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Ghrelin receptor (GhrR) is a promising drug target because of its central role in energy homeostasis. GhrR, known for high constitutive activity, is thought to display multi-state conformations during activation and signaling. We used genetically encoded unnatural amino acids and bioorthogonal labeling reactions to engineer multiple fluorescent donor-acceptor pairs to probe ligand-directed structural changes in GhrR. We demonstrate how conformational dynamics of a G-protein-coupled receptor can be measured in reconstituted systems.

Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers.

The first search for supersymmetry in the vector-boson fusion topology is presented. The search targets final states with at least two leptons, large missing transverse momentum, and two jets with a large separation in rapidity. The data sample corresponds to an integrated luminosity of 19.7 fb(-1) of proton-proton collisions at root s = 8TeV collected with the CMS detector at the CERN LHC. The observed dijet invariant mass spectrum is found to be consistent with the expected standard model prediction. Upper limits are set on the cross sections for chargino and neutralino production with two associated jets, assuming the supersymmetric partner of the tau lepton to be the lightest slepton and the lightest slepton to be lighter than the charginos. For a so-called compressed-mass-spectrum scenario in which the mass difference between the lightest supersymmetric particle (chi) over bar (0)(1) and the next lightest, mass-degenerate, gaugino particles (chi) over bar (0)(2) and (chi) over bar (+/-)(1) is 50 GeV, a mass lower limit of 170 GeV is set for these latter two particles.

IgG antibodies are actively produced in response to antigenic challenge or passively administered as an effective form of immunotherapy to confer immunity against foreign antigens. Their protective activity is mediated through their bifunctional nature: a variable Fab domain mediates antigen-binding specificity, whereas the constant Fc domain engages Fc receptors (FcRs) expressed on the surface of leukocytes to mediate effector functions. While traditionally considered the invariant domain of an IgG molecule, the Fc domain displays remarkable structural heterogeneity determined primarily by differences in the amino acid sequence of the various IgG subclasses and by the composition of the complex, Fc-associated biantennary N-linked glycan. These structural determinants regulate the conformational flexibility of the IgG Fc domain and affect its capacity to interact with distinct types of FcRs (type I or type II FcRs). FcR engagement activates diverse downstream immunomodulatory pathways with pleiotropic functional consequences including cytotoxicity and phagocytosis of IgG-coated targets, differentiation and activation of antigen presenting cells, modulation of T-cell activation, plasma cell survival, and regulation of antibody responses. These functions highlight the importance of FcR-mediated pathways in the modulation of adaptive immune responses and suggest a central role for IgG-FcR interactions during active and passive immunization.

Activation of Sirtuin (silent mating type information regulation 2 homolog) 1, or SIRT1, is an unexplored therapeutic approach for treatment of inflammatory diseases. We randomized 40 patients with moderate-to-severe psoriasis (4:1) to three escalating doses of SRT2104, a selective activator of SIRT1, or placebo. Across all SRT2104 groups, 35% of patients (p<0.0001) achieved good to excellent histological improvement based on skin biopsies taken at baseline and day 84 but was not consistently in agreement with PASI. Improvement in histology was associated with modulation of IL-17 and TNF-alpha signaling pathways and keratinocyte differentiation target genes. 27 subjects (69%) across all treatment groups, including placebo, experienced at least one treatment emergent adverse event. The majority of AEs were either mild or moderate. Most common were headache (8%), dizziness (8%), upper respiratory tract infection (8%), and psoriatic arthropathy (8%). Average drug exposure increased in a dose-dependent manner for escalating doses of SRT2104 and had high intra-subject variability in exposure (AUC %CV: 51-89%). Given the interesting signals of clinical activity, impact on gene expression and the generally favorable safety profile seen in this study, further investigation of SIRT1 activators for the treatment of psoriasis is warranted.

Hair cells, the mechanosensory receptors of the inner ear, underlie the senses of hearing and balance. Adult mammals cannot adequately replenish lost hair cells, whose loss often results in deafness or balance disorders. To determine the molecular basis of this deficiency, we investigated the development of a murine vestibular organ, the utricle. Here we show that two members of the SoxC family of transcription factors, Sox4 and Sox11, are down-regulated after the epoch of hair cell development. Conditional ablation of SoxC genes in vivo results in stunted sensory organs of the inner ear and loss of hair cells. Enhanced expression of SoxC genes in vitro conversely restores supporting cell proliferation and the production of new hair cells in adult sensory epithelia. These results imply that SoxC genes govern hair cell production and thus advance these genes as targets for the restoration of hearing and balance.

Cow's milk protein allergy in exclusively breastfed infants, the main cause of food intolerance during the first 6 months of life, is triggered by the mother's diet. beta-Lactoglobulin (BLG) present in cow's milk is one of the most potent allergens for newborns. Since no prophylactic treatment is available, finding ligands capable of binding BLG and reducing its allergenicity is currently the focus of research. In this work, an innovative methodology encompassing microfluidics based on fully automated chip-nanoelectrospray ionization (nanoESI), coupled with high-resolution mass spectrometry (MS) on a quadrupole time-of-flight (QTOF MS) instrument was developed. This platform was employed for the assessment of the noncovalent interactions between maltohexaose (Glc(6)) and beta-lactoglobulin extracted from human milk upon deliberate intake of cow's milk. The experiments were carried out in (+) ESI mode, using ammonium acetate (pH 6.0) as the buffer and also in pure water. In both cases, the MS analysis revealed the formation of BLG-Glc(6) complex, which was characterized by top-down fragmentation in tandem MS (MS/MS) using collision-induced dissociation (CID). Our findings have a significant biomedical impact, indicating that Glc(6) binds BLG under conditions mimicking the in vivo environment and therefore might represent a ligand, able to reduce its allergenicity.

A balanced interaction between dopaminergic and cholinergic signaling in the striatum is critical to goal-directed behavior. But how this interaction modulates corticostriatal synaptic plasticity underlying learned actions remains unclear-particularly in direct-pathway spiny projection neurons (dSPNs). Our studies show that in dSPNs, endogenous cholinergic signaling through M4 muscarinic receptors (M4Rs) promoted long-term depression of corticostriatal glutamatergic synapses, by suppressing regulator of G protein signaling type 4 (RGS4) activity, and blocked D1 dopamine receptor dependent long-term potentiation (LTP). Furthermore, in a mouse model of L-3,4-dihydroxyphenylalanine (L-DOPA)induced dyskinesia (LID) in Parkinson's disease (PD), boosting M4R signaling with positive allosteric modulator (PAM) blocked aberrant LTP in dSPNs, enabled LTP reversal, and attenuated dyskinetic behaviors. An M4R PAM also was effective in a primate LID model. Taken together, these studies identify an important signaling pathway controlling striatal synaptic plasticity and point to a novel pharmacological strategy for alleviating LID in PD patients.

Fibrolamellar hepatocellular carcinoma (FLHCC) tumors all carry a deletion of similar to 400 kb in chromosome 19, resulting in a fusion of the genes for the heat shock protein, DNAJ (Hsp40) homolog, subfamily B, member 1, DNAJB1, and the catalytic subunit of protein kinase A, PRKACA. The resulting chimeric transcript produces a fusion protein that retains kinase activity. No other recurrent genomic alterations have been identified. Here we characterize the molecular pathogenesis of FLHCC with transcriptome sequencing (RNA sequencing). Differential expression (tumor vs. adjacent normal tissue) was detected for more than 3,500 genes (log(2) fold change >= 1, false discovery rate <= 0.01), many of which were distinct from those found in hepatocellular carcinoma. Expression of several known oncogenes, such as ErbB2 and Aurora Kinase A, was increased in tumor samples. These and other dysregulated genes may serve as potential targets for therapeutic intervention.