Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels

In response to swelling, mammalian cells release chloride and organic osmolytes through VRAC volume-regulated anion channels. VRACs are heteromers of LRRC8A and other LRRC8 isoforms (B-E) which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and its dependence on particular LRRC8 isoforms remains largely unknown. We show that besides the osmolytes taurine and myo-inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and GABA and the co-activator D-serine. HEK293 cells engineered to express defined subsets of LRRC8 isoforms were used to elucidate the subunit-dependence of transport. Whereas LRRC8D was crucial for the translocation of overall neutral compounds like myo-inositol, taurine and GABA and sustained the transport of positively charged lysine, flux of negatively charged aspartate was equally well supported by LRRC8E. Disruption of LRRC8B or LRRC8C failed to decrease transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced VRAC's substrate preference. This suggested that individual VRACs can contain three or more different LRRC8 subunits, a conclusion confirmed by sequential co-immunoprecipitations. Our work suggests a composition-dependent role of VRACs in extracellular signal transduction

Knowledge-based gene expression classification via matrix factorization

Motivation: Modern machine learning methods based on matrix decomposition techniques, like independent component analysis (ICA) or non-negative matrix factorization (NMF), provide new and efficient analysis tools which are currently explored to analyze gene expression profiles. These exploratory feature extraction techniques yield expression modes (ICA) or metagenes (NMF). These extracted features are considered indicative of underlying regulatory processes. They can as well be applied to the classification of gene expression datasets by grouping samples into different categories for diagnostic purposes or group genes into functional categories for further investigation of related metabolic pathways and regulatory networks. Results: In this study we focus on unsupervised matrix factorization techniques and apply ICA and sparse NMF to microarray datasets. The latter monitor the gene expression levels of human peripheral blood cells during differentiation from monocytes to macrophages. We show that these tools are able to identify relevant signatures in the deduced component matrices and extract informative sets of marker genes from these gene expression profiles. The methods rely on the joint discriminative power of a set of marker genes rather than on single marker genes. With these sets of marker genes, corroborated by leave-one-out or random forest cross-validation, the datasets could easily be classified into related diagnostic categories. The latter correspond to either monocytes versus macrophages or healthy vs Niemann Pick C disease patients.Siemens AG, MunichDFG (Graduate College 638)DAAD (PPP Luso - Alem˜a and PPP Hispano - Alemanas

First identification of large electric monopole strength in well-deformed rare earth nuclei

Excited states in the well-deformed rare earth isotopes $^{154}$Sm and $^{166}$Er were populated via ``safe'' Coulomb excitation at the Munich MLL Tandem accelerator. Conversion electrons were registered in a cooled Si(Li) detector in conjunction with a magnetic transport and filter system, the Mini-Orange spectrometer. For the first excited $0^+$ state in $^{154}$Sm at 1099 keV a large value of the monopole strength for the transition to the ground state of $\rho^2(\text{E0}; 0^+_2 \to 0^+_\text{g}) = 96(42)\cdot 10^{-3}$ could be extracted. This confirms the interpretation of the lowest excited $0^+$ state in $^{154}$Sm as the collective $\beta$-vibrational excitation of the ground state. In $^{166}$Er the measured large electric monopole strength of $\rho^2(\text{E0}; 0^+_4 \to 0^+_1) = 127(60)\cdot 10^{-3}$ clearly identifies the $0_4^+$ state at 1934 keV to be the $\beta$-vibrational excitation of the ground state.Comment: submitted to Physics Letters

Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly

Amyloid fibrils are highly polymorphic structures formed by many different proteins. They provide biological function but also abnormally accumulate in numerous human diseases. The physicochemical principles of amyloid polymorphism are not understood due to lack of structural insights at the single-fibril level. To identify and classify different fibril polymorphs and to quantify the level of heterogeneity is essential to decipher the precise links between amyloid structures and their functional and disease associated properties such as toxicity, strains, propagation and spreading. Employing gentle, force-distance curve-based AFM, we produce detailed images, from which the 3D reconstruction of individual filaments in heterogeneous amyloid samples is achieved. Distinctive fibril polymorphs are then classified by hierarchical clustering, and sample heterogeneity is objectively quantified. These data demonstrate the polymorphic nature of fibril populations, provide important information regarding the energy landscape of amyloid self-assembly, and offer quantitative insights into the structural basis of polymorphism in amyloid populations

Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC

Regulation of cell volume is critical for many cellular and organismal functions, yet the molecular identity of a key player, the volume-regulated anion channel VRAC, has remained unknown. A genome-wide siRNA screen in mammalian cells identified LRRC8A as a VRAC component. LRRC8A formed heteromers with other LRRC8 multispan membrane proteins. Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A co-transfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Our work shows that VRAC defines a class of anion channels, suggests that VRAC is identical to the volume-sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents

Low-energy Coulomb excitation of 62^{62}Fe and 62^{62}Mn following in-beam decay of 62^{62}Mn

Sub-barrier Coulomb-excitation was performed on a mixed beam of $^{62}$Mn and $^{62}$Fe, following in-trap $\beta^{-}$ decay of $^{62}$Mn at REX-ISOLDE, CERN. The trapping and charge breeding times were varied in order to alter the composition of the beam, which was measured by means of an ionisation chamber at the zero-angle position of the Miniball array. A new transition was observed at 418~keV, which has been tentatively associated to a $(2^{+},3^{+})\rightarrow1^{+}_{g.s.}$ transition. This fixes the relative positions of the $\beta$-decaying $4^{+}$ and $1^{+}$ states in $^{62}$Mn for the first time. Population of the $2^{+}_{1}$ state was observed in $^{62}$Fe and the cross-section determined by normalisation to the $^{109}$Ag target excitation, confirming the $B(E2)$ value measured in recoil-distance lifetime experiments.Comment: 9 pages, 10 figure

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Tree water uptake enhances nitrogen acquisition in a fertilized boreal forest - but not under nitrogen-poor conditions

Understanding how plant water uptake interacts with acquisition of soil nitrogen (N) and other nutrients is fundamental for predicting plant responses to a changing environment, but it is an area where models disagree. We present a novel isotopic labelling approach which reveals spatial patterns of water and N uptake, and their interaction, by trees. The stable isotopes N-15 and H-2 were applied to a small area of the forest floor in stands with high and low soil N availability. Uptake by surrounding trees was measured. The sensitivity of N acquisition to water uptake was quantified by statistical modelling. Trees in the high-N stand acquired twice as much N-15 as in the low-N stand and around half of their N uptake was dependent on water uptake (H-2 enrichment). By contrast, in the low-N stand there was no positive effect of water uptake on N uptake. We conclude that tree N acquisition was only marginally dependent on water flux toward the root surface under low-N conditions whereas under high-N conditions, the water-associated N uptake was substantial. The results suggest a fundamental shift in N acquisition strategy under high-N conditions

First Results on In-Beam gamma Spectroscopy of Neutron-Rich Na and Mg Isotopes at REX-ISOLDE

After the successful commissioning of the radioactive beam experiment at ISOLDE (REX-ISOLDE) - an accelerator for exotic nuclei produced by ISOLDE - first physics experiments using these beams were performed. Initial experiments focused on the region of deformation in the vicinity of the neutron-rich Na and Mg isotopes. Preliminary results show the high potential and physics opportunities offered by the exotic isotope accelerator REX in conjunction with the modern Germanium gamma spectrometer MINIBALL.Comment: 7 pages, RNB6 conference contributio