Survival rates for males and females of the <i>Nothobranchius furzeri</i> strain MZM-04/10.

<p>Animals were maintained in 40 l tanks as males-only (A) or females-only groups (B) and in mixed-sex control groups. The graphs represent the lifespan of control males (n = 22), males-only (n = 28), control females (n = 24) and females-only (n = 30) recorded during 2 independent trials. Median (50% survival) and maximum lifespan (10% survival) is indicated.</p

Age dependent changes in mean body weight and size in gender separated and control animals.

<p>Determination of length (A) and body weight (B) of males-only and control males (left) and females-only and control females (right). Graphs show mean ± sem. Significance for given data points were calculated by Bonferroni post hoc test.</p

The BOR mutant L550P fails to translocate into the nucleus in presence of Six.

<p>(A) Eya domain of human Eya1 depicting the disease associated missense mutations included in this study. BOR branchio-oto-renal; BO branchio-otic; OD ocular defects. (B) Cellular localization of Eya1 and disease-associated Eya1 mutants. COS-7 cells were transfected with expression plasmids encoding EGFP-Eya1 or mutants either alone or in combination with Six2. Insets show counterstaining with Hoechst dye. (C) COS-7 cells were transfected with HA-tagged <i>Eya1</i> or mutants together with FLAG-Six2 or empty vector. Nuclear (N) and cytoplasmic (C) extracts were analyzed by immunoblotting.</p

Eya1 degradation occurs via the proteasome and is inhibited by Six proteins.

<p>(A) Eya1 and its mutants accumulate in the presence of the proteasome inhibitor lactacystin. COS-7 cells were transfected with wild type or mutant HA-Eya1 or empty vector. 24 h post-transfection cells were treated with lactacystin (1 µM). Cells were lysed and Eya1 was detected by immunoblotting. (B) Eya1 is ubiquitinated. COS-7 cells were co-transfected with HA-Eya1 and HA-ubiquitin or His-ubiquitin. 24 h later, cells were treated with MG132 (10 µM) or DMSO for 24 h. For <i>in vivo</i> ubiquitination assay, His-ubiquitin-labelled proteins were purified from cell lysates followed by immunoblotting and detection of Eya1 using anti-Eya1 antibody. Asterisks indicate unspecific signals. (C) Schematic representation of the Eya1 mutants used for <i>in vivo</i> ubiquitination assay. Position of lysine residues is indicated in the scheme of full-length Eya1. (D) Eya1 ubiquitination occurs in two distinct regions of the Eya domain. COS-7 cells were co-transfected with His-ubiquitin and the indicated <i>Eya1</i> expression constructs, treated with MG132 (10 µM), and subjected to <i>in vivo</i> ubiquitination assay followed by immunoblotting and detection of Eya1 using anti-HA antibody. (E) Six proteins inhibit ubiquitination of Eya1. COS-7 cells overexpressing wild type or mutant HA-Eya1, His-ubiquitin and Six2 or empty vector were subjected to <i>in vivo</i> ubiquitination assay. Ubiquitinated Eya1 protein was detected by immunoblotting using anti-HA antibody. β-actin was used as a loading control for total cell lysate.</p

Six proteins stabilize Eya1 and Eya1 mutants with the exception of L550P.

<p>(A) COS-7 cells were transfected with empty vector (control), HA-Eya1 or mutants, and FLAG-Six2. Equal amounts of protein were analyzed by SDS-PAGE and immunoblotting using anti-HA and anti-FLAG antibody. (B) Endogenous Eya1 is stabilized in presence of Six2. MK4 cells were transfected with <i>Eya1</i>-specific or non-target control siRNA and Eya1 protein levels were detected by immunoblotting using anti-Eya1 antibody (left). MK4 cells stably expressing FLAG-Six2 were subject to immunoblot analyses using Eya1-specific antibody (right). Asterisk indicates unspecific signal. (C) Six induces accumulation of Eya1 protein in different cell lines via the conserved Six and homeo domain. COS-7 and U2OS cells were transfected with HA-<i>Eya1</i> and expression vectors for <i>Six1</i>, <i>Six2</i>, C-terminal domain of Six2 (CD), <i>GDNF</i> and <i>H-Ras</i>. Eya1 protein levels were detected by immunoblotting using anti-HA antibody. (D) Schematic representation of the Six2 deletion constructs used for characterization of Eya1 stabilization (left). COS-7 cells were co-transfected with HA-Eya1, Six2 or deletion constructs as indicated. Eya1 protein levels were analyzed by immunoblotting.</p

Eya1 stabilization requires interaction with Six proteins.

<p>COS-7 cells were transfected with HA-Eya1 or mutants and FLAG-Six2 as indicated (left). In case of the mutant L550P cells were treated with MG132 (10 µM) to enhance protein levels (right). Six2 was immunoprecipitated using anti-FLAG antibody, and interacting Eya1 was detected by immunoblotting.</p

Linear Poly(ethylene imine)-Based Hydrogels for Effective Binding and Release of DNA

A series of copolymers containing both amine groups of linear poly­(ethylene imine) (LPEI) and double bonds of poly­(2-(3-butenyl)-2-oxazoline) (PButEnOx) was prepared. To this end, a poly­(2-ethyl-2-oxazoline) (PEtOx) precursor was hydrolyzed to the respective LPEI and functionalized in an amidation reaction with butenyl groups resulting in the double bond containing poly­(2-(3-butenyl-2-oxazoline)-<i>co</i>-ethylene imine) (P­(ButEnOx-<i>co</i>-EI)). Hydrogels were obtained by cross-linking with dithiols under UV-irradiation resulting in networks with different properties in dependence of the content of double bonds. The developed method allows the exact control of the amount of ethylene imine units within the copolymer and, thus, within the resulting hydrogels. The gel structures were characterized by solid state NMR and infrared spectroscopy. In addition the water uptake behavior from the liquid and the gas phase was investigated. It was shown by an ethidium bromide assay (EBA) that the copolymers and the respective hydrogels were able to bind and release DNA. Furthermore, the influence of the ethylene imine content on this interaction was investigated

Additional file 2: Table S1. of MiR-21 is required for efficient kidney regeneration in fish

Clustered differentially expressed genes after treatment of N. furzeri with gentamicin, antimir-21 orgentamicin/antimir-21. (XLSX 429 kb

Additional file 1: Figure S1. of MiR-21 is required for efficient kidney regeneration in fish

Trichrome staining of kidney samples treated with antimiR-21 at different time points after gentamicin injection. As positive control old fibrotic fish kidney was used. Blue indicates fibrotic tissue, hematoxylin and eosin was used as counterstain. (PPTX 67880 kb

Matrix Supported Poly(2-oxazoline)-Based Hydrogels for DNA Catch and Release

We describe the synthesis of matrix supported hydrogel structures based on amine containing poly­(2-oxazoline)­s and their use to bind and release genetic material for potential applications in diagnostics or pathogen detection. Amine containing poly­(2-oxazoline)­s were synthesized by copolymerization of 2-ethyl-2-oxazoline with a monomer bearing a <i>tert</i>-butyl oxycarbonyl (Boc) protected amine group in the 2-position and subsequent deprotection. The statistical copolymers were used to generate hydrogels and matrix supported hydrogels by cross-linking of a certain fraction of the amine groups with epichlorhydrin. Supported structures were prepared by soaking porous polyethylene (PE) or polypropylene (PP) filter materials in a copolymer/epichlorhydrin solution, which was cross-linked upon heating. Scanning electron microscopy (SEM) of the composites revealed a bead like structure of the gel phase, which could be attributed to a lower critical solution temperature (LCST) behavior of the initial polymer prior to gelation. The dependency of the LCST behavior on the content of amine groups was investigated. Swelling values and the ratio of hydrogel per composite was determined using water sorption analysis. Subsequently, the ability of the systems to absorb and release labeled DNA was tested. Uptake and stimulated release, triggered by changes in pH, temperature, and heparin concentration, were investigated using fluorescence microscopy. Polymerase chain reaction (PCR) proved the successful recovery of the DNA, demonstrating the potential of the presented system for a broad range of molecular biological applications