Low-oxygen response is triggered by an ATP-dependent shift in oleoyl-CoA in Arabidopsis

Plant response to environmental stimuli involves integration of multiple signals. Upon low-oxygen stress, plants initiate a set of adaptive responses to circumvent an energy crisis. Here, we reveal how these stress responses are induced by combining (i) energy-dependent changes in the composition of the acyl-CoA pool and (ii) the cellular oxygen concentration. A hypoxia-induced decline of cellular ATP levels reduces LONG-CHAIN ACYL-COA SYNTHETASE activity, which leads to a shift in the composition of the acyl-CoA pool. Subsequently, we show that different acyl-CoAs induce unique molecular responses. Altogether, our data disclose a role for acyl-CoAs acting in a cellular signaling pathway in plants. Upon hypoxia, high oleoyl-CoA levels provide the initial trigger to release the transcription factor RAP2.12 from its interaction partner ACYL-COA BINDING PROTEIN at the plasma membrane. Subsequently, according to the N-end rule for proteasomal degradation, oxygen concentration-dependent stabilization of the subgroup VII ETHYLENE-RESPONSE FACTOR transcription factor RAP2.12 determines the level of hypoxia-specific gene expression. This research unveils a specific mechanism activating low-oxygen stress responses only when a decrease in the oxygen concentration coincides with a drop in energy

Structure of the omalizumab Fab

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor FcɛRI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc Cɛ2–4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity

Plasma interferon-gamma-inducible protein 10 (IP-10) levels correlate with disease severity and paradoxical reactions in extrapulmonary tuberculosis

Background With 1.5 million deaths worldwide in 2018, tuberculosis (TB) remains a major global public health problem. While pulmonary TB (PTB) is the most common manifestation, the proportion of extrapulmonary TB (EPTB) is increasing in low-burden countries. EPTB is a heterogeneous disease entity posing diagnostic and management challenges due to the lack of reliable biomarkers. In this study, we prospectively evaluated clinical data and treatment response which were correlated with different biomarkers. Methods The study was conducted at the University Hospital of Cologne. 20 patients with EPTB were enrolled. We analyzed plasma interferon-gamma-inducible protein 10 (IP-10) levels in plasma by ELISA for up to 12 months of treatment. In addition, the QuantiFERON(R)-TB Gold Plus (QFT(R) Plus) test was performed during the course of treatment. Clinical data were assessed prospectively and correlated with QFT(R) Plus and IP-10 levels. Results Plasma IP-10 levels were found to be significantly increased (p < 0.001) in patients with extensive disease compared to patients with limited disease (cervical lymph node TB) or healthy controls. In patients with clinically confirmed paradoxical reaction (PR), a further increase of IP-10 was noted. IFN-gamma measured by the QFT(R) Plus test did not decrease significantly during the course of treatment. Of note, in four EPTB patients (20%) without radiographic pulmonary involvement, sputum culture was positive for Mycobacterium tuberculosis. Conclusion Our data demonstrate that IP-10 may be a valuable biomarker for estimation of disease severity in EPTB and monitoring of the disease course in extensive forms. However, IP-10 may be less suitable for diagnosis and monitoring of EPTB patients with limited disease. The QFT(R) Plus test does not appear to be a suitable marker for therapy monitoring. Sputum should be examined in EPTB patients even in case of normal diagnostic imaging of the chest

DNA sequence specificity of triplex-binding ligands

We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter- and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)(n) or (CCT)(n), but stabilizes triplexes that contain (TTC)(n). In contrast, BePI slightly destabilizes the triplexes that are formed at (TC)(n) (CCT)(n) and (TTC)(n). 2,7-Anthraquinone stabilizes (TC)(n) (CCT)(n) and (TTC)(n), although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT)(n) are stabilized by the 2,7-disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT)(n) and neither affects the complex with (CT)(n). We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C(+)GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C(+)GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues