Mycobacterium tuberculosis type VII secretion system effectors differentially impact the ESCRT endomembrane damage response

Mycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.Intracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case of Mycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and that M. tuberculosis could impair this host response. Indeed, we found that ESCRT-III proteins were recruited to M. tuberculosis phagosomes in an ESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context of M. tuberculosis infection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity

Measurement of the β\beta-asymmetry parameter of 67^{67}Cu in search for tensor type currents in the weak interaction

Precision measurements at low energy search for physics beyond the Standard Model in a way complementary to searches for new particles at colliders. In the weak sector the most general $\beta$ decay Hamiltonian contains, besides vector and axial-vector terms, also scalar, tensor and pseudoscalar terms. Current limits on the scalar and tensor coupling constants from neutron and nuclear $\beta$ decay are on the level of several percent. The goal of this paper is extracting new information on tensor coupling constants by measuring the $\beta$-asymmetry parameter in the pure Gamow-Teller decay of $^{67}$Cu, thereby testing the V-A structure of the weak interaction. An iron sample foil into which the radioactive nuclei were implanted was cooled down to milliKelvin temperatures in a $^3$He-$^4$He dilution refrigerator. An external magnetic field of 0.1 T, in combination with the internal hyperfine magnetic field, oriented the nuclei. The anisotropic $\beta$ radiation was observed with planar high purity germanium detectors operating at a temperature of about 10\,K. An on-line measurement of the $\beta$ asymmetry of $^{68}$Cu was performed as well for normalization purposes. Systematic effects were investigated using Geant4 simulations. The experimental value, $\tilde{A}$ = 0.587(14), is in agreement with the Standard Model value of 0.5991(2) and is interpreted in terms of physics beyond the Standard Model. The limits obtained on possible tensor type charged currents in the weak interaction hamiltonian are -0.045 $< (C_T+C'_T)/C_A <$ 0.159 (90\% C.L.). The obtained limits are comparable to limits from other correlation measurements in nuclear $\beta$ decay and contribute to further constraining tensor coupling constants

Post-translational modifications soften vimentin intermediate filaments

The mechanical properties of biological cells are determined by the cytoskeleton, a composite biopolymer network consisting of microtubules, actin filaments and intermediate filaments (IFs). By differential expression of cytoskeletal proteins, modulation of the network architecture and interactions between the filaments, cell mechanics may be adapted to varying requirements on the cell. Here, we focus on the intermediate filament protein vimentin and introduce post-translational modifications as an additional, much faster mechanism for mechanical modulation. We study the impact of phosphorylation on filament mechanics by recording force-strain curves using optical traps. Partial phosphorylation softens the filaments. We show that binding of the protein 14-3-3 to phosphorylated vimentin IFs further enhances this effect and speculate that in the cell 14-3-3 may serve to preserve the softening and thereby the altered cell mechanics. We explain our observation by the additional charges introduced during phosphorylation

Confirmation of Parity Violation in the Gamma Decay of 180Hfm^{180}Hf^{m}

This paper reports measurements using the technique of On Line Nuclear Orientation (OLNO) which reexamine the gamma decay of isomeric $^{\rm 180}$Hf$^{\rm m}$ and specifically the 501 keV 8$^{\rm -}$ -- 6$^{\rm +}$ transition. The irregular admixture of E2 to M2/E3 multipolarity in this transition, deduced from the forward-backward asymmetry of its angular distribution, has for decades stood as the prime evidence for parity mixing in nuclear states. The experiment, based on ion implantation of the newly developed mass-separated $^{\rm 180}$Hf$^{\rm m}$ beam at ISOLDE, CERN into an iron foil maintained at millikelvin temperatures, produces higher degrees of polarization than were achieved in previous studies of this system. The value found for the E2/M2 mixing ratio, $\epsilon$ = -0.0324(16)(17), is in close agreement with the previous published average value $\epsilon$ = - 0.030(2), in full confirmation of the presence of the irregular E2 admixture in the 501 keV transition. The temperature dependence of the forward-backward asymmetry has been measured over a more extended range of nuclear polarization than previously possible, giving further evidence for parity mixing of the 8$^{\rm -}$ and 8$^{\rm +}$ levels and the deduced E2/M2 mixing ratio.Comment: 28 pages, 9 figures, accepted for publication in Physical Review

Neodymium-140 DOTA-LM3:Evaluation of an <i>In Vivo</i> Generator for PET with a Non-Internalizing Vector

140Nd (t1/2 = 3.4 days), owing to its short-lived positron emitting daughter 140Pr (t1/2 = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140Nd is chemically disruptive to macrocycle-based radiolabeling, meaning that an in vivo redistribution of the daughter 140Pr is expected before positron emission. The purpose of this study was to determine how the delayed positron from the de-labeled 140Pr affects preclinical imaging with 140Nd. To explore the effect, 140Nd was produced at CERN-ISOLDE, reacted with the somatostatin analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(d-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)d-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify the in vivo redistribution of 140Pr following 140Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140Pr washed out of the pancreas before decay. Contrastingly, the liver, spleen, and lungs acted as strong sink organs for free 140Pr3+. Based upon these results, we conclude that 140Nd imaging with a non-internalizing vector convolutes the biodistribution of the tracer with the accumulation pattern of free 140Pr. This redistribution phenomenon may show promise as a probe of the cellular interaction with the vector, such as in determining tissue dependent internalization behavior

Ground-state spin of 59^{59}Mn

Beta decay of $^{59}$Mn has been studied at PSB-ISOLDE, CERN. The intense and pure Mn beam was produced using the Resonance Ionization Laser Ion Source (RILIS). Based on the measured $\beta$-decay rates the ground-state spin and parity are proposed to be $J^{\pi}$ = 5/2$^{-}$. This result is consistent with the systematic trend of the odd-A Mn nuclei and extends the systematics one step further towards the neutron drip line

Atomic spectroscopy studies of short-lived isotopes and nuclear isomer separation with the ISOLDE RILIS

The Resonance Ionization Laser Ion Source (RILIS) at the ISOLDE on-line isotope separator is based on the selective excitation of atomic transitions by tunable laser radiation. Ion beams of isotopes of 20 elements have been produced using the RILIS setup. Together with the mass separator and a particle detection system it represents a tool for high-sensitive laser spectroscopy of short-lived isotopes. By applying narrow-bandwidth lasers for the RILIS one can study isotope shifts (IS) and hyperfine structure (HFS) of atomic optical transitions. Such measurements are capable of providing data on nuclear charge radii, spins and magnetic moments of exotic nuclides far from stability. Although the Doppler broadening of the optical absorption lines limits the resolution of the technique, the accuracy of the HFS measurements examined in experiments with stable Tl isotopes approaches a value of 100 MHz. Due to the hyperfine splitting of atomic lines the RILIS gives an opportunity to separate nuclear isomers. Isomer selectivity of the RILIS has been used in studies of short-lived Ag, Cu and Pb isotopes

Measurement of the magnetic moment of the one-neutron halo nucleus 11^{11}Be

The magnetic moment of $^{11}$Be was measured by detecting nuclear magnetic resonance signals in a beryllium crystal lattice. The experimental technique applied to a $^{11}$Be$^+$ ion beam from a laser ion source includes in-beam optical polarization, implantation into a metallic single crystal and observation of rf resonances in the asymmetric angular distribution of the $\beta$-decay ($\beta$-NMR). The nuclear magnetic moment $\mu(^{11}{\rm Be}) = -1.6816(8)\,\mu_N$ provides a stringent test for theoretical models describing the structure of the 1/2$^+$ neutron halo state

Selective laser ionization of N ≥\geq 82 indium isotopes: the new r-process nuclide 135^{135}In

Production yields and beta-decay half-lives of very neutron-rich indium isotopes were determined at CERN/ISOLDE using isobaric selectivity of a resonance-ionization laser ion-source. Beta-delayed neutron multiscaling measurements have yielded improved half-lives for 206(6)~ms $^{132}$In, 165(3)~ms $^{133}$In and 141(5)~ms $^{134}$In. With 92(10)~ms $^{135}$In, a new r-process nuclide has been identified which acts as an important `waiting-point' in the In isotopic chain for neutron densities in the range n$_n \simeq 10^{24}$--10$^{26}$ n/cm$^3$, where the r-matter flow has already passed the ${\rm A} \simeq 130$ abundance-peak region

Early onset of ground-state deformation in the neutron-deficient polonium isotopes

In-source resonant ionization laser spectroscopy of the even-$A$ polonium isotopes $^{192-210,216,218}$Po has been performed using the $6p^37s$ $^5S_2$ to $6p^37p$ $^5P_2$ ($\lambda=843.38$ nm) transition in the polonium atom (Po-I) at the CERN ISOLDE facility. The comparison of the measured isotope shifts in $^{200-210}$Po with a previous data set allows to test for the first time recent large-scale atomic calculations that are essential to extract the changes in the mean-square charge radius of the atomic nucleus. When going to lighter masses, a surprisingly large and early departure from sphericity is observed, which is only partly reproduced by Beyond Mean Field calculations.Comment: As submitted to PR