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Set in tall, shady eucalypt forest, Mount Samaria State Park offers a cool and quiet retreat for beautiful walks, camping and picnicking. The Mount Samaria range incorporates a variety of scenery with spectacular vistas over the Victorian Alps, granite outcrops, high waterfalls and historic remains. Explore the park by foot, mountain bike or scenic drive and discover its tranquillity and beauty. Things to do- A range of camping and picnic areas, walking tracks and lookouts provide the visitor with many opportunities to experience this semi-remote park- During the spring and early summer the forest is alive with brilliant displays of wildflowers- Year-round, it provides a haven for an array of birds and wildlife and is ideal for nature observation and photography- The high altitude of the park provides spectacular views from Mount Samaria Summit, Rocky Point Lookout and Wild Dog Falls- Historic remains at Samaria Well, Spring Creek Sawmill, the timber tramway and braking station reflect past practices- A range of walking tracks cater for walkers of all abilities with opportunites for short walks through to full day and overnight hikesBefore you goConditions can change in parks for many reasons. For the latest information on changes to local conditions, please visit the relevant park page on the Parks Victoria website.Be bushfire ready in the great outdoors. Refer to the Bushfire Safety section on the Parks Victoria website for tips on how to stay safe.
Mount Samaria State Park is 130 km north-east of Melbourne, 28 km south of Benalla on the Samaria Road via Swanpool and 14 km north of Mansfield via the Tolmie Road. A gravel road traverses the range from north to south, and although steep and rocky in places, is open all year to most vehicles. Check on the condition of the road north of the old mill site before planning a through journey as it may become impassable in wet weather.


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My reasons for college are probably just like everyone else's i have to write an essay going to college essay for english class and i always have trouble with my opening. Use our papers to help you with going to college essay yours. Looking for reasons to consider going back to college? There are many reasons to consider. " Words like these are vague,. 1 through 30
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2005 Jeep Liberty
My driver side rear window stopped working for no reason. It went down and then would not go back up. How do I fix it? I took the inside door panel off to see if it is out of track. I can't see anything.
Friday, June 19th, 2009 AT 6:24 PM


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For folks who arent sure they like dance, theres MOMIX, a troupe so jaw-droppingly stunning and easy to appreciate that they were used in a Hanes underwear commercial. The companys famed choreographer and founder, Moses Pendleton, has once again produced a couple of trippy performances. If you didnt catch the first two weeks of his first show, Passion, you must see his second and final program, Lunar Sea, a surrealistic piece of playful transformation set to black lighting: Youll witness a troupe of dancers form mermaids, spiders, and crabs in this celestial under-the-sea setting.
Sun., June 1, 2 & 7:30 p.m., 2008
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In Time to Voices Import
|CDN$ 16.61 & FREE Shipping on orders over CDN$ 25. Details
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Track Listings
|1. In Time To Voices
|2. Lost Kids
|3. Cold
|4. Two Dead Minutes
|5. Silence And The Drones
|6. Night Light
|7. Je Me Oerds
|8. Stop Kicking
|9. Slip Into The Blue
|10. Down Here In The Dark
|11. 7 Years
Product Description
2012 album from Brighton boy/girl alt-rock duo consisting of Steven Ansell and Laura-Mary Carter. Carter described the album: ''With this album, we totally threw out the rulebook of how we write and record. We decided we wanted to make a really ambitious record, not something which reflects our live show but something which is only limited by our imaginations and not by how many instruments we use onstage. We figured the best way to push ourselves forward was to write the best possible songs and melodies we could - once you have that as your foundation, you have the freedom to go anywhere with the sounds you use. We feel like a totally different band now. We feel like we're shooting for the stars.'' Includes first single 'Cold'.
Customer Reviews
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Most Helpful Customer Reviews on Amazon.com (beta)
I get the impression upon reading other music reviews that I should discuss individual songs, which is difficult for me (I'll try though). I tend to listen to albums as full entities (showing my age, I know), and will progress as thus. IN TIME TO VOICES plays very well as an album, complete with and underlying theme. This is sadly uncommon in modern music, and welcome to those of us that grew up listening to such albums. The only semi-negative thing I can say about the album is that it starts a lot more energetically than it finishes, which may be (at first) off-putting to established fans. Longtime fans of the band will likely be drawn to the first three songs (title-track "In Time to Voices", "Lost", and "Cold"), and may have to warm up to later tracks. However, this does not mean that the latter songs of the album are not good. Quite the contrary actually, as some of the best songwriting is toward the end; "Night Light" is probably my favorite track in general, and is perhaps the slowest song on the album. The final track "7 Years" is lyrically among my favorites, alongside "The Silence and the Drones". Steven and Laura-Mary have not skimped on the album's sound while trying new things and exploring their influences; for example, the beginning of "Slip Into Blue" has a nice Pixies feel to it, which I find quite pleasing. There is not a single track on this album that I dislike, which says a lot.
In short, if you are a longtime fan of Blood Red Shoes and feel unsure upon first experiencing the album, give it a few listens. There is a very good chance that IN TIME TO VOICES will continue to grow on you with each listen.
NME made the album available for online listening several weeks before its release, and I listened to it quite a bit before purchasing it. This did not deter my playing of the album once acquired; I've owned it for about four months now, and it remains a constant in my vehicle (and it should be noted that I drive a lot for work). Great band, great album. A worthy addition to a young band's already strong catalog.
In the end, this is a very good album with some of their best songs but left this fan feeling a touch let down not by something done wrong, but by seeing the massive potential there merely touched and not fully grasped yet. This album gives me great hope for future efforts from Blood Red Shoes, including the soon to be released EP The Water.


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iHerb Customer Reviews
Savings of: $14.63 (49% off)
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Joint Synergy
Posted by Reviewer1786103 on Jan 05, 2012
helps with shoulder pain, has all recommended herbs in one formula, and this is not usual. All good
Didn't seem that effective
Posted by Reviewer2416129 on Jun 21, 2011
I can't really say this helped eliminate my arthritis pain. The roll-on does give temporary relief but unless you don't care about the odor you can't really use it and go out in public.
MRM Joint Synergy Roll-on
Posted by Reviewer1166508 on Dec 20, 2008
I bought this for my mother. She said the roll-on had a cooling effect, but the oder was not appealing. She would only use this product alone.
Average Product
Posted by Reviewer2924771 on Oct 04, 2009
No real difference in joint pain felt!!!
expected better results.
Posted by Reviewer2885021 on Nov 23, 2011
Needs to be stroner is formulation to ease more of the pain.
joint pain
Posted by 5660099391413790554 on May 27, 2012
it didnt effect me
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Women's Durango Boot® 11" Mai West Boot, Red
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LMT Communications, Inc. posted to How to Fabricate Winning Dentures Using Idiosyncratic Dentate Characterization
Check out the video version!
Heraeus Kulzer's clinic, Expanding Business Opportunities Presented with the PALA Digital Denture System, included an exciting LMT LAB DAY first: a live patient demonstration. Here, Dr. Lou Graham and Dental Assistant Yolanda Davila, take a patient's impression and bite registration while dozens of LAB DAY attendees observed and asked questions. Throughout the process, Graham and David Avery, AS, CDT, TE, David Avery Consulting Services, LLC, covered the clinical technique from the records appointment to the try-in and delivery of the definitive restoration, and also offered tips for labs to share with their dentist-clients.
With LMT's LAB DAY Chicago right around the corner—February 26 and 27—it's time to finalize your plans to get the most out of our industry's biggest, busiest show! Here's a look at some of the special promotions, raffles and demonstrations available inside the LAB DAY Exhibit Hall.
Read More 11 minute read
As part of our ongoing State of the Industry 2015 coverage, LMT Editors visited the world's largest dental show to bring you the scoop on the latest product and process innovations worldwide—including digital dentures, laser milling and more—and what else is coming down the pike for the U.S. market.
10 peopleSteven Touchie
Tais Clausen
Tim Thompson
Tillmann Steinbrecher
Carola Froelich
Andy Klein
Devon Howe
Avi Cohen
Justin Marks, CDT
Norbert Ulmer
Read More 21 minute read
The Pala Digital Denture System from Heraeus Kulzer uses CAD and 3D software to fabricate dentures with an ideal fit and esthetics with a processing time of 10 days or less. Here's how it works:
The dentist uses the Pala Digital Denture tray system which enables him to take the impression, vertical dimension and centric relation in a single visit.
The lab scans the impression and uploads it to Heraeus Kulzer's Pala Digital Design Center. The try-in is digitally articulated, the teeth are setup and customized, and an anatomically correct try-in is printed.
Once the try-in is approved by the patient and doctor, the lab sends it back to the Pala Digital Design Center to be processed by Heraeus Kulzer's proprietary injection process.
Labs must become a Pala Partner to offer Pala Digital Dentures. "As dental practices come to appreciate how powerfully our digital dentures can enhance both their productivity and their patient satisfaction, they'll want to...
EGS, which was acquired by Heraeus Kulzer GmbH in October, presented updates for its two core products:
DentalCad 4 software now features a new "DentalCad Touch Screen" working mode, powerful wax-up functions, angled implant support, and deformation and sculpting tools.
The latest generation of the DScan 3 scanner features a unique "ghost cover"—a clear, plastic cover that allows you to see the inner components of the unit. Its blue structured light technology allows for faster and more accurate 3D scanning of the model. It works with the verticulator tool in order to scan the models in occlusion and the entire verticulator in a certain and repeatable position. Due to its new high quality optical detectors and mechanical components and the latest-generation PC installed directly in the scanner case, it operates in all light conditions, even capturing semi-reflecting objects.
For more information, visit www.egsolutions.com/en/.
For nearly a decade, the North Carolina Missions of Mercy (NCMOM) has been serving dental patients in need. In 2012 alone, it provided free dental treatment to over 9,000 of the state's underserved residents.
In October, Hurricane Sandy hit the East Coast, devastating parts of New York and New Jersey. LMT checks in with Master-Touch Dental Laboratories and Valplast—both of which took a direct hit—and looks at how the industry has come together to help.
3 topicsNowak Dental Supplies, Inc.
Heraeus Kulzer
Valplast International Corp.
Read More 5 minute read
Attendees took advantage of two full days of clinics on Friday, May 4th and Saturday, May 5th, and were eager to shop exhibits on Saturday. In fact, the exhibit hall was already bustling with crowds
See the highlights from LMTâs LAB DAY East, where optimism was in the air and business was buzzing.
From bringing smiles to the underserved dental population to rebuilding the lives of our military veterans, technicians across the country are devoting their time and skills to making a difference.
6 topicsDENTSPLY Prosthetics
Heraeus Kulzer
Ivoclar Vivadent Inc.
Zahn Dental
Partial Dentures
LAB DAY 2012 could easily be labeled "The Reinvention Convention." Laboratory owners and managers are more engaged than ever in finding new ways to reinvigorate their businesses
To meet the needs of implant, geriatric and TMJ patients, Heraeus Kulzer launched its Mondial 8i-5° posterior tooth line. "New indication areas such as implant prosthetics are placing increased demands on the stability, function and esthetics of denture teeth," said Lesley Melvin, Heraeus Laboratory Product Manager. "That's why we've worked so closely with leading technicians to ensure that our Mondial 8i-5° posterior teeth complement our existing Mondial and Mondial i teeth to give dental practices and laboratories a comprehensive denture teeth line that is unsurpassed for its natural appearance, comfort and reliability."
Like Mondial and Mondial i teeth, Mondial 8i-5° posteriors feature Heraeus' NanoPearls® filler material that offers color stability and resistance to fracture, abrasion and plaque. Since the manufacturing process is identical, all three lines can be used in combination.
The key features of the new tooth line are:
•The flat occlusal morphology of...
Get the most "likes" of your photos of your upper or lower removable posted on this page and you could win $500. Contest ends July 31, 2012.
This case exemplifies how attention to detail, use of new materials and good communication with the dental team can result in restorations that disappear in the oral environment.
A supporter of educational initiatives, Heraeus offers instructional videos free to dental teams and patients, teaching effective techniques and procedures.
One of the laboratory procedures that has created stress has been investing and divesting partial dentures. Craig Nelson, AAS, CDT, has developed a technique to make this process easier.
The Argen Corp. acquired the Jelenko dental alloy business from Heraeus Kulzer, Inc. Argen is incorporating the business into its San Diego, California facility but will maintain the Jelenko brand name for the alloys.
8 topicsJelenko
Aurium Research USA
Leach & Dillon Dental Alloys
The Wilkinson Co.
Zahn Dental
Argen Corp.
Heraeus Kulzer
Dental Services Group's annual meeting is well-regarded for its open environment and sense of camaraderie among the laboratories. With the theme, Gateway to the Future, this year's event emphasized management and leadership, new technology and custom


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Features of Typing Tutor. Free typing lessons choice.
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WAR: Turkish Army Continues To Pound Syria For A 2nd Day In Response To Mortar Strike Across Border!!! Turkey Green-Lights Military Action...
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dont worry my friends it all over ım a turk and ı can tell the story from inside,my govorment announce that we make a few artilerry shot to syria and told us that they defend country and now from turkey everybody is relax and sleping in our beds and tomarrow morning only they planning to make out a decesion of act from parlement.and it will be samething to like that in my peaple's eye "if ever happen samething like that we will make more artilery shots"
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13 Important Elements To Look For In Every Staged Shooting Event
Gestapo In The USA: FCC Intimidates Press and Kills Free Speech at 5G Rollout
Democratic National Convention in Philly Is Not What We're Told About in the Media
Federal Judge Rules Hidden Microphones in Public Do Not Violate Constitution
Deutsche Bank's net income plummets
Sanders Supporters Walk Out Of Democratic Convention
Black Protesters at DNC SHOUTS: "Don't Vote For Hillary, She's Killing Black People"
Hillary Clinton will nominate justices to the Supreme Court who are prepared to overturn Citizens United."
Day After Clinton Cash Film's Go Worldwide, CEO Forced To Step Down
Ancient 'toilet paper' proves that Silk Road travelers spread parasites as they traveled
Sheriff David Clarke Says the DNC 'Seems to Be About Embracing Criminality'
Why News Junkies Are So Glum About Politics, Economics, and Everything Else
Here's What Happened When Democrats Recited 'The Pledge Of Allegiance' At The DNC
Superhuman Tech? Most Fear Worst..
Wikileaks Proves Primary Was Rigged: DNC Undermined Democracy
WIKILEAKS JULIAN ASSANGE Says He Has NEW Emails That Should Indict Hillary…And Why He's A Trump Fan
Undercover Video: Hillary Clinton Delegate Explains Her Deceptive Propaganda to Ban Guns
Is State Dept. Is Protecting Hillary Rotten Clinton's Presidential Bid?
Robots, drones fill police ranks
Companies around globe packing earth orbit with satellites to meet communications demands
Rise of black nationalist groups
Report: Mr. Robot's one-time only VR experience will return next week
'Kill More Cops' Graffiti Mural Appears on Freeway
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Should the Postal Service Handle Your Loans as Well as Your Mail? Elizabeth Warren Thinks So.
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"Astonishing...Shocking...Remarkable. Aleatha Romig is a master of words." - Author Cassy Roop
"Intense, intriguing, incredible... insidious." - International Bestselling Author, S.K. Hartley.
"Fans of dark, twisted erotic suspense will be VERY happy. INSIDIOUS is a five-star, must-read winner!" - Bestselling author Tia Louise
Kindle Crack Book Reviews is officially obsessed with INSIDIOUS!
"Jesus woman, You have done it once again. You gripped me right from the start and refused to let me go until the bitter end. I can not wait for fans of The Consequences series get their hands on this one. I knew you were going to top Tony, I just was not sure how and you have blown it out of the park. Insidious is Amazeballs!!!!!!" Chris BlogEmporium
"If you're a fan of suspense, raw emotion and thrilling ups and downs then you will not want to miss this dark and erotic tale from the evil wordsmith herself, Aleatha Romig. Insidious will creep into your mind, grab you tightly and not let go until The End. Even then you'll still be thinking about this story. A must read." Kathryn Perez, author of Therapy as well as Sex Unlimited.
From the Author
Duplicity (each book is a standalone tale) TBA
Consequences (Book #1)
Truth (Book #2)
Convicted (Book #3)
Revealed (Book #4)
Beyond the Consequences(book #5)
and The Consequences series reading companions, for those times when youwondered, "What was Tony thinking?"
Behind His Eyes Consequences
Behind His Eyes Truth


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Biodiesel: A Realistic Fuel Alternative for Diesel Engines / Edition 1by Ayhan Demirbas
View All Available Formats & Editions
Pub. Date: 03/20/2008
Publisher: Springer London
Environmental and political concerns are generating a growing interest in alternative engine fuels such as biodiesel. Biodiesel is a renewable energy source produced from natural oils and fats, which can be used as a substitute for petroleum diesel without the need for diesel engine modification. In addition to being biodegradable and non-toxic, biodiesel is also
Environmental and political concerns are generating a growing interest in alternative engine fuels such as biodiesel. Biodiesel is a renewable energy source produced from natural oils and fats, which can be used as a substitute for petroleum diesel without the need for diesel engine modification. In addition to being biodegradable and non-toxic, biodiesel is also essentially free of sulfur and aromatics, producing lower exhaust emissions than conventional gasoline whilst providing similar properties in terms of fuel efficiency. The greatest drawback of using pure vegetable oils as fuels are their high viscosity, although this can be reduced by techniques such as dilution, micro-emulsification, pyrolysis or transesterification. Of these processes, the transesterification of vegetable oil triglycerides in supercritical methanol has been shown to be particularly promising, producing high yields of low-viscosity methyl esters without the need of a catalyst. Furthermore, these methyl esters have considerably lower flash points than those of pure vegetable oils. Biodiesel: A Realistic Fuel Alternative for Diesel Engines describes the production and characterization of biodiesel, along with current experimental research work in the field. The book will be of great interest to advanced undergraduates, postgraduates and researchers in renewable energy, as well as to fuel engineers. Written for: Advanced undergraduates, postgraduates and researchers in renewable energy; fuel engineers
Product Details
- 9781846289941
- Publisher:
- Springer London
- Publication date:
- 03/20/2008
- Edition description:
- 2008
- Pages:
- 208
- Product dimensions:
- 6.20(w) x 9.30(h) x 0.70(d)
Table of ContentsIntroduction 1
Introduction to Energy Sources 1
Global Energy Sources and the Present Energy Situation 3
Renewable Energy Sources 6
Biomass Energy and Biomass Conversion Technologies 8
Hydropower 23
Geothermal Energy 24
Wind Energy 25
Solar Energy 27
Biohydrogen 28
Other Renewable Energy Sources 30
References 33
Biofuels 39
Introduction to Biofuels 39
Bioethanol 42
Biomethanol 45
Biohydrogen from Biomass by Steam Reforming 49
Steam-reforming Process 50
Fuels from Bio-syngas via Fischer-Tropsch Synthesis 51
Biodiesel 56
Bio-oil 57
Global Biofuel Scenarios 59
References 60
Vegetable Oils and Animal Fats 65
Use of Vegetable Oils and Animal Fats in Fuel Engines 65
Vegetable Oil Resources 67
Inedible Oil Resources 69
Vegetable Oil Processing 72
Recovery of Vegetable Oils from Plants 72
Vegetable Oil Refining 73
The Use of Vegetable Oils as Diesel Fuel 74
Physical and Chemical Properties of Vegetable Oils 75
Direct Use of Vegetable Oils in Diesel Engines 79
New Engine Fuels from Vegetable Oils 83
Pyrolysis of Vegetable Oils and Fats 83
Cracking of Vegetable Oils 85
Pyrolysis Mechanisms of Vegetable Oils 86
Gasoline-rich Liquid from Sunflower Oil by Alumina Catalytic Pyrolysis 88
Diesel-like Fuel from Tallow (Beef) by Pyrolysis and Steam Reforming 91
Converting Triglyceride-derived Synthetic Gas to Fuels via Fischer-Tropsch Synthesis 95
Triglyceride Analyses 99
Viscosity 99
Density 100
Cetane Number 100
Cloud and Pour Points 101
Distillation Range 101
Heat of Combustion 101
Water Content 102
Discussion of Fuel Properties of Triglycerides 102
Triglyceride Economy 105
References 105
Biodiesel 111
Introduction to Biodiesel Concept 111
History 112
Definitions 114
Biodiesel as an Alternative to Diesel Engine Fuel 115
Sources of Biodiesel 117
References 118
Biodiesel from Triglycerides via Transesterification 121
Biodiesel from Triglycerides via Transesterification 121
Catalytic Transesterification Methods 123
Supercritical Alcohol Transesterification 125
Biocatalytic Transesterification Methods 132
Recovery of Glycerine 133
General Reaction Mechanism of Transesterification 133
Esterification of Fatty Acids with Diazomethane 137
Non-catalytic Supercritical Alcohol Transesterification 137
Enzyme-catalyzed Processes 138
References 139
Fuel Properties of Biodiesels 141
Viscosity, Density, and Flash Point 141
Cetane Number, Cloud Point, and Pour Point 144
Characteristics of Distillation Curves 145
Higher Combustion Efficiency of Biodiesel 145
Water Content 146
Comparison of Fuel Properties and Combustion Characteristics of Methyl and Ethyl Alcohols and Their Esters 146
Advantages and Disadvantages of Biodiesels 151
Advantages of Biodiesel as Diesel Fuel 151
Availability and Renewability of Biodiesel 151
Lower Emissions from Biodiesel 152
Biodegradability of Biodiesel 155
Thermal Degradation of Fatty Acids During Biodiesel Production 156
Disadvantages of Biodiesel as Diesel Fuel 157
References 158
Current Technologies in Biodiesel Production 161
Biodiesel Production Processes 166
Primary Raw Materials Used in Biodiesel Production 166
Biodiesel Production with Batch Processing 167
Biodiesel Production with Continuous Process 168
Biodiesel Production with Non-catalyzed Transesterification 169
Basic Plant Equipment Used in Biodiesel Production 171
References 172
Engine Performance Tests 175
Engine Combustion Process and Combustion-related Concepts 177
Engine Performance Tests 179
Alcohol-diesel Emulsions 179
Using Microemulsions for Vegetable Oil 180
Diesel Engine Fumigation 180
Dual Injection 180
Injector Coking 181
Heated Surfaces 181
Torque Tests 181
Spark Ignition 181
Oxidation 182
References 182
Global Renewable Energy and Biofuel Scenarios 185
Global Renewable Energy Sources 187
Renewable Energy Scenarios 189
References 193
The Biodiesel Economy and Biodiesel Policy 195
Introduction to the Biodiesel Economy 195
Economic Benefits of Biodiesel 197
Biodiesel Costs 199
General Biodiesel Policy 201
European Biofuel Policy 202
References 203
Index 205
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Posted 03/14/2012 6:44 pm
Nice coin VF20 I agree, I see the hair lines but could that be the reflection off the plastic. That was my impression.
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The U.S. Senate was slated today to vote on its version of the economic-stimulus package that contains far less aid for schools than the version approved by the U.S. House. The Senate version eliminates $20 billion in funds to renovate school buildings and halves educational aid to states to $39 billion. Education Secretary Arne Duncan called the proposed $39 billion "not nearly as much as we need."
Full Story:NYTimes.com


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Free personalized radio that
plays the music you love
Failed Territory
Condemned pressure cooker, that explodes in your face
Another neighborhood gets destroyed by the drug deal
Staking a claim on an estate that is real
Features of This Trackhard rock roots
rap influences
the use of experimental sounds
a subtle use of vocal harmony
repetitive melodic phrasing
minor key tonality
a dirty electric guitar solo
a good dose of acoustic guitar pickin'
a gravelly male vocalist
an aggressive male vocalist
a partially spoken vocal delivery
mallet percussion
acoustic rhythm guitars
emphasis on instrumental arranging
intricate arranging
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LOL We Went to Russia: Think Thank narrowly makes it to the Caucasus Mountains
Max Warbington, Ted Borland, and Sean Lucey head to a Russian snowboard camp.Read More
Max Warbington, Ted Borland, and Sean Lucey head to a Russian snowboard camp.Read More
The trip that almost wasn't.Read More
There are few things better than the time-honored tradition of packing your bags, grabbing your buddies, and stuffing yourselves and the bags into a travel-ready vehicle for a road trip.Read More
The day every season when the new Think Thank teaser drops is one of our favorite days of the year. We've been eyeing the garbage lids and traffic cones, watchingRead More
Locator of unique spots, master of easy style, champion of the Bone Zone and haver of ender in Think Thank's Methods of Prediction, Ted Borland is a rider who defiesRead More
Ryan Paul, Brandon Reis, Phil Hansen, Mikey Swearingen, and "everyone else [they] met along the way."Read More
Mitch Richmond's hammer heavy part.Read More
From Think Thank's 2015 boarding blockbuster, Methods of Prediction, presenting the full part of Minnesota-grown Ryan Paul. RP is no stranger to the esoteric and creative side of snowboarding; heRead More
Bundy and Reis start off winter in Utah.Read More
The average day for Brandon Reis this winter. Wake up, wake up the crew staying at your house, make sure they are all happy, load all the gear in yourRead More


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Electra Heart
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2012 release, the sophomore album from Welsh singer songwriter Marina Lambrini Diamandis AKA Marina & The Diamonds. Electra Heart is not so much a creative leap forward, more an Olympian pole-vault over the bar of talented-newcomer into the global amphitheater of a cultivated Classic. Two years on from her top 5 debut The Family Jewels, the self-styled avant-garde D.I.Y artist has detonated her own experimental past and landed feet first in the future with Electra Heart, a stunningly ambitious, seamless, cohesive and confident sonic pulsar spinning between electro-pop euphoria and come-down melancholia. The album is produced by a cache of old school and A-List producers: Dr Luke (Katy Perry) and Liam Howe (Sneaker Pimps) but mostly ( nine out of 12 songs) Greg Kurstin (Lily Allen, Kylie) and Rick Nowels (Madonna, Stevie Nicks, Lykke Li).
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only down side was the case had a small crack in it, but i blame it on the fact it probably got dropped somewhere along the way.
Ironically, what the trouble seems to be is that she really DOES want to be Ms. Wonderful. She has been recorded as saying that she released Primadonna as the British single in a deliberate attempt to change her career, i.e. to BECOME A STAR (or more of one). Hence too the bland dance-pop of "Radioactive", released in the US but not officially part of this album (only on the deluxe version). The blonde dye-job and/or wig seems to be part of this campaign (and I for one don't find the new look flattering at all).
Ah, well, what can you do.Read more ›
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Marina & the Diamonds is a goddess. Though I liked this album a little less than Family Jewels, it is still a very good albumPublished 2 months ago by tangycandy


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Pacific officials made a phone call to Brush Creek Sewer District Director John Griesheimer offering to set up a meeting to discuss a lawsuit the city has filed against the sewer district, according to Mayor Herb Adams.
The directors of the sewer district are the three Franklin County commissioners — Griesheimer, Mike Schatz and Tim Brinker.
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Teespring has closed its Davol Square office, marking the last phase of a restructuring that began over the summer, a company spokeswoman said Monday. more
By Lori Stabile
(Updated 1:41 p.m.) A Canadian company wants to locate a cement distribution plant at the Port of Providence, which would act as a regional transfer center for its products through the Northeast. more
By Mary MacDonald
Passenger traffic fell 1.7 percent in July at T.F. Green Airport to 340,390 from 346,416 in July 2014, according to R.I. Airport Corp., which released data on Tuesday. more
By Lori Stabile
Rhode Island's economic momentum not only slowed this January compared with last year, it had been slowing during 2015 more than originally thought, according to University of Rhode Island economist Leonard Lardaro. more
By Patricia Daddona
Owner: Mott & Chace Sotheby's International Realty Architect: Saccoccio & Associates Architects, Cranston General Contractor: Case Construction Co., East Providence Cost: Not disclosed … more
PROVIDENCE – Single-family home sales in Rhode Island rose 24 percent in February, and median prices climbed as well, both indications of what the Rhode Island Association of Realtors described as a "robust market." The median sales price … more
For the 27th consecutive month on a year-over-year basis, foreclosure petitions rose in Massachusetts in May, according to figures released by The Warren Group on Thursday. more
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PVDFest, Rhode Island's signature outdoor art festival, is returning for a second year June 2 through 5, bringing four days of art, food and music to Providence's streets. more
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Rhode Island is being granted $17.8 million in federal funding to help Providence and Cranston hire and train new firefighters, the state's congressional delegation announced Wednesday. more
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JPMorgan Chase & Co. tumbled to No. 10 in an annual survey of customer satisfaction for mortgage-servicing companies, behind competitors such as Bank of America Corp., after being ranked second-best by homeowners last year. more
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Quite often, I need a wider board than what I have available. Usually, that means gluing up two or more boards edge to edge.
Since, in this situation, I am usually making a highly visible part such as a table top or cabinet side, I am very careful to match not only the grain pattern of the neighbouring boards, but the exact colour shading as well. 271 more words


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ANNAPOLIS, Md. — Anne Arundel County police say a 22-year-old Naval Academy midshipman has been charged with assault after threatening a cab driver with a butter knife.
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Welcome to Pittsburgh City Council District 2!
Kids Ride for $1 During Summer Holiday Weekends
All kids under the age of 12 are able to ride the Goodship Lollipop for just $1 during summer holiday weekends. These weekends include Memorial Day Weekend, Fourth of July Weekend and Labor Day Weekend. Grab the whole family for a fun holiday weekend aboard one of thier ships!
Contact Councilwoman Theresa Kail-Smith
Love Your Block Improves District 2's Memorials
Summer Musical Theater
Interested in singing, dancing, and acting? Check out our Musical Theater Summer Day Camp for ages 4-19.


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Carnegie Chamber of Commerce updated their cover photo. ... See MoreSee Less
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We are now accepting entries! As a treat this year we will have water, fruit and COOKIES at the finish line! What your favorite cookie? ... See MoreSee Less
Carnegie chamber meeting will be this coming Monday the 13th at noon at the Carnegie hospital. Hope to see everyone there!!! ... See MoreSee Less
The chamber meeting is today at noon! If you can make it out we will meet at the church of Christ. Hope to see you there! ... See MoreSee Less
Terri Johnson presents Carnegie School librarian Billie Fobes (left) with cookies provided by the chamber during Teacher Appreciation Day! Thanks teachers for all you do!! ... See MoreSee Less
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Under this market pressure can dividends and distributions hold in the markets and in FRO?
depends on what you define as hold. Will they still give a dividend, of course. Will they give out another $3 dividend. Probably not this quarter. Look at FROs distributions over the next few years and you will see that they have always been volatile.
As a percentage of share price .... easily!


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Presenting: A New Way To Bring Home The Bacon
Photo: Courtesy of Jetel Fogelson.
There's a new way to bring home the bacon that has nothing to do with cash. We're talking about the new Burke's Bacon Bar that just opened shop in The James Chicago. But be warned — if you're a vegetarian, you may want to skip this sizzling and crispy info.
While we'll always love David Burke's Primehouse, when a meal needs to be quick and dirty, there's nothing better than a sandwich to get the job done — especially when bacon is involved. Along with mini swine sammies ($4 each or three for $11), this bacon-influenced eatery offers satisfying (and non-calorie friendly) salads, sides, and desserts in a to-go fashion. Our picks? Crack Snacks, crispy tortilla strips with bacon and spices. And for your sweet-yet-savory tooth, there's bacon peanut brittle to motivate you to run an extra mile or two on the treadmill. Better yet, maybe you should get out those oversized sweaters a bit early.
Burke's Bacon Bar, 610 North Rush Street (at The James); 312-660-7200.
- Chicago | Burke's Bacon Bar Opening, James Hotel
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text:
12 Nov 2014:
The PLOS ONE Staff
Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.
Citation: Li W, Zhao Q, Deng W, Chen T, Liu M, Xie J (2014) Mycobacterium tuberculosis Rv3402c Enhances Mycobacterial Survival within Macrophages and Modulates the Host Pro-Inflammatory Cytokines Production via NF-Kappa B/ERK/p38 Signaling. PLoS ONE 9(4): e94418. doi:10.1371/journal.pone.0094418
Editor: Shamala Devi Sekaran, University of Malaya, Malaysia
Received: September 5, 2013; Accepted: March 16, 2014; Published: April 10, 2014
Copyright: © 2014 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Natural Science Foundation (grant numbers 81371851, 81071316, 81271882), National Megaprojects for Key Infectious Diseases (grant numbers 2008ZX10003-006, 2012ZX10003-003), New Century Excellent Talents in Universities (grant number NCET-11-0703), Excellent PhD thesis fellowship of Southwest University (grant numbers kb2010017, ky2011003), the Fundamental Research Funds for the Central Universities (Nos. XDJK2011D006, XDJK2012D011, XDJK2012D007 and XDJK2013D003), Natural Science Foundation Project of CQ CSTC (grant number CSTC 2010BB5002), the Chongqing Municipal Committee of Education for postgraduates excellence program (No. YJG123104), the undergraduates teaching reform program (No. 2011JY052). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Tuberculosis (TB), caused by the facultative intracellular pathogen Mycobacterium tuberculosis (Mtb), remains a major cause of morbidity and mortality around the world. Despite intense efforts to mitigate its toll on humanity, TB was still responsible for 1.3 million deaths, and caused 8.6 million new and relapse infections in 2012 (World Health Organization Tuberculosis Data and Statistics, 2013). In addition, the emergence of drug resistant Mtb strains and co-infection with HIV further highlighted the necessity of novel TB medications informed by in-depth understanding of Mtb biology.
Upon internalization by macrophages, Mtb is widely assumed to be confined within the phagosome, a specialized vacuole derived from the plasma membrane. Subsequently, the phagosome matures through fusion with late endosomes and lysosomes, creating an inhospitable environment for invading microorganisms, that includes phagosomal acidification, elevated levels of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), charged antimicrobial peptides [1] and iron deprivation [2]. However, Mtb has evolved a spectrum of subversive strategies to survive and thrive inside the macrophages [3].
The success of Mtb can be attributed to an array of virulence factors. These effectors can be classified as (a) enhancing the resistance to host toxic compounds, such as KatG [4], SodC [5], [6], AhpC [7], [8], (b) blocking phagosomes maturation, such as PtpA [9], [10], PknG [11], [12], SapM [13], [14], and (c) evading apoptosis through NuoG [15], [16], [17] and SodA [18], [19]. The identification and characterization of novel virulence factors can enrich our understanding of Mtb biology and facilitate better control measures.
Iron scavenging capacity is critical for pathogen proliferation and pathogenesis within host [20]. The Mtb ORF rv3402c, encoding an IdeR- and iron-repressed gene, was found to be significantly upregulated during Mtb growth under iron-limiting conditions [21]. Interestingly, rv3402c was further shown to be induced in human THP-1 [21] and primary bone marrow-derived murine macrophages [22], suggesting a role in the adaptation to intracellular niches.
In the following study, we investigated the role of Rv3402c through the construction of recombinant M. smegmatis expressing strains. We discovered that Rv3402c was able to enhance M. smegmatis' intracellular survival as well as alter the cytokine profile of infected macrophages.
Materials and Methods
Bacterial strains and growth conditions
Escherichia coli and M. smegmatis mc2 155 strains were provided by the Institute of Modern Biopharmaceuticals. Mtb H37Rv genomic DNA was provided by Chongqing Pulmonary Hospital. The human leukemic monocyte lymphoma cell line (U-937) was purchased from the Conservation Center in Wuhan University (China), and the murine macrophage cell line (RAW264.7) was a kind gift from Zhiren Zhang [23] (Third Military Medical University, China). Escherichia coli DH5α was routinely grown in LB medium for its use in DNA cloning procedures. M. smegmatis mc2 155 was grown at 37°C in Middlebrook (MB) 7H9 liquid medium or on MB 7H10 agar supplemented with 0.2% (w/v) glucose, 0.5% (v/v) glycerol and 0.05% (v/v) Tween 80. When required, kanamycin (20 μg/ml) or hygromycin (50 μg/ml) was added.
Gene amplification, plasmids construction, and recombinant M. smegmatis
The primers used in this study are listed in Table 1. The pNIT-1 plasmid used in this study has been previously described [24]. Briefly, pNIT-Myc was constructed from the pNIT-1 mycobacterial shuttle vector harboring a Myc-tag to the C-terminal of Rv3402c in order to perform Western blot. The full length rv3402c gene was amplified from Mtb H37Rv genomic DNA. Amplified rv3402c gene was then cloned into the pNIT-Myc vector. Constructs for the plasmid pALACE-Rv3402c (at the N terminus) were made by using the similar method as pNIT-Myc-Rv3402c. The gfp gene was excised from pSC301 [25] by digesting with BamHI and ClaI, then cloned into pALACE digested with the same restriction enzymes. The plasmids were electroporated into M. smegmatis mc2 155 according to standard procedures [26]. The recombinant M. smegmatis strains were selected on MB 7H10 agar containing kanamycin (for pNIT-Myc) or hygromycin (for pALACE). Gene expression was confirmed by PCR, and the recombinant strains were stored at −70°C until further use.
Expression of Rv3402c in M. smegmatis
The recombinant M. smegmatis strains expressing Myc-tagged Rv3402c (MS_Rv3402c) and the empty pNIT-Myc vector (MS_Vec) were cultured until an OD600 0.6–0.8 was reached. Protein expression was induced with 28 mM ε-caprolactam (Aladdin, China). Total bacterial RNA was isolated using RNAprep Pure Cell/Bacteria Kit (TIANGEN, China) after 16 h induction. An equal amount of total RNA was used as a template for reverse transcriptase (RT)-PCR with rv3402c specific primes and mycobacterium 16 S rRNA (rrsB gene) specific primers. The RT-PCR products were visualized using a 1% agarose gel.
For the detection of Myc-tagged Rv3402c bacterial pellets were harvested and disrupted by ultrasonication. Samples were subjected to SDS-PAGE and the Myc-tagged Rv3402c protein was detected by the mouse anti-Myc antibody (TIANGEN, China).
Subcellular fractionation of M. smegmatis
Subcellular fractionation was performed as described by Bashiri et al. [27], with minor modifications. In short, 200 ml of M. smegmatis was grown to an OD600 ∼0.8 and was induced with 28 mM ε-caprolactam. Next, the cells were harvested, resuspended in 9 ml lysis buffer (PBS, 1 mM PMSF, 0.6 mg/ml of both DNase and RNase) and then lysed by ultrasonication. Lysates were centrifuged at 3,000 g for 30 min to obtain the whole cell lysates (WCL) from the supernatant. WCL was centrifuged at 27,000 g for 30 min to obtain the cell wall pellet (CW). The supernatant from the WCL fraction was further centrifuged at 100,000 g for two hours to separate the cell membrane fraction (pellet, CM) from the soluble fraction (supernatant, SOL). CW and CM fractions were washed once with lysis buffer, re-centrifuged and subsequently re-suspended in 0.5 mL of lysis buffer. All centrifugation steps were performed at 4°C.
The above fractionated samples were subjected to SDS-PAGE, with the ratio of protein concentrations at 1∶0.36∶0.71∶0.91 (WCL∶CW∶CM∶SOL). After separation, proteins were transferred to a nitrocellulose membrane. The Myc-tagged Rv3402c protein was detected by the mouse anti-Myc antibody. Native M. smegmatis GroEL2, which contains a string of endogenous histidines [28], was detected by an anti-His mouse primary antibody (TIANGEN). The secondary antibody used was goat anti-mouse conjugated to HRP (TIANGEN). The signal was detected using Western Lighting ECL.
Proteinase K and trypsin sensitivity assays
Proteinase K and trypsin sensitivity assays were performed as previously described [29]. Briefly, M. smegmatis harboring pALACE, pALACE-Rv3402c or pALACE-GFP were induced with 0.2% acetamide for 10 h and harvested. Cells were washed and mixed with proteinase K or trypsin at a concentration of 100 μg/ml and incubated at 37°C for the indicated times. The reaction was stopped by adding 100 nM PMSF. The samples were dissolved in SDS loading buffer and analyzed by Western blotting using anti-His mouse primary antibody (Abm Inc.) or anti-GFP mouse primary antibody (Boehringer Mannheim Corp.). The secondary antibody used was Alexa Fluor 660 Goat Anti-Mouse IgG (Life technologies Corp.). The signal was detected using Odyssey CLx Infrared Imaging System.
Purification of recombinant Rv3402c protein
Recombinant Rv3402c (rRv3402c) was expressed in E. coli according to a published protocol [30]. Briefly, the full length rv3402c gene was cloned into the pET-28a(+) vector and the protein was purified by His-tag affinity chromatography on a Ni2+ - nitrilotriacetate column. Purified rRv3402c was dialyzed against PBS (pH 7.2), and then applied to an E-TOXATE Kit (Sigma, USA) to remove any endotoxin contamination prior to filter-sterilization, and then stored at −70°C until further use.
Intracellular survival assay
Suspension cultures of U-937 cells were seeded at 1×106 cells per well in 12-well tissue culture plates. Following 48 hours of treatment with 0.1 mg/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma), U-937 cells were transformed into an adherent state. Cells were infected with M. smegmatis transformants at a MOI of 10∶1 (bacteria-to-U-937 ratio) at 37°C in 5% CO2. Four hours after infection, the remaining bacteria in the culture were removed by washing three times with warm RPMI-1640. RPMI 1640–10% FCS containing 250 nM IVN (Sigma) in DMSO and hygromycin B (Roche, USA) (100 μg/ml) were then added. At 6 and 72 h after infection, the culture supernatants were collected, and stored at −70°C. The macrophages were then washed twice and lysed in sterile water containing 0.025% (w/v) SDS. The lysed macrophages were plated on MB 7H10 agar plates containing kanamycin and the colony forming units (CFUs) were determined as a measure of the intracellular survival of recombinant M. smegmatis. The intracellular survival assay of M. smegmatis in RAW264.7 cells was performed in a similar manner as described for the U-937 cells above. By comparison, RAW264.7 cells were seeded at 5×105 cells per well in 12-well tissue culture plates. Cells were infected with M. smegmatis transformants at a MOI of 10∶1 after 24 hours.
In vitro growth and stress assays
For in vitro growth curves, cultures were inoculated in triplicates with a starting absorbance (OD600) of ∼0.02. Broths were incubated at 37°C with shaking until an OD600 of ∼0.8 was reached. Twenty-eight micromolar of ε-caprolactam was added to the cultures, and the OD600 was measured at various time points over a 36 h growth period. To assess the growth curve of recombinant M. smegmatis in iron-depleted medium, the recombinant strains were grown with 100 μM 2′ 2′ dipyridyl [31]. Growth was monitored by determining the OD600 at various time points over 50 h.
To prepare for the pH stress assays [32], cells were harvested, washed with 7H9 (pH 3 or 5) and then resuspended to an OD600 of 0.5 in 5 ml 7H9 (pH 3 or 5). Cultures were incubated under stress conditions with agitation and 100 μl samples were removed for viable cell counts after 0, 3 or 6 h. To assess the survival of recombinant M. smegmatis after exposure to peroxide stress, the cells were washed and resuspended in 7H9 (pH 5). Five milliliters of each M. smegmatis strains, diluted to OD600 of 0.5 in 7H9 (pH 5), was exposed to 5 mM H2O2. At 0 and 6 h, 100 μl samples were removed to determine the viable count. All stress experiments were carried out in triplicates.
Measurement of LDH release from macrophage
Release of lactate dehydrogenase (LDH) by the U-937 infected with recombinant M. smegmatis was measured to evaluate the extent of cytolysis. Culture supernatants were harvested after infection of macrophages with MS_Rv3402c or MS_Vec for 6, 24, 48 or 72 h. LDH activity in culture supernatants was assayed with the CytoTox96 Non-radioactive Cytotoxicity Assay Kit (Promega, USA) as described in the manufacturer's instruction. The percentage of LDH release was calculated according to published methods [33].
Assay for cytokine production
U-937 cells were infected at 6 and 72 h time points, and processed for analysis by a sandwich ELISA. The concentration of cytokines in the culture supernatant was determined using commercially available ELISA kits for tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β) (eBioscience, USA). All assays were performed as recommended by the manufacturer's protocols.
After infection of macrophages with MS_Rv3402c or MS_Vec for 12 h, total RNA was extracted with the RNAprep Pure Cell/Bacteria Kit. The DNAase-treated total RNA (∼1 μg) was transcribed into cDNA with random hexamers using the Transcriptor First Strand cDNA Synthesis Kit (Roche, USA). Semi-quantitative RT-PCR was performed as mentioned above with primers described in Table 1 for the TNF-α, IL-1 β, and β-actin genes.
Statistical analysis
Data was analyzed using a Student's two-tailed t-test. Statistical significance was defined as a p- value of 0.05. Error bars are representative of standard deviation (SD).
Expression of rv3402c gene in M. smegmatis
The Mtb ORF rv3402c is approximately 1.2 kb in size and encodes for 45 kDa protein. In this study, we generated two recombinant M. smegmatis strains to investigate the role of Rv3402c in host interaction. The MS_Rv3402c strain was engineered to express a Myc-tagged Rv3402c protein from a recombinant pNIT-Myc vector, while the MS_Vec strain harbored the vector alone. Both MS_Rv3402c and MS_Vec, which were grown in MB 7H9 medium in the presence of kanamycin, expressed the aph gene [34]. The semi-RT-PCR results determined that only MS_Rv3402c was able to express the rv3402c gene (Fig. 1A). Western blot analysis using the anti-Myc antibody further confirmed the presence of the expressed ∼50-kDa Rv3402c-Myc fusion protein in the cell lysates of the recombinant strain, and its absence in the parental strain (Fig. 1B). These findings indicate that the Rv3402c protein from M. tuberculosis was successfully expressed in M. smegmatis.
(A). Ms_Rv3402c and Ms_Vec were grown at 37°C in MB 7H9 liquid medium to an OD600 of 0.6–1.0. Total bacterial RNA was isolated after 16 h induction and subjected to RT-PCR to detect expression of the rv3402c and rrsB genes. (B). Lysates were prepared from bacterial cells that were cultured as in (A) and subjected to Western blot analysis to detect Myc-tagged Rv3402c protein using mouse anti-Myc antibody.
Rv3402c is associated with the mycobacterial cell envelope
Rv3402c is predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules [21]. Moreover, Rv3402c probably involved in cell process, its sequence shows high similarity to the lipopolysaccharide biosynthesis protein from Bacillus cereus and a putative PLP-dependent enzyme from Rheinheimera sp., which is predicted to be involved in cell wall biogenesis. We therefore tested whether Rv3402c was present in mycobacterial cell wall and cell membrane fractions. We expressed Myc epitope-tagged Rv3402c in M. smegmatis; separated protein extracts into whole cell, cell wall, cytoplasmic, and cell membrane fractions; and analyzed in immunoblot. Although the majority of the Rv3402c proteins were detected in the soluble fraction, small amounts were also seen in the cell wall and cell membrane fractions (Fig. 2A), indicating that Rv3402c is associated with the cell envelope. However, Rv3402c was not detected in culture supernatants and is thus not likely to be secreted extracellularly (data not shown). As controls for fractionation, we show that cytoplasmic heat-shock protein, GroEL2 was detected only in the cytoplasm.
(A). Subcellular fractionation of M. smegmatis induced to express Rv3402c-myc showed localization to the cell wall and membrane fractions. Subcellular fractions were separated by SDS-PAGE and proteins were detected with an anti-Myc antibody. Native GroEL2 was detected as a cytoplasmic control. WCL, whole cell lysates (66 μg total protein); CW, cell wall fraction (24 μg total protein); CM, cell membrane fraction (47 μg total protein); SOL, soluble fraction (60 μg total protein). (B and C). M. smegmatis transformed with pALACE, pALACE-GFP or pALACE-Rv3402c were incubated with proteinase K or trypsin at different time points. Whole-cell lysates obtained by the recombinant bacteria were separated on SDS-PAGE. Immunoblots were performed using anti-His antibodies and anti-GFP antibodies. Similar results were obtained in two independent experiments.
To further confirm the cell surface association of Rv3402c, M. smegmatis overexpressing Rv3402c was subjected to proteinase K and trypsin sensitivity assays as previously described [29], [35]. In order to avoid the bias of plasmid pNIT-Myc-Rv3402c, we chose another widely used plasmid pALACE to perform this experiment. As shown in Fig. 2B and C, the His epitope-tagged Rv3402c protein overexpressed by M. smegmatis was partly digested by the proteinase K and trypsin treatment, respectively, even the reaction time prolonged to 30 min. Conversely, the recombinant M. smegmatis strain expressing only GFP was protected from digestion. Taken together, although the majority of the Rv3402c proteins are localized in the cytoplasm, these results demonstrate that Rv3402c protein is exposed at the mycobacterial cell surface and might gain access to the extracellular environment.
Rv3402c increases the survival of recombinant M. smegmatis in macrophages
Using comparative genomic analysis, no homologus gene could be identified for rv3402c in non-pathogenic species such as M. smegmatis, M. gilvum and M. vanbaalenii. Interestingly, in the attenuated vaccine BCG Pasteur strain, the rv3402c ortholog is disrupted into two fragments. One portion of the fragment is annotated as a pseudogene, the other, a 1053 bp gene known as BCG3472c (99% identity in DNA sequences). This comparative genomic analysis suggested that rv3402c might be a novel virulence factor as this gene is not present in non-pathogenic species. To gain further insight as to whether rv3402c is essential for Mtb pathogenesis, we analyzed the intracellular survival of M. smegamatis harboring the gene. To achieve this, we infected the mono-layers of U-937 with MS_Vec and MS_Rv3402c as described in the methods section (MOI = 10∶1). The two strains were easily dispersed into single cell suspensions, and the CFU determinations at six hours demonstrated that infection rates were similar in U-937 cells (data not shown). M. smegmatis expressing Rv3402c showed significantly higher bacillary counts in U-937 cells at 48 and 72 hours post infection (Fig. 3A), but differences in survival was only observed at 72 h in the RAW264.7 cells (Fig. 3B). Despite these differences, these results suggest that the expression of Rv3402c was able to enhance the intracellular survival of M. smegmatis in both human and murine macrophages.
U-937 cells (A) and RAW267.4 cells (B) were infected with MS_Vec and MS_Rv3402c as described in the methods section, respectively. Aliquots of infected cells were lysed with 0.025% SDS at indicated times, and serial dilutions were plated on 7H10 agar plates containing kanamycin. Recovered CFUs were enumerated after the incubation for 3–4 days at 37°C. Numbers of intracellular bacteria are shown as a percentage of the numbers detected at t = 6 h (U-937) or 4 h (RAW267.4) (% survival). Data are shown as means ± SD of triplicate wells. Similar results were obtained in three independent experiments.
Enhanced intracellular survival is not correlated with increased resistance to antimicrobial factors
To gain further insight as to how the M. smegmatis strain harbouring rv3402c confers greater survival in macrophages, we analyzed growth characteristics between the two strains. As seen in Fig. 4A, the two recombinant strains show the same growth kinetics in vitro, suggesting that enhanced survival is likely due to the interaction between mycobacteria and its host. Within the phagosome, the invading microbe is exposed to hostile environment, including iron-deprived conditions, reactive oxygen and reactive nitrogen compounds, and low pH conditions. As a result, we examined whether Rv3402c confers resistance to any of these intracellular stresses.
(A). Growth of MS-Vec and MS-Rv3402c at 37°C in MB 7H9 liquid medium was monitored by determining OD600 at intervals of 3 h. (B). Growth curve of MS-Vec and MS-Rv3402c in iron-depleted medium. Two recombinant M. smegmatis were grown in MB 7H9 medium supplemented with 100 μM 2′ 2′ dipyridyl. The growth of the strains were monitored by measuring OD600 at intervals of 6 h. (C). In vitro growth of MS-Vec and MS-Rv3402c at different pH. The bacteria were collected by centrifugation and resuspended to an OD600 of 0.5 in 5 ml MB 7H9 (pH 5 or 3). All cultures were again incubated at 37°C and 0.1 ml removed for viable count enumeration after 0, 3 and 6 h. (D). Survival of recombinant M. smegmatis after exposure to hydrogen peroxide. Aliquots (5 ml) of cultures at OD600 of 0.5 were exposed to 5 mM hydrogen peroxide (H2O2) for 6 h at 37°C. The cultures were serially diluted and plated onto MB 7H10 plates and the colonies counted after 3–4 days of incubation at 37°C.
Given that rv3402c is repressed by IdeR and induced by iron limited conditions [21], we explored whether increased survival was due to M. smegmatis' resistance to low iron conditions in the macrophage. We compared the ability of the MS_Rv3402c to that of the MS_Vec to grow in MB 7H9 medium in the presence of 2′ 2′ dipyridyl, an iron chelator. No significant differences were observed between the MS_Rv3402c and the empty vector strain in the presence of 100 μM 2′ 2′ dipyridyl (Fig. 4B).
To assess acid sensitivity, growth of the two recombinant M. smegmatis strains was monitored in MB 7H9 at pH 3.0 and 5.0, and was compared at three time points during a six hours incubation. No growth differences were observed between these two strains at any time (Fig. 4C). Similarly, susceptibility of the two recombinant strains to ROI was observed following six hours exposure to 5 mM H2O2. The percentage of survival of MS_Vec and MS_Rv3402c was 123.3% and 116.7%, respectively, with no difference observed between the two recombinants (Fig. 4D). Taken together, these results suggest that the increased survival of MS_Rv3402c in the macrophage cannot be attributed to the increased resistance to any of the tested stresses.
The effect of the recombinant M. smegmatis expressing Rv3402c on the viability of macrophages
One of the destinies of macrophages infected with Mtb is cell death due to the pathogen's ability to alter host cell signaling pathways. To test the effect of Rv3402c on macrophage viability, U-937 cell lines were infected by recombinant M. smegmatis containing Rv3402c or the control strain. The amount of LDH was measured in the culture supernatants to quantify cell lysis. Six hours following infection with MS_Rv3402c, there was a significant release of LDH that increased to a range of 49.8% and 50.4% over 72 hours in culture (Fig. 5). In contrast, the percentage of LDH release did not exceed 31% in cultures infected with MS_Vec. These results show that infection with the M. smegmatis strain harbouring rv3402c was able to induce macrophage lysis.
Culture supernatants were collected from mono-layers of U-937 infected at a MOI of 10∶1 with MS_Vec or MS_Rv3402c and the release of LDH as a measure of macrophage cell death was estimated at various time points. Data are shown as means ± SD of triplicate wells. Similar results were obtained in three independent experiments.
Rv3402c upregulates TNF-α and IL-1β production
To explore the potential role of Rv3402c in subverting the innate immune response, we investigated the levels of pro-inflammatory cytokines upon infection of U-937 with MS_Vec and MS_Rv3402c. Macrophages infected with MS_Rv3402c secreted significantly higher levels of TNF-α and IL-1β than the MS_Vec infected cells (Fig. 6A and C). To confirm these observations, the pro-inflammatory cytokine mRNA expression was determined using semi-RT-PCR. Compared to the MS_Vec, mRNA levels of TNF-α (Fig. 6B) and IL-1β (Fig. 6D) were significant upregulated in macrophages after 12 hours of infection with MS_Rv3402c. Similar results were obtained when U-937 cells were stimulated with rRv3402c (Fig. 7A and B). No difference was found for the levels of other inflammatory proteins, including IL-6, IL-10, and IL-12 p40 (data not shown). Collectively, our data suggests that Rv3402c might play a role in modulating the pro-inflammatory cytokine production of macrophages infected with M. smegmatis.
Culture supernatants were harvested after 6 or 72-α (A) and IL-1β (C) were determined. Cells were harvested after 12 h of infection and semi-quantitative RT-PCR analysis of TNF-α (B) and IL-1β (D) mRNA level was performed. Each three lanes in the RT-PCR analysis are replicates of a single time point. Similar results were obtained in three independent experiments.
Culture supernatants were harvested after 12 μg/ml and the concentrations of TNF-α (A) and IL-1β (B) were determined. U-937 cells were pre-treated with 30 μM TPCK (a NF-κB inhibitor) or with 20 μM PD 98059 (an ERK1/2 inhibitor) or with 10 μM SB 202190 (a p38 inhibitor). Treatment with DMSO served as a control for the inhibitor treatments. After 1 h, the macrophages were infected with MS_Vec at an MOI of 10 or treated with rRv3402c at the concentration of 5 μg/ml. Protease K (K) used to digest the recombinant protein. Culture supernatants were harvested after 24 h of infection and the concentrations of TNF-α (C, E) and IL-1β (D) were determined. (A), (B) and (E) are performed with recombinant protein; (C) and (D) are performed with M. smegmatis strains. The data are representative of two independent experiments.
Rv3402c robustly activates macrophage NF-κB, ERK and p38 signaling
NF-κB is a major transcription factor responsible for the transcription of TNF-α [36], [37], [38]. ERK 1/2 activation is essential for TNF-α production by macrophages [39], [40], and the MAPK pathways are crucial for macrophage signaling during mycobacterial infection [39], [41]. The increased TNF-α and IL-1β mRNA and protein expression (Fig. 6) raised the speculation that the activation of these signaling pathways might be altered within macrophages infected with MS_Rv3402c. To test this possibility, pharmacological inhibition experiments were used to confirm the requirement for NF-κB, ERK and p38 activities in the production of TNF-α and IL-1β in macrophages infected with MS_Rv3402c. MS_Rv3402c-induced TNF-α expression and protein production were significantly inhibited in U-937 cells pretreated with the specific NF-κB inhibitor TPCK, I kappa B-protease inhibitor [42], the ERK 1/2 inhibitor PD 98,059, and the p38 inhibitor SB 202190. However, IL-1β mRNA and protein expression were only inhibited by NF-κB specific inhibitor and the ERK 1/2 inhibitor (Fig. 7C and D). After stimulation with rRv3402c for the time points indicated, similar results were obtained (Fig. 7E). Pretreatment of U-937 cells with the NF-κB inhibitor, the ERK 1/2 inhibitor and the p38 inhibitor significantly reduced the production of TNF-α in a dose-dependent manner (Fig. 7E). Taken together, these results imply that the NF-κB, ERK and p38 pathways are required for Rv3402c-induced TNF-α and IL-1β production by macrophages.
The persistence of pathogenic mycobacteria within macrophages is in part due to the bacteria's ability to actively manipulate host signaling. Several mycobacterial secreted virulence factors have been well defined in the process of inhibiting phagosomal maturation, and such knowledge will unveil previously unknown host signaling pathways involved in the innate immunity against Mtb infection, which will lead to the development of more effective vaccines and drugs.
Our data suggests that the Rv3402c protein might play a role in intracellular survival of recombinant M. smegmatis by modulating the host innate immune response. M. smegmatis, a non-pathogenic mycobacterium species, does not multiply and is readily killed by the macrophage, enabling this bacteria to be an ideal surrogate for the identification of virulence factors implicated in intracellular survival [26], [32], [33], [43], [44], [45], [46], [47], [48], [49]. We found that M. smegmatis recombinant strain expressing Rv3402c showed enhanced survival both in the U-937 and the RAW264.7 cultures in vitro and impaired the viability of U-937. These results were not observed in the M. smegmatis strain containing the vector only. Since these two recombinant strains show the same growth kinetics in axenic culture or other stress conditions, the enhanced intracellular survival in macrophages of MS_Rv3402c was thought to be the result of the protein's interference with the innate immune response. Differences in viability between the U-937 and RAW264.7 cells infected with MS_3402c was possibly due to the inherent differences between mouse and human macrophages. For example, the Mtb ptpA deletion mutant was described to have reduced virulence in THP-1 cells [9], [10]. However, the same mutant was not found to have reduced survival in a mouse infection model [50]. Thus, definitive identification of the role of Rv3402c in these two models will require further functional studies which are beyond the scope of this study.
Despite recent evidence of the role of rv3402c in the interaction of recombinant M. smegmatis and host cells, the function of Rv3402c remains unknown. According to the TubercuList website (http://tuberculist.epfl.ch/index.html), rv3402c encodes a conserved hypothetical protein. Rv3402c is predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules, and its sequence shows >40% sequence similarity to three types of enzymes: an aminotransferase, a dehydratase and an enzyme involved in perosamine/O-antigen biosynthesis [21]. Moreover, using the NCBI BLAST server we found that rv3402c encodes a protein with high sequence similarity to the lipopolysaccharide biosynthesis protein from Bacillus cereus and a putative PLP-dependent enzyme from Rheinheimera sp., which is predicted to be involved in cell wall biogenesis. We therefore tested whether Rv3402c was altered the cell surface properties. We found that the overexpression in M. smegmatis of Rv3402c, which is found in the cell envelope of mycobacteria, does not impact on colony morphology (data not shown). Furthermore, bacterial fatty acids compositions of M. smegmatis was not affected by the overexpression of Rv3402c protein (data not shown).
Although the physiologic function of Rv3402c is poorly understood, based on the subcellular fractionation analysis in this study, this protein deserved to be investigated for its role as a membrane- or cell wall-associated components. In order to avoid the bias of plasmid pNIT-Myc-Rv3402c, we chose another widely used plasmid pALACE to perform proteinase K and trypsin sensitivity assays. These assays performed on live recombinant mycobacterial strains revealed that His-tagged Rv3402c protein is partly exposed on the surface of mycobacteria. Not surprisingly, there are many proteins detected both in the cytoplasm and cell envelope, such as Rv2224c [51], Rv0132c [27], Eis [52] PknI [53], et al. Together, these results provide evidence that the Rv3402c protein is present on the surface of mycobacteria and may be available for interaction with host components.
TNF-α, a critical pro-inflammatory cytokine, is essential for host protective immunity to contain M. tuberculosis infection [54], [55]. Compared to pathogenic mycobacteria, M. smegmatis is a potent inducer of TNF-α in macrophages [56], consistent with the elevated TNF-α levels in the supernatant of macrophages infection with MS_Vec instead of the non-infected groups. Elevated levels of TNF-α was regarded as one culprit for Mtb persistence and virulence within human macrophages [45]. Previous studies have shown that mycobacterial components can enhance the production of TNF-α in monocytes/macrophages [45], [57], [58], [59]. Other studies have shown that virulent growth of Mtb in human monocytes [60], [61] or alveolar macrophages [62] is associated with enhanced secretion of TNF, as well as cytotoxicity. This evidence may provide insight into the role of TNF in cytolysis and the enhanced survival of the M. smegmatis strain expressing Rv3402c. Finally, through the pharmacological inhibition experiments, we found that the NF-κB, ERK and p38 pathways are required for Rv3402c-induced TNF-α expression by macrophages. However, only the NF-κB and ERK1/2 pathways are required for Rv3402c-induced IL-1β expression and protein production. It is our observation that Rv3402c disrupts host signal transduction in an alternative manner, which ultimately enables the bacteria to subvert the host immune response.
In summary, the present study suggests that the expression of the Rv3402c protein in M. smegmatis provides the bacteria specific properties that enhance intracellular persistence, decrease viability and modify the pro-inflammatory cytokine response of macrophages. This might provide novel information in the complex interplay between the host and Mtb. A greater understanding of the effect of Rv3402c on host signaling pathways will enhance our knowledge of Mtb pathogenesis. Further experiments such as using Mtb knockout mutant to determine the precise function of Rv3402c during infection are worthwhile.
We would like to thank Dr. Yossef Av-Gay for providing facilities with the proteinase K and trypsin sensitivity assays. We thank Melissa Richard-Greenblatt (UBC, Canada) for her help with critical reading and revision of this manuscript.
Author Contributions
Conceived and designed the experiments: WL JX. Performed the experiments: WL QZ WD TC ML. Analyzed the data: WL. Contributed reagents/materials/analysis tools: WL QZ WD ML. Wrote the paper: WL JX.
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