Subtle Energy Transmission and Tao Calligraphy Mindfulness in Telomere Length in Peripheral Blood Leukocytes

OBJECTIVES The proposed research is a pilot project based on experiential data obtained from several previous pilot and formal studies by the investigators' research teams and by researchers from other institutions. It is intended to measure the effects of the transmission of subtle energy (blessing), combined with a specific form of meditation, on the length of human telomeres in peripheral blood leucocytes and on overall well-being. The investigators hope that the study will provide further data for individuals seeking an additional modality to prolong life expectancy that could be integrated into conventional medical protocols.

HYPOTHESIS The research null hypothesis is that individuals who receive a subtle energy transmission for improving telomere length in peripheral blood leucocytes and who will also then regularly practice mindfulness with Tao Calligraphy for nine months will (a) have no significant change in telomere length in follow-up analyses as measured by telomere length in peripheral blood leukocytes and will (b) show no improvement of well-being as assessed via the standardized scientific questionnaire Rand SF 36(s) at three and nine months.

Statistical test Anova (using Minitab version 14 and PSPP/SPSS) will be used to evaluate the null hypothesis. The p-value will represent how unlikely the observed data would be if the null hypothesis were actually true and the investigators will use them to reach conclusions. The investigators set the confidence level at 95% and if p <= 0.05, then H0 is rejected.

BACKGROUND Telomeres as Markers of Longevity Background: Telomeres are distinctive protein-DNA structures (repetitive (TTAGGG) sequences) at the end of the chromosomes that are essential for maintaining the integrity and stability of the genome. Telomere length shortens with age (due to multiple cell divisions). The rate of telomere shortening has been found to increase or decrease in association with specific lifestyle factors.

To counteract the loss of telomere repeats with each cell division, germline cells, highly proliferating cells from the skin, the gut, and hematopoietic tissues, as well as tumor cells, express DNAmAge telomerase, an enzyme complex with reverse transcriptase activity, which adds the necessary telomeric DNA (T2AG3 repeats) to the 3-end of the telomeres, protecting their degeneration. Despite expression of telomerase, the telomere length in highly proliferating cells from long-lived species decreases during development and with replication in vitro and with age in vivo. Most likely, telomerase levels in such cells are tightly regulated and are insufficient to make up for the net loss of telomere repeats with cell division.

The telomere length values reflect telomere shortening (resulting from cell divisions, oxidative damage, and other causes) and telomere elongation (mainly resulting from telomerase activity) of the chromosome-specific telomere length inherited in the gametes. Consequently, researchers have two main tools to analyze telomeres, the telomere length and the telomerase.

Although the ability of telomere length measured in peripheral blood to predict life expectancy or monitor mechanisms underlying the aging process is not entirely consistent across studies, telomere length currently remains one of the most widely used biomarkers in epidemiological and clinical studies of aging.

In the protocol of the study, the investigators have discussed several studies using either of these two markers.

The laboratory test used in this study to measure telomere length The average length of telomere repeats can be measured by in situ hybridization and flow cytometry [flow-FISH]. In order to measure the telomere length in granulocytes, naive T cells, memory T cells, B cells, and natural killer (NK)/NKT cells within a blood sample, flow-FISH can be combined with antibody-staining (Multicolor flow-FISH). Most steps in the staining protocol are automated using a 96-well micro-dispenser device. The minimum detectable difference in telomere length and the reproducibility of the method are in the range of 0.2-0.5 kb and measurements can be made with as few as a thousand cells. Automated multicolor flow-FISH greatly facilitates studies of telomere length regulation in subsets of nucleated blood cells, especially when only few cells are available and when differences in telomere length are small.

In Repeat Diagnostics, the laboratory being used for this study, the fluorescent signals from telomeres are assessed on a single cell basis by flow cytometry, with patient results reported for lymphocytes, granulocytes, B-cells, naïve and memory T-cells, and NK cells. All patient telomere length profiles are reported in relationship to age-matched controls and are available for all age ranges from pediatric to geriatric. This telomere reference curve was established by the study of over 800 healthy individuals between birth and 100 years of age. This informative technique allows analysis and comparisons of the telomere length between different cell types and within one cell type, e.g., in differentiating between constitutional deficiencies and specific cell type deficiencies.

The test used in this study to measure overall effect of mindfulness practice A 36-item short-form Quality of Life Questionnaire (SF-36) was constructed to survey health status in the Medical Outcomes Study. The SF-36 was designed for use in clinical practice and research, health policy evaluations, and general population surveys. Although the 12-item short-form of SF-36 would be easier to use, the investigators chose the longer 36-item version, as standard errors were described nearly always to be larger for the 12-item short-form.

The SF-36 includes one multi-item scale that assesses eight health concepts:

1. limitations in physical activities because of health problems;
2. limitations in social activities because of physical or emotional problems;
3. limitations in usual role activities because of physical health problems;
4. bodily pain;
5. general mental health (psychological distress and well-being);
6. limitations in usual role activities because of emotional problems;
7. vitality (energy and fatigue); and
8. general health perceptions.

Through a complex calculation, eight scores are derived from answers to 36 questions of SF-36. Finally, these eight scores are linearly transformed to a 0-to-100 scale, with 100 being the best possible outcome.

Chinese calligraphy Chinese calligraphic writing uses an ink-laden brush to create defined configurations of characters. Studies of Chinese calligraphic writing have confirmed its significant effects on psychosomatic conditions, post-traumatic hyper-arousal symptoms, hypertension, and type 2 diabetes.

The investigators chose a Tao Calligraphy practice, which is unique style of moving meditation, wherein mindfulness (heightened awareness) is achieved by the combination of movement and focus on a work of Tao Calligraphy art. In this practice, the subjects do not use an ink-laden brush to write on the paper, but trace the strokes of premade calligraphy with fingers and simultaneously repeat an affirmation (mantra) aloud or silently, which enables them to achieve deep concentration during wakefulness.

Results from the investigators of previous studies confirmed the efficacy of this practice, with significant results in improving overall well-being and chronic conditions as documented by SF MPQ (McGill Pain Questionnaire), by EORTC QLQ-30 (Cancer Quality of Life Questionnaire), and by SF-36 (Quality of Life Questionnaire).

METHOD The study will be performed as a pilot follow-up study. Should the null hypothesis be successfully rejected, this study will be followed by a formal Double Blind Randomized Crossover Study.

As this is a pilot study, there will be no randomization into intervention and control groups. As a control, the investigators will use the "normal" values from the general population. All subjects will receive a transmission and then will be practicing Tao Calligraphy mindfulness daily during the nine-month duration of the study.

The study is conducted on an outpatient basis and includes:

• Informational telephone or Zoom meeting to clarify inclusion/exclusion criteria, the study design, and the consent/information forms, with signing thereof
• Initial instructional Zoom meeting to teach the practice of Tao Calligraphy tracing meditation and to deliver the subtle energy transmission (blessing)
• Laboratory assessment of telomere length in peripheral blood leucocytes of participants
• Completion of the questionnaires by participants
• Completion of assessments by a study principal and co-investigators

Laboratory assessment of telomere length in peripheral blood leucocytes:

Laboratory assessment of telomere length in peripheral blood leucocytes will be performed at the entry time point (Time 0), at the end of three months (Time 1), and at the end of nine months (Time 2) for every participant.

Blood samples will be drawn at "Life Labs," a DAP-accredited institution in British Columbia and an accredited institution in the United States, and will be promptly sent by courier to Repeat Diagnostics, Suite 309 - 267 West Esplanade, North Vancouver, BC V7M 1A5 Canada for telomere assessment by Flow FISH.

Reports will be issued to clinical Principal Investigator, Dr. Peter Hudoba.

Improvement of general well-being or clinical well-being as measured by standardized questionnaires:

1. John Ware's SF-36 Quality of Life questionnaire
2. Simple follow-up questionnaire of the investigators' own design (to address any additional information participants may have).

Participants will complete both questionnaires online on the Sha Research Foundation website using unique IDs to secure confidentiality.

Each subject of the study will answer all 36 questions at the entry time point (Time 0), at the end of three months (Time 1), and at the end of nine months (Time 2).

From the SF-36 answers, the study statistician will derive 8 scores in a blind fashion. Then, the 8 scores from beginning and end data will be compared to each other and to "normal" values using Anova test. As a control, the investigators will use "normal" values corresponding to the age of each participant at the beginning and at the end of their participation.

The correlation coefficient will be used to determine any correlations between various factors (e.g., effects of age, sex, length, frequency of mindfulness practices, and others) on outcomes and regression analysis to determine the relationship between independent and dependent variables.

Data will be stored on an external hard drive to prevent unauthorized access through the internet.