The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration.

Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer.

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Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours—the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy.

The regulatory protein collybistin (CB) recruits the receptorscaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2 SH3؉ ) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally "opens" CB2 SH3؉ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.The function of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. The main scaffolding protein of inhibitory glycinergic and GABAergic postsynapses in mammals is gephyrin (1, 2), whose recruitment to the postsynaptic membrane is controlled by the adaptor protein collybistin (CB) 2 (3). Loss of CB results in a strong reduction of gephyrin and GABA A receptor clusters in several regions of the forebrain, which demonstrates the essential role of CB in the assembly and maintenance of GABAergic postsynaptic structures (4). CB belongs to the Dbl family of guanine nucleotide exchange factors. In mouse, four differently spliced CB mRNAs are present (CB1 SH3ϩ , CB2 SH3Ϫ , CB2 SH3ϩ , and CB3 SH3ϩ ). All four mRNAs encode a Dbl homology (DH) and a pleckstrin homology (PH) domain. The three major variants (CB1 SH3ϩ , CB2 SH3ϩ , and CB3 SH3ϩ ) encode CBs with an additional N-terminal Src homology 3 (SH3) domain but differ in their C termini. A fourth variant (CB2 SH3Ϫ ) encodes a CB2 isoform lacking the SH3 domain ( Fig. 1) but is very rare (5) as its protein product is not detectable in mouse brain (6). Importantly, the PH domain of the different CBs is required for proper function as indicated by the fact that its deletion abolishes the plasma membrane targeting of gephyrin-CB complexes when cotransfected in HEK293 cells and causes a loss of dendritic gephyrin clusters in dissociated rat cortical neurons (5).In vitro binding studies utilizing a variety of inositol headgroups, soluble phosphoinositide analogs, and liposomes containing phosphoinositides showed that PH domains bind phosphoinositides with a broad range of selectivity and affinity (7-10). An early membrane acti...

The phase III MIRROS trial (NCT02545283) evaluated the efficacy and safety of the small-molecule MDM2 antagonist idasanutlin plus cytarabine in patients with relapsed/refractory acute myeloid leukemia (R/R AML). Adults (N=447) with R/R AML whose disease relapsed or was refractory after ≤2 prior induction regimens as initial treatment or following salvage chemotherapy regimen, with Eastern Cooperative Oncology Group performance status ≤2 were enrolled regardless of TP53 mutation status and randomly assigned 2:1 to idasanutlin 300 mg or placebo orally twice daily plus cytarabine 1 g/m2 intravenously on days 1 to 5 of 28-day cycles. At primary analysis (cutoff, November 2019), 436 patients were enrolled, including 355 in the TP53 wild-type intention-to-treat (TP53WT-ITT) population. The primary endpoint, overall survival in the TP53WT-ITT population, was not met (median, 8.3 vs 9.1 months with idasanutlin-cytarabine vs placebo-cytarabine; stratified hazard ratio, 1.08; 95% CI, 0.81-1.45; p = .58). The complete remission (CR) rate, a key secondary endpoint, was 20.3% vs 17.1% (odds ratio [OR], 1.23; 95% CI, 0.70-2.18). The overall response rate (ORR) was 38.8% vs 22.0% (OR, 2.25; 95% CI, 1.36-3.72). Common any-grade adverse events (≥10% incidence in any arm) were diarrhea (87.0% vs 32.9%), febrile neutropenia (52.8% vs 49.3%), and nausea (52.5% vs 31.5%). In summary, despite improved ORR, adding idasanutlin to cytarabine did not improve overall survival or CR rates in patients with R/R AML.

We present a parallel matrix-free implicit finite volume scheme for the solution of unsteady three-dimensional advection-diffusion-reaction equations with smooth and Dirac-Delta source terms. The scheme is formally second order in space and a Newton-Krylov method is employed for the appearing nonlinear systems in the implicit time integration. The matrix-vector product required is hardcoded without any approximations, obtaining a matrix-free method that needs little storage and is well suited for parallel implementation. We describe the matrix-free implementation of the method in detail and give numerical evidence of its second order convergence in the presence of smooth source terms. For non-smooth source terms the convergence order drops to one half. Furthermore, we demonstrate the method's applicability for the long time simulation of calcium flow in heart cells and show its parallel scaling.Keywords: Finite volume method; Dirac delta distribution; Matrix-free Newton-Krylov method; Calcium waves; Parallel computing I INTRODUCTION Advection-diffusion-reaction systems occur in a wide variety of applications, as for instance heat transfer or transport-chemistry problems. Long-time simulations of such real-life applications often require implicit methods on very fine computational grids, to resolve the desired accuracy, especially in three dimensions. When performing such simulations with methods storing system matrices, the availability of memory becomes an issue already on relatively coarse meshes. One way to address this problem is the use of parallel methods, which distribute the workload among several CPUs and use the memory associated with these CPUs to solve larger systems. If this approach does not provide enough memory to obtain the desired resolution, parallel matrix-free methods are an excellent choice, since in general most of the memory is used to store system matrices. In addition, matrix-free methods require less communication compared to classical schemes, thus these are more suited for use on parallel architectures and allow for better scalability.To solve linear systems most matrix-free methods use Krylov subspace methods, which only require the results of matrix-vector products in every iteration. If these can be provided without storing the matrix, this leads to significant savings of memory and computations on high resolution meshes become feasible. A prominent example are Jacobian-free Newton Krylov (JFNK) methods, which approximate the Jacobian matrix by finite differences via function evaluations [1]. In the following we present another type of matrix-free Newton-Krylov method, which provides the matrix-vector products with the exact Jacobian by hardcoding the product. This method is thus specific to a given class of equations and grids and particularly suitable for computations on structured grids.

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