Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function

Molecular basis for Staphylococcus aureus-mediated platelet aggregate formation under arterial shear in vitro.

OBJECTIVE: Staphylococcus aureus is the most frequent causative organism of infective endocarditis (IE) and is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Previously, we showed that S. aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) can stimulate rapid platelet aggregation. METHODS AND RESULTS: In this study we investigate their relative roles in mediating aggregate formation under physiological shear conditions. Platelets failed to interact with immobilized wild-type S. aureus (Newman) at shear rates \u3c500\u3es(-1) but rapidly formed an aggregate at shear rates \u3e800 s(-1). Inactivation of the ClfA gene eliminated aggregate formation at any shear rate. Using surrogate hosts that do not interact with platelets bacteria overexpressing ClfA supported rapid aggregate formation under high shear with a similar profile to Newman whereas bacteria overexpressing FnBPA did not. Fibrinogen binding to ClfA was found to be essential for aggregate formation although fibrinogen-coated surfaces only allowed single-platelets to adhere under all shear conditions. Blockade of the platelet immunoglobulin receptor Fc gammaRIIa inhibited aggregate formation. CONCLUSIONS: Thus, fibrinogen and IgG binding to ClfA is essential for aggregate formation under arterial shear conditions and may explain why S. aureus is the major cause of IE

Staphylococcus aureus protein A plays a critical role in mediating bone destruction and bone loss in osteomyelitis.

Staphylococcus aureus is the most frequent causative organism of osteomyelitis. It is characterised by widespread bone loss and bone destruction. Previously we demonstrated that S. aureus protein A (SpA) is capable of binding to tumour necrosis factor receptor-1 expressed on pre-osteoblastic cells, which results in signal generation that leads to cell apoptosis resulting in bone loss. In the current report we demonstrate that upon S. aureus binding to osteoblasts it also inhibits de novo bone formation by preventing expression of key markers of osteoblast growth and division such as alkaline phosphatase, collagen type I, osteocalcin, osteopontin and osteocalcin. In addition, S. aureus induces secretion of soluble RANKL from osteoblasts which in turn recruits and activates the bone resorbing cells, osteoclasts. A strain of S. aureus defective in SpA failed to affect osteoblast growth or proliferation and most importantly failed to recruit or activate osteoclasts. These results suggest that S. aureus SpA binding to osteoblasts provides multiple coordinated signals that accounts for bone loss and bone destruction seen in osteomyelitis cases. A better understanding of the mechanisms through which S. aureus leads to bone infection may improve treatment or lead to the development of better therapeutic agents to treat this notoriously difficult disease

Timing of extension in the Pioneer metamorphic core complex with implications for the spatial-temporal pattern of Cenozoic extension and exhumation in the northern U.S. Cordillera

The Pioneer core complex (PCC) in central Idaho lies along a transition between Early Eocene and ca. 40 Ma core complexes to the north and south, respectively. Thus, the age of extensional development of the PCC is important in understanding the spatial-temporal patterns of core-complex development in the North American Cordillera. New results, including structural observations and U-Pb zircon (SHRIMP and ICPMS) geochronology, constrain the early extensional history of the footwall for the first time. High-temperature strain with a top-WNW shear-sense is pervasive throughout metamorphic rocks of the northwestern footwall. An isoclinally folded dike yields a crystallization age of ∼48-47 Ma, whereas a crosscutting dike yielded an age of 46 Ma. Metamorphic rocks are also intruded by the ∼50-48 Ma Pioneer intrusive suite (PIS), a W-dipping granodiorite sheet displaying a magmatic fabric. Northwest-trending lineations are locally visible and also defined by anisotropy of magnetic susceptibility, indicating that during emplacement, the PIS was undergoing similarly oriented extensional strain as the enclosing metamorphic rocks. Therefore, WNW-directed extension spanning this structural section occurred between ∼50 and 46 Ma. Following emplacement of crosscutting 46 Ma dikes, deformation was partitioned into the WNW-directed Wildhorse detachment. Motion on the detachment occurred between ∼38 and 33 Ma, as documented by previous 40Ar/ 39Ar thermochronology. It is not clear, however, whether extension was continuous through the interval between these two time periods. Although Early Eocene extension in the PCC was synchronous with extension in core complexes to the north, rates of footwall exhumation in central Idaho were much lower. This southward slowing is compatible with N-S differences in inferred subduction zone geometry/kinematics and in the internal character of the orogenic wedge

Evidence-Based intervention (Ebi) Mapping: a Systematic approach to Understanding the Components and Logic of Ebis

BACKGROUND: Despite the development of numerous evidence-based interventions (EBIs), many go unused in practice. Hesitations to use existing EBIs may be due to a lack of understanding about EBI components and what it would take to adapt it or implement it as designed. to improve the use of EBIs, program planners need to understand their goals, core components, and mechanisms of action. This paper presents EBI Mapping, a systematic approach based on Intervention Mapping, that can be used to understand and clearly describe EBIs, and help planners put them into practice. METHODS: We describe EBI Mapping tasks and provide an example of the process. EBI Mapping uses principles from Intervention Mapping, a systematic framework for planning multilevel health promotion interventions. EBI Mapping applies the Intervention Mapping steps retrospectively to help planners understand an existing EBI (rather than plan a new one). We explain each EBI Mapping task and demonstrate the process using the VERB Summer Scorecard (VSS), a multi-level community-based intervention to improve youth physical activity. RESULTS: EBI Mapping tasks are: 1) document EBI materials and activities, and their audiences, 2) identify the EBI goals, content, and mechanisms of action, 3) identify the theoretical change methods and practical applications of those methods, 4) describe design features and delivery channels, and 5) describe the implementers and their tasks, implementation strategies, and needed resources. By applying the EBI Mapping tasks, we created a logic model for the VSS intervention. The VSS logic model specifies the links between behavior change methods, practical applications, and determinants for both the at-risk population and environmental change agents. The logic model also links the respective determinants to the desired outcomes including the health behavior and environmental conditions to improve the health outcome in the at-risk population. CONCLUSIONS: EBI Mapping helps program planners understand the components and logic of an EBI. This information is important for selecting, adapting, and scaling-up EBIs. Accelerating and improving the use of existing EBIs can reduce the research-to-practice gap and improve population health

A web-based office climate control system using wireless sensors

Conventional heating, ventilating, and air conditioning (HVAC) systems usually achieve the desired control level by means of simple ON–OFF control, which can often result in high energy wastage. A potential solution to this issue is intelligent self-regulating HVAC controllers, which base their actions/decisions on sensor data. In this paper, an office climate monitoring and control system is designed and implemented. The system consists of various wireless sensor nodes and a control node. The sensor nodes provide the sensor data necessary to determine occupancy and the control node executes the algorithm, which decides whether to activate cooling or heating based on the sensor data. This system can serve as a controller and can be integrated into HVAC systems in smart buildings. It is shown that the developed control algorithm executed on the control node results in an improvement of up to 39% in energy efficiency over conventional ON–OFF controllers for HVAC systems.http://ieeexplore.ieee.org/xpl/RecentIssue.jsp?punumber=7361hb2017Electrical, Electronic and Computer Engineerin

The Optical Design of CHARIS: An Exoplanet IFS for the Subaru Telescope

High-contrast imaging techniques now make possible both imaging and spectroscopy of planets around nearby stars. We present the optical design for the Coronagraphic High Angular Resolution Imaging Spectrograph (CHARIS), a lenslet-based, cryogenic integral field spectrograph (IFS) for imaging exoplanets on the Subaru telescope. The IFS will provide spectral information for 138x138 spatial elements over a 2.07 arcsec x 2.07 arcsec field of view (FOV). CHARIS will operate in the near infrared (lambda = 1.15 - 2.5 microns) and will feature two spectral resolution modes of R = 18 (low-res mode) and R = 73 (high-res mode). Taking advantage of the Subaru telescope adaptive optics systems and coronagraphs (AO188 and SCExAO), CHARIS will provide sufficient contrast to obtain spectra of young self-luminous Jupiter-mass exoplanets. CHARIS will undergo CDR in October 2013 and is projected to have first light by the end of 2015. We report here on the current optical design of CHARIS and its unique innovations.Comment: 15 page

Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions.

Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing V(H)3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A-vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione-S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D\u27-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in V(H)3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized V(H)3 Fab had reduced binding to vWF A1 and D\u27-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site

Increase of Direct C-C Coupling Reaction Yield by Identifying Structural and Electronic Properties of High-Spin Iron Tetra-azamacrocyclic Complexes

Macrocyclic ligands have been explored extensively as scaffolds for transition metal catalysts for oxygen and hydrogen atom transfer reactions. C–C reactions facilitated using earth abundant metals bound to macrocyclic ligands have not been well-understood but could be a green alternative to replacing the current expensive and toxic precious metal systems most commonly used for these processes. Therefore, the yields from direct Suzuki–Miyaura C–C coupling of phenylboronic acid and pyrrole to produce 2-phenylpyrrole facilitated by eight high-spin iron complexes ([Fe3+L1(Cl)2]+, [Fe3+L4(Cl)2]+, [Fe2+L5(Cl)]+, [Fe2+L6(Cl)2], [Fe3+L7(Cl)2]+, [Fe3+L8(Cl)2]+, [Fe2+L9(Cl)]+, and [Fe2+L10(Cl)]+) were compared to identify the effect of structural and electronic properties on catalytic efficiency. Specifically, catalyst complexes were compared to evaluate the effect of five properties on catalyst reaction yields: (1) the coordination requirements of the catalyst, (2) redox half-potential of each complex, (3) topological constraint/rigidity, (4) N atom modification(s) increasing oxidative stability of the complex, and (5) geometric parameters. The need for two labile cis-coordination sites was confirmed based on a 42% decrease in catalytic reaction yield observed when complexes containing pentadentate ligands were used in place of complexes with tetradentate ligands. A strong correlation between iron(III/II) redox potential and catalytic reaction yields was also observed, with [Fe2+L6(Cl)2] providing the highest yield (81%, −405 mV). A Lorentzian fitting of redox potential versus yields predicts that these catalysts can undergo more fine-tuning to further increase yields. Interestingly, the remaining properties explored did not show a direct, strong relationship to catalytic reaction yields. Altogether, these results show that modifications to the ligand scaffold using fundamental concepts of inorganic coordination chemistry can be used to control the catalytic activity of macrocyclic iron complexes by controlling redox chemistry of the iron center. Furthermore, the data provide direction for the design of improved catalysts for this reaction and strategies to understand the impact of a ligand scaffold on catalytic activity of other reactions

Risk for Clostridium difficile Infection after Radical Cystectomy for Bladder Cancer: Analysis of a Contemporary Series

Introduction This study seeks to evaluate the incidence and associated risk factors of Clostridium difficile infection (CDI) in patients undergoing radical cystectomy (RC) for bladder cancer. Methods We retrospectively reviewed a single institution׳s bladder cancer database including all patients who underwent RC between 2010 and 2013. CDI was diagnosed by detection of Clostridium difficile toxin B gene using polymerase chain reaction–based stool assay in patients with clinically significant diarrhea within 90 days of the index operation. A multivariable logistic regression model was used to identify demographics and perioperative factors associated with developing CDI. Results Of the 552 patients who underwent RC, postoperative CDI occurred in 49 patients (8.8%) with a median time to diagnosis after RC of 7 days (interquartile range: 5–19). Of the 122 readmissions for postoperative complications, 10% (n = 12) were related to CDI; 2 patients died of sepsis directly related to severe CDI. On multivariate logistic regression, the use of chronic antacid therapy (odds ratio = 1.9, 95% CI: 1.02–3.68, P = 0.04) and antibiotic exposure greater than 7 days (odds ratio = 2.2, 95% CI: 1.11–4.44, P = 0.02) were independently associated with developing CDI. The use of preoperative antibiotics for positive findings on urine culture within 30 days before surgery was not statistically significantly associated with development of CDI (P = 0.06). Conclusions The development of CDI occurs in 8.8% of patients undergoing RC. Our study demonstrates that use of chronic antacid therapy and long duration of antimicrobial exposure are associated with development of CDI. Efforts focusing on minimizing antibiotic exposure in patients undergoing RC are needed, and perioperative antimicrobial prophylaxis guidelines should be followed