Single \pi^- production in np collisions for excess energies up to 90 MeV

The quasifree reaction np\to pp\pim was studied in a kinematically complete experiment by bombarding a liquid hydrogen target with a deuteron beam of momentum 1.85 GeV/c and analyzing the data along the lines of the spectator model. In addition to the three charged ejectiles the spectator proton was also detected in the large-acceptance time-of-flight spectrometer COSY-TOF. It was identified by its momentum and flight direction thus yielding access to the Fermi motion of the bound neutron and to the effective neutron 4-momentum vector $\mathbb{P}_n$ which differed from event to event. A range of almost 90 MeV excess energy above threshold was covered. Energy dependent angular distributions, invariant mass spectra as well as fully covered Dalitz plots were deduced. Sizeable $pp$ FSI effects were found as were contributions of $p$ and $d$ partial waves. The behavior of the elementary cross section $\sigma_{01}$ close to threshold is discussed in view of new cross section data. In comparison with existing literature data the results provide a sensitive test of the spectator model.Comment: 21 pages, 9 figures, 4 tables, submitted to EPJ

Observation of strong final-state effects in pi+ production in pp collisions at 400 MeV

Differential cross sections of the reactions $pp \to d\pi^+$ and $pp \to pn\pi^+$ have been measured at $T_p = 400$ MeV by detecting the charged ejectiles in the angular range $4^0 \leq \Theta_{Lab} \leq 21^\circ$. The deduced total cross sections agree well with those published previously for neighbouring energies. The invariant mass spectra are observed to be strongly affected by $\Delta$ production and $NN$ final-state interaction. The data are well described by Monte Carlo simulations including both these effects. The ratio of $pp \to pn\pi^+$ and $pp \to d\pi^+$ cross sections also compares favourably to a recent theoretical prediction which suggests a dominance of $np$-production in the relative $^3S_1$-state.Comment: 17 pages, 5 figure

The study of the negative pion production in neutron-proton collisions at beam momenta below 1.8 GeV/c

A detailed investigation of the reaction np -> pp\pi^{-} has been carried out using the data obtained with the continuous neutron beam produced by charge exchange scattering of protons off a deuterium target. A partial wave event-by-event based maximum likelihood analysis was applied to determine contributions of different partial waves to the pion production process. The combined analysis of the np -> pp\pi^{-} and pp -> pp\pi^{0} data measured in the same energy region allows us to determine the contribution of isoscalar partial waves (I=0) in the momentum range from 1.1 up to 1.8 GeV/c. The decay of isoscalar partial waves into (^1S_0)_{pp}\pi$ channel provides a good tool for a determination of the pp S-wave scalar scattering length in the final state which was found to be a_{pp}=-7.5\pm 0.3 fm.Comment: 6 pages, 6 figure

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Pathogenic p62/SQSTM1 mutations impair energy metabolism through limitation of mitochondrial substrates

Abnormal mitochondrial function has been found in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mutations in the p62 gene (also known as SQSTM1) which encodes the p62 protein have been reported in both disorders supporting the idea of an ALS/FTD continuum. In this work the role of p62 in energy metabolism was studied in fibroblasts from FTD patients carrying two independent pathogenic mutations in the p62 gene, and in a p62-knock-down (p62 KD) human dopaminergic neuroblastoma cell line (SH-SY5Y). We found that p62 deficiency is associated with inhibited complex I mitochondrial respiration due to lack of NADH for the electron transport chain. This deficiency was also associated with increased levels of NADPH reflecting a higher activation of pentose phosphate pathway as this is accompanied with higher cytosolic reduced glutathione (GSH) levels. Complex I inhibition resulted in lower mitochondrial membrane potential and higher cytosolic ROS production. Pharmacological activation of transcription factor Nrf2 increased mitochondrial NADH levels and restored mitochondrial membrane potential in p62-deficient cells. Our results suggest that the phenotype is caused by a loss-of-function effect, because similar alterations were found both in the mutant fibroblasts and the p62 KD model. These findings highlight the implication of energy metabolism in pathophysiological events associated with p62 deficiency

HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

The HIV-1 nucleocapsid is formed during protease (PR)-directed viral maturation, and is transformed into pre-integration complexes following reverse transcription in the cytoplasm of the infected cell. Here, we report a detailed transmission electron microscopy analysis of the impact of HIV-1 PR and reverse transcriptase (RT) on nucleocapsid plasticity, using in vitro reconstitutions. After binding to nucleic acids, NCp15, a proteolytic intermediate of nucleocapsid protein (NC), was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and p1. This allowed NC co-aggregation with its single-stranded nucleic-acid substrate. Examination of these co-aggregates for the ability of RT to catalyse reverse transcription showed an effective synthesis of double-stranded DNA that, remarkably, escaped from the aggregates more efficiently with NCp7 than with NCp9. These data offer a compelling explanation for results from previous virological studies that focused on i) Gag processing leading to nucleocapsid condensation, and ii) the disappearance of NCp7 from the HIV-1 pre-integration complexes. We propose that HIV-1 PR and RT, by controlling the nucleocapsid architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodelling process that spatiotemporally coordinates the pre-integration steps of HIV-1. Finally we suggest that nucleoprotein remodelling mechanisms are common features developed by mobile genetic elements to ensure successful replication

Intracellular expression of Tat alters mitochondrial functions in T cells: a potential mechanism to understand mitochondrial damage during HIV-1 replication

HIV-1 replication results in mitochondrial damage that is enhanced during antiretroviral therapy (ART). The onset of HIV-1 replication is regulated by viral protein Tat, a 101-residue protein codified by two exons that elongates viral transcripts. Although the first exon of Tat (aa 1–72) forms itself an active protein, the presence of the second exon (aa 73–101) results in a more competent transcriptional protein with additional functions. Results: Mitochondrial overall functions were analyzed in Jurkat cells stably expressing full-length Tat (Tat101) or one-exon Tat (Tat72). Representative results were confirmed in PBLs transiently expressing Tat101 and in HIV-infected Jurkat cells. The intracellular expression of Tat101 induced the deregulation of metabolism and cytoskeletal proteins which remodeled the function and distribution of mitochondria. Tat101 reduced the transcription of the mtDNA, resulting in low ATP production. The total amount of mitochondria increased likely to counteract their functional impairment. These effects were enhanced when Tat second exon was expressed. Conclusions: Intracellular Tat altered mtDNA transcription, mitochondrial content and distribution in CD4+ T cells. The importance of Tat second exon in non-transcriptional functions was confirmed. Tat101 may be responsible for mitochondrial dysfunctions found in HIV-1 infected patients.We greatly appreciate the secretarial assistance of Mrs Olga Palao. This work was supported by FIPSE (360924/10), Spanish Ministry of Economy and Competitiveness (SAF2010-18388), Spanish Ministry of Health (EC11- 285), AIDS Network ISCIII-RETIC (RD12/0017/0015), Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness (FIS PI12/00506). The work of Sara Rodríguez-Mora is supported by a fellowship of Sara Borrell from Spanish Ministry of Economy and Competitiveness (2013). The work of María Rosa López-Huertas is supported by a fellowship of the European Union Programme Health 2009 (CHAARM).S

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p