2. Brief Background
 Authors: Manoj Bhasin, Jeffery Dusek, Bei-Hung Chang,
Marie Joseph, John Denninger, Gregory Fricchione, Herbert
Benson, Towia Libermann
 Published: May 2013
 Grant Funding: H75/CCH123424 and R01 DP000339 from
the Centers for Disease Control and Prevention (CDC) (HB),
RO1 AT006464-01 from the National Center for
Complementary and Alternative Medicine (NCCAM)(HB),
M01 RR01032 from the NCRR, National Institutes of Health
(The Harvard-Thorndike GCRC). These grant funders had
“no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.”
 Conflicts of Interest: None.

3. Relaxation Response (RR)
 This is the opposite response of the stress response (better
known as “flight or fight” response) which produces specific
psychological and physiological reactions in the mind and body. This
response often activates the parasympathetic nervous system whereas
the stress response activates the sympathetic nervous system. Over
time, over heightened activity of the sympathetic nervous system can
activate the inflammatory response and lead to chronic disease where
as the activation of the parasympathetic response is responsible for
helping to bring the body and mind into a state of equilibrium and
health.
 The RR is activated by (1) focusing on a word, prayer, phrase,
sound, or movement, and (2) “disregarding everyday thoughts”
 Examples of activities that activate RR: meditation, yoga,
movement practices, biofeedback, breathing exercises, progressive
muscle relaxation
 Physiological changes associated with RR activation:
biochemical changes; < oxygen consumption, CO2 elimination, blood
pressure, heart and respiratory rate, and norepinephrine
responsiveness, >heart rate variability, and changes in cortical and
subcortical brain regions

4. Previous Research,
Purpose of This Study
Previous Research Findings:
 Activation of the RR has shown to be an effective therapeutic
intervention to counteract stress associated with hypertension,
anxiety, aging, diabetes, rheumatoid arthritis, and insomnia;
however, the cellular evidence for this effect has not been fully
defined.
 Previous study by authors established changes in genetic
expression with a similar design, specific with genes associated
with oxygen phosphorylation, antigen processing/presentation,
and apoptosis with short and long term practitioners when
compared to novices
 This same previous study also found psychobiological changes
only in long term practitioners during one session of RR
What is unique for this Research?
It is examining acute genetic changes within one RR session and how
the length of previous practices affects this genetic expression.

5. Hypotheses of Study
 (1) One Relaxation Response session for both short and
long-term practitioners would make specific changes in gene
expression that would be linked specific biological
pathways in comparison to a control group with no
experience
 (2) More significant genetic changes would be found in long
term practitioners of Relaxation Response sessions in
comparison to short term practitioners of RR.

6. Study Sample
 Study enrolled 26 healthy subjects with no previous
formal RR activation experience (ie. no formal practices of
RR) which were considered the control group, Novices (N1).
These subjects then took 8 weeks of RR training and then
became the first comparison as Short-term Practitioners (N2)
 A second group of 26 healthy subjects with prior practices
in RR (4-20 years experience) were to compared with the
Novice (N1) group and Short-term Practitioners (N2). This
group was named Long-term Practitioners (M).
 All subjects were recruited within Boston, MA

7.  N1 (control group): Listened to a 20 minute Health
Education CD on first visit
 N2 (control group which became N2) and M: Listened to
20 minute RR activating CD
 Blood samples (to be used for gene expression profiles) and
biological measurements were taken before the session (T0),
after the session (T1) and 15 minutes after the session was
complete.
 Fractional exhaled nitric oxide (FeNO) were collected at the
3 time points, which helps to assess physiological effects of
RR (such as reduction in blood pressure) and has shown
that RR typical increases FeNO levels which can influence
immune system responses.
Methods

8. Methods

9. Techniques
 RNA was isolated from peripheral blood mononuclear cells
(PBMCs) in blood samples
 Real-time FeNO (exhaled) was measured with a rapid response
chemoluminescent Nitric Oxide Analyzer before each session
 Transcriptional Profiling: Done by Affymetrix human genome
high throughput array plates. These plates contained 96 arrays
and 22,000 transcripts. Array images were analyzed with dChip.
 Types of Gene Analyses: The purpose was to identify RR
affected genes/sets of genes with a hierarchy of bioinformatic
techniques including Individual Gene Analysis, Gene Ontology
(GO) enrichment analysis, Gene Set Enrichment Analysis,
Pathways and Interactive network analysis, Systems Biology
analysis

10. Results Explained
 RR leads to qualitative and quantitative temporal transcriptome changes:
Individual Gene Analysis
 RR elicits distinct temporal patterns of differential gene expression: Self-
Organizing Map (SOM) Analysis
 RR progressively affected energy metabolism and inflammation pathways:
Canonical pathways: Gene Set Enrichment Analysis (GSEA)
 Upregulated Progressive changes induced by RR are linked to energy
production in mitochondria: Systems Biology Analysis
 Upregulated Long-term changes induced by RR are linked to telomerase
stability and maintenance: Systems Biology Analysis
 Progressive and Long-term Downregulated gene expression changes induced
by RR are linked to alteration of NF-kB-dependent pathways: Systems Biology
Analysis
 Immune response and telomere maintenance related pathways are affected
among Long-term RR practitioners: GSEA
 RR affected pathways are correlated with Fractional exhaled Nitric Oxide
(FeNO) levels : Correlation Analysis

11. Results
Across Group:
At T (0): Greatest differentiated genes between N1 vs. N2
At T (1): Greatest differentiated genes between N1 vs. M
At T (2): Greatest differentiated genes between N1 vs. M
What about Within Groups, what is the greatest differentiation?

12. Results

13. Results

14. Results:

15. Results: Gene Regulation

16. Results: Gene Regulation
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Table S1: Gene-ontology enrichment analysis of progressive and long-term
expression patterns.
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17. Results: FeNO Gene Regulation
Table S3: Correlation analysis of NO levels and Selected 10 pathways affected progressively or only in long term manner
by RR (Bold).
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T0, T1, T2) as well as changes in gene expression and FeNO levels within a group. The significance of the correlation
was determined on the basis of P value (P < 0.05) and FDR (<25%). The positive and negative correlations between
FeNO and gene expression levels are indicated by red and green color respectively.
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18. Results: Gene Hubs
How is this explained?

19. Discussion: Inflammation Relationships
 Significant research has shown that activation of the RR can reduce chronic stress
and promote wellness and these authors have provided some of the first evidence of
prolonged gene level changes that are opposite of the transcriptional changes that
accompany chronic stress.
 This research specifically looked at transient transcriptome changes in temporal
analysis because it focuses on indentifying genes affected by RR at time points and
reduces the likelihood of false positives.
 It is suspected that with upregulation of gene sets with RR for oxidation might
improve efficiency of oxidation-reduction reactions and reduce oxidative stress.
 Upregulation of the ATP synthase pathway with RR can play an important role in
understanding the biological supportive evidence of RR.
 Critical pro-inflammatory transcriptor factors were downregulated by RR thus
having an influential effect on the inflammatory response, which can reduce
oxidative stress, insulin resistance, and apoptosis.
 Psychological stress can cause chronic mitochondrial oxidative stress that can lead
to metabolic syndrome and activate NF-kB (pro-inflammatory factor) which can
make this worse.
 Understanding the NF-kB relationship in inflammation is important for
understanding the molecular/cellular mechanisms for health benefits for RR.

20.  Long term RR practice helped to upregulate pathways associated with gene
stability especially with telemere packing, telemere maintenance, and tight
junction interaction. Since telemere breakdown can affect mitochondria function
and lead to apoptosis.
 The psychological stress is linked to deregulated immune system function and
DNA repair that may be influenced by RR and thus RR may be able to reverse
stress related transcriptome changes.
 RR practice may create mitochondrial resiliency or mitochondrial reserve capacity
which can create cellular benefits in relationship to health and reducing
psychological stress and chronic disease.
 Mitochondria are considered the energy powerhouse of a cell and also „master
regulators of danger signaling‟ to help protect cellular health and life of an
organism.
 Certain mitochondria also experience differential reserve capacity to help work
with the different pathogenic effects of oxidative stress. Mitochondrial reserve
capacity and resiliency begins to fail when there is high cellular metabolic
demands, which contributes to high disease vulnerability.
The Future of This Research:
 To continue to clinically define the in-depth cellular/molecular
pathways and genetic connections related to RR with secondary
biochemical testing.
Discussion: Mitochondria Relationships

21. Reference
 Bhasin M., Dusek J., Chang B., Joseph M., Denninger J., Fricchione G.,
Benson H., Libermann T. (2013). Relaxation response induces temporal
transcriptome changes in energy metabolism, insulin secretion, and
inflammatory pathways. Retreived from PlosOne on March 6, 2014:
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.
0062817