Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.     

Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR

Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2-3 h after the material has arrived in the laboratory.     

Nanopore analysis reveals differences in structural stability of ovine PrPC proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP^C). Species barriers to transmission also reflect PrP^C sequence differences. Defining the structure-activity relationship of PrP^C/PrP^Sc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP^C corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP^Sc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP^Sc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP^Sc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.     

Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA₃, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

Noninvasive Imaging of In Vivo MuRF1 Expression during Muscle Atrophy

Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3′ untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.     

QTLs associated with dry matter intake,metabolic mid-test weight,growth and feed efficiency have little overlap across 4 beef cattle studies

Background

The identification of genetic markers associated with complex traits that are expensive to record such as feed intake or feed efficiency would allow these traits to be included in selection programs. To identify large-effect QTL, we performed a series of genome-wide association studies and functional analyses using 50 K and 770 K SNP genotypes scored in 5,133 animals from 4 independent beef cattle populations (Cycle VII, Angus, Hereford and Simmental × Angus) with phenotypes for average daily gain, dry matter intake, metabolic mid-test body weight and residual feed intake.

Results

A total of 5, 6, 11 and 10 significant QTL (defined as 1-Mb genome windows with Bonferroni-corrected P-value <0.05) were identified for average daily gain, dry matter intake, metabolic mid-test body weight and residual feed intake, respectively. The identified QTL were population-specific and had little overlap across the 4 populations. The pleiotropic or closely linked QTL on BTA 7 at 23 Mb identified in the Angus population harbours a promising candidate gene ACSL6 (acyl-CoA synthetase long-chain family member 6), and was the largest effect QTL associated with dry matter intake and mid-test body weight explaining 10.39% and 14.25% of the additive genetic variance, respectively. Pleiotropic or closely linked QTL associated with average daily gain and mid-test body weight were detected on BTA 6 at 38 Mb and BTA 7 at 93 Mb confirming previous reports. No QTL for residual feed intake explained more than 2.5% of the additive genetic variance in any population. Marker-based estimates of heritability ranged from 0.21 to 0.49 for residual feed intake across the 4 populations.

Conclusions

This GWAS study, which is the largest performed for feed efficiency and its component traits in beef cattle to date, identified several large-effect QTL that cumulatively explained a significant percentage of additive genetic variance within each population. Differences in the QTL identified among the different populations may be due to differences in power to detect QTL, environmental variation, or differences in the genetic architecture of trait variation among breeds. These results enhance our understanding of the biology of growth, feed intake and utilisation in beef cattle.     

A Probabilistic Analysis of Regional Mercury Impacts on Wildlife

We investigate the uncertainties associated with modeling the potential health effects on piscivorous animals of mercury released to the atmosphere. The multimedia modeling system combines an atmospheric fate and transport model, an aquatic cycling model, and a terrestrial food web model. First, the modeling system is used to calculate point values of the animals' hazard quotients (i.e., measures of toxic dose). Next, we use a simplified version of the modeling system to conduct a probabilistic analysis for the Great Lakes region that takes into account input uncertainty, variability, and uncertainty and variability combined. The use of two different software packages for the combined uncertainty/variability analysis led to similar results except for high values (>90th percentile) where some differences were evident. A sensitivity study was performed on the combined uncertainty and variability analysis. Regional variability caused more than 70% of the variance in the results, with the fish bioaccumulation factor accounting for the majority of the variability. The major sources of uncertainty were the speciation of the mercury emissions, the lake pH, and the sediment burial rate.     

Comparing forest structure and biodiversity on private and public land: secondary tropical dry forests in Costa Rica

Secondary forests constitute a substantial proportion of tropical forestlands. These forests occur on both public and private lands and different underlying environmental variables and management regimes may affect post‐abandonment successional processes and resultant forest structure and biodiversity. We examined whether differences in ownership led to differences in forest structure, tree diversity, and tree species composition across a gradient of soil fertility and forest age. We collected soil samples and surveyed all trees in 82 public and 66 private 0.1‐ha forest plots arrayed across forest age and soil gradients in Guanacaste, Costa Rica. We found that soil fertility appeared to drive the spatial structure of public vs. private ownership; public conservation lands appeared to be non‐randomly located on areas of lower soil fertility. On private lands, areas of crops/pasture appeared to be non‐randomly located on higher soil fertility areas while forests occupied areas of lower soil fertility. We found that forest structure and tree species diversity did not differ significantly between public and private ownership. However, public and private forests differed in tree species composition: 11 percent were more prevalent in public forest and 7 percent were more prevalent in private forest. Swietenia macrophylla, Cedrela odorata, and Astronium graveolens were more prevalent in public forests likely because public forests provide stronger protection for these highly prized timber species. Guazuma ulmifolia was the most abundant tree in private forests likely because this species is widely consumed and dispersed by cattle. Furthermore, some compositional differences appear to result from soil fertility differences due to non‐random placement of public and private land holdings with respect to soil fertility. Land ownership creates a distinctive species composition signature that is likely the result of differences in soil fertility and management between the ownership types. Both biophysical and social variables should be considered to advance understanding of tropical secondary forest structure and biodiversity.     

A work‐for‐food enrichment program increases exploration and decreases stereotypies in four species of bears

Zoo‐housed bears are prone to exhibiting stereotypic behaviors, generally considered indicators of negative welfare. We explored the effects of a variable‐time feeding enrichment schedule on behavioral indicators of welfare in four bear species at Cleveland Metroparks Zoo. We distributed the diets of eight bears in one of five enrichment items, for two consecutive days each, and monitored behavior throughout the day. In Experiment 1, we compared variable‐time to fixed‐time presentation of enrichment over two, 10‐day periods. Overall, bears performed more exploratory behavior when enriched (p < 0.0001). Furthermore, variable‐time enrichment was associated with a greater increase in exploratory behavior than fixed‐time enrichment when compared to baseline (p < 0.001). Both fixed‐time (p_unadjusted <0.05, p_adjusted = 0.07) and variable‐schedule (p_unadjusted <0.05, p_adjusted = 0.09) enrichment were also associated with similar decreases in abnormal behavior compared to baseline. For Experiment 2, we tested habituation to enrichment over 30 days using multiple items and a semi‐variable presentation schedule. Again during the enrichment period, bears exhibited increased exploratory behavior (p < 0.0001) and decreased abnormal behaviors compared to baseline (p_unadjusted = 0.05, p_adjusted = 0.09). We observed no habituation during the 30‐day sustained enrichment period for these behaviors. Collectively, these results suggest that daily, variable‐schedule feeding enrichment, with intermittent presentation of unique enrichment items, increases behavioral indicators of positive welfare and decreases behavioral indicators of negative welfare.