In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP–Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ−/− liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.

BackgroundImmunity decreases with age, which leads to reactivation of varicella zoster virus (VZV). In human subjects age-associated immune changes are usually measured in blood leukocytes; however, this might not reflect alterations in tissue-specific immunity.ObjectivesWe used a VZV antigen challenge system in the skin to investigate changes in tissue-specific mechanisms involved in the decreased response to this virus during aging.MethodsWe assessed cutaneous immunity based on the extent of erythema and induration after intradermal VZV antigen injection. We also performed immune histology and transcriptomic analyses on skin biopsy specimens taken from the challenge site in young (<40 years) and old (>65 years) subjects.ResultsOld human subjects exhibited decreased erythema and induration, CD4+ and CD8+ T-cell infiltration, and attenuated global gene activation at the site of cutaneous VZV antigen challenge compared with young subjects. This was associated with increased sterile inflammation in the skin in the same subjects related to p38 mitogen-activated protein kinase–related proinflammatory cytokine production (P < .0007). We inhibited systemic inflammation in old subjects by means of pretreatment with an oral small-molecule p38 mitogen-activated protein kinase inhibitor (Losmapimod; GlaxoSmithKline, Brentford, United Kingdom), which reduced both serum C-reactive protein levels and peripheral blood monocyte secretion of IL-6 and TNF-α. In contrast, cutaneous responses to VZV antigen challenge were increased significantly in the same subjects (P < .0003).ConclusionExcessive inflammation in the skin early after antigen challenge retards antigen-specific immunity. However, this can be reversed by inhibition of inflammatory cytokine production that can be used to promote vaccine efficacy and the treatment of infections and malignancy during aging.

BackgroundPsoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy.Methods and FindingsThe preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in ex vivo lesional psoriatic skin.ConclusionsOur work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.

Cystic fibrosis (CF) is a major lethal genetic disease caused by mutations in the CF transmembrane conductance regulator gene (CFTR). This encodes a chloride ion channel on the apical surface of epithelial cells. The most common mutation in CFTR (F508del-CFTR) generates a protein that is misfolded and retained in the endoplasmic reticulum. Identifying small molecules that correct this CFTR trafficking defect is a promising approach in CF therapy. However, to date only modest efficacy has been reported for correctors in clinical trials. We identified the marine sponge metabolite latonduine as a corrector. We have now developed a series of latonduine derivatives that are more potent F508del-CFTR correctors with one (MCG315 [2,3-dihydro-1H-2-benzazepin-1-one]) having 10-fold increased corrector activity and an EC 50 of 72.25 nM.We show that the latonduine analogs inhibit poly-ADP ribose polymerase (PARP) isozymes 1, 3, and 16. Further our molecular modeling studies point to the latonduine analogs binding to the PARP nicotinamide-binding domain. We established the relationship between the ability of the latonduine analogs to inhibit PARP-16 and their ability to correct F508del-CFTR trafficking. We show that latonduine can inhibit both PARP-3 and -16 and that this is necessary for CFTR correction. We demonstrate that latonduine triggers correction by regulating the activity of the unfolded protein response activator inositolrequiring enzyme (IRE-1) via modulation of the level of its ribosylation by PARP-16. These results establish latonduines novel site of action as well as its proteostatic mechanism of action.

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Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a

COMMUNICATIONSwhich allows structural information to be deduced from a measured vibrational spectrum. Strong hydrogen bonds with short 0-0 distances correlate with long, weakened 0 -H covalent bonds and low hydrogen stretching vibrations. If the 0-0 distance is increased. the length ofthe covalent bond approaches its unperturbed value approximately exponentially; the 0 -H stretching vibration increases almost linearly as the length of the covalent bond decreases. Aluminum-rich zeolites differ from their aluminum-poor counterparts both in their IR spectra and in their rea~tivity.'~. 16] In particular, the highest peak at 3500 cm-' seems to vanish as the Si:AI ratio approaches its lower limit of 1 ; 1. Therefore we investigated the interaction of methanol with two acid sites simultaneously. We find that the hydrogen bond between the methanol proton H, and an oxygen adjacent to another aluminum atom is shortened by about 5% and is therefore stronger than the hydrogen bond formed with an isolated acid site. Preliminary molecular dynamics simulations of I ps reveal a downward shift of the H,-0 vibration (located at 3500cm-' for the methanol hydroxyl group adsorbed on an isolated acid site) of roughly 400cm-', which could explain the disappearance of the former absorption band. However, it should be noted that aluminum-rich zeolites exhibit a substantially richer variety of structures for acid-site centers and adsorption complexes than aluminum-poor zeolites, a fact that has not yet been explored exhaustively.We studied sodalite to unravel processes that should also be relevant for the more complex zeolites actually used in technologically relevant processes. Therefore we should discuss to what extent our results apply to zeolites other than sodalite. We expect that the essential features of structure A are rather independent of the zeolite, as the tetrahedral angle on the Si and A1 atoms is largely conserved. Structure B, however, depends strongly on the local structure. Since it depends on the proximity of distant oxygen atoms, this structure can only occur in sufficiently small or highly deformed larger rings. Rings consisting of four atoms with tetrahedral coordination cannot be bridged, because the acid-site protons point outward. Since the structures A and B have similar vibrational patterns, we expect only quantitative changes in the IR spectra for zeolites that cannot form structure B. The largest deviations in the IR spectra will occur in the lower part (see Fig. 3), because this part is most sensitive to the strength of bond of the acid-site proton.Our computer experiments predicted adsorption structures of methanol in zeolites which, for the first time, are consistent with the available experimental data. This information forms the basis for the understanding of a large class of zeolite-catalyzed reactions. We have applied a theoretical approach t o zeolite chemistry, which allows us to study with a high level of accuracy dynamic behavior of molecules in zeolites at finite temperatures without the limitatio...

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