Deubiquitylating Enzymes and DNA Damage Response Pathways

Covalent post-translational modification of proteins by ubiquitin and ubiquitin-like factors has emerged as a general mechanism to regulate myriad intra-cellular processes. The addition and removal of ubiquitin or ubiquitin-like proteins from factors has recently been demonstrated as a key mechanism to modulate DNA damage response (DDR) pathways. It is thus, timely to evaluate the potential for ubiquitin pathway enzymes as DDR drug targets for therapeutic intervention. The synthetic lethal approach provides exciting opportunities for the development of targeted therapies to treat cancer: most tumours have lost critical DDR pathways, and thus rely more heavily on the remaining pathways, while normal tissues are still equipped with all DDR pathways. Here, we review key deubiquitylating enzymes (DUBs) involved in DDR pathways, and describe how targeting DUBs may lead to selective therapies to treat cancer patients

Postnatal Tshz3 Deletion Drives Altered Corticostriatal Function and Autism Spectrum Disorder–like Behavior

International audienceBACKGROUND: Heterozygous deletion of the TSHZ3 gene, encoding for the teashirt zinc-finger homeobox family member 3 (TSHZ3) transcription factor that is highly expressed in cortical projection neurons (CPNs), has been linked to an autism spectrum disorder (ASD) syndrome. Similarly, mice with Tshz3 haploinsufficiency show ASD-like behavior, paralleled by molecular changes in CPNs and corticostriatal synaptic dysfunctions. Here, we aimed at gaining more insight into "when" and "where" TSHZ3 is required for the proper development of the brain, and its deficiency crucial for developing this ASD syndrome. METHODS: We generated and characterized a novel mouse model of conditional Tshz3 deletion, obtained by crossing Tshz3 flox/flox with CaMKIIalpha-Cre mice, in which Tshz3 is deleted in CPNs from postnatal day 2 to 3 onward. We characterized these mice by a multilevel approach combining genetics, cell biology, electrophysiology, behavioral testing, and bioinformatics. RESULTS: These conditional Tshz3 knockout mice exhibit altered cortical expression of more than 1000 genes, w50% of which have their human orthologue involved in ASD, in particular genes encoding for glutamatergic syn-apse components. Consistently, we detected electrophysiological and synaptic changes in CPNs and impaired corticostriatal transmission and plasticity. Furthermore, these mice showed strong ASD-like behavioral deficits. CONCLUSIONS: Our study reveals a crucial postnatal role of TSHZ3 in the development and functioning of the corticostriatal circuitry and provides evidence that dysfunction in these circuits might be determinant for ASD pathogenesis. Our conditional Tshz3 knockout mouse constitutes a novel ASD model, opening the possibility for an early postnatal therapeutic window for the syndrome linked to TSHZ3 haploinsufficiency

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Ribosomal proteins RPL37, RPS15 and RPS20 regulate the Mdm2-p53-MdmX network.

Changes to the nucleolus, the site of ribosome production, have long been linked to cancer, and mutations in several ribosomal proteins (RPs) have been associated with an increased risk for cancer in human diseases. Relevantly, a number of RPs have been shown to bind to MDM2 and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and cell cycle arrest, thus revealing a RP-Mdm2-p53 signaling pathway that is critical for ribosome biogenesis surveillance. Here, we have identified RPL37, RPS15, and RPS20 as RPs that can also bind Mdm2 and activate p53. We found that each of the aforementioned RPs, when ectopically expressed, can stabilize both co-expressed Flag-tagged Mdm2 and HA-tagged p53 in p53-null cells as well as endogenous p53 in a p53-containing cell line. For each RP, the mechanism of Mdm2 and p53 stabilization appears to be through inhibiting the E3 ubiquitin ligase activity of Mdm2. Interestingly, although they are each capable of inducing cell death and cell cycle arrest, these RPs differ in the p53 target genes that are regulated upon their respective introduction into cells. Furthermore, each RP can downregulate MdmX levels but in distinct ways. Thus, RPL37, RPS15 and RPS20 regulate the Mdm2-p53-MdmX network but employ different mechanisms to do so

Detecting and Hunting Cyberthreats in a Maritime Environment: Specification and Experimentation of a Maritime Cybersecurity Operations Centre

International audienceThe vast majority of worldwide goods exchanges are made by sea. In some parts of the world, the concurrence for dominance at sea is very high and definitely seen as a main military goal. Meanwhile, new generation ships highly rely on information systems for communication, navigation and platform management. This ever-spreading attack surface and permanent satellite links have grown a concern about the potential impact of cyberattacks on a ship at sea or on naval shore infrastructures. Therefore, on top of the usual cyberprotection measures taken for safety reasons, it is essential to implement an ongoing cyber monitoring of ships in order to detect, react accordingly, and stop any incoming threat. In this paper, we explain the specific constraints when trying to assess the cyber situation awareness of maritime information systems. As we will demonstrate, those systems combine physical and logical constraints which complexify their cyber monitoring process and architecture. Gathering valuable data while having a limited and controlled impact on the satellite bandwidth, maintaining a high level of integrity on remote systems in production are, for instance, thriving challenges for both civilian and military ships. We have designed and set up a research platform which fulfils those specifications to streamline the cyber monitoring process.We will then describe the architecture used to detect cyber-threats and collect potential Indices of Compromise from naval systems, as well as the results we have currently achieved

RPL37, RPS15, and RPS20 increase levels of p53.

<p>(a) Stabilization of ectopically expressed p53 by RPs. U2OS cells were transfected with Flag-Mdm2 (1.2 µg), HA-p53 (0.3 µg), and Myc-RP (1.0–3.0 µg). (GFP was added as a control for transfection efficiency.) Ectopic Mdm2, p53, and RP levels were detected by immunoblotting with α-Flag, α-HA and α-Myc. (b–d) Stabilization of endogenously expressed p53 by RPs. U2OS cells were transfected with increasing amounts of Myc-RP (0–3.0 µg), and endogenous proteins were detected by immunoblotting with the indicated antibodies.</p

Knockdown of RPL37, RPS15, and RPS20 activate p53.

<p>(a–c) Increase in p53 and p21 by RPs. U2OS cells were transfected with siRNA targeting RPL37, RPS15, or RPS20 as indicated (0 µM, 50 µM, 100 µM, 200 µM). Cells were harvested and lysates were subjected to immunoblotting for Mdm2, MdmX, p53, p21, and the indicated RPs with the relevant antibodies. Immunoblots in panel (c) are taken from the same gel.</p

RPL37, RPS15, and RPS20 stabilize p53 protein.

<p>(a–c) Increase in half-life of p53 by RPs. U2OS cells were seeded in 35 mM tissue culture plates and transfected with empty vector (3.0 µg) or Myc-RP (3.0 µg). Approximately 22 hours after the initial transfection, 100 µg/mL cycloheximide was added to the culture medium and cells were harvested at the indicated timepoints. (d–f) Inhibition of Mdm2-mediated ubiquitination of p53 by RPs. H1299 cells were seeded in 60 mM tissue culture plates and transfected with HA-Ubiquitin (3.0 µg), p53 (0.75 µg), Flag-Mdm2 (7.5 µg), and Myc-RP (9.0 µg). MG132 was added for 6 hours, and ubiquitinated p53 species were assayed by immunoprecipitating with α-p53 and immunoblotting with α-HA. Inputs and IPs were run on separate gels.</p

Implicit Strategy and Parallelization of a High Order Residual Distribution Scheme

Adaptive Higher-Order Variational Methods for Aerodynamic Applications in Industr

RPL37, RPS15, and RPS20 bind to and regulate MdmX levels.

<p>(a–c) Association of RPs and MdmX. H1299 cells were transfected with HA-MdmX (0.5 µg), Myc-RP (1.2 µg), or both. (GFP was added as a control for transfection efficiency.) Ectopic MdmX and RP levels were detected by immunoblotting with α-MdmX and α-Myc. Inputs and IPs were run on separate gels. (d–f) Decrease in MdmX protein by RPs. U2OS cells were transfected with increasing amounts of Myc-RP (0–2.0 µg). Endogenous proteins were detected by immunoblotting with the indicated antibodies. (g) Decrease in MdmX mRNA by RPL37. U2OS cells were seeded in 60 mM tissue culture plates and transfected with Myc-RP (0–5.0 µg). Relative expression of MdmX mRNA was determined by quantitative RT-PCR and normalized to GAPDH. A representative experiment is plotted, and significant changes in mRNA levels were calculated using student’s t-test (* = p<0.05; ** = p<0.01; n <u>></u>3).</p