The rockefeller university

The gastric proton pump H+, K+-ATPase acidifies the gastric lumen, and thus its inhibitors, including the imidazo[ 1,2-alpha] pyridine class of K+-competitive acid blockers (P-CABs), have potential application as acid-suppressing drugs. We determined the electron crystallographic structure of H+, K+-ATPase at 6.5 angstrom resolution in thE2P state with bound BYK99, a potent P-Cwith a restricted ring structure. The BYK99 bound structure has an almost identical profile to that of a previously determined structure with bound SCH28080, the original P-Cprototype, but is significantly different from the previously reported P-CAB-free form, illustrating a common conformational change is required for P-Cbinding. The shared conformational changes include a distinct movement of transmembrane helix 2 (M2), from its position in the previously reported P-CAB-free form, to a location proximal to the P-Cbinding site in the present BYK99-bound structure. Site-specific mutagenesis within M2 revealed that D137 and N138, which face the P-Cbinding site in our model, significantly affect the inhibition constant (Ki) of P-CABs. We also found that A335 is likely to be near the bridging nitrogen at the restricted ring structure of the BYK99 inhibitor. These provide clues to elucidate the binding site parameters and mechanism of P-Cinhibition of gastric acid secretion.

Non-structural protein 3 (NS3) is an essential enzyme and a therapeutic target of hepatitis C virus (HCV). Compared to NS3-catalyzed nucleic acids unwinding, its translation on single stranded nucleic acids have received relatively little attention. To investigate the NS3h translocation with single-stranded nucleic acids substrates directly, we have applied a hybrid platform of single-molecule fluorescence detection combined with optical trapping. With the aid of mechanical manipulation and fluorescence localization, we probed the translocase activity of NS3h on laterally stretched, kilobase-size single-stranded DNA and RNA. We observed that the translocation rate of NS3h on ssDNA at a rate of 24.4 nucleotides per second, and NS3h translocates about three time faster on ssRNA, 74 nucleotides per second. The translocation speed was minimally affected by the applied force. A subpopulation of NS3h underwent a novel translocation mode on ssDNA where the stretched DNA shortened gradually and then recovers its original length abruptly before repeating the cycle repetitively. The speed of this mode of translocation was reduced with increasing force. With corroborating data from single-molecule fluorescence resonance energy transfer (smFRET) experiments, we proposed that NS3h can cause repetitive looping of DNA. The smFRET dwell time analysis showed similar translocation time between sole translocation mode versus repetitive looping mode, suggesting that the motor domain exhibits indistinguishable enzymatic activities between the two translocation modes. We propose a potential secondary nucleic acids binding site at NS3h which might function as an anchor point for translocation-coupled looping.

Alzheimer's disease (AD) is characterized by accumulation of the beta-amyloid peptide (A beta), which is generated through sequential proteolysis of the amyloid precursor protein (APP), first by the action of beta-secretase, generating the beta-C-terminal fragment (beta CTF), and then by the Presenilin 1 (PS1) enzyme in the gamma-secretase complex, generating A beta. gamma-Secretase is an intramembranous protein complex composed of Aph1, Pen2, Nicastrin, and Presenilin 1. Although it has a central role in the pathogenesis of AD, knowledge of the mechanisms that regulate PS1 function is limited. Here, we show that phosphorylation of PS1 at Ser367 does not affect gamma-secretase activity, but has a dramatic effect on A beta levels in vivo. We identified CK1 gamma 2 as the endogenous kinase responsible for the phosphorylation of PS1 at Ser367. Inhibition of CK1 gamma leads to a decrease in PS1 Ser367 phosphorylation and an increase in A beta levels in cultured cells. Transgenic mice in which Ser367 of PS1 was mutated to Ala, show dramatic increases in A beta peptide and in beta CTF levels in vivo. Finally, we show that this mutation impairs the autophagic degradation of beta CTF, resulting in its accumulation and increased levels of A beta peptide and plaque load in the brain. Our results demonstrate that PS1 regulates A beta levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the.-secretase complex, selective phosphorylation of PS1 on Ser367 also decreases A beta levels by increasing beta CTF degradation through autophagy. Elucidation of the mechanism by which PS1 regulates beta CTF degradation may aid in the development of potential therapies for Alzheimer's disease.

The visual photoreceptor rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that stabilizes its inverse agonist ligand, 11-cis-retinal (11CR), by a covalent, protonated Schiff base linkage. In the visual dark adaptation, the fundamental molecular event after photobleaching of rhodopsin is the recombination reaction between its apoprotein opsin and 11CR. Here we present a detailed analysis of the kinetics and thermodynamics of this reaction, also known as the "regeneration reaction". We compared the regeneration of purified rhodopsin reconstituted into phospholipid/detergent bicelles with rhodopsin reconstituted into detergent micelles. We found that the lipid bilayer of bicelles stabilized the chromophore-free opsin over the long timescale required for the regeneration experiments, and also facilitated the ligand reuptake binding reaction. We utilized genetic code expansion and site-specific bioorthogonal labeling of rhodopsin with Alexa488 to enable, to our knowledge, a novel fluorescence resonance energy transfer-based measurement of the binding kinetics between opsin and 11CR. Based on these results, we report a complete energy diagram for the regeneration reaction of rhodopsin. We show that the dissociation reaction of rhodopsin to 11CR and opsin has a 25-pM equilibrium dissociation constant, which corresponds to only 0.3 kcal/mol stabilization compared to the noncovalent, tightly bound antagonist-GPCR complex of iodopindolol and beta-adrenergic receptor. However, 11CR dissociates four orders-of-magnitude slower than iodopindolol, which corresponds to a 6-kcal/mol higher dissociation free energy barrier. We further used isothermal titration calorimetry to show that ligand binding in rhodopsin is enthalpy driven with 22 kcal/mol, which is 12 kcal/mol more stable than the antagonist-GPCR complex. Our data provide insights into the ligand-receptor binding reaction for rhodopsin in particular, and for GPCRs more broadly.

Spatial synchrony, defined as correlated temporal fluctuations among populations, is a fundamental feature of population dynamics, but many aspects of synchrony remain poorly understood. Few studies have examined detailed geographical patterns of synchrony; instead most focus on how synchrony declines with increasing linear distance between locations, making the simplifying assumption that distance decay is isotropic. By synthesising and extending prior work, we show how geography of synchrony, a term which we use to refer to detailed spatial variation in patterns of synchrony, can be leveraged to understand ecological processes including identification of drivers of synchrony, a long-standing challenge. We focus on three main objectives: (1) showing conceptually and theoretically four mechanisms that can generate geographies of synchrony; (2) documenting complex and pronounced geographies of synchrony in two important study systems; and (3) demonstrating a variety of methods capable of revealing the geography of synchrony and, through it, underlying organism ecology. For example, we introduce a new type of network, the synchrony network, the structure of which provides ecological insight. By documenting the importance of geographies of synchrony, advancing conceptual frameworks, and demonstrating powerful methods, we aim to help elevate the geography of synchrony into a mainstream area of study and application.

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of <= 0.25 mu g/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 mu g/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

A search for the production of heavy resonances decaying into top quark-antiquark pairs is presented. The analysis is performed in the lepton+jets and fully hadronic channels using data collected in proton-proton collisions at root s = 13 TeV using the CMS detector at the LHC, corresponding to an integrated luminosity of 2.6 fb(-1). The selection is optimized for massive resonances, where the top quarks have large Lorentz boosts. No evidence for resonant t (t) over bar production is found in the data, and upper limits on the production cross section of heavy resonances are set. The exclusion limits for resonances with masses above 2 TeV are significantly improved compared to those of previous analyses at root s = 8 TeV.

How morphologically complex cilia form is not well understood. A key regulator of ciliary shape has now been identified that links the establishment of neuronal fate with the formation of cell-specific ciliary structures in Caenorhabditis elegans.

Whole-genome sequencing of Staphylococcus xylosus strain JW2311 from bovine mastitis milk identified the novel 49.3-kb macrolide-lincosamide-streptogramin B (MLSB) resistance plasmid pJW2311. It contained the macrolide resistance gene mph(C), the macrolide-streptogramin B resistance gene msr(A), and the new MLSB resistance gene erm(48) and could be transformed into Staphylococcus aureus by electroporation. Functionality of erm(48) was demonstrated by cloning and expression in S. aureus.

In many contexts, the problem arises of determining which of many candidate mutations is the most likely to be causative for some phenotype. It is desirable to have a way to evaluate this probability that relies as little as possible on previous knowledge, to avoid bias against discovering new genes or functions. We have isolated mutants with blocked cell cycle progression in Chlamydomonas and determined mutant genome sequences. Due to the intensity of UV mutagenesis required for efficient mutant collection, the mutants contain multiple mutations altering coding sequence. To provide a quantitative estimate of probability that each individual mutation in a given mutant is the causative one, we developed a Bayesian approach. The approach employs four independent indicators: sequence conservation of the mutated coding sequence with Arabidopsis; severity of the mutation relative to Chlamydomonas wild-type based on Blosum62 scores; meiotic mapping information for location of the causative mutation relative to known molecular markers; and, for a subset of mutants, the transcriptional profile of the candidate wild-type genes through the mitotic cell cycle. These indicators are statistically independent, and so can be combined quantitatively into a single probability calculation. We validate this calculation: recently isolated mutations that were not in the training set for developing the indicators, with high calculated probability of causality, are confirmed in every case by additional genetic data to indeed be causative. Analysis of best reciprocal BLAST (BRB) relationships among Chlamydomonas and other eukaryotes indicate that the temperature sensitive-lethal (Ts-lethal) mutants that our procedure recovers are highly enriched for fundamental cell-essential functions conserved broadly across plants and other eukaryotes, accounting for the high information content of sequence alignment to Arabidopsis.