Screening of Bioactive Peptides Using an Embryonic Stem Cell-Based Neurodifferentiation Assay

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons

Developing rat testicular organoid models for assessing the reproductive toxicity of antidepression drugs in vitro

With the increasing incidence of depression worldwide, antidepressant medications are commonly used in males of reproductive age for long-term treatment of depression, as well as other disorders. Antidepressants are known to be associated with sexual side effects, such as decreased libido and impotence. Their effects on semen parameters and other markers of male fertility have been less thoroughly described, such as sperm motility and fertilization ability. Therefore, it is critical to determine the potential toxic effects of antidepressants on reproductive organs. A recent study systemically determined the effect of different drugs on the telomere-related DNA damage response of germ cells. In addition, another study showed that mirtazapine has less toxic effects than amitriptyline and other drugs. However, these studies used germ cell lines to determine the phenotype, which limits their potential translational value in toxicity studies in humans. Given the widespread and often long-term use of antidepressant medications, there is an urgent need for further data regarding their impact on semen quality and subsequent male fertility

TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors

Screening of bioactive peptides using an embryonic stem cell-based neurodifferentiation assay

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons

TYK2^K923E protein level is reduced and TYK2 differs organ-specifically.

<p>A. WT, <i>Tyk2^−/−</i> and <i>Tyk2^K923E</i> mice were used to prepare whole cell extracts from BMMΦs, T cells and various organs (as indicated). Levels of expression of TYK2 and JAK1 were determined by immunoprecipitation and Western blot analysis. NFκB-p65 was used as input control. TYK2^K923E protein levels were quantified using ImageJ software for Mac OS X (open source, <a href="http://rsb.info.nih.gov/ij/index.html" target="_blank">http://rsb.info.nih.gov/ij/index.html</a>) and were between 13% and 30% in BMMΦs and approximately 58% in T cells compared to WT. B. Total RNA was isolated from WT and <i>Tyk2^K923E</i> BMMΦs and cDNA was used to analyse Tyk2 mRNA expression normalized to the housekeeping gene <i>Ube2D2</i>. Results from 4 independent experiments are shown (n = 6 per genotype). C. BMMΦs were treated with the proteasomal inhibitor MG-132 (50 µM), the autophagy-lysosome inhibitor 3-MA (10 mM) or the lysosome-acidification inhibitor bafilomycin A₁ (80 nM) for the indicated period of time (upper panel), for 11 h (middle panel) or 48 h (lower panel). Whole cell extracts were used to determine TYK2 and JAK1 expression levels by immunoprecipitation and Western blot analysis. As a control, a Western blot for HO-1 was performed. D. From day 5 after isolation of WT BMMΦs, cells were treated with JAK inhibitor I (panJAK inhibitor; 15 nM upper panel and 300 nM lower panel) for the indicated period of time. TYK2 and JAK2 expression levels were analysed as described in (A and C); NFκB-p65 was used as input control.</p

Transcriptional induction of IFN-responsive genes is similar in <i>Tyk2^K923E</i> and <i>Tyk2^−/−</i> cells.

<p>A.-C. WT, <i>Tyk2^−/−</i> and <i>Tyk2^K923E</i> BMMΦs were treated with IFNα (500 U/ml), IFNβ (100 U/ml) or IFNγ (100 U/ml) for 6 h or left untreated. Total RNA was extracted, reverse-transcribed and analysed by RT-qPCR for expression of <i>Oas1a</i>, <i>Ifit1</i> (A), <i>Cxcl1</i>, <i>Socs1</i> (B) and <i>Irf7, Tap1</i> (C). <i>Ube2D2</i> was used for normalization and expression levels were calculated relative to untreated WT cells. Data are derived from three independent experiments and depicted as mean values (+/− SE). D. WT, <i>Tyk2^−/−</i> and <i>Tyk2^K923E</i> BMMΦs were treated with indicated doses of IFNβ for 6 h. Target gene expression was determined as described in A-C. Mean values (+/− SD) derived from two independent experiments are depicted. Note that due to sample size a statistical analysis was not performed.</p

<i>Tyk2^−/−</i> and <i>Tyk2^K923E</i> mice show similarly increased susceptibility to virus infection.

<p>VSV (A) was administered intranasally (i.n.) and EMCV (B) intraperitoneally (i.p.). Mice were monitored twice daily for survival over a two-week period. Data are derived from two independent experiments (n = 20/genotype and n = 21/genotype for A and B, respectively).</p

IFN treatment leads to similar activation of JAKs and STATs in <i>Tyk2^K923E</i> and <i>Tyk2</i>-deficient cells.

<p>BMMΦs were treated with IFNβ (500 U/ml) for 20 min or left untreated. Whole cell extracts were used to determine levels of JAK1 tyrosine phosphorylation and JAK1 expression (left panel) and of TYK2 tyrosine phosphorylation and TYK2 expression (right panel) by immunoprecipitation and Western blot analysis. B.-D. BMMΦs were treated with IFNα (500 U/ml), IFNβ (100 U/ml) or IFNγ (100 U/ml) for 20 min or left untreated. Whole cell extracts were used to determine STAT1α/β tyrosine phosphorylation and levels of STAT1α/β expression (B), levels of STAT2 tyrosine phosphorylation and STAT2 expression (C) and levels of STAT3 tyrosine phosphorylation and STAT3 expression (D) by Western blot analysis. E. BMMΦs were treated with IFNβ (10, 100 or 500 U/ml) for 20 min or left untreated and Western blot analysis performed as described in (B). F. NK cells were treated with the indicated doses of IFNβ for 20 min or left untreated. Levels of tyrosine phosphorylation and protein expression of STAT1α/β and STAT4 were analysed by Western blot. G. NK cells were treated with IFNα (500 U/ml) for the times indicated and STAT1 and 4 analysed as described in (F); H. NK cells were treated with IFNβ (100 U/ml) for the times indicated and STAT1 and 4 analysed as described in (F); ERK p85 (B, C, E-H) and ERK p42 (D) served as a loading control.</p