text:
Item Price

YSEA Corporate Platinum Membership

Platinum Corporate membership comes with

  • Complimentary registration to the YSEA Corporate Career Fair ($250 value), Platinum Sponsor recognition (2,500 value), social event with students and prescreen of Career Fair attendees
  • Platinum Sponsor recognition on the YSEA web site ($1,500 value)
  • Corporate Banner at YSEA functions ($2,000 value)
  • Recognition as sponsor of YSEA Industry and Alumni awards
  • Recognition as sponsor of one YSEA Faculty Grant ($2,000 value)
  • Eight tickets (one table) to the YSEA Annual Award dinner and four student sponsorships ($1200 value)
  • 15 subscriptions to the Yale Scientific Magazine ($300 value)
  • Corporate Profile (1/2 page) in YSM ($1,000 value)
  • 1/2 page ad (four times per year) in YSM as a YSEA corporate member ($1,000 value)
After registration, we will contact the corporate contact to gather additional information. Please include an email address, telephone number and mailing address where we can reach the corporate contact.

Total $10000.00

We accept VisaMasterCardAmerican ExpressDiscoverPayPal

The Yale Science & Engineering Association, Inc.

Powered by GroupSpaces · Terms · Privacy Policy · Cookie Use · Create Your Own Group


Content-Type: application/http; msgtype=response

WARC-Identified-Payload-Type: text/html

WARC-Type: response

text:
Yuma Sun

Now you can read Yuma Sun anytime, anywhere. Yuma Sun is available to you at home or at work, and is the same edition as the printed copy available at the newsstand.
Sections and supplements are laid out just as in the print edition, but complemented by a variety of digital tools which enhance the printed newspaper's look and feel.
We know you will appreciate the value and the freedom of choice that come with your subscription to Yuma Sun .

Yuma Sun : bookmarks

You may bookmark any article of Yuma Sun that you like. Come and see for yourself many other features and tools we have designed for Yuma Sun reading experience. Come and read your Yuma Sun here everyday!

Yuma Sun online

Yuma Sun        (19 Sep 2018) Yuma Sun
19 Sep 2018

Digital version of Yuma Sun

We knows how much people love to read Yuma Sun .

Yuma Sun in traveling

People are more mobile than ever before. How can their Yuma Sun be available to them anywhere in the world? Our service gives Yuma Sun the flexibility to follow readers wherever they travel.

Yuma Sun in digital format

We allow to read Yuma Sun online, anytime, anywhere.

Yuma Sun Market

Our service is changing the expectations of Yuma Sun readers everywhere. World newspapers at PressDisplay Newspapers from USA
Newspapers in English

warcinfo:
isPartOf: CC-MAIN-2018-39
publisher: Common Crawl
description: Wide crawl of the web for September 2018
operator: Common Crawl Admin (info@commoncrawl.org)
hostname: ip-10-37-160-190.ec2.internal
software: Apache Nutch 1.15 (modified, https://github.com/commoncrawl/nutch/)
robots: checked via crawler-commons 0.11-SNAPSHOT (https://github.com/crawler-commons/crawler-commons)
format: WARC File Format 1.1
conformsTo: http://iipc.github.io/warc-specifications/specifications/warc-format/warc-1.1/


text:
• +38 (050) 67-211-05
  • +38 (068) 67-211-05

period on a two point night

In a bit of an oddity, the teams exchanged two man advantage power play goals in the first period with rookie Matthew Phillips counting for Victoria at 6:10 and Kolesar for Seattle at 9:15. Barzal then unleashed a lacerating shot to the top of the net at 15:00 of the second period on a two point night. Nick Holowko made it 3 1 just 14 seconds later..

cheap nfl jerseys Yet there are some key differences when you get behind the wheel. For starters, the 109bhp 1.6 litre TDI engine isn’t as well insulated there’s a distinct diesel rattle at idle, and it sounds more strained when you accelerate, too.There’s a six speed manual or DSG gearbox available on the Octavia estate. The gear change itself is very smooth but opt for the automatic and you’ll sacrifice a little when it comes to economy. cheap nfl jerseys

cheap jerseys In addition to Lay, new recruits include all arounder Heather Fischer and time trial specialist Hannah Ross. Cyclocross racer Elle Anderson comes on board, along with sprint specialist Erica Allar and all arounder Jessica Prinner to headline the team’s criterium contingent. Also joining the team is a quartet of young up and coming riders; Sara Poidevin, Catherine Ouellette and Katherine Maine of Canada and junior World Championship medalist Emma White of the United States.. cheap jerseys

wholesale nfl jerseys Being a wedding photographer carries a huge responsibility. You are the person responsible for capturing all the precious, priceless memories of a couple’s biggest day. All those details that are meticulously planned, all the friends and family attending as guests, the emotional moments shared between the bride and groom, the tear slowly falling down the cheek of the father of the bride. wholesale nfl jerseys

Cheap Jerseys from china The 2008 09 Penguins beat Washington 6 2 in Game 7. The Capitals blew a 3 1 series lead against the Rangers in 2015. Overall, though, advancing to Round 2 and taking a stronger team to the limit isn’t the worst fate.They’ve only been upset a couple timesThe flipside of faring relatively well as the underdog is disappointing as the favourite, which Washington has admittedly done. Cheap Jerseys from china

wholesale jerseys from china Given enough time, the team will formulate a more extensive plan based on all the intel they have gathered. They will determine if there will be separate teams, where they will enter, the timing of the entry, what ordinance will be used and other details. There may be preliminary steps, such as drilling a small hole in a wall and using a pinhole camera to keep an eye on the suspect, or using a distraction to draw the suspect toward a certain location. wholesale jerseys from china

Cheap Jerseys free shipping As stated from «Influenza 1918»: «In 1918 America was caught up in the last horrific year of World War 1. Yet that had nothing to do with the extreme measures being taken. Deadly influenza, the so called «Spanish Flu», was sweeping the country, spreading terror everywhere. Cheap Jerseys free shipping

wholesale jerseys For that Moncler clothing is a professed labor to food dr. Jackets, the attribute of kids descending jackets is caretaker without incertitude. Even if it happened that you somebody one naughty boy, he loves performing up or kinds of sports, and you may worried whether he faculty wet himself in the hoodwink or prehend vasoconstrictor in the wintry winter,christian louboutin marketing saks, Moncler jackets may amend untold. wholesale jerseys

wholesale nfl jerseys from china The starting point described here is convenient for anyone completing Cleghorn Ridge. 2N33 to Pilot Rock near the Pinnacles. Road 2N17X which you’ll pass as you complete this route. There have been several well known brands that have fallen victim to their own success in this way. Aspirin, Band Aids, Xerox, Linoleum and others have all been ruled generic even though they started out as brand names. Just a few years ago, Google narrowly avoided its own genericide case after a trademark case led to claims that Google had become a verb interchangeable with searching for something on the internet regardless of search engine.. wholesale nfl jerseys from china

Cheap Jerseys china B m is a Vietnamese stuffed baguette, an ode to the influence introduced by the French during https://www.cheapjerseyseller.com/ its colonial period. The baguette is stuffed with all kinds of Vietnamese goodies wholesale jerseys mostly pork, chilies, herbs and other yummy things. In Hanoi, we loved the little street food cart Bahn Mi 25, selling the best tasting ones Cheap Jerseys china.

Залишити коментар

Все що з відмічино зірочкою має бути заповнене


text:
A Secret Weapon For engineering assignment help

When you are acquiring a great grade in the Java programming assignment, It is the the perfect time to acquire some action to transform your lousy grade.

This web site includes Recommendations for attaining and setting up access to ADOT's eCMS and instructions and recommendations for SOQ submission as well as the analysis plan.

We don’t utilize hidden or excess fees to your paper. Also, our company will work with extensively known payment programs to prevent money troubles and theft.

Our English language clientele contains people today for whom English is to start with language / mother-tongue, as well as All those for whom it really is analyzed as 2nd language (ESL). We cater towards the various amounts of English assignment prerequisites of learners from kindergarten, by way of highschool and the many technique to advanced university level English learners.

Just about every consumer that involves buy at our Site gets a private assignment writer. This writer will Focus on your get with or without our Handle. If you already have a chosen writer, you can find them to operate on your potential orders by specifying their ID range in the form.

In Java databases assignment, Every one of the assignment is principally contend with databases connectivity. I acquired numerous requests how to connect MySQL database with Java.

quite apparent, straight to the point, as well as the tempo was just right for me, that has not found any calculus or trig up to now 10 years and want to return up to speed quick."

Java Binary I/O Assignment consist the situation to examine and generate knowledge in binary documents.  In Java I/O Assignment, you've got specified some issue through which You will need to ask for enter from consumer and course of action the user enter and help you save the output in the binary file.

extremely very clear, straight to the point, along with the tempo was just right for me, who's got not noticed any calculus or trig up to now 10 years and need to come up to speed you can look here speedy."

It is possible to understand Java Programming by me or you may check with a person who could help you along with your programming assignment.

You could possibly print and distribute nearly two hundred copies of this document each year, at no demand, for private and classroom educational use. When printing this document, you may NOT modify it in any way. For any other use, please Get hold of Science Buddies.

I have great practical experience in java assignment/homework and venture. In Past 5 year. go to this website I have worked on quite a few Java IDE. I am skilled Java programmer, and I use these IDE commonly, so I am a master in these.

I am providing you one of the reliable online java homework help. As you use my Java programming homework help support, your grade in your java matter are going to be boosted.

Undecided what is anticipated with your assignment? No matter whether you’re composing an essay, report or literature assessment, our sample assignments will provide you with what markers are trying to find.

Leave a Reply

Your email address will not be published. Required fields are marked *


text:
AgustaWestland chopper scam: When will CBI interrogate ministers?

AgustaWestland bribe case is making headlines on the daily basis and many names are coming in light. But when the agencies will interrogate the Ministers? Zee News always stay ahead in bringing current affairs from all the valley of National interest, Politics, Entertainment, Sports and International happenings. We take you to the depth of every matter by providing every small detail and makes you familiar with all the happening around you. Zee News is the highly popular Hindi News channel of India’s largest television network ZMCL.

May 02, 2016, 00:02 AM IST

By continuing to use the site, you agree to the use of cookies. You can find out more by clicking this link


text:
Linezolid 600 Mg Iv Price

HyracotheriuMy we may say that Hyracotherium was the most general
linezolid for mrsa uti
cervical and submaxillaries were also involved and ulcers were found
canadian pharmacy zyvox 600mg
linezolid tablets 600 mg dosage
erudition of this society that before taking up the personal
zyvox price in egypt
be sent away without the order of the 7etcrinarian. When the Governor
linezolid 600 mg tablet in hindi
zyvox iv infiltration
living in tlie rural sanitary districts of tlie different towns.
zyvox pfizer product information
four feet. Under either of the long drop sj stems a criminal
linezolid injection spc
Unfortunately in many cases neither temporary nor pro
zyvox po dosage
toxine is quite harmless and ought to be used in the beginning
zyvox dose po
tears the saliva the urine and all the secretions were acid.
linezolid 600 mg iv price
here I will sandwich in my second proposition which will
zyvox generic brand
The opinion expressed in Piers Plowman s Vision was
what is the medication zyvox used for
Council of the College of Surgeons. That he would not again
where can i get linezolid in nigeria
harga zyvox linezolid
benefited. If this failed galvano punctui o might bo tried.
zyvox savings card
In the non suppurative cases treated by laparotomy the
zyvox 600 mg preco
Ihe pupils had contracted. About a quarter to eleven on my
retail price of zyvox
lion Blackwood Abdallah b. c four years by Homer dam by Blackwood
linezolid cost per pill
much does iv zyvox cost
tablishing new adhesions within the abdomen. The discharge
linezolid side effects usmle
was one of the most popular throughout T urtemburg as
linezolid dosage for uti
zyvox iv bag
I trust it will not be deemed a reflection on the dignity and
tab linezolid 600 mg cost
fection. In all these actions he has the law to sustain him. With
zyvoxam linezolid precio
trophic. The latter is easily recognized by the uneven thick
zyvox 600 costo
than thirty pounds a year Poor men says he I heartily
linezolid package insert south africa
jaws. The patient was a female aged 40.and the tumour had
zyvox discount card
linezolid iv administration
zyvoxid pfizer precio
a detailed account of the rise and progress of this move
zyvoxid renal dosage
To the Members of the Michigan State Medical Society
linezolid dosage iv
zyvox iv rate
pregnancy was the immediate factor and the case be classed
linezolid generic cost
zyvox oral price
scarcely any appreciable contraction of the true conjugate
purchase linezolid
said the first diagnosis is the important thing and we find
what type of drug is zyvox
Cruelty to a Horse Pukished. Two dissected fetlock joints of a horse s


text:
Getting Creative With Gear Advice

Interesting Facts about Glamping and Its Benefits

Many people have gone for camping in the past, but not all of them are impressed with the conditions that they encountered during that period. While some people say that camping is their best outdoor activity, some people do not share the same thought because the camping conditions are adverse. If you are not ready to share makeshift toilets and bathrooms and also sleep under canvas in the cold night, then camping might not be interesting to you. Camping does not necessarily have to be that awful, and the introduction of glamorous clamping which is also known as glamping provides the best alternative to camping for those who cannot keep up with camping conditions.

If you have a tight budget and you are planning to go for a holiday, choosing to visit another country and spending your days in a hotel could be quite expensive and out of your budget. Glamping provides an alternative to that and gives you a chance to appreciate nature in your locality with a budget that is manageable. For instance, you will have electricity to power your electronic gadgets and also get the best accommodation which is not usually available during regular camping.

With lots of glamping sites available today, it is for you to choose the best one that seems to suit your preferences. With is allowance, many people who do not love camping due to its conditions can have fun in better conditions that are tailored to suit their preferences. This is a common question but many people are getting interested in this activity but you can get references from people who had to some of the best glamping sites around or you can browse the internet to find a list of such places, and you can scrutinize their details to select the right one.

There are many benefits associated with glamping. Apart from being an affordable alternative to taking expensive holiday trips, you get to learn and appreciate the nature of your environment. In fact, you also get access to facilities such as hot water bath, electricity and other equipment which make your stay enjoyable. Additionally, you do not have to carry glamping equipment as you do with camping equipment because you find well-established accommodation facilities. However, glamping sites offer different experiences based on their location and other facilities and therefore, you must do extensive research before making a choice.

Glamping is a blend of luxurious accommodation in high-end hotels and traditional camping, and thus, you get both experiences. Therefore, do not stay at home because of the bad conditions at camping or lack of adequate finances to spend your holidays in luxurious hotels as glamping is affordable and exciting.

Suggested Post:


text:
Select your Subcategory

Zippy's Travel Scoop

Rating: 4 Stars4 Stars4 Stars4 Stars

Price: $4563.67

Travel Reviews

CruisesOnly Review - Review

Zippy's Review of CruisesOnly: This is one of my favorite sites even though it lacks depth in luxury cruise lines. It is a very well designed and easy to use site making your search effortless and fast. Cruisers have rated 142 ships and experts have rated 134 of the 193 cruise ships listed. The deficit is mostly in river cruise boats. Suspiciously, the reviews have an appearance of paid placement. You can filter the results by ratings, cost, cruise line and age, a terrific addition. There is also easy to find detailed information about each ship and cruise line.

Pros: very easy site to use with 7 search filters, best for big ship, lower cost cruises, some reviews are by authenticated passengers
Cons: only some ships have expert reviews (provided by Cruise Critic), expert reviews are not dated, not a site for luxury ship reviews
Best user: novice to wiz, anyone interested in large ship, mid or lower priced cruises

Cruise Critic Review - Review

Zippy's Review of Cruise The wealth of information on Cruise Critic almost overcomes the frustration of the site’s confusing navigation. Almost 400 ships sailing for 80 cruise lines are reviewed in depth by experts, creating the most comprehensive cruise review site online. The expert ship reviews are discriminating and well written but many are older than whitewall tires. Details include cabins, dining, public rooms, entertainment and five other areas. There is a 1 – 5 blue ribbon rating assigned to each ship and cruise line giving a sense of comparison against all others. Often the consumer reviews are peppered with silly comments requiring reading many reviews to find information of substance.

Pros: consumer and expert reviews, comprehensive information, thousands of reviews
Cons: not easy to maneuver around the site, consumer reviews are glowing or nasty with little middle ground, reviews listed by cruise line or cruise ship – confusing, reviews home page overwhelming
Best user: determined novice, average user

Expedia Review - Review

Zippy's Review of Expedia hotel reviews are most useful if you are searching for a specific hotel. The only reviews on the site are written by those who have booked through Expedia. Therefore bogus reviews should be non-existent. If you are looking within a city, you will have to wade through the site by each hotel listing.

Pros: reviews are written by travelers that have booked that hotel through Expedia within the last 6 months, no bogus reviews
Cons: no profiles of reviewers, difficult navigation on site
Best user: intermediate to wiz

Fodor's Review - Review

Zippy's Review of While reviews are not their mainstay, Fodor’s has so much added value and credibility that they should be taken seriously. The site is very easy to use and read with the added value of their experts doing reviews after hotel site visits.

Pros: Fodor’s expert reviews, hotel photos, site has additional great content
Cons: few, mostly outdated consumer reviews, limited number of hotels reviewed
Best user: novice to wiz

Frommer's Review - Review

Zippy's Review of Frommers only provides ratings using a 0 – 3 star scale. They differ in that they only provide ratings, even 0 star, for properties they recommend. All their listings are winners according to Frommers. There is a separate key for pricing listed as 1 – 4 dollar signs.

Pros: all listed hotels recommended,
Cons: no written reviews, only star ratings, limited number of hotels rated
Best user: novice to expert Review - Review

Zippy's Review of Sort and filter options almost create a personalized review list. guest reviews are noted eliminating bogus reviews. Reviewers from outside the US are grouped as are reviews taken from Trip Advisor.

Pros: reviews are dated, can be filtered and sorted
Best user: novice to wiz
How to use: click on (# reviews) for reviews pages

J.D. Power Review - Review

Zippy's Review of The stellar reputation of J.D. Power, normally associated with cars, has earned them a spot on Zippy Reviews. They can be trusted to provide accurate ratings. They do an admirable job of rating 36 European and 65 North American hotel brands. Their 1 – 5 dot ratings are based on 8 different factors ranging from check-in, food, cost, etc. European hotels are broken down into categories from economy to upscale. The North American properties include 6 categories adding in extended stay properties. This rating system is only as good as the individual hotel’s adherence to brand standards. The airline ratings, 1 - 5 dots, are spread between 5 low cost and 7 traditional US carriers. Again 8 factors are judged ranging from boarding experience, staff, aircraft, cost, service, overall, reservation, and check-in.

Pros: best for researching unfamiliar hotel chains
Cons: cannot research individual hotels, airport ratings are worthless, no international airlines
Best user: novice to expert
How to use: click on ratings category in Travel Ratings box, click on red “more” button for next page

Orbitz Review - Review

Zippy's Review of Orbitz is most useful for hotel reviews if you are quite specific in your search terms. Results include a 1 – 5 scale for 6 criteria but there are minimal comments from customers. If the reviewer booked through Orbitz, they are verified customers, which carries more credibility. Ships of 21 cruise lines, ocean and river, are reviewed by Cruise Critic experts and cruisers (profiles included). If the cruiser’s profile is similar to yours, the cruiser’s comments might be more valuable to you. Slide shows of the ship can be accessed from the review pages adding more interest to the review. There is a major deficit in luxury lines. Orbitz is for the budget to mid-range cruiser.

Pros: ease of use for cruise reviews, huge number of hotel reviews
Cons: expert cruise reviews (provided by Cruise Critic) are not dated, luxury ships lack consumer reviews, hotel reviews geared toward selling not toward providing information
Best user: novice to wiz (cruises), intermediate to wiz (hotels)
How to use: scroll down to cruise line, click on star in front of ship name, for hotels enter a city in the left hand pane “where” box leaving all other boxes blank, click “search,” click “# reviews” (such as “30 reviews”)in each hotel’s description

Skytrax Review - Review

Zippy's Review of Great go-to site for learning everything you ever wanted to know about an airline or airport. Airline safety, website, equipment, seats, food, service, and lounges are all rated or reviewed. Airport reviews cover ease of checking in, staff attentiveness, shops, and restaurants among many other criteria. Airport rankings contain maps, transportation options, parking guides – you name it. Because it is a combination of very scrupulous staff rankings and consumer reviews, the information is very expansive and reliable. The site also provides links to airlines, airports and various travel tools.

Pros: over 500 airlines listed, ease of use by alpha listings, ratings by star for each airport
Cons: cannot reserve flights on the site, no photos on the site
Best user: novice to expert

tripadvisor Review - Review

Zippy's Review of Probably the most famous and controversial of all Zippy review sites, it is difficult to quibble about the issues – are the reviews legitimate and honest? Truth is that the sheer number of hotel reviews compensates for the possible dilution by dishonest reviews, so forge forth. Read between the lines and read many reviews for each hotel. Skip the newest addition to the site, airlines ratings containing only 1 – 5 dot ratings with no text.

Pros: breadth and depth of reviews, over 45M reviews, consumer photos, possible hotel response to reviews, links to area information
Cons: possibility of some dishonest reviews, need to read large number of reviews to glean accurate information, highly subjective reviews, no experts
Best user: novice to wiz
How to use: in “Find Hotels Travelers Trust” put city/state/country in search box, click “leave dates blank,” click “Find Hotels”

  Do you know of a site or sites that do reviews? let us know about it!
(Multiple sites separated with commas,
(Letters are not case-sensitive.)

* Required Stuff


text:
Hot new heavy duty plastic moving crates with foldable lids

FOB Reference Price:Get Latest Price
US $5-10 / Pieces | 500 Piece/Pieces (Min. Order)
Report Suspicious Activity
Quick Details
Plastic, PP
Place of Origin:
Zhejiang, China (Mainland), Zhejiang, China
Brand Name:
new plastic moving crates
Model Number:
ZY-4030/26new plastic moving crates
heavy duty plastic moving crates with foldable lids
External Size:400X300X260mm
External Size:600X400X315mm
External Size:680X430X320mm
Single Loading:
Stack Loading:
Supply Ability
Supply Ability:
2000 Piece/Pieces per Week
Packaging & Delivery
Packaging Details
2014 hot new heavy duty plastic moving crates with foldable lidsSingle Loading:30kg
Stack Loading:150kg
Quantity Per Container:20':2150pcs 40':4350pcs
Lead Time :
15-20 days

2018 hot new heavy duty plastic moving crates with foldable lids

Product Description

   2014 hot new plastic moving crates

Have  3 SIZE:

20LExternal Size:400X300X260mm
50LExternal size : 600X400X330mm

External size : 680x430x320mm

    Item name:turnover crate.

    External Size:400*300*260mm.

    Internal Size:350*270*240mm.

    Packaging:in bulk.

    Single Loading:30kg Stack Loading:150kg.

    Quantity Per Container:20':2150pcs 40':4350pcs.

2> Description :50L

External size600*400*330mm
Internal size555*365*315mm
Single Load35KGS
Stack Load175KGS

3> Description :73L

    Austrial,Germany,UK,New Zealand....

DescriptionHinged Heavy-duty Plastic Moving Boxes with Foldable Lids
External Size680x430x320mm
Internal Size630x390x300mm
Materialcopolymer pp
Self Weight3.70kg
Packagingnested inside each other up to stacks and loaded into containers
Single Loading 35kgs
Stacking Loading175kgs
Container Quantity20ft: 789pcs / 40ft: 1680pcs / 40HC: 1920pcs

MOQ500pcs per color scheme

rose crate 3-_.jpg

Packaging & Shipping

2014 hot new heavy duty plastic moving crates with foldable lids

* Market Areas

It's been purchased a lot by customers in Australia, New Zealand, US, Canada, Netherlands and UK.

* More Photos for Easy Reference

1) Screen Printed Logo Example

2) A Few Stacked Inside Each Other

3) A Few on Top of Each Other

4) Grips Hands Could Be Placed While Holding & Textured Areas for Easy Label Removal

5) Eyelets on Each End of Lids

6) Large Support Ridges on Long Sides

7) Sitting on a Dolly

8) Anti-Mark PU Wheels of Dollies & A Braking Mechanism to Prevent Slides

9) Container Loading

10) Tags Supplied

11) Details

Hello, dear friend.

Thanks for your friendly visit.

Your kind inqury will be highly appreciated.

Welcome to email or telephone to us~

My Skype:gordonye1

My Iphone:0086-13484255249

Our Services

Sample collecting:

*When a sample order is placed, please also inform us of your account No. of courrier company, such as DHL or Fedex, full name of company, address, zip code, contact person and Tel.

Payment terms:

LC: at sight of BL copy.

T/T: 30% deposit in advance, the balance at the sight of B/L copy.

Other payment terms are negotiable

Certificate of ISO9001

Heavy duty large plastic crate with foldable lids

Our Dino Crate features:

- Our crate’s structure is designed according to “Honey Comb” style which will hold the best strength, uniqueness and exquisiteness.

- The handles –both solid and perforate—are easy to employ.

- You will be able to hot stamp or silk screen your ‘own’ logo on the side of the crates.

-  In order to maximize the utilization, every crate will have a small hole on its 4 corners which will allow users to hold the rope or string that increase ultimate usage out of the crate.

- Plastic container, plastic crate, stackable crate

The design focuses on the smoothness of the surface and structure in order to connect all the corners to be rounded therefore the crate will not carry the dust, powder or filthy articles. The design besides will let users to clean crates more easy.

Company Information

About us:

2014 hot new heavy duty plastic moving crates with foldable lids

Ningbo Zhenhai Zhongyi Industry Trade Co.,Ltd. is an importing and exporting

enterprise engaging in integrating mold research and development, plastic palletizing
 and plastic products producing to provide customers excellent products and services.
     The operation of the company is based on the market needs, so we focus on the
technology development and high quality of the products. We sincerely provide diverse
categories of products with low price to our customers all over the world. Our self-combined floor mats are developed on our own and can allow you to decorate your
backyard or balcony as you wish. This product's management right in Japan has been
uniquely owned by ADVAN. We also provide other products such as plastic pallets,
plastic boxes, tool boxes to customers from Germany, the Netherlands, Belgium, Japan,
 U.S., Hong Kong etc.
     We own some 20 equipments including 100-10000 gram computer-controlledplastic injectors, plastic blowers and plastic palletizers. Six departments are
established in the company, including production department, department of R&D,
export&import department, domestic business department, quality department and
storage&transportation department. Our motto is "people, Innovation and Quality".
We sincerely hope to cooperate with business friends all around the world.

We truely believe that quality service is what we sell beyond the product a collection research and development, production and sales as one integrated industrial company; Main products are folding or portable field/outdoor products---such as Folding Chairs/Stools,Folding Tables,Folding Dishes,Folding Baskets,and new Rice Containers,application areas includiong outdoor sports, adventure, travel, camping, military equipment, field defense, and other fields, in international enjoys a high reputation in the industry and users; The company has large-scale production ability and the world first-class production equipment, with wild products of top designers, outdoor equipment designers, materials engineers, engineers, product adaptation of human body engineering design engineers and many professional team and all kinds of professional talents, will bring you the best products and services; Our products have been sold to all over the world, in every corner of the globe; The company all staff will be dedicated to serve you, to provide you with the best products and the most preferential price, welcome you to be my best friend, and let us open up the future together!

1>What Color do you need?- We could have these crates made in any color scheme customers request as long as our MOQ is satisfied - 500pcs each size.
2>Do you need Screen Logo Printing? - We can also provide logo on crates. US$0.10 extra for 1 color branding on 1 you have sample logo show me.
3>Payment Terms: 30% deposit, the rest against the  copy of original documents.
4>Delivery Days:  20 days after received 30% deposit.
5> Mode of  Shipment: OCEAN TRANSPORTATION
6>Port of Loading :FOB (FOB Mean FREE ON BOARD)NingBo, China
7>FOB Mean Free on board,It doesnot includ the ocean freight,If you want the Ocean freight,pls let me know your cloest port name.let me check for you.
8>CIF Mean including the port of shipment to the port of destination of the freight.
9>If you need sample, I will on 3 ~ 5 days by Courier to your hands. and please give me the detailed receives an address, I can help you consulting Courier fee.
I realise its our first communication and we might need much more time to get to know each other before going any further, So please let me know if I can be of further assistance.


text:
09-Aug-2017 10:43 Free adult one on one chat no pay

dating location iphone app
Free adults webcam no apps no reg no fees

Eddy was the one who messaged me first, and initially I never got back to him.Then he was ready to quit online dating, and as a last-ditch effort he sent me a message, like, And I actually texted him back, because I looked at his profile and saw that he was Australian, and who doesn't want to go on a date with an Australian babe?!Our families don't know about us, because it's kind of a taboo.We're thinking about how and when we'll tell our families.Is it worth dating and building a relationship with Jackson? I was never really interested in dating Indian guys, but often white guys would try hitting on me by telling me how much they love Indian food. And then he joked that his parents were in town to get him an arranged marriage. 3, because I had planned four first dates in two days. So I saw Khalil like once a week for the first six weeks or so, but then we started getting a little more serious.This is actually my first serious relationship, and we're at almost a year now. The one thing that is a bit of a problem is, funnily enough, although we're both Indian, he's Muslim, and my background is Sikh.I knew from the first date that I really, really liked Matt.It was great, because I couldn't get out a lot at the time — I could get out maybe once a week, if I had a babysitter.

He had met my son, so we had to ask: Do we have a future? I don't know what I did to deserve this, but I'm just going with it. But I liked him back, and he messaged me right away. If he seemed normal, we'd have a drink, rather than building up this idea of who he is. On the site we used, they have a question that asks the things you can't live without, and I wrote was bad for the eyes and something about the evil eye. I actually forgot his name — I only remembered that he was no. When I got home, he texted me that he was deleting his account, and I was like, OK. It was the beginning of summer in New York, and every weekend was busy.I had been so conditioned to be blown off, I thought he was politely breaking up with me.So we didn't really talk for a couple days, which felt like a long time. I realized he wasn't trying to end things; he did want to keep dating.But [I] get panicked when I think about the fact that he lived literally one mile away and we had no friends in common. Six months after moving to New York, I started online dating.Not necessarily because I really wanted for find a boyfriend; it was just me just wanting to go on dates because they were so much fun.

12-Jan-2018 02:36 Camsex chat online

Private sex dating holland
Sex chart website without memebership

Cate Blanchett has been happily married for 18 years.

02-Feb-2018 19:27 Atlanta milf dating site

are we dating or just friends hanging out
most intimidating sports uniforms

In the course of his career, Levesque has held a total of 25 championships including nine reigns as WWF/WWE Champion and five as WWE's World Heavyweight Champion.

02-Nov-2017 17:52 dating nice france

Online camfree hot
cherry lewiss book the dating game

Having proven the concept - being certain there was both a need and commercial value - Matt went about building a team to assist him with the creation of the site.

16-Nov-2017 14:49 christian dating advice for middle age suddenly single men

honest dating
Girl chat wap

Despite the queen's claim that this is place where there are no happily-ever-afters, this movie does a good job of showcasing the magic that is New York. It continues what was, I think, Walt Disney's greatest talent, which was his ability to conjure up our collective imagination.

23-Sep-2017 00:50 dating texarkana

Skype hook up for cam sex
paul washe families dating and relationships

Finally, you’ll take the plunge and meet your new match in person.

29-Jan-2018 21:45 single dinner und power dating

disadvantages of dating an old man
How to hack into chatwithpornstars

With us you are dealing with experienced people who respect you and these wonderful ladies.

11-Dec-2017 02:42 mali america dating

Free sex dating face
Free chat rooms no registration in farmington mn

I am not married yet, but I want to prepare to be the best husband I can.

28-Nov-2017 16:33 mujeeb usa dating

Free private webcam sex chats
russiamore dating site

Les 6 canots de sauvetage sont bien insuffisant pour les 400 occupants.

10-Jul-2017 15:51 Good sexchat phrases

Onesexchat arab sex chat

Sometimes they are seeking only friendship, but more often than not, they are indulging in extra-marital affairs and infidelity.

11-Jan-2018 12:22 brother and sister dating another brother and sister

best dating site for canada
friends love dating

Terms of Use Agreement Welcome to Odnoklassnik.de, the service for adults to meet each other online operated by Odnoklassnik.de, (Odnoklassnik.de).


WARC-Identified-Payload-Type: application/xhtml+xml

text:
"The best way to have good ideas is to have a lot of ideas"

Through the mist and fog of rough ideas a Story was picked, but what of the once that didn't make it?
It's nice to look back at ideas, so this post is dedicated to the once that didn't make it...

Other Ideas
Concentration camp breakout -  You follow an orphan who successfully breaks free out of a concentration camp

A Jews Life - A day in a life of a Happy Jew

One Response so far.

  1. tutorphil says:

    This is a GREAT post - lovely to see all these shelved ideas and 'what might have been' concepts :)

Leave a Reply


text:
Find Your Passion...Follow Your Dream...Never Give Up

A great new Museum needs help funding their Opening Reception. Go to Kickstarter and donate what you can. Time is running out! And tell all your friends to donate as well! Then be sure to show up on April 12th, 2015 at the Millard Sheets Art Center at Fairplex in Pomona for the reception from 2:30 to 5:00 PM.

Along with the Contemporary Art from California’s Inland Empire & Beyond, they will be showcasing the museum's gallery for the "young at heart," the Young Eyes Gallery. It will be a fun time with a lot if interesting people. Look for us when you're there! More at:

Add comment

Security code


text:
Skip to content

  • Research article
  • Open Access
  • Open Peer Review

Spatial epidemiology and climatic predictors of paediatric dengue infections captured via sentinel site surveillance, Phnom Penh Cambodia 2011–2012

  • 1Email author,
  • 2,
  • 3,
  • 4,
  • 5,
  • 6,
  • 7,
  • 4,
  • 8,
  • 5,
  • 2,
  • 2,
  • 3,
  • 9,
  • 10 and
  • 11
BMC Public Health201414:658

  • Received: 19 January 2014
  • Accepted: 20 June 2014
  • Published:
Open Peer Review reports

Dengue is a major contributor to morbidity in children aged twelve and below throughout Cambodia; the 2012 epidemic season was the most severe in the country since 2007, with more than 42,000 reported (suspect or confirmed) cases.

We report basic epidemiological characteristics in a series of 701 patients at the National Paediatric Hospital in Cambodia, recruited during a prospective clinical study (2011–2012). To more fully explore this cohort, we examined climatic factors using multivariate negative binomial models and spatial clustering of cases using spatial scan statistics to place the clinical study within a larger epidemiological framework.

We identify statistically significant spatial clusters at the urban village scale, and find that the key climatic predictors of increasing cases are weekly minimum temperature, median relative humidity, but find a negative association with rainfall maximum, all at lag times of 1–6 weeks, with significant effects extending to 10 weeks.

Our results identify clustering of infections at the neighbourhood scale, suggesting points for targeted interventions, and we find that the complex interactions of vectors and climatic conditions in this setting may be best captured by rising minimum temperature, and median (as opposed to mean) relative humidity, with complex and limited effects from rainfall. These results suggest that real-time cluster detection during epidemics should be considered in Cambodia, and that improvements in weather data reporting could benefit national control programs by allow greater prioritization of limited health resources to both vulnerable populations and time periods of greatest risk. Finally, these results add to the increasing body of knowledge suggesting complex interactions between climate and dengue cases that require further targeted research.

  • Chikungunya
  • Syndromic Surveillance
  • Dengue Case
  • Epidemic Season
  • Dengue Transmission

Globally, dengue is the most widespread arbovirus, causing an estimated 390 million [95% Bayesian uncertainty interval 284–528] infections a year, of which only 96 million [67–136] are captured via surveillance systems [1]. Dengue is endemic throughout Cambodia, one of the poorest countries in Asia, which currently faces numerous health challenges with limited health infrastructure and poor economic indicators [2]. Morbidity from dengue is extensive due to a range of interconnected factors, including delays in care seeking, limited impacts of vector control, and limitations within the health sector [3, 4].

Historically, large epidemics have occurred on a 3–4 year cycle, and generally all four dengue virus serotypes circulate annually; DENV-2 and DENV-3 predominated until recently, but have been largely displaced by DENV-1 since 2009 [5]. Dengue cases are reported throughout the year, but with a sharp increase during the rainy season (May to November).

A national syndromic surveillance program was established in 1980, which includes passive reporting of clinically diagnosed cases by public sector hospitals. Dengue is included in the list of the 12 priority diseases that are reported weekly to the Communicable Disease Control Department of the Ministry of Health (MOH); this reporting system contains only aggregated data on new cases and deaths for the diseases under syndromic surveillance. To supplement this system, in 2001 the National Dengue Control Program (NDCP) of the MOH implemented a hospital-based surveillance in seven paediatric hospitals or paediatric wards within referral hospitals. Weekly case reporting from these sentinel hospitals includes basic demographic indicators (age, gender, place of residence) clinical findings on admission, and status at discharge. Subsequently, this program was enhanced by the inclusion of virological and serological surveillance at five sentinel hospital sites. During the period 2002–2008, the National Dengue Control Program (NDCP) has reported on average 103 cases per 100,000 with an annual case fatality rate (CFR) ranging from 0.7 to 1.7% [3]. However, these sentinel sites are not able to capture detailed epidemiological, clinical or laboratory data, and reporting is generally incomplete; a capture-recapture study in Cambodia indicated a 4- to 29-fold underreporting rate [6]. The 2012 epidemic season was the most severe in the country since 2007, with more than 42,000 suspect or confirmed cases reported, and serves to highlight the serious epidemic potential within Cambodia.

To help address these limitations, our clinical study was designed to capture comprehensive data for a prospective series of paediatric dengue cases. The global objectives were to fully explore the epidemiology and clinical spectrum of clinical paediatric dengue in Cambodia; this portion of the study examines the captured clinical cases within the larger epidemiological context of spatial and climatic trends.

As a vector-borne disease, dengue transmission is inherently tied to weather patterns and climatic cycles, and epidemics generally peak during the wet season, beginning in the ‘hot wet’ season from May-August, and then tapering off through the ‘cold wet’ season in September. However, the fine-scale interactions of these climatic conditions on Aedes vector populations are poorly understood. Recent years have seen progressively larger epidemics throughout Cambodia and SE Asia; detailed spatial and epidemiological data are therefore increasingly critical to maximize the effectiveness of control programs within inherently limited budgets [7].

Sampling plan

Enrolment was organized weekly from September 20, 2011 to January 15, 2013 during specific surveillance days in both the internal medicine and emergency wards of the National Paediatric Hospital, Phnom Penh. All children aged 1–15 years and presenting with fever, or history of fever within seven days, on admission were screened for eligibility criteria using the 1997 and the 2009 WHO dengue case definitions [8, 9]. Informed consent was obtained from parents or legal guardians for all eligible children. Demographics and clinical data on admission and during hospitalization were reported on a study-designed Case Report Form (CRF). Paired sera were collected on admission and at discharge and shipped via courier the same day for serologic (IgM Capture ELISA and haemagglutination inhibition assays) and virological (virus isolation on 2 cell lines, real time RT-PCR, and NS1 antigen detection by commercial ELISA (BioRad®)) tests for dengue and other arboviral infections (i.e., Japanese encephalitis and chikungunya) as reported previously [10]. Anonymized data were entered into an electronic password-protected database by a trained data manager, using Epi-Data 3.1 (Odense, Denmark) with range-checks; data monitoring was performed by a clinician who checked the concordance between the clinical notes, the study CRF and the electronic files; discrepancies were then corrected in both the CRF and the dataset.

Data sources

All clinical data in this study were collected by National Paediatric Hospital staff (Phnom Penh) and all serolological and virological testing were performed at the virology department of Institut Pasteur, Phnom Penh. Temperature and humidity data were obtained for the weather station in Phnom Penh [11], and precipitation was obtained from satellite estimations [12]. Population data and population density were obtained from published reports from the Ministry of Statistics (2008) [13]. Approximately 60% of the 558 dengue-positive patients resided in the greater Phnom Penh area; 216 patients were geocoded to the sangkat (commune/urban village) level. National-level reporting data was provided by the National Dengue Control Program at the Ministry of Health, Phnom Penh; open source base map layers were obtained from Open Development Cambodia (March 2013), see

Data analysis- regressions

Analysis of the relationship between weather events and national reported disease counts utilized Poisson regression for over-dispersed data (negative binomial), and examined the time period January 2011 to January 2013. All time-series models utilized Newey-West error corrections for autocorrelated and heteroskedastic data [14]. Explanatory factors examined were the weekly mean, median, minimum, and maximum of available weather data, including temperature, relative humidity and rainfall, plus the rainfall index defined as [(cumulative rainfall X days with rainfall)/days in reporting period] [15]. Lag periods of 1–15 weeks were examined for all variables. Optimal models were identified by Akaike information (AIC) and Bayesian information (BIC) criteria; model fit was assessed using standardized deviance residuals [16]. Stata 13.1 (College Station, Texas) was used for all analyses and all tests were two-tailed, with α = 0.05.

Data analysis- spatial

Visualization of captured cases data utilized ArcGIS 10.1 and QGIS 1.8 ‘Lisboa.’ Detailed mapping and analysis included only patients residing within greater Phnom Penh; cases were geo-referenced to the centroid of the respective sangkat (commune). ‘Hot-spot’ detection used SaTScan version 9.1 [17]; spatial and spatio-temporal clusters were examined with Poisson models including the underlying population density by commune. Bernoulli models were not utilized due to the limited number of dengue-negative patients. Clusters were limited to 25% of the total population, and estimations used 999 replications for Monte Carlo inference.

Ethics review

The study was conducted according to the ethical principles of the Declaration of Helsinki of October 2002. The Cambodian National Ethics Committee for Health Research approved the overall study protocol after institutional review (approval number 123-NECHR, 22 August 2011).

From September 20, 2011 to January 15, 2013 a total of 1,228 febrile cases were admitted to the two participating wards. An eligibility form was completed for more than 96% of these admissions (1,185/1,228); the proportion of eligible patients was 59% (703/1,185) and the study refusal rate was less than 1% (2/703). A total of 701 suspected dengue patients were enrolled during the study period, and of these 80% (558/701) had a laboratory confirmation of dengue (Figure 1).
Figure 1
Figure 1

Patient inclusion flow, National Paediatric Hospital, Phnom Penh, January 2011- January 2013.

All four serotypes were identified during the study, but DENV-1 predominated (520/558; 93%) (Table 1). Our sentinel surveillance also detected a noteworthy chikungunya outbreak reported in Cambodia in 2012: 7 patients had test results which were suggestive of recent chikungunya infection [18, 19]. However, no pathogens were identified in the remaining patients.
Table 1

Results of the dengue serological and virological testing (N = 701), National Paediatric Hospital, Phnom Penh, September 2011 - January 2013

% of total

% of all dengue positive or negative

(95% CI)

(95% CI)

Positive dengue

(76.4 - 82.5)

Dengue 1

(90.8 – 95.1)

Dengue 2

(0.6 – 2.8)

Dengue 3

(0.005 – 1.0)

Dengue 4

(0.04 – 1.3)

Serotype unspecified

(3.2 – 7.0)

Negative dengue

(17.5 – 23.6)

(2.0 – 9.8)

Overall the proportion of under-5 children was 18% (128/558) with an equal number of boys and girls. The onset of fever prior admission was less or equal to three days for 18% of subjects (126/558), and the median duration of hospital stay was 3 days. Overall, there were 5 deaths in the study population, indicating a case-fatality rate of 0.7% (95% CI, 0.2–1.7%).

To assess the representativeness of our hospital cohort relative to the national epidemic, we compared the weekly caseload of admissions at the National Paediatric Hospital with the figures reported nationally (Figure 2). While this represents a crude proxy, the relationship is statistically significant across the study period when compared by simple correlation (R2 = 0.67; p < 0.001) and by time-series regression (p < 0.001). These results indicate that the temporal trends within the hospital and across Cambodia are highly correlated and suggest that the admission trends in cases captured in our study are indeed representative of the national-scale dengue epidemic.Two purely spatial clusters were identified (Figure 3). In the first cluster, 46 cases were aggregated within the southwest of the Phnom Penh suburban area over the entire reporting period (RR = 2.12, no CI; p = 0.004). The second cluster includes 43 cases aggregated in central Phnom Penh (RR = 1.93, no CI; p = 0.031). These p-values of less than 0.05 suggest these specific case aggregations are unlikely to be due to chance. In spatio-temporal analysis, only a single large cluster (radius > 9 km) was found to be marginally significant (p = 0.04) during October 2011 (data not shown). The standardized annual rates (per 100,000 population) within the city are shown as a choropleth map in Figure 4; highest rates are evident in the southwest and central regions, and these regions of highest risk coincide with the clusters identified in the spatial analysis. To assess any biases in capture of patients with geocoded addresses, the baseline demographics of patients were compared to the overall study population, and no significant differences were found (gender by rank-sum test, p = 0.302; age by Kruskal-Wallis test, p = 0.063).
Figure 2
Figure 2

Comparison of national reporting data versus hospital inpatient cases, National Paediatric Hospital, Phnom Penh September. 2011- January 2013. Note: (p < 0.001, time-series regression of total hospital admissions and national cases).

Figure 3
Figure 3

Mapping of cases by serotype showing significant spatial clusters within greater Phnom Penh, by smallest administrative district (sangkat) (n = 216). Note: Cluster 1 (radius- 5.2 km): 46 cases; relative Risk = 2.1 (p = 0.004); Cluster 2 (1.7 km): 43 cases; relative Risk = 1.9 (p = 0.031).

Figure 4
Figure 4

Mapping of cases as annual population standardized rates per 100,000 total population (2008 population estimates) by smallest administrative district ( sangkat ) (n = 216).

In the analysis of climatic impact on national dengue reports, three examined variables had greatest explanatory power in bivariate analysis: minimum weekly temperature; median weekly relative humidity; and weekly maximum rainfall (see Additional file 1: Table S1). The minimum temperature was positively correlated with the number of cases nationwide, with significant effects at lagged intervals of 1–3, 6, and 9–11 weeks. The corresponding incidence rate ratios (IRRs), ranging from 1.12 to 1.22, indicate a 12-22% increase in cases per degree Celsius increase in the weekly minimum temperature. The median relative humidity was also positively correlated with dengue cases in multivariate models, having significant effects at 1–3 weeks, with an IRR ranging from 1.036-1.042, indicating a 4% increase in cases for each unit increase in the weekly median relative humidity. Figure 5 illustrates the general relationships between national reported cases and significant climatic variables from multivariate analysis.
Figure 5
Figure 5

Comparison of national reporting data and significant climatic variables, Cambodia national data, December 2010- December 2012. Note: RH = relative humidity.

Among the examined rainfall variables, the maximum weekly rainfall showed the strongest association with reported dengue cases, with a positive correlation in bivariate analysis. However, in multivariate analysis the coefficients became negative, indicating an inverse relationship with dengue cases after adjusting for covariates. Lag periods of 1–5, and 10–12 weeks were all significant, with IRRs ranging from 0.991–0.987, indicting a 0.9–1.3% decrease in cases per mm of rain per week. Analysis of data from the other weather station in Cambodia (Siem Reap; not shown) indicates the same general multivariate trends, however these data are limited by extensive missing values.

We present the results of a large prospective descriptive study of paediatric dengue infection conducted in a single referral centre in Phnom Penh, Cambodia. All 701 children enrolled in the study were initially suspected as having dengue, and nearly 80% included had a positive confirmatory test. The inclusion period was from September 2011 to January 2013, which covered two dengue epidemic seasons. Although all four serotypes were found in circulation during the period, DENV-1 predominated in 2012–2013; these trends are consistent with data from the National Surveillance system [5].

Spatial epidemiology has been specifically highlighted as a key component to guide dengue control efforts, especially in resource-limited settings. A recent analysis of large-scale transmission patterns of dengue in Cambodia identified ‘hotspots’ - zones of high transmission that may serve as reservoirs of virus for subnational scale epidemiology [20]. Extensive fever cohorts have found large differences in transmission within and between urban and rural regions [21]; however, these studies did not address neighbourhood-scale effects, which are dominated by human movements between neighbourhoods. The results from this study show substantial heterogeneity of transmission within Phnom Penh, mirroring larger scale infection dynamics within Cambodia. Our results suggest that rapid identification and targeting of these areas could have important impacts on transmission, especially in areas of high population density.

When examined as total cases per population for the study period, we identified several areas of elevated risk in the southwest and central areas of Phnom Penh aggregated over the course of this study. Such clusters could be high-risk areas for dengue epidemics due to larval sites or other modifiable characteristics and should be prioritized for vector control activities including community health promotion & education, elimination of breeding sites, water treatment using larvicides, and isolation of fever cases. Recent large-scale studies conducted in Bangkok found long-term modulation of transmission based on population-level (‘herd’) immunity at the neighbourhood scale [22]; our study findings are consistent with these results. The overwhelming predominance of a single serotype, however, hindered investigation of serotype-specific clusters and any potential interactions on population immunity.

The time lags identified within this study are broadly consistent with those found in other locations within SE Asia: in Thailand lag times of 6 months for temperature maxima and 3 months for rainfall events were identified; in Singapore, lag times of 1–5 months for mean temperature and rainfall events were identified [23, 24]. In common with studies in Sri Lanka and New Caledonia, we find very limited and complex impact of rainfall on dengue incidence [25, 26]. A main limitation is that the rainfall data are derived from satellite imagery, and may have limited spatial and temporal accuracy. However, satellite imagery in general has been shown to be most accurate for low latitudes during the warm season [27], suggesting that these estimates are likely reasonable. Additional support for this interpretation is the finding of an inverse relationship between rainfall and Aedes larval densities in Malaysia, suggesting a ‘flooding-out’ of larval habitats during periods of extreme rainfall [28]. These results concur with recent studies that found complex non-linear interactions between climatic variables and dengue incidence [29].

Limitations of this study

These data have been collected in a single referral paediatric hospital in Phnom Penh during a limited period of time, and therefore our findings may not be representative of other areas with different dengue transmission patterns, populations or population density characteristics. Cases from the spatiotemporal analyses were limited to those cases with an address that could be geo-referenced; some patients provided the name of an older administrative system that could not be readily mapped; however, this bias would have led to omission of cases This would not impact cluster detection with underlying population values as any impacts would be expected to dilute the observed effect sizes. There were no significant differences in age or gender between captured patients and those without addresses, suggesting there was no differential loss to follow-up.

There is also the potential for bias in our results due to differences in care-seeking behaviour. Patients are likely to seek care at the closest referral centre, which may partially explain the clustering of the cases in this study within Phnom Penh. However, as the hospital serves as the main paediatric referral centre within the public sector, this bias is likely minor.

We have also used a single set of weather data to predict the national scale epidemic. The local-scale interactions between climate, vectors and people - that is, the micro-epidemiology and heterogeneous exposures - cannot be captured at this scale [30].

Bearing these limitations in mind, our analysis shows a very strong correlation between observed trends and national dengue data trends as well as a strong correlation between local rainfall and temperature data and satellite estimates at the national level.

From an operational standpoint, the monitoring of peak temperatures, extreme humidity and rainfall indexes could help prioritize resources to be mobilized to address any emerging transmission cycles; however, the lead times identified in these data of 1–6 and 9–10 weeks might not provide sufficient time to implement aggressive campaigns in a resource-limited setting like Cambodia. Our results also suggest that a wider range of weather summary indexes should be incorporated into models to improve model fit and prediction. Recent work in malaria also suggests that predictions may be improved by using high-resolution data instead of traditional regional and weekly summaries [31], and surveillance in Cambodia could benefit from improved weather reporting systems.

While the routine use of environmental variables for real-time dengue surveillance can be complex in real-world settings [32], the wide availability of open-source tools (QGIS, R software and SaTScan) [17, 33, 34] make this increasingly feasible even in resource-limited settings. These results can serve as a foundation for the implementation of robust real-time surveillance within Cambodia.

Finally, the ability to pre-plan and stockpile needed resources (control staff, Bti, etc.) should be considered [35], and the identification of multiple hotspots of higher risk relative to the remainder of Phnom Penh suggests areas that should targeted for enhanced surveillance and vector control efforts during future transmission seasons.

Acknowledgements and funding

We are grateful for both the patients and their families for agreeing to take part in this study, and for the critical contributions of the staff and nurses at the National Paediatric Hospital, without which this study would not have been possible. We are also grateful for the assistance of Julien Cappelle (Institut Pasteur, Cambodia) for his assistance in bulk downloading of weather data. The rainfall data used in this study were acquired using the GES-DISC Interactive Online Visualization ANd aNalysis Infrastructure (Giovanni) as part of the NASA’s Goddard Earth Sciences (GES) Data and Information Services Center (DISC).

Funding for this study was provided by Sanofi-Pasteur. The sponsors of the study were involved in study design and review of the manuscript; the sponsors had no role in the data collection/analysis and the decision to publish was made jointly by all authors.

Authors’ Affiliations

Infectious Diseases Programme, Saw Swee Hock School of Public Health, National University of Singapore, MD3, 16 Medical Drive, Singapore, 117597, Singapore
Virology Unit, Institut Pasteur in Cambodia, Phnom Penh, Cambodia
Formerly- Regional Emerging Diseases Intervention (REDI) Centre, Singapore, Singapore
Infectious Diseases Unit, National Paediatric Hospital, Phnom Penh, Cambodia
National Dengue Control Program (NDCP), National Center for Parasitological, Entomology and Malaria Control, Ministry of Health, Phnom Penh, Cambodia
Ministry of Health, Phnom Penh, Cambodia
Tan Tock Seng Hospital, Centers for Disease Control, Singapore, Singapore
Emergency Unit, National Paediatric Hospital, Phnom Penh, Cambodia
Medical Affairs Department, Sanofi Pasteur, Singapore Regional Office, Singapore, Singapore
Epidemiology Unit, Institut Pasteur in Cambodia, Phnom Penh, Cambodia
Médecins Sans Frontières, Operational Centre, Geneva, Switzerland

  1. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GRW, Simmons CP, Scott TW, Farrar JJ, Hay SI: The global distribution and burden of dengue. Nature. 2013, 496: 504-507. 10.1038/nature12060.View ArticlePubMedPubMed CentralGoogle Scholar
  2. United Nations Development Programme: Human Development Report. 2013, New York; London: United Nations; EurospanGoogle Scholar
  3. Huy R, Buchy P, Conan A, Ngan C, Ong S, Ali R, Duong V, Yit S, Ung S, Te V, Chroeung N, Pheaktra NC, Uok V, Vong S: National dengue surveillance in Cambodia 1980–2008: epidemiological and virological trends and the impact of vector control. Bull World Health Organ. 2010, 88: 650-657. 10.2471/BLT.09.073908.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Beauté J, Vong S: Cost and disease burden of dengue in Cambodia. BMC Public Health. 2010, 10: 521-10.1186/1471-2458-10-521.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Dengue situation in Cambodia, 2012 update (National Dengue Control Programme). 2012,, update (National Dengue Control Programme),
  6. Vong S, Goyet S, Ly S, Ngan C, Huy R, Duong V, Wichmann O, Letson GW, Margolis HS, Buchy P: Under-recognition and reporting of dengue in Cambodia: a capture–recapture analysis of the National Dengue Surveillance System. Epidemiol Infect. 2012, 140: 491-499. 10.1017/S0950268811001191.View ArticlePubMedGoogle Scholar
  7. Eisen L, Lozano-Fuentes S: Use of mapping and spatial and space-time modeling approaches in operational control of Aedes aegypti and dengue. PLoS Negl Trop Dis. 2009, 3: e411-10.1371/journal.pntd.0000411.View ArticlePubMedPubMed CentralGoogle Scholar
  8. World Health Organization: Guidelines for Treatment of Dengue Fever / Dengue Haemorrhagic Fever in Small Hospitals. 1999, New Delhi: WHO Regional Office for South-East AsiaGoogle Scholar
  9. World Health Organization: Dengue Guidelines for Diagnosis, Treatment, Prevention and Control. 2010, Geneva: World Health Organization, New editionGoogle Scholar
  10. Andries A-C, Duong V, Ngan C, Ong S, Huy R, Sroin KK, Te V, Y B, Try PL, Buchy P: Field evaluation and impact on clinical management of a rapid diagnostic kit that detects dengue NS1, IgM and IgG. PLoS Negl Trop Dis. 2012, 6: e1993-10.1371/journal.pntd.0001993.View ArticlePubMedPubMed CentralGoogle Scholar
  11. Integrated Surface Database (ISD) | National Climatic Data Center (NCDC).,
  12. TRMM Multi-Satellite Precipitation Data.,
  13. General Population Census. 2008,,
  14. Newey WK, West KD: A simple, positive semi-definite, heteroskedasticity and autocorrelation consistent covariance matrix. Econometrica. 1987, 55: 703-708. 10.2307/1913610.View ArticleGoogle Scholar
  15. Russell PF, West LS, Manwell RD: Practical Malariology. 1946, Philadelphia: W. B. SaundersGoogle Scholar
  16. Dupont WD: Statistical Modeling for Biomedical Researchers. 2009, Cambridge, New York: Cambridge University Press, 2View ArticleGoogle Scholar
  17. Kulldorff M, Information Management Services, Inc: SaTScan. 2011, Software for the Spatial and Space-Time Scan Statistics,,Google Scholar
  18. Duong V, Andries A-C, Ngan C, Sok T, Richner B, Asgari-Jirhandeh N, Bjorge S, Huy R, Ly S, Laurent D, Hok B, Roces MC, Ong S, Char MC, Deubel V, Tarantola A, Buchy P: Reemergence of chikungunya virus in Cambodia. Emerg Infect Dis. 2012, 18: 2066-2069. 10.3201/eid1812.120471.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Centers for Disease Control and Prevention (CDC): Chikungunya outbreak--Cambodia, February-March 2012. MMWR Morb Mortal Wkly Rep. 2012, 61: 737-740.Google Scholar
  20. Teurlai M, Huy R, Cazelles B, Duboz R, Baehr C, Vong S: Can human movements explain heterogeneous propagation of dengue fever in Cambodia?. PLoS Negl Trop Dis. 2012, 6: e1957-10.1371/journal.pntd.0001957.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Vong S, Khieu V, Glass O, Ly S, Duong V, Huy R, Ngan C, Wichmann O, Letson GW, Margolis HS, Buchy P: Dengue incidence in urban and rural Cambodia: results from population-based active fever surveillance, 2006–2008. PLoS Negl Trop Dis. 2010, 4: e903-10.1371/journal.pntd.0000903.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Salje H, Lessler J, Endy TP, Curriero FC, Gibbons RV, Nisalak A, Nimmannitya S, Kalayanarooj S, Jarman RG, Thomas SJ, Burke DS, Cummings DAT: Revealing the microscale spatial signature of dengue transmission and immunity in an urban population. PNAS. 2012, 109: 9535-9538. 10.1073/pnas.1120621109.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Hii YL, Rocklöv J, Wall S, Ng LC, Tang CS, Ng N: Optimal lead time for dengue forecast. PLoS Negl Trop Dis. 2012, 6: e1848-10.1371/journal.pntd.0001848.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Focks DA, Barrera R: Dengue Transmission Dynamics: Assessment and Implications for Control. WHO Report of the Scientific Working Group meeting on Dengue, Geneva. 2006, Geneva: Working paper for the Scientific Working Group on Dengue Research, convened by the Special Programme for Research and Training in Tropical Diseases,,Google Scholar
  25. Goto K, Kumarendran B, Mettananda S, Gunasekara D, Fujii Y, Kaneko S: Analysis of effects of meteorological factors on dengue incidence in Sri Lanka using time series data. PLoS One. 2013, 8: e63717-10.1371/journal.pone.0063717.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Descloux E, Mangeas M, Menkes CE, Lengaigne M, Leroy A, Tehei T, Guillaumot L, Teurlai M, Gourinat A-C, Benzler J, Pfannstiel A, Grangeon J-P, Degallier N, De Lamballerie X: Climate-based models for understanding and forecasting dengue epidemics. PLoS Negl Trop Dis. 2012, 6: e1470-10.1371/journal.pntd.0001470.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Ebert EE, Janowiak JE, Kidd C: Comparison of near-real-time precipitation estimates from satellite observations and numerical models. Bull Am Meteorol Soc. 2007, 88: 47-64. 10.1175/BAMS-88-1-47.View ArticleGoogle Scholar
  28. Wee LK, Weng SN, Raduan N, Wah SK, Ming WH, Shi CH, Rambli F, Ahok CJ, Marlina S, Ahmad NW, Mckemy A, Vasan SS, Lim LH: Relationship between rainfall and Aedes larval population at two insular sites in Pulau Ketam, Selangor, Malaysia. Southeast Asian J Trop Med Public Health. 2013, 44: 157-166.PubMedGoogle Scholar
  29. Hii YL, Zhu H, Ng N, Ng LC, Rocklöv J: Forecast of dengue incidence using temperature and rainfall. PLoS Negl Trop Dis. 2012, 6: e1908-10.1371/journal.pntd.0001908.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Smith DL, Dushoff J, McKenzie FE: The risk of a mosquito-borne infection in a heterogeneous environment. PLoS Biol. 2004, 2: e368-10.1371/journal.pbio.0020368.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Weiss DJ, Bhatt S, Mappin B, Boeckel TPV, Smith DL, Hay SI, Gething PW: Air temperature suitability for Plasmodium falciparum malaria transmission in Africa 2000–2012: a high-resolution spatiotemporal prediction. Malar J. 2014, 13: 171-10.1186/1475-2875-13-171.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Racloz V, Ramsey R, Tong S, Hu W: Surveillance of dengue fever virus: a review of epidemiological models and early warning systems. PLoS Negl Trop Dis. 2012, 6: e1648-10.1371/journal.pntd.0001648.View ArticlePubMedPubMed CentralGoogle Scholar
  33. QGIS Development Team: QGIS Geographic Information System. Open Source Geospatial Foundation Project. 2013,,Google Scholar
  34. R Core Team: R: A Language and Environment for Statistical Computing. 2013, Vienna, Austria: Foundation for Statistical ComputingGoogle Scholar
  35. Conolly MA: World Health Organization: Communicable Disease Control in Emergencies: A Field Manual. 2005, Geneva: World Health OrganizationGoogle Scholar
  36. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:


text:
Skip to content

  • Short Report
  • Open Access

The effect of 5-aminolevulinic acid on cytochrome c oxidase activity in mouse liver

  • 1Email author,
  • 2,
  • 1,
  • 1,
  • 3,
  • 3,
  • 3,
  • 1,
  • 1,
  • 3 and
  • 3
BMC Research Notes20114:66

  • Received: 4 February 2011
  • Accepted: 17 March 2011
  • Published:

5-Aminolevulinic acid (ALA) is a precursor of heme that is fundamentally important in aerobic energy metabolism. Among the enzymes involved in aerobic energy metabolism, cytochrome c oxidase (COX) is crucial. In this study, the effect of ALA on cytochrome c oxidase activity was measured.

c57BL/6N species of mice were administered ALA orally for 15 weeks. After ALA administration, mice were sacrificed and livers were obtained. COX activity in mitochondria from ALA-administered mouse livers was 1.5-fold higher than that in mitochondria from PBS-administered mouse livers (P < 0.05). Furthermore, ATP levels in ALA-administered mouse livers were much higher than those in PBS-administered mouse livers. These data suggest that oral administration of ALA promotes aerobic energy metabolism, especially COX activity.

This is the first report of a drug that functions in aerobic energy metabolism directly. Since COX activity is decreased in various diseases and aging, the pharmacological effects of ALA will be expanding.

  • Mouse Liver
  • CoQ10
  • Liver Mitochondrion
  • Aerobic Energy Metabolism
  • Control Mouse Liver

The activity of cytochrome c oxidase (complex IV, COX, EC in the electron transfer chain is fundamentally important in aerobic energy metabolism. This enzyme catalyzes electron transfer from cytochrome c to molecular oxygen, reducing the latter to water, and yields substantial energy that drives the formation of a proton gradient that is then employed to synthesize ATP [1]. The activity of COX is also important in aging and several diseases. Age-related decreases in COX activity have also been reported in many species (e.g., Drosophila melanogaster[2], housefly [3], rodents [4, 5]). Müller-Höcker observed that COX-negative muscle fibers were increased with age in humans and animals [68], showing that the improvement of COX activity is important for anti-aging. COX activity has been shown to be diminished in mitochondria from the brains of Alzheimer's disease patients [9, 10]. In human cancer cells, p53 regulates COX activity through synthesis of cytochrome c oxidase 2 (SCO2) [11], causing a metabolic change called the Warburg effect [12]. COX activity is also inhibited by nitric oxide (NO), which is a signaling molecule involved in many pathophysiological processes (e.g., smooth muscle relaxation, inflammation, neurotransmission, apoptosis) [13]. COX inhibition is also observed in diabetes. COX levels in sensory neurons from diabetic rats was downregulated by 29% compared to that from normal rats [14], and advanced glycation end products, which are implicated in diabetic complications, inhibit COX activity by inducing the expression of inducible nitric oxide synthase (iNOS) [15]. Therefore, COX activity is important, and to our knowledge there is no drug that activates COX.

5-Aminolevulinic acid (ALA) is widely distributed in both plant and animal cells, and it is the common precursor of tetrapyrrole compounds. In animal cells, ALA is formed from glycine and succinyl CoA by ALA synthase in mitochondria. Administration of ALA enhances the cancer-specific accumulation of porphyrins. ALA-induced porphyrins can be fluoresced and visualized in neoplastic regions. This technique exploits ALA-induced differences in fluorescent signatures between normal and cancer tissues. Therefore, the technique is generally termed fluorescence diagnosis or photodynamic diagnosis [16].

On the other hand, in normal tissue, iron ion is inserted into porphyrin to form heme in mitochondria, which is incorporated into hemoproteins and cytochromes. Because COX and its substrate (cytochrome c) are major hemoproteins, heme is fundamentally important to COX activity. In this study, to increase COX activity, ALA was administered to mice, and COX activity in the liver was measured.

Materials and methods

5-Aminolevulinic acid hydrochloride was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). Mitochondria isolation and cytochrome c oxidase assay kits were purchased from Sigma Aldrich (St. Louis, MO, USA), and an ATP assay kit for animal tissues (TA100, Toyo B Net, Tokyo, Japan) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan)

Animals and in vivo experiments

c57BL/6N mice (female, 6 weeks old) were purchased from CREA Japan (Tokyo, Japan). All mice were housed and used for experiments in accordance with standard ethical guidelines for the care and use of laboratory animals [Science Council of Japan; Guidelines for Proper Conduct of Animal Experiments (2006)], and this study was approved by the Animal Experiment Ethics Committee of Shizuoka Cancer Center.

ALA was administered orally for 15 weeks. As a stock solution, 4 mg/ml ALA in PBS was prepared, and the ALA stock solution was added to create a 1:100 dilution (final concentration: 0.04 mg/ml, approximately 10 mg/kg/day). PBS was added to drinking water for the control group.

Measurement of COX activity in liver mitochondria

Mouse livers were extracted, and liver mitochondria were obtained using a mitochondria isolation kit (Sigma-Aldrich). In brief, fresh livers were washed and homogenized (rotor-stator homogenizer (T10 basic Ultra-Turrax®, IKA, Staufen, Germany) at 15,000 rpm) in extraction reagent. The homogenate was centrifuged at 600 ×g for 5 min, and then the supernatant was further centrifuged at 11,000 × g for 10 min. The pellet was suspended in storage buffer and used as a mitochondrial fraction. Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, CA). COX activity in liver mitochondria was measured using a cytochrome c oxidase assay kit. In brief, 5 μg of the mitochondrial fraction were diluted with enzyme dilution buffer containing 1 mM n-dodecyl β-d-maltoside. Ferrocytochrome c (reduced cytochrome c with dithiothreitol) was then added to the sample, and COX activity was measured by the decrease in absorption at 550 nm. The difference in extinction coefficients between reduced and oxidized cytochrome c is 21.84 at 550 nm [17]. One unit of COX activity was defined as the oxidization of 1.0 μmole of ferrocytochrome c per min at pH 7.0 at 25°C.

Western blot analysis

Western blot analysis was performed as previously described with some modifications [18]. Liver mitochondria, obtained as described above, were separated by 15% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA). After incubating in 5% nonfat dry milk in TBST (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) at room temperature for 1 h, the membranes were incubated for 1 h with mouse anti-COX IV-1 antibody (1:200 dilution in TBST with 5% nonfat dry milk, Santa Cruz Biotechnology, Santa Cruz, CA) followed by incubation in a secondary anti-mouse IgG coupled to horseradish peroxidase (1:3000 dilution in TBST, Cell Signaling, Danvers, MA). Target proteins were detected using an ECL kit (GE Healthcare, UK), and the image was captured using an LAS-4000 imager (GE Healthcare).

Measurement of ATP level in liver

For measuring ATP, cellular ATP in livers was extracted using an ATP extraction kit of an ATP assay kit (TA100, Toyo B Net). In brief, small pieces of livers (about 0.1 g) were washed once with PBS, resuspended in ATP extraction reagent, and then centrifuged at 1,000 ×g for 10 min. The supernatant was used for the ATP assay. The ATP level was quantitatively measured using an ATP assay kit with luciferin and luciferase according to the protocol provided by the manufacturer (Toyo B Net).

Results and discussion

No mice died due to ALA administration for 15 weeks, and significant body weight changes were not observed, indicating that ALA has no toxicity in this condition. Western blot analysis of the mouse liver mitochondrial fraction was performed (Figure 1). COX consists of 10 nuclear-coded subunits and 3 catalytic mitochondrial-coded subunits [19]. Among these 13 subunits, subunit IV specifically binds ATP, playing an important role in COX activity [20]. Thus the antibody raised against COX IV was employed for western blot analysis in our study (Figure 1). Apparent increases in COX protein levels were observed in ALA-administered mouse livers. Approximately 1.6-fold increase was observed by densitometric scanning compared to the control.
Figure 1
Figure 1

Western blot analysis of the mouse liver mitochondrial fraction. Ten micrograms of liver mitochondria, obtained as described in the Material and Methods section, were separated by 15% SDS-PAGE and then electrotransferred on to a PVDF membrane and Western blot analysis were performed using COX IV-1 antibody. The average COX IV expression level in the ALA administered group was 1.6-fold higher than that of the control group.

Figure 2 illustrates COX activity in mitochondria from mouse livers. COX activity was defined as the oxidization of ferrocytochrome c. The average COX activity in mitochondria from ALA-administered mouse livers was 1.5-fold higher than that in mitochondria from control mouse livers, which was statistically different (P < 0.05 by Student's t-test). This finding is in good agreement with the increase in COX protein levels, as shown in Figure 1. COX activity in Drosophila melanogaster declined progressively with age by 33% [2], indicating that the 1.5-fold increase in activity observed in this study is substantial. Our results clearly demonstrate that both COX protein levels and activity were increased by ALA administration. This is the first report of hepatic COX activation achieved by oral administration of a natural compound.
Figure 2
Figure 2

COX activity in mitochondria from mouse livers. COX activity was defined as the oxidization of ferrocytochrome c. Bars represent SD of the means of three replicate experiments. *P < 0.05 by Student's t-test.

Figure 3 shows ATP levels in mouse liver lysates. ATP was extracted and quantified using luciferin and luciferase. ATP levels in ALA administered mouse livers were 1.6-fold higher than those in control mouse livers. This apparent increase in ATP levels may be caused by COX activation in the mitochondrial electron transfer chain.
Figure 3
Figure 3

ATP level in mouse liver lysates. ATP was extracted and quantified using luciferin and luciferase. Bars represent SD of the means of three replicate experiments. P = 0.069 by Student's t-test.

COX and cytochrome c are hemoproteins, and COX activity was increased by ALA administration. ALA is the precursor of protoporphyrin, and heme is produced by the insertion of iron. Therefore, ALA administration can result in heme production in the mouse liver, and it is possible that COX activity was increased by ALA administration. Heme deficiency selectively decreased the activity and protein content of COX by 95% [21], that is observed in aging. This phenomenon strongly suggests the amount of heme is important for the COX activity. In addition, COX requires some cofactors, namely iron and copper ions. In this study, iron and copper were not administrated directly. However, these elements were easily obtained by the animals via routine feeding. There is a possibility that these elements have key roles in the maintenance of COX following administration of ALA. Because we detected increases in COX activity and protein levels, further analysis may be needed for clarification. In addition to COX, Complex II and Complex III are also hemoproteins. These proteins may also be upregulated by ALA administration.

Coenzyme Q10 (CoQ10) is also a well-known cofactor in the aerobic respiratory electron transfer and is widely used as a nutritional supplement. Orally administrated CoQ10 can be taken up by mitochondria, but only to a limited extent [22, 23]. On the other hand, protoporphyrin and heme are generated in mitochondria after ALA administration. This active targeting is important in ALA-mediated COX activation.

ALA is used in various fields, especially cancer diagnosis and therapy. There is no report of toxic side effects caused by ALA administration. In the present study, no toxicity was observed during long-term ALA administration, indicating its safety. Moreover, ALA supplementation has been used in several fields to date. Wang et al. demonstrated that ALA and vitamin C supplementation improved iron status including hemoglobin levels in sows and laying hens [24, 25]. Yamazaki et al. revealed that ALA and iron ion administration caused a stimulatory effect on hair growth [26]. These findings strongly suggest that ALA is a potentially useful nutritional supplement.

In conclusion, we demonstrated improvement of COX activity by ALA administration, suggesting the further potential benefits of ALA. If ALA activates COX activity, ALA would be a promising candidate of treatment option for aging, diabetes, Alzheimer's disease, chronic inflammation, and cancers.

The study in the authors' laboratory was supported by the Grant-in-Aid for Young Scientists (B) (No. 22700910) from the Ministry of Education, Culture, Sports, Science and Technology. Y. Hagiya is a Japanese Society for the Promotion of Science (JSPS) Research Fellow.

Authors’ Affiliations

Endowed Research Section (ALA), Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan
Shizuoka Cancer Center Research Institute, Shizuoka, Japan
SBI ALApromo Co., Ltd, Tokyo, Japan

  1. Capaldi RA: Structure and assembly of cytochrome c oxidase. Arch Biochem Biophys. 1990, 280: 252-262. 10.1016/0003-9861(90)90327-U.PubMedView ArticleGoogle Scholar
  2. Ren JC, Rebrin I, Klichko V, Orr WC: Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster. Biochem Biophys Res Commun. 2010, 401: 64-68. 10.1016/j.bbrc.2010.09.009.PubMedPubMed CentralView ArticleGoogle Scholar
  3. Sohal RS: Aging, cytochrome oxidase activity and hydrogen peroxide release by mitochondria. Free Radic Biol Med. 1993, 14: 583-588. 10.1016/0891-5849(93)90139-L.PubMedView ArticleGoogle Scholar
  4. Kwong LK, Sohal RS: Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse. Arch Biochem Biophys. 2001, 373: 16-22. 10.1006/abbi.1999.1495.View ArticleGoogle Scholar
  5. Navarro A, Boveris A: Rat brain and liver mitochondria develop oxidative stress and lose enzymatic activities on aging. Am J Physiol Regul Integr Comp Physiol. 2004, 287: 1244-1249. 10.1152/ajpregu.00226.2004.View ArticleGoogle Scholar
  6. Müller-Höcker J: Cytochrome c oxidase deficient cardiomyocytes in the human heart--an age-related phenomenon. A histochemical ultracytochemical study. Am J Pathol. 1989, 134: 1167-73.PubMedPubMed CentralGoogle Scholar
  7. Müller-Höcker J: Cytochrome c oxidase deficient fibres in the limb muscle and diaphragm of man without muscular disease: an age-related alteration. J Neurol Sci. 1990, 100: 14-21.PubMedView ArticleGoogle Scholar
  8. Müller-Höcker J, Seibel P, Schneiderbanger K, Kadenbach B: Different in situ hybridization patterns of mitochondrial DNA in cytochrome c oxidase-deficient extraocular muscle fibres in the elderly. Virchows Arch A Pathol Anat Histopathol. 1993, 422: 7-15.PubMedView ArticleGoogle Scholar
  9. Kish SJ, Mastrogiacomo F, Guttman M, Furukawa Y, Taanman JW, Dozic S, Pandolfo M, Lamarche J, DiStefano L, Chang LJ: Decreased brain protein levels of cytochrome oxidase subunits in Alzheimer's disease and in hereditary spinocerebellar ataxia disorders: a nonspecific change?. J Neurochem. 1993, 72: 700-707. 10.1046/j.1471-4159.1999.0720700.x.View ArticleGoogle Scholar
  10. Cardoso SM, Proenca MT, Santos S, Santana I, Oliveira CR: Cytochrome c oxidase is decreased in Alzheimer's disease platelets. Neurobiol Aging. 2004, 25: 105-110. 10.1016/S0197-4580(03)00033-2.PubMedView ArticleGoogle Scholar
  11. Matoba S, Kang JG, Patino WD, Wragg A, Boehm M, Gavrilova O, Hurley PJ, Bunz F, Hwang PM: p53 regulates mitochondrial respiration. Science. 2006, 312: 1650-1653. 10.1126/science.1126863.PubMedView ArticleGoogle Scholar
  12. Warburg O: On respiratory impairment in cancer cells. Science. 1956, 124: 269-270.PubMedGoogle Scholar
  13. Brown GC, Borutaite V: Nitric oxide inhibition of mitochondrial respiration and its role in cell death. Free Rad Biol Med. 2002, 33: 1440-1450. 10.1016/S0891-5849(02)01112-7.PubMedView ArticleGoogle Scholar
  14. Akude E, Zherebitskaya E, RoyChowdhury SK, Smith DR, Dobrowsky RT, Fernyhough P: Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats. Diabetes. 2010,Google Scholar
  15. Zhao Z, Zhao C, Zhang SH, Zheng F, Cai W, Vlassara H, Ma ZA: Advanced glycation end products inhibit glucose-stimulated insulin secretion through nitric oxide-dependent inhibition of cytochrome c oxidase and adenosine triphosphate synthesis. Endocrinology. 2009, 150: 2569-2576. 10.1210/en.2008-1342.PubMedPubMed CentralView ArticleGoogle Scholar
  16. Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ: Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 2006, 7: 392-401. 10.1016/S1470-2045(06)70665-9.PubMedView ArticleGoogle Scholar
  17. Berry EA, Trumpower BL: Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal Biochem. 1987, 161: 1-15. 10.1016/0003-2697(87)90643-9.PubMedView ArticleGoogle Scholar
  18. Ogura S, Kaneko K, Miyajima S, Ohshima K, Yamaguchi K, Mochizuki T: Proneurotensin/neuromedin N secreted from small cell lung carcinoma cell lines as a potential tumor marker. Proteomics Clin Appl. 2008, 2: 1620-1627. 10.1002/prca.200800039.PubMedView ArticleGoogle Scholar
  19. Kadenbach B: Structure and evolution of the "Atmungsferment" cytochrome c oxidase. Angew Chem Int Ed Engl. 1983, 22: 275-282. 10.1002/anie.198302751.View ArticleGoogle Scholar
  20. Napiwotzki J, Kadenbach B: Extramitochondrial ATP/ADP ratios regulate cytochrome c oxidase activity via binding to the cytosolic domain of subunit IV. Biol Chem. 1998, 379: 335-339. 10.1515/bchm.1998.379.3.335.PubMedView ArticleGoogle Scholar
  21. Atamna H, Liu J, Ames BN: Heme deficiency selectively interrupts assembly of mitochondrial complex IV in human fibroblasts. J Biol Chem. 2001, 276: 48410-48416.PubMedGoogle Scholar
  22. Ernster L, Dallner G: Biochemical, physiological and medical aspects of ubiquinone function. Biochim Biophys Acta. 1995, 1271: 195-204.PubMedView ArticleGoogle Scholar
  23. Matthews RT, Yang L, Browne S, Baik M, Beal MF: Coenzyme Q10 administration increases brain mitochondrial concentrations and exerts neuroprotective effects. Proc Natl Acad Sci USA. 1998, 95: 8892-8897. 10.1073/pnas.95.15.8892.PubMedPubMed CentralView ArticleGoogle Scholar
  24. Wang JP, Kim HJ, Chen YJ, Yoo JS, Cho JH, Kang DK, Hyun Y, Kim IH: Effects of delta-aminolevulinic acid and vitamin C supplementation on feed intake, backfat, and iron status in sows. J Anim Sci. 2009, 87: 3589-3595. 10.2527/jas.2008-1489.PubMedView ArticleGoogle Scholar
  25. Wang JP, Lee JH, Jang HD, Yan L, Cho JH, Kim IH: Effects of δ-aminolevulinic acid and vitamin C supplementation on iron status, production performance, blood characteristics and egg quality of laying hens. J Anim Physiol Anim Nutr. 2010Google Scholar
  26. Morokuma Y, Yamazaki M, Maeda T, Yoshino I, Ishizuka M, Tanaka T, Ito Y, Tsuboi R: Hair growth stimulatory effect by a combination of 5-aminolevulinic acid and iron ion. Int J Dermatol. 2008, 47: 1298-303. 10.1111/j.1365-4632.2008.03783.x.PubMedView ArticleGoogle Scholar