text: We have implemented a more accurate physical system representation, a strip-area system model (SASM), for improved fan-beam collimator (FBC) SPECT reconstruction. This approach required implementation of modified ray tracing and attenuation compensation in comparison to a line-length system model (LLSM). We have compared performance of SASM with LLSM using Monte Carlo and analytical simulations of FBC SPECT from a thorax phantom. OSEM reconstruction was performed with OS=3 in a 64×64 matrix with attenuation compensation (assuming uniform attenuation of 0.13 cm -1). Scatter correction and smoothing were not applied. We observe overall improvement in SPECT image bias, visual image quality and an improved hot myocardium contrast for SASM vs. LLSM. In contrast to LLSM, the sensitivity pattern artifacts are not present in the SASM reconstruction. In both reconstruction methods, cross-talk image artifacts (e.g. inverse images of the lungs) can be observed, due to the uniform attenuation map used. SASM applied to fan-beam collimator SPECT results in better image quality and improved hot target contrast, as compared to LLSM, but at the expense of 1.5-fold increase in reconstruction time.


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Tom, November 21, 2011
84 / 100 by 450 users
FrogCityCheese. Destinations: Macau's Best Five Places Must be Visited - Top Interesting Tourist Destination. Senado SquareWhile enjoying my egg tart, I walked along Avenoda de Almeida Ribeiro for about 300 meters. Senado Square was directly in sight with a pool and fountain in the centre of the square and becomes. Macau Tower, China, Macau Travel Destinations, Casino Building, Guan Yin Statue.
Tom, March 15, 2011
96 / 100 by 288 users
FrogCityCheese. Lodging: Find Best Hotels to Enjoy Your Vacation. Visiting the city that has a high culture as well as a business center featuring a dynamic community lifestyle can be an interesting experience. Leipzig could be a choice destination you visit. You can. Leipzig, Dusseldorf, Hotels, German Vacation, Cologne.
Tom, March 08, 2011
79 / 100 by 237 users
FrogCityCheese. Transportation: Comfortable Ride. Traveling in buses and trains are so boring and privacy of the passengers is lost. This time, my father has booked a trip with the car rental facilities Alquiler de coches because they are very reliable. Transportation, Alquiler De Coches,
Tom, February 23, 2011
76 / 100 by 69 users
FrogCityCheese. Amazing Places: Amazing Unusual Restaurants (Location). 2. Da Pancrazio RestaurantDa Pancrazio restaurant located in Rome, Italy. A restaurant which is built over the ruins of Theatre of Pompey, and also one of the most original buildings in the city. A lovely. Amazing Unusual Restaurants, Amazing Places, Restaurants,
Tom, March 19, 2011
99 / 100 by 127 users
FrogCityCheese. Festival: Cultural Vacation Ideas for March. South by Southwest, Austin, TXSouth by Southwest, Austin, TX is a set of interactive and music festivals, conferences, and films that are usually held every spring, exactly around March. This event is. March Vacation Ideas, Monthly Vacation Ideas, Cultural Vacation Ideas, Travel Destination, Festival.
Tom, March 08, 2011
65 / 100 by 195 users
FrogCityCheese. Transportation: A Memorable Moment. As we reached the airport we found the car waiting for us. The driver explained about the country, the nuances of the local language and cuisine. The driver was fluent in English and helped us, and it. Reader Experiences, Greek Island, Hyrbil,
Tom, March 03, 2011
84 / 100 by 252 users
FrogCityCheese. Destinations: Senggigi Beach Traveling. This place offer many facilities that will be great for you. Besides, those good things can be enjoyed with spending affordable budget for you so you can be sure that it is really great to run. Also, this. Travel Destination, Beach, Senggigi Beach, Indonesia,
Tom, March 13, 2011
92 / 100 by 276 users
FrogCityCheese. Destinations: Antarctica for Summer Vacation?. This place is able to warm faster and cool down faster because the Ocean water brings heat from equator to the poles. Here, you can see the changes of atmosphere that can happen so fast. Besides, you need. Summer Vacation, Summer Vacation Ideas, Antarctica,
We love holiday! Vacation every time with beloved ones, traveling to the entire world capturing how beautiful this earth provides. Those we colect into a small yet beautiful and comfy blog, yes, the one you are currently on.
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domain: travel


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st 70 steel properties, NV A shipbuilding steel,Hot sell st 70 steel properties, NV A shipbuilding steel stock in China,China st 70 steel properties, NV A shipbuilding steel factory. BBN steel keeps more than 10,000 tons hot rolled and cold rolled st 70 steel properties, NV A shipbuilding steel in stock each month. BBN steel can meet our customer's any quantity request. Based on these stock source, our advantage industry spread into steel process like : cutting ( any size and any shape ) drilling, welding, milling, machining and steel structure. BBN steel also have a high quality logistics team who make sure the fast delivery time and shipment. Anyway, if you want to find st 70 steel properties, NV A shipbuilding steel plate/sheet/angle bar/bulb flat bar/ L section steel bar supplier, st 70 steel properties, NV A shipbuilding steel plate/sheet/angle bar/bulb flat bar/ L section steel bar manufacturer, st 70 steel properties, NV A shipbuilding steel plate/sheet/angle bar/bulb flat bar/ L section steel bar exporter, please contact BBN steel's sales team. We will give you the st 70 steel properties, NV A shipbuilding steel plate/sheet/angle bar/bulb flat bar/ L section steel bar competitive price, st 70 steel properties, NV A shipbuilding steel plate/sheet/angle bar/bulb flat bar/ L section steel bar good quality and best service from us.
Henan BEBON international co.,ltd - steel-plate …the chemical composition and the mechanical properties of EN ST 70-2 steel. ST 70-2 steel is according to DIN 17100 standard and it is equivalent to EN: Fe690-2, DIN17100:St70-2,NFA: A70-2 and DIN 17100:E360 steel grades.
St 70-2 steel plate - BEBON INTERNATIONALST 70-2 steel plate/sheet, DIN 17100 ST 70-2 steel plate/sheet, under DIN standard, we can regard ST 70-2 steel plate/sheet for general Construction steels.DIN 17100 ST 70-2 steel plate/sheet is for general Construction steels. The followings are the chemical composition and the mechanical properties of …
Equivalents of Carbon Steel Qualities - MESTEEL.COMIt Provides Steel And Construction Related Companies In The Middle East Information, News And An Extensive Interactive Database, Inclusive Offers And Inquiries. Equivalents of …
DIN 17100 ST 70,DIN 17100 ST 70 steel plate,DIN 17100 ST ...ST 70 steel plate can be offered by Katalor Industry,Katalor Industry has DIN 17100 ST 70 steel plate price and application ,you can contact us if you need DIN 17100 ST 70 steel plate specification .
St70-2 - Steel grades, Properties and Global StandardsThis page provides St70-2 datasheet download, these include St70-2 mechanical properties, chemical element St70-2, technical specifications of St70-2 steel material properties. St70 …
maechanical properties st 70 2 -BEBON Steel ManufacturingBBN STEEL main supply maechanical properties st 70 2 , Inaddition, we specialized in producing are shipbuilding steel, weathering resistant steel, boiler and pressure vessel steel, pipeline steel ans so on. we can also arrange producition according to the following standards - EN10025, EN10028, DIN 17100, ASTM A131, JISG3101, JISG3106...
MEsteel - Qualities German DIN 17100 Carbon Steel ...St.70-2 Mechanical and technological properties of the steels in the as-delivered condition and/or condition of treatment according to section 18.104.22.168 Steel grade according to the above table
Steel material properties - Steelconstruction.infoSteel material properties. From Steelconstruction.info. Jump to: navigation, search. The properties of structural steel result from both its chemical composition and its method of manufacture, including processing during fabrication. Product standards define the limits for composition, quality and performance and these limits are used or ...
Stainless Steel Data - Fuller FastenersStainless Steel Data. Designation System for Stainless Steel (ISO 3506 1979) ... Different grades of steel may be used as long as all physical and mechanical properties and the corresponding corrosion resistance of the finished product are achieved. ... Guideline values for screws in steel groups A2-70 and A4-70 with standard metric threads to ...
ASTM A516 Grade 70 and ASME SA516 Grade 70 Carbon Steel PlateASTM A516 Grade 70 / ASME SA516 Grade 70 - Mechanical Properties The mechanical properties of ASTM A516 Grade 70 and ASME SA516 Grade 70 carbon steel plate are outlined in table 3. Table 3.
ASTM A27 Steel | Metal Casting ResourcesDepending on the mechanical and chemical properties required, steel (Fe) will contain varying amounts of foreign elements (alloys) in addition to carbon (C). ... ASTM A27 includes carbon steel castings for applications requiring up to 70 ksi (485 MPa) minimum tensile strength. The chemical analysis requirements show the percentage of carbon ...
Mechanical Properties - Fuller FastenersDIN 127B Spring Steel Bare Steel/Zinc Plated/Yellow Zinc Plated; DIN 127B Spring Steel Hot Dipped Galvanized; ... Mechanical Properties for Socket Products. DIN 912 Socket Cap Screws. Property Class 12.9; Hardness (HRC) 39-44: Tensile Strength (Mpa) 1,220 min. Yield Strength at …
Mencinger's WebStandards/Comparison; Steel for Offshore structures : Grade 355 / API 2H and 2W - Chemical Compositions Grade 355 N - Comparison of Tensile Strength, Yield Strength and Toughness
A St 52 steel chemical composition, A St 52 steel ...Application:A St 52 steel grade is one of grade in DIN 17135 , which is non alloy and alloy steels with specified elevated temperature properties. A St 52 grades are used above all for manufacturing boilers, pressure vessels and pipes transporting hot liquids.
Magnetic Properties of 304 & 316 Stainless SteelThe magnetic properties of stainless steel are very dependent on the elements added into the alloy. A basic stainless steel has a ‘ferritic’ structure and is magnetic, formed from the addition of chromium – it can be hardened through the addition of carbon, making it ‘martensitic’.
Different Steel Types and Properties - The BalanceAccording to the World Steel Association, there are over 3,500 different grades of steel, encompassing unique physical, chemical, and environmental properties. In essence, steel is composed of iron and carbon, although it is the amount of carbon, as well as the level of impurities and additional ...
AISI Type 304 Stainless Steel - MatWebMaterial Notes: Austenitic Cr-Ni stainless steel. Better corrosion resistance than Type 302. High ductility, excellent drawing, forming, and spinning properties.
China Steel - SteelfromChinaCheck the China Steel designations and grades below for a variety of China Steel products. If you can’t find the type of China Steel comparison you’re looking for, send us a message with your inquiry and we’ll get back to you with an answer shortly.
Stainless steel - WikipediaOne of the more popular stainless steel grades used in 3D printing is 316L stainless steel. Due to the high temperature gradient and fast rate of solidification, stainless steel products manufactured via 3D printing tend to have a more refined microstructure; this in turn results in better mechanical properties.Corrosion resistance· [PDF]
Carbon Steel Comparison Tabel - Norsk Ståls185 s185 fe 310-0 st 33 a 33 fe 320 a 320 13 00-00 ss 330 fe 310 s235jr fe 360b st 37-2 e24-2 fe 360 b ae 235-b 13 11-00 s235jrg1 fe 360bfu ust 37-2
AISI 4140 Alloy Steel (UNS G41400) - AZoMAISI 4140 Alloy Steel (UNS G41400) Download PDF Copy; Request Quote; Written by AZoM Sep 13 2012. Topics Covered. ... The physical properties of AISI 4140 alloy steel are highlighted in the following table. Properties Metric ... 25.70%: Hardness, Brinell: 197: 197:
shipbuilding steel stock
ABS,LR shipbuilding steel
steel for shipbuilding and oil platform:
Henan BEBON Iron & Steel Co., Ltd , is professional in exporting the steel for shipbuilding and oil platform. Our product have the following several feature
1.The size of steel plates we can supply is 3mm-180mm * 1250mm-4000mm * 3000mm-18000mm
2.The main standard our steel is according to ASTM A131,API.
3.The main classification society we cooperate are: ABS ,GL, LR, BV, NK,DNV,KR,RINA.
4.Productive technology: HR,CR,TMCP,N,Q&T,IMPACT TEST,Z15,Z25,Z35.
5.Hard stamp about the brand of manufacturer, Heat No.,Batch No., steel grade, steel size, and the brand of Classification Society.
6.Blasting according to SA2.5 standard, and the shop primed on the steel, coating thickness is 15-35 micrometer.
- Carbon low alloy steel
- weathering steel grade
- Shipbuilding steel
- Hot steel plate product

domain: chemistry


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When some people think about homemade baby food they think that it's too much work, but I am here to tell you that it doesn't have to be. With the right things you can seriously make baby food in less than ten minutes. Not to mention it is sooooo much cheaper than buying baby food already made.
To make this quick and easy you need the following things: steamable vegetables, a food processor or a way to puree the food. I HIGHLY recommend investing in a Ninja Blender. A regular food processor is big and bulky and has so many parts to it. With a Ninja you can use the cup that is used to make smoothies to puree the food. It's two parts, super easy to put together and to clean, annnnndddd it only takes 30 seconds to puree the food. I literally get excited just thinking about it (I know I might be going crazy). If you have a regular food processor don't worry you can still make baby food in less than 10 minutes.
For this batch of food I picked up some Steamables from Publix because they had them 50% off. I didn't realize the butternut squash wasn't a Steamable until I got home but it wasn't a problem and didn't add on any time. I also buy Steamables at ALDI because they are even cheaper!
The first step is to steam your vegetables in the microwave by following the directions on the package. While that is happening you can be putting together whatever kitchen gadget you are using to puree the food and prepare your ice cub trays. In this case I also boiled the butternut squash while the other veggies were cooking. It took me about 8 minutes to cook all of these using the microwave and stove top.
Once your veggies are cooked it's time to puree them. I did one at a time. Add the vegetable to the blender. Puree until food is smooth. You may need to add water (1 tbsp at a time) to reach desired consistency. **For added nutrition add breastmilk or formula instead of water.** If you have a Ninja this part is super easy because you can rinse out the cup in between purees and it just takes a few seconds. Once all veggies are pureed pour them into ice cube trays.
Once the cubes are frozen remove them from the trays and place in ziplock freezer bags. When you're little one is ready to eat, heat cubes in the microwave until warm and melted.
That's it! Super easy and quick AND you didn't have to break the bank!
Green Peas and Carrots:

domain: food


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Investigating the Impact of Information & Communication Technology (ICT) and International Protocols on Co2 Emissions
thesisposted on 2021-06-15, 01:42 authored by Samin Shaaban Nejad
Our life hangs in the delicate balance of the natural environment, on which many of us would agree that human-induced activities are promptly degrading it. Businesses as the engine of an economy play a crucial role in both enhancing and degrading the natural environment. This study aims to investigate the role of international protocols and Information and Communication Technology (ICT) in reducing carbon emissions. For this study, the author has conducted quantitative research in which both micro and macro level approaches were used. The findings indicate that both international treaties and ICT have an inverse impact on the overall model, while they were found to be statistically significant factors in reducing carbon emissions globally. The author has further narrowed down the findings to a micro level to conduct a comparative study between Canada and Norway to evaluate the performance of these two countries on climate mitigation initiatives. This study has further investigated some of the reasons that have caused Canada to perform insufficiently in meeting its climate change targets despite its active role in the negotiations that led to both the Kyoto Protocol and Paris Agreement.

domain: literature


|Listed in: Outdoors > Scuba|
"World of Scuba Diving" webring members enjoy exploring the ocean or freshwater lakes for sunken ships, archeological treasures, or the flora and fauna of these bodies of water. They may encounter other scuba divers or the bigger predatory animals of the sea. These websites display underwater photography and SCUBA trip reports from around the world. They document the critters found in the locations they visit. Other websites offer equipment for scuba diving or snorkeling.
Sponsor this Ring


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Wake Commissioners Approve Funding For Mental Health Center
Posted June 7, 2005 9:04 a.m. EDT
RALEIGH, N.C. — Wake County commissioners approved $10 million of funding for a mental health crisis in-patient facility.
Dozens of people turned out Monday night to express their concerns about what they call a looming mental health care crisis.
For years, the county has relied on Dorothea Dix hospital for patients with serious psychiatric needs, but the facility is closing in 2007. It will be replaced by a facility in Granville County.
Mental health advocates were hoping commissioners would put more up even more money than they did.
The county's amount would only cover a third of the cost of a new clinic.

domain: sociology


Middle School Principal of the Year: Phil Ambrose
The Illinois Principals Association is pleased to announce that Phillip Ambrose, Principal of Thurgood Marshall Learning Center, has been named as the 2012-2013 Middle School Principal of the Year. The award recognized middle school principals who have demonstrated a positive impact on their students and learning community. Criteria for the award include:
- Demonstrates positive impact on education and advocacy for children
- Ensures the school climate is positive and reflects high staff and student morale
- Demonstrates creativity and imagination in bringing about positive change
- Willing to take risks to improve student learning
- Moves actively to implement the goals and objectives of the school
- Works collaboratively with teachers and other staff to improve the educational program and student achievement
- Anticipates emerging problems and acts effectively to resolve them
- Involves the community in the life of the school and uses community resources for students
Mr. Ambrose has brought his curriculum into the 21st Century by offering a great deal of online instruction that can be tailored to fit the students’ needs and abilities. Through his calm demeanor, his belief that every student can learn, and his leadership in creating the most positive atmosphere possible for his students, Mr. Ambrose has created a school environment where students can find success.
Mr. Ambrose was recognized at a regional Illinois Principal Association meeting on Feb. 21, and will also be recognized at the annual IPA Principals Professional Conference held in October in Peoria.
“Principal Ambrose is a great leader and takes responsibility for the school’s success.” said Superintendent Mike Oberhaus. “His top priority is in the best interest of the students at Thurgood Marshall Learning Center.”


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NEW Regulation Size Bean Bag Toss Cornhole Game w/Bags
- Source: eBay
BRAND NEW CORNHOLE GAME!!!!
(24 in. X 48 in.)
BAGS INCLUDED Over 3000 sets sold!!!!
****We have auctions for two different size games - READ THE AUCTION COMPLETELY BEFORE BIDDING!!
Feel free to email with questions about the products to:
See below for recent Feedback!!!!
This auction is for a Regulation Cornhole Game that includes: (2) Boards - 24" X 48", (8) 6" square bean bags (1lb. corn)(4 red and 4 blue).
(We may have to occasionally substitute bags colors to expedite shipping)
Bean bags are made from durable cotton duck material, double stiched and are virtually indestructable.
Face is 1/2" MDF material and frame/legs are 2X3 construction. We chose MDF (medium density fiberboard) because it has the right amount of bounce and will not warp like plywood. The legs fold up for flat storage. Don't be fooled by the "light weight" games. They can't stand up to ours - ours are built to last.
Item has been carefully constructed to game specifications for cornhole games. Game comes READY TO PLAY. Item will come painted white so that you can decorate it however you choose or use it as-is.
Buyer pays actual shipping, $20 and up (depending on location - use the shipping calculator provided). Thank you and good luck!... read more

domain: hobby


Viewpoints in Homeland Defense and Security: Top Threats to America's Infrastructure [video] [open html - 0B]
From the Video Description: "For two grading periods in a row, the Army Corps of Engineers has awarded our nation's infrastructure a 'D'. But what are the top threats to our infrastructure? In this interview, Tom Mackin, instructor at the NPS [Naval Postgraduate School] Center for Homeland Defense and Security and Professor at Cal Poly, discusses the enormous needs in rebuilding our dams, waterways, airports, bridges and roadways, and the reasons why the political will to do so is so hard to find." The duration of this video is 14 minutes and 9 seconds.
Center for Homeland Defense and Security: http://www.chds.us/


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One of the big challenges in cancer treatment is resistance, when cells never react or stop reacting to a chemotherapy drug’s lethal effects. For ovarian cancer, resistance can occur with the commonly-used drug cisplatin.
The phytochemicals found in plant foods may help, a new study suggests. The study is preliminary, conducted on cancer cells, yet it suggests that a phytochemical called kaempferol may help this ovarian treatment be more effective.
Kaempferol is a flavonoid, one of the largest groups of phytochemicals and studied previously for its role in protecting against cancer. It’s found in many plant-foods, including berries, tea, and broccoli.
You can read the study here.
AICR has funded many studies on the effects of dietary components on cancer treatments: We featured a couple of them on breast cancer treatment in this months eNews.
Of the days’ worth of food below, which do you think would be more filling?
The food on the right is certainly more colorful and offers a lot more to eat than the few choices on the left.
A new AICR review of the research on calorie (or energy) density and weight loss has found that diets low in calorie density can play an important role in efforts to achieve and maintain a healthy weight.
The photo above is a great example of how calorie density works. Both 1575 calories, the food on the right contains more low calorie-dense foods – fewer calories, but more food weight.
Low calorie dense foods, such as fruits and non-starchy vegetables have a lot of water, so by filling up on these, you satisfy your hunger, but eat fewer calories.
Read more about how to make your plate heavier, but with fewer calories.
The AICR brochure More Food, Fewer Calories contains strategies on following a low-calorie dense diet along with more information on the health benefits.
After you’ve tried some of these ideas, share your successes with us.
Photos: Dr. Barbara Rolls, Penn State University, used with permission.
Pancreatic cancer is one of the leading causes of cancer-related deaths because it is often diagnosed only in its advanced stages. This week, two new studies on pancreatic cancer suggest there are lifestyle habits that can prevent this disease.
Source: NCI. Unfortunately, incidence (and mortality) have changed little in recent years.
The first study, highlighted in today’s issue of Cancer Research Update, supports findings from AICR’s expert report linking higher body weight and waist circumference to increased risk. The study used pooled data from a major National Cancer Institute group of participants, which included about 4,400 individuals – half with the disease and half without. You can read more about the study in CRU.
The second study linked heavy alcohol use and binge drinking to increased risk of pancreatic cancer in men. Previous research has produced conflicting findings on the alcohol-pancreatic cancer link.
But this study – which you can read here – found that the more men drank, the higher their increased risk. This held true even if the men consumed the alcoholic drinks years or decades prior to diagnosis. The study included about 500 individuals with the cancer and 1,700 controls, and the participants reported their history of alcohol consumption. There was no connection between alcohol consumption and pancreatic cancer risk among women.
AICR’s expert report found that foods containing folate — such as beans, leafy greens, and peanuts — probably protect against pancreatic cancer. Want to add some folate to your day? Spinach will help. Try making AICR’s Turkey, Spinach and Apple Wrap.

domain: biology


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NORRISTOWN, PA – The Montgomery County Commissioners announced today that five projects across the county will receive Montgomery County Transportation Program (CTP) grants for the 2022 funding cycle. These grants are part of the fifth round for the program, which was established to fund transportation projects using the county’s $5 Vehicle Registration Revenue Fee.
Each year municipalities apply for funds to implement projects that include road construction and repair, traffic sign and signal installation, crosswalk markings, curb ramps, pedestrian trails within public right-of-way, and street lighting.
This year’s grant awards total $1,034,320 across five projects. Each project was evaluated in terms of the benefit and visibility to the overall public, county and local planning consistency, readiness, funding support, and focus area. The selection committee consists of members of the Montgomery County Planning Commission Board, the Montgomery County Transportation Authority, and interdepartmental county staff.
This year’s grant recipients include two boroughs and three townships. Two of the municipalities do not contain county-owned roadways or bridges. This brings the total number of municipalities that have received County Transportation Program grants over the five years of the program to twenty-seven.
“Because we have a seamless transportation network that crosses every municipal boundary, it’s critically important that governments work together to keep our roads, bridges, and trails in good condition,” said Dr. Valerie A. Arkoosh, Chair of the Montgomery County Board of Commissioners. “That’s why we are pleased this year to award County Transportation Program grants to five of our partner municipalities to help fund needed improvements to our bridges, intersections, and traffic signals.”
The five projects that received 2022 grants are highlighted below:
Lower Providence Township – Germantown Pike and Evansburg Road Signal Improvements
Lower Providence will fund the installation of advanced detection to provide left-turn arrow phase and pedestrian countdown to existing signals at the intersection of Germantown Pike and Evansburg Road.
CTP Grant Award: $72,844
Montgomery Township – Spring Valley Park and Windlestrae Park Rapid Flashing Beacons
Montgomery Township will fund the installation of Rectangular Rapid Flashing Beacons, signage, and pavement markings on Upper State Road at Spring Valley Drive and Kenas Road at Davis Drive.
CTP Grant Award: $271,072
Pottstown Borough – Moser Bridge Rehabilitation
Pottstown Borough will fund the rehabilitation of Moser Road Bridge over the Norfolk Southern Railroad to address high-priority and complementary low-priority items highlighted in the corresponding safety inspection report.
CTP Grant Award: $448,750
Royersford Borough – 7th Avenue and Washington Street Pedestrian Crossing
Royersford Borough will fund the installation of a flashing beacon and audible alerts on both sides of Washington Street at 7th Avenue near Spring-Ford Middle School.
CTP Grant Award: $92,598
Upper Merion Township – Henderson Road Pedestrian Crossing
Upper Merion Township will fund the installation of a Rectangular Rapid Flashing Beacon, ADA curb ramps, and pavement markings on Henderson Road at Crossfield Road—connecting the township’s municipal complex with the surrounding residential neighborhoods.
CTP Grant Award: $149,056
Visit www.montcopa.org/CountyTransportationProgram for more on the Montgomery County Transportation Grant Program. For questions, contact Matthew Popek at (610) 278-3730 or at firstname.lastname@example.org.
Media Contact: Kelly Cofrancisco, email@example.com

domain: geography


text:
As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (https://www.ibridgenetwork.org/wustl/splinter). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.
19 Related JoVE Articles!
Pyrosequencing: A Simple Method for Accurate Genotyping
Institutions: Washington University in St. Louis.
Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.
Cellular Biology, Issue 11, Springer Protocols, Pyrosequencing, genotype, polymorphism, SNP, pharmacogenetics, pharmacogenomics, PCR
Collection and Extraction of Saliva DNA for Next Generation Sequencing
Institutions: The Research Institute at Nationwide Children's Hospital, The Ohio State University, The Ohio State University.
The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.
Medicine, Issue 90,
DNA collection, saliva, DNA extraction, Next generation sequencing, DNA purification, DNA
Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Institutions: National Research Council Canada.
One of the major questions in microbial ecology is “who is there?” This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.
Molecular Biology, Issue 90, Metagenomics, Bacteria, 16S ribosomal RNA gene, Amplicon sequencing, Next-generation sequencing, benchtop sequencers
Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis
Institutions: The University of Texas Graduate School of Biomedical Sciences at Houston.
Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro
. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro
and in vivo
Genetics, Issue 93, EML Cells, Self-renewal, Differentiation, Hematopoietic precursor cell, RNA-Sequencing, Data analysis
Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Institutions: San Diego State University, DOE Joint Genome Institute, University of Colorado, University of Colorado.
The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.
Molecular Biology, Issue 94, virome, microbiome, metagenomics, metatranscriptomics, cystic fibrosis, mucosal-surface
Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells
Institutions: Diagenode S.A., Diagenode Inc..
Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.
Molecular Biology, Issue 94, Automation, chromatin immunoprecipitation, low DNA amounts, histone antibodies, sequencing, library preparation
Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution
Institutions: Weill Cornell Medical College, Weill Cornell Medical College, Weill Cornell Medical College, University of Michigan.
DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.
Genetics, Issue 96, Epigenetics, bisulfite sequencing, DNA methylation, genomic DNA, 5-methylcytosine, high-throughput
A Multi-detection Assay for Malaria Transmitting Mosquitoes
Institutions: School of Veterinary Medicine, University of California - Davis, School of Veterinary Medicine, University of California, Davis.
The Anopheles gambiae
species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae
complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium
parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g.
, human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium
detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.
Infectious Diseases, Issue 96, Mosquito, SNP genotyping, multiplex assay, iPLEX, MALDI-TOF, insecticide resistance, speciation islands, species diagnosis, parasite detection, blood source detection, host preference, infection status
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
Institutions: Memorial Sloan-Kettering Cancer Center, Memorial Sloan-Kettering Cancer Center.
Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.
Molecular Biology, Issue 80, Molecular Diagnostic Techniques, High-Throughput Nucleotide Sequencing, Genetics, Neoplasms, Diagnosis, Massively parallel sequencing, targeted exon sequencing, hybridization capture, cancer, FFPE, DNA mutations
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)
Institutions: Children's Hospital of Philadelphia Research Institute, University of Pennsylvania .
ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo
. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.
Molecular Biology, Issue 74, Genetics, Biochemistry, Microbiology, Medicine, Proteins, DNA-Binding Proteins, Transcription Factors, Chromatin Immunoprecipitation, Genes, chromatin, immunoprecipitation, ChIP, DNA, PCR, sequencing, antibody, cross-link, cell culture, assay
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Institutions: Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g.
drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2
. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4
in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Genetics, Issue 72, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'
Institutions: The J. Craig Venter Institute.
Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3
. Viruses, which are ubiquitous and the most numerous entities on our planet 4
and important in all environments 5
, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.
Genetics, Issue 75, Microbiology, Immunology, Virology, Molecular Biology, Environmental Sciences, Genomics, environmental genomics, Single virus, single virus genomics, SVG, whole genome amplification, flow cytometry, viral ecology, virion, genome analysis, DNA, PCR, sequencing
Genotyping of Plant and Animal Samples without Prior DNA Purification
Institutions: Thermo Fisher Scientific.
The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1
). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2
. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4
is performed directly from Arabidopsis
plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5
. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1
). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins.
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7
. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.
Genetics, Issue 67, Molecular Biology, Plant Biology, Medicine, Direct PCR, DNA amplification, DNA purification, dCAPS, PCR-based target DNA detection, genotyping, Arabidopsis, oak, mouse tissues
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
Institutions: Michigan State University.
This protocol describes a fluorescence microscope-based screening of Arabidopsis
seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis
is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2
), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.
Genetics, Issue 62, EMS mutagenesis, secretory pathway, mapping, confocal screening
Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation
Institutions: Agency for Science, Technology and Research, Singapore, A*STAR-Duke-NUS Neuroscience Research Partnership, Singapore, National University of Singapore, Singapore.
Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12
. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13
. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6
. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.
Genetics, Issue 62, ChIP, ChIA-PET, Chromatin Interactions, Genomics, Next-Generation Sequencing
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
Targeted DNA Methylation Analysis by Next-generation Sequencing
Institutions: University of Oklahoma College of Medicine, University of Oklahoma College of Medicine.
The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.
Molecular Biology, Issue 96, Epigenetics, DNA methylation, next-generation sequencing, bioinformatics, gene expression, cytosine, CpG, gene expression regulation


text:
Research translation, community environmental health
Laura Senier’s research interests include the sociology of medicine and public health, community environmental health, and environmental justice. Her research identifies political, social, and economic barriers in research translation, or the effort to migrate scientific discoveries into clinical and public health practice. She has also studied community mobilization in communities burdened by environmental injustices, in the Boston area and in the upper Midwest. Her work has appeared in Social Science & Medicine; Genetics in Medicine; Sociological Inquiry; Organization & Environment; and Environmental Science & Technology. She teaches graduate and undergraduate courses in sociology of health and illness, environmental health, environmental justice, and qualitative research methods.
Senier, Laura, Colleen M. McBride, Alex Ramsey, Vence L. Bonham, and David Chambers. In press. “Blending insights from implementation science and the social sciences to mitigate disparities in screening for hereditary cancer syndromes.” International Journal of Environmental Research and Public Health.
Senier, Laura, *Leandra Smollin, *Rachael Lee, *Lauren Nicoll, *Michael Shields, and *Catherine Tan. 2018. “Navigating the evidentiary turn in public health: sensemaking strategies to integrate genomics into state-level chronic disease prevention programs.” Social Science & Medicine 211: 207-215. PMCID: PMC6067973
Senier, Laura, Phil Brown, Sara Shostak, and *Bridget Hanna. 2017. “The socio-exposome: advancing exposure science and environmental justice in a postgenomic era.” Environmental Sociology 3: 107-121. PMCID: PMC5604315
PHTH 3250, Fundamentals of Qualitative Research
PHTH 6320, Qualitative Methods in Health and Illness
Distribution Committee, Metrowest Health Foundation (2014-present)


text:
Introduction for The Gift of Therapy
It is dark. I come to your office but can’t find you. Your office is empty. I enter and look around. The only thing there is your Panama hat. And it is all filled with cobwebs.
My patients’ dreams have changed. Cobwebs fill my hat. My office is dark and deserted. I am nowhere to be found.
My patients worry about my health: will I be there for the long haul of therapy? When I leave for vacation, they fear I will never return. They imagine attending my funeral or visiting my grave.
My patients do not let me forget that I grow old. But they are only doing their job: have I not asked them to disclose all feelings, thoughts, and dreams? Even potential new patients join the chorus and, without fail, greet me with the question: “are you still taking on patients?”
One of our chief modes of death-denial is a belief in personal specialness, a conviction that we are exempt from biological necessity and that life will not deal with us in the same harsh way it deals with everyone else. I remember, many years ago, visiting an optometrist because of diminishing vision. He asked my age and then responded: “Forty-eight, eh? Yep, you’re right on schedule!”
Of course I knew, consciously, that he was entirely correct, but from deep within me a cry welled up: “What schedule? Who’s on schedule. It is altogether right that you and others may be on schedule but certainly not I!”
And so it is daunting for me to realize that I am entering a designated later era of life. My goals, interests, and ambitions are changing in predictable fashion. In his study of the life cycle, Erik Erickson described the late life stage of generativity, a post-narcissism era when attention turns from expansion of oneself toward care and concern for succeeding generations. Now, approaching seventy, I appreciate the clarity of Erickson’s vision. His concept of generativity feels right to me. I want to pass on what I have learned. And to do it as soon as possible.
But offering guidance and inspiration to the next generation of psychotherapists is exceedingly problematic today because our field is in such crises. An economically-driven health care system mandates a radical modification in psychological treatment and psychotherapy is now obliged to be streamlined — that is, above all, inexpensive and, perforce, brief, superficial, and insubstantial.
I worry where the next generation of effective psychotherapists will be trained. Not in psychiatry residency training programs. Psychiatry is on the verge of abandoning the field of psychotherapy. Young psychiatrists are forced to specialize in psycho-pharmacology because third-party payers now reimburse for psychotherapy only if it is delivered by low-fee (in other words, minimally trained) practitioners. It seems certain that the present generation of psychiatric clinicians, skilled in both dynamic psychotherapy and in pharmacological treatment, is an endangered species.
What about clinical psychology training programs — the obvious choice to fill the gap? Unfortunately, clinical psychologists face the same market pressures and doctorate-granting schools of psychology are responding by teaching a therapy which is symptom-oriented, brief, and, hence, reimbursable.
So I worry about psychotherapy — about its deformation by economic pressures and about its impoverishment by radically abbreviated training programs. Nonetheless I am confident that, in the future, a cohort of therapists coming from a variety of educational disciplines (psychology, counseling, social work, pastoral counseling, clinical philosophy) will continue to pursue rigorous post-graduate training and, even in the crush of HMO reality, will find patients desiring extensive growth and change who are willing to make an open-ended commitment to therapy. It is for these therapists and these patients that I write The Gift of Therapy.
Throughout these pages I advise students against sectarianism and suggest a therapeutic pluralism in which effective interventions are drawn from several different therapy approaches. Still, for the most part, I work from an interpersonal and from an existential frame of reference. Hence, the bulk of the advice which follows issues from one or the other of these two perspectives.
Since I first entered the field of psychiatry I have had two abiding interests: group therapy and existential therapy. These are parallel but separate interests: that is, I do not practice ‘existential group therapy’ — in fact, I don’t know what that would be. The two modes are different not only because of the format (that is, a group of approximately six to nine members versus a one-to-one setting for existential psychotherapy) but differ fundamentally in their frame of reference. When I see patients in group therapy I work from an interpersonal frame of reference and I make the assumption that patients fall into despair because of their inability to develop and sustain gratifying interpersonal relationships.
When, however, I operate from an existential frame of reference, I make a very different assumption: that patients fall into despair as a result of a confrontation with harsh facts of the human condition — the “givens” of existence. Since many of the offerings in this book issue from an existential framework that is unfamiliar to many readers, a brief introduction is in order.
Definition of existential psychotherapy: Existential psychotherapy is a dynamic therapeutic approach which focuses on concerns that are rooted in existence.
Let me dilate this terse definition by clarifying the phrase “dynamic approach.” Dynamic has both a lay and a technical definition. The lay meaning of dynamic (derived from the Greek root, dunasthi — to have power or strength) implying forcefulness or vitality (to wit, dynamo, a dynamic football runner or political orator) is obviously not relevant here. But, if that were the meaning of dynamic, then where is the therapist who would claim to be other than a dynamic therapist, in other words, a sluggish, or inert therapist?
No, I use “dynamic” in its technical sense which retains the idea of force but is rooted in Freud’s model of mental functioning which posits that forces in conflict within the individual generate the individual’s thought, emotion, and behavior. Furthermore — and this is a crucial point — these conflicting forces exist at varying levels of awareness; indeed some are entirely unconscious.
So, existential psychotherapy is a dynamic therapy which, like the various psychoanalytic therapies, assumes the presence of unconscious forces which influence conscious functioning. However, it parts company from the various psychoanalytic ideologies when we ask the next question: what is the nature of the conflicting internal forces?
The existential psychotherapy approach posits that the inner conflict bedeviling us issues not only from our struggle with suppressed instinctual strivings or internalized significant adults or shards of forgotten traumatic memories, but also from our confrontation with the “givens” of existence.
And what are these “givens” of existence? If we permit ourselves to screen out or ‘bracket’ the everyday concerns of life and reflect deeply upon our situation in the world, we inevitably arrive at the deep structures of existence (the “ultimate concerns,” to use theologian Paul Tillich’s term). Four ultimate concerns, to my view, are highly salient to psychotherapy: death, isolation, meaning in life, and freedom. (Each of these ultimate concerns will be defined and discussed in a designated section.)
Students have often asked why I don’t advocate training programs in existential psychotherapy. The reason is that I’ve never considered existential psychotherapy to be a discrete free-standing ideological school. Rather than attempt to develop existential psychotherapy curriculums, I believe it is preferable to supplement the education of all well-trained dynamic therapists by increasing their sensibility to existential issues.
Process and content. What does Existential therapy look like in practice? To answer that question one must attend to both “content” and “process” — the two major aspects of therapy discourse. “Content” is just what it says — the precise words spoken, the substantive issues addressed. “Process” refers to an entirely different and enormously important dimension: the interpersonal relationship between the patient and therapist. When we ask about the ‘process’ of an interaction, we mean: what do the words (and the nonverbal behavior as well) tell us about the nature of the relationship between the parties engaged in the interaction?
If my therapy sessions were observed, one might often look in vain for lengthy explicit discussions of death, freedom, meaning, or existential isolation. Such existential content may only be salient for some patients (but not all patients) at some stages (but not all stages) of therapy. In fact, the effective therapist should never try to force discussion of any content area: therapy should not be theory-driven but relationship-driven.
But observe these same sessions for some characteristic process deriving from an existential orientation and one will encounter another story entirely. A heightened sensibility to existential issues deeply influences the nature of the relationship of the therapist and patient and affects every single therapy session.
I am myself surprised by the particular form this book has taken. I never expected to author a book containing a sequence of tips for therapists. But, looking back, I know the precise moment of inception. Two years ago after viewing the Huntington Japanese gardens in Pasadena I noted the Huntington Library’s exhibit of best selling books of the Renaissance in Great Britain and wandered in. Three of the ten exhibited volumes were books of numbered “tips” — on animal husbandry, sewing, gardening. I was struck that even then, hundreds of years ago, just after the introduction of the printing press, lists of tips attracted the attention of the multitudes.
Years ago, I treated a writer who, having flagged in the writing of two consecutive novels, resolved never to undertake another book until one came along and bit her on the ass. I chuckled at her remark but I didn’t really comprehend what she meant until that moment in the Huntington Library when the idea of a book of tips bit me on the ass. I remember that, on the spot, I resolved to put away other writing projects, to begin looting my clinical notes and journals, and to write a book of tips for beginning therapists.
Rainer Maria Rilke’s ghost hovered over the writing of this volume. Shortly before my experience in the Huntington library, I had reread his Letters to a Young Poet and I consciously attempted raise myself to his standards of honesty, inclusiveness, and generosity of spirit.
The advice in this book is drawn from notes of forty-five years of clinical practice. It is an idiosyncratic m�lange of ideas and techniques that I have found useful in my work. These ideas are so personal, opinionated and occasionally original that the reader is unlikely to encounter them elsewhere. Hence, this volume, in no way, is meant to be a systematic manual; instead, I intend it as a supplement to a comprehensive training program. I selected the eighty-five categories in this volume randomly, guided by my passion for the task rather than any particular order or system. I began with a list of over two hundred pieces of advice and ultimately pruned away those for which I felt too little enthusiasm.
One other factor influenced my selection of these eighty-five items. My recent novels and stories contain many descriptions of therapy procedures I’ve found useful in my clinical work but, since my fiction has a comic, often burlesque tone, many readers are unclear if I am serious or not about the therapy procedures I describe. The Gift of Therapy offers me an opportunity to set the record straight.
As a nuts and bolts collection of favorite interventions or statements, this volume is long on technique and short on theory. Readers seeking more theoretical background may wish to read my texts, Existential Psychotherapy and The Theory and Practice of Group Psychotherapy, the mother books for this work.
Being trained in medicine and psychiatry, I have grown accustomed to the term patient (from the Latin patiens — one who suffers or endures) but I use it synonymously with client, the common appellation of psychology and counseling traditions. To some, the term “patient” suggests an aloof, disinterested, unengaged, authoritarian therapist stance. But read on — I intend throughout to encourage a therapeutic relationship based on engagement, openness, and equalitarianism.
Many books, my own included, consist of a limited number of substantive points and then considerable filler to connect the points in a graceful manner. Because I have selected a large number of suggestions, many free-standing, and omitted much filler and transitions, the text will have an episodic, lurching quality.
Though I selected these suggestions haphazardly and expect many readers to sample these offerings in an unsystematic manner, I have tried, as an afterthought, to group them in a reader-friendly fashion.
The first section ( #1 - 41) addresses the nature of the therapist-patient relationship, with particular emphasis on the here-and-now, the therapist’s use of the self, therapist self-disclosure.
The next section (#41-51) turns from process to contentand suggests methods of exploring the ultimate concerns of death, meaning in life, and freedom (encompassing responsibility and decision.)
The third section (#52 - 76) addresses a variety of issues arising in the everyday conduct of therapy.
In the fourth section (#77-84) I address the use of dreams in therapy.
The final section (#84-85) discuss the hazards and the privileges of being a therapist.
This text is sprinkled with many of my favorite specific phrases and interventions. At the same time I encourage spontaneity and creativity. Hence do not view my idiosyncratic interventions as a specific procedural recipe; they represent my own perspective and my attempt to reach inside to find my own style and my own voice. Many students will find that other theoretical positions and technical styles will prove more compatible for them. The advice in this book derives from my clinical practice with moderately high to high functioning patients (rather than those who are psychotic or markedly disabled) meeting once or, less commonly, twice a week, for a few months to two to three years. My therapy goals with these patients are ambitious: in addition to symptom removal and alleviation of pain, I strive to facilitate personal growth and basic character change. I know that many of my readers may have a different clinical situation: a different setting with a different patient population and a briefer duration of therapy. Still it is my hope that readers may find their own creative way to adapt and apply what I have learned to their own particular work situation.


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Great news, and further momentum today in Australia. Ian Hunter MLC – A Labor member of the South Australian Parliament – is introducing a same sex marriage bill into that state parliament. It will be co-sponsored by Tammy Franks MLC from the Greens.
It’s great news from both of them, but especially significant for the pressure it puts on others in the Labor party across Australia to declare their hand. From the unions to the right-wing heavyweights, to the national Left faction and now to individual MPs like Ian Hunter – there is support everywhere.
Do we have a majority in Canberra? Not yet – but the point is the debate cannot be stopped now. It’s been happening and now it will keep happening in state parliaments …. Until Julia Gillard wakes up and realises she doesn’t have the power to stop people talking about matters that dramatically affect, and harm, their daily lives.
“I sense the mood for change in the country and the I sense the mood for change in the Labor party,” yes indeed Ian Hunter.
PS. Ian has been supporting Rainbow Labor from the outset – good to see him follow it through to the end.


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Market prices, erratic weather and increasing input costs are making coffee cultivation unviable, it notes
Coffee farmers in Karnataka are either selling their estates or killing themselves as growing the cash crop is becoming increasingly unviable, a new report released on December 6, 2019, said.
The Status on Indian Coffee 2019 prepared by Karnataka Growers Federation and released in Bengaluru also noted that Karnataka’s coffee production had fallen by around 40 per cent even as input costs had increased.
Consistent, non-remuneration for drought, high incidence of pests and diseases, non-availability of credit and the exorbitant cost of labour had forced traditional coffee farmers to abandon their plantations, the report said.
It said that from 1999 to 2005 and from 2008 to 2018, the market price of coffee was depressed. Prices improved only in 2011, 2014 and 2015, when one kilogram of Arabica coffee cost more than Rs 250.
Besides market factors, the other major factor making coffee cultivation unviable was erratic weather.
During 2002-05 and 2008-16, farmers suffered due to drought. In 2006-07, 2007-08 and 2018-19, they suffered damages due to heavy rainfall.
During the 2019 Kharif season, the Karnataka government declared 80 taluks of 17 districts as flood-affected. These included the three coffee-growing districts of Kodagu (three taluks), Hassan (three taluks) and Chikmagalur (four taluks).
The three districts together produce more than 70 per cent of India’s coffee. They reported crop losses between 33 and 50 per cent. Around 1,20,000 million tonnes of coffee, valued at Rs 2,200 crore was destroyed due to flooding.
In 2006-07, 2007-08 and 2018-19, heavy rainfall caused severe infestation of pests and diseases like white stem borer and leaf rust, which resulted in about 30-80 per cent of plant loss, according to the report.
Between 2000 and 2008, area under coffee production increased by around two per cent whereas yield reduced by around 3 per cent. Between 2008 and 2018, yield reduced by around 39 per cent and cropping area reduced by 0.6 per cent. The decrease in yield directly impacted the growers’ income.
In addition to market prices and erratic weather, increase in cost of inputs like fertilisers has also caused misery among coffee farmers.
Input costs increased by 2.6 times in the last eight years. The cost of fertilisers including Diammonium Phosphate (DAP), Urea, Rock Phosphate, Suphala and Potash, was around Rs 1,540 in 2011. This increased to Rs 4,030 in 2019.
The cost of manures like murate of potash has risen from Rs 250 per bag in 2011 to Rs 945 per bag in 2019 is extremely worrying as potash is essential for formation of coffee berries. DAP cost has risen from Rs 500 per bag in 2011 to Rs 1,440 per bag.
Those who left coffee cultivation, were being forced in many cases to sell their estates at distress prices. They usually shifted to growing and selling silver timber for meeting their immediate needs.
According to the report, 150 coffee farmers killed themselves between 2001 and 2011.
It called for complete loan waiver, interest on loan being reduced to below 3 per cent, support for coffee growers collectives and cheap supply of fertilisers and pesticides.
They also demanded government assistance in re-plantation, water augmentation, eco-certification and support for small growers’ collectives or co-operatives for coffee marketing.
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domain: agronomy


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If a person suffers from tinnitus, your first step should get your hearing checked. Even slight hearing difficulties can do to cause an oncoming tinnitus. In the event that you get hearing loss, simply getting hearing aids can eliminate your tinnitus symptoms. In an few extreme cases, surgery may be necessary.
Reduce your consumption of caffeine and salt. Caffeine is a stimulant that doesn’t only increases your beat and breathing but also elevates tinnitus levels. Salt acts similarly by elevating blood pressure and increasing aggravating noise levels in your head. Making dietary changes will reduce tinnitus levels and get you a better night’s stay.
If the suffering from hearing a ringing sound in your ear, you need to know the root tinnitus contributes to. There is no cure to do this problem. Doctors can recommend medications only temporary. Some would seek the guidance of natural and alternative cure. You may also find others offering cure. There are a lot of tinnitus consequences. Some of them are myths while other people the real thing. There are treatments that can work much better than others and also the only difference is source.
Vitamins and herbs. If meds aren’t for and also your you’d like to know how to stop ear ringing using natural methods, you consider vitamins and herbs as opposed to. Gingko biloba has been used by the Chinese popular – it functions to get considerably more circulation in the childs body and brain, and prevents tinnitus. Vitamins include niacin, lecithin, zinc, and magnesium.
The most at risk group from tinnitus would be older people who had hearing diminishment. That has now changed dramatically and people in their twenties and even teenagers are enduring it. This is a of experience with loud noise at concerts, nightclubs along with mp3 competitors.
Reduce shock. One way to do this is through regular exercise, doing yoga or mediation or even going for their long run. As stress increase so does the hub bub.
The rules you remember all the time, never overlook, become the golden rules that frequently apply. Like stars to guide us, they shine brilliantly, keeping us on course and always going in an direction.

domain: medical