Characterization of neurite dystrophy after trauma by high speed structured illumination microscopy and lattice light sheet microscopy.

BACKGROUND: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma. NEW METHOD: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented. RESULTS: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another. COMPARISON WITH EXISTING METHODS: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite. CONCLUSIONS: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.

Measurement equivalence of child feeding and eating measures across gender, ethnicity, and household food security.

BACKGROUND: Although there have been extensive studies that make group comparisons on child eating and feeding practices, few studies have examined measurement equivalence to ensure that measures used to make such group comparisons are equivalent across important group characteristics related to childhood obesity. METHODS: Using a sample of 243 caregivers with children between the ages of 4 to 6 years, we conducted a measurement equivalence analysis across gender, ethnicity (Latino versus non-Latino White), and household food security. The subscales of the Child Feeding Questionnaire (CFQ) and the Child Eating Behaviour Questionnaire (CEBQ) were examined separately using a one factor multi-group confirmatory factor analysis. RESULTS: For the CFQ, Concern about Child Weight and Parental Responsibility subscales were consistent across all groups examined. In contrast, Pressure to Eat, Restriction, and Perceived Parent Weight subscales varied or fit poorly across the groups. For the CEBQ, Emotional Overeating, Enjoyment of Food, and Satiety Responsiveness performed consistently across the groups. On the other hand, Food Fussiness, Desire to Drink, Slowness in Eating, and Emotional Undereating subscales varied or fit poorly across the groups. CONCLUSIONS: Findings from this study suggest both of these measures need continued psychometric work, and group comparisons using some subscales should be interpreted cautiously. Some subscales such as Food Responsiveness and Parental Restriction may be assessing behaviors that occur in food secure households and are less applicable to food insecure environments.

Model-free quantification and visualization of colocalization in fluorescence images.

The spatial association between fluorescently tagged biomolecules in situ provides valuable insight into their biological relationship. Within the limits of diffraction, such association can be measured using either Pearson's Correlation Coefficient (PCC) or Spearman's Rank Coefficient (SRC), which are designed to measure linear and monotonic correlations, respectively. However, the relationship between real biological signals is often more complex than these measures assume, rendering their results difficult to interpret. Here, we have adapted methods from the field of information theory to measure the association between two probes' concentrations based on their statistical dependence. Our approach is mathematically more general than PCC or SRC, making no assumptions about the type of relationship between the probes. We show that when applied to biological images, our measures provide more intuitive results that are also more robust to outliers and the presence of multiple relationships than PCC or SRC. We also devise a display technique to highlight regions in the input images where the probes' association is higher versus lower. We expect that our methods will allow biologists to more accurately and robustly quantify and visualize the association between two probes in a pair of fluorescence images. © 2018 International Society for Advancement of Cytometry.

Image co-localization - co-occurrence versus correlation.

Fluorescence image co-localization analysis is widely utilized to suggest biomolecular interaction. However, there exists some confusion as to its correct implementation and interpretation. In reality, co-localization analysis consists of at least two distinct sets of methods, termed co-occurrence and correlation. Each approach has inherent and often contrasting strengths and weaknesses. Yet, neither one can be considered to always be preferable for any given application. Rather, each method is most appropriate for answering different types of biological question. This Review discusses the main factors affecting multicolor image co-occurrence and correlation analysis, while giving insight into the types of biological behavior that are better suited to one approach or the other. Further, the limits of pixel-based co-localization analysis are discussed in the context of increasingly popular super-resolution imaging techniques.

Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels.IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells-presumably a prerequisite for alphavirus maintenance in nature.

Predictive Validity of Curriculum-Embedded Measures on Outcomes of Kindergarteners Identified as At Risk for Reading Difficulty.

This study examined the predictive validity of formative assessments embedded in a Tier 2 intervention curriculum for kindergarten students identified as at risk for reading difficulty. We examined when (i.e., months during the school year) measures could predict reading outcomes gathered at the end of kindergarten and whether the predictive validity of measures changed across the kindergarten year. Participants consisted of 137 kindergarten students whose reading development was assessed four times from October to February. Measures aligned with content taught in the curriculum and assessed a range of phonologic, alphabetic, and word-reading skills. Results from structural equation modeling indicate that 36.3% to 65.2% of the variance was explained on the latent decoding outcome and 62.0% to 86.8% on the latent phonological outcome across the four time points. Furthermore, the predictive validity of specific skills increased over the kindergarten year, with more complicated tasks (e.g., word segmentation) becoming more predictive at subsequent measurement occasions. Results suggest that curriculum-embedded measures may be viable tools for assessing and predicting reading performance.

Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells.

Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions.

The Shelf Life of a Safety Climate Assessment: How Long Until the Relationship with Safety-Critical Incidents Expires?

PURPOSE: This study investigates safety climate as both a leading (climate â†’ incident) and a lagging (incident â†’ climate) indicator of safety-critical incidents. This study examines the "shelf life" of a safety climate assessment and its relationships with incidents, both past and future, by examining series of incident rates in order to determine when these predictive relationships expire. DESIGN/METHODOLOGY/APPROACH: A survey was conducted at a large, multinational chemical manufacturing company, with 7,467 responses at 42 worksites in 12 countries linked to over 14,000 incident records during the 2 years prior and 2 years following the survey period. Regressions revealed that safety climate predicts incidents of varying levels of severity, but it predicts the most severe incidents over the shortest period of time. The same is true for incidents predicting safety climate, with more severe incidents having a shorter predictive window. For the most critical relationship (climate predicting more severe incidents), the ability of a safety climate assessment to predict incidents expires after 3 months. IMPLICATIONS: The choice of aggregation period in constructing incident rates is essential in understanding the safety climate-incident relationship. The common yearly count of incidents would make it seem that more severe incidents cannot be predicted by safety climate and also fails to show the strongest predictive effects of less severe incidents. ORIGINALITY/VALUE: This research is the first to examine assumptions regarding aggregation periods when constructing safety-related incident rates. Our work guides organizations in planning their survey program, recommending more frequent measurement of safety climate.

Monitoring early first-grade reading progress: a comparison of two measures.

This study compared the validity of progress monitoring slope of nonsense word fluency (NWF) and word identification fluency (WIF) with early first-grade readers. Students (N = 80) considered to be at risk for reading difficulty were monitored with NWF and WIF on a 1-2 week basis across 11 weeks. Reading skills at the end of first grade were assessed using measures of passage reading fluency, real and pseudoword reading efficiency, and basic comprehension. Latent growth models indicated that although slope on both measures significantly predicted year-end reading skills, models including WIF accounted for more variance in spring reading skills than NWF, and WIF slope was more strongly associated with reading outcomes than NWF slope. Analyses of student growth plots suggested that WIF slope was more positively associated with later reading skills and discriminated more clearly between students according to successful or unsuccessful year-end reading outcomes. Although both measures may be used to monitor reading growth of at-risk students in early first grade, WIF may provide a clearer index of reading growth. Implications for data-based decision-making are discussed.

An alternative approach to analysis of mental states in experimental social cognition research.

Establishing the mental states that affect human behavior is a primary goal of experiments on social cognitive processes. Such mental states can be manipulated only indirectly; therefore, after delivering a manipulation, researchers attempt to verify that the mental state of interest, the representation of a mental state, was in fact changed by the manipulation and that this change caused the observed effect. The usual procedure is to examine mean differences in a measure of the mental state of interest (a manipulation check) among experimental conditions and to infer whether the manipulation was effective. We describe a procedure that strengthens the construct validity of manipulations and, hence, causal inferences in experiments that focus on mental states using analyses familiar to most researchers. This procedure employs a traditional manipulation check that assesses the relationship between manipulations and mental states but, additionally, tests the relationship between the manipulation check and dependent measure.

Fast Count-Regulated OSEM Reconstruction With Adaptive Resolution Recovery.

Ordered subsets expectation maximization (OSEM) is widely used to accelerate tomographic reconstruction. Speed-up of OSEM over maximum likelihood expectation maximization (MLEM) is close to the number of subsets (NS). Recently we significantly increased the speed-up achievable with OSEM by specific subset choice (pixel-based OSEM). However, a high NS can cause undesirable noise levels, quantitative inaccuracy or even disappearance of lesions in low-activity image regions, while a low NS leads to prohibitively long reconstructions or unrecovered details in highly active regions. Here, we introduce count-regulated OSEM (CROSEM) which locally adapts the effective NS based on the estimated amount of detected photons originating from individual voxels. CROSEM was tested using multi-pinhole SPECT simulations and in vivo imaging. With the maximum NS set to 128, CROSEM attained acceleration factors close to 128 in high-activity regions and kept quantitative accuracy in low-activity regions close to that of MLEM. At equal cold-lesion contrast in high-activity regions, CROSEM exhibited lower noise than MLEM in low-activity regions. CROSEM is a fast and stable alternative to OSEM, preventing excessive image noise and quantitative errors in low-activity regions while achieving high-resolution recovery in structures with high activity uptake.

Four applications of permutation methods to testing a single-mediator model.

Four applications of permutation tests to the single-mediator model are described and evaluated in this study. Permutation tests work by rearranging data in many possible ways in order to estimate the sampling distribution for the test statistic. The four applications to mediation evaluated here are the permutation test of ab, the permutation joint significance test, and the noniterative and iterative permutation confidence intervals for ab. A Monte Carlo simulation study was used to compare these four tests with the four best available tests for mediation found in previous research: the joint significance test, the distribution of the product test, and the percentile and bias-corrected bootstrap tests. We compared the different methods on Type I error, power, and confidence interval coverage. The noniterative permutation confidence interval for ab was the best performer among the new methods. It successfully controlled Type I error, had power nearly as good as the most powerful existing methods, and had better coverage than any existing method. The iterative permutation confidence interval for ab had lower power than do some existing methods, but it performed better than any other method in terms of coverage. The permutation confidence interval methods are recommended when estimating a confidence interval is a primary concern. SPSS and SAS macros that estimate these confidence intervals are provided.

Explanation of Two Anomalous Results in Statistical Mediation Analysis.

Previous studies of different methods of testing mediation models have consistently found two anomalous results. The first result is elevated Type I error rates for the bias-corrected and accelerated bias-corrected bootstrap tests not found in nonresampling tests or in resampling tests that did not include a bias correction. This is of special concern as the bias-corrected bootstrap is often recommended and used due to its higher statistical power compared with other tests. The second result is statistical power reaching an asymptote far below 1.0 and in some conditions even declining slightly as the size of the relationship between X and M, a, increased. Two computer simulations were conducted to examine these findings in greater detail. Results from the first simulation found that the increased Type I error rates for the bias-corrected and accelerated bias-corrected bootstrap are a function of an interaction between the size of the individual paths making up the mediated effect and the sample size, such that elevated Type I error rates occur when the sample size is small and the effect size of the nonzero path is medium or larger. Results from the second simulation found that stagnation and decreases in statistical power as a function of the effect size of the a path occurred primarily when the path between M and Y, b, was small. Two empirical mediation examples are provided using data from a steroid prevention and health promotion program aimed at high school football players (Athletes Training and Learning to Avoid Steroids; Goldberg et al., 1996), one to illustrate a possible Type I error for the bias-corrected bootstrap test and a second to illustrate a loss in power related to the size of a. Implications of these findings are discussed.

Dopaminergic modulation of risky decision-making.

Many psychiatric disorders are characterized by abnormal risky decision-making and dysregulated dopamine receptor expression. The current study was designed to determine how different dopamine receptor subtypes modulate risk-taking in young adult rats, using a "Risky Decision-making Task" that involves choices between small "safe" rewards and large "risky" rewards accompanied by adverse consequences. Rats showed considerable, stable individual differences in risk preference in the task, which were not related to multiple measures of reward motivation, anxiety, or pain sensitivity. Systemic activation of D2-like receptors robustly attenuated risk-taking, whereas drugs acting on D1-like receptors had no effect. Systemic amphetamine also reduced risk-taking, an effect which was attenuated by D2-like (but not D1-like) receptor blockade. Dopamine receptor mRNA expression was evaluated in a separate cohort of drug-naive rats characterized in the task. D1 mRNA expression in both nucleus accumbens shell and insular cortex was positively associated with risk-taking, while D2 mRNA expression in orbitofrontal and medial prefrontal cortex predicted risk preference in opposing nonlinear patterns. Additionally, lower levels of D2 mRNA in dorsal striatum were associated with greater risk-taking. These data strongly implicate dopamine signaling in prefrontal cortical-striatal circuitry in modulating decision-making processes involving integration of reward information with risks of adverse consequences.

Evaluating model fit for growth curve models: Integration of fit indices from SEM and MLM frameworks.

Evaluating overall model fit for growth curve models involves 3 challenging issues. (a) Three types of longitudinal data with different implications for model fit may be distinguished: balanced on time with complete data, balanced on time with data missing at random, and unbalanced on time. (b) Traditional work on fit from the structural equation modeling (SEM) perspective has focused only on the covariance structure, but growth curve models have four potential sources of misspecification: within-individual covariance matrix, between-individuals covariance matrix, marginal mean structure, and conditional mean structure. (c) Growth curve models can be estimated in both the SEM and multilevel modeling (MLM) frameworks; these have different emphases for the evaluation of model fit. In this article, the authors discuss the challenges presented by these 3 issues in the calculation and interpretation of SEM- and MLM-based fit indices for growth curve models and conclude by identifying some lines for future research.

R2 effect-size measures for mediation analysis.

R(2) effect-size measures are presented to assess variance accounted for in mediation models. The measures offer a means to evaluate both component paths and the overall mediated effect in mediation models. Statistical simulation results indicate acceptable bias across varying parameter and sample-size combinations. The measures are applied to a real-world example using data from a team-based health promotion program to improve the nutrition and exercise habits of firefighters. SAS and SPSS computer code are also provided for researchers to compute the measures in their own data.

Effects of a health behavior change model--based HIV/STI prevention intervention on condom use among heterosexual couples: a randomized trial.

This study examines an intervention for heterosexual couples to prevent human immunodeficiency virus/sexually transmitted infections. It also evaluates the effect of the intervention, which is based on current models of health behavior change, on intermediate outcomes (individual and relationship factors) and consistency of condom use. Eligible couples were administered a baseline interview and randomized to either a 3-session theory-based intervention or a 1-session standard of care comparison condition. Men and women completed 3-month interviews; only women completed 6-month interviews. No significant intervention effect on condom use was found among couples at 3 months (n = 212) or among women (n = 178) at 6 months. However, condom use increased significantly between baseline and 3 months and baseline and 6 months for participants in both treatment conditions. Intervention effects on condom use self-efficacy were found at 3 months and 6 months and on health-protective communication at 3 months. These findings provide valuable information for the design of future studies to help disentangle the effects of intervening with couples.

Dendrites contain a spacing pattern.

The distinctive branching patterns of dendritic arbors are essential for neuronal information processing. The final shape of an arbor is the result of intrinsic and extrinsic factors. However, the cellular mechanisms that underlie branch patterning are unknown. In many biological systems, locally acting factors are intrinsically organized into spacing patterns that guide patterned morphogenesis. Here, we show that neurons contain two types of periodic and regular elements (PADREN1s and PADREN2s) that are arranged into a spacing pattern. The wavelength of the pattern is approximately 20 microm. Dendritic branches occur preferentially within PADREN1s, and specific PADREN lengths correspond to specific arbor types. The lengths of the PADRENs also change over time and can be modified by activity. However, PADRENs are intrinsically organized, possibly by a reaction-diffusion process. PADRENs reveal a previously unrecognized level of neuronal organization that may provide insight into how the distinct branching patterns of the dendrites are intrinsically organized.