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

Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia

A single-cell screening approach identifies targets for CAR-T cells in acute myeloid leukemia. Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development

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

Complex general stress response regulation in Sphingomonas melonis Fr1 revealed by transcriptional analyses

The general stress response (GSR) represents an important trait to survive in the environment by leading to multiple stress resistance. In alphaproteobacteria, the GSR is under the transcriptional control of the alternative sigma factor EcfG. Here we performed transcriptome analyses to investigate the genes controlled by EcfG of Sphingomonas melonis Fr1 and the plasticity of this regulation under stress conditions. We found that EcfG regulates genes for proteins that are typically associated with stress responses. Moreover, EcfG controls regulatory proteins, which likely fine-tune the GSR. Among these, we identified a novel negative GSR feedback regulator, termed NepR2, on the basis of gene reporter assays, phenotypic analyses, and biochemical assays. Transcriptional profiling of signaling components upstream of EcfG under complex stress conditions showed an overall congruence with EcfG-regulated genes. Interestingly however, we found that the GSR is transcriptionally linked to the regulation of motility and biofilm formation via the single domain response regulator SdrG and GSR-activating histidine kinases. Altogether, our findings indicate that the GSR in S. melonis Fr1 underlies a complex regulation to optimize resource allocation and resilience in stressful and changing environments.ISSN:2045-232

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

PhyT is a negative regulator of GSR <i>in vivo</i>.

<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>phyT</i> from the cumate-inducible pQH vector with 25 μM cumate. Empty pQH vector was used as a negative 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). Values are given as mean ±SD of three independent experiments.</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