The use of statistical tools in field testing of putative effects of genetically modified plants on nontarget organisms

To fulfill existing guidelines, applicants that aim to place their genetically modified (GM) insect‐resistant crop plants on the market are required to provide data from field experiments that address the potential impacts of the GM plants on nontarget organisms (NTO's). Such data may be based on varied experimental designs. The recent EFSA guidance document for environmental risk assessment (2010) does not provide clear and structured suggestions that address the statistics of field trials on effects on NTO's. This review examines existing practices in GM plant field testing such as the way of randomization, replication, and pseudoreplication. Emphasis is placed on the importance of design features used for the field trials in which effects on NTO's are assessed. The importance of statistical power and the positive and negative aspects of various statistical models are discussed. Equivalence and difference testing are compared, and the importance of checking the distribution of experimental data is stressed to decide on the selection of the proper statistical model. While for continuous data (e.g., pH and temperature) classical statistical approaches – for example, analysis of variance (ANOVA) – are appropriate, for discontinuous data (counts) only generalized linear models (GLM) are shown to be efficient. There is no golden rule as to which statistical test is the most appropriate for any experimental situation. In particular, in experiments in which block designs are used and covariates play a role GLMs should be used. Generic advice is offered that will help in both the setting up of field testing and the interpretation and data analysis of the data obtained in this testing. The combination of decision trees and a checklist for field trials, which are provided, will help in the interpretation of the statistical analyses of field trials and to assess whether such analyses were correctly applied.     

SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

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Highlights? SorLA is a sorting receptor for GDNF and its signaling receptors GFRa1 and RET ? The SorLA/GFRa1 complex targets GDNF for lysosomal degradation, while GFRa1 is recycled ? SorLA/GFRa1 targets RET for endocytosis and influences GDNF-induced neurotrophic effects ? SorLA knockout mice display altered dopaminergic function and an ADHD-like phenotype     

Subliminal Salience Search Illustrated: EEG Identity and Deception Detection on the Fringe of Awareness

We propose a novel deception detection system based on Rapid Serial Visual Presentation (RSVP). One motivation for the new method is to present stimuli on the fringe of awareness, such that it is more difficult for deceivers to confound the deception test using countermeasures. The proposed system is able to detect identity deception (by using the first names of participants) with a 100% hit rate (at an alpha level of 0.05). To achieve this, we extended the classic Event-Related Potential (ERP) techniques (such as peak-to-peak) by applying Randomisation, a form of Monte Carlo resampling, which we used to detect deception at an individual level. In order to make the deployment of the system simple and rapid, we utilised data from three electrodes only: Fz, Cz and Pz. We then combined data from the three electrodes using Fisher''s method so that each participant was assigned a single p-value, which represents the combined probability that a specific participant was being deceptive. We also present subliminal salience search as a general method to determine what participants find salient by detecting breakthrough into conscious awareness using EEG.     

Activating and Relaxing Music Entrains the Speed of Beat Synchronized Walking

Inspired by a theory of embodied music cognition, we investigate whether music can entrain the speed of beat synchronized walking. If human walking is in synchrony with the beat and all musical stimuli have the same duration and the same tempo, then differences in walking speed can only be the result of music-induced differences in stride length, thus reflecting the vigor or physical strength of the movement. Participants walked in an open field in synchrony with the beat of 52 different musical stimuli all having a tempo of 130 beats per minute and a meter of 4 beats. The walking speed was measured as the walked distance during a time interval of 30 seconds. The results reveal that some music is ‘activating’ in the sense that it increases the speed, and some music is ‘relaxing’ in the sense that it decreases the speed, compared to the spontaneous walked speed in response to metronome stimuli. Participants are consistent in their observation of qualitative differences between the relaxing and activating musical stimuli. Using regression analysis, it was possible to set up a predictive model using only four sonic features that explain 60% of the variance. The sonic features capture variation in loudness and pitch patterns at periods of three, four and six beats, suggesting that expressive patterns in music are responsible for the effect. The mechanism may be attributed to an attentional shift, a subliminal audio-motor entrainment mechanism, or an arousal effect, but further study is needed to figure this out. Overall, the study supports the hypothesis that recurrent patterns of fluctuation affecting the binary meter strength of the music may entrain the vigor of the movement. The study opens up new perspectives for understanding the relationship between entrainment and expressiveness, with the possibility to develop applications that can be used in domains such as sports and physical rehabilitation.     

Metabotropic Glutamate Receptor 1 Expression and Its Polymorphic Variants Associate with Breast Cancer Phenotypes

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

High Fat Diet Accelerates Pathogenesis of Murine Crohn’s Disease-Like Ileitis Independently of Obesity

Background

Obesity has been associated with a more severe disease course in inflammatory bowel disease (IBD) and epidemiological data identified dietary fats but not obesity as risk factors for the development of IBD. Crohn’s disease is one of the two major IBD phenotypes and mostly affects the terminal ileum. Despite recent observations that high fat diets (HFD) impair intestinal barrier functions and drive pathobiont selection relevant for chronic inflammation in the colon, mechanisms of high fat diets in the pathogenesis of Crohn’s disease are not known. The aim of this study was to characterize the effect of HFD on the development of chronic ileal inflammation in a murine model of Crohn’s disease-like ileitis.

Methods

TNF^ΔARE/WT mice and wildtype C57BL/6 littermates were fed a HFD compared to control diet for different durations. Intestinal pathology and metabolic parameters (glucose tolerance, mesenteric tissue characteristics) were assessed. Intestinal barrier integrity was characterized at different levels including polyethylene glycol (PEG) translocation, endotoxin in portal vein plasma and cellular markers of barrier function. Inflammatory activation of epithelial cells as well as immune cell infiltration into ileal tissue were determined and related to luminal factors.

Results

HFD aggravated ileal inflammation but did not induce significant overweight or typical metabolic disorders in TNF^ΔARE/WT. Expression of the tight junction protein Occludin was markedly reduced in the ileal epithelium of HFD mice independently of inflammation, and translocation of endotoxin was increased. Epithelial cells showed enhanced expression of inflammation-related activation markers, along with enhanced luminal factors-driven recruitment of dendritic cells and Th17-biased lymphocyte infiltration into the lamina propria.

Conclusions

HFD feeding, independently of obesity, accelerated disease onset of small intestinal inflammation in Crohn’s disease-relevant mouse model through mechanisms that involve increased intestinal permeability and altered luminal factors, leading to enhanced dendritic cell recruitment and promoted Th17 immune responses.     

Suppressed Retinal Degeneration in Aged Wild Type and APPswe/PS1ΔE9 Mice by Bone Marrow Transplantation

Alzheimer''s disease (AD) is an age-related condition characterized by accumulation of neurotoxic amyloid β peptides (Aβ) in brain and retina. Because bone marrow transplantation (BMT) results in decreased cerebral Aβ in experimental AD, we hypothesized that BMT would mitigate retinal neurotoxicity through decreased retinal Aβ. To test this, we performed BMT in APPswe/PS1ΔE9 double transgenic mice using green fluorescent protein expressing wild type (wt) mice as marrow donors. We first examined retinas from control, non-transplanted, aged AD mice and found a two-fold increase in microglia compared with wt mice, prominent inner retinal Aβ and paired helical filament-tau, and decreased retinal ganglion cell layer neurons. BMT resulted in near complete replacement of host retinal microglia with BMT-derived cells and normalized total AD retinal microglia to non-transplanted wt levels. Aβ and paired helical filament-tau were reduced (61.0% and 44.1% respectively) in BMT-recipient AD mice, which had 20.8% more retinal ganglion cell layer neurons than non-transplanted AD controls. Interestingly, aged wt BMT recipients also had significantly more neurons (25.4%) compared with non-transplanted aged wt controls. Quantitation of retinal ganglion cell layer neurons in young mice confirmed age-related retinal degeneration was mitigated by BMT. We found increased MHC class II expression in BMT-derived microglia and decreased oxidative damage in retinal ganglion cell layer neurons. Thus, BMT is neuroprotective in age-related as well as AD-related retinal degeneration, and may be a result of alterations in innate immune function and oxidative stress in BMT recipient mice.