Oscillatory Kelvin-Helmholtz instability. Part 2. An experiment in fluids with a large viscosity contrast

International audienceThe stability of two-layer oscillatory flows was studied experimentally in a cylindrical container with a vertical axis. Two superposed immiscible liquids, differing greatly in viscosity, were set in relative oscillatory motion by alternating container rotation. Waves arising beyond a threshold were observed in detail for small oscillation frequencies ranging from 0.1 to 6 Hz. Measurements were performed on the growth rate and the wavenumber of these waves. The instability threshold was determined from the growth rate data. It was found that the threshold and the wavenumber varied with the frequency. In particular, significantly lower thresholds and longer waves were found than those predicted by the inviscid theory of the oscillatory Kelvin-Helmholtz instability. Favourable agreement with the predictions of an existing viscous theory for small oscillation amplitude flows indicates the important role of viscosity, even at the highest frequency, and suggests a similar mechanism behind the instability as that for the short wave instability in steady Couette flows. A semi-numerical stability determination for finite amplitude flows was also performed to improve the prediction in experiments with a frequency lower than 1 Hz

Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC

Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long ultracentrifugation to collecting and analyzing the fractions. Each of these steps in the SDG workflow can introduce potential technical variation that affects the reproducibility of gradient profiles between samples. To address these limitations, we have developed a flexible, alternative approach for analyzing polysomes and ribosomal subunits based on size-exclusion chromatography (SEC), termed ‘Ribo Mega-SEC.’ In comparison with the SDG method, Ribo Mega-SEC involves a single step using ultra-high-performance liquid chromatography (uHPLC). The entire workflow, from injecting the lysate to collecting the fractions, can be performed in as little as 15 min, with high reproducibility. By varying the pore size of the SEC column, polysomes and ribosomal subunits can be separated using extracts from either human or mouse cultured cell lines or from tissue samples, Drosophila embryos, or budding yeast. The resulting separated fractions are suitable for analysis using a wide range of subsequent analytical techniques including mass spectrometry (MS)-based proteomics, RNA-Seq, electron microscopy (EM), and multiple biochemical assays

Convection thermique d'un fluide diélectrique confiné dans un anneau cylindrique en rotation auquel sont appliqué un gradient de température et une tension électrique alternative

Le champ électrique couplé à un gradient de température sur un fluide diélectrique est un outil de génération de la convection thermique, en particulier dans des conditions de microgravité où la poussée d'Archimède devient négligeable [1]. On considère un fluide diélectrique incompressible dans un anneau cylindrique tournant autour de son axe en condition d'apesanteur. Les surfaces cylindriques sont des électrodes maintenues à des températures différentes et auxquelles est appliquée une tension électrique alternative de haute fréquence. Le champ électrique radial agit sur la stratification de permittivité électrique pour donner lieu à la force diélectrophorétique (DEP). Dans ce cas, deux poussées thermiques sont effectives : la poussée liée à l'action de l'accélération centrifuge sur la stratification de masse volumique, et la force DEP qui s'apparente à l'action d'une gravité centripète de nature électrique (gravité électrique) sur la stratification de masse volumique [2]. A l'aide de l'analyse de stabilité linéaire, l'instabilité pilotée par ces deux poussées thermiques protagonistes est caractérisée par le nombre de Rayleigh associé à chacune d'entre elles. Les cellules de convections sont sous formes de vortex contrarotatifs hélicoïdaux pour les faibles taux de rotation, et prennent la forme de colonnes alignées à l'axe de l'anneau lorsque le taux de rotation est suffisamment important [3]. La gravité électrique et l'accélération centrifuge étant de directions opposées, le sens du gradient de température a un fort impact sur les mécanismes de l'instabilité. Une attention particulière est portée sur la nature instationnaire des modes critiques.   [1] B. Futterer, A. Krebs, A.C. Plesa, F. Zaussinger, R. Hollerbach, D. Breuer, C. Egbers, Sheet-like and plume-like thermal flow in a spherical convection experiment performed under microgravity, J. Fluid Mech. 735, 647 (2013). [2] H.N. Yoshikawa, O. Crumeyrolle & I. Mutabazi, Dielectrophoretic force-driven thermal convection in annular geometry, Phys. Fluids 25, 024106 (2013). [3] A. Meyer, M. Jongmans, M. Meier, C. Egbers & I. Mutabazi, Thermal convection in a cylindrical annulus under a combined effect of the radial and vertical gravity, C.R. Mecanique 2016 (http://dx.doi.org/10.1016/j.crme.2016.10.003)

Application of two-color LIF thermometry to nucleate boiling

International audienceThe laser-induced fluorescence (LIF) thermometry is applied to measure the temperature field surrounding a single vapor bubble growing at an artificial nucleation site. In order to correct measurement errors due to the non-uniformity of the incident laser intensity, the two-color LIF thermometry technique is used in this nucleate boiling experiment. This technique is based on the use of two fluorescent dyes: the temperature sensitive dye Rhodamine B and the temperature insensitive dye Sulforhodamine-101. The concentration of the dyes is optimized by analyzing the behavior of fluorescence intensities. The mapping between the two images is determined through a geometrical calibration procedure. This technique presents a success in correcting the non uniformities due to the reflection of the light at the bubble surface and to the temperature gradient. The obtained temperature fields show that the two-color LIF is a promising technique in the investigation of nucleate boiling

Collaborator of alternative reading frame protein (CARF) regulates early processing of pre-ribosomal RNA by retaining XRN2 (5′-3′ exoribonuclease) in the nucleoplasm

Collaborator of alternative reading frame protein (CARF) associates directly with ARF, p53, and/or human double minute 2 protein (HDM2), a ubiquitin-protein ligase, without cofactors and regulates cell proliferation by forming a negative feedback loop. Although ARF, p53, and HDM2 also participate in the regulation of ribosome biogenesis, the involvement of CARF in this process remains unexplored. In this study, we demonstrate that CARF associates with 5′-3′ exoribonuclease 2 (XRN2), which plays a major role in both the maturation of rRNA and the degradation of a variety of discarded pre-rRNA species. We show that overexpression of CARF increases the localization of XRN2 in the nucleoplasm and a concomitant suppression of pre-rRNA processing that leads to accumulation of the 5′ extended from of 45S/47S pre-rRNA and 5′-01, A0-1 and E-2 fragments of pre-rRNA transcript in the nucleolus. This was also observed upon XRN2 knockdown. Knockdown of CARF increased the amount of XRN2 in the nucleolar fraction as determined by cell fractionation and by immnocytochemical analysis. These observations suggest that CARF regulates early steps of pre-rRNA processing during ribosome biogenesis by controlling spatial distribution of XRN2 between the nucleoplasm and nucleolus

Chtop (Chromatin target of Prmt1) auto-regulates its expression level via intron retention and nonsense-mediated decay of its own mRNA

Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5′ end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine-rich (RG) domain, and with intron 2 via its N-terminal (N1) domain; both are required for retention of intron 2. In addition, we show that hnRNP H accelerates intron 2 splicing of Chtop mRNA in a manner dependent on Chtop expression level, suggesting that Chtop and hnRNP H regulate intron 2 retention of Chtop mRNA antagonistically. Thus, the present study provides a novel molecular mechanism by which mRNA and protein levels are constitutively regulated by intron retention

Poly(A)-specific ribonuclease regulates the processing of small-subunit rRNAs in human cells

Ribosome biogenesis occurs successively in the nucleolus, nucleoplasm, and cytoplasm. Maturation of the ribosomal small subunit is completed in the cytoplasm by incorporation of a particular class of ribosomal proteins and final cleavage of 18S-E pre-rRNA (18S-E). Here, we show that poly(A)-specific ribonuclease (PARN) participates in steps leading to 18S-E maturation in human cells. We found PARN as a novel component of the pre-40S particle pulled down with the pre-ribosome factor LTV1 or Bystin. Reverse pull-down analysis revealed that PARN is a constitutive component of the Bystin-associated pre-40S particle. Knockdown of PARN or exogenous expression of an enzyme-dead PARN mutant (D28A) accumulated 18S-E in both the cytoplasm and nucleus. Moreover, expression of D28A accumulated 18S-E in Bystin-associated pre-40S particles, suggesting that the enzymatic activity of PARN is necessary for the release of 18S-E from Bystin-associated pre-40S particles. Finally, RNase H–based fragmentation analysis and 3΄-sequence analysis of 18S-E species present in cells expressing wild-type PARN or D28A suggested that PARN degrades the extended regions encompassing nucleotides 5–44 at the 3΄ end of mature 18S rRNA. Our results reveal a novel role for PARN in ribosome biogenesis in human cells

An allele-selective inter-chromosomal protein bridge supports monogenic antigen expression in the African trypanosome

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion

Thermoelectric convection in a planar capacitor: theoretical studies and experiments in parabolic flights

International audienceNous avons étudié la convection thermoélectrique dans un condensateur plan placé dans un environnement de microgravité et dans des conditions terrestres avec une stratification thermique instable ou stable. L’analyse énergétique montre que dans le cas de stratification stable, la convection thermoélectrique est retardée alors que dans le cas de stratification thermique instable, elle est amplifiée par la poussée d’Archimède avant que cette dernière ne prenne le dessus et pilote la convection thermique naturelle. Une expérience réalisée lors des vols paraboliques illustre la formation de la convection thermoélectrique à la fin de la phase de microgravité