Consolidated Research on IA Haridwar 2014

Researches Consolidated Researches on IA Haridwar 2014

IA-2010, 2011, 2014 ANTI AGEING RESEARCH • Quantitative study of stem cells • Telomerase activity Telomere, Telomerase and Ageing • Telomeres are like end caps protecting the chromosome which contains DNA , the genetic material of the individual organism • The enzyme responsible for maintaining the telomere length is called Telomerase

Telomere, Telomerase and Ageing • Adults have negligible telomerase activity ad therefore their cells can divide for a limited time and die. • This is called the Hayflick limit Theories of Ageing • Deterioration theory • Maintenance and repair theory • Programmed ageing • Regulated programmed ageing • Disposable Soma theory Key Stone • Each cell has a sophisticated molecular circuitry monitor damage in DNA and Mitochondria • When damage reaches a particular threshold, cell death happens leading to ageing • The total energy of a human system is devoted to either growth and reproduction (Grihastashram – reduced life span or maintenance and repair ( Sanyasashram)- increased life span Evidence Based medicine • Blackburn and Grieder won the nobel prize for linking ageing to telomere and telomerase • Biotime inc , California, is currently producing pharmaceutical aid to increase telomerase activity • SkQ project ,Russia, is marketing drugs to increase the mitochondria • Exceter life Sum, Arizona, is currently developing stem cell injections for anti ageing

Medical Based Evidence

• Boston research published in PLoS ONE, has shown that ,anxiety, phobia and other similar emotions (incompletion) ,lead to short life span. • The somatic cell of a new born can divide about 80 – 90 times, where as the somatic cell of a 70 year old can divide only 20 – 30 times.

STEM CELLS

• Stem cells are niche cells, which theoretically are immortal until they decide to differentiate in to a specific cell type, after which they have to follow the rules of the Hayflick Limit. • In theory, stem cells are immortal self renewing pools, which facilitate repair and maintenance. • However, it is now shown that in reality , even stem cells have a limited life span and self renewal cycles. • Journal of clinical medicine- Jan 2014- Page 88-134 – Adult stem cells and diseases of ageing Haridwar Study -2014 • 120 participants • Blood samples taken from peripheral veins and processed • For quantitative stem cell analysis, 100 samples were acceptable and 20 rejected due to technical reasons • For telomerase activity, 120 samples have been processed , however, we currently have results for 20 participants

Statistical Methods • Descriptive values were reported as mean and SD for the continuous variables, number and percentages for the categorical variables. Paired t test was used to compare the pre and post cell counts and treatment values. P value less than 5% was considered statistically significant. Analyses were carried out using SPSS version 18.0 . Results: • Study1: The mean age of the study subjects was 43 ± 14 years and 63% were females. The mean cell count at baseline and end of the study were 30.9 ± 9.3 and 39.2 ± 10.9 respectively. Cell counts were significantly increased at post from the baseline (p<0.001). Analysis was carried out separately among younger and elder age groups. In both the age groups, there was a significant increase in the cell count at post from the baseline (p<0.001). • Study2: The pre and post treatment mean values were 0.61± 0.17 and 1.22 ± 0.28 respectively. There was a significant improvement in the mean values at the end of the study from baseline (p<0.001). A Human Cell & Mitochondria

Mitochondria 1.Responsible for respiration and energy production 2.Reduction in performance is thought to lead to onset of diabetes, stroke, cancer, diabetes etc. 3.A major factor in ageing of the body 4.Methods to increase energy in this tiny generators are allopathic medicine, exercise etc. (Success rate is 25% to 30%)

Ageing and Anti ageing • A damaged mitochondria will produce less energy and more reactive oxygen species (ROS) which lead to cell damage and ageing. • Thus, a highly energized and efficient mitochondria is certainly likely to produce pure ATP and least likely to produce toxic free radicals ( which are a sequelae of unrepaired mitochondrial DNA). • This certainly could be one of the key factors in anti-ageing

Study of Mitochondria • Samples of IA participants, randomized, above 50 yrs of age, double blinded -- Day 0 and day 18 • Samples of control ( Non IA ), but volunteering in the ashram , above 50 yrs of age, double blinded -- Day 0 and 18 • All samples from day 0 were transported to laboratory immediately as well as on day 18. • Testing was done (MTT assay) at , Post Graduate Department of Biotechnology, St Aloysius College, Mangalore University.

Results of the research

1300% increase in Mitochondria Activity and cell viablity in the program participants

DNA Gene expression study

• Human beings have about 25,000 genes, of which only 5% are actively expressed and functional.

• The present study was structured as a combination of a wet lab study using the latest microarray chips to study gene expression changes before and after the inner awakening program in April 2011 and a dry lab bioinformatics analysis using sophisticated soft wares which specializes in identifying relevant genes , their pathways and possible end result, in order to narrow down further the data generated from the previous study

DNA Gene expression study

• The current study during Inner Awakening, was based on a similar study done at Harvard Medical school, by Jeffery A.Dusck, Hasan Out etc on genomic counter stress changes, induced by the relaxation response, published in PLOS1 , vol 3 , issue 7, July 2008. • 40 Healthy adult volunteers male and female enrolled for Inner Awakening Yoga for 21 days has volunteered for giving blood samples with informed consent for differential gene expression study before and after Inner Awakening Yoga.

Gene Expression Study

• 40 Healthy adult volunteers male and female enrolled for Inner Awakening Yoga for 21 days has volunteered for giving blood samples with informed consent for differential gene expression study before and after Inner Awakening Yoga. The objective of the study was to explore Indian traditional yoga and meditation techniques by new generative experimental biology and bioinformatics. • • In the study we compare gene expression profiles in whole blood between Test and Control samples. Samples collected from volunteers before program were names as BIAY samples and after the program were named as AIAY samples. BIAY were considered as controls and AIAY were considered as test samples.

Study design & Labelling

• Sample numbers 1-10 were pooled in as BIAY1 matched against sample numbers 1-10 of after samples as AIAY1 and

• BIAY1 vs AIAY1 • Same is been done from 10-20, 20-30 & 30-40 and they were name and matched respectively as below:

• BIAY2 vs AIAY2 BIAY3 vs AIAY3 BIAY4 vs AIAY4 Human whole genome HG-U133_Plus_2 chips form Affymatrix been used for the study.

Results

(a) 420 Up regulated and 165 Down regulated entities are obtained

(b) Gene ontology has shown significantly enriched GO terms, and pathway analysis has shown genes significant gene expression differences in the following 22 pathways

• p38 signalling mediated by MAPKAP kinases P38 MAPK signaling pathway Validate targets of C-MYC transcriptional activation VEGFR1 specific signals

• TNF alpha/NF-kB TGFBR Hypoxic and oxygen homeostasis regualtion of HIF-1-alpha Endogenous TLR signaling Direct Interactions Cellular roles of Antrax toxin C-MYC pathway BMP receptor signaling Androgen Receptor Androgen-Mediate signaling ATF-2 transcription factor network Signaling mediated by p38-alpha and p38-beta Regulation of p38-alpha and p38-beta Regulation of Androgen receptor activity N0cadherin signaling events IL27-mediated signaling events IL12-mediated signaling events • (c)GSEA analysis for the dataset wherein Kaposi_Liver_cancer_Poor_Survival_up was found to be significant. • (d)Further analysis on disease wise been done for cancer, autoimmune & neurotransmission and results are as below.

MeSH pathway analysis on following terms: 1. Autoimmunity 2. Neurotransmitter: Neurotransmitter Uptake Inhibitors, Receptors and Synaptic Transmission together. 3. Cancer: Brain, Liver and Neoplastic Stem Cells seperately. We limited the network building to these terms because including all of the cancer MeSH terms lead to large network.

Following are the observations: Significant genes from Pathways which are also 2 fold up or down regulated: • •with Autoimmunity: 18 • •with Neurotransmitter: 17 • •with Brain cancer: 40 • •with Liver cancer: 59 • •with Neoplastic Stem cells: 18 • Lists with Fold change values and Gene Symbol are enclosed. • Interestingly 8 genes were common in all the cancer lists. 3(EIF4E, BMI1, TMEF2) of these were significant in C-MYC pathway as well. • 5 out of 59 significant genes from liver cancer network are also found in IL 12 mediated signalling pathways, 7 in BMP receptor signalling, 3 in IL27, 5 in TGFBR, 4 in Androgen receptor and 8 in c-myc pathway. Image for C-MYC pathway with Fold change overlaid for these genes (EIF4E, EP300, BMI1, PKN2, HSP90AA1, TMEFF2, IREB2, CREB1) also enclosed.

Summary • Current medical evidence suggests that the mitochondria ,stem cells, emotions and telomerase are directly linked to the ageing process • Current medical therapy is aimed at pharmacological correction of the same . • The first direct evidence of non pharmacological enhancement of mitochondria, stem cells by changing gene expression pathways, correcting the adverse impact of emotions through the completion process and a significant increase in stem cells , all of which happen simultaneously during IA, has been established from 2010 till today through a series of independent projects Conclusion • Immortality of the body is not in the plan of the cosmos • However, it is clear that, it is possible to increase the lifespan of an individual with good health and fitness, through appropriate processes developed in our shastras