Application of the Virtual Fields Method to determine dynamic properties at intermediate strain rates

Crash analysis simulation is now very important in automotive industry to assess automotive crashworthiness and safety. In order to acquire reliable crash simulation results, precise material behaviors at intermediate strain rates should be used as input data. To determine the stress-strain curves at various strain rates, the number of expensive and complicated experiments is large. The present study aims at determining the stress-strain curves of sheet metals at various strain rates from a single dynamic experiment. A new type of high speed tensile tester for sheet metal specimens was built and high speed tensile tests were carried out. Full-field heterogeneous strain fields were measured by a digital image correlation technique using a highspeed camera. The load data was acquired from strain gauges attached to the elastic deformation region on the specimen. Then, an inverse identification scheme with a rate dependent hardening law was applied to retrieve dynamic parameters. The stress-strain curves of advanced high strength steel at intermediate strain rates (100 /s - 300 /s) were successfully obtained from a single experiment.11Ysciescopu

Characterization of dynamic hardening behavior using acceleration information

Crash analysis simulation is very important in automotive industry to assess automotive crashworthiness and safety. In the FE simulation, accurate dynamic hardening behavior should be used as input data to provide reliable results. But, it is difficult to obtain precise hardening properties at intermediate or high strain rates due to inaccurate measurement of load caused by the inertial effect. In this study, a new methodology was applied to retrieve dynamic strain hardening properties of sheet metal specimens. The virtual fields method (VFM) was adopted as an inverse method to identify hardening parameters without load information. As an initial study, Swift model for a rate independent hardening law was selected for an elasto-plastic constitutive model. In order to validate the proposed methodology in the experiments, a new type of high speed tensile tester for sheet metal specimens was built and high speed tensile tests were performed. Digital image correlation technique using a high-speed camera was utilized to measure strain and acceleration fields so that the identification is carried out from the measured quantities. The validation of the proposed VFM identification procedure using the acceleration will be performed by comparing with the conventional procedure using a load-cell. (C) 2017 The Authors. Published by Elsevier Ltd.110Ysciescopu

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria–localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death–inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics

Re-viewing the 3D Organization of mRNPs

International audienc

Physical relationship between a gene and its origin of replication in Physarum polycephalum

International audienceTaking advantage of the natural synchrony of the Sphasewithin the plasmodium of Physarum polycephakm,we extracted highly synchronous DNA samples atprecise time points in early S-phase. We then separated,by electrophoresis under denaturating conditions, thenewly synthesized DNA strands of the nascent chromosomalreplicons from the parental DNA template. Usingthe cDNA clone of the early-replicating LAVl-2 geneas a probe, we could establish by filter hybridizationthat the elongation rate of the replicon which encompassesthis gene is constant, at a rate of 1 kb/min duringthe first 30 min of S-phase. The smallest replication intermediate(RI) that we have detected by probing withthe LAVl-2 cDNA was 5 kb long, suggesting that theLAVl-2 gene and its origin of replication are closelyassociated within the chromosome. This procedureshould facilitate the mapping of replication originswithin the genome of Physaru

SWI/SNF chromatin-remodeling complexes function in noncoding RNA-dependent assembly of nuclear bodies

Paraspeckles are subnuclear structures that form around nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding RNA (lncRNA). Recently, paraspeckles were shown to be functional nuclear bodies involved in stress responses and the development of specific organs. Paraspeckle formation is initiated by transcription of the NEAT1 chromosomal locus and proceeds in conjunction with NEAT1 lncRNA biogenesis and a subsequent assembly step involving >40 paraspeckle proteins (PSPs). In this study, subunits of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complexes were identified as paraspeckle components that interact with PSPs and NEAT1 lncRNA. EM observations revealed that SWI/SNF complexes were enriched in paraspeckle subdomains depleted of chromatin. Knockdown of SWI/SNF components resulted in paraspeckle disintegration, but mutation of the ATPase domain of the catalytic subunit BRG1 did not affect paraspeckle integrity, indicating that the essential role of SWI/SNF complexes in paraspeckle formation does not require their canonical activity. Knockdown of SWI/SNF complexes barely affected the levels of known essential paraspeckle components, but markedly diminished the interactions between essential PSPs, suggesting that SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. The interactions between SWI/SNF components and essential PSPs were maintained in NEAT1-depleted cells, suggesting that SWI/SNF complexes not only facilitate interactions between PSPs, but also recruit PSPs during paraspeckle assembly. SWI/SNF complexes were also required for Satellite III lncRNA-dependent formation of nuclear stress bodies under heat-shock conditions. Our data suggest the existence of a common mechanism underlying the formation of lncRNA-dependent nuclear body architectures in mammalian cells.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1423819112/-/DCSupplemental.http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1423819112/-/DCSupplementa

Determination of the dynamic strain hardening parameters from acceleration fields

The aim of the present study is to determine dynamic strain hardening parameters accurately using an inverse method so that accurate hardening properties at intermediate or high strain rates can be provided for automotive crash analysis simulations. In order to validate the identification procedure, a proper constitutive model was selected and a finite element (FE) analysis was carried out to obtain simulated measurements. The same identification procedure as that which will be implemented in the experiments was adopted. The virtual fields method (VFM) was used as an inverse method to retrieve the constitutive parameters. Since precise measurement of the applied force is not an easy task at high strain rates due to the inertial effect, the identification was performed without using the force information. Instead, the acceleration fields were used for the identification. The obtained parameters using the VFM were compared with the reference ones, which were fed into the FE simulations and very promising results were acquired from the VFM. In addition, a novel high speed tensile tester for sheet metal specimens was developed to validate the proposed methodology in the experiments.11sciescopu

NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies

Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MEN epsilon/beta long noncoding RNA (IncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno-electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1(-/-) fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles

Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation

A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation