An anisotropic interaction model for simulating fingerprints.

Evidence suggests that both the interaction of so-called Merkel cells and the epidermal stress distribution play an important role in the formation of fingerprint patterns during pregnancy. To model the formation of fingerprint patterns in a biologically meaningful way these patterns have to become stationary. For the creation of synthetic fingerprints it is also very desirable that rescaling the model parameters leads to rescaled distances between the stationary fingerprint ridges. Based on these observations, as well as the model introduced by Kücken and Champod we propose a new model for the formation of fingerprint patterns during pregnancy. In this anisotropic interaction model the interaction forces not only depend on the distance vector between the cells and the model parameters, but additionally on an underlying tensor field, representing a stress field. This dependence on the tensor field leads to complex, anisotropic patterns. We study the resulting stationary patterns both analytically and numerically. In particular, we show that fingerprint patterns can be modeled as stationary solutions by choosing the underlying tensor field appropriately

Phosphorelay through the bifunctional phosphotransferase PhyT controls the general stress response in an alphaproteobacterium.

Two-component systems constitute phosphotransfer signaling pathways and enable adaptation to environmental changes, an essential feature for bacterial survival. The general stress response (GSR) in the plant-protecting alphaproteobacterium Sphingomonas melonis Fr1 involves a two-component system consisting of multiple stress-sensing histidine kinases (Paks) and the response regulator PhyR; PhyR in turn regulates the alternative sigma factor EcfG, which controls expression of the GSR regulon. While Paks had been shown to phosphorylate PhyR in vitro, it remained unclear if and under which conditions direct phosphorylation happens in the cell, as Paks also phosphorylate the single domain response regulator SdrG, an essential yet enigmatic component of the GSR signaling pathway. Here, we analyze the role of SdrG and investigate an alternative function of the membrane-bound PhyP (here re-designated PhyT), previously assumed to act as a PhyR phosphatase. In vitro assays show that PhyT transfers a phosphoryl group from SdrG to PhyR via phosphoryl transfer on a conserved His residue. This finding, as well as complementary GSR reporter assays, indicate the participation of SdrG and PhyT in a Pak-SdrG-PhyT-PhyR phosphorelay. Furthermore, we demonstrate complex formation between PhyT and PhyR. This finding is substantiated by PhyT-dependent membrane association of PhyR in unstressed cells, while the response regulator is released from the membrane upon stress induction. Our data support a model in which PhyT sequesters PhyR, thereby favoring Pak-dependent phosphorylation of SdrG. In addition, PhyT assumes the role of the SdrG-phosphotransferase to activate PhyR. Our results place SdrG into the GSR signaling cascade and uncover a dual role of PhyT in the GSR

Phosphorylatable SdrG is essential for its positive regulatory role.

<p>β-galactosidase activity of the EcfG-dependent <i>nhaA2p-lacZ</i> fusion in indicated <i>S</i>. <i>melonis</i> Fr1 mutant backgrounds upon overnight overexpression of <i>sdrG</i> and variants from the vanillate-inducible pVH vector with 250 μM vanillate. pVH was used as empty-vector control. Black bars and gray bars represent β-galactosidase activity pre- and 1 h post-induction with the chemical stress mixture (1% ethanol, 80 mM NaCl and 50 μM TBHP), respectively. Values are given as mean ±SD of three independent experiments.</p

PhyT forms a complex with PhyR.

<p>(A) BACTH assay with bacteria spotted onto LB plates containing X-Gal (40 μg/mL), IPTG (0.5 mM) and antibiotics for selection. Wild-type PhyR and PhyR derivatives carrying either a D194A, a E235A or a combination of both mutations, were analyzed as C-terminal T18 fusions, while wild-type NepR was fused N-terminally and PhyT C-terminally to the T25 fragment of the <i>B</i>. <i>pertussis</i> CyaA protein to investigate interaction. Pictures were taken after 24 h of incubation at 30°C. Blue colonies indicate protein interaction. This image is a representative of three independent experiments. (B) β-galactosidase assays were performed for quantification in three biological replicates. Overnight cultures containing 0.5 mM IPTG and antibiotics for selection, were inoculated from single colonies of the co-transformed bacteria and incubated at 30°C. (C) Adenylate cyclase T18 fusions to PhyR proteins were detected in the samples used for quantitative analysis with Western blot analysis with CyaA monoclonal antibody (3D1) (1:2.000) (Santa Cruz Biotechnology) and a goat α-mouse antibody (1:3.000) with an exposure time of 2 min.</p

PhyT (formerly PhyP) transfers phosphoryl groups from SdrG~P to PhyR.

<p><i>In vitro</i> phosphotransfer from SdrG~P to PhyT and further to PhyR in absence and presence of NepR over time. SdrG (10 μM) was phosphorylated using PakF (autophosphorylated with [γ-32P] ATP) on Ni-NTA columns. PhyR (5 μM), NepR (7.5 μM) and <i>E</i>. <i>coli</i> membrane particles (5 mg membrane fraction/mL) harboring either wild-type PhyT or the PhyT (H341A) derivative as a control were added as indicated. For confirmation of comparable amounts of PhyT and PhyT (H341A), Western blot analysis was conducted (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.s002" target="_blank">S2A Fig</a>).</p

Model for GSR regulation in <i>S</i>. <i>melonis</i> Fr1, which involves SdrG and PhyT.

<p>Upon stress induction, the Paks autophosphorylate and transfer the phosphoryl group to the SDRR SdrG. Direct phosphorylation of PhyR by the Paks is inhibited due to PhyT-PhyR complex formation. PhyT transfers the phosphoryl group from SdrG~P to PhyR. PhyR~P dissociates from PhyT to bind the anti-sigma factor NepR and thereby releases the alternative sigma-factor EcfG, which initiates transcription of the GSR regulon by binding to RNA polymerase. For further details on the PhyR-NepR-EcfG cascade and Paks see [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.ref019" target="_blank">19</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.ref021" target="_blank">21</a>].</p

Membrane localization of PhyR depends on stress level in a H341-PhyT dependent fashion.

<p>Spinning-disc confocal images of different <i>S</i>. <i>melonis</i> Fr1 knockout mutants (A) upon production of sfGFP-PhyR, which was induced by addition of 25 μM cumate for 12 min. The chemical stress mixture (1% ethanol, 80 mM NaCl and 50 μM TBHP) was applied for 60 min. (B) Bacteria were imaged under unstressed conditions upon production of sfGFP-PhyR, which was induced by addition of 25 μM cumate for 12 min. (C) Bacteria were imaged under unstressed conditions upon overnight production of PhyT or the PhyT (H341A) derivative, which was induced by addition of 25 μM cumate and production of sfGFP-PhyR, induced by addition of 250 μM vanillate for 12 min. Scale bar, 5 μm. Comparable production of PhyT and the PhyT (H341A) derivative was confirmed using Western blot analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.s002" target="_blank">S2C Fig</a>).</p

Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2/neu or EpCAM as target receptors

To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass

The circadian clock circuitry modulates leukemia initiating cell activity in T-cell acute lymphoblastic leukemia

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, characterized by restricted cellular subsets with asymmetrically enriched leukemia initiating cell (LIC) activity. Nonetheless, it is still unclear which signaling programs promote LIC maintenance and progression. Methods Here, we evaluated the role of the biological clock in the regulation of the molecular mechanisms and signaling pathways impacting the cellular dynamics in T-ALL through an integrated experimental approach including gene expression profiling of shRNA-modified T-ALL cell lines and Chromatin Immunoprecipitation Sequencing (ChIP-Seq) of leukemic cells. Patient-derived xenograft (PDXs) cell subsets were also genetically manipulated in order to assess the LIC activity modulated by the loss of biological clock in human T-ALL. Results We report that the disruption of the circadian clock circuitry obtained through shRNA-mediated knockdown of CLOCK and BMAL1 genes negatively impacted the growth in vitro as well as the activity in vivo of LIC derived from PDXs after transplantation into immunodeficient recipient mice. Additionally, gene expression data integrated with ChIP-Seq profiles of leukemic cells revealed that the circadian clock directly promotes the expression of genes, such as IL20RB, crucially involved in JAK/STAT signaling, making the T-ALL cells more responsive to Interleukin 20 (IL20). Conclusion Taken together, our data support the concept that the biological clock drives the expression of IL20R prompting JAK/STAT signaling and promoting LIC activity in T-ALL and suggest that the selective targeting of circadian components could be therapeutically relevant for the treatment of T-ALL patients