An Integrated Approach for Analyzing Clinical Genomic Variant Data from Next-Generation Sequencing

Next-generation sequencing (NGS) technologies provide the potential for developing high-throughput and low-cost platforms for clinical diagnostics. A limiting factor to clinical applications of genomic NGS is downstream bioinformatics analysis for data interpretation. We have developed an integrated approach for end-to-end clinical NGS data analysis from variant detection to functional profiling. Robust bioinformatics pipelines were implemented for genome alignment, single nucleotide polymorphism (SNP), small insertion/deletion (InDel), and copy number variation (CNV) detection of whole exome sequencing (WES) data from the Illumina platform. Quality-control metrics were analyzed at each step of the pipeline by use of a validated training dataset to ensure data integrity for clinical applications. We annotate the variants with data regarding the disease population and variant impact. Custom algorithms were developed to filter variants based on criteria, such as quality of variant, inheritance pattern, and impact of variant on protein function. The developed clinical variant pipeline links the identified rare variants to Integrated Genome Viewer for visualization in a genomic context and to the Protein Information Resource’s iProXpress for rich protein and disease information. With the application of our system of annotations, prioritizations, inheritance filters, and functional profiling and analysis, we have created a unique methodology for downstream variant filtering that empowers clinicians and researchers to interpret more effectively the relevance of genomic alterations within a rare genetic disease.     

Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings

In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.     

A Chemical and Enzymatic Approach to Study Site-Specific Sumoylation

A variety of cellular pathways are regulated by protein modifications with ubiquitin-family proteins. SUMO, the Small Ubiquitin-like MOdifier, is covalently attached to lysine on target proteins via a cascade reaction catalyzed by E1, E2, and E3 enzymes. A major barrier to understanding the diverse regulatory roles of SUMO has been a lack of suitable methods to identify protein sumoylation sites. Here we developed a mass-spectrometry (MS) based approach combining chemical and enzymatic modifications to identify sumoylation sites. We applied this method to analyze the auto-sumoylation of the E1 enzyme in vitro and compared it to the GG-remnant method using Smt3-I96R as a substrate. We further examined the effect of smt3-I96R mutation in vivo and performed a proteome-wide analysis of protein sumoylation sites in Saccharomyces cerevisiae. To validate these findings, we confirmed several sumoylation sites of Aos1 and Uba2 in vivo. Together, these results demonstrate that our chemical and enzymatic method for identifying protein sumoylation sites provides a useful tool and that a combination of methods allows a detailed analysis of protein sumoylation sites.     

Effect of Roadside Vegetation Cutting on Moose Browsing

Moose (Alces americanus ) vehicle collisions (MVCs) are an issue throughout the distribution of moose. Many mitigation strategies have been tested and implemented to reduce the number of MVCs, but there have been few empirical analyses of the effectiveness of roadside vegetation cutting. The goal of this study was to determine if roadside vegetation cutting attracted moose into roadside areas to browse on the vegetation regrowth. We hypothesized that moose would be attracted to roadside areas with cut vegetation. Consequently, we predicted that there would be higher levels of browsing in cut areas compared to uncut areas. To determine if moose were browsing more in cut or uncut areas, we measured the number of plants browsed by moose in paired treatment (cut on or after 2008) and control (not cut since at least 2008) sites, along with a suite of potential environmental covariates. Using a model selection approach, we fit generalized linear mixed-effects models to determine the most parsimonious set of environmental variables to explain variation in the proportion of moose browse among sites. In contrast to our hypothesis, our results show that the proportion of moose browse in the uncut control areas was significantly higher than in the cut treatment areas. The results of this study suggest that recently cut roadside areas (7 years or less based on our work) may create a less attractive foraging habitat for moose. The majority of the variance in the proportion of moose browse among sites was explained by treatment type and nested plot number within site identification (34.16%), with additional variance explained by traffic region (5.00%) and moose density (4.35%). Based on our study, we recommend that vegetation cutting be continued in roadside areas in Newfoundland as recently cut areas may be less attractive browsing sites for moose.     

Elevated temperature and periodic water stress alter growth and quality of common milkweed (<Emphasis Type="Italic">Asclepias syriaca</Emphasis>) and monarch (<Emphasis Type="Italic">Danaus plexippus</Emphasis>) larval performance

In this study, we examined the independent and interactive effects of temperature and water availability on the growth and foliar traits of common milkweed (Asclepias syriaca) and performance of a specialist herbivore, larvae of the monarch butterfly (Danaus plexippus). Milkweed from multiple population sources collected across a latitudinal gradient in Wisconsin, USA, were grown under all combinations of ambient or elevated temperature and the presence or absence of periodic water stress. Elevated temperature marginally increased, while water stress decreased plant growth. Milkweed from more northerly latitudes experienced larger growth responses to elevated temperature and were more resistant to water stress, especially under higher temperatures. Elevated temperature and water stress also altered milkweed composite foliar trait profiles. Elevated temperature generally increased leaf nitrogen and structural compounds, and decreased leaf mass per area. Water stress also elevated foliar nitrogen, but reduced defensive traits. Monarch larvae performed well on milkweed under elevated temperature and water stress, but gained the most mass on plants exposed to both treatments in combination. Our findings suggest that milkweed populations from more northerly latitudes in the upper Midwest may benefit more from rising temperatures than those in southerly locations, but that these beneficial effects depend on water availability. Monarch larvae grew larger on plants from all experimental treatments relative to ambient condition controls, indicating that future changes in milkweed presence on the landscape will likely influence monarch populations more than the effects of future changes in plant quality on larval performance.     

Effects of Pharmacotherapy on Combat-Related PTSD,Anxiety, and Depression: A Systematic Review and Meta-Regression Analysis

ObjectivesTo estimate the effect of pharmacotherapy on PTSD, anxiety, and depression among combat veterans; to determine whether the effects varied according to patient and intervention characteristics; and to examine differential effects of pharmacotherapy on outcomes.ResultsPharmacotherapy significantly reduced (Δ, 95%CI) PTSD (0.38, 0.23-0.52), anxiety (0.42, 0.30-0.54), and depressive symptoms (0.52, 0.35-0.70). The effects of SSRIs and tricyclic antidepressants on PTSD were greater than other medications independent of treatment duration. The effect of SSRIs and tricyclic antidepressants were greater than other medications up to 5.2 and 13.6 weeks for anxiety and depression, respectively. The magnitude of the effect of pharmacotherapy on concurrently-measured PTSD, anxiety, and depression did not significantly differ.ConclusionsPharmacotherapy reduced PTSD, anxiety, and depressive symptoms in combat veterans. The effects of SSRIs and tricyclic antidepressants were greater for PTSD and occurred quicker for anxiety and depression than other medications.     

Orientation Transfer in Vernier and Stereoacuity Training

Human performance on various visual tasks can be improved substantially via training. However, the enhancements are frequently specific to relatively low-level stimulus dimensions. While such specificity has often been thought to be indicative of a low-level neural locus of learning, recent research suggests that these same effects can be accounted for by changes in higher-level areas–in particular in the way higher-level areas read out information from lower-level areas in the service of highly practiced decisions. Here we contrast the degree of orientation transfer seen after training on two different tasks—vernier acuity and stereoacuity. Importantly, while the decision rule that could improve vernier acuity (i.e. a discriminant in the image plane) would not be transferable across orientations, the simplest rule that could be learned to solve the stereoacuity task (i.e. a discriminant in the depth plane) would be insensitive to changes in orientation. Thus, given a read-out hypothesis, more substantial transfer would be expected as a result of stereoacuity than vernier acuity training. To test this prediction, participants were trained (7500 total trials) on either a stereoacuity (N = 9) or vernier acuity (N = 7) task with the stimuli in either a vertical or horizontal configuration (balanced across participants). Following training, transfer to the untrained orientation was assessed. As predicted, evidence for relatively orientation specific learning was observed in vernier trained participants, while no evidence of specificity was observed in stereo trained participants. These results build upon the emerging view that perceptual learning (even very specific learning effects) may reflect changes in inferences made by high-level areas, rather than necessarily fully reflecting changes in the receptive field properties of low-level areas.     

Adolescent Intermittent Alcohol Exposure: Deficits in Object Recognition Memory and Forebrain Cholinergic Markers

The long-term effects of intermittent ethanol exposure during adolescence (AIE) are of intensive interest and investigation. The effects of AIE on learning and memory and the neural functions that drive them are of particular interest as clinical findings suggest enduring deficits in those cognitive domains in humans after ethanol abuse during adolescence. Although studies of such deficits after AIE hold much promise for identifying mechanisms and therapeutic interventions, the findings are sparse and inconclusive. The present results identify a specific deficit in memory function after AIE and establish a possible neural mechanism of that deficit that may be of translational significance. Male rats (starting at PND-30) received exposure to AIE (5g/kg, i.g.) or vehicle and were allowed to mature into adulthood. At PND-71, one group of animals was assessed using the spatial-temporal object recognition (stOR) test to evaluate memory function. A separate group of animals was used to assess the density of cholinergic neurons in forebrain areas Ch1-4 using immunohistochemistry. AIE exposed animals manifested deficits in the temporal component of the stOR task relative to controls, and a significant decrease in the number of ChAT labeled neurons in forebrain areas Ch1-4. These findings add to the growing literature indicating long-lasting neural and behavioral effects of AIE that persist into adulthood and indicate that memory-related deficits after AIE depend upon the tasks employed, and possibly their degree of complexity. Finally, the parallel finding of diminished cholinergic neuron density suggests a possible mechanism underlying the effects of AIE on memory and hippocampal function as well as possible therapeutic or preventive strategies for AIE.