Architecture and dynamics of a desmosome-endoplasmic reticulum complex.

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

Superresolution microscopy reveals actomyosin dynamics in medioapical arrays.

Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, superresolution approaches (grazing incidence structured illumination, GI-SIM, and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in Drosophila. In expanded cells, F-actin and myosin form loose, apically domed meshworks at the plasma membrane. The arrays condense as cells contract, drawing the domes into the plane of the junctional belts. As condensation continues, individual filaments are no longer uniformly apparent. As cells expand, arrays of actomyosin are again resolved-some F-actin turnover likely occurs, but a large fraction of existing filaments rearrange. In morphologically isotropic cells, actin filaments are randomly oriented and during contraction are drawn together but remain essentially randomly oriented. In anisotropic cells, largely parallel actin filaments are drawn closer to one another. Our images offer unparalleled resolution of F-actin in embryonic tissue, show that medioapical arrays are tightly apposed to the plasma membrane and are continuous with meshworks of lamellar F-actin. Medioapical arrays thereby constitute modified cell cortex. In concert with other tagged array components, superresolution imaging of live specimens will offer new understanding of cortical architecture and function.

Whole-cell organelle segmentation in volume electron microscopy.

Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes-ranging from endoplasmic reticulum to microtubules to ribosomes-in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM)1. We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, 'OpenOrganelle', to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.

An open-access volume electron microscopy atlas of whole cells and tissues.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome.

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite's satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.

YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

Waddell (Nonorganic) Signs and Their Association With Interventional Treatment Outcomes for Low Back Pain.

BACKGROUND: The rising use of injections to treat low back pain (LBP) has led to efforts to improve selection. Nonorganic (Waddell) signs have been shown to portend treatment failure for surgery and other therapies but have not been studied for minimally invasive interventions. METHODS: We prospectively evaluated the association between Waddell signs and treatment outcome in 3 cohorts: epidural steroid injections (ESI) for leg pain and sacroiliac joint (SIJ) injections and facet interventions for LBP. Categories of Waddell signs included nonanatomic tenderness, pain during sham stimulation, discrepancy in physical examination, overreaction, and regional disturbances divulging from neuroanatomy. The primary outcome was change in patient-reported "average" numerical rating scale for pain intensity (average NRS-PI), modeled as a function of the number of Waddell signs using simple linear regression. Secondary outcomes included a binary indicator of treatment response. We conducted secondary and sensitivity analyses to account for potential confounders. RESULTS: We enrolled 318 patients: 152 in the ESI cohort, 102 in the facet cohort, and 64 in the SIJ cohort, having sufficient data for primary analysis on 308 patients. Among these, 62% (n = 192) had no Waddell signs, 18% (n = 54) had 1 sign, 11% (n = 33) had 2, 5% (n = 16) had 3, 2% (n = 7) had 4, and about 2% (n = 6) had all 5 signs. The mean change in average NRS-PI in each of these 6 groups was -1.6 ± 2.6, -1.1 ± 2.7, -1.5 ± 2.5, -1.6 ± 2.6, -1 ± 1.5, and 0.7 ± 2.1, respectively, and their corresponding treatment failure rates were 54% (102 of 192), 67% (36 of 54), 70% (23 of 33), 75% (12 of 16), 71% (5 of 7), and 83% (5 of 6). In the primary analysis, an increasing number of Waddell signs were not associated with a significant decrease in average NRS-PI (coefficient [Coef] = 0.19; 95% confidence interval [CI], -0.43 to 0.05; P = .12). A higher number of Waddell signs were associated with treatment failure, with a 1.35 increased odds of treatment failure per cumulative number of signs (P = .008). CONCLUSIONS: Whereas this study found no consistent relationship between Waddell signs and decreased mean pain scores, a significant relationship between the number of Waddell signs and treatment failure was observed.

Correction to Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging.

[This corrects the article DOI: 10.1021/acscentsci.9b00676.].

Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging.

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant (K L-Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF526)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

Evaluating an Online Training Course to Develop and Sustain Cancer Navigation and Survivorship Programs.

OBJECTIVES: To evaluate the impact of the Executive Training on Navigation and Survivorship (Executive Training) online training course, designed by the George Washington University Cancer Center, on nurses and other healthcare professionals. SAMPLE & SETTING: A volunteer sample of 499 healthcare professionals, including nurses and patient navigators, were recruited through multiple Internet-based channels. METHODS & VARIABLES: Participants completed questionnaires before and after each module and at the end of the training. Descriptive statistics were calculated, and paired t tests were used to assess pre- and post-test learning confidence gains for each module. Qualitative feedback from participants was also summarized. RESULTS: From pre- to post-test, each group demonstrated statistically significant improvements in confidence (p < 0.05) for all seven training modules. Confidence gains were statistically significant for 19 of 20 learning objectives (p < 0.05). Overall rating scores and qualitative feedback were positive. IMPLICATIONS FOR NURSING: The Executive Training course prepares healthcare professionals from diverse backgrounds to establish navigation and survivorship programs. In addition, the training content addresses gaps in nursing education on planning and budgeting that can improve success.

Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III.

Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin's dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.

Oncology healthcare provider perspectives on caring for diverse patients fifteen years after Unequal Treatment.

OBJECTIVE: The purpose of this study was to provide a snapshot of U.S. oncology provider perspectives on caring for diverse patients, including self-rated awareness, comfort, skills, practices, challenges, facilitators, and barriers. METHODS: An online survey was administered to a convenience sample of multidisciplinary oncology providers. Descriptive statistics and bivariate analyses were computed for Likert-style items to investigate differences by level of past diversity training. Qualitative content analysis was conducted on open-response questions. RESULTS: Roughly one-third (36.7%) of the 406 survey respondents reported receiving high levels of past diversity training, with statistically significant differences by training amount for self-rated skills and select awareness and practice items (p < 0.05). Key challenges qualitatively described included language barriers (n = 143) and alternative health beliefs (n = 52). Knowledge and training (n = 62), interpretation services (n = 53), and staff attitudes (n = 46) were the most frequently mentioned factors affecting culturally sensitive care. CONCLUSION: Fifteen years after the publication of Unequal Treatment, the National Academies' landmark report on healthcare disparities, oncology healthcare providers have ongoing challenges caring for diverse patients and opportunities to implement recommendations from the report. PRACTICE IMPLICATIONS: Content of diversity trainings should focus on identified gaps and practical challenges. Multi-level supports are needed, including resources and training for oncology providers.

Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial ß-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

Prognostic value of hypersensitivity reactions on epidural steroid injection outcomes: a phenotypic signature? A prospective cohort study.

BACKGROUND: Studies have found that diffuse pain, indicative of central sensitization, portends poor interventional outcomes. Multiple chemical sensitivities are associated with signs of central sensitization. We sought to prospectively determine whether hypersensitivity reactions (HR) were associated with epidural steroid injection (ESI) outcomes. METHODS: HR were classified as immune-related or non-immune-related and categorized by number (0=low, 1 or 2=intermediate, ≥3=high). The primary outcome measure was mean reduction in average leg pain score 1 month post-procedure. A positive outcome was defined as a two-point or greater decrease in average leg pain accompanied by satisfaction 1 month post-procedure. RESULTS: The mean number of immune-mediated and non-immune-mediated HR were 0.6±1.2 and 0.8±1.4, respectively. Individuals in the high (n=24) total HR group had a mean reduction in average leg pain of 0.1±2.7, compared with those in the low (n=61; 1.8±2.1, p=0.025) and intermediate groups (n=52; 1.6±3.1, p=0.060). For back pain and categorical successful outcome, those with fewer HR experienced greater benefit. There were no differences in outcomes when patients were stratified by immune-related HR. Among participants in the low, intermediate and high non-immune-mediated HR groups, the mean reductions in average leg pain scores were 1.7±2.5, 1.6±3.0, and -0.2±2.3, respectively (p = 0.002). 51%, 35%, and 12% of people with low, intermediate and high numbers of non-immune-mediated HR experienced a positive categorical outcome, respectively (p=0.007). CONCLUSIONS: Non-immune-related HR were inversely correlated with some ESI outcome measures.

Healthy eating interventions in adults living with and beyond colorectal cancer: a systematic review.

BACKGROUND: Rates of cancer survival are increasing, with more people living with and beyond cancer. Lifestyle recommendations for cancer survivors are based largely on extrapolation from cancer prevention recommendations. The present study aimed to systematically review the literature on randomised controlled trials (RCTs) focusing on healthy eating interventions in people with colorectal cancer (CRC). METHODS: A structured search of electronic databases was conducted in March 2018 using medical subject headings (MeSH) and text words related to CRC and diet. The results of the literature searches were uploaded to online software for data management. Titles and abstracts were screened based on the inclusion and exclusion criteria and data were extracted. Quality of data was assessed using the Cochrane Handbook. RESULTS: Seven studies were identified, including six RCTs and one RCT protocol, with a total of 2233 participants from six studies, of whom 1010 (45%) had CRC. Three studies assessed anthropometrics demonstrating participants who received dietary intervention had a greater reduction in measurements. Six studies assessed changes in dietary components; however, only one demonstrated an increase in dietary fibre. Two studies reported improvements in quality of life favouring dietary intervention groups. CONCLUSIONS: The quality of identified studies was variable, with limited evidence to support dietary intervention improving dietary intake in people living with or after CRC. Studies to date have not been based on robust study design that has combined all dietary interventions linked to CRC. As a result of the heterogeneity of the studies identified, it was difficult to draw strong conclusions.

In their own words: patient navigator roles in culturally sensitive cancer care.

PURPOSE: Patient navigation has emerged as a promising strategy in reducing disparities among diverse cancer patients. However, little is known about navigators' own perspectives on their roles in providing culturally competent care. The purpose of the present study is to describe these self-identified roles. METHODS: Data were collected from an online survey with a convenience sample of cancer patient navigators. Using NVivo 10, qualitative content analysis was conducted on free text responses to the question: "In your opinion, what is the role of a patient navigator or nurse navigator in the provision of culturally sensitive care to patients?" Frequencies of each navigator-identified role mentioned were tabulated. RESULTS: Of 294 respondents, 50.7% (n = 149) provided a response to the question of interest. Respondents described the following 11 interrelated navigator roles in the provision of culturally competent care: (1) assess and understand patient needs, (2) tailor care to patient, (3) build rapport/open communication, (4) facilitate communication between patient and health care team, (5) educate/provide resources to the patient, (6) advocate, (7) self-motivated learning, (8) address barriers to care, (9) involve/meet the needs of family or support people, (10) educate/support health care team, and (11) support patient empowerment in care. CONCLUSIONS: Patient navigators are uniquely well-positioned to improve cultural competence of cancer care given their role as liaison to patients and providers. Cancer care settings should use navigators with direct knowledge of patient culture whenever possible; however, communication and cultural competence training is highly recommended for all navigators given the diversity of patient needs.

Transport and sorting in the Golgi complex: multiple mechanisms sort diverse cargo.

At the center of the secretory pathway, the Golgi complex ensures correct processing and sorting of cargos toward their final destination. Cargos are diverse in topology, function and destination. A remarkable feature of the Golgi complex is its ability to sort and process these diverse cargos destined for secretion, the cell surface, the lysosome, or retained within the secretory pathway. Just as these cargos are diverse so also are their sorting requirements and thus, their trafficking route. There is no one-size-fits-all sorting scheme in the Golgi. We propose a coexistence of models to reconcile these diverse needs. We review examples of differential sorting mediated by proteins and lipids. Additionally, we highlight recent technological developments that have potential to uncover new modes of transport.