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Personal Statement
Lybrate.com has a number of highly qualified Gynaecologists in India. You will find Gynaecologists with more than 32 years of experience on Lybrate.com. You can find Gynaecologists online in Bangalore and from across India. View the profile of medical specialists and their reviews from other patients to make an informed decision.
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NRV Hospital#28/3, Vidhya Nagar Cross, Chikkajala. Landmark: Opposite To Reliance Petrol Bunk, Bangalore Get Directions
NRV Hospital#28/3, Vidhya Nagar Cross, Chikkajala. Landmark: Opposite To Reliance Petrol Bunk., Bangalore Get Directions
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Endometrial Ablation Procedure
Treatment of Treatment of Breast Cancer
Management of Abortion
Hormonal Replacement Therapy Treatment
Caesarean Section Procedure
Contraceptive Advice
Delivery Procedure
Treatment of Gynae Problems
Gynecology Laparoscopy Procedures
Hpv Vaccination
Treatment Of Female Sexual Problems
Treatment Of Menopause Related Issues
Treatment Of Menstrual Problems
Treatment of Mirena (Hormonal Iud)
Pap Smear Procedure
Polycystic Ovary Syndrome Treatment
Ureteroscopy Procedure
Treatment of Uterine Bleeding
Antenatal And Postnatal Exercise
Antenatal Checkup
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Submit a review for Dr. SasmithaYour feedback matters!
Is doing crunches bad for females? I have heard a few people saying that it causes severe cramps during periods and damages reproductive system. Is it true?
Is there any side effects to use contraception pill? Like I pill. Any other medicine for having safe sex?
I ve been married from 4 years, we are trying for baby and are unsuccessful. I have PCOD and thyroid. Thyroid is in normal range .But due to Pcod, have irregular periods. How can I successfully conceive. Any suggestions?
Man is different from the other species in that we are social beings. One of the main differentiators is our constant need to 'connect' with our fellow beings. Be it family or friends or society at large, we constantly need to be connected to our near and dear ones and want attention, affection, and assurance.
We need to respect this basic desire and appreciate that there is nothing wrong in feeling needed. Problems arise when this feeling to be needed gets excessive. There is a significant constant demand from the needy person, especially from those who are close to them.
Some cases where neediness is demonstrated are in new relationships. In the beginning, there is a great urge to please and therefore a greater focus on interacting with them. There is a fear of losing attention, there is insatiable excessive demand for attention - be it pleas, accusations, or demands - so as to not feel abandoned and deserted. Neediness is also quite evident in people with depression. Again, an attempt to not be abandoned by their near and dear ones.
By making a conscious effort, the neediness can be reduced, thereby making the relationships smoother and less painful to both the parties involved. As the saying goes - 'acknowledging the problem is half the problem solved'. Needy people should realize or accept if told that there is an issue and make a conscious effort to work on reducing neediness.
1. Divert attention - As simple as it might sound, it is equally effective. Indulge in whatever interests you - exercise, meditation, dance, sing, etc. The idea is to divert attention so that the urge to feel needy and act on that instinct is reduced. This reduces the fear of being lonely and works magic. The busier you get, the less bored you are. As they say, be alone but not lonely.
2. Clarity in communication - One of the intricacies of neediness is making unclear demands. Sadly, quite often, the person does not know what he wants, and so are the demands or requests which are equally unclear. Once you are aware that your neediness is making you do this, make an effort to clearly communicate what you actually want. This reduces the stress on both the parties involved and leads to a happier engagement.
3. Clarity in relationships - Take a step back and see if your neediness is affecting any major relationships. If yes, revisit and clarify. Make an attempt and reestablish the relationships. If it is your truly near and dear ones, they will be very forthcoming and understanding.
4. Move on - In cases where your neediness has affected another person, be wise to accept their wishes and not be more 'needy' or clingy. This acceptance will make way for a lot of healthier relationships as you learn to deal with accepting your issues.
5. Socialize - Use your hobbies to make more friends and socialize more. Having more people to interact with reduces your need to be accepted by the same people. You have more people to rely on and might make more friends for keeps.
These are some simple ways to reduce neediness on others, but as mentioned, the first step is to accept and then work on ways to reduce it.
Related Tip: Are You an Extremely Dependent Individual? Here's How to Overcome it.?
Could you please suggest me. What duet should be taken during pregnancy for a easy normal delivery?
Hello doctor. I'm 28 years old and got married 1.5 years before. Since I have been affected by hbv, we were postponed the pregnancy. Now my husband is vaccinated and we are planning for a baby. Suggest me some tips to get healthy pregnancy with boy baby.
A woman is the primary health care taker in a family. She is the first person a child consults when he or she is sick; she looks after the nutrition of the entire family, she is the one who dresses your wounds at home. According to a study conducted on 500 mumbai families the monitoring of a disease, the nutrition and proper intake of medicines is monitored by the lady of the house. This women's day let us look into the health problem she faces. Let us take care of our daughters, sisters, wives, mothers and grandmothers.
A woman faces many changes in her life, firstly at puberty, during pregnancy and child birth and menopause. During each of this change her body goes through many changes physically, mentally and emotionally which can cause a deviation in her state of health. It is here that homoeopathy comes to be of great aid for her.
Let us look at few homoeopathic medicines that can give the woman in your family a disease free life:
1) Dysmenorrhoea that is painful menses: every woman has faced this problem in her life. This pain can incapacitate her from her work. Mag phos is an excellent remedy for dysmenorrhoea. Severe cramping pain in lower abdomen relieved by warm fomentation is relieved when mag phos is taken in 6x potency.
2) Remedies such as calc carb, bovista etc are of good use in leucorrhoea that is white discharge from vagina but these remedies are best taken on prescription of registered homoeopathic practitioner.
3) A common complaint a woman faces during pregnancy is vomiting during pregnancy. Medicines such as nux vomica and sepia will help a great deal in this complaint. A pregnant lady must take care that if she is vomiting more than 3 times a day and cannot retain slightest solid or liquid then she must consult the practitioner immediately.
4) Another change that comes in a woman's life is at menopause. As the name it self suggests that it means the end of her fertility cycle. During this period a woman complaints of palpitations, weakness, irritability, hot flashes etc. The medicine belladonna will help you against the hot flashes and palpitation. Women rapidly lose bone density after menopause increasing their risk of osteoporosis. Calc phos taken in 6x potency will help in such case.
The health authorities all over the world are trying to educate and inform people regarding the woman's health. It is time that we wake up to the reality of healthy women being the harbingers of a healthy nation and work individually and collectively in this regard.
Almost every man and woman craves to have a child they can call their own. This makes an inability to procreate one of the most dreaded situations. Infertility can be defined as the inability to conceive a child within 12 months of biologically appropriately timed intercourse. Infertility exacts a huge psychological toll on the person.
Infertility often triggers depression in a person. This may be acute and result from specific events such as the holidays, family celebrations of the news of a someone else having conceived a child or could be cyclic and coincide with phases of their fertility cycle. This depression could lead to problems at work and communication with the spouse.
Infertility can also make a person feel helpless and affect their self esteem. Infertility accompanied by depression can trigger the feeling of having lost control over one's life and can make people withdraw into themselves.
An infertile person may also often feel guilty and make them blame themselves and their prior actions for the condition. This leads them to question their worthiness of being a good spouse and potential parent. A woman who has suffered from an STD earlier or has undergone an abortion earlier can become obsessed with blaming herself for being infertile.
This affects their daily lives and ability to work. An infertile person may also feel guilty for having robbed their partner of the chance to become a parent. This can put a strain on their relationship and in many cases lead to a broken marriage.
When they find themselves unable to conceive a child, a couple may try to time their intercourse with ovulation tools, such as an ovulation thermometer etc. and may try different positions, some of which may be fairly uncomfortable. They may also end up having intercourse more often than desired. In order to prevent sperm from leaking out of the vagina, a woman may also find herself lying in an awkward supine position after intercourse. A combination of all these factors makes the act a chore and with time can lead to a loss of libido.
Infertility can also trigger anger against life for having been treated unfairly or towards other parents. This may also be displaced towards their family and friends leading to situations where they feel ostracized and unsupported.
When dealing with infertility, it is important to understand that neither partner is to blame. Infertility is a physiological condition and can be treated in most cases. It is important for both partners to support each other during this process and if need be consult a counselor.


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YP - The Real Yellow PagesSM - helps you find the right local businesses to meet your specific needs. Search results are sorted by a combination of factors to give you a set of choices in response to your search criteria. These factors are similar to those you might use to determine which business to select from a local Yellow Pages directory, including proximity to where you are searching, expertise in the specific services or products you need, and comprehensive business information to help evaluate a business's suitability for you. "Preferred" listings, or those with featured website buttons, indicate YP advertisers who directly provide information about their businesses to help consumers make more informed buying decisions. YP advertisers receive higher placement in the default ordering of search results and may appear in sponsored listings on the top, side, or bottom of the search results page.
Bulk Mulch in Orlando, FL
1.Sunrise Landscape Supply Inc
6670 E Colonial Dr
5.46 mi
MulchesSand & GravelLandscaping Equipment & SuppliesLawn & Garden Equipment & Supplies
2.Global Rental Dumpsters
1920 Silver Star Rd
3.26 mi
MulchesRecycling CentersGarbage CollectionConstruction Site-Clean-Up
GRD is exceptional in the timeliness of its scheduled deliveries. And Violet is ... perfect. Her warm voice and professional and caring demeanor is inviting at a time when most vendors act like they care little for your business. We have used GRD over the past two years and are pleased to write this review. The drivers have always done a great job by contacting us by cell phone immediately prior to the delivery and pick up. Our satisfaction with GRD will keep us coming back whenever we require their services.
3.Sunrise Landscape Supply, Inc
2916 Apopka Blvd
8.15 mi
MulchesLandscape ContractorsLandscaping & Lawn ServicesLandscaping Equipment & Supplies
4.A Bliss Outdoors
Landscaping Equipment & SuppliesIrrigation ConsultantsSprinklers-Garden & LawnIrrigation Systems & Equipment
Richard is very professional and takes pride in his work! Not only is he proficient in repairing and helping you maintain your sprinkler system, but we helps make your yard look nice when he trims and mulches. He puts everything back the way or even better than he found it, it's like he was never even there! I would definitely recommend him 10/10 times!
5.Kempfer Sawmill IncServing the Orlando, FL area
MulchesWood ProductsLumber
KEMPFER SAWMILL, INC. traces its roots back to the Union Cypress Company, which was formed in the early 1900's south of Melbourne in the town of Hopkins. George W. Hopkins ran Union Cypress Company until it burned down in 1919. Now, George W. Hopkins's great-grandsons Reed and Billy Kempfer run Kempfer Sawmill. Please give us a call at 407-892-1169 if you have any questions. Kempfer Sawmill, Inc. can help you with all of your lumber needs.
6.Larsen Landscape & Tree Service
Landscape Designers & ConsultantsStump Removal & GrindingMulchesTree Service
Welcome to Larsen Landscape & Tree Service. We have been providing a complete tree, landscape design, and installation within the Orlando area since 1977. Larsen Landscape & Tree Service is licensed, insured, and covered by worker?s compensation. Call us today for a free estimate.
7.Florida's Mulch Express Orlando
8300 E Colonial Dr
6.84 mi
MulchesLandscaping & Lawn Services
8.Orlando Outdoors Landscape Nursery
5001 W Sand Lake Rd
7.43 mi
MulchesSod & Sodding Service
Very nice group running this business. Great selection of landscaping supplies with sod deliveries every morning. They seem to bend over backwards to get you what you need. Location is a bit cramped for space but they will get you loaded and out asap.
9.Complete Pro Maintnenace
4650 Raintree Ridge Rd
13.39 mi
MulchesLandscaping Equipment & SuppliesLawn & Garden Equipment & Supplies
Help keeping your neighborhood beautiful.
6832 Narcoossee Rd
7.93 mi
11.Rubber Mulch Products
2500 Kunze Ave
1.66 mi
12.Major Mulch Installations
Landscape ContractorsMulches
Major Mulch Installations is widely regarded in the landscape and mulch industry as the most professional and reliable blow-on mulch installation company in Florida. Because Major Mulch Installations is the industry's technological leader in mulch installation, your projects are mulched quickly and efficiently with state-of-the-art mulch blowing trucks. As a result, your landscape is given a very elegant, finished look - something that is extremely difficult to obtain by hand spreading the material. Nothing compares to the beautiful look and results of a project mulched by Major Mulch Installations.
13.Uptown Lawn Services, Inc
P.O.Box 677083
0.12 mi
Landscaping & Lawn ServicesSprinklers-Garden & LawnWeed Control ServiceMulches
We have had Uptown Lawn Service for the last 5 years handle our home and have been so happy with their work. Our yard always looked great and many of our neighbors began to use them as well. Recently we moved and we actually asked them to come with us to our house in Windermere. They did a great job working with us to get our new yard in good shape. They not only do our weekly service, they have also been great with extra jobs like landscaping, sodding and mulching. Javier (the owner) takes great pride in his work and it shows. Uptown is hands down the best landscape and lawn care company we have ever had in Florida. We highly recommend them for residential properties.
14.Creeds Tree Service
Tree ServiceMulchesStump Removal & Grinding
Creeds Tree Service did a great job on my tree trimming and removal!!! They arrived on time and cleaned everything up as expected plus more!!! I recommend them for any service that you need regarding tree services. http:/www.creedstreeservicesorlando.com
15.T & J's Lawn Care Service
PO Box 721437
5.92 mi
Landscaping & Lawn ServicesLawn MaintenanceTree ServiceSod & Sodding Service
Our services include: Grass Cutting Edging Blowing Tree and Shrub Care Flower Beds Yard Cleanup and Removal Debris Cleanup General Maintenance and more!
16.Sod Depot & Orange Blossom Landscape Supply
4929 N Orange Blossom Trl
5.13 mi
Sod & Sodding ServiceLandscaping Equipment & SuppliesLawn & Garden Equipment & SuppliesMulches
17.greenfeld & Yauger power Wash & Landscape designs
6110 Edgewater Dr
5.58 mi
Landscaping & Lawn ServicesIrrigation Systems & EquipmentMulchesWater Pressure Cleaning
18.Seay Tree Service, Inc.
4885 Clarion Dr
6.53 mi
Tree ServiceMulchesStump Removal & GrindingGrading Contractors
14566 Cheever St
15.10 mi
Tree ServiceStump Removal & GrindingArboristsGrading Contractors
20.Tree Work Now
Tree ServiceArboristsGrading ContractorsLandscaping & Lawn Services
21.Root's Complete Lawn And Tree Care
3515 Dr Love Rd
6.38 mi
Landscaping & Lawn ServicesSod & Sodding ServiceTree ServiceLandscape Contractors
laudable services and absolutely flexible payment alternatives that will fit your budgetary considerations. professional savvy shows in their workmanship and approach carefully configuring your lawn's issues and requirements up to the last tiny sprinkler. thanks to Ivan and Vicki and family. We are impressed!!!
22.A Total Lawn Care Landscaping Solutions
938 Windmill Grove Cir
13.12 mi
Landscaping & Lawn ServicesLawn MaintenanceLawn MowersMulches
We provide the best quality Lawn Care Service at the Best Prices. We offer Complete lawn care, fertilizing, weed control, mulching, pressure washing, Sod work, plant design and installation.
23.Bolling Forest Products Inc
371 W Mitchell Hammock Rd
13.17 mi
MulchesLandscape Designers & ConsultantsLandscape ContractorsGarden Centers
Mulch, Bark & Soil for all your Landscaping Needs!
24.Sod Depot Of Orlando
523 Douglas Ave
Altamonte Springs,FL
9.21 mi
25.Mulch For You
371 W Mitchell Hammock Rd
13.17 mi
MulchesLandscape Designers & ConsultantsLandscape Contractors
26.Cowart Mulch
2320 W Orange Blossom Trl
14.83 mi
27.Gardner's Mobile Mulch Blowing
Lady Lake,FL
MulchesGarden CentersNurseries-Plants & Trees
28.Millers Sod Sales
944 S Orange Blossom Trl
11.11 mi
Sod & Sodding ServiceMulchesLandscaping Equipment & SuppliesTrucking-Heavy Hauling
What a pleasure to sit out in the back yard now!! They were prompt and so nice, grass is absolutely beautiful, thank you! I definatley will be referring them to my family and friends
29.A Sunstate Tree Service
1580 S Us Highway 17 92
10.79 mi
Tree ServiceArboristsWaste ReductionEnvironmental Services-Site Remediation
They were on time, worked quickly and cleaned up as they went. Cut down 3 big trees in less than 2 hours and shredded the branches, cut the trunk into smaller pieces that they came and picked up later. Good work, the best price in all the quotes we got. Highly recommended. Our sales rep was Chris and he was friendly and courteous.
30.Gator Tree Experts
1635 Minnesota Ave
Winter Park,FL
3.58 mi
Tree ServiceGrading ContractorsMulchesLawn Maintenance
The guys at Gator Tree service did a great job on our trees and also carefully removed a nest with 4 baby squirrels so they would not get hurt during the job. They gave us a number for a wildlife rescue organization that came to take them and hand raise them until they were ready to be released. The mother squirrel was scared away and would not come back to get her babies. The guys at Gator Tree Service went above and beyond to save the babies.
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Monin Stirs Up Sales with Email Segmentation
The premium syrup and concentrate manufacturer uses purchase, engagement, and preference data to deliver the right message to the right customer
FTC Warns Influencers and Brands to Make Sponsorships Clear
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How Collaborative Data Helps Marketers Read the Clouds
The Weather Company and LiveRamp collaborate for a first of its kind data integration
Identifying Data Bias Early
How to guard against biases that can wreck your analytics results and machine learning models
Programmatic Display Spending to Reach Nearly $33 Billion
Programmatic advertising appears to be stronger than ever
One on One: Zeta Can Compete Everywhere, says Jeffry Nimeroff
Zeta is lining up with the big marketing cloud players, says CIO Jeffry Nimeroff
Striking Matches with MaxPoint
The CRM and matching software vendor catalyses audiences for the marketing eco-system
LIDS Tips its Hat to Social Media
The popular headwear brand wanted to update its social marketing strategy, and found success doing so through Salesforce Marketing Cloud.
The Portland Trail Blazers Score ROI with Facebook Advertising
How the professional basketball team uses data and segmentation to target the right fans
Company of the Week
Brightcove is the world's leading video platform. The most innovative and respected brands confidently rely on Brightcove to solve their most demanding communication challenges because of the unmatched performance and flexibility of our platform, our global scale and reliability, and our award-winning service. With thousands of customers and an industry-leading suite of cloud video products, Brightcove enables customers to drive compelling business results.
Career Center
Explore careers in digital marketing, sales, eCommerce, marketing communications, IT, data strategies, and much more. And don't forget to update your resume so employers can contact you privately about job opportunities.


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Former patient donates toys to children's hospital
Posted April 16
LEBANON, N.H. — The toy room at the Children's Hospital at Dartmouth-Hitchcock will be well stocked for the next six to nine months thanks to the generosity of a former patient.
Nicholas Koloski says he remembers that playing with the toys was the only relief he felt during the scary experience of being in the hospital. Now a firefighter in Claremont, he recently used his annual firefighters' stipend and contributions from the community to purchase toys and stuffed animals and recently delivered a truckload to the hospital.
Hospital officials say the toys help normalize the experience for children and to provide them comfort and an opportunity to play while they are away from home.


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The following email was passed to me by a Jewish supporter:
"Re: Holocaust Remembrance Day - May 2,
Please wait 20 Seconds before you close this e-mail.
This message asks you to do one small act to remember the six million (6,000,000) Jewish lives that were lost during the Holocaust.
Send this message to everyone you know who is Jewish.
If we reach the goal of reaching six million e-mail names before May 2, we will fulfill and give back to God what He gave to us:
6 million Jews who are alive today who remember those who perished.
Please send this message and Ask them to also forward it to others.
Thank you"
Despite the fact that I am an ex Jew, notwithstanding the fact that I want nothing to do with Jewish politics or religion, I really wanted to follow up the request and to send the email to all my 'Jewish' friends. I thought to myself that the memory of the Holocaust is indeed important and crucial, especially due to the embarrassing fact that just 3 years after the liberation of Auschwitz, the newly born Jewish state ethnically cleansed the vast majority of the indigenous Palestinians from their cities and villages.
Just five years after the liberation of Auschwitz the newly born Jewish State set racist discriminating laws (not at all different from infamous Nazi Nuremberg Laws) to stop Palestinians from returning to their land. 62 years after the brutal expulsion of the Palestinian people, Palestinians are still dispossessed and watching Israelis living in their houses, on their land and spreading emails about the Nazi brutality and Jewish survival. The memory of the Holocaust is indeed important because for the last 62 years the Jewish state is committing crimes against humanity in the name of the Jewish people and in the name of the Holocaust.
The memory of the Holocaust is important because as some Israelis are brave enough to admit, Israelis are the Nazis of our time.
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Home > News > 29 Hot Businesses to Start in 2006
November 21st, 2005
29 Hot Businesses to Start in 2006
Nanotech: Some say nanotechnology - the creation of materials at the molecular level, enhancing their capabilities - is this generation's "Plastics." Ecology Coatings of Akron, Ohio, for example, is already warping what's possible with the creation of "liquid solid" coatings, which flow like liquid and stay "wet" without drying or evaporating until they're hit with ultraviolet light - then they dry in just three seconds. There are other applications: Apollo Diamond cultures real diamonds one atom at a time. Clothing, lotion, diapers and medicine are all on the same path. What, you thought your wrinkle-free pants were magic?
(Ed.'s note: this part of the article is way down towards the bottom.)
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Our Bedroom Makeover: The Painting Dilemma
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text:
Assessing histone demethylase inhibitors in cells: lessons learned
- Stephanie B. Hatch†1, 2,
- Clarence Yapp†1, 2,
- Raquel C. Montenegro1, 2, 3,
- Pavel Savitsky1,
- Vicki Gamble1, 2,
- Anthony Tumber1, 2,
- Gian Filippo Ruda1, 2,
- Vassilios Bavetsias4,
- Oleg Fedorov1, 2,
- Butrus Atrash4,
- Florence Raynaud4,
- Rachel Lanigan4,
- LeAnne Carmichael4,
- Kathy Tomlin4,
- Rosemary Burke4,
- Susan M. Westaway5,
- Jack A. Brown5,
- Rab K. Prinjha5,
- Elisabeth D. Martinez6,
- Udo Oppermann1, 7,
- Christopher J. Schofield8,
- Chas Bountra1, 2,
- Akane Kawamura8, 9,
- Julian Blagg4,
- Paul E. Brennan1, 2,
- Olivia Rossanese4 and
- Susanne Müller1, 2, 10Email author
DOI: 10.1186/s13072-017-0116-6
© The Author(s) 2017
Received: 13 December 2016
Accepted: 21 February 2017
Published: 1 March 2017
Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active 'probe' molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells.
A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition.
High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.
KeywordsHistone lysine demethylase Chromatin Immunofluorescence Cell proliferation Apoptosis Toxicity Epigenetics 2-Oxoglutarate oxygenases
Histones are highly conserved proteins that play important roles, not only in compacting DNA in the cell, but which also have dynamic functions in many physiological and molecular processes. Covalent modifications of the histone tail, in particular on lysine residues, are an essential part of epigenetic mechanisms to regulate processes like DNA repair and gene transcription [1, 2]. The complex code of histone tail modifications is tightly regulated and includes a variety of modifications, most prominently acetylation and methylation of lysine and arginine residues. Enzymes that modify or bind to these residues have been implicated in a variety of diseases including cancer, inflammatory diseases, heart disease and neurodegenerative diseases [3–5]. Methylation of histone lysines was once thought by many to be irreversible, but two classes of histone lysine demethylases (KDMs) have now been identified that catalyse the removal of these methylation marks. Based on their enzymatic mechanism, the flavin-dependent demethylases of the KDM1 subfamily (lysine-specific demethylases or LSDs) are distinguished from the Jumonji C (JmjC) family of KDMs, comprising the KDM2 to KDM7 subfamilies, which belong to the superfamily of Fe(II)- and 2-oxoglutarate (2-OG)-dependent oxygenases. Demethylation of different marks has been described thereby influencing the ratios of mono-, di- and tri-methylation on H3K4, H3K9, H3K27, H3K36 and H4K20, resulting in differential effects on gene transcription. For example, H3K4me2/3 and H3K36me3 are preferentially associated with transcriptionally active genes, while H3K9me2/3, H3K27me2/3 and H4K20me3 correlate with transcriptional repression [6–8].
There is growing interest in exploring the biology and disease relevance of the KDMs with the help of well-characterized 'probe molecules' that show potent activity in biochemical and cellular assays as well as selectivity over other KDMs [9]. Several inhibitors for the KDM1 family have been described, with the most advanced compounds entering clinical trials [6]. A number of inhibitors have also recently been discovered for the JmjC family of demethylases, e.g. [6, 10–17]. In order to draw conclusions regarding the biological activity of new inhibitors, it must first be confirmed that their observed cellular effect is due to the inhibition of the respective enzyme rather than uncharacterized off-target activity or nonspecific effects. We have developed and evaluated an immunofluorescence-based high-content imaging assay to assess the on-target effect of demethylase inhibitors and describe here the lessons learned.
In vitro and cellular IC50 of selected KDM inhibitors tested
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
IC50 (µM)
0.010 ± 0.0030 (6)
0.007 ± 0.0030 (12)
0.019 ± 0.0050 (10)
0.019 ± 0.0040 (6)
2.2 ± 0.37 (2)
1.2 ± 0.91 (4)
0.67 ± 0.23 (2)
0.099 (1)
0.19 ± 0.081 (6)
1.4 ± 0.45 (2)
In vitro
In cells
0.068 ± 0.010 (2)
0.094 ± 0.065 (6)
0.17 ± 0.073 (6)
19 ± 10 (2)
2.6 ± 1.3 (4)
11 ± 4.5 (2)
2.5 (1)
2.8 ± 1.3 (4)
11 ± 4.6 (2)
In vitro
In cells
>10 (2)
In vitro
In cells
0.040 ± 0.016 (2)
0.063 ± 0.028 (2)
0.26 ± 0.039 (2)
0.23 ± 0.039 (2)
>100 (2)
>100 (2)
3.9 ± 1.4 (3)
1.4 ± 0.47 (3)
5.1 ± 0.062 (4)
>100 (2)
In vitro
In cells
0.015 ± 0.0041 (4)
0.014 ± 0.0046 (4)
0.024 ± 0.0044 (4)
0.024 ± 0.0038 (4)
8.6 ± 5.9 (2)
6.0 ± 3.9 (4)
0.126 ± 0.013 (2)
0.050 ± 0.0038 (2)
0.10 ± 0.031 (4)
44 ± 17 (3)
In vitro
In cells
0.16 ± 0.068 (2)
0.26 ± 0.064 (2)
7.8 ± 1.6 (2)
23 ± 3.5 (2)
>100 (2)
31 (1)
44 (1)
11 ± 2.3 (2)
>100 (2)
In vitro
iN cells
0.102 ± 0.058
0.031 ± 0.012
15% at 100 µMc
In vitroa
In cells
>10 (2)
>10 (2)
In vitroa
In cells
20.6 ± 9.0 (10)
30.4 ± 13.2 (13)
5 ± 3.8 (4)
0.2 ± 0.22 (30)
0.5 ± 0.4 (2)
3.5 ± 2.9 (16)
1.2 ± 1.3 (55)
In vitroa
In cells
0.561 (1)
0.622 ± 0.05 (2)
1.2 ± 0.01 (3)d
0.65 ± 0.05 (3)
0.62 ± 0.02 (2)
1.04 ± 0.6 (4)
In vitroa
In cells
7.01 (1)
>10 (3)
>10 (3)
>10 (2)
>10 (4)
In vitro
In cells
HeLa cells transiently overexpressing WT KDM5B were treated with the ester KDOAM-21 for 24 h. At the highest compound concentration tested, the KDM5 enzymes were completely inhibited and H3K4me3 levels reached that of the cells transfected with the MUT KDM5 enzyme (Fig. 2a (i–iii), b). In line with the in vitro biochemical potency of the parent carboxylic acid (KDOAM-20), KDOAM-21 showed potent inhibition of H3K4me3 demethylation caused by overexpressed KDM5B with an EC50 < 1 µM. Surprisingly, KDOAM-20 also showed activity against KDM5B in cells with an EC50 of 20 µM despite having low cell permeability in Caco-2 assays (Additional file 4: Table S2). The other KDM5 family members tested (KDM5A, KDM5C and KDM5D) were also potently inhibited by KDOAM-21 with EC50 values in the 3–5 μM range in line with the measured in vitro potency of the parent carboxylic acid KDOAM-20 (Fig. 2b; Table 1), but showed little inhibition upon treatment with KDOAM-20 consistent with poor cell penetrance of the parent carboxylic acid (Additional file 4: Table S2). KDOAM-20/21 also showed inhibition of the KDM4 family, which was assessed by increase of the H3K9me3 mark. KDM4B was inhibited most potently with an EC50 value of 10 μM for KDOAM-21 (Fig. 2c; Table 1). For the KDM4 subfamily members tested, we also observed inhibition of the overexpressed enzymes by the compounds within the same potency rank order as observed in enzyme kinetic assays (Fig. 2c; Table 1; Additional file 4: Table S2). None of the inhibitors showed activity on members of the other demethylase families tested (KDM3A activity on H3K9me2, KDM6B activity on H3K27me3) (Fig. 2c; Table 1) consistent with the weak in vitro enzymatic potency of these compounds versus the respective demethylase assays.
Interestingly, at higher KDOAM-20/21 inhibitor concentrations, a dose-dependent effect on the H3K4me3 mark was observed in cells overexpressing MUT KDM5. This effect was not seen in cells expressing WT or MUT KDM5 treated with the control compound KDOAM-32, possibly indicating that the observed effects were either due to inhibition of the endogenous enzyme(s) or due to a nonspecific effect of KDOAM-21 and KDOAM-20 (Fig. 2b). We observed no toxicity as measured by cell count using HeLa cells at any of the measured concentrations, indicated by a constant number of cells assessed in the high-content screen (Additional file 5: Figure S3). In order to further study inhibition of endogenous H3K4me3 levels, nontransfected HeLa cells were treated with KDOAM-20, KDOAM-21 and the inactive control KDOAM-32 at the same concentrations used in assays where the protein was ectopically expressed. H3K4me3 was monitored after 72 h by IF staining. Observed EC50 (sub-μM potency for KDOAM-21, ~50 μM for KDOAM-20 and no inhibition observed for KDOAM-32) were in good agreement with EC50 values measured using the overexpressed enzyme (Fig. 2d; Table 1). Similar results were obtained after treatment of nontransfected HeLa cells for 48 h, whereas at 24 h the observed EC50 values were slightly lower (Hatch et al. unpublished results). We therefore have established a robust panel of cell assays to test inhibitors for the Jumonji family of KDMs.
The KDM6A/B inhibitor GSK-J4 [21] is reported to also inhibit KDM5 at higher concentrations in vitro [31]. We tested GSK-J4 together with its inactive control compound GSK-J5 and also observed an increase of the H3K4me3 mark upon treatment with the active KDM6A/B inhibitor, but not the inactive control. These two compounds showed only minor toxicity at the highest concentrations used, which is well above the effective doses used for KDM6A/B inhibition (~1 µM) (Fig. 4b; Additional file 6: Figure S4). Assessment of inhibitors in the high-content assay therefore needs careful consideration of all parameters, WT and MUT data. In addition, steep-slope increases of histone methylation mark are often accompanied by a reduction in cell number pointing to cell toxicity of the compound. The results imply that simultaneous inhibition of several KDMs does not seem to be tolerated at higher compound concentrations by the tested cells.
Almost all currently available JmjC-KDM inhibitors act via metal chelation and compete with the 2-OG co-substrate for binding at the active site. Poor cellular activity has been a problem; however, cell permeability is not the only reason for the poor cellular activities of the 2-OG competitive KDM inhibitors [11]. The results presented here further inform on the cellular activities of KDM inhibitors. They indicate that the currently used in vitro assays may overestimate the cellular effects due to the use of lower than physiologically relevant 2-OG concentration.
A number of methods have been described to assess cellular activity of KDM inhibitors, each with advantages as well as disadvantages [20]. We have described a panel of cellular assays to assess the direct inhibition of KDMs as well as selectivity of KDM inhibitors in cultured human cells using a high-content-based overexpression system. We assessed several known KDM inhibitors as well as newly described inhibitors and demonstrate the usefulness of structure-matched inactive control compounds to assess effects related to specific KDM inhibition. In addition, the results obtained with overexpression of the MUT protein in combination with assessment of cell numbers provide a useful guide to enzyme inhibition in cells, helping to identify on-target effects. It is now our observation across multiple compounds that, in our hands, a spike in histone mark methylation is characteristic of cytotoxicity. The results obtained using this system are in good agreement with data obtained assaying endogenous demethylases, but offer the additional advantage of distinguishing between a direct effect on KDM inhibition and changes to histone methylation upon induction of cell death.
Cell death observed upon treatment with toxic compounds (e.g. doxorubicin or paclitaxel) resulted in a dose-dependent increase in histone methylation, in particular of the repressive H3K27me3 mark, but also of other marks such as H3K4me3 and H3K9me2. A recent study reported the induction of global changes in histone lysine and arginine methylation and altered expression of lysine demethylases upon treatment of human cancer cells with HDAC inhibitors pointing to a complex network of histone crosstalk [34]. Increased methylation of histone H3K27 associated with apoptosis induced by staurosporine has also been described in osteosarcoma cells, but the study did not examine any changes in dimethyl or trimethyl histone H3K4 and H3K9 [35].
Although not addressed in the current study, one can speculate regarding the underlying physiological mechanisms of these histone mark changes in apoptosis and general cell death. Little is known about changes in histone methylation upon induction of cell death in humans although a global decrease in histone acetylation has been observed during apoptosis [36, 37]. Loss of histone H3 methylation at lysine 4 has, however, been shown to trigger apoptosis in Saccharomyces cerevisiae [38]. In melanoma cells, short-term treatment (4 h) with doxorubicin decreased H3K4me3 methylation but did not have an effect on H3K27me3 [39]. Detailed chromatin immunoprecipitation-based assays and gene expression analyses, as well as time-resolved experiments, will be necessary to investigate in more detail the interplay between cell death and chromatin changes addressing questions of specific mechanisms—for example, if cells first activate genes through increased density of H3K4me3 activating marks before increasing repressive marks like H3K27me3 when cell death cannot be avoided.
In summary, we have developed a series of overexpression IF assays to assess the target engagement of KDM inhibitors in cells. The presented assays cover most major branches of the 2-OG JmjC-KDMs. Nonspecific toxicity of tested inhibitors, in particular those resulting in apoptosis, leads to changes in histone tail modifications unrelated to the expected cellular activity of these compounds. The presented assay provides a suitable method to distinguish between direct and indirect effects of histone methylation.
Full-length cDNA for KDM5A (JARID1A, P29375), KDM5B (JARID1B, Q9UGL1, cDNA), KDM5C (JARID1C, P41229, IMAGE 5492114), KDM4A (O75164) containing A482E substitution (natural variant rs586339), KDM4B (JMJD2B, O94953, gift from ICR), KDM4C (JMJD2C, Q9H3R0, 8143862), KDM3A (JMJD1A, Q9Y4C1, 4823253), KDM2A (FBXL11, Q9Y2K7, 5534384) and KDM6B (JMJD3, O15054, cDNA) were amplified by PCR from either a MGC clone or commercial cDNA source or received as a gift from collaborators and cloned into pDONR-221 vector using Gateway BP reaction producing Gateway entry clones.
To produce catalytically inactive KDMs residues involved in iron coordination were substituted for alanine (KDM5A H483A/E485A, KDM5B H499A/E501A, KDM5C H513A/E515A, KDM5D H510A/E512A, KDM4A H188A/E190A, KDM4B H189A/E191A, KDM4C H190A/E192A, KDM3A H1120Y, KDM2A H212A/D214A and KDM6B H1390A/E1392A). Mutations were introduced into full-length KDM Gateway entry clones using 15 cycles QuikChange II PCR protocol (Agilent Technologies).
Mammalian expression constructs encoding for N-terminal 3*FLAG potencies were constructed using the Gateway LR recombination reaction between pCDNA5-FRT/TO-3FLAG destination vector [40] and the wild-type or mutated KDM Gateway entry clone.
All constructs described are available upon request.
Cell culture/transfection for 24-h IF assay
HeLa cells (ATCC) were cultured at 37 °C and 5% CO2 in Minimal Essential Media—Eagle (Sigma-Aldrich, UK) supplemented with 10% heat-inactivated foetal calf serum (FCS) (Gibco, UK), and 1% l-glutaMAX (Gibco, UK) in T75 flasks (Sarstedt, UK) and passaged regularly with TrypLE (Life Technologies, UK) before cells reached confluence. On day 0, 7000 cells per well were plated on a 96-well clear bottom CellCarrier plate (PerkinElmer, UK). The next day the cells were transfected with Lipofectamine 3000 (ThermoFisher Scientific, UK) following the manufacturer's instructions. Briefly, 0.1 µg of plasmid DNA (encoding for the respective demethylase) and 0.15 µL of Lipofectamine 3000 were diluted in separate tubes containing 5 µL OptiMEM. 0.15 µL of P3000 was added to the tube with the DNA. The two solutions were then mixed gently and allowed to incubate for 5 min at room temperature. Next, the DNA-lipid complex was added to the cells for 4 h at 37 °C and 5% CO2.
Compound dilutions were made in a 96-well PCR plate (Starlab, UK). The culture medium was supplemented with 0.4% DMSO so that the solvent concentration was maintained throughout. Compounds were serially diluted at a 1:2 or 1:3 dilution ratio. Next, the cell culture plate was removed from the incubator and the medium was gently aspirated and replaced with the fresh medium containing diluted compounds.
After 24 h of compound incubation, cells were stained using the following protocol. First, the cells were washed once with Phosphate Buffered Saline (PBS) (Sigma-Aldrich, UK) and fixed for 20 min at room temperature using formaldehyde diluted in PBS to 4% w/v. Next, cells were rinsed once with PBS and permeabilized for 5 min at room temperature using TritonX-100 diluted in PBS at 0.5% v/v. Then cells were rinsed once with PBS and blocked for 30 min at room temperature using 3% v/v foetal calf serum diluted in PBS. This mixture was then replaced with the appropriate primary antibodies for the histone mark and FLAG-tag diluted in blocking solution and left on overnight at 4 °C. The following day, cells were rinsed three times with PBS and stained with the secondary antibody for 1 h at room temperature. Before imaging, cells were rinsed once with PBS, stained with DAPI (LifeTechnologies, UK) for 5 min and rinsed again with PBS at room temperature.
Endogenous IF assay
Antibody specifications for each demethylase IF assay
Histone mark
Primary antibody for histone mark (cat. no.)
Secondary antibody for histone mark (cat. no.)
Primary antibody for FLAG-tag (cat. no.)
Secondary antibody for FLAG-tag (cat. no.)
Abcam (ab1220)
Alexafluor 488 (MG2A20)
Abcam (ab8898-100)
Alexafluor 488 (A-11070)
Millipore (07-449)
Alexafluor 488 (A-11070)
Diagenode (C15410003-50)
Alexafluor 488 (A-11070)
Widefield fluorescence microscopy
Transfection of cells was assessed on a Zeiss AxioObserver Z1 inverted fluorescence microscope fitted with an Axiocam 506 monochrome camera, Colibri.2 LED system (385, 475, 561, 647 nm), 10× 0.45 N.A. and 20× 0.8 N.A. Plan Apochromat objectives. The system PC ran Windows 7 Ultimate 64-bit and had an Intel Core i5-2500 @3.30 GHz processor with 8 GB RAM and an integrated video card. The microscope was controlled with ZEN Blue.
High-content analysis
Immunofluorescence images of 20 fields (if not indicated otherwise) were captured through a 20× objective on a PerkinElmer Operetta or the GE IN Cell 6000 using the appropriate filters and light sources for exciting DAPI, Alexafluor 488, and Alexafluor 568 or Alexafluor 647. The images were imported into Columbus (PerkinElmer) for further analysis. From the DAPI channel, a binary mask was created to exclude individual nuclei smaller than 100 µm2 and those overlapping the edge of the field. The remaining nuclei were used for further analysis. The average nuclear intensity of the anti-Flag staining (Alexafluor 568 or Alexafluor 647) was measured, and then the intensity of the anti-methylated histone mark (Alexafluor 488) was analysed only in those cells that strongly overexpressed the FLAG-tagged demethylase. The total number of nuclei was used as a measure of cell survival and to calculate transfection efficiency. For the endogenous assay, the intensity of Alexafluor 488 from all cells in 9–10 fields was used for analysis. The average intensity and standard deviation of Alexafluor 488 cells was measured for each well and plotted in a dose-dependent manner in Prism (GraphPad, USA).
Hoechst 33342/Yo-Pro 3/Annexin triple staining and live cell death pattern analysis
Cells exposed to the inhibitors for 24 h were stained with Hoechst 33342 (1 µM), Yo-Pro 3 (1 µM) and Annexin V (0.3 μL per well) for 1 h. Cellular fluorescence was measured using the IN Cell 6000 (GE) using the following set-up parameters: bright-field transmitted light at 50% for 15 ms; Hoechst 33342 was excited by 35-ms exposure(Ex 360–400 nm/Em 410–480 nm), Yo-Pro 3 by 35-ms exposure(Ex 560–580 nm/Em 650–760 nm) and Annexin V (Alexa 488) by 45-ms exposure(Ex 460–490 nm/Em 500–550 nm). All the generated data were analysed using the IN Cell Analyser software, and three categories were distinguished: healthy cells, apoptosis and necrosis—calculated as percentage of each class for every concentration used.
Caco-2 permeability
P app (apparent permeability) was determined in the Caco-2 human colon carcinoma cell line essentially as described in [11].
IC50 values were determined as previously described [11]. The following amendments were made to the protocol for carrying out 2-OG competition assays.
For each compound, a 10 mM stock concentration in 100% DMSO was used. Compounds (100 nL) were dispensed with an ECHO® 550 acoustic dispenser (Labcyte Inc™, Sunnyvale, CA, USA) to generate 10–12 pt dilution curves directly into 384-well Proxiplates (#6008289, PerkinElmer, Waltham, MA, USA) to give final assay concentrations in the range 0.00015–30 µM in 2%(v/v) DMSO where appropriate. Compounds were pre-incubated with their enzymes (2.5 µL, 1.5 nM KDM4A, 0.5 nM KDM4B or for 2 nM KDM5B final assay concentration) for 10 min at room temperature before the addition of their peptide substrates (2.5 µL).
Peptide substrates (60 nM ARTKQTARK(me3)STGGKAPRKQLA-GGK-biotin for KDM4A and KDM4B or 200 nM ARTK(me3)QTARKSTGGKAPRKQLA-GGK-biotin for KDM5B) were made up in peptide buffer consisting of HEPES (50 mM, pH 7.5), BSA (0.1%), sodium ascorbate (200 µM), ammonium iron(II) sulphate hexahydrate (2 µM for KDM4A/KDM4B and 10 µM for KDM5B), Tween 20 (0.01%) and 11 2-OG concentrations ranging from 2000 to 0.5 µM. Peptide and buffer components were diluted twofold upon the addition to the compound plate containing enzyme in assay buffer [HEPES (50 mM, pH 7.5), BSA (0.1%) and Tween 20 (0.01%)]. The plate was sealed, then centrifuged at 1000 rpm for 1 min before being left for 15 min at room temperature. The reaction was stopped with the addition of 2.5 µL EDTA (30 mM EDTA in water for KDM4A and KDM4B or 30 mM EDTA, 1600 mM NaCl, 50 mM HEPES pH 7.5 and 0.01% Tween 20 for KDM5B) before the addition of 2.5 µL AlphaScreen detection reagents and antibody appropriate for the substrate being detected (mouse IgG beads (#6760606M, PerkinElmer) and anti-dimethyl H3K9 antibody (#Ab1220, Abcam) for KDM4A and KDM4B and Protein A beads (#6760617M, PerkinElmer) and anti-dimethyl H3K4 antibody (#9725S, Cell Signalling Technology) for KDM5B). AlphaScreen beads and antibody mixes were prepared and pre-incubated for 1 h prior to addition to the plate. Plates were left for 1.5 h in the dark before being read on the EnVision® Multilabel Reader (PerkinElmer Life Sciences). All assays were run at a final assay reaction volume of 5 µL and a total assay volume of 10 µL.
Data were normalized to the controls without 2-OG and DMSO wells containing all reagents as the totals. IC50 values at each 2-OG concentration were determined using a nonlinear regression fit of the log (inhibitor) versus response with variable slope equation in GraphPad Prism 6.0.
Chemical synthesis
Additional file 10 Methods.
histone lysine demethylase
- JmjC:
Jumonji C
high-content assay
Authors' contributions
SBH and CY set up and performed IF assays and contributed to drafting of the manuscript. RCM set up and performed cytotoxicity assays and contributed to manuscript writing. PS cloned and sequenced all KDM constructs and contributed to manuscript writing. VG performed cytotoxicity assays. AT performed in vitro screening of compounds and evaluation of data. GFR and RL performed chemical synthesis. VB designed chemical inhibitors used in the study and contributed to drafting the manuscript. OF contributed to in vitro assays and evaluation of data. BA designed and synthesized chemical inhibitors. FR evaluated cell permeability data. LAC performed IF assay. KT performed in vitro 2-OG-dependent assay. RB evaluated in vitro assay data and contributed to manuscript writing. SMW designed chemical synthesis and contributed to manuscript writing. JAB, RKP, EDM UO, CJS, AK, JEB, CB and PEB contributed to manuscript writing. OR contributed to study design and participated in coordination and manuscript writing. SM devised the study and wrote the manuscript. All authors read and approved the final manuscript.
We thank Stefan Knapp for carefully reading of the manuscript and Adam Donovan for the provision of Caco-2 cell permeability assay data.
Competing interests
The authors declare that they have no competing interests.
The research has received support from the Innovative Medicines Initiative Joint Undertaking under Grant Agreement No. 115766, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. Authors from the Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research are supported by Cancer Research UK. Grant C309/A11566.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Chapter 6: apprenticeship.
- The village of East Poultney -- Horace applies for the place -- scene in the garden -- he makes an impression -- a difficulty arises and is overcome -- he enters the office -- rite of Initiation -- Horace the Victor—his employer's recollections of him -- the pack of cards -- Horace begins to paragraph -- joins the Debating Society -- his manner of Debating -- Horace and the Dandy -- his noble conduct to his father -- his first glimpse of Saratoga -- his manners at the table -- becomes the town -- Encyclopedia -- the doctor's story -- recollections of one of his fellow apprentices -- Horace's favorite poets -- politics of the time -- the anti-mason excitement -- the Northern Spectator stops -- the apprentice is Free.
East Poultney is not, decidedly not, a place which a traveler if, by any extraordinary chance, a traveler should ever visit it would naturally suspect of a newspaper. But, in one of the most densely-populated parts of the city of New York, there is a field! —a veritable, indubitable field, with a cow in it, a rough wooden fence around it, and a small, low, wooden house in the middle of it, where an old gentleman lives, who lived there when all was rural around him, and who means to live there all his days, pasturing his cow and raising his potatoes on ground which he could sell—but won—at a considerable number of dollars per foot. The field in the metropolis we can account for. But that a newspaper should ever have been published at East Poultney, Rutland county, Vermont, seems, at the first view of it, inexplicable. Vermont, however, is a land of villages; and the business which is elsewhere done only in large towns is, in that State, divided among the villages in the country. Thus, the stranger is astonished at seeing among the few signboards of mere hamlets, one or two containing most unexpected and metropolitan announcements, such as, 'Silversmith,' 'organ factory,' 'Piano Fortes,' 'print-ing office,' or 'Patent melodeons.' East Poultney, for example, is little more than a hamlet, yet it once had a newspaper, and boasts a small factory of melodeons at this moment. A foreigner


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Located in the northeast corner of New London County, Voluntown is a rural community consisting of 39.8 square miles with two-thirds of the town made up of state forest. Pachaug State Forest offers many outdoor activities; such as hunting, camping, hiking and horseback riding. The town hall and library are located in the Village Center, along with the PreK-8 school, Constitution Field, the walking track, and gazebo. Voluntown was Incorporated in 1721 and has a rich history of textile mills, historic buildings, the Civilian Conservation Corps. (CCC Camp), folklore and was originally known as "Volunteer Town".
Free Electronics Recycling for Voluntown & Sterling Residents
First Selectman Robert A. Sirpenski would like to remind residents that they may, free of charge, bring their unwanted electronics to the Voluntown/Sterling Transfer Station located at 514 Brown Rd in Voluntown, CT. Hours are Wednesday through Saturday, from 7:00am-2:45pm. Reminder: May – September, Thursday hours change to 11:00am-6:45pm.
All electronic equipment is safely packaged by Take 2 and transported to their facility for responsible and secure recycling.
What is and isn't accepted?
Accepted materials include, but are not limited to: computers, laptops, fax machines, monitors, TVs, cell phones, tablets, video games, game consoles, VCR/DVD machines, remotes, keyboards, computer mice, modems, small appliances, and stereo equipment.
Unaccepted materials include: smoke detectors, ballasts, propane tanks, pain, household hazardous waste, and appliances containing freon- air conditioners, refrigerators, and dehumidifiers.
About Take 2 INC
Take 2 Inc is a Waterbury based electronics recycler. Take 2 is approved by the Connecticut Department of Energy and Environmental Protection as a Covered Electronics Recycler in conjunction with the Connecticut's Electronics Recycling Law, RCSA 22a-638-1.
Residents may also, free of charge, bring their unwanted electronics to Take 2's facility located at 567 South Leonard Street, Building One, Waterbury, CT. Hours are Monday through Friday, from 7:00am-3:30pm and Saturdays, from 8:00-noon. Additionally, residents may bring their electronics to any of Take 2 Inc's electronics recycling events. For a listing of upcoming events go to: www.ewaste-recycle-ct.com. For your convenience at events and at Take 2's facility, Take 2 employees will unload electronics from your vehicle for you.
For questions and more information
Email: firstname.lastname@example.com Phone: 1-800-209-9322 Website: Take2Recycle.com
Voluntown EDC Hosting SBA 101 Workshop, May 18
SBA 101 Workshop
Hosted by the Voluntown Economic Development Commission.
The U.S. Small Business Administration (SBA) is presenting a free SBA 101 workshop for existing and start-up businesses on Thursday, May 18, 2017 from 6:30 pm to 8:00 pm at the Voluntown Town Hall Meeting Room. Light refreshments will be served.
This free workshop is open to the public and will give an overview of the capital, counseling and contracting programs and services available from the SBA and its resource partners to help entrepreneurs start and grow a small business. Attendees of SBA 101 will learn more about:
- SBA loan programs
- CT Procurement Technical Assistance Program (CPTAP) – Government contracting
- Counseling and training services from SCORE.
Speakers include:
- Lisa Powell, Small Business Association (SBA) Economic Development Specialist
- Walter Shephard of the Southeastern CT SCORE Chapter
- Lisa Wood of the CT Procurement Technical Assistance Center (PTAP)
SPACE IS LIMITED! Please register by May 11.
To register or for more information, contact Lisa Powell at 860.240.4892 or firstname.lastname@example.com
Upcoming Road Closings Expected the First Week of May
The following roads will be closed at times during the first week of May, May 1st- May 6th, for paving:
Ten Rod Road
Tanglewood Lane
Lakeside Terrace
Valley Drive
Voluntown Youth Service Bureau Upcoming Events
The Voluntown Youth Service Bureau has many events coming up in April and May. For more info and updates visit the Voluntown Youth Service Bureau Facebook page and webpage.
Super 5K Run/Walk/Stroller Race Fundraiser
Saturday, May 13, 2017 at 9am at the Pachaug State Forest in Voluntown. ChipTiming by Platt Systems
Please fill out the registration form (under forms) and return it to Voluntown Elementary School, 195 Main Street, Voluntown, CT 06384. Registration fee is as follows:
Ages (0 – 18) $10 $15 w/shirt
Ages (19+) $25 $30 w/shirt
Please come join us for a fun-filled morning in the Pachaug State Forest!
Voluntown Volunteer Fire Department is Seeking Volunteers
A message from the Voluntown Volunteer Fire Department:
Volunteerism is on the fall. We are a town that was built on volunteerism! Our town residents can get involved. It is a commitment that is worthwhile in helping your fellow neighbors.
The Voluntown Volunteer Fire Department is made up of 40 members that are trained as either Fire Fighters, Emergency Medical Technicians or Fire Police. Our responsibilities range from controlling traffic during emergencies related to fire such as car fires and structure fires. We provide patient care for the sick, traumatic injuries, domestic violence, and falls. We conduct rescues in water, forest emergencies such as search and rescue, recreational vehicle accidents and hunting accidents. We also respond to hazmat emergencies.
We work in unison with advanced life support (Paramedics and LifeStar). We transport our patients to regional hospitals. Our department works closely with the Connecticut State Police (CSP), DEEP, and Eversource.
We provide all the training and gear necessary to provide volunteer support to our department!
If you are unable to commit to our department you can still volunteer to help at our fundraising events or support us by purchasing tickets to our fundraising dinners.
For more information, contact the Voluntown Volunteer Fire Department at 860-376-0475, or visit the firehouse Monday evenings 7-9.
Meals-On-Wheels Delivery Available to Voluntown Senior Residents
Serving persons sixty years and older and their spouses, Meals-on-Wheels delivery is available on a referral basis by the Visiting Nurse, Social Worker, Doctor office, Hospital, municipal agent, family, friends, and clients themselves. Meals are delivered to Voluntown residents Monday through Thursday with frozen meals provided for weekends. Meals provided are nutritionally balanced and within the guidelines of a no-added-salt and no-added-sugar diet. More info in the link below or contact Voluntown elderly municipal agent, Beth Taylor, at Town Hall (860) 376-4089
Voluntown Farmers' Market Volunteers Needed
The Voluntown Economic Development Commission is seeking volunteers to help recruit vendors and plan activities for the 2017 season of the Voluntown Farmers' Market. If you are interested in volunteering, or would like more information, please send an email to firstname.lastname@example.com, with your contact information.


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Galway Festivals
Galway Food Festival
Celebrating Galway as a good food destination, 2017 will be the 5th year of the Galway Food Festival. The festival will promote good food and good food outlets in Galway, as well as showcasing food producers based in Galway and in the West. The festival takes place in April primarily as it is the time of year when some of the best local ingredients become available, such as Connemara Spring Lamb, and trout and salmon caught locally.
Cuirt International Festival of Literature
April 2017
The Cúirt International Festival of Literature is Ireland's premier literary event. The festival annually brings approximately sixty authors of local, national and international repute to Galway for readings, workshops, discussions, book signings and much, much more.
Galway Film Fleadh
July 2017
Film festival for feature films and shorts. 2017 marks the 29th Galway Film Fleadh, and the central goal of the festival remains unchanged: to bring together audiences and filmmakers within an intimate environment and share a common experience.
Galway Fringe Festival
July 2017
Running in parallel with the Galway International Arts Festival, the Galway Fringe Festival aims to provide a platform for local artists, writers, playwrights, poets, actors and musicians to perform and showcase new work.
Galway International Arts Festival
17th - 30th July 2017
One of Europe's most inventive, vibrant & colorful events. A truly international celebration of the performing and visual arts which takes place in and around Galway City each July.


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DALLAS (AP) – Although U.S. Sen. Ted Cruz isn't a candidate this election season, he remains on the minds of Republican primary voters deciding the highly charged race for lieutenant governor.
Susan Cartwright cast her ballot Tuesday in Aledo, west of Fort Worth, for state Sen. Dan Patrick, saying if he's elected to replace incumbent David Dewhurst then she expects Patrick to follow in Cruz's footsteps. She believes constitutional rights are being "whittled away" and firebrands like Cruz and Patrick are needed to maintain them.
Tim Shell of The Woodlands describes himself as a "Cruz guy" who voted for Patrick because it's time for "patriots" to sweep out ineffective political leaders.
But Charles Jacobus of Houston says although he supports Cruz, he voted for Dewhurst because he believes Patrick sounds "like a mad old man."
(© Copyright 2014 The Associated Press. All Rights Reserved. This material may not be published, broadcast, rewritten or redistributed.)
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text:
+ Today's opening hours
- Ashburton 10am-5pm
- Balwyn 10am-5pm
- Camberwell 10am-5pm
- Hawthorn 10am-5pm
- Kew 10am-5pm
+ Search
Australian Heritage Festival 18 April - 21 May 2017- Having a voice
Join us for a series of events brought to you by Boroondara Library Service.
Free events, bookings essential.
Events in Australian Heritage Festival 2017
6:30pm to 8:00pmThursday 11 May 2017
at Camberwell Library
Remember the family slide night? Join us for a fun 'slide night' with pizza. We'll have a 35mm projector and selected slides from the Boroondara collection, and beyond. Dig out some of your own family slides and bring them along to share.


text:
Independence, OH State Parks
YP - The Real Yellow PagesSM - helps you find the right local businesses to meet your specific needs. Search results are sorted by a combination of factors to give you a set of choices in response to your search criteria. These factors are similar to those you might use to determine which business to select from a local Yellow Pages directory, including proximity to where you are searching, expertise in the specific services or products you need, and comprehensive business information to help evaluate a business's suitability for you. "Preferred" listings, or those with featured website buttons, indicate YP advertisers who directly provide information about their businesses to help consumers make more informed buying decisions. YP advertisers receive higher placement in the default ordering of search results and may appear in sponsored listings on the top, side, or bottom of the search results page.
1. City of Independence
6363 Selig BlvdIndependence, OH 44131
2. Independence Elmwood Park Ofc
6200 Elmwood AveIndependence, OH 44131
3. Cleveland Metroparks
100 E Pleasant Valley RdIndependence, OH 44131
4. HQ Global Workplaces
6100 Oak Tree BlvdIndependence, OH 44131
5. Metro Parks
7301 Dunham RdBedford, OH 44146
6. Cuyahoga Valley National Park - Canal Visitor Center
7104 Canal RdCleveland, OH 44125
7. Dan Kostel Recreation Ctr
5411 Turney RdCleveland, OH 44125
8. Cleveland Metroparks
1969 W Ridgewood DrCleveland, OH 44134
9. National Park Svc
1510 Vaughn RdBrecksville, OH 44141
10. Brecksville Purchasing Dept
1 Community DrBrecksville, OH 44141
11. National Park Service Contracting
1113 W Aurora RdNorthfield, OH 44067
12. Us Government
9329 Riverview RdBrecksville, OH 44141
13. Broadview Heights Recreation Department
8938 Broadview RdBroadview Heights, OH 44147
14. Maple Heights Recreation Dept
15901 Libby RdMaple Heights, OH 44137
15. Coventry Village Special Improvement District
Cleveland, OH 44102
16. Cleveland Botanical Gardens
Cleveland, OH 44106
From Business: Founded in 1930, Cleveland Botanical Garden, formerly known as the Garden Center of Greater Cleveland, is one of the leading urban horticulture and botanical educ…
17. Memphis Kiddie Park
I was born and raised in Cleveland. I now live in Las Vegas. But when I was a kid my parents took me there every year. I always had lots of fun with…
18. Cuyahoga Valley National Park
Over Labor day weekend had the pleasure of taking the Cuyahoga Valley Scenic Railroad in thru the valley it was fun. We went to and from Hale Farm …
19. Cleveland Metroparks
4101 Fulton PkwyCleveland, OH 44144
From Business: Hop on a bicycle, lace up your running shoes or take your time strolling through any part of this scenic 60-mile long string of parks and garden areas.
20. Twinsburg Parks Dept
10260 Ravenna RdTwinsburg, OH 44087
From Business: The Twinsburg Parks and Recreation Department offers a variety of recreational amenities and programs. It maintains a range of facilities that includes parks, fit…
21. Thomas Nenadovich
4920 E 85th StCleveland, OH 44125
22. Cleveland Metropark Mntnc Ctr
11410 Broadway AveCleveland, OH 44125
23. Institute Of Great Outdoors
11350 Broadway AveCleveland, OH 44125
24. Veterans Memorial Park
6328 State RdCleveland, OH 44134
25. Ohio & Erie Canal Park Mntnc
4704 E 49th StCleveland, OH 44125
26. Parma Parks & Recreation Dept
7335 Ridge RdCleveland, OH 44129
27. Parma Parks & Recreation Dept
5983 W 54th StCleveland, OH 44129
28. 49th Annex Park Station
4150 E 49th StNewburgh Heights, OH 44105
29. Parma Parks & Recreation Dept
7912 Day DrCleveland, OH 44129
30. Cleveland Johnston Parkway Pk
4071 Johnston PkwyCleveland, OH 44128


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The recanvass for Kentucky's Republican nominees for governor and agriculture commissioner in the county is complete and results are the same as the May 19 figures, which came as no surprise to Hardin County Clerk Debbie Donnelly.
As part of the Hawkins steel basin stream improvements, motorists are encouraged to choose alternate routes beginning Monday to avoid the intersections of Hodgenville Road at Hawkins Drive and Par Lane.
A worker fell Wednesday afternoon from a water tower in Vine Grove, suffering "significant" and "life-threatening" injuries, a Hardin County EMS official said.
With a little more than a week before his murder trial, a judge will address a defense motion today to move the trial of Michael Todd Hilton.
A two-part collision involving six tractor-trailers shut down the southbound lanes of Interstate 65 near Radcliff for seven hours Wednesday afternoon and evening and sent two to the hospital.
Associated Railroad will be closing Ky. 84 at the P&L Crossing near Eastview for reconstruction beginning at 7 a.m. Monday.
Electronic message boards have been in place for several days to alert motorists of the upcoming closure, according to a Kentucky Transportation Cabinet news release. A signed detour will direct traffic around the closure.
As seniors around Hardin County head into their final day of high school, many may have participated in a longstanding local tradition Wednesday night: senior sign.
Some would say the annual party is a rite of passage for many and at some of the parties, alcohol is consumed.
The Hardin County Animal Shelter soon will microchip all adopted dogs.
According to Hardin County animal control director Jerry Foley, the new program, approved Tuesday by Hardin Fiscal Court, is a "win-win" situation for the county and dog owners.
Radcliff Mayor Mike Weaver said his search for money to hire an events coordinator is over despite amending and resubmitting a request for $25,000 from the Radcliff/Fort Knox Convention & Tourism Commission.
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Browse Article by Topicsemail email templates conditional emails translate emails translate email autoresponder email language
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How to Change Email Template Sent Based on the Language Used?Last Update: April 9, 2017
To set this all up you will first need to set up two things:
1. Set up all the languages that you want to have on your jotform
2. Set up email autoresponders for each language
Once you have these two all set up and ready it is time to use conditional logic and set the email templates to match the selected forms language. To do this, all we need to do is following.
Click SETTINGS at the top > CONDITIONS on the left > then CHANGE EMAIL RECIPIENT.
Then setup your Condition like so:
If you're still using the previous Form Builder (V3), refer to the steps and screenshots below:
Go to Conditions Wizard by clicking on Conditions option in the toolbar.
Once you do, select the Change Email Recipient option
And configure your conditional logic like this:
As you can see on the screenshot above we have selected Language to match its content and check what language is selected on the form. Following that, we have the action set to send US English (name of) autoresponder template to the email entered in the email field.
As soon as you select the email autoresponder template the email to field is filled out automatically. If you have multiple email fields, then it is best to double check if what is the name / tag of the email field and enter it manually. You can check this by following this guide: How to find Field IDs & Names.
We repeat the same for all languages that we will use and that is it.
The best thing now is to go directly to your jotform and submit it like that, testing it few times by changing the languages. This will allow you to quickly and simply see if there are any issues with your conditions setup.
You can see a demo form used when creating this guide here: http://form.jotformpro.com/form/51364668890972
If you wish to apply the same steps to your thank you message as well, we would suggest taking a look at this guide: How to change thank you message depending on the language used?.
If you have any problems or questions in regards to this, do let us know in the comments below and we would be happy to answer them for you.
1 Comment...
|(May 25, 2016 at 10:45 AM)


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We will be visiting Baltimore (inner harbor area and Fells Point) this month on a Saturday (day trip). We will be coming from the north and are hoping to park and ride at a suburban Baltimore area Light Rail Station. I was wondering what station is convenient to I-83, has plenty of parking, and what the cost to park on Saturdays in the rail station lot may be, Or would it be best to drive into the city and possibly pay high parking rates?
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Baltimore Light Rail
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Even if you are not coming to an Orioles game, this website will give you the options for downtown Baltimore.
I am not aware of any light rail.
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