View source for Anti-aging Effects of Inner Awakening® program

==Title:==
Anti-aging Effects of Inner Awakening® program

==Researches:==
Anti-aging Effects of an Intensive Mind and Body Therapeutic Program through Enhancement of Telomerase activity and Adult Stem cell Counts Note: Results of these studies were published in the below journals: 
1. Journal of Stem Cells, Volume 10, Issue 2 (pp. 107-125) Link: https://www.novapublishers.com/ catalog/product_info.php?products_ id=56446&osCsid=5c086307d4ddb51ff5cfbe6d8498ee99
 2. Journal: Advances in Aging Research, Vol. 4, No.3, May 2015 

Paper: An Intensive Mind and Body Therapeutic Program Leads to Alteration in Gene Expression Critical to Aging Process in Peripheral Blood Stem Cells 

Links: http://www.scirp.org/journal/aar http://dx.doi.org/10.4236/aar.2015.43011 

Objective Key modalities of integrative medicine known to rejuvenate the mind and body are meditation, yoga, completion process, energy initiation by Paramahamsa Nithyananda every day and unrestricted satvic diet. It has been shown previously that intensive or prolonged mind and body therapies (MBT) may have beneficial effects on the wellbeing of both healthy people and patients. Telomerase activity and levels of peripheral blood adult pluripotent stem cells (PB-APSC) are reliable markers of long-term well-being that are known to decrease with age. The objective of this study is to understand the effect of our MBT program on telomerase activity and stem cells in blood collected from the participants.

Design 
Here, we have investigated the effects of an intensive three-week Inner Awakening® spiritual program on telomerase activity and the peripheral blood stem cells in participants before and after the MBT. A total of 108 people were enrolled in the study; 38 men and 70 women (aged 18-90) randomly assigned for the study.

Introduction Aging represents the accumulation of physiological, biological and associated alterations in a person for a longer period of time [1,2]. Aging is one of the most critical known risk factors for many human disease [3]. Roughly 0.1 million people worldwide die each day of age-related complications [4]. 

Integrative mind-body therapies (MBTs), which include meditation, pranayama, other kinds of breath control exercises, and yoga have gained importance and intense attention from the scientific community over the last two decades. Among the various types of MBTs, meditation has still been considered as one of the most common practiced techniques. Many scientific studies have been carried out to understand the efficacy of meditation on physical and mental well-being [5]. Meditation, yoga with breath control exercises, and controlled diet with right nutritional supplementation exert the best anti-aging effects including reduced anxiety and illness, slow-down of the aging process, and improved cognitive function [6,7,8]

During these years the scientific community has been consistently trying to understand the safety and efficacy of meditation on physical and mental well-being. Recent studies show that meditation with breath and diet control, can lead to stable changes in brain pattern for a longer period of time as compared to non-meditating human beings, thereby indicating the importance of intentional practices. These studies may help in understanding the basic unifying mechanisms of the brain, mind and body that enhance our capacity to adapt to stressful and uncertain conditions [9]. 

There are several cellular, molecular, biochemical, and clinical markers to evaluate the rate of aging in an individual. These markers are extensively being used to evaluate anti-aging potential of any MBT program. Telomerase activity levels in a cell and evaluation of the circulating adult stem cells in an individual are two such molecular and cellular parameters for detecting aging [10].

Telomeres are DNA sequences that are essential for protecting chromosome ends and ensuring chromosome stability during replication and also allow cells to distinguish chromosome ends. However, their lengths get reduced with each cell division and further shortened during oxidative stress [11]. 

Below a critical telomere length, cell division can no longer occur which causes cells to enter in a state of senescence that can further enhance the tissue aging process [12,13,14,15,16,17].

Nevertheless, a recent longitudinal study indicates that telomere length of peripheral blood mononuclear cells can increase over time [18-20]. This recent finding thus emphasize the need for identifying potential regulator(s) that control telomere length shortening. The rate of telomere shortening or lengthening depends primarily on the activity and the levels of telomerase [21], which adds DNA sequences back to telomeres and thereby increases their length and restore normal cellular function [22]. 

Lately telomere length has been proposed as a useful “psychobiomarker” [18,21,23,24] and the new findings indicate that telomere length can be regulated in part by psychological stress [25,26]. Additionally, literature in the Hindu (and later Buddhist) traditions indicate the role of meditation in the reduction of psychological stress, which may be due to upregulation of telomerase activity [27].

Adult stem cells are capable of renewal and facilitate regeneration of lost tissues and also repair damaged tissues in the body during stress/insult [28]. Importantly, maintenance of stem cell quiescence is essential for preserving the long-term self-renewal potential of the stem cell pool [28] such as in the brain, bone marrow, musculoskeletal system, and the skin. Furthermore, there is an emerging body of evidence that signals such as metabolic stress can lead to impairment of adult stem cells function in vivo [29]

Materials and Methods Study Design and Subject 

The study determined effect of telomere shortening on aging by detecting peripheral blood adult regenerative stem cell (ARSS) and telomerase activity in peripheral blood mononuclear cells (PBMC). All samples were collected at the site of the study. The study originally included 110 participants. The samples from 2 participants could not be used since either they missed a session or were not good enough for further analysis. Hence total of 108 patients were enrolled in the study, 38 men and 70 women (aged 18-90) . 

The Inner Awakening® program consisted of completion process, meditation, yoga and pranayama in the morning, and energy initiations by Paramahamsa Nithyananda. Interactive sessions were held between mentors and participants throughout the day. Diet was strictly vegetarian with no calorie restriction. The participants of the Inner Awakening® program were from Indian and international locations with different ethnicities, social backgrounds and represented unique lifestyles, with stress and physiological problems at varying degrees. Most of them had never undergone any similar or the same MBT programs previously. Participant’s blood samples were assessed before and after the MBT. Inclusion criteria were: age 18–90 years, no chronic diseases such as CMV infection, heart diseases, diabetes, HIV etc, and good psychological health, for example lack of anxiety and depression, lack of bipolar disorders or schizophrenia.  

Ethics Statement 
The study protocol and the design have been approved by Institutional Ethics Committee. Informed consents in writing were obtained from all subjects prior to start-date of experimentation. 

Intervention 
On day 1 of the program, 5 ml of peripheral blood was collected from each subject in anticoagulant EDTA vacutainer (BD Biosciences, San Jose, USA). After 21 days (i.e., end of the program) again 5ml blood was collected in anticoagulant vacutainer and forwarded through validated clinical cold chain. 

Stem cell isolation process 
Viability measurement was done by using Trypan blue (0.4% Trypan blue stain, Invitrogen, Grand Island, NY, USA). Trypan blue exclusion method [30] was used to count viable PBMCs. Stem cell count was done using hemocytometry. Adult regenerative stem cell (ARSS) was collected from the peripheral blood by density gradient centrifugation [31] and sedimentation process [32]. Our approved protocol on the isolation of PMBC was initially established by examining key pluripotency markers such as OCT4, SOX2, NANOG by reverse transcriptase–polymerase chain reaction (RT-PCR) [33]. 

Telomerase activity measurement in PBMCs 
Telomerase relative activity assessment was done as per the instruction of Millipore’s TRAPeze telomerase detection kit (Billerica, MA, USA) with each sample being analyzed in triplicate. These assays were done in the peripheral blood mononuclear cell (PBMC) counts. Protein estimation assay was done using Lowry method [34] and equal amount of protein lysate (0.4μg) was used for TRAP reaction. 

PBMCs were isolated by centrifugation using Lympho-Ficoll (Ficoll-Paque Plus; GE Healthcare, Little Chalfont, UK) gradient density centrifugation for 30 minutes at 400g. The mononuclear layer was collected and washed 3 times in PBS supplemented with 2% FBS (Stemcell Technologies, Vancouver, BC. Canada), and finally an aliquot containing 0.2 million cells was transferred into an Eppendorf tube (Eppendorf, Hamburg, Germany) and centrifuged at 3000g for 5 min. The pelleted cells were preserved at -80°C until use. Heat-denatured telomerase was used as a negative control for TRAP reaction. 

The positive control was done using one million telomerase positive cells supplied in the kit. The relative telomerase activity was calculated by taking negative control values as 1 and the data were normalized based on exact protein content of the samples

Statistical Analysis 
Each patient served as their own control. We tested the differences in pre and post values for stem cell and telomerase. Day 0 served as control for both telomerase and stemcell measurement and Day 21 post meditation program served as the test sample. We tested for differences in pre versus post using the Wilcoxon Signed Rank Test. We found that for both changes in stem cells and telomerase, there was a significant increase, P < 0.01. 

Results
A proper randomization of patients was done before the start of the treatment. As shown in Table 1 (page 148), all patients participated in this study were randomized in terms of age, sex, stem cells, and telomerase activity prior to meditation. The randomization was successful as evidenced by age-range, sex of the patients just to be sure increased telomerase activity in the treated group were not due to inclusion of more young people.

Mind-Body Therapy (MBT) program did not affect the viability of stem cells
 Out of 110 patients, 2 patients’ samples were found to be contaminated and hence rejected for the study. Using Trypan blue exclusion method 80-90% viability was observed in all subjects (101 - 208). The results are presented in Table 2 (page 150). A close examination of the table revealed that the retreat did not affect the viability of the stem cells among all subjects. Substantially large numbers of stem cells (80-90%) were found to be viable before and after the meditation. 

MBT augmented stem cells numbers in peripheral blood isolated from the participants 
Having established that the retreat did not have much effect on the viability of stem cells, we asked the question whether meditation affected the number of total stem cell in subjects. Table 3 (page 151) shows the stem cell fold changes in subjects before and after the meditation. Thus program group participants showed higher fold changes in stem cell numbers, indicating a possible role of stem cells on the well-being of the subjects undergoing meditation. In addition, a substantial percentage of subjects (about 90%) exhibited increased stem cell counts after meditation as shown in Table 5 

MBT augmented telomerase activity in stem cells isolated from peripheral blood from the participants 
Furthermore, having established that meditation had a marked effect on the stem cell fold changes among the majority of the people undergoing MBT as opposed to pre-MBT group, we went on to investigate a key molecular parameter of well-being: telomerase activity. Table 4 (page 152-153) represents the telomerase activity fold changes in subjects before and after the meditation. Thus telomerase activity was augmented in the majority of the subjects after MBT, thereby indicating better well-being status. In addition, as shown in Table 5, about 45% of people showed more than one-fold of telomerase activity after MBT. Furthermore, about 27% of people showed higher fold changes (2-fold) in telomerase activity after MBT retreat. 

Discussion 
Telomerase activity is critical to prevent early telomere shortening which can delay the aging process [21,14,15]. Interventions that can augment telomerase activity are clinically important because [27, 35, 36, 37, 38, 39] there are no known pharmacological or behavioral interventions that have this beneficial effect to date. Also there is an inverse relationship between telomerase activity and perceived stress [25] that can be reduced by meditative practice [40,41].  

To our knowledge, ours is the first study to date where simultaneous investigation of the critical cellular and molecular parameters of measuring human well-being were investigated. Most earlier studies had looked at the effect of meditation on telomerase activity only. However, in this particular study we looked at the effect of the MBT on stem cell population along with telomerase activity

Furthermore, in this study, we examined if there is any interrelationship between the telomerase activity and the augmentation of stem cell counts in subjects. Since stem cells are responsible for new cell generation, higher stem cell counts could facilitate any age-related damaged tissue healing processes. We found after intensive MBT training, retreat participants had significantly higher telomerase activity compared to the premeditated group. Interestingly, increased telomerase activity among the program participants was associated with increased stem cells count in the blood, thereby indicating an inter-relationship between increased stem cells numbers and telomerase activity. 

The reasons for the positive correlation between the increased stem cell count and increased telomerase activity could be twofold. First, increased telomerase activity could be due to intrinsic telomerase activity in the stem cells. So the higher the stem cell number the higher is the overall telomerase activity. 

Second, since the stem cells have a potent healing capacity, they could migrate and home to the damaged tissue, due to agerelated processes, and stabilize the telomere length by activating telomerase enzyme through an unknown mechanism. However, we don’t know for sure whether the positive correlation between stem cell number and telomerase activity is direct or indirect. 

Furthermore, a recent pilot study on early-stage prostate cancer patients [26], in which a modest increase in telomerase activity (2.22 as opposed to 2.0 before MBT) occurred in response to lifestyle changes including a small amount of meditation or yoga. This is consistent with the positive effect of MBT on the enhancement of telomerase activity

This increase was due to a reduction in ‘‘intrusive thoughts’’ as reported by the patients. Given that meditative practice and negative impact of intrusive thoughts are inversely co-related, it is plausible that the increase in telomerase activity reported in that study might be in part due to meditation-induced changes. Importantly, the association between changes in control preMBT and post-retreat telomerase activity in the present study is consistent with the previous report showing an inverse relationship between perceived stress and immune cell telomerase activity in caregivers for their chronically ill children

Conclusion 
Statistical analysis reveals that more than 45% participants showed more than one-fold change post the Inner Awakening® program and more than 27% participants showed more than two-fold change post the Inner Awakening® program. This is a significant increase in telomerase activity in as short a period as 3 weeks
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Longevity and Health 

♦ The ultimate goal of any life form is to preserve life, increase life span without reducing productivity. This can be achieved by the prevention of diseases most of which are lifestyle-related and by the reduction of stress and cellular inflammation. 

♦ The detailed scientific studies conducted during the Inner Awakening® program, from December 2010 till date, have encompassed several different aspects of human molecular physiology. There is ample evidence in scientific literature that malfunctions of mitochondria, increase in proinflammatory genes and regulatory pathways, decrease in telomerase enzyme and decrease in the stem cell numbers in the body are responsible for premature ageing, diseases and death.

♦ The remarkable evidence gathered so far in Inner Awakening® studies indicate that Mitochondria levels increase several-fold.

♦ Pro-inflammatory genes are down regulated. 

♦ Gene and signaling pathways which enhance production of mitochondria and stem cells are up regulated. 

♦ The number of stem cells is increased. 

♦ Telomerase activity is significantly increased. 

♦ Questionnaire-based evaluations suggest significant decrease in lifestyle diseases, psychosomatic conditions and autoimmune diseases.

♦ The expected life span of a human was declared as 120 years in yogic scientific literature, which has now reduced due to poor lifestyle, etc. 

♦ With programs like Inner Awakening®, involving powerful initiations from an Avatar, it is possible to reestablish the Vedic life span of 120 years, with an improved quality of life, and avoiding preventable diseases.


[[Category: Researches]]