here we used genetically modified rats and mice of both sexes to show that p75NTR regulates the duration of the GCP cell cycle, requiring activation of RhoA. Rats and mice lacking p75NTR have dysregulated GCP proliferation, with deleterious effects on cerebellar circuit development and behavioral consequences persisting into adulthood. In the absence of p75NTR, the GCP cell cycle is accelerated, leading to delayed cell cycle exit, prolonged GCP proliferation, increased glutamatergic input to Purkinje cells, and a deficit in delay eyeblink conditioning, a cerebellum-dependent form of learning. These results demonstrate the necessity of appropriate developmental timing of the cell cycle for establishment of proper connectivity and associated behavior.SIGNIFICANCE STATEMENT The cerebellum has been shown to be involved in numerous behaviors in addition to its classic association with motor function. Cerebellar function is disrupted in a variety of psychiatric disorders, including those on the autism spectrum. Here we show that the p75 neurotrophin receptor, which is abundantly expressed in the proliferating cerebellar granule cell progenitors, regulates the cell cycle of these progenitors. In the absence of this receptor, the cell cycle is dysregulated, leading to excessive progenitor proliferation, which alters the balance of inputs to Purkinje cells, disrupting the circuitry and leading to functional deficits that persist into adulthood.

Is there an optimal timing between radiotherapy and surgery to reduce wound complications in metastatic spine disease? A systematic review.

PURPOSE: Surgery with radiation therapy (RT) is more effective in treating spinal metastases, than RT alone. However, RT when administered in close proximity to surgery may predispose to wound complications. There exist limited guidelines on the optimal timing between RT and surgery. The purpose of this systematic review is to: (1) address whether pre-operative RT (preop-RT) and/or post-operative RT (postop-RT) is associated with wound complications and (2) define the safe interval between RT and surgery or vice versa. METHODS: PubMed, Embase and Scopus databases were systematically searched for articles dealing with spinal metastases, treated with surgery and RT, and discussing wound status. RESULTS: We obtained 2332 articles from all databases, and after applying exclusion criteria, removing duplicates and reading the full text, we identified 27 relevant articles. Fourteen additional articles were identified by hand-search, leading to a total of 41 articles. All 41 mentioned wound complications/healing. Sixteen articles discussed preop-RT, 8 postop-RT, 15 both, and 2 mentioned intraoperative-RT with additional pre/postop-RT. Twenty studies mentioned surgery-RT time interval; one concluded that wound complications were higher when RT-surgery interval was ≤ 7 days. Seven studies reported significant association between preop-RT and wound complications. CONCLUSIONS: Evidence is insufficient to draw definitive conclusion about optimal RT-surgery interval. However, based on published literature and expert opinions, we conclude that an interval of 2 weeks, the minimum being 7 days, is optimum between RT-surgery or vice versa; this can be reduced further by postop-stereotactic body RT. If RT-surgery window is > 12 months, wound-complications rise. Postop-RT has fewer wound complications versus preop-RT.

PRE-WORKOUT SUPPLEMENT INDUCED CARDIAC ISCHAEMIA IN A YOUNG FEMALE.

The popularity of pre-workout supplements is rising amongst professional athletes and fitness enthusiasts. Despite increased usage, the safety profile of pre-workout supplements is likely to be not well understood. Additionally, many different brands use various undisclosed proprietary blends of active ingredients creating safety regulation difficulties. This lack of oversight could prove unsafe for certain patients. This patient MK is a 33-year-old healthy housewife who presented with central chest tightness, pre-syncope and mild dyspnoea to the emergency department via ambulance. The presentation was in the context of recent strenuous exercise and ingestion of a pre-workout supplement (Alpha Lean-7). Most striking in her presentation was a troponin rise of 50 ng/L, while not very high it is unusual given her lack of cardiac risk factors. She had a 3-day uneventful admission with a downtrending troponin prior to discharge. This case highlights the possible dangers of pharmacologically active ingredients in pre-workout supplements.