Wednesday, March 30, 2016

Swiss to recognise homeopathy as legitimate medicine

The interior ministry has announced its intention to elevate five complementary therapies including homeopathy to the same level as conventional medicine. 



Homeopathy, holistic medicine, herbal medicine, acupuncture and traditional Chinese medicine will acquire the same status as conventional medicine by May 2017.
After being rejected in 2005 by the authorities for lack of scientific proof of their efficacy, complementary and alternative medicines made a comeback in 2009 when two-thirds of Swiss backed their inclusion on the constitutional list of paid health services.
As a result of the vote, these treatments are covered by basic compulsory insurance as part of six-year trial period from 2012-2017, during which their efficacy would be examined.
The ministry has come to the conclusion that it is impossible to verify the efficacy of these therapies in their entirety. It has therefore opted to accept them on par with other medical disciplines. It plans to continue allowing reimbursements of treatment costs by compulsory health insurance, provided they are administered by certified medical professionals. 
However, as is the practice for conventional medicine, certain controversial practices under these complementary therapies will be subjected to further scrutiny. The ministry has initiated a consultation process - open until June 30, 2016 - on the proposed modification of the regulations.
swissinfo.ch

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Please feel free to contact if need a homeopathic treatment for you or your family.
Hussain Kaisrani is a Homeopathic Physician at Bahria Homeopahic Clinic Safari Villas, Bahria Town Lahore Pakistan and could be contacted through email or phone for an appointment.
kaisrani@gmail.com ; 03002000210

Monday, March 28, 2016

Homeopathic Treatment of Hay Fever and Pollen Allergy related problems

Last week, I presented here a case of homeopathic treatment of hoarseness which went very successful. The lady suffering from hoarseness persuaded her husband (Mr GA) to consider homeopathy for his issues like Hay Fever / Pollen Allergy.


Mr GA - 48 years age - is a professional accountant in East London. Before moving to UK, he worked on high positions in health department of Punjab Government so has vast relations with senior doctors. Tried his level best to find out a proper solution of his problem. The final advise from each consultant was to take anti-allergy pills as per requirement. After discussing the matter with many consultants he concluded that there is no other solution of his problem but to use anti-allergy drugs.

The weather condition in the UK was compelling him to increase the intake of anti allergy tablets. Further more, he was facing side effects like dizziness and dullness. He was also aware that this is only a management of problem not the treatment. Quality of life was seriously disturbed as there was irritation, disturbance and bad mood along with constant sneezing, bland watery discharge from both the nose and eyes. High aversion to noise and conversation was witnessed. There was no significant temperature noticed when his case was taken.

After taking the anti allergy tablet, he used to get relief from sneezing and watery discharge. The feeling of irritation, bad mood and dullness was not being managed even after taking anti allergy.

On March 23, 2016 at 7PM, Mr GA visited me for consultation. It was his second day in Lahore when he developed continuous sneezing and watery discharge from nose and eyes. His feelings were slightly better when in open air. He was bit reluctant to take homeopathic medicine as he thought that it will take too much time to work IF it really works.

After taking complete history, I short listed two remedies -- Allium Cepa and Natrum Muriaticum -which seemed best suitable considering the totality of Mr GA.

Though Natrum Muriaticum is not one of the main remedy for Hey Fever / Pollen Allergy type problems yet I preferred it on Allium Cepa for following reasons:
  1. It was covering the main symptoms of patient. Hay Fever or Pollen Allergy related symptoms are not always the same for every individual.
  2. It was the simplest selection. Biochemic remedies are always my first choice 
  3. Biochemic remedies work amazingly nice if indicated. 
  4. Frequent repetition is normal for all biochemic remedies including Natrum Muriaticum
  5. It is not a deep acting medicine if used in X
  6. Finally, I decided to treat the patient constitutionally
Single dose of Natrum Muriaticum 6X was provided to the patient at 7PM. It was a great surprise for him that a soothing affect started within minutes. He was provided 6 more doses to take only if problem remains there or starts again.

After an hour, he informed that his problem is fully cured. The feeling of wellness is simply amazing. He was advised not to take any further dose.

He had some commitments in Islamabad and was sure that problem was start there again. 

On March 28, 2016 (late night) he responded to my message as following:
Sir everything was 100 percent under control I did not take other tablets



His next appointment is due on April 06, 2016 before his departure to UK. He already has few tablets with him. It will be updated if any further change is noticed. Quite possibly, he will need Natrum Muriaticum or some Nosode (Psorinum) in high potency for complete and long term cure if he remained connected with homeopathy. But it will totally depend upon the symptoms and changes witnessed in his system.


UPDATE and Follow Up (April 06, 2016)
Mr GA updated that his problem is fully controlled which is still unbelievable for him. He traveled to Islamabad area and then to villages of Layyah side. He witnessed all the weather and climate changes yet he did not felt any problem. Due to too much change in diet and intake, he developed stomach problem for a day or so.
He still has few doses of Natrum Muriaticum 6x as he only taken one dose. He is also provided one dose of Psorinum 200 to keep with him. If he informs about any further change in his health after reaching UK, will be shared here. Otherwise, his problem will be considered cured. 
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This is how a properly selected Homeopathic remedy works for acute problems - A speedy and gentle cure without any side affects. 
Please feel free to contact if need a homeopathic treatment for you or your family.
Hussain Kaisrani is a Homeopathic Physician at Bahria Homeopathic Clinic Safari Villa, Bahria Town Lahore and could be contacted through email or phone for an appointment.
kaisrani@gmail.com ; 03002000210

Monday, March 21, 2016

A homeopathic treatment of Hoarseness - My latest case

A lady - age 35 years - from London (UK) developed a problem of hoarseness آواز یا گلا بیٹھنا soon after reaching Pakistan. She is already a patient of diabetes since 5 years along with tonsil problem which is being managed by gargling. The recent issue of hoarseness was severe. Due to it, she was having problem in socializing for which she came to Pakistan.


On March 19, 2016 she visited me and all the detail of her case was taken. No other issue directly related to hoarseness was found. She was taking good amount of conventional medicines prescribed and provided by NHS, EnglandThere were no symptoms of cold, flu or inflammation found. She was neither a speaker nor too much talkative.  No cold water or item was taken which would have caused this. The physical condition of throat was also quite normal.

There were more than 20 remedies suggested in the books under acute hoarseness. Rai Bahadur Bishambar Das has given a list of 36 remedies in his book Select Your Remedy

I prescribed Causticum though it was not clearly indicated in the case except that it works great if some problem seems only localised. It also has an impact on local paralysis, vocal cords and muscles of tongue. (Homeopathic Materia Medica, William Boericke, M.D.)

Soon after taking first dose (when started sharing her other health issues), she surprisingly said to her husband that she feels a significant betterment. It was just unbelievable for her though not for me. She was given two more doses with a suggestion to take only if needed.

March 20 night -- she updated that she had to take both doses and now feeling almost fine but if one more dose is provided, her problem will be fully cured. She was advised to wait one more day and see the results.

March 21 afternoon -- she wanted to have a dose (which was provided and suggested to take only if needed) though she was feeling almost fine.

This is how a properly selected Homeopathic remedy works for acute problems - A speedy and gentle cure without any side affects. 

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This is how a properly selected Homeopathic remedy addresses the health problems - A speedy and gentle cure without any side affects. 
Please feel free to contact if need a homeopathic treatment for you or your family.
Hussain Kaisrani is a Homeopathic Physician at Bahria Homeopathic Clinic Safari Villa, Bahria Town Lahore and could be contacted through email or phone for an appointment.
kaisrani@gmail.com ; 03002000210

Saturday, March 19, 2016

‪‎Homeopathic Tissue Salts Usages

Have you heard about ‪‎Homeopathic Tissue Salts‬?



They are potentised micro-doses of the 12 essential minerals your body needs to repair and maintain itself. They are prepared in homeopathic 6X and 12x potencies.

Calcium fluoride
Loss of elasticity, varicose veins, hemorrhoids, deficient tooth enamel, hard or cracked skin

Calcium phosphate
Poor digestion, anemia, bone diseases

Calcium sulphate
Spots, pimples, skin disorders and slow healing wounds

Ferrum phosphate
Inflammation, redness, throbbing, first stage of respiratory complaints, inflammations, injuries

Potassium chloride
Mucous congestion, glandular swellings, coughs, tonsillitis, ulcerated sore throat, digestive disturbances, swollen joints

Potassium phosphate
Nervous tension, irritability, tension headaches, depression, lethargy, forgetfulness

Potassium sulphate
Dry scaly skin, dandruff, skin eruptions, inflammations with yellow discharges, shifting pains

Magnesium phosphate
Muscle cramps, spasms & twitches, spasmodic pain or colic, migraine

Sodium chloride (Natrum muritecum)
Excessive dryness or edema of tissues, runny nose, loss of smell or taste, sadness

Sodium phosphate (Nat phos)
Acidity, indigestion or heartburn, rheumatism

Sodium sulphate (Nat sulph)
Biliousness, liver upsets and fluid retention, constipation, leg ulcers

Silicea
Hair, skin and nail complaints – pimples, boils, ulcers, styes, excessive perspiration

Courtesy: Homeopathic Care

==============
Please feel free to contact if need a homeopathic treatment for you or your family.
Hussain Kaisrani is a Homeopathic Physician at Bahria Homeopahic Clinic Safari Villas, Bahria Town Lahore Pakistan and could be contacted through email or phone for an appointment.
kaisrani@gmail.com ; 03002000210

Thursday, March 17, 2016

The Masters advice for Successful results in Homeopathic Cases

It is always a good to be reminded of the truth in homeopathy. Many masters have given us real pearls of wisdom and truth. Hold these close and you will have many more successful cases.

  • We have no long acting drugs; the action is immediate, continued favorable condition depends on the quality of the vital force and it’s harmonious action. (H.A. Roberts)
  • If the symptoms for which a remedy is given are removed and a new symptom appears, with hold the hand if you wish the case to go on to recovery. (Lippe)
  • Susceptibility exists in the vital force, not in the tissues. (J.T.Kent)
  • Ultimately the constitutional peculiarity is bound to reveal itself in a form pointing to it’s remedial counterpart. Nature calls for relief in her own language which it behoves us to learn properly. It is contained in the symptom picture but many times we are forced to look for it elsewhere. (C.M. Boger)
  • On uterine Contractions and Pulsatilla – It will often cause in five minutes a very strong contraction of the uterus, sometimes almost in a painless way. (J.T.Kent)
  • Never leave a remedy until you have tested it in a higher potency if it has benefited the patient. (J.T.Kent)
  • Do not dip into a chronic state when dealing with an acute condition, and vise versa. (H.A. Roberts)
  • Why prescribe for a part of the patient when you have the whole patient with you? The patient was sick before the glands were. (Hayes)
  • Some have been confused by primary and secondary effects of medicine. You need not worry over this. You only need to know that certain symptoms follow each other. Primary and secondary action reverse themselves in different individuals. (J.T.Kent)
  • The constitutional remedy is found by a series of symptoms absolutely new to that patient. (C.T.Boger)
  • There is no better evidence of the good action of remedy than mental improvement. (J.T.Kent)
  • Keep a symptom, don’t follow a remedy. (H.A. Roberts)
  • In a cure, the discharge may not come back at the original place but from some other mucous membrane. (G. Miller)
  • The bond between two miasms can be broken only by a prescription that will meet the totality of the most active one. (J.H. Allen)
  • All maladies which show skin eruptions are always present internally before showing local symptoms externally. (S. Hahnemann)
  • Don’t leave your inter-current remedy too soon, it may be the curative remedy. (F.E. Gladwin)
  • Look for the picture of the chronic following recovery from an acute condition. (H.A. Roberts)
  • If we could accept opinion we should have to go back to Allopathy, because we find there only a record of man’s experiments; a mass of heterogeneous opinions. (J.T. Kent)
  • Man must be studied as he is, as he was, everything of man and of the human race in general, in order to understand disease. (J. T. Kent)
  • Minutes or hours in acute; days, weeks, or months in chronic diseases, never repeat while amelioration holds. (M.L. Tyler)
  • The principle of Homeopathy is applicable to any range of potency. (C.M. Boger)
  • Hahnemann’s central idea is fundamental that the further an outstanding symptom seems removed from the ordinary course of disease, the greater is that symptoms value in determining the remedy. (C.M. Boger)
  • If you love homeopathy, it will love you. Such is natural charity. (J.T. )
  • It is impossible to learn homeopathy except from a master. (G. Miller)
  • The homeopathic principles, when known, are plain, simple, and easily comprehended. They are in harmony with all things known to be true. (J.T. Kent)
  • When you make failures you may be sure that they are within yourself. If you think the failure is in homeopathy you will begin your corrections on the wrong side of the ledger. (J.T. Kent)

Tuesday, March 15, 2016

The Importance of Peculiar Symptoms in Homeopathic Case Taking (Dr. Banaras Khan Notes)


ایسی علامات جنہیں ایک ایلوپیتھ درخورِ اِعتنا نہیں سمجھتا لیکن ایک ہومیوپیتھ اِنہیں قدر کی نگاہ سے دیکھتا ہے۔ مثلاً اسہال میں مبتلامریض کا بیان، مجھے شدید پیاس لگتی ہے اور جوں ہی پانی پیتا ہوں کپکپاہٹ طاری ہو جاتی ہے اور ساتھ ہی واش روم جانے کی حاجت ہوتی ہے، ایک ایلوپیتھ کے نزدیک چنداں اہمیت کا حامل نہیں۔
(کیپسیکم)

گردے فیل مریض ۔۔ جو عموماً اختلاجِ قلب، تنگئ تنفس اور استقائی سوجن کا شکار ہوتا ہے ۔۔ اگر یہ کہے کہ میرا چکناہٹ کھانے کو جی کرتا ہے، پیشاب ٹھنڈا اور تیز بُو والا آتا ہے تو مریض کا یہ بیان شاید ایلوپیتھ کے علم و تشخیص میں کوئی اضافہ نہ کر سکے لیکن ہومیوپیتھ کے لیے گراں قدر اہمیت کا حامل ہوتا ہے۔
(نائٹرک ایسڈ)
دمہ میں مبتلا مریض جب ایلوپیتھک ڈاکٹر کو یہ بتا کر اُس کی معلومات میں اضافہ کرتا ہے کہ اگر میں سجدے کی حالت میں چلا جاؤں تو سانس لینے میں آسانی ہوتی ہے تو وہ بےاختیار مسکرا اٹھتا ہے لیکن ایک ہومیوپیتھ ایسی علامت پا کرمطمئن ہو جاتا ہے کہ درست سمت میں رہنمائی ہو گئی۔ (میڈورینم)۔

کینتھرس کے مریض کا بیان کہ سرِذَکر کو دبانے سے درد گردہ کم ہو جاتا ہے ایلوپیتھ کے نزدیک بے سروپا ہو سکتاہے لیکن ایک ہومیوپیتھ اسے موتی سمجھ کر چُن لیتا ہے۔ایسی علامات اُس کے نزدیک نشانِ راہ ہیں جو منزل کا پتا دیتی ہیں۔



Monday, March 14, 2016

Observing the patient after prescribing the homeopathic Remedy


After prescribing the remedy, the physician shall very carefully observe his patient in every respect;
  • his general condition,
  • aggravations and amelioration of symptoms,
  • any change in the symptoms or in the patient himself, etc.



The Observations
  1. After giving the medicine to the patient if the physician's observation is a prolonged aggravation and final decline of the patient, the case is incurable.
  2. The second observation is, the long aggravation, but final and slow improvement. It is a hopeful case with prolonged repetition of the same remedy at longer intervals. For many years you may go along with prolonged aggravations followed by amelioration untill an outward manifestation appears whereby the patient will attain final recovery.
  3. The third observation after administering the remedy is, quick aggravation, short and strong with rapid improvement of the patient. Whenever you find an aggravation comes quickly, is short, and has been more or less vigorous, then you will find improvement of the patient will be long, the structural changes many appear in the surface organ, and with the passage of time the surface organs regain their natural structure after they have dragged out all that was destroying internal organs.
  4. The fourth observation relates to cases in which we have no aggravation with recovery patient. The remedy administered was correct according to the symptoms, so was the potency suitable to the vitality of the patient. In such cases there is no organic disease, and no tendency to organic disease. It is simple functional disorder of the nerves.
  5. The fifth observation is, the amelioration comes first and the aggravation comes afterwards. This condition is unfavourable. Either the remedy was only a superficial remedy, and could only act as a palliative, or the patient was incurable and the remedy was somewhat suitable. The physician should re-examine the patient and find out whether the symptoms relate to that remedy. If the remedy is found to be correct then the case is incurable. If the remedy selected was not correct, the Physician will take up the case again, and very carefully select the remedy according to the symptoms.
  6. The sixth observation is too short relief of symptoms. It is because of some conditions that interferes with the action of the remedy. The physician has to find out that condition - whether the patient is causing that condition or there is some deep rooted destructive process going on in some vital organ. It is not difficult to find out the first cause and remove it by cautioning the patient, but it is quite difficult to arrive at the depth of the second cause; in spite of the difficulty it is the duty of the physician to know that cause and remove it.
  7. A full time amelioration of the the symptoms, yet no special relief of the patient is the seventh observation. Such patients have some permanent deformity or an organic bson that prevents improvement beyond a certain stage. A patient with one kidney can only improve to a certain degree; patients with fibrous structural change in certain places, tubercles, that have become encysted and lungs capable of doing limited work, will be ameliorated from time to time with remedies, but the patient is only curable to certain extent, he/she can never be cured. The patient is palliated in this instance.
  8. The eighth observation is, some patients prove every remedy they get; patients inclined to be hysterical, overweight, oversensitive, to all things. The patient is said to have an idiosyncrasy to everything, and these oversensitive patients are often incurable. They are born with this sensitivity and the will die with it.
  9. The ninth observation relates to new symptoms appear after the remedy. If a great number of new symptoms appear after the administration of a remedy, the prescription will generally prove an unfavourable one. After these new symptoms have passed away, the patient will settle down to he original state and no improvement take place, the case is incurable.
  10. The tenth observation is when old symptoms are observed to reappear. After the aggravation has come and symptoms, we observe, disappearing in the reverse order of their coming, the disease is curable. The medicine must be let alone. If the old symptoms come back to stay, the repetition of the dose is often necessary.

Saturday, March 12, 2016

Homeopathic Treatment of Toothache and Oral Health Issues - A Case

A patient called to discuss her problem yesterday (March 11, 2016) at around 1 PM. She was having pain in teeth, jaw and gums. There was swelling in the mouth and also redness. An excessive saliva was also another problem. She was not clear whether pain is actually in the teeth, gums or throat. Before contacting me she went to see a dentist who prescribed her an antibiotic course for five days and advised for wisdom teeth extraction once swelling and pain gets controlled.


The patient was not ready for extraction of tooth so instead of taking antibiotic she preferred to contact Bahria Homeopathic Clinic for consultation.

After getting symptoms on phone (which were not clear though), she was advised to take Belladonna 30. 

At 4PM, she informed that redness seems less but pain is not controlled. Second dose of Belladonna 30 was suggested to take which lessened the redness but the pain and overall condition did not get better.

At 7pm she was able to understand that pain is not in the teeth or jaw but it is in the gums and throat area. It was hurting a lot when she touched the area. Swelling along with disturbing pain was still there. After a thorough case taking Mercurius Solubilis was found indicated. A dose in 30 potency was given around 7 pm. After 2 hours or so, the patient informed that her pain increased a lot and it has moved to the throat area. Tongue and mouth area had sore feelings as well. 

It was a clear aggravation as system started reacting properly. She was advised to wait.

Till March 12, 2016 morning her problem got better upto 30%. At 2PM she reported that she is 60% better but no further improvement is being felt.

At 6PM, second dose of Mercurius Solubilis was given which helped her to become almost normal - Pain fully controlled, No more redness, Overall feeling is fine.
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This is how a properly selected Homeopathic remedy works for acute problems - A speedy and gentle cure without any side affects. 

Please feel free to contact if need a homeopathic treatment for you or your family.

Hussain Kaisrani is a Homeopathic Physician at Bahria Homeopahic Clinic and could be contacted through email or phone for an appointment.
kaisrani@gmail.com ; 03002000210

Pharmacoeconomic comparison between homeopathic and antibiotic treatment strategies in recurrent acute rhinopharyngitis in children.



Trichard M1, Chaufferin G, Nicoloyannis N.
Abstract

OBJECTIVES:
A pharmacoeconomic study to compare, in terms of: medical effectiveness, quality of life and costs two treatment strategies (‘homeopathic strategy’ vs ‘antibiotic strategy’) used in routine medical practice by allopathic and homeopathic GPs in the treatment of recurrent acute rhinopharyngitis in 18-month to 4-year-old children.



METHODS:
Statistical analysis of data obtained from a population of 499 patients included in a previous 6-month prospective, pragmatic study. The patients were regrouped according to type of drug prescribed. Medical effectiveness was assessed in terms of (i) episodes of acute rhinopharyngitis, (ii) complications, (iii) adverse effects. Quality of life was assessed using the Par-Ent-Qol scale. Direct medical costs (medical consultations, drug prescriptions, prescriptions for further tests) and indirect medical costs (sick-leave) were evaluated from three viewpoints (society, patient, Social Security) using public prices and French Social Security tariffs.
RESULTS:
The ‘homeopathic strategy’ yielded significantly better results than the ‘antibiotic strategy’ in terms of medical effectiveness (number of episodes of rhinopharyngitis: 2.71 vs 3.97, P<0 .001="" 1.25="" 1.95="" 21.38="" 30.43="" 31.6="" 99="" and="" by="" complications:="" costs="" covered="" direct="" euros="" global="" less="" life="" lower="" medical="" number="" of="" p="" parents="" quality="" score:="" security="" sick-leave="" significantly="" social="" span="" vs="" with="">
CONCLUSIONS:
Homeopathy may be a cost-effective alternative to antibiotics in the treatment of recurrent infantile rhinopharyngitis.

Courtesy: http://www.ncbi.nlm.nih.gov/pubmed/15751328

Thursday, March 10, 2016

Reversing Disease with Homeopathy at the onset of the disease



Homeopathy has been of tremendous value in reversing diseases such as diabetes, arthritis, bronchial asthma, epilepsy, skin eruptions, allergic conditions, mental or emotional disorders, especially if applied at the onset of the disease.


(Prof. George Vithoulkas)

https://www.facebook.com/bahria.homeopathy/

Tuesday, March 8, 2016

Highly dilute homeopathic remedies modify gene expression in cervical cancer cells



Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro
1.Santu Kumar Saha (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India )
2.Sourav Roy (Department of Entomology and Institute for Integrative Genome Biology, University of California Riverside, CA 92521, USA )
3.Anisur Rahman Khuda-Bukhsh (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India )
ABSTRACT Objective: Methylation-specific epigenetic process and gene expression profiles of HeLa cells treated with ultra-high dilutions (HDs) of two plant extracts, Hydrastis canadensis (HC-30) and Marsdenia condurango (Condu-30), diluted 1060 times, were analyzed against placebo 30C (Pl-30) for alterations in gene profiles linked to epigenetic modifications. Methods: Separate groups of cells were subjected to treatment of Condu-30, HC-30, and Pl-30 prepared by serial dilutions and succussions. Global microarray data recorded on Affymetrix platform, using 25-mer probes were provided by iLifeDiscoveries, India. Slides were scanned with 3000 7G microarray scanner and raw data sets were extracted from Cel (raw intensity) files. Analyses of global microarray data profile, differential gene expression, fold change and clusters were made using GeneSpring GX12.5 software and standard normalization procedure. Before microarray study, concentration of RNA (ng/μL), RIN value and rRNA ratio for all the samples were analysed by Agilant Bioanalyzer 2100. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR were done for analyzing SMAD-4 expression. Fluorescence-activated cell sorting study was further made to elucidate fate of cells at divisional stages. Methylation-specific restriction enzyme assay was conducted for ascertaining methylation status of DNA at specific sites. Results: HDs of HC-30 and Condu-30 differentially altered methylation in specific regions of DNA and expression profiles of certain genes linked to carcinogenesis, as compared to Pl-30. Two separate cut sites were found in genomic DNA of untreated and placebo-treated HeLa cells when digested with McrBC, compared to a single cut observed in Condu-30-treated genomic DNA. SMAD-4 gene expression validated the expression pattern observed in microarray profile. Methylation-specific restriction enzyme assay elucidated differential epigenetic modifications in drug-treated and control cells. Conclusion: HDs triggered epigenetic modifications and alterations in microarray gene expression profiles of many genes associated with carcinogenesis in HeLa cells in vitro.
http://dx.doi.org/10.1016/S2095-4964(15)60201-1 Received February 17, 2015; accepted July 9, 2015. Correspondence: Prof. Anisur Rahman Khuda-Bukhsh; Tel: +91-33-25828750 extn. 315; E-mail: prof_arkb@yahoo.co.in, khudabukhsh_48@rediffmail.com
  
1 Introduction 

In the development of pharmacological drugs, some of the most common barriers include poor pharmacokinetics, insufficient therapeutic activity, drug toxicity, and poor bioavailability[1]. Given these issues, complementary and alternative medicines (CAMs), which often use plant extracts and other natural products that are relatively less cytotoxic, have gained increasing popularity. Various scientific methods are now being applied to learn about their efficacy, proper dosage, and mechanisms of action. Homeopathy, a popular branch of CAM, uses both crude and ultra-highly diluted (potentized) forms of remedies. Homeopathy is often criticized for its failure to explain the mechanism of action of the ultra-high dilutions (HDs) and therefore needs closer examination with a modern scientific approach[2]. An exhaustive survey in Britain suggests that 70% of all oncology departments employ at least one form of CAM treatment in cancer care[3]. More recently, the National Center for Complementary and Alternative Medicine (NCCAM), under the National Institutes of Health (NIH), USA, approved homeopathy as an alternative medicinal approach under the regulation of Food, Drug and Cosmetic Act (FDCA)[4]. However, the dearth of information on homeopathy’s scientific mechanisms of action makes its acceptance rather difficult. In recent years, the documentation of the existence of nano-particles[5,6] of the original drug substance(s) in some highly diluted remedies used in homeopathy has given a new impetus for conducting more rigorous research utilizing state-of-the-art techniques[7—9].
Other studies have used in vitro and in vivo conditions in models like Escherichia coli, yeast, bacteriophage and plant-based experimental models to understand exact molecular mechanisms of highly diluted homeopathic remedies[10—16]. Human tumor-derived cell line models used in cancer therapeutics programs are effective for the evaluation of potential new therapeutic agents. These cell line-based models are integrated with efforts to identify biomarkers of cancer progression and mechanisms of drug action[17]
Human cervical cancer cell line HeLa belongs to HPV-18-positive cell line, which carries approximately 10–50 viral integrated copies[18]. This is an ideal model to study genetic and epigenetic modifications by therapeutic agents. The epigenetic phenomenon is defined as a heritable reversible phenotype, resulting from changes in a chromosome without alteration in the DNA sequence. DNA methylation, histone modifications, nucleosome positioning, micro-RNAs and non-coding RNAs are the hallmark traits of epigenetics[8,9,19].
Previously, the notable hypotheses put forth to explain the efficacy of homeopathic remedies include molecular imprints or memory of water, similia principle, hormesis, nano-silicon principle, electromagnetic transfer through DNA wave principle, thermo luminiscence, and gene regulatory and epigenetic hypotheses[9,11,20—26]. Of these, the gene regulatory hypothesis of Khuda-Bukhsh[11] is gradually gaining wide attention for its ability to explain the biological action of HDs in all living organisms, from prokaryotes through human beings; this hypothesis is being studied at the molecular level. 
This study has been designed to examine the cervical cancer HeLa cell line, a suitable in-vitro model, to get insight into whether the HDs of two plant extracts, Hydrastis canadensis (HC-30) and Marsdenia condurango (Condu-30), can manifest any demonstrable changes in the global microarray profiles of HeLa cells by induction through epigenetic alterations. We also conducted experiments on functional validation of SMAD4 biomarker in cancer and methylation specific restriction enzyme (RE) digestion in order to make differential gene expression analysis of two HDs generally used against cancer, namely, HC-30 and Condu-30, versus placebo 30C (Pl-30), the vehicle of the HDs, and if epigenetic alteration is one of the main pathways for bringing about necessary changes in gene expression profiles, to elucidate underlying mechanisms of action of the highly diluted homeopathic remedies at the molecular level.
 
  
2 Materials and methods

2.1 Chemicals and reagents
Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin and ethylene diamine tetra-acetic acid (EDTA) were purchased from Gibco BRL (Carlsbad, CA, USA). Penicillin-streptomycin-amphotericin (PSA) antibiotic and Hipur A RNA-Xpress reagent were obtained from Himedia (Mumbai, India). Tissue culture plastic wares were obtained from Tarsons (Kolkata, India). Propidium iodide (PI) was purchased from Sigma Chemical Co. (St Louis, USA). Oligonucleotide primers were obtained from Imperial Life Sciences, India. Agarose was procured from Lonza, USA. Taq DNA polymerase, deoxynucleoside triphosphates (dNTPs) and reverse transcriptase enzymes were procured from Chromous Biotech (Bangalore, India). Methylation-specific restriction enzyme McrBC was procured from New England Biolabs, USA. All other chemicals used were procured either from Sigma, USA or from Merck, Germany, if not mentioned otherwise.
2.2 Preparation of HC-30, Condu-30 and Pl-30
Crude ethanolic root extract of Hydrastis canadensis and Marsdenia condurango were dynamized to the 30th potency by following the standard serial dilution and succussion method as advocated in the European Homeopathic Pharmacopeia (http://www.echamp.eu/news/newsletter/newsletter-archive/2013/september/homeopathy-in-the-european-pharmacopoeia.html; see Khuda Bukhsh[27] for further detail by Boiron Laboratories (Lyon, France)). In brief, 1 mL of the crude ethanolic plant extract is mixed with 99 mL of 70% ethanol and given 10 mechanical jerks (succusions) of equal magnitude and the potency 1 is obtained. Again 1 mL of potency 1 is mixed with 99 mL of 70% ethanol and given 10 jerks to produce the potency 2 and so on. We procured both the HDs (diluted 10–60), HC-30 and Condu-30, as well as the Pl-30, in which the same stock of ethanol as in drugs was used, from Boiron Laboratories, Lyon, France.
2.3 Cell culture and treatment 
HeLa cells were obtained from National Centre for Cell Science (NCCS), Pune, India. Cells were routinely maintained in DMEM supplemented with 10% FBS and 1% antibiotic at 37 ℃ in a humidified incubator containing 5% CO2. Cells were treated with 4% (v/v) of either HC-30, Condu-30 or Pl-30 and incubated for 48 h in CO2 incubator. Cells without any treatment were considered as negative control.
2.4 Microarray experiment
Separate groups of cells were subjected to the treatment of Condu-30, HC-30, and Pl-30. Cells were sent to iLifeDiscoveries, Gurgaon, India for providing us global microarray data conducted on Affymetrix platform, using 25-mer probes. The total number of probes detected for the experiment was 49 395; hybridization was done at 45 ℃ for 16 h at 60×g.
Slides were scanned with 3000 7G microarray scanner and raw data sets were extracted from the Cel (raw intensity) files. Microarray data analysis, differential gene expression analysis, fold change analysis and cluster analysis were performed using GeneSpring GX12.5 software.
2.5 Experimental grouping
For microarray gene expression study, samples of untreated HeLa cells, and HC-30-, Condu-30- and Pl-30 -treated series were termed as control, SET I, SET II and SET III respectively. All sets were taken in triplicates. 
2.6 RNA quality control before microarray experiment
Before the microarray gene expression study, concentration of RNA (ng/μL), RIN value and rRNA ratio for all the samples were analysed by Agilant Bioanalyzer 2100 .
2.7 Data pre-processing and normalization
All the original microarray data (CEL files) for the experiment were pre-processed using robust multichip average (RMA) algorithm that consists of three steps: a background adjustment, quantile normalization and finally summarization. All above procedures were done by selecting RMA algorithm in GeneSpring GX12.5. Genes of low-intensity information content in each data set were filtered as follows: the probes of intensities less than the 10.0 percentile in the raw data were excluded from the analysis. The step used in pre-processing and normalization is given below.
2.7.1 Raw signal values
The term “raw” signal values refer to the linear data after thresholding and summarization. Summarization is performed by computing the geometric mean.
2.7.2 Normalized value
“Normalized” value is the value generated after log transformation and normalization (scale) and baseline transformation.
2.7.3 Treatment of control probes
The control probes were included while performing normalization. However, there should be an exact match between the control probes in the technology and the sample for the probes to be utilized.
2.7.4 Sequence of events
The sequence of events involved in the processing of the data files was: thresholding > summarization (summarization is performed by computing the geometric mean) > log transformation > normalization > baseline transformation.
2.7.5 Baseline to median of all samples
For each probe the median of the log summarized values from all the samples was calculated and subtracted from each of the samples.
2.8 Box whisker and profile plot
The box-whisker plot presents the normalized microarray expression data visualization summary. Further data are also distributed on conditions in the active interpretation with respect to the active entity or gene list in the experiment. The box-whisker plots are created between normalized intensity values and all probes.
The profile plot describes the summary of overall expression patterns of microarray experimental data.
2.9 Principle component analysis plot
After data normalization, quality control (QC) on samples was performed to remove the unreliable data from further analysis. The QC results are shown in the form of principle component analysis (PCA) in a 3D scatter plot. The scores are used to check data quality. It shows one point per array and is colored by the experiment factors or conditions. This allows viewing of separations between groups of replicates. Ideally, replicates within a group should cluster together but be separate from arrays in other groups.
2.10 Hybridization and correlation plot
Hybridization quality was checked by comparison with “hybridization control”. Hybridization controls were composed of a mixture of biotin-labelled cRNA transcripts of bioB, bioC and bioD, and prepared in staggered concentrations (1.5, 5, 25, and 100 parts per million (PPM), respectively). This mixture was spiked into the hybridization cocktail. BioB was at the level of assay sensitivity and should be considered at least “50% present” at the time. BioC, bioD and cre must be “present” all of the time and must appear in increasing concentrations. 
2.11 Gene expression analysis
A total number of 1 345 genes were found to be oppositely expressed in SET I (HC-30) vs. control and SET III (Pl-30 or placebo) vs. control. Number of genes with ≥ 1.5-fold differential expression was found to be 23. The 1.5-fold change was used because during the fold change analysis, 1 out of 3 of the compared conditions was considered valid. This means if any gene showed up-or down-regulation ≥ 1.5 in any 1 out of the 3 conditions, this would be included in the fold change results. This is important for the gene regulation pattern identification. If one gene is expressed below 1.5 in two conditions and the same gene has expressed more than 1.5 fold in 1 condition, the gene could be considered as showing a change in expression. If we selected the condition 3 out 3 then we would filter out only those genes that have consistence in fold change ≥ 1.5 in all three compared conditions. So fold change 1.5 in 1 out of 3 was deemed optimal. This parameter was also suggested for time series, dose and drug response microarray experiments. How this particular analysis was carried out includes the chance that we may have missed identifying some important gene information from our data, because of the possibility that some gene expressions were instantly decreased/increased in any conditions (less than 1.5 fold) after drug treatment, and these were not counted. 
A total number of 650 genes were identified as oppositely expressed between SET II (Condu-30) vs. control and SET III (ethanol-30 or placebo) vs. control. The number of genes with ≥ 1.5-fold differential expression was found to be 12.
A total number of 1 182 genes were identified to be oppositely expressed between SET I (HC-30) vs. control and SET II (Condu-30) vs. control. The number of genes with ≥ 1.5-fold differential expression was found to be 36. 
2.12 Hierarchical clustering
Cluster analysis was performed for the identification of similar type of experiments or co-expressed gene sets across the sample for the differentially expressed genes. Clustering can group the genes having similar type of expression. Unfortunately, because of the limited number of experiments run (largely due to prohibitive costs), the cluster analysis did not yield significant results. But gene alteration using heatmap image of hierarchical clustering was more revealing, showing more clear differential expression of genes for individual experiment sets.
Hierarchical clustering is one of the simplest and widely used unsupervised clustering techniques for the analysis of gene expression data. The method follows an agglomerative approach, where the most similar expression profiles are joined together to form a group. These profiles are further joined in a tree structure, until all data form a single group. The dendrogram is the most intuitive view of results of this clustering method. There are several important parameters, which control the order of merging entities and sub-clusters in the dendrogram. The most important of these is the linkage rule. After two most similar entities (clusters) are clubbed together, this group is treated as a single entity and its distances from the remaining groups (or entities) have to be recalculated.
2.13 Qualitative reverse transcriptase-polymerase chain reaction and quantitative reverse transcriptase-polymerase chain reaction analysis of SMAD4
For quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis the method described by Saha and Khuda-Bukhsh[28] was followed. Equal amounts of total RNA extracted with RNA expression reagent (Himedia, Mumbai, India) were reverse-transcribed using random hexamer primer and then subjected to PCR with enzymes and reagents of the reverse transcription system using Techne PCR system (Staffordshire, UK). Sequences of primers used in the study are given in Table 1.
Quantitative measure of SMAD4 gene expression analysis was further done by qRT-PCR on ABI 7900HT by relative quantification using the comparative CT method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as an internal control for normalization. For all the samples, the median CT values for the target genes and GAPDH were taken and the expression of target genes was normalized with that of GAPDH. The primer used in the assay is of the same sequence as used in qualitative RT-PCR study.
2.14 Epigenetic study by RE digestion assay
In order to determine methylation status of genomic DNA of HeLa cells in HC-30-, Condu-30-and Pl-30- treated and untreated series, the RE digestion method was employed. First, DNA samples were prepared for RE study by following the rapid isolation method[29] consciously to avoid the use of phenol-chloroform. In brief, cells were harvested and dissolved in ice-cold lysis buffer containing 10 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA pH 8.0 and 0.1% SDS with proteinase K (20 mg/mL) followed by incubation at 55 ℃ for 3 h. The digest was then incubated further at 37 ℃ for 1 h with RNase (4 mg/mL). Next, potassium acetate solution (60 mL of 5 mol/L potassium acetate, 11.5 mL of glacial acetic acid and 28.5 mL of water) was added and mixed by vortexing. Protein-SDS complex was precipitated by centrifugation at 8 000 × g for 15 min. The supernatant was transferred to a fresh tube containing isopropanol. It was then mixed and again centrifuged at 8 000 × g for 15 min. The DNA pellet was washed with 70% ethanol. The semi-dried DNA was dissolved in TE buffer at pH 7.0. Further quality checking was carried out by measuring optical density (OD) at 260/280 nm (ratio maintained at 1.6–1.8) to confirm that DNA was free of protein contamination. After confirmation of quality, DNA was used for further experiments. Methylation-dependent enzyme McrBC (New England Biolabs) was used in the study. Genomic DNA (1.0 μg) was digested with 10 units of RE, in 50 μL total reaction mixtures supplemented with reaction buffers and accessory reagents for overnight at 37 ℃. The digested DNAs were electrophoresed on 1.5% agarose gel followed by ethylene bromide staining to observe differential band patterns, if any.
2.15 DNA content or ploidy analysis in terms of cell cycle distribution by flow cytometry
HeLa cells were grown at an equal density of 2 × 106 cells in 90 mm culture plate and treated with HC-30, Condu-30 and Pl-30. The cells were harvested and fixed in ice-chilled 80% ethanol with constant vortexing and stored at -20 ℃ for future use. For flow cytometric analysis, cells were separated from the fixative by centrifugation and washed in staining buffer (1× phosphate buffered saline, 2% FBS, 0.1% sodium azide at pH 7.1–7.4). RNase (100 μg/mL) was added and the cells were incubated at 37 ℃ for 2 h. After incubation 50 μg/ml PI was added to the material and kept for 30 min at 4 ℃. Cell cycle analysis was performed using the BD FACS VerseTM system and data were analyzed using the BD FACS DivaTM software. For each sample equal numbers of cells (10 000) were counted. Cell cycle distribution analysis was done by gating the cell population[30].
2.16 Blinding
The observer was kept “blinded” during the main part of observation, after which the codes were deciphered for data analysis.
  
3 Results 

3.1 RNA quality analysis data
All samples showed RIN values above 6.0, considered highly satisfactory for microarray analysis. These samples were therefore further processed for gene expression microarray experiment.
3.2 Box-whisker and profile plot data
The total number of probe sets detected for the experiment was 49 395. After data pre-processing, normalization and quality control on data, 40 678 probe sets remained out of 49 395. Baseline transformation was performed by taking median of all probe sets. Transformation was shifted from 20% to 75% for raw intensity data in GeneSpring tool on the basis of median value. The box-whisker plots were created between normalized intensity values and all probes. The box-whisker plots showed the median in the middle of the box, the 25th quartile and the 75th quartile. Box whisker plots had been generated for all samples for expression pattern visualization. The same replicate experiment set had similar expression pattern in the whisker plot (Figures 1A and 1B). All experiment entities (probe sets and genes) were represented separately in the form of line graphs. The experiment name is represented at X-axis, and Y-axis shows the normalized intensity value for the experiment (Figure 1C).
3.3 PCA plot
The PCA components, represented in the X, Y and Z axes, are numbered 1, 2 and 3, according to their decreasing significance (Figure 2).
3.4 Hybridization and correlation plot
The X-axis in this graph represents the controls and the Y-axis, the log of the normalized signal values. The correlation plot shows the correlation analysis across arrays. It finds the correlation coefficient for each pair of arrays and then displays them in a textual form as a table as well as in the form of a heatmap. The correlation coefficient is calculated using pearson correlation coefficient (Figures 3A and 3B).
3.5 Cluster analysis
In the current study hcl (average linkage) was performed on “conditions” and “genes” to explore the co-expression or co-regulation of genes in three groups. First cluster analysis performed on all experiment sets (SET I, SET II, SET-III and control) tried to explore biological similarity among experiment sets. The hcl expression image was presented in the form of a dendrogram. The expression image tree was further characterized on the basis of color. The red color shows over-expression, blue color shows under- expression and yellow color shows normal expression of genes. In the condition tree, experiments having similar expression profiles clustered adjacently in the tree. The genes having similar type of expression profiles also clustered adjacently or on the same tree node (Figure 4).
3.6 Functional validation of SMAD4 gene
There was down-regulation observed in Pl-30-, HC-30-and Condu-30-treated series compared to the untreated control. Fold change values were as follows: for placebo it was 1.76, for HC-30C the fold change was 3.867 and for Condu-30C it was 4.72 (Figures 5A and 5B).
3.7 Alteration in methylation status: an event of epigenetic alteration
Alteration of methylation-status of DNA was analyzed using methylation-dependent enzyme McrBC digestion. There appeared to be two separate cut sites in genomic DNA obtained from untreated and placebo-treated HeLa cells when digested with McrBC, in contrast to a single cut observed in DNA of Condu-30-treated genomic DNA after proper normalization (Figure 5C).
Changes observed were marginal when DNA was digested with HC-30 used for the methylation-dependent enzyme McrBC digestion (Figure 5C).
3.8 Cell cycle distribution analysis
There was increased accumulation of cells at G0/G1 population by 10.25% in HC-30-treated HeLa cells, compared to 6.62% in untreated HeLa cells and 4.59% in Pl-30-treated HeLa cells (Figure 6). In case of HC-30-treated HeLa cells the G0/G1 population was found to be 2.83%.
  
4 Discussion

Our previous study[7] showed that the expression profiles of certain genes of HeLa cells treated with HC-30 and Condu-30, respectively, were significantly different from that of the Pl-30-treated cells. Both the drugs and placebo differed in their ability to trigger gene responses, some of which were implicated in cancer. Thus, an analysis of data obtained in this global microarray study would be able to demonstrate whether the HDs can trigger gene responses in a cascade of reactions consistent with the hypothesis first proposed by Khuda-Bukhsh[11,27]. Ideally, the microarray data would have been tested by making qualitative and quantitative analyses of expressions of many candidate genes, but due to financial and resource constraint, we could validate the microarray data by RT-PCR and qRT-PCR studies of only one important cancer-related candidate gene, SMAD4. Incidentally, Belavitte et al[29] also obtained evidence through transcriptome analysis that HDs of Gelsemium semperverens could alter expression profiles of genes in neurocytes. Very recently, Bigagli et al[30] also demonstrated the effects of homeopathic Apis mellifica preparations on altered gene expression profiles of human prostate cells. The ability of HDs to trigger gene expression profiles is not limited to animal models. Marotti et al[31] documented clear evidence of change in expression profiles of wheat seedlings in the plant kingdom following treatment with ultra-high dilutions of arsenic trioxide used as a homeopathic remedy against symptoms of arsenic poisoning. Thus substantial recent evidence on microarray analysis, which is an accepted modern tool of studying large scale gene expression profiles, clearly depicts that homeopathic remedies in ultra-high dilutions can trigger altered gene expressions presumably through epigenetic modifications, validating the “gene regulatory hypothesis” first advocated by Khuda-Bukhsh[11,27]
Epigenetic modifications are a hallmark of cancer, and a large number of genes remain in modified state of expression in cancer cells. Our present study of alteration in methylation status of DNA by Condu-30 by McrBc RE supports the previous findings obtained by Bishayee et al[8]. Additionally, the results of the present study would further lend support to the gene regulatory hypothesis[11,27,32] that can effectively be achieved by the epigenetic modification triggered by the HDs. Epigenetic modification is one way through which the expression of genes can be strongly influenced and regulated as per need and condition of the organism; it is particularly useful at abnormal physiological states or in disease states, when the regulatory systems are often error-prone.
Our present study revealed the increase in G0/G1 population by HC-30-treated set compared against untreated control and placebo-treated HeLa cells. Increase in G0/G1 population is an indication of apoptosis induction. The present findings thus confirm the apoptosis-inducing ability of Condu-30 observed in HeLa cells and H460 lung cancer cells, also previously reported by our group[8,9]. As cell cycle events are very much related to the structural and functional states of DNA, and the ability of the HDs to induce changes in DNA methylation status adds an important clue in the induction of apoptosis[28,33],thus, various mechanisms of action shown by the HDs indicate the need for further in-depth studies to see if these phenomena can also be observed in other in vitro and in vivo experimentations. Our results give further credence for the gene regulatory hypothesis, which can explain the molecular mechanism of action of the HDs in a scientifically validated way. The ability of the HDs in modulation of various genes in the cell cultures indicates that they can have direct influence on the expression of relevant genes, particularly when the HDs have been reported to elicit responses in unicellular organisms such as yeast[34], bacteria and bacetriophages[12—14].
  
5 Acknowledgements

Part of the work was financially supported by Boiron Laboratories, Lyon, France and part by Emeritus Fellowship of UGC granted to ARKB.
  
6 Competing interests

The authors declare that there are no competing interests.
  

 
6 Competing interests

The authors declare that there are no competing interests.
Figure 1 Box-whisker and profile plot (A) Box-whisker plot of different groups. (B) Box-whisker plot of individuals. Data are robust multichip average normalized from all experiment sets (data align well from baseline to median, and microarray experiment was successful). It is also clear from the image that same replicate experiment has closely related expression pattern. (C) The profile plot shows over all expression patterns of genes. The Y-axis shows normalized ratio and X-axis shows experiment set name. The normalized expression value >0 shows over-expressed genes and <0 br="" genes.="" shows="" style="margin: 0px; padding: 0px;" under-expressed="">


Figure 2 Principle component analysis plot



Figure 3 Hybridization and correlation plot (A) Pair-wise correlation plot for each experiment; (B) Hybridization plot with control probes.



Figure 4 Cluster analysis The snap shot of the heatmap image on experiment conditions and genes. The heatmap image was generated on the experiment conditions and classified on the basis of gene expression. Red color shows over-expressed genes (>0) and blue color shows under-expressed genes (<0 br="" style="margin: 0px; padding: 0px;">




 
Figure 5 Functional validation of SMAD4 and methylation-dependent restriction enzyme digestion?(A) Qualitative RT-PCR study of SMAD4; (B) Quantitative RT-PCR study of _optD4; (C) Methylation-dependent McrBc digestion of genomic DNA obtained from untreated, HC-30-, Condu-30- and placebo-treated HeLa cells. RT-RCR: reversed transcriptase-polynerase chain reaction.
Figure 6 Cell cycle distribution study



Figure 5 Functional validation of SMAD4 and methylation-dependent restriction enzyme digestion (A) Qualitative RT-PCR study of SMAD4; (B) Quantitative RT-PCR study of _optD4; (C) Methylation-dependent McrBc digestion of genomic DNA obtained from untreated, HC-30-, Condu-30- and placebo-treated HeLa cells. RT-RCR: reversed transcriptase-polynerase chain reaction. Table 1 Primer sequences for qualitative RT-PCR and quantitative RT-PCR analyses of SMAD4



 

  
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