Reconstruction of neocortex: Organelles, compartments, cells, circuits, and activity.

We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from ∼250 × 140 × 90 µm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.

Functional characterization of the dural sinuses as a neuroimmune interface.

Despite the established dogma of central nervous system (CNS) immune privilege, neuroimmune interactions play an active role in diverse neurological disorders. However, the precise mechanisms underlying CNS immune surveillance remain elusive; particularly, the anatomical sites where peripheral adaptive immunity can sample CNS-derived antigens and the cellular and molecular mediators orchestrating this surveillance. Here, we demonstrate that CNS-derived antigens in the cerebrospinal fluid (CSF) accumulate around the dural sinuses, are captured by local antigen-presenting cells, and are presented to patrolling T cells. This surveillance is enabled by endothelial and mural cells forming the sinus stromal niche. T cell recognition of CSF-derived antigens at this site promoted tissue resident phenotypes and effector functions within the dural meninges. These findings highlight the critical role of dural sinuses as a neuroimmune interface, where brain antigens are surveyed under steady-state conditions, and shed light on age-related dysfunction and neuroinflammatory attack in animal models of multiple sclerosis.

The Mind of a Mouse.

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.

A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC.

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.

Oligodendrocyte precursor cells ingest axons in the mouse neocortex.

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

A first look at consistency of documentation across care settings during emergency transitions of long-term care residents.

BACKGROUND: Documentation during resident transitions from long-term care (LTC) to the emergency department (ED) can be inconsistent, leading to inappropriate care. Inconsistent documentation can lead to undertreatment, inefficiencies and adverse patient outcomes. Many individuals residing in LTC have some form of cognitive impairment and may not be able to advocate for themselves, making accurate and consistent documentation vital to ensuring they receive safe care. We examined documentation consistency related to reason for transfer across care settings during these transitions. METHODS: We included residents of LTC aged 65 or over who experienced an emergency transition from LTC to the ED via emergency medical services. We used a standardized and pilot-tested tracking tool to collect resident chart/patient record data. We collected data from 38 participating LTC facilities to two participating EDs in Western Canadian provinces. Using qualitative directed content analysis, we categorized documentation from LTC to the ED by sufficiency and clinical consistency. RESULTS: We included 591 eligible transitions in this analysis. Documentation was coded as consistent, inconsistent, or ambiguous. We identified the most common reasons for transition for consistent cases (falls), ambiguous cases (sudden change in condition) and inconsistent cases (falls). Among inconsistent cases, three subcategories were identified: insufficient reporting, potential progression of a condition during transition and unclear reasons for inconsistency. CONCLUSIONS: Shared continuing education on documentation across care settings should result in documentation supports geriatric emergency care; on-the-job training needs to support reporting of specific signs and symptoms that warrant an emergent response, and discourage the use of vague descriptors.

Evidence for a feeding related association between melanocortin in the NTS and Neuropeptide-Y in the PVN.

Melanocortin and neuropeptide-Y (NPY) are both involved in feeding and energy regulation, and they have opposite effects in the paraventricular nucleus of the hypothalamus (PVN). The present study examined an interaction between melanocortin in the nucleus of the solitary tract (NTS) and NPY in the PVN. Male Sprague-Dawley rats were implanted with cannulae in the injection sites of interest. In Experiment 1, subjects received either the melanocortin 3/4-receptor (MC3/4) antagonist SHU9119 (0, 10, 50 and 100 pmol/0.5 µl) or the MC3/4 agonist MTII (0, 10, 50, 100 and 200 pmol/0.5 µl) into the NTS. Food intake was measured at 1, 2, 4, 6 and 24-h post-injection. Administration of SHU9119 into the NTS significantly and dose-dependently increased food intake at 1, 2, 4, 6 and 6-24-h, and administration of MTII into the NTS significantly and dose-dependently decreased 24-h free feeding. In Experiment 2, subjects received the MC3/4 agonist MTII (0, 10, 50, 100 and 200 pmol/0.5 µl) into the NTS just prior to NPY (0 and 1µg/0.5 µl) in the PVN. PVN injection of NPY stimulated feeding, and administration of MTII (50, 100 and 200 pmol) into the NTS significantly and dose-dependently decreased NPY-induced feeding at 2, 4, 6 and 6-24-h. These data suggest that there could be a neuronal association between melanocortin in the NTS and NPY in the PVN, and that the melanocortin system in the NTS has an antagonistic effect on NPY-induced feeding in the PVN.

Incidence of Pressure Injury Among Older Adults Transitioning from Long-term Care to the ED.

OBJECTIVE: To identify sociodemographic, health condition, and organizational/process factors associated with pressure injury (PI) incidence during older adults' emergency transitions from long-term care (LTC) to the ED. METHODS: Emergency transitions were tracked for older adults within included LTC facilities to participating EDs in two urban centers located in provinces in Canada. Binary logistic regression was used to examine the influence of sociodemographic, service use, and client health and function factors on the incidence of PIs during transitions from LTC facilities to EDs. RESULTS: Having a mobility issue (odds ratio [OR], 4.318; 95% CI, 1.344-13.870), transitioning from a publicly owned versus a nonprofit volunteer LTC facility (OR, 4.886; 95% CI, 1.157-20.634), and time from ED arrival to return to LTC being 7 to 9 days (OR, 41.327; 95% CI, 2.691-634.574) or greater than 9 days (OR, 77.639; 95% CI, 5.727-1,052.485) significantly increased the odds of experiencing a new skin injury upon return to LTC. A higher number of reported reasons for emergency transition (up to 4) significantly decreased the odds of a new PI upon return to LTC (OR, 0.315; 95% CI, 0.113-0.880). CONCLUSIONS: The study findings can be used to identify LTC residents at increased risk for developing new skin injuries during an emergency transition, namely, those with mobility impairment, those requiring inpatient care for 6 or more days, and those transitioning from publicly owned LTC facilities. Evaluating the uptake and effectiveness of single-pronged and multipronged interventions such as visual cues for patient turning through online monitoring, consistent risk assessments, and improved nutrition in all care settings are vital next steps in preventing skin injuries in this population.

Drivers of fungal diversity and community biogeography differ between green roofs and adjacent ground-level green space.

Green roof soils are usually engineered for purposes other than urban biodiversity, which may impact their fungal communities, and in turn impact the health of plants in the urban ecosystem. We examined the drivers of fungal diversity and community composition in soil of green roofs and adjacent ground-level green spaces in three Midwestern USA cities-Chicago, Cleveland, and Minneapolis. Overall, fungal communities on green roofs were more diverse than ground-level green spaces and were correlated with plant cover (positively) and roof age (negatively) rather than abiotic soil properties. Fungal community composition was distinct between roof and ground environments, among cities, and between sampling sites, but green roofs and their immediately surrounding ground-level green space showed some similarity. This suggests dispersal limitation may result in geographic structuring at large spatial scales, but dispersal between roofs and their neighbouring sites may be occurring. Different fungal taxonomic and functional groups were better explained when roofs were classified either by depth (extensive or intensive) or functional intent of the roof design (i.e. stormwater/energy, biodiversity, or aesthetics/recreation). Our results demonstrate that green roofs are an important reservoir of fungal diversity in the urban landscape, which should be considered in future green roof design.

Anatomy and function of an excitatory network in the visual cortex.

Circuits in the cerebral cortex consist of thousands of neurons connected by millions of synapses. A precise understanding of these local networks requires relating circuit activity with the underlying network structure. For pyramidal cells in superficial mouse visual cortex (V1), a consensus is emerging that neurons with similar visual response properties excite each other, but the anatomical basis of this recurrent synaptic network is unknown. Here we combined physiological imaging and large-scale electron microscopy to study an excitatory network in V1. We found that layer 2/3 neurons organized into subnetworks defined by anatomical connectivity, with more connections within than between groups. More specifically, we found that pyramidal neurons with similar orientation selectivity preferentially formed synapses with each other, despite the fact that axons and dendrites of all orientation selectivities pass near (<5 µm) each other with roughly equal probability. Therefore, we predict that mechanisms of functionally specific connectivity take place at the length scale of spines. Neurons with similar orientation tuning formed larger synapses, potentially enhancing the net effect of synaptic specificity. With the ability to study thousands of connections in a single circuit, functional connectomics is proving a powerful method to uncover the organizational logic of cortical networks.

Influences of post-implementation factors on the sustainability, sustainment, and intra-organizational spread of complex interventions.

BACKGROUND: Complex interventions are increasingly applied to healthcare problems. Understanding of post-implementation sustainment, sustainability, and spread of interventions is limited. We examine these phenomena for a complex quality improvement initiative led by care aides in 7 care homes (long-term care homes) in Manitoba, Canada. We report on factors influencing these phenomena two years after implementation. METHODS: Data were collected in 2019 via small group interviews with unit- and care home-level managers (n = 11) from 6 of the 7 homes using the intervention. Interview participants discussed post-implementation factors that influenced continuing or abandoning core intervention elements (processes, behaviors) and key intervention benefits (outcomes, impact). Interviews were audio-recorded, transcribed verbatim, and analyzed with thematic analysis. RESULTS: Sustainment of core elements and sustainability of key benefits were observed in 5 of the 6 participating care homes. Intra-unit intervention spread occurred in 3 of 6 homes. Factors influencing sustainment, sustainability, and spread related to intervention teams, unit and care home, and the long-term care system. CONCLUSIONS: Our findings contribute understanding on the importance of micro-, meso-, and macro-level factors to sustainability of key benefits and sustainment of some core processes. Inter-unit spread relates exclusively to meso-level factors of observability and practice change institutionalization. Interventions should be developed with post-implementation sustainability in mind and measures taken to protect against influences such as workforce instability and competing internal and external demands. Design should anticipate need to adapt interventions to strengthen post-implementation traction.

The Symptom Experience of Patients With Atrial Fibrillation Before Their Initial Diagnosis.

BACKGROUND: Atrial fibrillation (AF), the most common form of dysrhythmia, steadily increases in prevalence with age. If left untreated, AF significantly increases the risk of stroke, heart failure, and death. Despite the increasing prevalence, there are significant research gaps in the prediagnosis symptom experiences of patients with AF. OBJECTIVE: The purpose of this qualitative descriptive study was to explore the prediagnosis symptom experience of patients with AF. METHODS: Participants 19 years or older with AF diagnosed in the previous year were recruited (n = 26) from outpatient cardiac rehabilitation and AF clinics. Semistructured interviews, broadly guided by the Symptom Experience Model, explored perceptions, evaluations, and responses to AF symptoms. Thematic analysis used a 2-step approach, deductively coding for participants' symptom perceptions, evaluations, and responses and inductively coding within these broader Symptom Experience Model concepts. RESULTS: Perception involved awareness of bodily sensations, ranging from imperceptible noticing to commanding attention, heightened by rest and activity. In evaluation, participants used self-derived theories to explain their symptoms, gathered evidence to support/refute their theories, and formulated alternative theories as symptoms changed over time. Responses consisted of nontreatment, self-treatment, and health seeking; most participants needed repeated healthcare visits before diagnosis. CONCLUSIONS: The current study identified challenges participants experienced in developing awareness of their AF symptoms, the complex cognitive processes associated with evaluation, and barriers that made it difficult to respond to AF symptoms in a timely manner. Understanding the prediagnosis symptom experience from the patient's perspective is essential for the enhancement of current AF screening practices.

Visual physiology of the layer 4 cortical circuit in silico.

Despite advances in experimental techniques and accumulation of large datasets concerning the composition and properties of the cortex, quantitative modeling of cortical circuits under in-vivo-like conditions remains challenging. Here we report and publicly release a biophysically detailed circuit model of layer 4 in the mouse primary visual cortex, receiving thalamo-cortical visual inputs. The 45,000-neuron model was subjected to a battery of visual stimuli, and results were compared to published work and new in vivo experiments. Simulations reproduced a variety of observations, including effects of optogenetic perturbations. Critical to the agreement between responses in silico and in vivo were the rules of functional synaptic connectivity between neurons. Interestingly, after extreme simplification the model still performed satisfactorily on many measurements, although quantitative agreement with experiments suffered. These results emphasize the importance of functional rules of cortical wiring and enable a next generation of data-driven models of in vivo neural activity and computations.

STRIDER NZAus: a multicentre randomised controlled trial of sildenafil therapy in early-onset fetal growth restriction.

OBJECTIVE: To assess the effect of maternal sildenafil therapy on fetal growth in pregnancies with early-onset fetal growth restriction. DESIGN: A randomised placebo-controlled trial. SETTING: Thirteen maternal-fetal medicine units across New Zealand and Australia. POPULATION: Women with singleton pregnancies affected by fetal growth restriction at 22+0 to 29+6 weeks. METHODS: Women were randomised to oral administration of 25 mg sildenafil citrate or visually matching placebo three times daily until 32+0 weeks, birth or fetal death (whichever occurred first). MAIN OUTCOME MEASURES: The primary outcome was the proportion of pregnancies with an increase in fetal growth velocity. Secondary outcomes included live birth, survival to hospital discharge free of major neonatal morbidity and pre-eclampsia. RESULTS: Sildenafil did not affect the proportion of pregnancies with an increase in fetal growth velocity; 32/61 (52.5%) sildenafil-treated, 39/57 (68.4%) placebo-treated [adjusted odds ratio (OR) 0.49, 95% CI 0.23-1.05] and had no effect on abdominal circumference Z-scores (P = 0.61). Sildenafil use was associated with a lower mean uterine artery pulsatility index after 48 hours of treatment (1.56 versus 1.81; P = 0.02). The live birth rate was 56/63 (88.9%) for sildenafil-treated and 47/59 (79.7%) for placebo-treated (adjusted OR 2.50, 95% CI 0.80-7.79); survival to hospital discharge free of major neonatal morbidity was 42/63 (66.7%) for sildenafil-treated and 33/59 (55.9%) for placebo-treated (adjusted OR 1.93, 95% CI 0.84-4.45); and new-onset pre-eclampsia was 9/51 (17.7%) for sildenafil-treated and 14/55 (25.5%) for placebo-treated (OR 0.67, 95% CI 0.26-1.75). CONCLUSIONS: Maternal sildenafil use had no effect on fetal growth velocity. Prospectively planned meta-analyses will determine whether sildenafil exerts other effects on maternal and fetal/neonatal wellbeing. TWEETABLE ABSTRACT: Maternal sildenafil use has no beneficial effect on growth in early-onset FGR, but also no evidence of harm.

Regulatory T Cells Promote Myositis and Muscle Damage in Toxoplasma gondii Infection.

The coordination of macrophage polarization is essential for the robust regenerative potential of skeletal muscle. Repair begins with a phase mediated by inflammatory monocytes (IM) and proinflammatory macrophages (M1), followed by polarization to a proregenerative macrophage (M2) phenotype. Recently, regulatory T cells (Tregs) were described as necessary for this M1 to M2 transition. We report that chronic infection with the protozoan parasite Toxoplasma gondii causes a nonresolving Th1 myositis with prolonged tissue damage associated with persistent M1 accumulation. Surprisingly, Treg ablation during chronic infection rescues macrophage homeostasis and skeletal muscle fiber regeneration, showing that Tregs can directly contribute to muscle damage. This study provides evidence that the tissue environment established by the parasite could lead to a paradoxical pathogenic role for Tregs. As such, these findings should be considered when tailoring therapies directed at Tregs in inflammatory settings.

Hormone therapy and breast cancer: risk communication and the 'perfect storm'.

Many factors are considered when a woman estimates her personal risk of breast cancer. Common to most decisions are four separate influences that have convinced the public and many health-care providers that breast cancer is the greatest concern for menopausal women and that menopausal hormone therapy (MHT) is generally responsible. Historically there have been well-documented situations in which big pharma and doctors have not put patient interests first. Conflicting reports about the safety of MHT and the media imperative to always increase readership by presenting a compelling scary story have created an underlying distrust of science, doctors, and MHT. Numerical and statistical illiteracy in the general population creates a situation where lotteries succeed despite astronomical odds and the risks of medical interventions are exaggerated by their description using relative, rather than absolute, risks. Finally, mammographic overdiagnosis contributing to improved breast cancer survival has contributed to the 'popularity paradox' (more screening - more enthusiasm) especially among survivors and advocacy groups. As a result, worry about breast cancer has overshadowed concern about cardiovascular diseases as the major cause of death and disability in the later years. The ongoing challenge for clinicians dealing with menopausal women is to bridge the gap in risk perception with evidence-based common-sense advice.

Inferring cortical function in the mouse visual system through large-scale systems neuroscience.

The scientific mission of the Project MindScope is to understand neocortex, the part of the mammalian brain that gives rise to perception, memory, intelligence, and consciousness. We seek to quantitatively evaluate the hypothesis that neocortex is a relatively homogeneous tissue, with smaller functional modules that perform a common computational function replicated across regions. We here focus on the mouse as a mammalian model organism with genetics, physiology, and behavior that can be readily studied and manipulated in the laboratory. We seek to describe the operation of cortical circuitry at the computational level by comprehensively cataloging and characterizing its cellular building blocks along with their dynamics and their cell type-specific connectivities. The project is also building large-scale experimental platforms (i.e., brain observatories) to record the activity of large populations of cortical neurons in behaving mice subject to visual stimuli. A primary goal is to understand the series of operations from visual input in the retina to behavior by observing and modeling the physical transformations of signals in the corticothalamic system. We here focus on the contribution that computer modeling and theory make to this long-term effort.

Evolution of models to support community and policy action with science: Balancing pastoral livelihoods and wildlife conservation in savannas of East Africa.

We developed a "continual engagement" model to better integrate knowledge from policy makers, communities, and researchers with the goal of promoting more effective action to balance poverty alleviation and wildlife conservation in 4 pastoral ecosystems of East Africa. The model involved the creation of a core boundary-spanning team, including community facilitators, a policy facilitator, and transdisciplinary researchers, responsible for linking with a wide range of actors from local to global scales. Collaborative researcher-facilitator community teams integrated local and scientific knowledge to help communities and policy makers improve herd quality and health, expand biodiversity payment schemes, develop land-use plans, and fully engage together in pastoral and wildlife policy development. This model focused on the creation of hybrid scientific-local knowledge highly relevant to community and policy maker needs. The facilitation team learned to be more effective by focusing on noncontroversial livelihood issues before addressing more difficult wildlife issues, using strategic and periodic engagement with most partners instead of continual engagement, and reducing costs by providing new scientific information only when deemed essential. We conclude by examining the role of facilitation in redressing asymmetries in power in researcher-community-policy maker teams, the role of individual values and character in establishing trust, and how to sustain knowledge-action links when project funding ends.

Glucose regulates protein turnover and growth-related mechanisms in rainbow trout myogenic precursor cells.

Rainbow trout are considered glucose intolerant because they are poor utilizers of glucose, despite having functional insulin receptors and glucose transporters. Following high carbohydrate meals, rainbow trout are persistently hyperglycemic, which is likely due to low glucose utilization in peripheral tissues including the muscle. Also, rainbow trout myogenic precursor cells (MPCs) treated in vitro with insulin and IGF1 increase glucose uptake and protein synthesis, whereas protein degradation is decreased. Given our understanding of glucose regulation in trout, we sought to understand how glucose concentrations affect protein synthesis, protein degradation; and expression of genes associated with muscle growth and proteolysis in MPCs. We found that following 24 h and 48 h of treatment with low glucose media (5.6 mM), myoblasts had significant decreases in protein synthesis. Also, low glucose treatments affected the expression of both mstn2a and igfbp5. These findings support that glucose is a direct regulator of protein synthesis and growth-related mechanisms in rainbow trout muscle.