An acto-myosin II constricting ring initiates the fission of activity-dependent bulk endosomes in neurosecretory cells.

Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact-GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells.

Coping measurement: Creating short form of Coping and Adaptation Processing Scale using item response theory and patients dealing with chronic and acute health conditions.

PURPOSE: The purpose of this study was to enhance the CAPS tool by clarifying the concept of coping, using item response theory (IRT) to shorten and assess the metric equivalence of the scale, and testing the preliminary validity of the resulting shortened scale. METHODS: A descriptive design of participants from different ethnic backgrounds was employed (USA n = 347 and Panama n = 327). To select items for the shortened CAPS, a well-established multi-step process grounded in IRT was used. Further, a coping ladder was created to approximate the a priori perceived location/difficulty of each item along the coping trait scale. Items for the shortened scale were selected based on considerations central to the middle range theory of coping and adaptation processing and the results of the item calibration and model testing. RESULTS: A total of 15 items were selected. The selected items were well distributed on the coping ladder and all basic subconcepts of the middle range theory were included. Further the sum of the DIF size for the selected short form items is − 0.01, so the overall bias of the total score is minimal. Finally, concurrent and divergent validity of the new scale was demonstrated in two separate correlational studies. CONCLUSION: The 15-item Coping and Adaptation Processing Scale (CAPS)--Short-Form can be a practical tool to effectively and efficiently measure coping and adaptation in both practice and research for people dealing with both chronic and acute health conditions.

Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

Modern Measurement Approaches to Health Literacy Scale Development and Refinement: Overview, Current Uses, and Next Steps.

There are currently more than 100 health literacy instruments. The procedures used to develop and test the measures are primarily guided by classical test theory. However, a small and growing number (n = 13) of health literacy measures are guided by modern measurement theories such as item response theory. This article briefly describes (a) the benefits of using modern measurement approaches for the development of health literacy measures, (b) how these approaches have been used with existing health literacy measures, and (c) some considerations for how modern measurement theory can help strengthen future work in health literacy measurement. Ultimately, this article provides evidence to support an assertive shift toward the use of modern measurement approaches in health literacy instrument development.

Community-based cardiovascular health interventions in vulnerable populations: a systematic review.

BACKGROUND: Although cardiovascular health has been improving for many Americans, this is not true of those in "vulnerable populations." To address this growing disparity, communities and researchers have worked for decades, and as a result of their work, a growing body of literature supports the use of community engagement as a component of successful interventions. However, little literature synthesizes community-based interventions that address this disparity among a wide range of vulnerable populations. OBJECTIVE: This article provides a critical review of community-based cardiovascular disease interventions to improve cardiovascular health behaviors and factors among vulnerable populations based on the American Heart Association's 7 metrics of ideal cardiovascular health. METHODS: In February 2011, 4 databases (PubMed, PsychInfo, CINAHL, and Scopus) were searched using the following keywords: vulnerable populations OR healthcare disparities AND cardiovascular disease AND clinical trials OR public health practice AND English. RESULTS: This search strategy resulted in the retrieval of 7120 abstracts. Each abstract was reviewed by at least 2 authors, and eligibility for the systematic review was confirmed after reading the full article. Thirty-two studies met eligibility criteria. Education was the most common intervention (41%), followed by counseling or support (38%) and exercise classes (28%). Half of the interventions were multicomponent. Healthcare providers were the most frequent interventionists. Interventions aimed at decreasing blood pressure were the most promising, whereas behavior change interventions were the most challenging. Almost all of the interventions were at the individual level and were proof-of-concept or efficacy trials. CONCLUSIONS: This analysis provides a step toward understanding the current literature on cardiovascular interventions for vulnerable population. The next step should be integrating the identified successful interventions into larger health systems and/or social policies.

Synthetic self-assembling clostridial chimera for modulation of sensory functions.

Clostridial neurotoxins reversibly block neuronal communication for weeks and months. While these proteolytic neurotoxins hold great promise for clinical applications and the investigation of brain function, their paralytic activity at neuromuscular junctions is a stumbling block. To redirect the clostridial activity to neuronal populations other than motor neurons, we used a new self-assembling method to combine the botulinum type A protease with the tetanus binding domain, which natively targets central neurons. The two parts were produced separately and then assembled in a site-specific way using a newly introduced 'protein stapling' technology. Atomic force microscopy imaging revealed dumbbell shaped particles which measure ∼23 nm. The stapled chimera inhibited mechanical hypersensitivity in a rat model of inflammatory pain without causing either flaccid or spastic paralysis. Moreover, the synthetic clostridial molecule was able to block neuronal activity in a defined area of visual cortex. Overall, we provide the first evidence that the protein stapling technology allows assembly of distinct proteins yielding new biomedical properties.

Topological optimization for designing patient-specific large craniofacial segmental bone replacements.

Restoring normal function and appearance after massive facial injuries with bone loss is an important unsolved problem in surgery. An important limitation of the current methods is heuristic ad hoc design of bone replacements by the operating surgeon at the time of surgery. This problem might be addressed by incorporating a computational method known as topological optimization into routine surgical planning. We tested the feasibility of using a multiresolution three-dimensional topological optimization to design replacements for massive midface injuries with bone loss. The final solution to meet functional requirements may be shaped differently than the natural human bone but be optimized for functional needs sufficient to support full restoration using a combination of soft tissue repair and synthetic prosthetics. Topological optimization for designing facial bone tissue replacements might improve current clinical methods and provide essential enabling technology to translate generic bone tissue engineering methods into specific solutions for individual patients.

In vivo inhibition of nuclear ACE2 translocation protects against SARS-CoV-2 replication and lung damage through epigenetic imprinting.

In vitro, ACE2 translocates to the nucleus to induce SARS-CoV-2 replication. Here, using digital spatial profiling of lung tissues from SARS-CoV-2-infected golden Syrian hamsters, we show that a specific and selective peptide inhibitor of nuclear ACE2 (NACE2i) inhibits viral replication two days after SARS-CoV-2 infection. Moreover, the peptide also prevents inflammation and macrophage infiltration, and increases NK cell infiltration in bronchioles. NACE2i treatment increases the levels of the active histone mark, H3K27ac, restores host translation in infected hamster bronchiolar cells, and leads to an enrichment in methylated ACE2 in hamster bronchioles and lung macrophages, a signature associated with virus protection. In addition, ACE2 methylation is increased in myeloid cells from vaccinated patients and associated with reduced SARS-CoV-2 spike protein expression in monocytes from individuals who have recovered from infection. This protective epigenetic scarring of ACE2 is associated with a reduced latent viral reservoir in monocytes/macrophages and enhanced immune protection against SARS-CoV-2. Nuclear ACE2 may represent a therapeutic target independent of the variant and strain of viruses that use the ACE2 receptor for host cell entry.

Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism.

The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.

Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin.

Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Ca(v)2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca(2+) oscillations sensitive to the inhibitor omega-conotoxin GVIA at the amphibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.

Using item response theory to develop and refine patient-reported outcome measures.

There is a long tradition of incorporating patient-reported outcomes (PROs) into nursing research and practice. Classical Test Theory (CTT) has been the traditional approach used to develop and refine PROs. Item response theory (IRT) offers promise for addressing measurement problems that have been difficult to solve using CTT. This paper presents foundational concepts in IRT to illustrate how it can be used to improve the development and refinement of PRO measures, with emphasis on interpreting key IRT parameters.

Landscape of Epidermal Growth Factor Receptor Heterodimers in Brain Metastases.

HER2+ breast cancer patients have an elevated risk of developing brain metastases (BM), despite adjuvant HER2-targeted therapy. The mechanisms underpinning this reduced intracranial efficacy are unclear. We optimised the in situ proximity ligation assay (PLA) for detection of the high-affinity neuregulin-1 receptor, HER2-HER3 (a key target of pertuzumab), in archival tissue samples and developed a pipeline for high throughput extraction of PLA data from fluorescent microscope image files. Applying this to a large BM sample cohort (n = 159) showed that BM from breast, ovarian, lung and kidney cancers have higher HER2-HER3 levels than other primary tumour types (melanoma, colorectal and prostate cancers). HER2 status, and tumour cell membrane expression of pHER2(Y1221/1222) and pHER3(Y1222) were positively, but not exclusively, associated with HER2-HER3 frequency. In an independent cohort (n = 78), BM had significantly higher HER2-HER3 levels than matching primary tumours (p = 0.0002). For patients who had two craniotomy procedures, HER2-HER3 dimer levels were lower in the consecutive lesion (n = 7; p = 0.006). We also investigated the effects of trastuzumab and pertuzumab on five different heterodimers in vitro: HER2-EGFR, HER2-HER4, HER2-HER3, HER3-HER4, HER3-EGFR. Treatment significantly altered the absolute frequencies of individual complexes in SKBr3 and/or MDA-MB-361 cells, but in the presence of neuregulin-1, the overall distribution was not markedly altered, with HER2-HER3 and HER2-HER4 remaining predominant. Together, these findings suggest that markers of HER2 and HER3 expression are not always indicative of dimerization, and that pertuzumab may be less effective at reducing HER2-HER3 dimerization in the context of excess neuregulin.

Dimensions of Community Assets for Health. A Systematised Review and Meta-Synthesis.

Since Aaron Antonovsky's salutogenesis theory and Morgan and Ziglio's health assets model were first proposed, there has been a growing concern to define the resources available to the individual and the community to maintain or improve health and well-being. The aim of the present study was to identify the dimensions that characterise community assets for health. To this end, we conducted a systematised review with a meta-synthesis and content analysis of research or projects involving asset mapping in the community. Articles that met our eligibility criteria were: (1) based on the salutogenic approach and (2) described an assets mapping process and among their results, explained what, how and why particular community assets for health had been selected. The search included primary studies in the published and grey literature which were selected from websites and electronic databases (Web of Science, MEDLINE, Scopus, EBSCOhost, Dialnet, SciELO). Of the 607 records examined by a single reviewer, 34 were included in the content analysis and 14 in the qualitative synthesis. Using an inductive process, we identified 14 dimensions with 24 categories, for which in-depth literature reviews were then carried out to define specific indicators and items. These dimensions were: utility, intention, previous use, accessibility ("circumstances-opportunity-affordability"), proximity-walkability, connectivity, intelligibility (visibility, transparency), identity (uniqueness, appropriability, attachment), design (configuration, functionality, comfort), safety (objective/subjective), diversity, the dimension of public and private, and sustainability (which includes maintenance, profitability or economic sustainability, environmental sustainability, centrality-participation and equity-inclusiveness).

Association between Life Skills and Academic Performance in Adolescents in the Autonomous Community of Aragon (Spain).

BACKGROUND: Learning and socio-emotional development is promoted through the creation and nurturing of an optimal school climate. This study aims to analyze the relationship between life skills and academic performance in a large sample of adolescents from the autonomous community of Aragón (Spain). METHODS: A cross-sectional study was conducted on the life skills and academic performance of a sample of 7th and 8th grade middle school students during the academic year 2018-2019. A sample of 43 middle schools were randomly selected; the final sample comprised 1745 students. The following data were collected through an anonymized, previously validated questionnaire: sociodemographic variables, social skills, self-efficacy, affective balance, and academic performance. RESULTS: We found a statistically significant association between life skills and academic performance (p < 0.001) in our sample. We also observed significant gender differences in life skills, with boys obtaining higher scores in cognitive skills and affective balance, and with girls achieving higher scores in social skills. CONCLUSION: We argue that life skills should be integrated into educational policies in order to improve the academic performance and health outcomes of students.

Characterization of Immune Cell Subsets of Tumor Infiltrating Lymphocytes in Brain Metastases.

The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p < 0.01) as well as in their microenvironment (p < 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA (p < 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 (p < 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.

Development of a Diabetes-Focused Print Health Literacy Scale Using the Rapid Estimate of Adult Literacy in Medicine Model.

BACKGROUND: A diabetes mellitus (DM)-specific health literacy (HL) measure that focuses on both oral and print HL is needed in clinical and research settings. OBJECTIVE: The present study developed a psychometrically sound DM-specific HL instrument that measures oral and print HL. METHODS: We developed the measure in three steps. First, we reviewed clinical guidelines and conducted focus groups with experts to generate items. Next, we conducted a psychometric evaluation of the scale in three language versions (English, Spanish, and Korean). Lastly, we identified and removed items with potential cultural bias and duplicate functions to produce shorter versions of the scale, using item response theory (IRT). KEY RESULTS: We initially developed an 82-item DM-specific oral HL scale using the Rapid Estimate of Adult Literacy in Medicine (DM-REALM) model. To improve the clinical utility of the DM-REALM, we created shorter forms, a 40-item and 20-item version, and evaluated them by using IRT. All DM-REALM versions had high Cronbach alphas (.985, .974, and .945, respectively) and yielded sufficient convergent validity by positive correlations with existing functional HL scale (r = .49, p < .001), education (r = .14, p = .14 to r = .54, p < .001), and DM knowledge (r = .04, p = .70 to r = .36, p < .001). DM-REALM also demonstrated adequate sensitivity as an intervention evaluation tool that captures the changes induced by an intervention. CONCLUSIONS: All forms of the DM-REALM tool were reliable, valid, and clinically useful measures of HL in the context of DM care. Both researchers and clinicians can use this tool to assess DM-specific HL across multiple racial and ethnic populations. [HLRP: Health Literacy Research and Practice. 2020;4(4):e237-e249.] PLAIN LANGUAGE SUMMARY: This article reported the process and findings of a newly developed health literacy scale for people with diabetes mellitus using three different language versions. Both long and short versions of the scale demonstrated adequate validity and reliability.

Descriptive Study of Children's Nutritional Status and Identification of Community-Level Nursing Diagnoses in a School Community in Africa.

Effectively responding to children's nutritional status and eating behaviors in Mozambique requires a community-based care approach grounded in sound nursing research that is evidence-based. The Community Assessment, Intervention, and Empowerment Model (MAIEC) is a nursing theoretical model that is based upon clinical decision-making for community health nurses using communities as a unit of care. We used the MAIEC to identify a community-based nursing diagnosis to address children's nutritional status and eating behaviors in Mozambique. OBJECTIVES: (1) to conduct a descriptive study of children's nutritional status and eating behaviors in a school community in Mavalane, Mozambique, and (2) to identify a community-based nursing diagnosis using the MAIEC clinical decision-making matrix in the same school community. METHOD: a cross-sectional, quantitative study was conducted to assess the nutritional status of children using anthropometric data, including brachial perimeter and the tricipital skinfold, and standard deviation for the relation of weight-height, in a sample of 227 children. To assess community management of the problem and identify a community-based nursing diagnosis, we surveyed 176 parents/guardians and 49 education professionals, using a questionnaire based on the MAIEC clinical decision matrix as a reference. RESULTS: malnutrition was identified in more than half of the children (51.3%). We also identified a community-based nursing diagnosis of impaired community management related to the promotion of child health and healthy eating evidenced by the lack of community leadership, participation, and processing among more than 70% of the community members (parents/guardians and education professionals). CONCLUSION: a nursing diagnosis and diagnostic criteria for nutritional status and community management were identified. The need to intervene using a multidisciplinary public health approach is imperative, with the school community as the unit of care. In addition, reliable anthropometric data were identified as important criteria to complement the nursing diagnosis and guide future public health interventions.

Qualitative Analysis by Experts of the Essential Elements of the Nursing Practice Environments Proposed by the TOP10 Questionnaire of Assessment of Environments in Primary Health Care.

Background: A short TOP10 scale based on the Practice Environment Scale-Nursing Work Index questionnaire measures the characteristics of nursing work environments. Positive environments result in better quality care and health outcomes. Objective: To identify a small number of core elements that would facilitate more effective interventions by nurse managers, and compare them with the essential elements proposed by the TOP10. Method: Qualitative research by a nominal group of eight experts. The content analysis was combined with descriptive data. Results: Ten most important items were selected and analyzed by the expert group. A high level of consensus in four items (2, 15, 20, 31) and an acceptable consensus in five items was reached (6, 11, 14, 18, 26). The tenth item in the top ten was selected from content analysis (19). The expert group agreed 90% with the elements selected as essential to the TOP10. Conclusion: The expert group achieved a high level of consensus that supports 90% of the essential elements of primary care settings proposed by the TOP10 questionnaire. Organizational changes implemented by managers to improve working environments must be prioritized following our results, so care delivery and health outcomes can be further improved.