Achieving LDL cholesterol target levels <1.81 mmol/L may provide extra cardiovascular protection in patients at high risk: Exploratory analysis of the Standard Versus Intensive Statin Therapy for Patients with Hypercholesterolaemia and Diabetic Retinopathy study

Aims To assess the benefits of intensive statin therapy on reducing cardiovascular (CV) events in patients with type 2 diabetes complicated with hyperlipidaemia and retinopathy in a primary prevention setting in Japan. In the intension-to-treat population, intensive therapy [targeting LDL cholesterol = 2.59 to = 100 to = 2.59 to <3.10 mmol/L in patients with hypercholesterolaemia and diabetic retinopathy

Male Courtship Behavior of Tylototriton (Echinotriton) andersoni Boulenger under Laboratory Conditions

We observed male courtship behavior of Tylototriton (Echinotriton) andersoni in the laboratory. In some males, the cloaca swelled and became wet with mucous secretions from December to May. A male in this condition crept around a female, sniffing around her body and drawing a thread of mucus from his cloaca, so that the female was surrounded by spiderweb-like string of mucous attached to the substrate. The male then deposited a spermatophore by rubbing his cloaca against the substratum while swaying his body to and fro. The male behaved similarly towards females from different localities, though we could not observe any female reaction to the male. Under a phase-contrast microscope, the gross morphology of sperms from a male from Amamioshima Island was similar to that reported for males from Okinawajima

Functional Domains of ZFP809 Essential for Nuclear Localization and Gene Silencing

<div><p>Zinc finger protein 809 (ZFP809) is a member of the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family, and is highly expressed in mouse immature cells. ZFP809 is known to inhibit the expression of transduced genes driven by Moloney murine leukemia virus (MoMLV)-typed retroviral vectors by binding to the primer binding site (PBS) located downstream of the MLV-long terminal repeat (LTR) of the vectors and recruiting protein complexes that introduce epigenetic silencing marks such as histone modifications and DNA methylation at the MLV-LTR. However, it remains undetermined what domains of ZFP809 among the KRAB domain at N-terminus and the seven zinc fingers are critical for gene silencing. In this study, we assessed subcellular localization, gene silencing ability, and binding ability to the PBS of a series of truncated and mutated ZFP809 proteins. We revealed the essential role of the KRAB A box for all functions assessed, together with the accessory roles of a subset of zinc fingers. Our data also suggest that interaction between KAP1 and the KRAB A box of ZFP809 is critical in KAP1-dependent control of gene silencing for ZFP809 targets.</p></div

Identification of ZFP809 sub-domains required for the PBS-dependent gene silencing of MLV-LTR driven transgene expression.

<p>(A) The structures of luciferase reporter vectors used are shown. Black and grey boxes at downstream of the CMV promoter represent the MLV- and dl587rev-derived PBS, respectively. The five nucleotide positions whose base sequence is different between two PBSs are underlined. 293FT cells were co-transfected with one of the reporter vectors and one of the pCMV/flag-X vectors (X denotes either of ZFP809, ΔSD, ΔZF, ZF1, ZF1-2, ZF1-3, ZF1-4, ZF1-5, ZF1-6, ΔKRAB_A, or mtKRAB) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.s001" target="_blank">S1B Fig</a>). RRR (relative response ratio) represents the value of the firefly luciferase activity relative to that of the <i>Renilla</i> luciferase activity (control) at 24 hours after transfection. Error bars show the standard deviation obtained from four independent experiments. Asterisks indicate statistical significance (<i>P</i> <0.05) with <i>Bonferroni</i> correction. (B) The structures of the retroviral GFP reporter vectors. When the RNA genome derived from retroviral vectors is reverse transcribed into an RNA-DNA duplex and then into double-stranded DNA, a part of 5' LTR is recombined to a part of 3' LTR. (C) 293FT cells transduced with one of the retroviral vectors (MLV/EGFP or MSCV/EGFP) were sorted based on EGFP expression, and transduced with one of the pLVSIN/CMV/flag-X/IRES/mCherry vectors or the control vector (pLVSIN/CMV/flag/IRES/mCherry). Then, the expression levels of EGFP and mCherry were analyzed at day 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139274#pone.0139274.s004" target="_blank">S4 Fig</a>) and day 15 (Fig 3C) after transduction.</p

KAP1 dependent biding ability of ZFP809 to MLV-PBS.

<p>(A) Confirmation of knockdown efficiency for KAP1. 293FT cells transduced with lentivirus from shRNA vectors for NC or KAP1 were sorted based on EGFP expression. The sorted cells were subjected to Western blot analysis using anti-KAP1 and Anti-β-Actin antibodies. (B) and (C) EMSA of the MLV-PBS sequence with the intact ZFP809 and ΔKRAB_A proteins in the presence of cold competitor DNA on an 8% gel (B) and a 10% gel (C). Asterisks indicate non-specific bands.</p

Models for ZFP809 sub-domains for nuclear localization, gene silencing and binding ability to MLV-PBS.

<p>ZFP809 contains a KRAB domain at the N-terminus and seven zinc fingers at the C-terminus. The nucleolar exclusion (under the nuclear localization) of ZFP809 is dependent of KAP1- KRAB_A domain interaction. Five (first to fifth) zinc fingers can bind to MLV-PBS in the absence of the KRAB_A domain (accordingly, without KAP1 interaction). However, the intact ZFP809 protein binds to MLV-PBS only when KAP1 interacts with the KRAB_A domain. Therefore, the interaction of KAP1 with the KRAB_A domain is essential for the PBS-dependent gene silencing primed by ZFP809.</p