July 2013 003

Researches

Impact of Kundalini Awakening on Gene Function Introduction Health:
Health is defined as not merely absence of illness, but also a sense of positive feeling in physical, physiological, psychological and emotional states. It is a core process affecting all individual cells of the body (50 trillion cells) and their relationship with one another to function as a whole (human being). The relationship of this whole, its interaction with physical and environmental factors (society), and the influence of this interdependence on the collective consciousness (spiritual health) are likewise aspects of the domain of health. There are four different aspects of health: • Physical • Physiological • Psychological • Spiritual Life: The ability of any structure, whether molecule, organelle, cell or multi-cellular organism, to function in its environment by actively processing signals, responding to stimuli, preserving its systems, repairing damage and reproducing itself to further propagate its species, is known as Life. Life processes also include undergoing quantum adaptive changes to deal with a dynamic and changing environment. The primordial universe consisted initially of sub atomic particles (energy), which combined together to form atoms, the smallest organized unit of life - for example, the atom of hydrogen. Hydrogen in turn can be further combined to form molecules through chemical bonds, for example, the molecule of water and so on, which is hydrogen bonded to oxygen.

Gene Gene is a functional hereditary unit made up of DNA, which occupies a fixed location on a chromosome. Chromosome A chromosome is a long strand of DNA, found within the nucleus of a cell. A human being has 23 pairs of chromosomes in each cell of which 22 are autosomal (not sex-related). The last pair are the sex chromosomes, which determine our gender.

Molecular Basis of Life Of all the possible combinations of elements, the formation of a specially designed molecule called DNA (Deoxy ribo nucleic acid), became the fundamental mechanism of giving life to otherwise inanimate molecules. The DNA is a poly-nucleotide chain containing a repetitive backbone of sugar-phosphate units with small organic bases - Adenine, Thymine, Guanine and Cytosine - attached to each sugar. In human beings, DNA is a long strand measuring almost 2 meters in length, but coiled inside a cell, which is only a few microns in diameter

Genome The complete set of chromosomes (with their genes) in an organism is called its “Genome”. The Human Genome contains between 25,000 and 30,000 genes similar to that of a mouse and less than a grain of rice, which contains 50-60,000 genes. Hence it is clear that it is not the number of genes, but what a gene can do that determines the complexity of a living organism. Genes not only transmit information from generation to generation, but also act on a minute-to-minute basis, reacting to external needs, synthesizing amino acids and proteins which carry out the various functions of the body, whether structural, enzymatic or defensive mechanisms. Comparative Genomics The human and chimpanzee genomes are 96% identical. The 4% gap makes a profound difference in the physical and psychological presentation (phenotype) of these two species. For example, language, which is unique to humans, has been tracked down to a double mutation in a gene called ‘FOX P2’. This single factor forms the entire basis for verbal communication in the human species.

Genes and Disease Some diseases involve a single gene mutation such as sickle-cell anemia, which affects only a small subset of humanity. Other diseases involve multiple genes. Over 100 genes are implicated in susceptibility to asthma and cancer, causing disease due to genomic instability often involving the mutation (change) of genes from their normally programmed behavior. Gene-related metabolic pathways such as MAP-K, which stands for Mitogen Associated Protein Kinase, are implicated in the processing of long-term potentiation (LTP), the basis for learning and memory. In human beings, DNA is present in: ♦ The nucleus of the cell as chromosomes, completely covered (protected) by a histone coat. ♦ The organelle called the mitochondrion, the powerhouse or energy-producing battery of the cell. Mitochondrial DNA is naked, unprotected by the histone coat. This makes the mitochondrial DNA more susceptible to damage. Built-in mechanisms function to constantly repair the DNA when errors occur. Gene Expression It is estimated that only 10,000 of the 30,000 human genes are expressed at any given time (redundancy). Since many genes are repeated and many parts of the chromosomes do not contain genetic material, it is estimated that only 5% of the human genome is actively expressed and has functional genes. There is a parallel to this in the Cosmos, where the sum total of all known matter makes up only 5% of the universe, the remaining 95% as yet unknown.

Expression, Up Regulation and Down Regulation of Gene, Or Its End Product - The Receptor Gene Expression is the process by which information from a gene is used in the synthesis of a functional gene product. The process is used by all known life to generate the macromolecular machinery for life. For example, depending on environmental conditions, the common yeast undergoes a quantum shift in metabolic pathway from fermentation to oxidative metabolism, upon exhaustion of glucose as an energy source. This is called dioxy or double growth. Experiments in fruit flies demonstrated differential expression of genes during waking and sleeping states. During the waking state, genes and encoding proteins involved in detoxification and defense mechanisms are highly expressed. During sleep, nerve-related genes are expressed, which leads to consolidation of memory. (It is significant to note here that His Holiness Paramahamsa Nithyananda routinely advises spiritual aspirants to take a bath and meditate just before sleep, to avoid the consolidation of negative energies/ emotions accumulated during the day into the bio-memory.) Up Regulation In times of shortage, certain genes or their end products (specific receptors) are up regulated or increased because of an apparent shortage of access to specific materials. Example: In Type 2 Diabetes Mellitus, there is an up regulation of insulin receptors, in the hope of capturing passing Insulin molecules for glucose metabolism in the cell. This phenomenon is called ‘Insulin Resistance’. Down Regulation Certain hormones can also down regulate or decrease the receptors in order to balance the internal environment of the cell, as happens with excess availability of substances

Review of Literature Several papers have studied the role of meditation (relaxation response), yoga, tai chi and other eastern techniques and their effects on human physiology. ♦ Benson and Steiner (1975) studied the role of hypoxia, O2 consumption and CO2 elimination during meditation. This was published in the Journal of Human Stress, 1, 37-44. ♦ Peng and Henry (2004) studied the heart rate dynamics during meditation, which was published in the International Journal of Cardiology, 95, 19-27 ♦ Zheng (2007) studied the modulation of immune functions and oxidative status, induced by noise stress, published in Journal of Occupational Heath, 49, 32-38 ♦ Glaser (1990) studied psychological stress-induced modulation of IL2 receptor gene expression, published in the Archives of General Psychiatry, 47, 707-712 The current study during Inner Awakening® was based on a similar study done at Harvard Medical School by Dusck, et al. (2008) on genomic counter stress changes, induced by the relaxation response, published in Public Library Of Science One, 3 (7). The Study Control sample collection date: April 12, 2011 Test Sample collection date: May 2, 2011 Venue: Nithyananda Peetham, Bengaluru Aadheenam, Bidadi, INDIA Contract scientific services were carried out by Leads Clin, Bangalore. Principal Investigator: Dr. Krishna Shama Rao, Yenapoya Medical University Forty healthy adult volunteers, male and female enrolled for the 21 day Inner Awakening® program, volunteered to give blood samples with informed consent for differential gene expression study before and after Inner Awakening®.

The objective of the study was to explore Indian traditional yoga and meditation techniques through new experimental biology and bioinformatics. ♦ Gene expression changes including up regulation and down regulation were assessed by micro array analysis, using affymetrix HG-U 133+ 2.O gene chips. ♦ Gene Ontology (GO) categories were identified using expression analysis systematic explorer (EASE Analysis). Study Design & Labeling In the study, we compared gene expression profiles in whole blood between Test and Control samples. Samples collected from volunteers before program were named as BIAY samples and after the program were named as AIAY samples. BIAY were considered as controls and AIAY were considered as test samples. Sample numbers 1-10 were pooled in as BIAY1 matched against sample numbers 1-10 of after samples as AIAY1 and studied BIAY1 vs AIAY1 Similarly, samples 10-20, 20-30 and 30-40 were named and matched respectively as BIAY2 vs AIAY2, BIAY3 vs AIAY3 and BIAY4 vs AIAY4. Human whole genome HG-U133_Plus_2 chips form affymetrix have been used for the study. Materials and Methods Blood sample collection and RNA storing: Five milliliters of blood was collected from volunteers in EDTA vacutainers, mixed with 25ml of Erythrocyte Lysis Buffer, Qiagen, and incubated for 10 minutes on ice, then mixed by vortexing briefly 2 times during incubation and kept for 10-20 minutes and centrifuged at 4000 rpm for 10 minutes at 4°C and the supernatant was completely removed and discarded. After 10-20 minutes visible pellets were re-suspended in 10ml of solution. RNA Precipitation Procedure:

1. Add 1/10 volume 3M NaOH, pH 5.2 and 2.5 volumes ethanol. 2. Mix and incubate at -20°C for at least 1 hour. 3. Centrifuge at ≥ 12,000 x g in a micro-centrifuge for 20 minutes at 4°C. 4. Wash pellet twice with 80% ethanol. 5. Air dry pellet. Check for dryness before proceeding. 6. Re-suspend pellet in DEPC-treated water. Quantification of RNA Yield by Spectrophotometric Analysis Absorbance of the sample was checked in 260 and 280 nm. The ratio of A260/A280 was 1.9 which is in an acceptable range. cDNA and cRNA Synthesis, Hybridization, Scanning, Data Extraction were done. a) Single cycle labeling process with cDNA using a T4 promoter— dT24 oligonucleotide as primer and second strand cDNA synthesis and incubation with T4 DNA polymerase has been done and the products were purified using affymetrix clean up module. Biotinylated cRNA is made using affy IVT kit. b) cRNA is purified using Qiagen, quantified and then fragmented by incubation at high temperature with Magnesium. Biotinylated cRNA is added to a total hybridization cocktail of 300 microliters and 200 microliters is hybridized to gene chip after adding control oligo-nucleotides, overnight. c) Hybridization has been done at 45o C, 17 hours overnight with constant rotation. The hybridization mixture is then removed and the Gene Chips are washed, stained with phycoerythrin-labeled straptavidin, washed, incubated with biotinylated anti-streptavidin and then restrained with phycoerythrin-labeled straptavidin to amplify the signals. d) Arrays are then scanned using dedicated scanner, controlled by GCOS software. Images are examined for defects. The Affy Micro Array Suite 5 (MAS%) algorithm analyzed the hybridization intensity data of gene chip expression probe arrays checked normalized and CEL files were stored for further analysis. e) Microarray Data Analysis was done using the latest version software Gene Spring 11.5. The CEL files are imported in Affymetrix Expression workflow using Technology for HG-U133_Plus_2. Summarization: We opted for MAS 5 summarization. It also provides flag information for the probe sets. Baseline to median of all samples was applied for this data. Analysis: A t test unpaired was used to identify the differentially expressing entities. Fold change analysis at the cut-off of 2 was performed. Hierarchical clustering on the output was performed. Gene Ontology Analysis was done by using fold change output. NCI to find significant pathways was used for pathway analysis. 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 significant gene expression differences in the following 22 pathways: p38 signalling mediated by MAPKAP kinases, P38 MAPK signalling pathway, validate targets of C-MYC transcriptional activation, VEGFR1 specific signals, TNF alpha/NF-kB, TGFBR, Hypoxic and oxygen homeostasis, regulation of HIF-1-alpha, Endogenous TLR signalling, Direct Interactions, Cellular roles of Anthrax toxin, C-MYC pathway, BMP receptor signalling, Androgen Receptor Androgen-mediated signalling, ATF-2 transcription factor network, signalling mediated by p38-alpha and p38-beta, Regulation of p38-alpha and p38-beta, Regulation of Androgen receptor activity, N0 cadherin signalling events, IL27-mediated signalling events and IL12-mediated signalling events. c) GSEA analysis for the dataset wherein Kaposi Liver cancer Poor Survival was found to be significant. d) Further analysis was done for cancer, autoimmune and neurotransmission related diseases and results are as below. MeSH pathway analysis was done for the following terms: a) Autoimmunity b) Neurotransmitter: Neurotransmitter Uptake, Inhibitors, Receptors and Synaptic Transmission together. c) Cancer: Brain, Liver and Neoplastic Stem Cells separately. We limited the network building to these terms because including all of the cancer MeSH terms leads to large network. Following are the observations made: Significant genes from Pathways which are two fold up or down regulated: • with Autoimmunity: 18 • with Neurotransmitter: 17 • with Brain cancer: 40 • with Liver cancer: 59 • with Neoplastic Stem cells: 18 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

Discussion & Summary 1. The control group was the participants of Inner Awakening® on day 0, and the study group was the same participants post Inner Awakening®, day 21. Hence, human variations of gene expression between control and study groups have been completely avoided. 2. 420 up regulated and 165 down regulated entities have been demonstrated within a short period of 21 days. The shortest previous study done at Harvard Medical School was of 8 weeks duration. 3. Gene ontology studies and pathway analysis has shown significant gene expression differences in 22 pathways. 4. MeSH pathway analysis, further subdivided the GO pathways under the following: • Auto Immunity • Neurotransmitter • Malignancy • Androgen related • Mitochondria There is a 7-fold increase in pathways related to Immune modulation, Inflammation, Hypoxia regulation etc. This matches with the clinical relief of inflammatory symptoms in the Inner Awakening® participants, both in relation to autoimmune disorders like rheumatoid arthritis, fibromyalgia and other chronic pain conditions. MAP-K signalling pathways are largely implicated in long term potentiation involved in learning and memory. Eight children participated in the Inner Awakening® for Kids program in parallel with the Inner Awakening® program for adults, and their memory and ability to recall were tested and recorded before and after 21 days. The significant improvement in memory correlates well with the MAP-K gene up regulation. Almost 117 gene expression changes with relationship to various known malignancies were noticed following MeSH analysis; however none of the registered participants had any recorded malignant conditions during the Inner Awakening® processes. There was more than a 7-fold increase in Androgen receptor activity and other Androgen mediated signalling events. It is well known in Vedic/yogic literatures that the intense practice of certain Kriyas and meditation can lead to a natural suppression of sex-related desires (Muladhara Chakra), which, similar to insulin resistance, would lead to up-regulation of androgen receptors in peripheral cells. The only other condition where such upregulation of androgen receptors is seen is in prostate cancer. However, none of the participants had any history of cancer of the prostate. Previous study on the mitochondrial activity of Inner Awakening® participants using the MTT assay techniques revealed a 967% increase of mitochondrial activity. This correlates well with the current finding of a significant increase of MAP-K and other related pathways, which are essential in increasing both the number and size of mitochondria. Techniques of pranayama, which include various forms of bandhas and kumbhakas, would naturally be expected to affect oxygen homeostasis under hypoxic conditions, and the up regulation of the HIF-1-alpha signifies the same. Further studies in collaboration with Medical Universities, may provide greater information on changes in mitochondrial activity in relation to age, health and disease. Studies are being structured to do concomitant fMRI and qEEG studies to correlate with neurotransmitter activity in relation to normal brain physiology and psychiatric illnesses. The study was conducted at Nithyananda Peetham, using processes under the guidance of His Holiness Paramahamsa Nithyananda. This study was funded by Nithyananda Peetham, and the data, results and the copyright are the sole property of Nithyananda Peetham.

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