In vivo imaging of cell behaviors and F-actin reveals LIM-HD transcription factor regulation of peripheral versus central sensory axon development

<p>Abstract</p> <p>Background</p> <p>Development of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Dynamic remodeling of the filamentous actin (F-actin) cytoskeleton is critical for these processes; however, little is known about the mechanisms controlling motile axon behaviors and F-actin dynamics <it>in vivo</it>. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. Zebrafish Rohon-Beard (RB) sensory neurons have a unique morphology, with central axons that extend in the spinal cord and a peripheral axon that innervates the skin. LIM homeodomain (LIM-HD) transcription factor activity is required for formation of peripheral RB axons. To understand how neuronal morphogenesis is controlled <it>in vivo </it>and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution <it>in vivo</it>.</p> <p>Results</p> <p>We used an F-actin biosensor containing the actin-binding domain of utrophin to characterize actin rearrangements during specific developmental processes <it>in vivo</it>, including axon initiation, consolidation and branching. We found that peripheral axons initiate from a specific cellular compartment and that F-actin accumulation and protrusive activity precede peripheral axon initiation. Moreover, disruption of LIM-HD transcriptional activity has different effects on the motility of peripheral versus central axons; it inhibits peripheral axon initiation, growth and branching, while increasing the growth rate of central axons. Our imaging revealed that LIM-HD transcription factor activity is not required for F-actin based protrusive activity or F-actin accumulation during peripheral axon initiation, but can affect positioning of F-actin accumulation and axon formation.</p> <p>Conclusion</p> <p>Our ability to image the dynamics of F-actin distribution during neuronal morphogenesis <it>in vivo </it>is unprecedented, and our experiments provide insight into the regulation of cell motility as neurons develop in the intact embryo. We identify specific motile cell behaviors affected by LIM-HD transcription factor activity and reveal how transcription factors differentially control the formation and growth of two axons from the same neuron.</p

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons

The Structural and Macroeconomic Determinants of Manufacturing Export-Value Performance in ASEAN Countries

This study aims to scrutinize the determinants of manufacturing exports in several ASEAN countries, specifically: Indonesia, Malaysia, Thailand, Philippines, and Vietnam. It adopts a panel data regression using the random effects model to predict manufacturing export value using structural (economic complexity and human capital) and macroeconomic (real effective exchange rate, foreign direct investment, and inflation) variables. The research finds that foreign direct investment, human capital, real effective exchange rate, and inflation are positive and statistically significant predictors of manufacturing exports in these ASEAN countries. However, the positive correlation between the real effective exchange rate and manufacturing exports is against previous literature arguing that a currency’s depreciation drives export competitiveness. The findings suggest that currency appreciation can enhance a country’s export performance as exports’ input products are cheaper than before. Additionally, the positive influence of inflation on exports can be explained by the subsequent increase in consumption from foreign countries. Therefore, in addition to managing their exchange rates, countries must develop their human capital and attract more foreign investments to enhance their export performance

A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin

The molecular basis for Toll-like receptor (TLR) recognition of microbial ligands is unknown. We demonstrate that mouse and human TLR5 discriminate between different flagellins, and we use this difference to map the flagellin recognition site on TLR5 to 228 amino acids of the extracellular domain. Through molecular modeling of the TLR5 ectodomain, we identify two conserved surface-exposed regions. Mutagenesis studies demonstrate that naturally occurring amino acid variation in TLR5 residue 268 is responsible for human and mouse discrimination between flagellin molecules. Mutations within one conserved surface identify residues D295 and D367 as important for flagellin recognition. These studies localize flagellin recognition to a conserved surface on the modeled TLR5 structure, providing detailed analysis of the interaction of a TLR with its ligand. These findings suggest that ligand binding at the β sheets results in TLR activation and provide a new framework for understanding TLR–agonist interactions

The contribution of schools to supporting the well being of children affected by HIV in eastern Zimbabwe

OBJECTIVES: Schools are often cited as a source of support for orphans and children affected by HIV/AIDS in populations experiencing generalized HIV epidemics and severe poverty. Here we investigate the success of schools at including and supporting the well being of vulnerable children in rural Zimbabwe. DESIGN: Data from a cross-sectional household survey of 4577 children (aged 6–17 years), conducted between 2009 and 2011, were linked to data on the characteristics of 28 primary schools and 18 secondary schools from a parallel monitoring and evaluation facility survey. METHODS: We construct two measures of school quality (one general and one HIV-specific) and use multivariable regression to test whether these were associated with improved educational outcomes and well being for vulnerable children. RESULTS: School quality was not associated with primary or secondary school attendance, but was associated with children’s being in the correct grade for age [adjusted odds ratio 2.0, 95% confidence interval (CI) 1.2–3.5, P = 0.01]. General and HIV-specific school quality had significant positive effects on well being in the primary school-age children (coefficient 5.1, 95% CI 2.4–7.7, P < 0.01 and coefficient 3.0, 95% CI 0.4–5.6, P = 0.02, respectively), but not in the secondary school-age children (P > 0.2). There was no evidence that school quality provided an additional benefit to the well being of vulnerable children. Community HIV prevalence was negatively associated with well being in the secondary school-age children (coefficient −0.7, 95% CI −1.3 to −0.1, P = 0.03). CONCLUSIONS: General and HIV-specific school quality may enhance the well being of primary school-age children in eastern Zimbabwe. Local community context also plays an important role in child well being

Effects of Salicornia-Based Skin Cream Application on Healthy Humans’ Experimental Model of Pain and Itching

Halophyte plants are salt-tolerant and are acclimated for growth in saline soils such as along coastal areas. Among the halophytes, the Salicornia species have been used as both folk medicine and functional food for many years due to their high levels of bioactive compounds with supposed anti-inflammatory and antioxidative effects. However, the properties of Salicornia bioactive extracts on pain and itching still remain unclear. In this study, 30 healthy volunteers were randomized to treatments with 10% Salicornia-based cream or placebo cream for 24 or 48 h. On day 0, and 24 or 48 h post cream application, cold/heat detection and pain thresholds, mechanical pain thresholds and sensitivity, trans-epidermal water loss, histamine- and cowhage-evoked itch, and micro-vascular reactivity (neurogenic inflammation) were assessed to evaluate the analgesic, anti-pruritogenic and vasomotor effects. Skin permeability was reduced in the Salicornia-treated area for 48 h compared with 24 h application (p-value < 0.05). After 48 h of application, a decrease in mechanical-evoked itching (hyperkinesis) compared with 24 h treatment (p-value < 0.05) and increased warm detection and heat pain thresholds (p-value < 0.05) was found. Histamine-induced neurogenic inflammation showed a significant reduction in the cream-treated areas after 48 h compared with 24 h (p-value < 0.05). The results of this study indicate the overall inhibitory effect of Salicornia on hyperkinesis (mechanically evoked itch), the analgesic effect on thermal sensation, and modulation of the skin barrier architecture. Further studies are needed for the assessment of the long-term effects

Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 20132016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP\u27s roles in protein structure, virion budding, and transcription and replication

Copy number variant discrepancy resolution using the ClinGen dosage sensitivity map results in updated clinical interpretations in ClinVar

Conflict resolution in genomic variant interpretation is a critical step toward improving patient care. Evaluating interpretation discrepancies in copy number variants (CNVs) typically involves assessing overlapping genomic content with focus on genes/regions that may be subject to dosage sensitivity (haploinsufficiency (HI) and/or triplosensitivity (TS)). CNVs containing dosage sensitive genes/regions are generally interpreted as â likely pathogenicâ (LP) or â pathogenicâ (P), and CNVs involving the same known dosage sensitive gene(s) should receive the same clinical interpretation. We compared the Clinical Genome Resource (ClinGen) Dosage Map, a publicly available resource documenting known HI and TS genes/regions, against germline, clinical CNV interpretations within the ClinVar database. We identified 251 CNVs overlapping known dosage sensitive genes/regions but not classified as LP or P; these were sent back to their original submitting laboratories for reâ evaluation. Of 246 CNVs reâ evaluated, an updated clinical classification was warranted in 157 cases (63.8%); no change was made to the current classification in 79 cases (32.1%); and 10 cases (4.1%) resulted in other types of updates to ClinVar records. This effort will add curated interpretation data into the public domain and allow laboratories to focus attention on more complex discrepancies.The ClinGen Dosage Sensitivity (DS) Map provides evidenceâ based assessments of the haploinsufficiency and triplosensitivity of genes/genomic regions. We identified 251 clinical copy number variants (CNVs) in ClinVar that overlapped known DS genes/regions but were not interpreted as â likely pathogenicâ or â pathogenic;â these were sent back to their original laboratories for reâ evaluation. Of the 246 that were reâ evaluated, 63.0% resulted in updated classifications, showing that the ClinGen DS Map can be an effective initial step in CNV classification discrepancy resolution.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146425/1/humu23610_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146425/2/humu23610.pd