An Infralimbic Cortex Neuronal Ensemble Encoded During Learning Attenuates Fear Generalization Expression.

Generalization allows for experience to flexibly guide behavior when conditions change. A basic physical unit of memory storage and expression in the brain are sparse, distributed groups of neurons known as ensembles (i.e., the engram). The infralimbic (IL) subregion of the ventral medial prefrontal cortex plays a key role in modulating conditioned defensive responses. How IL neuronal ensembles established during learning contribute to generalized responses is unknown. In this set of experiments, generalization was tested in male and female mice by presenting a novel, ambiguous, tone generalization stimulus following Pavlovian defensive (fear) conditioning. The first experiment was designed to test a role for IL in generalization using chemogenetic manipulations. Results show IL bidirectionally regulates defensive behavior. IL silencing promotes a switch in defensive state from vigilant scanning to generalized freezing, while IL stimulation reduces freezing in favor of scanning. Leveraging activity-dependent tagging technology (ArcCreERT2 x eYFP system), a neuronal ensemble, preferentially located in IL superficial layer 2/3, was associated with the generalization stimulus. Remarkably, in the identical discrete location, fewer reactivated neurons were associated with the generalization stimulus at the remote timepoint (30 days) following learning. When an IL neuronal ensemble established during learning was selectively chemogenetically silenced, generalization increased. Conversely, IL neuronal ensemble stimulation reduced generalization. Overall, these data identify a crucial role for IL in suppressing generalized responses. Further, we uncover an IL neuronal ensemble, formed during learning, functions to later attenuate the expression of generalization in the presence of ambiguous threat stimuli.

Feasibility of a Child-Friendly 2-Minute Walk Test: A Crossover Randomized Controlled Trial.

AIMS: Most studies that use the NIH Toolbox 2-Minute Walk Test with young children, modify the protocol, compromising the generalizability of outcomes. A standardizable protocol is needed. The purpose of this study was to compare the 2MWT performance of children ages 3-6 years on the standard NIH Toolbox protocol and on a modified protocol designed to support young children. METHODS: Cross-over randomized controlled trial. Fifteen typically developing children ages 3-6 years were randomly assigned to the performance order of the NIH toolbox 2MWT protocol and the Modified Accessibility Path (MAP) 2MWT protocol. Outcome variables and statistical analyses included test completion (McNemar test), distance walked (Wilcoxon signed-rank test), and accuracy (general estimating equation model with Poisson distribution). RESULTS: All children completed 2 min of walking with the MAP protocol. Only 40% of children completed the NIH Toolbox protocol, with 83% of these NIH completers bolstered by previous exposure to the MAP protocol. Collapsed across the order, children also had significantly fewer errors per lap with the MAP protocol (p < 0.0001), despite walking a significantly greater distance (p = 0.006). CONCLUSIONS: These findings lend preliminary support for standardized application of a 2MWT with young children when the protocol is designed to be child-friendly.

Hand-rim biomechanics during geared manual wheelchair propulsion over different ground conditions in individuals with spinal cord injury.

Geared manual wheelchair wheels, a recently developed alternative propulsion mechanism, have the potential to alleviate the high upper extremity demands required for wheelchair propulsion and help decrease the risk of secondary injuries in manual wheelchair users. The objective of this study was to investigate the effects of using geared manual wheelchairs on hand-rim biomechanics of wheelchair propulsion in individuals with spinal cord injury (SCI). Seven manual wheelchair users with SCI propelled their wheelchairs equipped with geared wheels over tile, carpet, and up a ramp in low gear (gear ratio 1.5:1) and standard gear (gear ratio 1:1) conditions. Hand-rim kinetics and stroke cycle characteristics were measured using a custom instrumented geared wheel. Using the geared wheels in the low gear condition, propulsion speed (P = 0.013), peak resultant force (P = 0.005), peak propulsive moment (P < 0.006), and peak rate of rise of the resultant force (P = 0.035) decreased significantly in comparison with the standard gear condition. The significant increase in the number of stroke cycles when normalized to distance (P = 0.004) and decrease in the normalized integrated moment (P = 0.030) indicated that although a higher number of stroke cycles are required for travelling a given distance in the low gear than the standard gear condition, the low gear condition might be less demanding for the upper extremity. These results suggest that geared wheels could be a useful technology for manual wheelchair users to independently accomplish strenuous propulsion tasks including mobility on carpeted floors and ramp ascension, while reducing the risk factors contributing to the incidence of secondary upper extremity injuries.

Validation of a series of walking and stepping tests to predict maximal oxygen consumption in adults aged 18-79 years.

INTRODUCTION: Field tests to estimate maximal oxygen consumption (VO2max) are an alternative to traditional exercise testing methods. Published field tests and their accompanying estimation equations account for up to 80% of the variance in VO2max with an error rate of ~4.5 ml.kg-1.min-1. These tests are limited to very specific age-range populations. The purpose of this study was to create and validate a series of easily administered walking and stepping field equations to predict VO2max across a range of healthy 18-79-year-old adults. METHODS: One-hundred-fifty-seven adults completed a graded maximal exercise test to assess VO2max. Five separate walking and three separate stepping tests of varying durations, number of stages, and intensities were completed. VO2max estimation equations were created using hierarchal multiple regression. Covariates including age, sex, body mass, resting heart rate, distance walked, gait speed, stepping cadence, and recovery heart rate were entered into each model using a stepwise approach. Each full model created had the same base model consisting of age, sex, and body mass. Validity of each model was assessed using a Jackknife cross-validation analysis, and percent bias and root mean square error (RMSE) were calculated. RESULTS: Base models accounted for ~72% of the total variance of VO2max. Full model variance ranged from ~79-83% and bias was minimal (<±1.0%) across models. RMSE for all models were approximately 4.5 ml.kg-1.min-1. Stepping tests performed better than walking tests by explaining ~2.5% more of the variance and displayed smaller RMSE. CONCLUSION: All eight models accounted for a large percentage of VO2max variance (~81%) with a RMSE of ~4.5 ml.kg-1.min-1. The variance and level of error of models examined highlight good group mean prediction with greater error expected at the individual level. All the models perform similarly across a broad age range, highlighting flexibility in application of these tests to a more general population.

Molecular determinants in Frizzled, Reck, and Wnt7a for ligand-specific signaling in neurovascular development.

The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.

Dlg1 activates beta-catenin signaling to regulate retinal angiogenesis and the blood-retina and blood-brain barriers.

Beta-catenin (i.e., canonical Wnt) signaling controls CNS angiogenesis and the blood-brain and blood-retina barriers. To explore the role of the Discs large/membrane-associated guanylate kinase (Dlg/MAGUK) family of scaffolding proteins in beta-catenin signaling, we studied vascular endothelial cell (EC)-specific knockout of Dlg1/SAP97. EC-specific loss of Dlg1 produces a retinal vascular phenotype that closely matches the phenotype associated with reduced beta-catenin signaling, synergizes with genetically-directed reductions in beta-catenin signaling components, and can be rescued by stabilizing beta-catenin in ECs. In reporter cells with CRISPR/Cas9-mediated inactivation of Dlg1, transfection of Dlg1 enhances beta-catenin signaling ~4 fold. Surprisingly, Frizzled4, which contains a C-terminal PDZ-binding motif that can bind to Dlg1 PDZ domains, appears to function independently of Dlg1 in vivo. These data expand the repertoire of Dlg/MAGUK family functions to include a role in beta-catenin signaling, and they suggest that proteins other than Frizzled receptors interact with Dlg1 to enhance beta-catenin signaling.

Technology Implementation and Associated Pharmacy Interruptions.

This study focuses on interruptions in an inpatient pharmacy setting and the impact of CPOE implementation on the types, frequency, and duration of interruptions. A cross-sectional observation study of pharmacy employees in an inpatient pharmacy was conducted. The independent variables included day of week, time of day, job position of the person interrupted, and description of each interruption. A total of 552 interruptions were observed with a mean frequency of 10.6 interruptions per hour lasting a mean (SD) duration of 1.34 (1.43) minutes. Incoming calls were the most frequent interruption type across all phases. Pharmacy employees spend almost a quarter of their time on interruptions, and pharmacists have longer interruptions than technicians. Immediately after CPOE implementation, durations tend to be one-and-a-half times longer than before. CPOE implementation did not affect the frequency of interruptions. Recommendations included redesign of work processes and job responsibilities.

Detection of Age-Related Macular Degeneration by Portable Optical Coherence Tomography Operated by Nonexpert Personnel: Potential Use for Screenings.

PURPOSE: The purpose of this study is to determine the sensitivity and specificity of detecting age-related macular degeneration (AMD) using portable optical coherence tomography (OCT) operated by nonexpert photographers on undilated patients. METHODS: In this case-control study, 92 individuals were recruited from the glaucoma and retina clinics at the Wilmer Eye Institute (Johns Hopkins University, Baltimore, MD). Using the portable iVue (Optovue, Inc, Fremont, CA) spectral-domain OCT (SD-OCT), 2 nonexpert photographers acquired retina map scans on undilated eyes of all participants. In total, 33 AMD eyes and 105 control eyes were evaluated and graded by ophthalmologists masked to the diagnoses. RESULTS: Detection of AMD on the portable OCT by ophthalmologists exhibited sensitivities of 0.91 and 0.94 and specificities of 0.88 and 0.89, for graders 1 and 2, respectively. A strong intergrader agreement was observed (κ = 0.87). CONCLUSIONS: Nonexpert photographers can use a portable OCT on undilated eyes to acquire images for the detection of AMD. These findings present the potential utility of implementing a portable OCT in community screenings for earlier detection and treatment of disease.

Interplay of the Norrin and Wnt7a/Wnt7b signaling systems in blood-brain barrier and blood-retina barrier development and maintenance.

ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.

Validation of the English Version of the HeartQoL Health-Related Quality of Life Questionnaire in Patients With Coronary Heart Disease.

PURPOSE: The aim of this study was to validate the English version of the HeartQoL health-related quality of life questionnaire for use in patients with angina or myocardial infarction. METHODS: Patients living in the United States and referred, either for percutaneous coronary intervention or to cardiac rehabilitation, completed the HeartQoL, the Short Form-36 Health Survey, and the Hospital Anxiety and Depression Scale at baseline and 3-months later. The data were analyzed for validity, reliability, and responsiveness. RESULTS: Patients (n = 313 with angina and n = 97 with myocardial infarction) who were referred either for percutaneous coronary intervention (n = 164) or to cardiac rehabilitation (n = 246) completed baseline questionnaires. Patients with angina had significantly lower HeartQoL scores (poorer health-related quality of life) compared with patients with myocardial infarction. Exploratory factor analysis largely supported the 2-factor structure of the HeartQoL in both diagnoses, but further investigation is warranted. Internal consistency reliability was adequate, convergent validity correlations were significant, and discriminative validity was fully confirmed in patients with angina and largely confirmed in patients with myocardial infarction. Responsiveness was largely confirmed in patients who underwent percutaneous coronary intervention (n = 67) and those referred to cardiac rehabilitation (n = 167) with conventional statistical tests and clinically with the effect size, a standardized measure of change. CONCLUSIONS: The English HeartQoL health-related quality of life questionnaire is valid, reliable, and responsive in patients with angina and myocardial infarction allowing (1) assessment of baseline, (2) between-diagnosis comparisons, and (3) evaluation of change over time.

Use of Optical Coherence Tomography by Nonexpert Personnel as a Screening Approach for Glaucoma.

PURPOSE: This pilot study was conducted to assess optical coherence tomography (OCT) as a screening tool for glaucoma when used by nonexpert personnel. METHODS: This prospective case-control study included 54 patients with open-angle glaucoma and 54 age-matched comparison individuals. Optovue iVue SD-OCT imaging was performed by nonprofessional photographers on undilated patients. The sensitivity, specificity, negative predictive value, and positive predictive value of iVue scan parameters for detecting open-angle glaucoma were evaluated. RESULTS: The iVue cup to disc vertical ratio had a sensitivity of 0.96 [95% confidence interval (CI), 0.90-1.00] at 90% specificity and was strongly correlated with both the Cirrus HD-OCT cup to disc vertical ratio (Pearson coefficient=0.84) and the cup to disc ratio observed on dilated clinical examination by faculty ophthalmologists (Pearson coefficient=0.80). The retinal nerve fiber layer (RNFL) parameters performed robustly, but the ganglion cell complex parameters showed limited diagnostic value. The inferior quadrant thickness was among the best performing RNFL parameters, with a sensitivity of 0.87 (95% CI, 0.78-0.96) and a specificity of 0.88 (95% CI, 0.80-0.97) using the iVue normative database thresholds for abnormality. CONCLUSIONS: OCT imaging may be performed by nonprofessional photographers on undilated patients, and quantitative parameters derived from the resultant images, particularly the vertical cup to disc ratio and the RNFL inferior quadrant thickness, demonstrate sensitivities and specificities that may be adequately robust for glaucoma screening in the community setting.

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation.

Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck, combined with loss of Norrin, impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck, and a soluble Reck(CC1-5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB.

Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/ß-catenin pathway during brain angiogenesis.

Despite the critical role of endothelial Wnt/ß-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/ß-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/ß-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/ß-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function.

Protein aggregation caused by aminoglycoside action is prevented by a hydrogen peroxide scavenger.

Protein mistranslation causes growth arrest in bacteria, mitochondrial dysfunction in yeast, and neurodegeneration in mammals. It remains poorly understood how mistranslated proteins cause such cellular defects. Here we demonstrate that streptomycin, a bactericidal aminoglycoside that increases ribosomal mistranslation, induces transient protein aggregation in wild-type Escherichia coli. We further determined the aggregated proteome using label-free quantitative mass spectrometry. To identify genes that reduce cellular mistranslation toxicity, we selected from an overexpression library protein products that increased resistance against streptomycin and kanamycin. The selected proteins were significantly enriched in members of the oxidation-reduction pathway. Overexpressing one of these proteins, alkyl hydroperoxide reductase subunit F (a protein defending bacteria against hydrogen peroxide), but not its inactive mutant suppressed aggregated protein formation upon streptomycin treatment and increased aminoglycoside resistance. This work provides in-depth analyses of an aggregated proteome caused by streptomycin and suggests that cellular defense against hydrogen peroxide lowers the toxicity of mistranslation.

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment.

Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular tRNA(2Thr) with a threonine (Thr) anticodon, MST1 also recognizes an unusual tRNA(1Thr), which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for tRNA(1Thr) recognition, whereas the anticodon sequence is essential for aminoacylation of tRNA(2Thr). The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate tRNA(1Thr), reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix α11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.

Modifications of ribonuclease A induced by p-benzoquinone.

The nature of ribonuclease A (RNase) modifications induced by p-benzoquinone (pBQ) was investigated using several analysis methods. SDS-PAGE experiments revealed that pBQ was efficient in producing oligomers and polymeric aggregates when RNase was incubated with pBQ. The fluorescence behavior and anisotropy changes of the modified RNase were monitored for a series of incubation reactions where RNase (0.050 mM) was incubated with pBQ (0.050, 0.25, 0.50, 1.50 mM) at 37 °C in phosphate buffer (pH 7.0, 50 mM). The modified RNase exhibited less intense fluorescence and slightly higher anisotropy than the unmodified RNase. UV-Vis spectroscopy indicated that pBQ formed covalent bonds to the modified RNase. Confocal imaging analysis confirmed the formation of the polymeric RNase aggregates with different sizes upon exposure of RNase to high concentrations of pBQ. The interaction between the modified RNase and salts affecting biomineralization of salts was also investigated by scanning electron microscopy. Overall, our results show that pBQ can induce formation of both RNase adducts and aggregates thus providing a better understanding of its biological activity.

Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid emulsion inhibits lung tumors in mice.

MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer.