Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development,Function or Stress Response

The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive Cxadr knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, Cxadr is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of Cxadr for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific Cxadr knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of Cxadr on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of Cxadr.     

Pleiotropic and Sex-Specific Effects of Cancer GWAS SNPs on Melanoma Risk in the Population Architecture Using Genomics and Epidemiology (PAGE) Study

Background

Several regions of the genome show pleiotropic associations with multiple cancers. We sought to evaluate whether 181 single-nucleotide polymorphisms previously associated with various cancers in genome-wide association studies were also associated with melanoma risk.

Methods

We evaluated 2,131 melanoma cases and 20,353 controls from three studies in the Population Architecture using Genomics and Epidemiology (PAGE) study (EAGLE-BioVU, MEC, WHI) and two collaborating studies (HPFS, NHS). Overall and sex-stratified analyses were performed across studies.

Results

We observed statistically significant associations with melanoma for two lung cancer SNPs in the TERT-CLPTM1L locus (Bonferroni-corrected p<2.8x10^-4), replicating known pleiotropic effects at this locus. In sex-stratified analyses, we also observed a potential male-specific association between prostate cancer risk variant rs12418451 and melanoma risk (OR=1.22, p=8.0x10^-4). No other variants in our study were associated with melanoma after multiple comparisons adjustment (p>2.8e^-4).

Conclusions

We provide confirmatory evidence of pleiotropic associations with melanoma for two SNPs previously associated with lung cancer, and provide suggestive evidence for a male-specific association with melanoma for prostate cancer variant rs12418451. This SNP is located near TPCN2, an ion transport gene containing SNPs which have been previously associated with hair pigmentation but not melanoma risk. Previous evidence provides biological plausibility for this association, and suggests a complex interplay between ion transport, pigmentation, and melanoma risk that may vary by sex. If confirmed, these pleiotropic relationships may help elucidate shared molecular pathways between cancers and related phenotypes.     

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca²⁺-imaging using the superior green Ca²⁺-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.     

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.     

The Magical Activation of Left Amygdala when Reading Harry Potter: An fMRI Study on How Descriptions of Supra-Natural Events Entertain and Enchant

Literature containing supra-natural, or magical events has enchanted generations of readers. When reading narratives describing such events, readers mentally simulate a text world different from the real one. The corresponding violation of world-knowledge during this simulation likely increases cognitive processing demands for ongoing discourse integration, catches readers’ attention, and might thus contribute to the pleasure and deep emotional experience associated with ludic immersive reading. In the present study, we presented participants in an MR scanner with passages selected from the Harry Potter book series, half of which described magical events, while the other half served as control condition. Passages in both conditions were closely matched for relevant psycholinguistic variables including, e.g., emotional valence and arousal, passage-wise mean word imageability and frequency, and syntactic complexity. Post-hoc ratings showed that readers considered supra-natural contents more surprising and more strongly associated with reading pleasure than control passages. In the fMRI data, we found stronger neural activation for the supra-natural than the control condition in bilateral inferior frontal gyri, bilateral inferior parietal lobules, left fusiform gyrus, and left amygdala. The increased activation in the amygdala (part of the salience and emotion processing network) appears to be associated with feelings of surprise and the reading pleasure, which supra-natural events, full of novelty and unexpectedness, brought about. The involvement of bilateral inferior frontal gyri likely reflects higher cognitive processing demand due to world knowledge violations, whereas increased attention to supra-natural events is reflected in inferior frontal gyri and inferior parietal lobules that are part of the fronto-parietal attention network.     

Tissue Distribution of the Ehrlichia muris-Like Agent in a Tick Vector

Human pathogens transmitted by ticks undergo complex life cycles alternating between the arthropod vector and a mammalian host. While the latter has been investigated to a greater extent, examination of the biological interactions between microbes and the ticks that carry them presents an equally important opportunity for disruption of the disease cycle. In this study, we used in situ hybridization to demonstrate infection by the Ehrlichia muris-like organism, a newly recognized human pathogen, of Ixodes scapularis ticks, a primary vector for several important human disease agents. This allowed us to assess whole sectioned ticks for the patterns of tissue invasion, and demonstrate generalized dissemination of ehrlichiae in a variety of cell types and organs within ticks infected naturally via blood feeding. Electron microscopy was used to confirm these results. Here we describe a strong ehrlichial affinity for epithelial cells, neuronal cells of the synganglion, salivary glands, and male accessory glands.     

New Mutation in the Mouse Xpd/Ercc2 Gene Leads to Recessive Cataracts

Cataracts are the major eye disorder and have been associated mainly with mutations in lens-specific genes, but cataracts are also frequently associated with complex syndromes. In a large-scale high-throughput ENU mutagenesis screen we analyzed the offspring of paternally treated C3HeB/FeJ mice for obvious dysmorphologies. We identified a mutant suffering from rough coat and small eyes only in homozygotes; homozygous females turned out to be sterile. The mutation was mapped to chromosome 7 between the markers 116J6.1 and D7Mit294;4 other markers within this interval did not show any recombination among 160 F2-mutants. The critical interval (8.6 Mb) contains 3 candidate genes (Apoe, Six5, Opa3); none of them showed a mutation. Using exome sequencing, we identified a c.2209T>C mutation in the Xpd/Ercc2 gene leading to a Ser737Pro exchange. During embryonic development, the mutant eyes did not show major changes. Postnatal histological analyses demonstrated small cortical vacuoles; later, cortical cataracts developed. Since XPD/ERCC2 is involved in DNA repair, we checked also for the presence of the repair-associated histone γH2AX in the lens. During the time, when primary lens fiber cell nuclei are degraded, γH2AX was strongly expressed in the cell nuclei; later, it demarcates clearly the border of the lens cortex to the organelle-free zone. Moreover, we analyzed also whether seemingly healthy heterozygotes might be less efficient in repair of DNA damage induced by ionizing radiation than wild types. Peripheral lymphocytes irradiated by 1Gy Cs¹³⁷ showed 6 hrs after irradiation significantly more γH2AX foci in heterozygotes than in wild types. These findings demonstrate the importance of XPD/ERCC2 not only for lens fiber cell differentiation, but also for the sensitivity to ionizing radiation. Based upon these data, we hypothesize that variations in the human XPD/ERCC2 gene might increase the susceptibility for several disorders besides Xeroderma pigmentosum in heterozygotes under particular environmental conditions.     

Single Doses up to 800 mg of E-52862 Do Not Prolong the QTc Interval – A Retrospective Validation by Pharmacokinetic-Pharmacodynamic Modelling of Electrocardiography Data Utilising the Effects of a Meal on QTc to Demonstrate ECG Assay Sensitivity

Background

E-52862 is a Sigma-1 receptor antagonist (S1RA) currently under investigation as a potential analgesic medicine. We successfully applied a concentration-effect model retrospectively to a four-way crossover Phase I single ascending dose study and utilized the QTc shortening effects of a meal to demonstrate assay sensitivity by establishing the time course effects from baseline in all four periods, independently from any potential drug effects.

Methods

Thirty two healthy male and female subjects were included in four treatment periods to receive single ascending doses of 500 mg, 600 mg or 800 mg of E-52862 or placebo. PK was linear over the dose range investigated and doses up to 600 mg were well tolerated. The baseline electrocardiography (ECG) measurements on Day-1 were time-matched with ECG and pharmacokinetic (PK) samples on Day 1 (dosing day).

Results

In this conventional mean change to time-matched placebo analysis, the largest time-matched difference to placebo QTcI was 1.44 ms (90% CI: -4.04, 6.93 ms) for 500 mg; -0.39 ms (90% CI: -3.91, 3.13 ms) for 600 mg and 1.32 ms (90% CI: -1.89, 4.53 ms) for 800 mg of E-52862, thereby showing the absence of any QTc prolonging effect at the doses tested. In addition concentration-effect models, one based on the placebo corrected change from baseline and one for the change of QTcI from average baseline with time as fixed effect were fitted to the data confirming the results of the time course analysis.

Conclusion

The sensitivity of this study to detect small changes in the QTc interval was confirmed by demonstrating a shortening of QTcF of -8.1 (90% CI: -10.4, -5.9) one hour and -7.2 (90% CI: -9.4, -5.0) three hours after a standardised meal.

Trial Registration

EU Clinical Trials Register EudraCT 2010 020343 13