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Keywords: electromagnetic ball valve, cylindrical seat, functional diagram, modeling, MATLAB,Simscape/SimHydraulics.
Abstract. The main objective of this work is to perform numerical analysis of an electromagneticnormally-closed direct-acting ball valve with cylindrical seat. A functional diagram is developedusing the MATLAB/Simscape/SimHydraulics programming language. The principal elements ofthe functional diagram are: the fixed-displacement hydraulic pump, the ball valve which is drivenby an electromagnetic actuator, the pressure-relief valve, pressure and volumetric flow ratemeasuring devices and the hydraulic pipes. Two sets of analyses are performed, the first one for anideal case, without the effects that are associated with the hydraulic pipes and the second one for thereal case, with these effects. The valve flow factor is presented, comparatively, for both cases, aswell as the volumetric flow rates through the ball valve and the pressure relief valve, and finally thepressure drop in the valve.
Introduction The electromagnetic valves are wide used as control devices by switching on or off the fluidflow in hydraulic systems. There are many classification criterions of electromagnetic valves, suchas: the variable orifice geometry (ball with cylindrical seat, ball with conical seat, needle andpoppet); the valve type according to the de-energized state (normally-closed valve NC, normally-open valve NO); the acting type (direct-acting valve, internally piloted valve). Recent developmentsare focused on optimization models of the valves [1], as well as on the cavitations flow through thevalve [2]. All the relevant electromagnetic ball valve specific data (including the valve flow factor,Kv ) can be found on the product data sheet. However, there is a strong correlation between theelectromagnetic actuator characteristics, the ball valve parameters and the global parameters of thehydraulic system. In this paper a functional diagram for modeling an electromagnetic ball valvewith cylindrical seat will be presented. The simulation will be performed with and without thehydraulic system effects in order to observe the influence of these effects on the ball valvefunctional characteristics. The most important effects associated with the hydraulic circuit are: thefluid inertia, the fluid compressibility, the viscoelastic properties relates with the pipe internaldiameter to pressure variations, and the pressure losses caused by the linear and local resistances.
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TransTech Publications, www.ttp.net. (ID: 220.127.116.11, Purdue University Libraries, West Lafayette, USA-30/05/15,06:04:47) Applied Mechanics and Materials Vol. 657 625
Valv e Open
Electromagnetic Actuator
Q Qbv S A B I B E A Discharge Pipe Flow Rate Sensor BV Ball Valve
Differential Manometer A pA p pB pA-pB dp pA Results Manometer
A Motor Q Qprv P A B I E S S Return Pipe T By-pass Pipe Flow Rate Sensor PRV
B Fixed-Displacement Pump
Pressure Relief f(x)=0 Valve
B Suction Pipe SolverConfiguration A
Hydraulic Fluid R V
Fig. 1. Functional diagram for modeling the electromagnetic ball valve with cylindrical seat.
The hydraulic power unit is composed by the fixed displacement pump, the tank and the pressurerelief valve. The parameters of the hydraulic pump are: the fixed displacement D = 510-6 m3/rad,the volumetric efficiency v =0.92, the total efficiency t =0.8, the nominal pressure pn =100 bar, thenominal speed nn =1500 rpm, the nominal kinematic viscosity n =18 cSt. The inlet section of thehydraulic pump is connected with the tank through the suction pipe with the pipe diameter ds =10mm, the pipe length Ls =1 m, the aggregate equivalent length of local resistances Lse =0.1 m. The fixed displacement pump is driven by an ideal motor that will generate constant velocityregardless of the torque exerted on the system. The functional diagram of the ideal motor ispresented in Fig. 2. The motor speed will start from zero and will rise to 1500 rpm at 0.3 s, havingthree characteristic intermediate values: 500 rpm at 0.1 s, 1000 rpm at 0.15 s and 1400 rpm at 0.2 s.The speed measuring unit is set to [rpm] using the Simulink-PS Converter n. The motor speedsignal is generated using the Signal Builder block and the pump shaft speed is simulated using theIdeal Angular Velocity Source functional element. The pressure-relief valve will protect the hydraulic elements when the pressure exceeds thedesign limit allowing the fluid to be diverted through the by-pass pine back to the tank. Theprincipal parameters of the pressure relief valve are: the relief valve pressure setting is 90 bar, therelief valve regulation range is 5 bar, and the maximum passage area is equivalent with a 5 mmdiameter orifice. When the relief valve pressure setting is exceeded, the relief valve will open andtherefore the fluid will be discharged to the tank through the by-pass pipe with the pipe diameterdb =10 mm, the pipe length Lb =1 m, the aggregate equivalent length of local resistances Lbe =0.1 m. In order to measure the fluid flow rate diverted through the by-pass pipe, a fluid flow rate sensoridentified by the name Flow Rate Sensor PRV is installed on that pipe. The functional diagram ofthe flow rate sensor is presented in Fig. 3. The measuring unit of the fluid flow rate, which is set inthis case to [m3/h], is controlled using the PS-Simulink Converter Q. When the pressure inside the hydraulic system is smaller than the set pressure of the relief valve,the pressure relief valve will close and the fluid discharged by the pump will flow through the mainhydraulic circuit composed by: the discharge pipe, the ball valve and after that, back to the tank626 Engineering Solutions and Technologies in Manufacturing
through the return pipe. The principal parameters of the discharge pipe are: the pipe diameter dd =10mm, the pipe length Ld =5 m, the aggregate equivalent length of local resistances Lde =0.1 m. Theprincipal parameters of the return pipe are: the pipe diameter dr =10 mm, the pipe length Lr =5 m, theaggregate equivalent length of local resistances Lre =0.1 m. The second fluid flow rate sensoridentified by the name Flow Rate Sensor BV is installed on the discharge pipe. The functionaldiagram of this second flow rate sensor is identical with that one presented in Fig. 3, including themeasuring unit which is set also to [m3/h]. Ideal Angular Velocity Source PS S 1 Group 1 Signal 1 S PS S Q R 1 PS-Simulink Simulink-PS C Converter Q Signal Builder Q Converter n S 1 A B 2 I Hydraulic Flow Rate E MRR Sensor Fig. 2. Functional diagram for modeling the pump motor. Fig. 3. Functional diagram for modeling the Flow Rate Sensor PRV.
As for the ball valve, the main characteristic parameters are: the valve ball diameter=6 mm, thecylindrical seat diameter=5 mm, the initial opening=0.01 mm and the leakage area=10-12 m2. Theball valve opening is defined using the Electromagnetic Actuator subsystem presented in Fig. 4.The Pulse Generator block will generate a DC electric signal having the amplitude of 24 V, theperiod of 8 s and the pulse width of 50% of signal period. This signal applied on the 2-PositionValve Actuator, will generate the valve opening signal. The main characteristic of the actuator are:the push-pin stroke=3 mm (and, as consequence, the ball valve travel will be 3 mm as well), theswitching-on time=1 s, the switching-off time=1 s, the nominal signal value=24 V. U x S PS
PS-Simulink Converter S PS 1 Valve Open Pulse Simulink-PS 2-Position Valve Generator Converter Actuator
Measuring the discharge pressure and the pressure drop in the ball valve is performed using onemanometer and one differential manometer, which are presented in Fig. 5. Both manometers havethe measuring unit set to [bar] by means of the PS-Simulink Converter p. 1 P PS S 1 1 A pB B pA A PS-Simulink B Hydraulic Pressure Converter p 2 A P PS S 1 Sensor pA Hydraulic Reference Hydraulic Pressure pA-pB PS-Simulink Sensor Converter p
Numerical Results There are performed two sets of analyses, the first one for an ideal case, without the effects thatare associated with the hydraulic circuit and the second one for the real case, with these effects. Oneof the most important characteristic parameter of the ball valve is the flow factor, Kv :
Kv =Q 1000p , (1)
where Q [m3/h] is the volumetric flow rate, [kg/m3] is the fluid density and p [bar] is thepressure drop in the ball valve. Applied Mechanics and Materials Vol. 657 627
In Fig. 6 is presented the dimensionless valve flow factor Kv Kvmax with respect to thedimensionless ball valve travel xxmax 100 [%], where 0% travel coresspond to the valve full closeand 100% travel coresspond to the valve full open and Kvmax and xmax are the maximum values ofthe valve flow factor and ball valve travel. The first diagram, Fig. 6.a, has been obtained for amaximum ball valve travel xmax =3 mm. It can be observed that even at xxmax 60%, the valveflow factor reaches its maximum value, which leads to the conclusion that the ball valve travel isoversized. Thats why the maximum ball valve travel has been reduced from xmax =3 mm toxmax =1.8 mm, Fig. 6.b. 1 1 0.75 0.75 Kv/Kvmax
Kv/Kvmax 0.5 0.5 0.25 0.25 0 0 0 25 50 75 100 0 25 50 75 100 x/xmax [%] x/xmax [%] a) travel 3 mm b) travel 1.8 mm Fig. 6. Dimensionless valve flow factor (ideal case).
x [mm] U [V]
ideal 3
overshoots. In Fig. 11 the influence of the hydraulic system 2effects on the valve flow factor can be observed.The case presented in Fig. 11,a, correspond to theinitial case with ball valve travel xmax =3 mm. 0 0 2 4 6 8Reducing the ball valve travel to xmax =1.8 mm t [s]will improve the ball valve behavior, Fig. 11,b; but Fig. 9. Volumetric flow rate through thestill can be observed a relevant difference with pressure relief valve.respect to the ideal case, Fig. 6,b.628 Engineering Solutions and Technologies in Manufacturing
p [bar] ideal On the other hand, if the hydraulic system 50effects are considered, as shown in Fig. 11, ahysteresis-like phenomenon can be observedrelated to the valve flow factor, because there are 0two curves, one for opening travel time and the 0 2 4 6 8other for the closing travel time. t [s] Fig. 10. Pressure drop in the ball valve. 1 1 opening opening 0.75 0.75 Kv/Kvmax
Kv/Kvmax 0.5 0.5 closing closing 0.25 0.25 0 0 0 25 50 75 100 0 25 50 75 100 x/xmax [%] x/xmax [%] a) travel 3 mm b) travel 1.8 mm Fig. 11. Dimensionless valve flow factor (real case).
Conclusions The valve flow factor diagram obtained for the ideal case corresponds to a valve with quickopening travel time, Fig. 6. However, when the same valve is analyzed on a real hydraulic circuit itcan be observed a considerable delay, approximately 2530%, in the volumetric fluid flow risingprocess, Fig. 11.b. The hysteresis-like phenomenon obtained on the valve flow factor analysis isrelated strictly with the hydraulic circuit and implies further investigations. Considering thehydraulic system effects, has the tendency to move the valve flow factor curve away from the idealshape, toward a square root characteristic. The oversizing of the ball valve travel time can be easily detected using the valve flow factorcharacteristic, with or without the piping effects, Fig. 11 and Fig. 6. The under-damped response has been observed on two critical points: at the end of valve traveltime on the opening phase for the volumetric flow rate through the valve, Fig. 8; as well as on theend of valve closing travel time, for the pressure drop in the valve, Fig. 10, and for the volumetricflow rate through the pressure relief valve, Fig. 9.
References[1] W. Chen, The Optimal Design of Electromagnetic Ball Valve with MATLAB OptimizationToolbox, Advanced Material Research. 510 (2012) 277-282.[2] J.R. Valdes, J.M. Rodriguez, R. Monge, J.C. Pena, T. Putz, Numerical Simulation andExperimental Validation of the Cavitating Flow Through a Ball Check Valve, Energy Conversionand Management. 78, (2014) 776-786.[3] D. Zahariea, Simulation of Physical Systems in MATLAB, (in Romanian), PIM PublishingHouse, Iai, 2010.[4] Mathworks, Simscape Documentation, available at: http://www.mathworks.com/help/physmod/simscape/index.html, accesed 18.01.2014.[5] Mathworks, SimHydraulics Documentation, available at: http://www.mathworks.com/help/physmod/hydro/index.html, accesed 18.01.2014.Engineering Solutions and Technologies in Manufacturing10.4028/www.scientific.net/AMM.657
Functional Diagram for Modeling the Electromagnetic Ball Valve with Cylindrical Seat10.4028/www.scientific.net/AMM.657.624
DOI References[1] W. Chen, The Optimal Design of Electromagnetic Ball Valve with MATLAB Optimization Toolbox,Advanced Material Research. 510 (2012) 277-282.http://dx.doi.org/10.4028/www.scientific.net/AMR.510.277


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THE WOODLANDS, Texas, June 27, 2019 /PRNewswire/ -- Summit Midstream Partners, LP (NYSE: SMLP) announced today a final investment decision ("FID") to proceed with the Double E Pipeline Project ("Double E" or the "Project"), having secured sufficient commitments pursuant to binding precedent agreements for long-term, firm transportation service and having executed definitive joint venture agreements with an affiliate of Double E's Foundation Shipper (the "JV Partner"). Concurrent with the FID, Double E's Foundation Shipper amended its previously executed precedent agreement. Together with firm commitments from other shippers, Double E has secured 10-year take-or-pay volume commitments for a substantial majority of its initial throughput capacity of 1.35 billion cubic feet per day ("Bcf/d"). Throughput capacity on Double E could be expanded at a later date to 1.85 Bcf/d with the installation of compression facilities. Commercial discussions with additional potential shippers are ongoing and SMLP will look for opportunities to subscribe the limited remaining capacity prior to commissioning.
Double E will provide natural gas transportation service from multiple receipt points in the Delaware Basin to various delivery points in and around the Waha Hub in Texas. SMLP will own a majority of the Project, will lead the development, permitting and construction of the Project, and will operate Double E upon commissioning. SMLP estimates that its share of the capital expenditures required to develop Double E will total approximately $350 million, and that more than 90% of those capital expenditures will be incurred in 2020 and 2021.
SMLP intends to fund its share of the capital expenditures for Double E with a combination of internally generated cash flow and proceeds from potential non-core asset divestitures. In addition, given the take-or-pay volume commitments underpinning the Project, SMLP and its lenders have amended SMLP's revolving credit facility to provide additional flexibility with respect to its financial performance metrics during the construction of the Project. SMLP will continue to evaluate opportunities to further optimize its financing plan for Double E as it continues to develop the Project.
"We are excited to announce our FID on the Double E Pipeline Project, which will provide critical energy infrastructure for rapidly growing residue natural gas volumes in the Northern Delaware Basin," said Leonard Mallett, interim President and Chief Executive Officer of SMLP. "The Double E Project will serve an infrastructure-constrained area, alleviate natural gas flaring, and provide access to growing markets along the U.S. Gulf Coast. Double E is a marquee project for SMLP, and when combined with our existing gathering and processing operations in the Permian Basin, promotes our strategy of achieving scale, diversifying our operations downstream of wellhead gathering, and becoming more integrated in our Core Focus Areas."
Double E expects to file its section 7(c) application with the Federal Energy Regulatory Commission in the third quarter of 2019, and estimates a third quarter of 2021 in-service date, assuming timely receipt of the requisite regulatory approvals.
About Summit Midstream Partners, LPSMLP is a growth-oriented limited partnership focused on developing, owning and operating midstream energy infrastructure assets that are strategically located in the core producing areas of unconventional resource basins, primarily shale formations, in the continental United States. SMLP provides natural gas, crude oil and produced water gathering services pursuant to primarily long-term and fee-based gathering and processing agreements with customers and counterparties in six unconventional resource basins: (i) the Appalachian Basin, which includes the Utica and Marcellus shale formations in Ohio and West Virginia; (ii) the Williston Basin, which includes the Bakken and Three Forks shale formations in North Dakota; (iii) the Denver-Julesburg Basin, which includes the Niobrara and Codell shale formations in Colorado and Wyoming; (iv) the Permian Basin, which includes the Bone Spring and Wolfcamp formations in New Mexico; (v) the Fort Worth Basin, which includes the Barnett Shale formation in Texas; and (vi) the Piceance Basin, which includes the Mesaverde formation as well as the Mancos and Niobrara shale formations in Colorado and Utah. SMLP also owns an ownership interest in Ohio Gathering, which owns and operates extensive natural gas gathering and condensate stabilization infrastructure in the Utica Shale in Ohio. SMLP is headquartered in The Woodlands, Texas.
Forward-Looking StatementsThis press release includes certain statements concerning expectations for the future that are forward-looking within the meaning of the federal securities laws. Forward-looking statements contain known and unknown risks and uncertainties (many of which are difficult to predict and beyond management's control) that may cause SMLP's actual results in future periods to differ materially from anticipated or projected results. An extensive list of specific material risks and uncertainties affecting SMLP is contained in its 2018 Annual Report on Form 10-K filed with the Securities and Exchange Commission on February 26, 2019, and as amended and updated from time to time. Any forward-looking statements in this press release, including forward-looking statements regarding 2019 financial guidance or financial or operating expectations for 2019, are made as of the date of this press release and SMLP undertakes no obligation to update or revise any forward-looking statements to reflect new information or events.
View original content to download multimedia:http://www.prnewswire.com/news-releases/summit-midstream-partners-lp-makes-final-investment-decision-on-double-e-pipeline-project--secures-joint-venture-partner-300875854.html
SOURCE Summit Midstream Partners, LP
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Tell Democratic 2020 hopefuls: Lead now. No wall. No boots. No beds.
Donald Trump's two-year tantrum over a border wall is about to cost the country another government shutdown.1
Any Democrat even entertaining the thought of running to unseat Trump in 2020 should be leading the fight to block every part of his white supremacist wish list, especially the wall.
Now is the time for every Democratic 2020 hopeful to prove to the American electorate that they have what it takes to stand up to Trump's hate; lead with moral clarity; and stop attacks on immigrants, refugees and all communities that Trump and his collaborators are terrorizing.
Tell Democratic 2020 hopefuls: Lead now. No wall. No boots. No beds.
The list of 2020 presidential hopefuls includes many of our progressive champions and rising Democratic stars.2 From Sens. Sanders and Harris to Reps. O'Rourke and Gabbard, the list of party leaders hoping to replace Trump is growing. But to take down Trump and heal the hurt his administration is inflicting on millions of Americans, we need bold leadership. We need a leader who refuses to compromise with white supremacists. A leader who puts people's health and wellbeing over profits. A leader who stands up for children, people experiencing poverty, the sick and elderly. A leader who protects our democracy and the right to vote. And a leader who will fight with urgency to make the promise of freedom and justice for all real for every person in the country.
Yesterday, Trump met with Sen. Schumer and Rep. Pelosi to demand $5 billion for his border wall and funding to expand his rogue deportation force, add more beds to detention facilities and build new prisons.3 These aren't just line items in a budget, they are funding requests to supercharge state-sanctioned violence against immigrants at the border and in communities across the country. Our advocacy worked to make sure Leaders Schumer and Pelosi rejected Trump's funding proposal, but their milquetoast statements after the meeting showed that they are still playing it safe. With their record of caving to Trump's anti-immigrant agenda, this is a cause for concern.
After meeting with Trump, Sen. Schumer told reporters that since Trump is happy with what's happening at the border, he should also be happy with a bill that keeps DHS running with its current level of funding.4 This was an opportunity for Sen. Schumer to set the national narrative and frame this fight as a moral crisis. He failed to do that, and it's not the first time.
We need new leaders who speak truth to power and take bold action to stop Trump and Republicans from institutionalizing hate. The future of our country depends on it.
Tell Democratic 2020 hopefuls: Lead now. No wall. No boots. No beds.
Democratic voters just elected the youngest, most progressive and diverse group of congresspeople in history.5 They also ended Republican control of many state legislatures. The most effective and groundbreaking campaigns were the ones of candidates like Rep. Alexandria Ocasio-Cortez who championed progressive issues like Medicare for All and publicly condemned Trump's racism and hate.
If Democrats who are planning to run for president want to win, they need to pay attention to what is activating the base. They must lead with a bold progressive vision and confront Trump's white supremacy head on.
DHS will run out of funding on December 21. This budget fight will be a testing ground for any Democrat who is planning to run for president. Speak out now to demand that they step up and lead.
Thanks for your activism.
- Kathryn Watson, "'I am proud to shut down the government for border security,' Trump says in Oval Office "tantrum" with Schumer, Pelosi," CBS, Dec. 11, 2018.
- Colby Itkowitz, "An 'unprecedented' number of Democrats in Congress want to be president," The Washington Post, Dec. 7, 2018.
- Jacob Pramuk, "Trump, Pelosi and Schumer get into a heated Oval Office fight over border wall and shutdown," CNBC, Dec. 11, 2018.
- Watson, "'I am proud to shut down the government for border security,' Trump says in Oval Office "tantrum" with Schumer, Pelosi."
- Katie Edmondson and Jasmine Lee, "Meet the New Freshmen in Congress:More Democrats, Diversity and Women," The New York Times, Nov. 28, 2018.


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The Devil Wears Prada - Vengeance album FLAC
- Performer: The Devil Wears Prada
- Title: Vengeance
- Genre: Rock
- Formats: MOD VQF MP1 MPC AHX MP4 APE
- Released: 2011
- Style: Hardcore
- MP3 album: 1452 mb
- FLAC album: 1473 mb
- Rating: 4.6/5
- Votes: 216
The Devil Wears Prada discography, an American Christian melodic metalcore band from Dayton, Ohio, consists of six studio albums, one demo, two extended plays, seventeen singles and ten music videos. Punk Goes Crunk – "Still Fly" (Big Tymers cover; 2008). Warped Tour 2008 – "Reptar King of the Ozone" (2008). Warped Tour 2009 – "Sassafras" (2009). Warped Tour 2011 – "Anatomy" (2011). Warped Tour 2014 – "Sailor's Prayer" (2014). Patterns of a Horizon (The Foundation Recording Studios, 2005).
The Devil Wears Prada. Featuring Jeremy McKinnon. You are a sickness, my only sincere plague Is this what you've worked for? Running your false empire to the ground What is this substance that made your very own morals change? It's gone a little too far to turn back now, turn back now It's gone a little too far to turn back Always running I am the outcast Keep running, keep running away. Vengeance" Track Info. Release Date September 13, 2011. Dead Throne The Devil Wears Prada. Follow The Devil Wears Prada and others on SoundCloud. The Devil Wears Prada.
The Devil Wears Prada - Vengeance Tab. Can't play "Vengeance"? Improve your playing via easy step-by-step video lessons! Standard guitar tuning . there is no tab for this song so i gave it a listen. iv basically tabbed out the rhythm guitar. picked up the lead riff in the chorus). 0:00-0:16) this strumming pattern is weird. the guitars switch turns on playing (this repeats till next riff). American Christian metalcore band from Dayton, Ohio, formed in 2005. It consists of members Mike Hranica, Jeremy DePoyster, Kyle Sipress, and Andy Trick. The band had maintained its original lineup until keyboardist James Baney left the band. To date, the group has released six full-length albums: Dear Love: A Beautiful Discord, Plagues, With Roots Above and Branches Below, Dead Throne, 8:18 and Transit Blues as well as two EPs, one DVD, sixteen singles, and thirteen music videos. The Devil Wears Prada - Sour Breath (03:08, 320Kb/s)
The Devil Wears Prada. The Devil Wears Prada – Vengeance. lol This song and the rest of the album is amazing, maybe as good as Plagues.
THE DEVIL WEARS PRADA: 'Vengeance' Video Released. 23 фев 2012 : THE DEVIL WEARS PRADA Parts Ways With Keyboardist. THE DEVIL WEARS PRADA: Footage From New Album Writing Sessions. com receive instant free MP3 downloads of two new tracks, "Home For Grave" and "Martyrs".
The Devil Wears Prada: Vengeance Meaning.


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The densely populated Campi Flegrei resurgent caldera is one of the widest known, best studied and highly dangerous volcanoes of the world. Campi Flegrei will synthesize the current knowledge of this volcano, through different review chapters. Campi Flegrei consists of about 15 chapters, each on a specific aspect, authored by well-recognized experts. Campi Flegrei attempts to cross the barriers between the volcanological, geological, geochemical and geophysical perspectives, with the aim of offering a comprehensive and up-to-date reference to earth-science scholars, as well as land planners and civil defence officers.
The articles will cover the following general topics:
- Geological and geophysical structure of the caldera
- Magmatic and hydrothermal systems: architecture and dynamics
- Caldera current state, monitoring and modelling
- Volcanic and related hazards assessment
- Urban inhabitation, present urbanization and geology
- Introduction
- Geological and structural setting and evolution
- Geophysical structure
- Architecture and evolution of the magmatic feeding system I
- Architecture and evolution of the magmatic feeding system II
- Geochemistry of distal tephra and its implications on tephra chronology
- Rheological properties of magmas and their influence on mixing processes and eruption dynamics
- Magma chamber dynamics
- Geochemistry of the hydrothermal system
- Current degassing and hydrothermal dynamics
- Current seismicity
- Ground deformation and source modelling
- Reconstruction of the last eruption from chronicles
- Slope instability in pyroclastic terrains
- Millenary human inhabitation and present urbanization
- Campi Flegrei volcanic hazards assessment


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Famille de produits
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Christian Michael L'Abbe
A slightly adenoidal pubescent voice sans voice cracks with a slight western accent ( I can speak fluently without it) I am able throw my voice in a few different ways ranging from gravelly to silvery and honeyed however when it comes to accents I can do about nine but my best ones are German and russian
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Runs voice acting YouTube channel doing comic read overs from the game Overwatch (Website hidden)
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Garmin wired gaming headset (Using software to polish audio)
Computer & Software
Acer Windows 7 Home Premium OA/WavePad Audio Recorder/Editor
Special Equipment
Delivery Methods
.Wav MP3
Turnaround Time
5-8 days
Service Description
I am very flexible and dedicated towards the project able to take on multiple roles so i am capable of pull double duty if you are short staffed however the roles must be on the lighter to medium side dialogue wise as I am often taken with external affairs and if you wish to accept me into a larger role the end date must be farther away than average
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Arsenal have approached Paulo Sousa about their current managerial vacancy (Picture: Getty) Arsenal have made contact with the representatives of Bordeaux manager Paulo Sousa as the north London club ... [Read More]
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'Love him', 'Prem ain't ready', some Arsenal fans are delighted with 24-year-old's display after West Ham win
A section of the Arsenal faithful have heaped praise on Nicolas Pepe, who bagged his first club goal from open play against West Ham United on Monday night. The Ivorian has been in-and-out of the star... [Read More]
Ian Wright makes Gabriel Martinelli prediction as 'brilliant' Arsenal star posts delight on Instagram
Gabriel Martinelli has posted on Instagram after scoring his first Premier League goal for Arsenal on Monday night as the Gunners came from behind to beat West Ham – and Ian Wright has commented... [Read More]
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Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galβ-GlcNAc-Galβ-Glcβ-Hep2 (GlcNAc, Glcα) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A; 1,4′ bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.
Neisseria meningitidis is a frequent inhabitant of the upper respiratory tract of humans and is a major cause of meningitis and fulminant septicemia in otherwise healthy individuals. Among the important virulence factors involved in meningococcal pathogenesis, endotoxin, or lipooligosaccharide (LOS), is a major component inducing proinflammatory responses in meningococcal sepsis and meningitis (15, 36). Furthermore, the morbidity and mortality of meningococcal sepsis are directly correlated with levels of circulating meningococcal LOS (3, 4).
Meningococcal LOS interacts with human cells, resulting in the production of proinflammatory cytokines and chemokines, including interleukin 1 (IL-1), IL-6, and tumor necrosis factor (TNF), that are important in the pathogenesis of meningococcal disease (3, 42). While pili and Opa and Opc outer membrane proteins are also critical, LOS is one of the structures important in mediating meningococcal attachment to (23, 24) and invasion into (30) epithelial cells. The role of LOS in these events is further substantiated by the findings that LOS-deficient meningococcal mutants show impaired adherence (2) and reduced induction of serum cytokines (35) compared to the wild-type strain.
The structure of N. meningitidis LOS has been characterized both immunologically (33) and biochemically (6, 8, 19, 20, 40). Meningococcal LOS lacks the repeating O antigens of enteric lipopolysaccharide but maintains a conserved inner core composed of heptose and 3-deoxy-d-manno-2-octulosonic acid bound to lipid A, to which variable α and β chain saccharides are attached (15). Lipid A is the active moiety through its ability to upregulate the inflammatory response. Changes in the configuration or the conformational structure of lipid A affect the biological response (27). Variation in the composition of meningococcal LOS is also postulated to mediate changes in host immune responses and bacterial virulence (28, 37).
In order to further define the role of LOS in meningococcal pathogenesis, each of a series of genetically and structurally defined LOS mutants of the serogroup B meningococcal strain NMB was examined for its capacity to adhere to and invade epithelial cells in comparison to the wild-type parent strain. Also, the pathogenicities of the parent and of each of the mutants were examined by use of a mouse model of meningococcal disease (9), and survival in the blood of these mice was assessed. Our results show that structural variations of LOS affect attachment and invasion of epithelial cells, access to and survival in the bloodstream, and the subsequent innate immune response and morbidity in vivo.
Bacterial strains.Neisseria meningitidis serogroup B strain NMB (encapsulated; L2/4 immunotype) and the genetically defined mutants of this strain (pgm, rfaK, and gmhB mutants) are shown in Fig. 1 and described in references 12, 15, 25, 29, 34, and 41. The meningococcal strain NMB and the pgm, rfaK, and gmhB mutants were grown on GC agar with Kellogg's supplement (16) at 37°C in 5% CO2. The antibiotics for LOS mutants were used at the following concentrations: for the pgm and gmhB mutants, tetracycline at 5 μg/ml; and for the rfaK mutant, spectinomycin at 60 μg/ml. The M7 strain used in the whole-cell enzyme-linked immunosorbent assay (ELISA) is an unencapsulated mutant (NMB synA::Tn916 mutant) (32).
Cells.Detroit 562 human pharyngeal cells (ATCC CCL 138) were maintained in modified Eagle medium (Sigma) supplemented with 10% inactivated fetal bovine serum (Sigma), 2 mM l-glutamine (Sigma), 1 mM sodium pyruvate (Gibco), and 1× nonessential amino acids (Gibco) at 37°C in 5% CO2.
Electron microscopy.Bacteria were resuspended to an optical density at 600 nm (OD600) of 0.3 in Tris-Mg buffer (10 mM Tris-HCl [pH 7.4] and 10 mM MgCl2) and mounted on copper grids. The grids were stained with 1% ammonium molybdate and analyzed with a JEOL 1230 electron microscope.
Western blotting.Bacterial protein extracts (750 ng) were separated by 10% (PilC expression) and 15% (Opa expression) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by standard methods and transferred to polyvinylidene difluoride membranes. The membranes were blocked overnight with 5% nonfat dried milk in Tris-buffered saline (TBS; 0.05 M Tris and 2 M NaCl, pH 7.4) containing 0.05% Tween 20. PilC was identified by incubation with the K3 antibody that recognizes both PilC1 and PilC2 (polyclonal rabbit antibody diluted 1:5,000). The Opa proteins were detected with the 4B12/C11 antibody, which recognizes all Opa proteins (1) (monoclonal mouse antibody diluted 1:5,000). Incubation with primary antibodies was followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad; diluted 1:10,000) or horseradish peroxidase-conjugated goat anti-mouse antibody (Santa Cruz Biotechnologies; diluted 1:10,000). A chemiluminescence kit from Perkin Elmer Life Sciences was used for detection.
Examination of LOS expression.Tricine SDS-PAGE was used to examine the LOS expression of all bacterial mutants. Whole-cell lysates of bacterial strains were prepared by incubation of bacterial suspensions (8 mg) with proteinase K (2.5 μg/ml), Tris-HCl (50 mM), CaCl2 (1 mM), and glycerol (2%) at 60°C for 18 h. Following digestion, loading buffer (1 M Tris-HCl, 10% glycerol, 2% SDS, 0.5% 2-β-mercaptoethanol, and 0.0125% bromophenol blue) was added, 200 ng of sample was separated on a 16.5% gel, and LOS was detected by silver staining (39).
Capsule quantification by whole-cell ELISA.The expression of capsule by various mutants used in this study was quantified by a whole-cell ELISA method as described previously (31). Briefly, cells were harvested from overnight growth on GC plates and suspended in phosphate-buffered saline. A 50-μl aliquot of a 1:3 dilution of cell suspensions at an OD650 of 0.1 was applied and dried at 37°C overnight. Antibodies specific for serogroup B (2-2-B) and serogroup A (14-1-A) were used at 1:2,000 and 1:30,000 dilutions, respectively. The M7 strain does not express capsule due to the synA mutation and was used as a negative control for the whole-cell ELISA.
Adhesion and invasion assay.Detroit 562 cells were grown in 24-well tissue culture plates to full confluence. Suspensions of meningococci were added to triplicate wells at a multiplicity of infection of 100. Infected cells were incubated at 37°C in 5% CO2 for 2 h and washed five times with media to remove unbound bacteria. Adhered bacteria were quantified after 2 h by lysing the cells for 5 min with 1% saponin and serially diluting the samples and spreading them on GC agar plates. The number of invaded bacteria was determined after 3, 4, 5, and 6 h by incubating cells in medium containing 100 μg/ml gentamicin for 1 h to kill extracellular bacteria before the saponin treatment. Both assays were conducted in at least three independent experiments, and data were normalized to results for the original inocula. The sensitivity of all bacterial strains to gentamicin was determined in cell media by recovery of viable bacteria following a 1-h incubation in gentamicin at 0, 0.1, 1, 10, and 100 μg/ml.
Growth curve.To monitor the growth of bacteria in cell culture medium, 100-μl suspensions of bacteria (OD600 = 0.1) were inoculated into 2 ml of cell culture medium and incubated at 37°C in 5% CO2. The optical density at 600 nm was measured at various time points after inoculation.
Animal infection assay.A mouse model of meningococcal infection (9, 10) was used to examine the pathogenicity of the LOS mutants. Briefly, 5- to 8-week-old CD46 transgenic mice (n = 17) were infected intraperitoneally (i.p.) with 3 × 108 bacteria in two independent experiments. Blood samples were taken from the tail 1, 4, 8, and 24 h after infection and spread on GC agar plates to determine the bacterial load in blood. At 4, 8, and 24 h postinfection, blood smears were made for analysis of neutrophil infiltration. Serum was collected 24 h postchallenge for analysis of proinflammatory cytokine levels in blood. The cisterna magna was punctured 24 h postinfection, and the cerebrospinal fluid (CSF) was checked for absence of red blood cells and spread on GC plates for a viable count.
Survival assay.Blood from CD46 transgenic mice was collected by retro-orbital bleeding and heparin treated. Whole blood was diluted 1:1 with bacterial suspension in Detroit cell media (1 × 107 bacteria) in a final volume of 300 μl. Bacterial survival was determined by serial dilutions and plating of the blood-bacteria mixture at 3, 6, 9, and 12 h postinfection. Survival was monitored in freshly isolated blood in two independent experiments.
Immunoassays for cytokines in serum.Concentrations of murine IL-6 and TNF were measured in the sera of N. meningitidis-challenged mice at 24 h postinjection by using sandwich ELISAs according to the manufacturer's recommendations (Diaclone).
Blood smears.Samples (approximately 5 μl) of blood were obtained from the tail vein of infected mice, and smears were made on glass slides. The slides were fixed with methanol and left to dry. The smear samples were stained with Wright's stain according to the manufacturer's recommendations (Sigma). Samples were analyzed by light microscopy.
Statistical analysis.Experiments were evaluated with Microsoft Excel. A two-tailed Student's t test was used to assess significance in the adhesion and invasion assays and in the cytokine assays. Lethality experiments were assessed using Fisher's exact test, and bacteremia and in vitro blood survival was monitored with a nonparametric Mann-Whitney test. All experiments were performed in sets of at least two independent experiments.
Expression of virulence factors by LOS mutants.N. meningitidis strain NMB and the LOS mutants have been previously described and are shown in Fig. 1 (12, 15, 25, 29, 34, 41). The mutant strains showed growth rates similar to that of the wild-type strain, with the rfaK and gmhB mutants having lower final bacterial numbers in the stationary phase (Fig. 2A); however, growth rates were not significantly different (P > 0.05). N. meningitidis NMB and each mutant expressed the SDS-PAGE LOS profiles expected (Fig. 2B) on the basis of their nuclear magnetic resonance-defined structures (Fig. 1), and Western blotting of whole-cell preparations revealed that the parent and the mutants expressed PilC (Fig. 2B). The wild-type strain expressed a two-band Opa repertoire and the pgm and rfaK mutants expressed a third Opa of higher molecular weight, while the gmhB mutant expressed one Opa in common with the parent and a unique Opa of higher molecular weight (Fig. 2B). All strains showed levels of capsule expression that were not significantly different (P < 0.05) from that of the wild-type strain, NMB (Fig. 2C), as determined by a whole-cell ELISA (34). Electron microscopy analysis showed that all strains were piliated (Fig. 2D).
Mutations in LOS change the capacity of meningococci to adhere to and invade epithelial cells.An assay to assess adhesion of the wild-type strain and LOS mutants to the epithelial cell line Detroit 562 revealed that mutations resulting in a truncated structure of LOS modulated the capacity of the meningococci to associate with these cells (Fig. 3A). The wild-type strain NMB showed a level of adherence to Detroit 562 cells significantly lower than those of the mutants (P < 0.05), with approximately five associated bacteria per cell at 2 h postinfection. Although the NMB strain appeared to express less PilC (Fig. 2B), it is unlikely that this difference alone mediated the decrease in adhesion, since the mutant strains expressed similar levels of PilC but also had different capacities to adhere. The pgm [Hep2(GlcNAc) 3-deoxy-d-manno-2-octulosonic acid (KDO2)-lipid A] and gmhB (KDO2-lipid A) mutants demonstrated that between 50 and 80 bacteria adhered to each cell, which was a level significantly enhanced (P < 0.05) compared to that seen for the rfaK mutant (Hep2-KDO2-lipid A), which adhered at levels of approximately 20 bacteria per cell. The percentage of associated meningococci that entered and survived within cells was also examined using gentamicin to kill extracellular meningococci. The wild-type strain showed a steady increase in bacterial counts with time, with significantly greater numbers of bacteria within cells at 3, 4, and 5 h (P < 0.05). The pgm and rfaK mutants showed decreased bacterial counts compared to the wild-type strain at the early time points (Fig. 3B), but at 6 h postinfection, the numbers of these mutants in epithelial cells were not different from that of the parent. The gmhB mutant was unable to enter and survive within epithelial cells. The differences in intracellular numbers of bacteria were not related to differences in sensitivity to gentamicin (data not shown).
LOS structure affects the pathogenicity of meningococci and mortality in a mouse model of infection.The pathogenicities of the wild-type strain and LOS mutants were further examined in a mouse model of meningococcal infection (9). Adult CD46 transgenic mice, which have increased susceptibility to meningococci in the absence of additional supplements, such as iron, were infected intraperitoneally with 3 × 108 meningococci/mouse. The wild-type strain and the rfaK mutant showed similar mortalities, with significant fatality noted for the wild-type strain at 4 days postinfection and for the rfaK mutant at 3 days postinfection (Fig. 4). The pgm mutant showed a mortality significantly lower than the that of the wild-type strain and that of the rfaK mutant. The gmhB mutant was avirulent in the mouse model.
LOS structure affects the levels of meningococci in the bloodstream.At 1 and 4 h postinfection, mice infected with the wild-type strain NMB and the rfaK mutant had bacterial blood counts (P < 0.05) higher than those for to the pgm and gmhB mutant-infected mice (Fig. 5). At 24 h postinfection, no significant difference in bacteremia levels in the pgm mutant- and rfaK mutant-infected mice was observed, but the mice infected with the wild-type strain still showed significantly enhanced bacteremia (P < 0.05). Hence, it appears that a correlation between the level of bacteremia at the early time points and mouse mortality exists. Meningococci were detected in the CSF of all mice infected with the wild-type strain NMB and in 12.5% of the mice infected with the pgm mutant and 25% of mice infected with the rfaK mutant (data not shown). No bacteria were detected in the CSF of mice infected with the gmhB mutant. These data suggest that LOS structure, including a requirement for inner core heptose, affects the level of bacteremia seen following infection and the ability of meningococci to enter into the CSF. It also suggests that the mortality associated with the rfaK mutant is caused by sepsis rather than meningitis, whereas the mortality associated with the wild-type strain is probably due to both sepsis and meningitis.
In vitro survival in whole mouse blood.In order to evaluate if the wild-type strain and the LOS mutants differed in their abilities to survive in blood, bacteria were incubated with heparin-treated mouse blood in vitro. As shown in Fig. 6, each of the mutations resulting in truncation of meningococcal LOS structure was associated with a decrease in the capacity of meningococci to survive in whole blood taken from CD46 mice. These data suggest that the wild-type LOS structure best promoted the survival of meningococci in blood and correlated with the persistent bacteremia of the wild-type strain in the mouse model. However, pathogenesis in the mouse model was not solely mediated by the capacity to survive in blood, since no significant differences were observed in the survival rates of the LOS mutants in the mouse blood in vitro (P > 0.05), in contrast to the differences observed in mouse mortality rates.
Mouse mortality is associated with high serum cytokine levels.Meningococcal sepsis-associated death is often due to hypotension and organ failure characteristic of septic shock, which occurs as a result of uncontrolled release of proinflammatory cytokines, such as IL-6 and TNF. Host cytokine responses were analyzed by measuring cytokine levels in mouse serum 24 h postinfection by ELISA. As shown in Fig. 7A, IL-6 increased significantly in serum after i.p. challenge with the wild-type strain NMB and the rfaK mutant. IL-6 was not elevated in sera of mice at 24 h after infection with the pgm mutant or the gmhB mutant. The wild-type strain, the rfaK mutant, and the gmhB mutant induced serum TNF (Fig. 7B). TNF was not induced in serum following i.p. infection of the pgm mutant. Cytokine levels did not correlate with a shift of neutrophil levels in blood samples following challenge (Fig. 7C), since all strains induced comparable influxes of neutrophils into the blood at 8 h postinfection. Interestingly, the gmhB mutant, which was avirulent in the mouse model, did induce TNF but did not induce IL-6 and also caused increased neutrophil infiltration after infection.
LOS, or endotoxin, including the ability to express a full repertoire of LOS structures, is an important virulence factor involved in multiple steps in the pathogenesis of N. meningitidis and is a significant contributor to the pathologies of meningococcal disease (2-4, 15, 23, 36). In this study, wild-type meningococci of serogroup B strain NMB (LOS immunotype L2/4) and mutants of this strain with defined mutations in enzymes required for LOS assembly (12, 15, 25, 29, 34, 41) were used to further determine the role of the LOS structure in meningococcal virulence. Variations of meningococcal LOS α chain and inner core structures are known to occur at high frequencies (15) and to influence pathogenesis. Immunotypes with full-length, sialylated α chain L3/7/9 and L2/4 immunotypes are those most frequently associated with invasive disease, while LOS with a truncated α chain {L8 immunotype [Galβ-Glcβ-Hep2 (GlcNAc, Glcα) KDO2-lipid A]} are more often found on carrier isolates (11). However, recent evidence indicates the L2/4 immunotype (strain NMB) has the genetic capability to express all other immunotypes (13).
Piliation is known to be critically important in the initial steps of the adhesion cascade of pathogenic Neisseria spp. (21). The wild-type parent and all mutants showed similar levels of piliation; however, the wild-type strain had slightly decreased PilC expression compared to the mutant strains. Other important meningococcal surface components influencing adherence and invasion are capsule, Opa, and Opc (2, 5, 38). The parent and mutants expressed the same amounts of capsule, although the Opa repertoire expressed by all the mutants was different from that expressed by the parent strain. To analyze the interaction between bacteria and host cells, in vitro adhesion and invasion of human pharyngeal cells by meningococci with LOS truncation was studied. As anticipated on the basis of previous studies (5, 18, 35), the capacity to express a full-length LOS with a sialylated α chain and inner core structure, as in the wild-type strain, was associated with the lowest levels of adhesion to the epithelial cells. The very truncated gmhB mutant (KDO2-lipid A) was, in contrast, quite adherent. This may be explained by the expression of different Opa proteins or better exposure of these or other adherence ligands due to the marked truncation in LOS structure. The rfaK mutant (Hep2-KDO2-lipid A) and the pgm mutant [Hep2(GlcNAc)-KDO2-lipid A] were also more adherent than the wild-type parent. Interestingly, meningococci expressing the Hep2-KDO2-lipid A LOS inner core structure were less adherent than meningococci expressing a Hep2(GlcNAc)-KDO2-lipid A structure, suggesting a possible ancillary role of the inner core GlcNAc in the adherence pathway. Changes in LOS structure may also enhance charge and other nonspecific interactions that facilitate adhesion; however, the mutants used in this study did not show visibly different levels of autoaggregation when observed microscopically.
In contrast to adhesion, invasion of meningococci into epithelial cells was best correlated with the capacity to produce α chain LOS structures and a requirement for the heptose disaccharide in the LOS inner core. As shown by the kinetics of invasion of epithelial cells, meningococci capable of expressing the LOS α chain appeared to invade epithelial cells rapidly. Although poorly adherent, the wild-type strain showed a steady increase in epithelial cell invasion over time. This may be due to the capacity of the wild-type strain to express the α chain, nonsialylated L3, L8 structure, which can facilitate invasion (5). Mutants unable to express or modify the α chain exhibited slower kinetics but were eventually as invasive as the wild-type strain. However, the strongly adherent gmhB mutant lacking the heptose disaccharide in the inner core did not invade epithelial cells. The data suggest that the minimal LOS structure required for the invasion of epithelial cells was Hep2-KDO2-lipid A. Similar observations have also been made in regard to the invasion of human nasopharyngeal epithelial cells in an organ culture model (D. S. Stephens, unpublished). The Opa protein expression differences may also have contributed to the low levels of meningococcal invasion of cells observed with the gmhB mutant in this study.
Other studies have indicated a role of LOS in bacterial cell invasion (24, 30). Harvey et al. (7) showed that the lacto-N-neotetraose-terminal LOS of N. gonorrhoeae facilitated gonococcal entry via the asialoglycoprotein receptors into primary human urethral epithelial cells. LOS α chain structures are known to bind asialoglycoprotein receptors on host cell surfaces (23, 24), and Opc-mediated cell invasion is best observed in meningococci expressing nonsialylated LOS structures (38). Furthermore, it was recently demonstrated that complementation of an rfaE Salmonella mutant (KDO2-lipid A) to wild-type lipopolysaccharide permitted the invasion of different epithelial cells that were not seen with the mutant (17).
Infection of CD46 transgenic mice with the wild-type strain and LOS mutants revealed that the wild-type strain and the rfaK mutant caused mortality in 77 to 82% of mice and in 0 to 12% of mice infected with the pgm and gmhB mutants. Mortality in the mouse model (Fig. 4) was correlated with significantly higher levels of bacteremia at early time points after peritoneal infection (Fig. 5). The detection of meningococci in the CSF of infected mice also correlated with mortality in the model. All mice infected with the wild-type NMB strain showed bacteria in the CSF.
The link between high levels of bacteremia early in infection and mortality was also associated with the induction of cytokine responses in the mouse model. TNF and IL-6 were not induced by infection with the pgm mutant and were induced at low levels following infection with the gmhB strain. The wild-type NMB strain and the rfaK mutant both induced high levels of IL-6 and increased levels of TNF. The greater-than-twofold increase in the number of meningococci in blood at these early time points is reminiscent of the studies of Brandtzaeg et al. (3, 4) defining the association of human morbidity and mortality to meningococcemia with serum levels of circulating meningococcal LOS. Thus, the ability of meningococci to rapidly enter and survive in the bloodstream after invasion and cause early high-level bacteremia is an important virulence factor, and this effect is related to LOS structure. The molecular basis for differences between the LOS structures is not clear. Equal molar quantities of endotoxins from the parent and each of these mutants are not different in terms of mouse or human macrophage cytokine activation (5). However, changes in LOS inner core structure do influence meningococcal survival in blood (26), and differences in interactions of the LOS with a variety of other host cells in vivo might also contribute to the differences observed. For example, the lack of cytokine response by the pgm mutant might be due to stimulation of a negative inhibitory pathway through C-lectin or other receptor recognition facilitated by the inner core structure. The difference in responses warrants further investigation into the mechanisms involved.
LOS structure is important in the resistance of meningococci to serum bactericidal activity (18, 22). The wild-type strain showed the greatest resistance to killing by whole blood taken from CD46 transgenic mice. The LOS mutants all showed decreased survival compared to the wild-type strain. Previous work by Kahler et al. (14) showed the pgm and rfaK mutants were more susceptible to normal human serum than the wild-type parent, supporting the findings of this study.
An encapsulated serogroup B wild-type strain capable of expressing a sialylated α chain or other structures containing α chain sugars was highly virulent in all the assays elucidated in this study. This strain had the best invasive capacity and had the highest serum resistance in whole CD46 transgenic mouse blood. A mutant with complete loss of inner core and α chain structures, although capable of avid adherence and bloodstream infection in the mouse model, was completely avirulent. Structural variations resulting in the loss of α chain structures were shown to exhibit increased adherence and decreased rates of cellular invasion in in vitro studies. However, meningococcal mutants deleted for LOS α chain structures but retaining inner core heptoses, although more sensitive to whole mouse blood, remained significantly virulent, providing evidence that virulence strongly associated with meningococcal LOS structure may be quite specific and not simply due to in vivo strain viability.
We gratefully acknowledge Margareta Hagelin and Maj-Britt Alter for excellent assistance with animal experiments and Ann von Euler for the electron microscopy.
This work was supported by grants from the Swedish Medical Research Council (Dnr 10846), the Swedish Cancer Society, the Swedish Society for Medicine, Lars Hiertas Stiftelse, Magnus Bergvalls Stiftelse, Åke Wibergs Stiftelse, Wenner-Gren foundations, Stiftelsen Wenner-Grenska Samfundet, and Karolinska Institutet. This work was also supported by Public Health Service grant AI-033517 (to D.S.S.) from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health.
- Received 19 September 2005.
- Returned for modification 7 November 2005.
- Accepted 22 November 2005.
Editor: J. N. Weiser
- American Society for Microbiology


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Employees at Siemens love to give back, and the company is sure to provide them with that opportunity!
On May 15th, a group of Siemens staff members arrived at the home of a Clermont Senior Services customer who was in desperate need of yard work and window washing.
The tasks of the group were many, and very much appreciated by the homeowner. The flower beds were weeded, the shrubs were trimmed, leaves were removed, debris was removed, a giant tree limb was removed from the roof and the windows were washed! To say this team put in a full day is an understatement.
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text: Job ID: 8876 Locations: Bayan Lepas, Penang, Malaysia Qualifications: - Bachelor degree in Accounting or Finance and 2-5 yrs experience in general accounting or auditing. Other Requirements: - Knowledge of Oracle financial accounting system, Excel skills and ability to learn and use applications. - Good communication skills both written and verbal. Must be able to communicate clearly, timely and accurately. - Ability to work in a team oriented environment as well as work independently in a time sensitive environment. - Ability to organize workload, adapt quickly to change and ability to meet deadlines and execute on commitments. Job Descriptions: - Process daily fixed assets transactions/ activities (capitalization, retirement, adjustment, transfer of assets between different legal entities within the group & dispatch asset tags) accurately in fixed asset ledgers. - Works with other departments to ensure timely communication of documents related to the acquisition and disposition of fixed assets. - Run depreciation and close fixed asset module for worldwide entities, - Assist in the monthly reconciliations between fixed assets sub ledger to General ledger. - Ensure compliance to Standard Operating Procedures (SOP) which is designed in accordance with SOX process. - Assist in identification of process improvements and other duties as assigned.


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My web developer @shiriamram rocks. She likes to tell me about things that might make sense to me. How many vendors do that for you?
Check out the bottom of the site that you are reading. The WordPress plug-in from Wibya allows for easy collaboration and a whole host of other features. It's very simple to add chat, translation, and social networking functionality (and a bunch of other incredibly cool stuff) to your site.
I was amazed at the translation with one click, make possible via Google.
This is just sick.
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Samuel Marshall "Sam" Raimi is an American film director, producer, writer, and actor, best known for directing the horror trilogy of The Evil Dead, the 1990 superhero film, Darkman, the original Spider-Man film trilogy (2002-2007), and the 2004 horror film, The Grudge, the 2009 horror film, Drag Me to Hell.
For Disney, he played Stick Coder in the 1993 Touchstone Pictures film, Indian Summer and directed the 2013 film, Oz the Great and Powerful. He also voiced a Giant Squirrel in the 2016 live-action remake of The Jungle Book.
Community content is available under CC-BY-SA unless otherwise noted.


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- Liked: Cleanliness, staff & service, amenities, property conditions & facilities
Very clean place. Staff was very friendly. There was a store across the street. And was very close to everything I needed
Book online or call:1-800-238-0767
Located in Carcar, Ashley and Bill's Suites 'N Garden is in the city center. Local points of interest include San Fernando Sports Complex and Monastery of the Holy Eucharist.
You'll be asked to pay the following charges at the property:
We have included all charges provided to us by the property. However, charges can vary, for example, based on length of stay or the room you book.
The following fees and deposits are charged by the property at time of service, check-in, or check-out.
The above list may not be comprehensive. Fees and deposits may not include tax and are subject to change.
Very clean place. Staff was very friendly. There was a store across the street. And was very close to everything I needed
I came to stay at this hotel as my family live nearby. This small property is tucked away just off the main road in Carcar, and reminds me of the townhomes in America. Our room was small but since we opted for the economy room, it was a good bang for buck. It also had everything we needed; bed, dinette, full restroom with hot shower, and A/C for when you need to get out of the heat. Where this place shines is the customer service. The staff was kind, friendly, and always smiling. They kept checking on us to see if we needed anything, which was comforting. They are also very knowledgeable of the area so getting directions was simple. There is also is also a very delicious breakfast served daily that is homemade. It is a full course meal with full service staff served outside under the pavilion that felt like the royal treatment. Romantic like as well. We also got the tasty basket with free bottled water. There aren't many tourists attractions in Carcar but don't be afraid to stay here when tourist area is full on the southern side of the island as there are local services nearby to take you down to have a good time. We will be staying here again in the near future.
Location is good and staff pleasent.
Nice rooms in the heart of Carcar. Secure location and 24 hour staff
Nice room near several hubs of activity, on-site staff provided us with excellent native foods breakfasts two of our mornings there... real treats! Additionally, a welcome basket full of fruit and local confections as well as complimentary drinks greeted our arrival.
Dubay Panglao Beachfront Resort
Studio Apartment at Taguig Manila
Makati Shangri-la Hotel, Manila
Microtel by Wyndham UP Technohub
Sofitel Philippine Plaza Manila
Costabella Tropical Beach Hotel
Hyatt Regency Manila, City of Dreams


text: Luxy Balloons DC works with many non-profit agencies on races, walks, and benefits. Luxy Balloons DC has the experienced staff to use the right balloons décor for your race walk or benefit. Outdoor balloons and balloons that people may walk or run under need to be set by professionals We will be there at 5am to set-up balloons décor for your race walk or benefit before registration begins. We utilize 6 foot balloons, banners, cloudbusters, arches and other décor to relay information about your event to participants while making the event look great!


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Computer Science News
In spite of all of the advanced features person nonetheless come throughout error message whereas accessing the reminiscence card from the digital digital camera. The error message is Built-in Reminiscence Card Error. As a consequence of this cause it disallows the users to access and store the images. This error message generally arises when the digital camera screen exhibits some mishap with the digicam. Sometimes users turns into unable to observe the issue and retains utilizing the digital camera continuously. After that all of the sudden removes the memory card from the camera while digital camera remains to be working. Resulting from this purpose the digital digicam becomes unresponsive and after that the digicam fail completely to carry out. After this if consumer reinsert the memory card then they expertise the constructed-in reminiscence card error message.
Record Audio From Computer Mac
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In the 21st century, many firms have gone international. The advent of trading on the internet, or e-commerce, has created the necessity for a method to report and store these quite a few transactions. Previously, firms spent 1000's on backup tapes paper and paper copies of every assembly, determination and transaction. Those days are behind us.
Article Disclosure
The next tips will enable you to navigate among the commonest problems and options that come up when protecting your data protected. Up to date onerous drives use a voice coil actuator, which controls the motion of a coil toward or away from a everlasting magnet primarily based on the quantity of current flowing by it. Elementary structures of all exhausting disk are same, and are composed of the identical physical options, but their performance is determined by the standard of their inner elements.
About Writer
39 yr old Petroleum Engineer Jaimes from Guelph, usually spends time with passions including internet, . and train spotting. Recently took some time to journey to Mana Pools National Park.


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By Canon USA
For Macintosh, Windows USB
If you closed your eyes and tried to imagine the best possible flatbed scanner for the home or small office, you might very well conjure up this sleek little machine, a dream-come-true for those of us with relatively light needs to copy images and scan in documents.
The N676U is about 11/2 inches thick and roughly as long and wide as a 14-inch legal pad, so it fits well in a backpack or briefcase. It draws all of its power from the USB port on a laptop or desktop, so there is no need to lug about a power supply.
Laptop users could easily tote this along to the library. The 3.3-pound scanner also lends itself to moving between USB-equipped Macs and PCs running Windows 98 and above, and handles resolutions up to 600 by 1,200 dots per inch in 48-bit color.
While this is a tad less than serious photograph-scanning machines, it produces elegant color pictures suitable for most uses and probably should be more than adequate for the great bulk of home applications.
The ScanGear Toolbox, the core scanning software that comes with the N676U, is particularly intuitive and allows one to make quick work of both scanning in a preview image and then cropping it for a serious high-resolution scan.
A collection of companion software, including ArcSoft PhotoBase, allows a user to keep track of scanned images.
The software lets users type in descriptive key words for pictures and also permits quick browsing through thumbnails of all images.


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With a shift of personnel rivaling the biggest of all-time, it's a very different NBA from the one that finished the 2018-19 season. So what's ahead?
The offseason in the NBA sent shock waves through the hierarchy of the league, with so many players changing jerseys that program sales may hit an all-time high in 2019-20.
These haven't been run-of-the-mill rotation guys switching clubs, either. For the first time since the award was first handed out in 1969, the NBA Finals MVP from last season will be with a new club, as Kawhi Leonard jumped from the Toronto Raptors to sign with the LA Clippers.
The only other Finals MVP not to return to his old club was Michael Jordan, who retired from the Bulls a second time after leading the club to a third straight title in 1998.
And that's just the tip of the iceberg.
Former NBA MVP's Kevin Durant and Russell Westbrook will be modeling different uniforms in 2019-20, with Durant leaving the Golden State Warriors to sign with the Brooklyn Nets and Westbrook being traded from the Oklahoma City Thunder to the Houston Rockets.
A third former MVP was also on the move, with 2010-11 winner Derrick Rose signing with the Detroit Pistons after a season with the Minnesota Timberwolves.
The big shift has been away from the superteam and toward the dynamic duo. Durant and Kyrie Irving will eventually pair up in Brooklyn. Westbrook and James Harden are together again for the first time since the 2011-12 season, now in Houston.
Leonard convinced Paul George to request a trade from the Thunder to join forced with the Clippers. The Los Angeles Lakers have combined Anthony Davis with holdover superstar LeBron James.
Beyond that, clubs such as the Utah Jazz and Philadelphia 76ers made moves that — on paper — make them much stronger.
The Warriors are one of the losers in all the transition as are the defending champion Raptors.
It is an offseason that might bring the NBA as much of a sense of parity as it has had since the crazy days following the merger with the ABA in 1976, another time when a lot of players were changing teams and new dynamics were emerging that led to a league that from 1976-79 produced:
- A 44-win NBA champion.
- Three straight seasons without a team posting 60 wins and only one team losing 60 games in a season (the 1976-77 New York Nets went 22-60).
- Three first-time title winners in the Portland Trail Blazers, Washington Bullets and Seattle SuperSonics.
- At a time when only six teams per conference reached the playoffs, 19 of the NBA's then-22 teams reached the postseason at least once.
The NBA is as wide-open as it has been in years and after five straight seasons of Warrior domination of the Western Conference, it opens the door to one of the most highly anticipated regular seasons in decades.
With that, there are the 10 big questions entering the 2019-20 season.


text: I know nothing about video, but would like to play with a video camera. The one hurdle I could never get over is which media storage type to go with ... DVD, mini DVD, tape, CF ... ? What are the advantages/disadvantages of the various digital video media storage systems?


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The long-lasting stick light with 300 lumens in the 18V category
The GLI 18V-300 Professional is Bosch's long-lasting hand-held stick light with 300 lumens in the 18V category. It includes a hanging hook and 5 adjustment angles for flexibility in illumination. Its shape and the 6 powerful LEDs make it ideal for lighting up narrow spaces. This handheld stick light is designed with an ergonomic shape and soft grip for comfortable handling.
It is compatible with all Bosch Professional 18 V batteries (Professional 18V System).
Order number: 0 601 4A1 100
Selected version
Price includes:
* Battery and charger not included


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Let All 16 Candidates Debate
What happens when you try to rerun the last election? Answer: You lose.
That's precisely what the Republican National Committee is setting itself up to do with its presidential debate gambit. The RNC, in league with Fox News and CNN, is limiting the participants in the first two Republican debates, on Aug. 6 and Sept. 16, to only the top candidates in national polls, half a year before any voters cast a ballot.
Let's speak the truth out loud, just for fun. The big donors to the Republican Party were mad after Mitt Romney lost to President Obama in 2012. I don't blame them: I was mad too, and the country has paid the price for its choice.
These donors' theory is that one of the big reasons Mr. Romney lost is that he was forced to argue with a bunch of inferior life-forms in countless debates, which forced him to claim that he was "severely conservative" and in favor of "self-deportation" for illegal immigrants, whatever that is. News flash: A competitive primary did not cause Mitt Romney to lose.
But that was then and this is now. By all accounts, the Republican field boasts a deep and impressive array of candidates, much stronger than that of 2012. The Democrats, by contrast, are less about primaries or debates than about a coronation.
The Republican Party should be looking forward instead of backward—and seeking every opportunity to feature its roster of excellent candidates, rather than trying to find ways to constrict the field. The voters will do that, as is their prerogative. The simple truth is that competitive primaries usually make a party stronger, not weaker.
It's also true that whenever the smart guys in Washington get together and try to shortcut the democratic process by imposing a candidate from the top down, it generally goes poorly. Whatever happened to the idea of freedom? Or democracy? Or robust argument? As a Republican, I wonder: When did we start fearing debates? And if we do fear debates, what business do we have trying to win elections?
The plan to limit the participants in these debates is ridiculous in almost every respect:
• National polling would be a decent way to measure candidates' support in a national primary. But we don't have a national primary. We have a series of elections by state over an extended period, so this measurement has nothing to do with the reality. Other than that, it's just fine.
• The policy favors retread candidates and political dynasties based solely on name identification. If your brother or father ran, you get rewarded. If you ran before, you get rewarded. But if you are a new face, you are penalized.
• The move also benefits personalities and circus acts. If you have a TV show, you will easily have enough name recognition to get into the debates. Doesn't matter if you'd make a plausible president of the United States. It is a certainty that under this approach if Tom Brady or Jimmy Fallon announced this week that they were running, they'd make the cut. A certainty.
• Other beneficiaries: camera hounds from the political class who live in the Washington and New York media capitals. If you're a senator or congressman, you can get yourself on cable news four times a week. Or if you're the governor of New Jersey, you can schlep across the bridge, traffic permitting, and do a bunch of national TV shows.
• The RNC's approach circumvents the political process. In the past two elections, the winners of the Iowa caucuses, Mike Huckabee and Rick Santorum, were nowhere to be found in national polling the summer before. The same would be true for two little-known governors of Georgia and Arkansas, both of whom went on to call 1600 Pennsylvania Ave. their home.
• Relying on national polling the summer before the election gives candidates an incentive to run much longer campaigns and penalizes candidates who don't. Is that what we really want? Campaigns that begin two years before voters will actually head to the polls?
• Finally, there are extreme methodological flaws in these surveys, making the entire thing a charade. Most of these so-called national polls contact roughly 1,000 random voters, and thus end up with Republican sample sizes of only a few hundred people. That is a complete joke: Every reputable polling firm demands at least a 400-person sample for a survey of even a single congressional district. Furthermore, most media surveys inexplicably fail to screen for likely primary voters, which is a basic necessity for accurate results.
The Republican Party's leaders vowed to take control of the debate process, but they're fighting the last war—and inviting trouble. The RNC seems to know that it has created a mess, and last week laughably attempted to reverse course by advancing a bizarre argument that it is forbidden by Federal Election Commission rules from influencing the debate process. That line was instantly dismissed by legal experts, but it's worth noting the logic: The people who vowed to take control of the debate process now say they are legally forbidden from doing so. Go figure.
Here's a wild and controversial idea: Trust the voters. Let candidates debate whenever and wherever they want. Don't try to control the process from Washington. Let freedom ring.
This op-ed originally ran in the Wall Street Journal on July 22, 2015.


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Realme is launching
Immediately, Realme's phones work on ColorOS, which is built with the help of Oppo. Confirming this information, the CEO of the agency Madhav Seth has also shared some facts about RealmeOS.
What can be unique in reality os
According to media reviews, Madhav Seth has said that the organization has been taking comments from customers for a long time.
At the same time, Seth advised that the Reality study and improvement team was engaged in preparing that specific feature for the operating machine, the document states that this OS of Reality could be primarily based on Android Q.
Similar to OnePlus's OxygenOs
Although Madhav Seth has not given nearly complete records of this OS now, it is being said that the company will release the stock version of the new OS.
It is estimated that Realme's new operating system may be very similar to OnePlus's OxygenOS. The document states that the company will participate in the improvement of its OS after the release of Android Q.


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12-06-2019, 05:54 PMOnly three countries among the 36 countries in OECD. https://finance.yahoo.com/news/trump-cuts-us-taxes-fourth-233815750.html39 replies | 537 view(s)
12-05-2019, 08:30 PMShe definitely chose the right sport. And given her post collegiate success, it's hard to question the choice Williams made (although she could have...33 replies | 552 view(s)
12-05-2019, 05:36 PMThere are many people who think it could be bad. Here is one example. ...39 replies | 537 view(s)
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12-03-2019, 02:51 AMMercy rule in baseball and softball exists so that a lopsided game does not go on for too long. Tennis does not have that problem, because the more...321 replies | 19320 view(s)
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12-01-2019, 03:15 AMJust found out aboutthis game between Stanford and Prairie View A&M. No mercy at all. The question is, did they celebrate after the 15th goal? ...321 replies | 19320 view(s)
12-01-2019, 01:58 AMThat article also should be read with some grains of salt, given the authors' agenda. In any case, my point was that P5 schools and the rest of D1...54 replies | 3405 view(s)
12-01-2019, 01:55 AMTN1965 replied to a thread NYC Marathon 2019 in Current EventsA story on Girma Bekele Gebre and West Side Runners Club. https://news.yahoo.com/ny-team-helping-immigrant-runners-032626192.html32 replies | 3080 view(s)
11-29-2019, 02:24 AMOkay. So I found the answer to my own question. (Data are from 2016.) P5 schools. 64/65 make profit on football. 54/65 make profit on men's...54 replies | 3405 view(s)
11-27-2019, 08:41 PMTN1965 replied to a thread tips on manipulating list data in Current EventsI don't have any proficiency with Excel, but how hard is it to understand that 977 performers have produced 8634 performances? In fact, it is hard...27 replies | 1349 view(s)
11-27-2019, 08:31 PMHow many D1 schools actually make net profit from football?54 replies | 3405 view(s)
11-26-2019, 02:19 AMTN1965 replied to a thread the 5 most memorable days in your life in Things Not T&Fit is kind of sad to think the deaths of Sadat and Rabin made bigger impressions on me than Camp David or Oslo Accord. So I will post about a...63 replies | 1558 view(s)
11-24-2019, 01:49 AMTN1965 replied to a thread the 5 most memorable days in your life in Things Not T&F+1 on this. The break-up of the USSR was also a question of when by the time it happened. The same for the end of Apartheid. I had not expected any...63 replies | 1558 view(s)
11-24-2019, 01:28 AMTN1965 replied to a thread Clueless writing about our sport in Things Not T&F"Muhammad beat fellow nominees Brigid Kosgei, who broke the women's marathon world record, and Sifan Hassan, who won world gold in the 5,000 and...635 replies | 208626 view(s)
11-22-2019, 05:34 PMAnd Hasay didn't cut her hair. She decided to change her ponytail to braids to reduce the drag instead. One big difference is that Hasay was...245 replies | 21324 view(s)
11-18-2019, 05:40 PMTN1965 replied to a thread Hard to believe the 44:20 15K by Letesenbet Gidey. in Current EventsVDOT calculator predicts 13:53 / 28:54 / 1:03:37 / 2:13:19. Inconceivable? Depends on what you are wearing... ...28 replies | 3561 view(s)
11-18-2019, 05:32 PMTN1965 replied to a thread wPV Chloe Cunliffe (WaHS) 14-9i (4.50) AJR HSR [14-8 AJr out] in Current EventsAre you talking about someone like Stefanidi, who won a World Youth title and a World Junior medal? She bothered to come to the US to attend Stanford.94 replies | 11992 view(s)
11-15-2019, 05:10 PMTN1965 replied to a thread US Debt in Things Not T&FBut the US government no longer has the AAA rating (at least not from S&P). https://tradingeconomics.com/country-list/rating This is a more...14 replies | 450 view(s)
11-12-2019, 09:33 PMI don't know how rich Mr. Zhao was (is), or how he earned his income. But based on his background, I suspect most of that comes from his investment....39 replies | 537 view(s)
11-12-2019, 05:00 PMTN1965 replied to a thread Losing Vice Presidential Candidate Trivia in Things Not T&FHere is the visual evidence. https://commons.wikimedia.org/wiki/File:Dan_Quayle_presidential_campaign,_2000.png20 replies | 997 view(s)
11-12-2019, 01:32 AMTN1965 replied to a thread '20 DL schedule released [and event distribution] in Current EventsThis has already happened with wHT, and the WR was indeed broken multiple times.130 replies | 13497 view(s)
11-11-2019, 05:38 PMTN1965 replied to a thread IAAF getting complaints about Vaporfly? in Current EventsA new pair of the same model needs significantly less "breaking in" than a new model.204 replies | 13481 view(s)
11-10-2019, 02:40 AMNozomi Takamatsu. (2014 Youth Olympic champion. She was 4th in the World Junior race that Cain won.) ...245 replies | 21324 view(s)
11-10-2019, 02:35 AMThis kind of behavior is pretty common in Japan. Not just in track and field but in many other sports as well.245 replies | 21324 view(s)
11-09-2019, 03:41 AMTN1965 replied to a thread IAAF getting complaints about Vaporfly? in Current EventsIsn't Mary Keitany sponsored by Adidas?204 replies | 13481 view(s)
11-08-2019, 05:23 PMTN1965 replied to a thread Track without Nike in Current Events"Track" may not matter, but road racing does. Casual rec runners who cannot name more than a few elite marathon runners know about Vaporfly, and the...28 replies | 2808 view(s)
11-07-2019, 06:23 PMTN1965 replied to a thread Standing HJ WR in Things Not T&FThere are many WRs in track & field like this. Krause's 2000mSC is the most recent example. Beatrice Chepkoech can easily break that record. But she...14 replies | 668 view(s)
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มีดพับ Spyderco MGGYP Manbug VG10 Plain Blade, Gray G10 Handles (MGGYP)
Men and women are different. Spyderco's Manbug, compared with the classic Ladybug, contrast these gender related differences. Manbugs are thicker, more muscular and bulky than the thinner, lighter Ladybug. Their flat-ground VG-10 blades are equal in length to the Ladybug but wider with a robust modified clip-point. Fabricated with pinned dove-gray G-10 scales, the handle has stainless bolsters and a mid-spine positioned back-lock with a David Boye Dent. Alignment of the Spyderco Round Hole, relative to the pivot and to the back-lock adds fluidity and ease to the action of opening/closing the blade.


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A man driving home late at night in his Volkswagen beetle car was stopped by by policemen on patrol.
The police asked the man to produce his car document.
When they could not fault the document, the next question to the man was: "My friend, do you realize that you committed a criminal offense by driving alone in this car at late night?"
The man became angry and responded: "How could you say that? God the father, the son and holy spirit, prophet Elijah and Angels Micheal and Gabriel are all with me in the Car."
The policeman replied: "You mean, all these people are in this small car? I charge you for overloading!"
Really funny jokes-Overloading
send by Fadi Sikas
Categories Good Jokes
|it is
1 التعليقات:
- Afsheen said...
nice funny jokes
Chuck Norris
- August 1, 2012 at 9:42 AM
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Dunkelgrünes sofa Fantastisches Wohnen Für Ihre Heimat Inspiration
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