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.     

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.     

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.     

Predator‐driven elemental cycling: the impact of predation and risk effects on ecosystem stoichiometry

Empirical evidence is beginning to show that predators can be important drivers of elemental cycling within ecosystems by propagating indirect effects that determine the distribution of elements among trophic levels as well as determine the chemical content of organic matter that becomes decomposed by microbes. These indirect effects can be propagated by predator consumptive effects on prey, nonconsumptive (risk) effects, or a combination of both. Currently, there is insufficient theory to predict how such predator effects should propagate throughout ecosystems. We present here a theoretical framework for exploring predator effects on ecosystem elemental cycling to encourage further empirical quantification. We use a classic ecosystem trophic compartment model as a basis for our analyses but infuse principles from ecological stoichiometry into the analyses of elemental cycling. Using a combined analytical‐numerical approach, we compare how predators affect cycling through consumptive effects in which they control the flux of nutrients up trophic chains; through risk effects in which they change the homeostatic elemental balance of herbivore prey which accordingly changes the element ratio herbivores select from plants; and through a combination of both effects. Our analysis reveals that predators can have quantitatively important effects on elemental cycling, relative to a model formalism that excludes predator effects. Furthermore, the feedbacks due to predator nonconsumptive effects often have the quantitatively strongest impact on whole ecosystem elemental stocks, production and efficiency rates, and recycling fluxes by changing the stoichiometric balance of all trophic levels. Our modeling framework predictably shows how bottom‐up control by microbes and top‐down control by predators on ecosystems become interdependent when top predator effects permeate ecosystems.     

Assessing biodiversity and endemism using phylogenetic methods across multiple taxonomic groups

Identifying geographical areas with the greatest representation of the tree of life is an important goal for the management and conservation of biodiversity. While there are methods available for using a single phylogenetic tree to assess spatial patterns of biodiversity, there has been limited exploration of how separate phylogenies from multiple taxonomic groups can be used jointly to map diversity and endemism. Here, we demonstrate how to apply different phylogenetic approaches to assess biodiversity across multiple taxonomic groups. We map spatial patterns of phylogenetic diversity/endemism to identify concordant areas with the greatest representation of biodiversity across multiple taxa and demonstrate the approach by applying it to the Murray–Darling basin region of southeastern Australia. The areas with significant centers of phylogenetic diversity and endemism were distributed differently for the five taxonomic groups studied (plant genera, fish, tree frogs, acacias, and eucalypts); no strong shared patterns across all five groups emerged. However, congruence was apparent between some groups in some parts of the basin. The northern region of the basin emerges from the analysis as a priority area for future conservation initiatives focused on eucalypts and tree frogs. The southern region is particularly important for conservation of the evolutionary heritage of plants and fishes.     

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.     

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.     

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.     

Gβ1 controls collective cell migration by regulating the protrusive activity of leader cells in the posterior lateral line primordium

Collective cell migration is critical for normal development, tissue repair and cancer metastasis. Migration of the posterior lateral line primordium (pLLP) generates the zebrafish sensory organs (neuromasts, NMs). This migration is promoted by the leader cells at the leading edge of the pLLP, which express the G protein-coupled chemokine receptor Cxcr4b and respond to the chemokine Cxcl12a. However, the mechanism by which Cxc112a/Cxcr4b signaling regulates pLLP migration remains unclear. Here we report that signal transduction by the heterotrimeric G protein subunit Gβ1 is essential for proper pLLP migration. Although both Gβ1 and Gβ4 are expressed in the pLLP and NMs, depletion of Gβ1 but not Gβ4 resulted in an arrest of pLLP migration. In embryos deficient for Gβ1, the pLLP cells migrated in an uncoordinated fashion and were unable to extend protrusions at the leading front, phenocopying those in embryos deficient for Cxcl12a or Cxcr4b. A transplantation assay showed that, like Cxcr4b, Gβ1 is required only in the leader cells of the pLLP. Analysis of F-actin dynamics in the pLLP revealed that whereas wild-type leader cells display extensive actin polymerization in the direction of pLLP migration, counterparts defective for Gβ1, Cxcr4b or Cxcl12a do not. Finally, synergy experiments revealed that Gβ1 and Cxcr4b interact genetically in regulating pLLP migration. Collectively, our data indicate that Gβ1 controls migration of the pLLP, likely by acting downstream of the Cxcl12a/Cxcr4b signaling. This study also provides compelling evidence for functional specificity among Gβ isoforms in vivo.