The rockefeller university

Staphylococcus aureus virulence is regulated when secreted autoinducing peptides (AIPs) are recognized by a membrane-bound receptor histidine kinase (RHK), AgrC. Some AIPs are agonists of virulence gene expression, while others are antagonists. It is unclear how AIP binding regulates AgrC activity. Here, we reconstitute an AgrC family member, AgrC-I, using nanometer-scale lipid bilayer discs. We show that AgrC-I requires membranes rich in anionic lipids to function. The agonist, AIP-I, binds AgrC-I noncooperatively in a 2: 2 stoichiometry, while an antagonist ligand, AIP-II, functions as an inverse agonist of the kinase activity. We also demonstrate the kinase and sensor domains in AgrC are connected by a helical linker whose conformational state exercises rheostat-like control over the kinase activity. Binding of agonist or inverse-agonist peptides results in twisting of the linker in different directions. These two observations provide a view of the molecular motions triggered by ligand binding in an intact membrane-bound RHK.

Background: Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown. Results: Here, we show that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence of Slit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance in vivo. Conclusions: These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain.

Toe clipping is used to identify and genotype preweanling mice, but the procedure generates concerns relevant to pain and distress. The few pertinent studies available evaluated mice between postnatal days (PND) 3 and 7, advocate the use of toe clipping in mice PND 7 or younger, and identify handling as the most distressing aspect of the procedure. Because both toe and tail clipping may be necessary in older mice to obtain sufficient DNA for genotyping, we surmised that performing these procedures concurrently to minimize handling would be beneficial. We also examined reflex development until PND 21 and adult behavior at 8 to 10 wk of age in mice toe clipped at PND 7 or 17 and the benefits of using topical vapocoolant anesthesia. C57BL/6J pups at PND 7 and 17 were assigned to 1 of 4 groups: 1) clipping of digit 3 of contralateral fore- and hindpaws; 2) toe clipping after topical vapocoolant anesthesia; 3) unclipped, unsprayed controls; and, 4) unclipped and vapocoolant-sprayed. Compared with unanesthetized pups, those sprayed with vapocoolant vocalized and struggled more when handled and had more bleeding, erythema, and swelling, which persisted for as long as 12 h after toe clipping. Reflex development, anxiety, locomotion, and motor coordination were not different among groups or with regard to the age of toe clipping. No tissue reaction was noted microscopically in paws collected at 10 wk of age. We conclude that the use of vapocoolant cannot be recommended due to its harmful effects and that toe clipping at PND 7 or 17 does not significantly affect the long-term welfare of mice.

A search for forbidden and exotic Z boson decays in the diphoton mass spectrum is presented for the first time in hadron collisions, based on data corresponding to 10.0 fb(-1) of integrated luminosity from proton-antiproton collisions at root s = 1.96 TeV collected by the CDF experiment. No evidence of signal is observed, and 95% credibility level Bayesian upper limits are set on the branching ratios of decays of the Z boson to a photon and neutral pion (which is detected as a photon), a pair of photons, and a pair of neutral pions. The observed branching ratio limits are 2.01 x 10(-5) for Z -> pi(0)gamma, 1.46 x 10(-5) for Z -> gamma gamma, and 1.52 x 10(-5) for Z -> pi(0)pi(0). The Z -> pi(0)gamma and Z -> gamma gamma limits improve the most stringent results from other experiments by factors of 2.6 and 3.6, respectively. The Z -> pi(0)pi(0) branching ratio limit is the first experimental result on this decay.

Pneumococcal disease is frequent at the extremes of age. While several studies have looked at colonization among young children, much less is known among the elderly. We aimed to evaluate pneumococcal carriage among elderly adults living in Portugal. Between April 2010 and December 2012, nasopharyngeal and oropharyngeal swabs of adults over 60 years of age, living in an urban area (n = 1,945) or in a rural area (n = 1,416), were obtained. Pneumococci were isolated by culture-based standard procedures, identified by optochin susceptibility, bile solubility and PCR screening for lytA and cpsA, and characterized by antibiotype, serotype, and MLST. Associations between pneumococcal carriage, socio-demographic and clinical characteristics were evaluated by univariate analysis and multiple logistic regression. The global prevalence of carriage was 2.3% (95% CI: 1.8-2.8). In the multiple logistic regression analysis, smoking, being at a retirement home, and living in a rural area increased the odds of being a pneumococcal carrier by 4.4-fold (95% CI: 1.9-9.2), 2.0-fold (95% CI: 1.1-3.6) and 2.0-fold (95% CI: 1.2-3.5), respectively. Among the 77 pneumococcal isolates, 26 serotypes and 40 STs were identified. The most prevalent serotypes were (in decreasing order) 19A, 6C, 22F, 23A, 35F, 11A, and 23B, which accounted, in total, for 60.0% of the isolates. Most isolates (93.5%) had STs previously described in the MLST database. Resistance to macrolides, non-susceptibility to penicillin and multidrug resistance were found in 19.5%, 11.7%, and 15.6% of the isolates, respectively. We conclude that the prevalence of pneumococcal carriage in the elderly, in Portugal, as determined by culture-based methods, is low. Serotype and genotype diversity is high. Living in a rural area, in a retirement home, and being a smoker increased the risk of pneumococcal carriage. This study contributes to the establishment of a baseline that may be used to monitor how novel pneumococcal vaccines impact on colonization among the elderly.

While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting, and cell-cycle inhibitors reveal that closure does not require overlying periderm, proliferation, or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA- and alpha 5 beta 1 integrin/fibronectin-mediated migration and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue.

Background: Triacylglycerols (TAGs) are the most abundant form of storage oil in plants. They consist of three fatty acid chains (usually C16 or C18) covalently linked to glycerol. SDP1 is a specific lipase for the first step of TAG catabolism in Arabidopsis seeds. Arabidopsis mutants deficient in SDP1 accumulate high levels of oils, probably due to blockage in TAG degradation. We applied this knowledge from the model plant, Arabidopsis thaliana, to engineer increased seed oil content in the biodiesel plant Jatropha curcas using RNA interference (RNAi) technology. Results: As Jatropha is a biodiesel crop, any significant increase in its seed oil content would be an important agronomic trait. Using A. thaliana as a model plant, we found that a deficiency of SDP1 led to higher TAG accumulation and a larger number of oil bodies in seeds compared with wild type (Columbia-0; Col-0). We cloned Jatropha JcSDP1, and verified its function by complementation of the Arabidopsis sdp1-5 mutant. Taking advantage of the observation with Arabidopsis, we used RNAi technology to generate JcSDP1 deficiency in transgenic Jatropha. We found that Jatropha JcSDP1-RNAi plants accumulated 13 to 30% higher total seed storage lipid, along with a 7% compensatory decrease in protein content, compared with control (CK; 35S: GFP) plants. Free fatty acid (FFA) content in seeds was reduced from 27% in control plants to 8.5% in JcSDP1-RNAi plants. Conclusion: Here, we showed that SDP1 deficiency enhances seed oil accumulation in Arabidopsis. Based on this result, we generated SDP1-deficient transgenic Jatropha plants using by RNAi technology with a native JcSDP1 promoter to silence endogenous JcSDP1 expression. Seeds of Jatropha JcSDP1-RNAi plants accumulated up to 30% higher total lipid and had reduced FFA content compared with control (CK; 35S: GFP) plants. Our strategy of improving an important agronomic trait of Jatropha can be extended to other oil crops to yield higher seed oil.

Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K+ channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K+ (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the "force-from-lipid" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K+ channels.

A search for three-jet hadronic resonance production in pp collisions at a centre-of-mass energy of 8 TeV has been conducted by the CMS Collaboration at the LHC with a data sample corresponding to an integrated luminosity of 19.4 fb(-1). The search method is model independent, and events are selected that have high jet multiplicity and large values of jet transverse momenta. The signal models explored assume R-parity-violating supersymmetric gluino pair production and have final states with either only light-flavour jets or both light- and heavy-flavour jets. No significant deviation is found between the selected events and the expected standard model multijet and t (t) over bar background. For a gluino decaying into light-flavour jets, a lower limit of 650 GeV on the gluino mass is set at a 95% confidence level, and for a gluino decaying into one heavy- and two light-flavour jets, gluino masses between 200 and 835 GeV are, for the first time, likewise excluded. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

Background: Phenethyl isothiocyanate (PEITC), present naturally in cruciferous vegetables, is a chemopreventive agent. It blocks initiation and post-initiation progression of carcinogenesis. Mechanism study in human prostate cancer cells revealed that PEITC is a dual inhibitor of aberrant DNA hypermethylation and histone deacetylases, reactivating silenced genes and regulating the androgen-mediated growth of tumor cells. The identity of the cellular organelle that initially interacts with PEITC has not been fully described. Methods: Human prostate cancer LNCaP cells were exposed to PEITC and the effects on cellular fine structure examined by transmission electron microscopic studies. Alteration of mitochondrial membrane potential and cytochrome c release were evaluated as early events of apoptosis, and the TUNEL method for quantifying apoptotic cells. Mitochondria were isolated for determining their protein expression. Results: Ultrastructural analyses have revealed condensed mitochondria and a perturbed mitochondrial cristae structure, which assumed a rounded and dilated shape within 4-hours of PEITC contact, and became more pronounced with longer PEITC exposure. They presented as the most prominent intracellular alterations in the early hours. Mitochondria structure alterations were demonstrated, for the first time, with the isothiocyanates. An increase in the number of smooth endoplasmic reticulum and vacuoles were also noted that is consistent with the presence of autophagy. Early events of apoptosis were detected, with cytochrome c released along with the appearance of mitochondrial alteration. Mitochondrial membrane potential was disrupted within 18 hours of PEITC exposure, preceding the appearance of apoptotic cells with DNA strand breaks. In parallel, the expression of the mitochondrial class III beta-tubulin in the outer membrane, which associates with the permeability transition pore, was significantly reduced as examined with isolated mitochondria. Conclusion: Mitochondria may represent the organelle target of the isothiocyanates, indicating that the isothiocyanates may be mitochondria-interacting agents to inhibit carcinogenesis.