Comparative Methylation of ERVWE1/Syncytin-1 and Other Human Endogenous Retrovirus LTRs in Placenta Tissues

Human endogenous retroviruses (HERVs) are globally silent in somatic cells. However, some HERVs display high transcription in physiological conditions. In particular, ERVWE1, ERVFRDE1 and ERV3, three proviruses of distinct families, are highly transcribed in placenta and produce envelope proteins associated with placenta development. As silencing of repeated elements is thought to occur mainly by DNA methylation, we compared the methylation of ERVWE1 and related HERVs to appreciate whether HERV methylation relies upon the family, the integration site, the tissue, the long terminal repeat (LTR) function or the associated gene function. CpG methylation of HERV-W LTRs in placenta-associated tissues was heterogeneous but a joint epigenetic control was found for ERVWE1 5′LTR and its juxtaposed enhancer, a mammalian apparent LTR retrotransposon. Additionally, ERVWE1, ERVFRDE1 and ERV3 5′LTRs were all essentially hypomethylated in cytotrophoblasts during pregnancy, but showed distinct and stage-dependent methylation profiles. In non-cytotrophoblastic cells, they also exhibited different methylation profiles, compatible with their respective transcriptional activities. Comparative analyses of transcriptional activity and LTR methylation in cell lines further sustained a role for methylation in the control of functional LTRs. These results suggest that HERV methylation might not be family related but copy-specific, and related to the LTR function and the tissue. In particular, ERVWE1 and ERV3 could be developmentally epigenetically regulated HERVs

A prospective comparison of ER, PR, Ki67 and gene expression in paired sequential core biopsies of primary, untreated breast cancer

BACKGROUND: Sequential biopsy of breast cancer is used to assess biomarker effects and drug efficacy. The preoperative "window of opportunity" setting is advantageous to test biomarker changes in response to therapeutic agents in previously untreated primary cancers. This study tested the consistency over time of paired, sequential biomarker measurements on primary, operable breast cancer in the absence of drug therapy. METHODS: Immunohistochemistry was performed for ER, PR and Ki67 on paired preoperative/operative tumor samples taken from untreated patients within 2 weeks of each other. Microarray analysis on mRNA extracted from formalin fixed paraffin embedded cores was performed using Affymetrix based arrays on paired core biopsies analysed using Ingenuity Pathway Analysis (IPA) and Gene Set Analysis (GSA). RESULTS: In 41 core/resection pairs, the recognised trend to lower ER, PR and Ki67 score on resected material was confirmed. Concordance for ER, PR and Ki67 without changing biomarker status (e.g. ER+ to ER-) was 90, 74 and 80 % respectively. However, in 23 paired core samples (diagnostic core v on table core), Ki67 using a cut off of 13.25 % was concordant in 22/23 (96 %) and differences in ER and PR immunohistochemistry by Allred or Quickscore between the pairs did not impact hormone receptor status. IPA and GSA demonstrated substantial gene expression changes between paired cores at the mRNA level, including reduced expression of ER pathway analysis on the second core, despite the absence of drug intervention. CONCLUSIONS: Sequential core biopsies of primary breast cancer (but not core versus resection) was consistent and is appropriate to assess the effects of drug therapy in vivo on ER, PR and Ki67 using immunohistochemistry. Conversely, studies utilising mRNA expression may require non-treatment controls to distinguish therapeutic from biopsy differences

Experimental demonstration of a predictable single photon source with variable photon flux

We present a predictable single-photon source (SPS) based on a silicon vacancy centre in nanodiamond which is optically excited by a pulsed laser. At an excitation rate of 70 MHz the source delivers a photon flux large enough to be measured by a low optical flux detector (LOFD). The directly measured photon flux constitutes an absolute reference. By changing the repetition rate of the pulsed laser, we are able to change the photon flux of our SPS in a controllable way which in turn can act as a reference. The advantage of our method is that it does not require precise knowledge of the source efficiency, but the source is calibrated by the LOFD and can be used for detector responsivity characterizations at the few-photon level

Experimental realization of an absolute single-photon source based on a single nitrogen vacancy center in a nanodiamond

We report on the experimental realization of an absolute single-photon source based on a single nitrogen vacancy (NV) center in a nanodiamond at room temperature and on the calculation of its absolute spectral photon flux from experimental data. The single-photon source was calibrated with respect to its photon flux and its spectral photon rate density. The photon flux was measured with a low-noise silicon photodiode traceable to the primary standard for optical flux, taking into account the absolute spectral power distribution using a calibrated spectroradiometer. The optical radiant flux is adjustable from 55 fW, which is almost the lowest detection limit for the silicon photodiode, and 75 fW, which is the saturation power of the NV center. These fluxes correspond to total photon flux rates between 190,000 photons per second and 260,000 photons per second, respectively. The single-photon emission purity is indicated by a g((2))(0) value, which is between 0.10 and 0.23, depending on the excitation power. To our knowledge, this is the first single-photon source absolutely calibrated with respect to its absolute optical radiant flux and spectral power distribution, traceable to the corresponding national standards via an unbroken traceability chain. The prospects for its application, e.g., for the detection efficiency calibration of single-photon detectors as well as for use as a standard photon source in the low photon flux regime, are promising. (C) 2017 Optical Society of Americ

Loss of cone photoreceptors caused by chromophore depletion is partially prevented by the artificial chromophore pro-drug, 9-cis-retinyl acetate

Inactivating mutations in the retinoid isomerase (RPE65) or lecithin:retinol acyltransferase (LRAT) genes cause Leber congenital amaurosis (LCA), a severe visual impairment in humans. Both enzymes participate in the retinoid (visual) cycle, the enzymatic pathway that continuously generates 11-cis-retinal, the chromophore of visual pigments in rod and cone photoreceptor cells needed for vision. We investigated human RPE65–LCA patients and mice with visual cycle abnormalities to determine the impact of chronic chromophore deprivation on cones. Young patients with RPE65 mutations showed foveal cone loss along with shortened inner and outer segments of remaining cones; cone cell loss also was dramatic in young mice lacking Rpe65 or Lrat gene function. To selectively evaluate cone pathophysiology, we eliminated the rod contribution to electroretinographic (ERG) responses by generating double knockout mice lacking Lrat or Rpe65 together with an inactivated rod-specific G protein transducin gene (Gnat1−/−). Cone ERG responses were absent in Gnat1−/−Lrat−/− mice which also showed progressive degeneration of cones. Cone ERG responses in Gnat1−/−Rpe65−/− mice were markedly reduced and declined over weeks. Treatment of these mice with the artificial chromophore pro-drug, 9-cis-retinyl acetate, partially protected inferior retinal cones as evidenced by improved ERGs and retinal histochemistry. Gnat1−/− mice chronically treated with retinylamine, a selective inhibitor of RPE65, also showed a decline in the number of cones that was ameliorated by 9-cis-retinyl acetate. These results suggest that chronic lack of chromophore leads to progressive loss of cones in mice and humans. Therapy for LCA patients should be geared toward early adequate delivery of chromophore to cone photoreceptors