Hypercapnia induces cleavage and nuclear localization of RelB protein, giving insight into CO2 sensing and signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-?B pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-?B family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3ß or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-?B family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

REST is a hypoxia-responsive transcriptional repressor

Cellular exposure to hypoxia results in altered gene expression in a range of physiologic and pathophysiologic states. Discrete cohorts of genes can be either up- or down-regulated in response to hypoxia. While the Hypoxia-Inducible Factor (HIF) is the primary driver of hypoxia-induced adaptive gene expression, less is known about the signalling mechanisms regulating hypoxiadependent gene repression. Using RNA-seq, we demonstrate that equivalent numbers of genes are induced and repressed in human embryonic kidney (HEK293) cells. We demonstrate that nuclear localization of the Repressor Element 1-Silencing Transcription factor (REST) is induced in hypoxia and that REST is responsible for regulating approximately 20% of the hypoxia-repressed genes. Using chromatin immunoprecipitation assays we demonstrate that REST-dependent gene repression is at least in part mediated by direct binding to the promoters of target genes. Based on these data, we propose that REST is a key mediator of gene repression in hypoxia.

The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.

Auditory stream segregation in cochlear implant listeners:measures based on temporal discrimination and interleaved melody recognition

The evidence that cochlear implant listeners routinely experience stream segregation is limited and equivocal. Streaming in these listeners was explored using tone sequences matched to the center frequencies of the implant’s 22 electrodes. Experiment 1 measured temporal discrimination for short (ABA triplet) and longer (12 AB cycles) sequences (tone/silence durations = 60/40 ms). Tone A stimulated electrode 11; tone B stimulated one of 14 electrodes. On each trial, one sequence remained isochronous, and tone B was delayed in the other; listeners had to identify the anisochronous interval. The delay was introduced in the second half of the longer sequences. Prior build-up of streaming should cause thresholds to rise more steeply with increasing electrode separation, but no interaction with sequence length was found. Experiment 2 required listeners to identify which of two target sequences was present when interleaved with distractors (tone/silence durations = 120/80 ms). Accuracy was high for isolated targets, but most listeners performed near chance when loudness-matched distractors were added, even when remote from the target. Only a substantial reduction in distractor level improved performance, and this effect did not interact with target-distractor separation. These results indicate that implantees often do not achieve stream segregation, even in relatively unchallenging tasks.

Solid-supported boronic acid conjugates for sugar and glycopeptide recognition

The Fmoc synthetic strategy was employed to synthesise two identical combinatorial peptide libraries on a hydrophilic PEG-PS resin. One library was appended with boronic acid moieties at two positionally-fixed locations. Successful inclusion of the boronic acid units was confirmed using a novel UV fluorescent colorimetric assay employing carminic acid as the dye compound. A study of the effect had by the resin-bound peptides bearing boronic acid groups on the binding characteristics of vancomycin, a medically relevant antibiotic glycoprotein, was conducted. In all, 132 library compounds were tested for their binding affinity with vancomycin, via immobilisation of the glycopeptide onto the solid support through hydrogen bonding or complexation with the boronic acid moieties. Subsequent cleavage via acidolysis afforded vancomycin containing solutions which were quantified by growth inhibition of methicillin susceptible Staphylococcus aureus. Comparison of the diameters of the resultant zones of inhibition and those produced by vancomycin of known concentrations afforded a means of calculating the vancomycin concentration of the cleavage solutions, and thereby determining the binding affinity of vancomycin to each peptide sequence. Five peptide sequences and twenty one of the peptidyl-boronic acid sequences showed zones of inhibition, demonstrating their reversible affinity for vancomycin. Three peptide sequences showed zones of inhibition in both libraries. The presence of boronic acid was therefore shown to impart, enhance, detract and remove the affinity of vancomycin to a range of resin-bound peptide sequences.

Source localization of reaction-diffusion models for brain tumors

We propose a mathematically well-founded approach for locating the source (initial state) of density functions evolved within a nonlinear reaction-diffusion model. The reconstruction of the initial source is an ill-posed inverse problem since the solution is highly unstable with respect to measurement noise. To address this instability problem, we introduce a regularization procedure based on the nonlinear Landweber method for the stable determination of the source location. This amounts to solving a sequence of well-posed forward reaction-diffusion problems. The developed framework is general, and as a special instance we consider the problem of source localization of brain tumors. We show numerically that the source of the initial densities of tumor cells are reconstructed well on both imaging data consisting of simple and complex geometric structures.