Berberine Supplementation, Sprint Interval Training, and Immune Function Study

Whole blood single cell suspension, 200 μL, will be stained for CD14 (anti-human CD14-FITC) and CD16 (anti-human CD16-APC) (BioLegend, San Diego CA) following lysis of red blood cells with 1X Red Blood Cell Lysis Buffer (BioLegend, San Diego CA) and blocking of Fc receptors with Fc-Blocker (BioLegend, San Diego CA). Prepared samples will be run on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA) following fixation and permeabilization.

100 µL of cells will be aliquoted into 12 x 75 mm tubes alongside 150 μL of Cyto-Fast™ Fix/Perm Solution (BioLegend, San Diego CA), then incubated for 20 minutes at room temperature. Then, 1 mL of 1X Cyto-Fast™ Perm Wash Solution (BioLegend, San Diego CA) will be added, then centrifuged at 350xg for 5 minutes and the supernatant discarded, repeated twice. Cells will then be stained with optimal concentrations of PE anti-human IL-6 antibody (BioLegend, San Diego CA) prepared as a master mix of 100 μL with 1X Cyto-Fast™ Perm Wash, then incubated for 20 minutes at room temperature in the dark. Then, the cells will be washed with 1 mL of 1X Cyto-Fast™ Perm Wash, then centrifuged at 350xg for 5 minutes. Discard supernatant, add 1 mL of Cell Staining Buffer (BioLegend, San Diego CA), then centrifuge at 350xg for 5 minutes. Discard supernatant, resuspend cells in 600 uL of Cell Staining Buffer (BioLegend, San Diego CA) and run on Attune NxT (Thermo Fisher Scientific, Waltham, MA).

Approximately 60mL of whole blood will be collected into serum separator tubes (Beckton Dickinson, East Rutherford, NJ, USA), allowed to clot for 30-min at room temperature, then centrifuged at 2000 RPM for 15 min. The serum will be pipetted into 1.5 mL microcentrifuge tubes (Eppendorf AG, Hamburg, Germany) and immediately stored in a -80 °C freezer. Serum concentrations of CRP will be determined with a commercially available enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH, USA). Microplates will be read with an ELx800 BioTek microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at the recommended wavelength of 450 nanometers.

Approximately 60mL of whole blood will be collected into serum separator tubes (Beckton Dickinson, East Rutherford, NJ, USA), allowed to clot for 30-min at room temperature, then centrifuged at 2000 RPM for 15 min. The serum will be pipetted into 1.5 mL microcentrifuge tubes (Eppendorf AG, Hamburg, Germany) and immediately stored in a -80 °C freezer. Serum concentrations of IL-6 will be determined with a commercially available enzyme-linked immunosorbent assay (BioLegend, San Diego CA). Microplates will be read with an ELx800 BioTek microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at the recommended wavelength of 450 nanometers.

Participants will complete a measure of anaerobic fitness using the Wingate anaerobic power test on a cycle ergometer (Monark Ergomedic 894E, Monark, Varberg, Sweden). Participants will warm-up between 60-75 revolutions per minute (RPM) at a self-selected resistance for 5 minutes. Upon completion of the warm-up, resistance will be dropped to 0, and the participant will then be instructed to begin pedaling at their max cadence. Once participants reach their max pedal cadence, 7.5% of the participant's body mass will be added to the cycle ergometer, and the test will begin. Participants will cycle for a total of 30 seconds at a resistance of 7.5% body mass. At the cessation of the 30 second max test, participants will cycle for an additional 5 minutes at a self-selected resistance for their cool-down. Measurements of peak anaerobic power, mean anaerobic power, relative peak anaerobic power, total work, fatigue index, and heart rate max will be recorded.

Participants will be asked to complete a paper version of the 21-item Depression Anxiety and Stress Scale (DASS-21). The DASS-21 consists of 21 questions using a 0-3 Likert scale assessing the participant's subjective mental health and stress state with a "0" indicating "Did not apply to me at all" and "3" indicating "Applied to me very much or most of the time." The DASS-21 contains 3 sub scales assessing Depression, Anxiety, and Stress consisting of 7 questions for each sub scale, where a high score of 28+ indicates "Extremely Severe" levels of Depression, a high score of 20+ indicates "Extremely Severe" levels of Anxiety, and a high score of 34+ indicates "Extremely Severe" levels of Stress, while low scores of 0-9 indicates "Normal" levels of Depression, scores of 0-7 indicates "Normal" levels of Anxiety, and scores of 0-14 indicates "Normal" levels of Stress.

Lean body mass (LBM) will be evaluated using air displacement plethysmography using a calibrated BODPOD (COSMED USA Inc., Concord, CA). Participants will be instructed to remove their shoes, socks, jewelry, and all additional clothing other than their base layer. Participants will then be given a swim cap to wear and body composition analysis will be performed per the manufacturer's guidelines.

Body fat percentage (BF%) will be evaluated using air displacement plethysmography using a calibrated BODPOD (COSMED USA Inc., Concord, CA). Participants will be instructed to remove their shoes, socks, jewelry, and all additional clothing other than their base layer. Participants will then be given a swim cap to wear and body composition analysis will be performed per the manufacturer's guidelines.

Participants will be asked to complete an online version of the psychological wellbeing scale (PWB). The PWB is an 18-item, 6-minute measurement of six subscales of wellbeing and happiness: Autonomy, Environmental Mastery, Personal Growth, Positive Relation with Others, Purpose in Life, and Self-Acceptance. They will be instructed to rate how strongly they disagree or agree with each subscale on a 7-point Likert scale, where scores of 1 indicate "strongly agree" and scores of 7 indicate "strongly disagree." Subscale scores for Autonomy, Environmental Mastery, Personal Growth, Positive Relations with Others, Purpose in Life, and Self-Acceptance are added together with higher scores indicating higher levels of psychological well-being in each category, although no classifications between scores are provided.

Participants will be asked to complete the online version of the Piper Fatigue Scale (PFS). The PFS is a 22-question fatigue screening tool using a 0-10 visual analogue scale where scores of 0 indicate "little to no fatigue" and scores of 10 indicate "maximal fatigue" symptoms, with 5 additional questions for qualitative characterization of fatigue symptoms and causes. The questionnaire is broken into 4 subscales: behavior (6 items), affect (5 items), sensory (5 items), and cognition/mood (6 items). Sub-scale scores are obtained by dividing the scores from the sub-scale items by the number of the items in the sub-scale. The same method is used to calculate the total score with higher scores indicating more fatigue.

Participants will be asked to complete a online version of the Pittsburgh Sleep Quality Index (PSQI). The PSQI is a 19-question self-reported sleep quality tool broken up into 7 subcategories for scoring, such as subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction. Scoring for each subsection is as follows: Sleep Duration (0 = better, 3 = worse), Sleep Disturbance (0 = better, 3 = worse), Sleep Latency (0 = better, 3 = worse), Daytime Dysfunction (0 = better, 3 = worse), Sleep Efficiency (0 = better, 3 = worse), Sleep Quality (0 = better, 3 = worse), Sleep Medications (0 = better, 3 = worse). For total scoring, the sum of each subsection scores are added together, where total scores ≤ 5 are associated with "good sleep quality," while scores ≥ 5 are associated with "poor sleep quality."

Participants will be asked to complete a online version of the Immune Status Questionnaire (ISQ). The ISQ consists of 7 items rating the respondent's past year's immune status, by inquiring about the incidence of specific immune-related complaints. The ISQ helps to establish participant's perceived immune fitness based on the previous 12 months incidence of disease symptoms. The ISQ utilizes a 0-4 Likert scale to evaluate incidence of these symptoms where a 0 indicates "Never" and a 4 indicates "Always (Almost)." The raw scores are added and converted into a final score where a final score of 0 indicates "very poor perceived immune status," while a final score of 10 indicates "excellent perceived immune status." Final scores less than 6 will be classified as reduced perceived immune function.

Participants will be asked to complete an online version of the Gastrointestinal Symptoms Questionnaire (GSQ). The GSQ is a 29-question self-reported gastrointestinal (GI) screening tool broken up into two subcategories for scoring, one for their GI symptoms experienced today and the other for their GI symptoms experienced in the last 2 weeks. There are also two open-ended question at the end of the survey, one describing any other GI symptoms not addressed in the questionnaire and another addressing normal food consumption. Scoring of GI symptom incidence is based on a 4-point likert scale with scores of 0 indicating "absent" symptoms while scores of 3 indicate "severe" presence and frequency of symptoms. This survey will be utilized to characterize the frequency and severity of common GI symptoms.

Participants will be asked to complete an online version of the Adult Eating Behavior Questionnaire (AEBQ) (Hunot et al., 2016). The AEBQ is a 35-item self-reported tool designed to assess individual differences in eating behavior across eight distinct appetite traits: Enjoyment of food, Emotional over-eating, Emotional under-eating, Food fussiness, Food responsiveness, Slowness in eating, Hunger, Satiety responsiveness. Each item is rated on a 5-point Likert scale where scores of 1 indicate "strongly disagree" and scores of 5 indicate "strongly agree" with higher scores indicating worse eating habits (represented by emotional over-eating and emotional under-eating) or better dietary habits (represented by food responsiveness, enjoyment of food, satiety responsiveness, food fussiness and slowness in eating).