Understanding the Role of Gut Microbiota in Hyperphagia in Prader-Willi Syndrome
Phase: PHASE2
Conditions: Prader-Willi Syndrome, Obesity
Sponsor: Rutgers, The State University of New Jersey

 
 
 
 
COVER PAGE 
 
Title: Understanding the Role of Gut Microbiota in Hyperphagia in Prader-Willi Syndrome 
NCT number: NCT05541003 
Document date: 4/12/2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
Protocol Title: Gut microbiota and hyperphagia in PWS 
Protocol Version Date: October 7, 2022 
 
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INTERVENTIONAL 
RESEARCH PROTOCOL TEMPLATE 
(HRP-503a) 
 
 
 
 
STUDY INFORMATION 
 
▪ 
Title of Project: 
            Understanding the role of gut microbiota in hyperphagia in Prader-Willi syndrome 
 
▪ 
Principal Investigator Name 
Keerthana Kesavarapu, DO 
Liping Zhao, PhD 
 
▪ 
Principal Investigator Div. & Dept. 
Department of Gastroenterology and Hepatology, School of Medicine 
Department of Biochemistry and Microbiology, School of Environmental and Biological 
Sciences 
 
▪ 
Principal Investigator Contact Info:  
           kk1132@rwjms.rutgers.edu 
           125 Paterson St, New Brunswick, NJ 08901 
           (678) 469-0425 
 
liping.zhao@rutgers.edu  
76 Lipman Drive, New Brunswick, NJ 08901 
           (848) 932-5675  
            
▪ 
Protocol Version and Date: 
Version 6, October 7, 2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
Protocol Title: Gut microbiota and hyperphagia in PWS 
Protocol Version Date: October 7, 2022 
 
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Protocol Title: Gut microbiota and hyperphagia in PWS 
Protocol Version Date: October 7, 2022 
 
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Table of Contents 
Skip To Section: Hold CTRL + Click (Below) To Follow Link in Blue 
 
1.0 
Research Design 
 
 
1.1 
Purpose/Specific Aims 
 
 
1.2 
Research Significance 
 
 
1.3 
Research Design and Methods 
 
 
1.4 
Preliminary Data 
 
 
1.5 
Sample Size Justification 
 
 
1.6 
Study Variables 
 
 
1.7 
Drugs/Devices/Biologics 
 
 
1.8 
Specimen Collection 
 
 
1.9 
Data Collection 
 
 
1.10 
Timetable/Schedule of Events 
 
 
2.0 
Project Management 
 
 
2.1 
Research Staff and Qualifications 
 
 
2.2 
Research Staff Training 
 
 
2.3 
Other Resources 
 
 
2.4 
Research Sites 
 
 
3.0 
Multi-Center Research 
 
 
4.0 
Subject Considerations 
 
 
4.1 
Subject Selection and Enrollment Considerations 
 
 
4.2 
Obtaining Identifiable Information About Non-Subjects 
 
 
4.3 
Number of Subjects 
 
 
4.4 
Consent Procedures 
 
 
4.5 
Special Consent Populations 
 
 
4.6 
Economic Burden and/or Compensation For Subjects 
 
 
4.7 
Risks of Harm/Potential for Benefits to Subjects 
 
 
5.0 
Special Considerations 
 
 
5.1 
Health Insurance Portability and Accountability Act (HIPAA) 
 
 
5.2 
Family Educational Rights and Privacy Act (FERPA) 
 
 
5.3 
Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations) 
 
 
5.4 
General Data Protection Regulation (GDPR) 
 
 
5.5 
 NJ Access to Medical Research Act (Surrogate Consent)  
 
 
6.0 
Data Management Plan 
 
 
6.1 
Data Analysis 
 
 
6.2 
Data Security 
 
 
6.3 
Data Safety And Monitoring 
 
 
6.4 
Reporting Results 
 
 
6.5 
Secondary Use of Data 
 
 
7.0 
Research Repositories – Specimens and/or Data 
 
 
8.0 
Approvals/Authorizations 
 
 
9.0 
Bibliography 
 
 
 
 
 
 
 
 
 

 
 
 
 
Protocol Title: Gut microbiota and hyperphagia in PWS 
Protocol Version Date: October 7, 2022 
 
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1.0 Research Design 
 
1.1 Purpose/Specific Aims 
To examine clinical relevance and test for a causative role of gut microbiota in the context of appetite 
regulation and food-related behavior in patients with hyperphagia syndrome 
A.  Objectives  
To use high-fiber supplementation, an intervention known to create shifts in the gut microbiota 
 
towards a healthier structure, to explore the relationship between gut microbiota, appetite control 
 
and feeding behavior in patients with hyperphagia syndrome. 
B.  Hypotheses / Research Question(s) 
We hypothesize that the gut microbiota in patients with  hyperphagia is deficient in carbohydrate-utilizing 
short-chain fatty acid (SCFA) producers and dominated by bacteria that produce detrimental compounds. 
Such dysbiosis produces distinct profiles of bioactive compounds, including the proinflammatory 
lipopolysaccharide (LPS), that contribute to altered signaling in central appetite regulation. 
 
1.2 Research Significance 
Hyperphagia is a hallmark feature in PWS patients, with obsessive preoccupation with food and extreme 
overeating (energy intake of PWS patients may be 3-6 times that of controls (1, 2)) leading to obesity and 
the related co-morbidities that become one of the leading causes of death in this patient population (3). 
There is some evidence for hypothalamic dysfunction as an underlying mechanism for PWS-associated 
hyperphagia (4) but there is no effective treatment to normalize food-related behaviors. The emerging 
literature on the role of gut microbiota in the gut-brain axis may provide a novel perspective of feeding 
dysregulation associated with PWS and facilitate novel therapeutic approaches. In our previous study, high-
fiber intervention induced significant shifts in the gut microbiota structure in PWS patients, an effect 
associated with improvements in metabolic parameters including body weight and glucose homeostasis 
and a modest improvement in the overall food-related behavior (5). Data from animal studies further support 
a plausible mechanism by which gut microbiota participates in the gut-brain axis to regulate food-related 
behavior (6): SCFAs generated from bacterial fermentation signal enteroendocrine cells for the production 
of appetite-regulating hormones including glucagon-like peptide-1 and peptide YY; LPS from Gram-
negative bacteria disrupt the blood-brain barrier and thus enhances the effect of circulating cytokines on 
central feeding pathways. Other mechanisms that gut microbiota may regulate appetite include the 
production of neurotransmitters and neuromodulators (e.g., γ-amino butyric acid from Bifidobacterium and 
serotonin from Enterococcus spp.) that act on afferent axons locally or interact with the intestinal cells to 
modify neural signaling to the central nervous system. 
 
1.3 Research Design and Methods 
 
A. Research Procedures 
 
RECRUITMENT. We are recruiting patients with hyperphagia aged 18-45 years who have not 
received growth hormone treatment in the previous 6 months. Study candidates will be recruited 
from Robert Wood Johnson Medical School (RWJMS) pediatric endocrine clinic as well as DEEP6, 
which applies artificial intelligence to identify patients meeting study criteria in the RWJMS 
outpatient electronic medical record.  
 
ELIGIBILITY SCREENING.  
For recruitment, individuals with patients with hyperphagia will be identified via Dr. Ian Marshalls 
RWJMS clinic and DEEP6. Patients identified on the DEEP6 list will be considered for the study if 
they are patients of Dr. Marshall. Once identified, the research staff in Rutgers will contact the 
patient and guardian via a phone call to provide an overview of the study, requirements for research 
participation, and review eligibility for the study (Appendix A). The study candidates and guardians 
will be informed whether they may be eligible for the study, and if so, a virtual informed consent 

 
 
 
 
Protocol Title: Gut microbiota and hyperphagia in PWS 
Protocol Version Date: October 7, 2022 
 
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interview with the research team will be scheduled (Appendix B). Female candidates will be given 
two pregnancy test kits (Rapid Detection Pregnancy Test, Clearblue, Geneva, Switzerland) via mail 
and will perform the test on the day of the fMRI.  
 
INFORMED CONSENT AND ELIGIBILITY SCREENING PART 2. The informed consent process 
will be conducted virtually through Rutgers Zoom, which is HIPPA protected. The study coordinator 
will explain the rationale, aim, expected outcomes significance of the research, the experimental 
procedures, what is required from the participants, and the benefits and risks associated with the 
study. Informed consent form and consent for health information release will be signed during the 
interview using DocuSign, a HIPPA compliant platform during the interview (Appendix B). The 
study candidates will receive copies of both signed documents. Upon completing these documents, 
the study coordinator will ask some additional questions regarding their medical history and other 
conditions to establish eligibility. Candidates will perform and present proof of a negative pregnancy 
test result the first study session.  
 
TESTING AND INTERVENTION. Participants will attend a baseline visit at the Center for Advanced 
Human Brain Imaging Research (CAHBIR) at Rutgers University, during which functional magnetic 
resonance imaging (fMRI) coupled with a meal test will be performed to assess peripheral and 
central feeding pathways (7, 8). fMRI scans will be performed during resting state to assess 
functional connectivity of feeding related networks, with a specific focus on connectivity between 
the hypothalamus, insula, anterior cingulate, ventromedial/orbitofrontal prefrontal cortex and 
amygdala (9, 10). Activation to food (vs non-food) images will be assessed to index responsivity to 
appetitive feeding networks including the above regions and the ventral striatum using paradigms 
that have previously been found to be sensitive to trait and state in multiple studies (11-13). 
Thereafter participants will consume a liquid meal (525 kcal; Ensure Plus, Abbott). fMRI will be 
repeated immediately after the meal. Participants will attend a second visit at the IFNH, during 
which laboratory work coupled with a meal test will be performed to assess satiety hormones and 
inflammatory markers. Fasting blood draw will be taken, and the participants will consume a liquid 
meal (525 kcal; Ensure Plus, Abbot) followed by blood draws at 30, 60, 120, 180 and 240 min post-
meal. Upon completion of baseline testing and providing a fecal sample, participants will consume 
NBT-NM108 (a mixture of inulin, Fibersol-2, and brans of oat, wheat, corn and sorghum; Notitia 
Biotechnologies) daily for 4 weeks. At the completion of the 4-week intervention, all sampling and 
testing will be repeated as per baseline. At baseline and end of intervention, participants will be 
interviewed with appetite and physical activity questionnaires (Appendix C and G) and requested 
to complete a 24-h food recall using MyFitnessPal, a mobile health app on their phone. 
 
B. Data Points 
- 
Gut microbiota composition: fecal samples will be collected at weeks 0 and 4. 
- 
fMRI scans will be completed at baseline and end of intervention. 
- 
Blood parameters (satiety hormones and inflammatory markers) at minutes 0, 30, 60, 120, 180, 
and 240 post-meal at week 0 and week 4. 
- 
Appetite and physical activity questionnaires at weeks 0 and 4.  
- 
Calorie count calculated via 24-h food recall on MyFitnessPal at weeks 0 and 4. 
 
C. Study Duration 
It will take each participant 28 days to complete the study. The overall duration of the study is one 
year.  
 
D. Endpoints 
1. Primary Outcome  
- 
Gut microbiota composition at Weeks 0 and 4 
2. Secondary outcomes:  

 
 
 
 
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- 
Weeks 0, 1, 2, 3, and 4 
a. Weight measured in pounds  
- 
Weeks 0 and 4 
a. Hormones related to satiety and hunger include ghrelin, peptide YY (PYY), leptin, glp-
1/GIP, insulin, glucose 
b. Inflammatory markers include c-reactive protein (CRP), interleukin 6 (IL-6) and tumor 
necrosis factor-alpha (TNF-), adiponectin   
c. 
Outcome of fMRI showing responsivity of appetite feeding networks in the brain 
d. Caloric intake recorded in MyFitnessPal application 
e. Appetite behavior measured by hyperphagia questionnaire and visual analog scale (VAS) 
f. 
Physical activity measured by physical activity questionnaire 
 
1.4 Preliminary Data 
 
Dietary fibers modulated gut microbiota and 
conferred clinical benefits in PWS patients. 
Children aged 3-16 years old (N=17 with PWS 
and N=12 with simple obesity) were prescribed 
a diet enriched with wholegrains, traditional 
Chinese medicinal foods and prebiotics (WTP 
diet) for 30-90 days (5). This diet was 
specifically formulated to include a large 
amount 
of 
dietary 
fibers 
with 
diverse 
physicochemical 
structures 
to 
maximize 
carbohydrate 
fermentation 
by 
the 
gut 
microbiota. The diet induced significant and yet 
similar shifts in the overall gut microbiota structure in both groups, an effect associated with improvements 
in metabolic parameters including body weight and glucose homeostasis (Figure 1). Importantly, based on 
a hyperphagia questionnaire, we observed a modest but non-significant improvement in the overall food-
related behavior in PWS patients (P=0.074).  
Our network analysis identified guild-level 
structure in the gut microbiota (Figure 2). When 
supplemented with dietary fiber, gut bacteria 
exhibited co-abundance patterns and formed 
genome interaction groups (GIGs) or “guilds” 
(an ecological group in which members exploit 
an environmental resource in a similar way and 
show co-abundant behavior). Three guilds, 
which included strains of beneficial bacteria 
such as Bifidobacterium were negatively 
correlated with body weight and HbA1c, while 9 
guilds which contained many opportunistic 
pathogens such as LPS producers were 
positively associated with disease parameters. 
Taken together, we showed that high-fiber diet 
was a feasible and effective way to modulate 
gut 
microbiota 
in 
PWS 
patients, 
which 
conferred clinical improvements and may also 
alleviate hyperphagia.  
Interestingly, transplantation of the pre- and post-intervention gut microbiota from the same patient to germ-
free mice induced opposite responses in the recipient mice. While these mice had no genetic defects and 
were maintained on a standard chow diet, receiving the pre-intervention PWS gut microbiota alone was 
sufficient to induce inflammation, excessive fat accumulation and liver steatosis whereas the post-

 
 
 
 
Protocol Title: Gut microbiota and hyperphagia in PWS 
Protocol Version Date: October 7, 2022 
 
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intervention gut microbiota was without effect. This piece of causality evidence showed that at least some 
of the major metabolic phenotypes associated with PWS can be transferred to germ-free mice along with 
the transplanted gut microbiota (5, 15).  
 
Dietary fibers selectively 
promoted 
acetate 
and 
butyrate producers and 
structured a healthier gut 
ecosystem. Patients with 
type 
2 
diabetes 
were 
randomized to receive either 
the high-fiber WTP diet (W 
group; N=27) or usual care 
(U group; N=16) for 84 days 
(16). 
We 
identified 
141 
bacterial genomes that had 
the 
genetic 
capacity 
for 
fermenting 
dietary 
fibers. 
Intriguingly, only 15 of them 
were able to take advantage 
of the fibers and increased 
their abundance, while 79 
remained unchanged and 47 
were suppressed during the 
high-fiber intervention. All 15 
positive 
responders 
possessed the pathways to 
produce acetate, and some 
could also produce butyrate. 
When the gut microbiota was 
dominated by these bacteria, there was a concomitant reduction in bacteria that produced metabolically 
detrimental compounds (e.g. LPS, hydrogen sulfide and indole) and collectively these microbial changes 
led to improved metabolic outcomes (Figure 3). Acetate and butyrate can stimulate the production of GLP-
1 and PYY; LPS can induce systemic inflammation; indole and hydrogen sulfide can inhibit the production 
of GLP-1 and PYY (17, 18). Shifting of the abundance of producers of these bioactive compounds may 
drive the improvement of metabolic phenotypes. It would be interesting to assess if appetite regulation and 
feeding behavior are also modulated by these high fermentable fiber-induced changes in gut microbiota.  
 
1.5 Sample Size Justification 
No power calculation is performed for this pilot study. We aim to recruit 10 patients with hyperphagia(5 
male and 5 female) to capture a reasonable degree of variations in gut microbiota composition.  
 
1.6 Study Variables 
 
A.   Independent Variables, Interventions, or Predictor Variables  
NBT-NM108 
 
B.  Dependent Variables or Outcome Measures  
Gut microbiota composition, weight, fMRI, total caloric intake, hyperphagia behavior, physical 
activity behavior and hormones of satiety  
 
 
1.7 Drugs/Devices/Biologics  

 
 
 
 
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Protocol Version Date: October 7, 2022 
 
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A. Schedule and Administration 
Participants will receive NBT-NM108 prepared as muffin (each contains 30 g of the product) for 4 
weeks. The dosage will be 2 muffins a day. This dosage of NBT-NM108 will provide 24 g/day of 
dietary fibers of diverse physicochemical structures as sources of fermentable carbohydrates for 
the gut microbiota. Participants will be given a supplement diary to keep track of consumption of 
muffins along with potential side effects (Appendix H). 
  
B. Drug/Device Accountability and Storage Methods 
NBT-NM108 will be manufactured by Safe Sterilization USA (138 Sylvania Place, South Plainfield, 
NJ), which is an FDA FSMA compliant GMP/NSF certified third party toll manufacturing facility and 
supplied by Notitia Biotechnologies. NBT-NM108 will be prepared into muffins by a ready to eat 
manufacturer and provided to participants. 
 
1.8 Specimen Collection 
 
A. Primary Specimen Collection 
▪ 
Types of Specimens:  
1) Stool. Each participant will be provided with kits to collect fecal samples at home in Week 
0 and Week 4. The fecal sample collection kit has been cleared by FDA for at-home use. 
Instructions and supplies will be provided to collect these samples at home (Appendix D). 
2) Blood. Venous blood draws (10 ml each) will be taken during the baseline and Week 4 
post-intervention meal tests (T=0, 30, 60, 120, 180 and 240 min of each test) at IFNH by 
qualified professionals including phlebotomists, registered nurses and physicians. 
 
▪ 
Annotation: Data to be annotated or associated with each specimen will include participant 
code, age, sex, ethnicity, medical history, and medications. 
 
▪ 
Transport:  
1) Stool. Participants will return the samples by mailing them to the research team in prepaid 
shipping boxes. They will either schedule a FedEx pickup or drop off the package at a local 
FedEx office. The shipping packages are DOT (Department of Transportation) and IATA 
(International Air Transport Association) compliant. 
2) Blood. The samples will be processed and analyzed by Dr. Malin’s lab located at the IFNH.  
 
▪ 
Processing:  
1) Stool. Samples will be processed by members of PI Dr. Zhao’s lab listed in this protocol. 
2) Blood. Samples will be processed by  Dr.Malin’s laboratory. 
 
▪ 
Storage: All samples will be stored in locked -80°C freezers in laboratories in Lipman Hall (76 
Lipman Dr, New Brunswick, NJ 08901) and New Jersey Institute for Food, Nutrition and Health 
(61 Dudley Rd, New Brunswick, NJ 08901) of Rutgers University until analysis. Samples will 
only be accessible by authorized research personnel named in this protocol. Specimens will 
be stored until they are no longer in a fit state for scientific analyses. 
 
▪ 
Disposition: When specimens are no longer in a fit state for scientific analyses, they will be 
destroyed by incineration by authorized research personnel named in this protocol. 
 
B. Secondary Specimen Collection 
Not applicable.  
 
1.9 Data Collection 
 

 
 
 
 
Protocol Title: Gut microbiota and hyperphagia in PWS 
Protocol Version Date: October 7, 2022 
 
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A. Primary Data Collection 
▪ 
Location:  
o 
Participants will be interviewed with Appetite and physical activity questionnaires and 
diet 24-hour recall at the clinical room at IFNH building. 
o 
fMRI will be conducted at the CAHBIR (116 RWJMS Staged Research Building).  
o 
Lab draws will be performed at IFNH or CAHBIR  
▪ 
Process of Data Collection:  
o 
Study coordinators will interview participants with questions in the appetite and 
physical activity questionnaires and for 24-hour recall. 
o 
fMRI Session: Participants will complete an online MRI safety screening form prior to 
the study day (Appendix E). The form must be reviewed by the certified MRI 
Technologist prior to entry into the scanner room. The individual will be positioned in 
the magnet and will receive an anatomical scan followed by resting state and food 
picture fMRI scans. They will then be taken out of the scanner, consume a meal and 
then have a brief anatomical scan for purposes of alignment with their high-resolution 
MRI and a repeat of their fMRI scans. 
▪ 
Timing and Frequency: The interviews and scanning will be conducted at Week 0 and Week 
4. 
▪ 
Procedures for Audio/Visual Recording: N/A 
 
Study Instruments:  
▪ 
See Appendix C for the appetite questionnaire  
▪ 
See Appendix G for the physical activity questionnaire 
▪ 
MyFitnessPal application will be used to calculate caloric intake from 24-h diet recall 
 
Ethnographic Studies, Interviews, Or Observation: N/A 
 
Subject Identifiers: participants' names and birthday will be linked with the data collected. 
 
B. Secondary Data Collection  
NA 
▪ 
Type of Records: NA 
▪ 
Location: NA 
▪ 
Inclusion/Exclusion: NA 
▪ 
Data Abstraction Form(s): NA 
 
1.10 Timetable/Schedule of Events 
We expect to complete this trial within one year upon institutional approval. Detailed timeline is as follows: 
 
 
Preparations 
M1 
M2 
M3 
M4 
M5 
M6 
M7 
M8 
M9 
M10 
M11 
M12 
Institutional 
approval, staff 
training and material 
development 
 
 
Recruitment 
 
 
 
Intervention, sample 
measurement and 
clinical data 
collection 
 
 
 
Bioinformatics and 
data analysis 
 
 
 
Results 
dissemination 
 
 
 

 
 
 
 
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2.0 Project Management 
 
2.1 Research Staff and Qualifications 
PI Dr. Keerthana Kesavarapu (DO) is the Director of Medical Obesity and Associate Professor of 
Gastroenterology and Hepatology at Rutgers-Robert Wood Johnson Medical School. She is a board-
certified specialist in adult gastroenterology and obesity. Dr. Kesavarapu will actively recruit patients, 
participate in helping the study team design the assessment visit protocol, problem-solve with the study 
team on any issues that may arise and assist in analysis and interpretation of data. 
 
Co-principal investigator Dr. Liping Zhao (BSc, MSc, PhD) is the Eveleigh-Fenton Chair of Applied 
Microbiology in the School of Environmental and Biological Sciences and the Director of Center for Nutrition, 
Microbiome & Health at New Jersey Institute for Food, Nutrition and Health (IFNH). Dr. Zhao’s research 
applies molecular and genomic tools in understanding systems biology and predictive manipulation of the 
gut microbial communities, with his recent research primarily focuses on using dietary interventions to 
restore and maintain a health gut microbiota as a key strategy to improve metabolic health. With extensive 
experience in using high-fiber diets in clinical trials to modulate the gut microbiota, Dr. Zhao leads 
experimental design and will contribute to data analysis and interpretation.  
 
Co-Investigator Dr. Steven Malin (PhD) is an Associate Professor in the Department of Kinesiology & 
Health with a dual appointment in the Department of Medicine. He is also the Director of the Applied 
Metabolism & Physiology Laboratory. He has published several studies that examined the effects of 
exercise, diet, pharmacology and weight loss surgery on insulin sensitivity, appetite regulation and clinical 
outcomes. 
 
Co-Investigator Dr. David Zald (PhD) is the Director of CAHBIR at Rutgers Brain Health Institute. His 
research focuses on understanding the neural and neuropharmacological substrates of emotion and 
cognition, and the manner in which individual differences in the functioning of these systems impacts 
temperament, personality and psychopathology. Dr. Zald will provide supervision of stimulation paradigms 
and analysis and lead the interpretation of neuroimaging results. 
 
Co-Investigator Dr. Ian Marshall (MD) is the Chief of the Division of Pediatric Endocrinology Division and 
Associate Professor of Pediatrics at Rutgers-Robert Wood Johnson Medical School. He is a board-certified 
specialist in Pediatric Endocrinology and have spent over 15 years in the care of pediatric and adult patients 
with PWS. Dr. Marshall will actively recruit patients, participate in helping the study team design the 
assessment visit protocol, problem-solve with the study team on any issues that may arise regarding any 
aspects of the study, and assist in the analysis and interpretation of data 
 
Dr. Guojun Wu (BSc, PhD) is a Postdoctoral Research Associate of the Zhao Lab at the Department of 
Biochemistry and Microbiology. Dr. Wu was awarded his PhD in Microbiology from Shanghai Jiao Tong 
University (China) in March 2018. With initial training in bioinformatics, his research primarily focuses on 
mining high-throughput sequencing datasets to dissect the interactions between gut microbial community, 
diet and human health. Dr. Wu will lead bioinformatics and statistical analysis.  
 
Dr. Shreya Ghosh (BSc, MSc, PhD) is a Postdoctoral Research Associate in the Zhao Lab at the 
Department of Biochemistry and Microbiology. Dr. Ghosh was awarded her PhD in Microbiology from 
Shanghai Jiao Tong University (China) in March 2022. She has experience in designing and conducting 
animal experiments to investigate the role of dietary interventions in metabolic health by interacting with the 
gut microbiota.  
 
Graduate Students Tristan Ragland, Ying Wang, Rishika Navlani, Matt Pelton and Yongjia Gong 
contribute to experimental design and research material development. They will assist with screening, 
informed consent, data monitoring, follow-up phone calls, sample processing and data analysis. 

 
 
 
 
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2.2 Research Staff Training 
All investigators have successfully completed the Collaborative Institutional Training Initiative (CITI) 
training.  
2.3 Other Resources  
Gut microbiome analysis will be performed at the Microbiome Core of the New Jersey Institute for Food, 
Nutrition and Health of Rutgers University. The Core owns a next-generation sequencing platform (Ion 
GeneStudio S5, ThermoFisher Scientific) to perform 16S rRNA gene sequencing and a 2X Intel Xeon 
Skylake 6130 Node (22M Cache, 2.666 MHz, 192 GB RAM, 5 TB storage) of the high-performance 
computing cluster Amarel at Rutgers University for statistical analyses and bioinformatics. fMRI will be 
performed at CAHBIR of the Robert Wood Johnson Medical Center. The proposed study will have access 
to a state-of-the art Siemens 3T MRI scanner which allows for high resolution multi-band collection 
optimized for imaging regions in ventral brain regions involved in feeding. The image processing facilities 
at CAHBIR is connected to Rutgers’ high-performance computing facilities to allow rapid parallel processing 
of imaging data. 
 
2.4 Research Sites 
- 
Brain Health Institute (683 Hoes Lane West, Piscataway, NJ 08854) 
- 
New Jersey Institute for Food, Nutrition and Health (61 Dudley Rd, New Brunswick, NJ 
08901) 
- 
Zhao Lab (76 Lipman Dr, New Brunswick, NJ 08901) 
 
3.0 Multi-Center Research 
Not applicable 
 
4.0 Subject Considerations 
 
4.1 Subject Selection and Enrollment Considerations  
 
A. Recruitment Details 
Patients with the diagnosis of PWS and/or hyperphagia will be recruited from Dr. Ian Marshall’s 
pediatric endocrinology RWJMS clinic and DEEP6 software.  
 
B. Subject Screening 
▪ 
Inclusion Criteria 
o 
Aged between 18-45 (inclusive) 
o 
Confirmed PWS with genetic testing or documented ICD9/10 coding for hyperphagia 
o 
No growth hormone treatment in the previous 6 months  
o 
Body weight < 300 lbs.  
▪ 
Exclusion Criteria 
o 
History of other gastrointestinal disorders such as small intestinal bacterial 
overgrowth, celiac disease, inflammatory bowel disease, or irritable bowel syndrome. 
o 
Pregnancy or breastfeeding 
o 
Prior gastrointestinal or bariatric surgery 
o 
Immunocompromised e.g., cancer treatment, bone marrow/organ transplant, immune 
deficiency, poorly controlled HIV/AIDS, prolonged use of steroids or other 
immunosuppressant medications 
o 
Antibiotic administration in the previous 30 days 
o 
Administration of pre/probiotic supplements or antibiotics.  
o 
Growth hormone administration in the previous 6 months 
o 
Have access to a smartphone, tablet, computer, or other qualifying internet-enabled 
device and be able to follow instructions. 

 
 
 
 
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o 
Individuals who are not proficient in English 
o 
Contraindications for MRI scanning, including Ferrous material implanted in or on the 
body, including flakes or filings, surgical clips, bullets, or electrical devices such as a 
pacemaker, or nonremovable ferrous jewelry (fillings in teeth and permanent 
retainers are permitted). Individuals with surgical pins or plates above the neck are 
excluded. Surgical pins or plates below the neck are exclusions, except when the 
material is fixed to bone, and considered acceptable by the Reference Manual for 
Magnetic Resonance Safety. Implants and Devices, 2020 Edition. Almost all recent 
orthopedic implants are made of materials that are not ferromagnetic and therefore 
are safe for scanning, and even though some screws are still made of ferromagnetic 
materials these are firmly screwed into bone. In cases where the material is unknown 
or deemed unsafe for scanning by the Reference Manual for Magnetic Resonance 
Safety. Implants and Devices, the participant will be excluded. History of eye injury 
involving metallic materials, shavings in eyes, or welding without a face mask. 
Lead/iron tattoos and tattoos performed by a nonprofessional artist if the pigment 
material is unknown. Claustrophobia (history of significant anxiety in closed places).  
Back problem that would prevent the subject from laying still comfortably for up to 60 
minutes.  
 
C. Privacy Protections 
Should any participants desire not to interact with or provide personal information to specific 
members of the research team, they may report such a need to the PI and the arrangement of the 
research personnel will be adjusted accordingly. Participants will not be required to provide a 
reason if they desire not to do so. If changing the research personnel is not possible, the PI will 
discuss with the subjects about alternative solutions.  
  
Each participant will be assigned a unique identification code. A master code identifier which links 
the identification code and personal information will be maintained separately from the study data. 
Subject information is only accessible to named investigators in this protocol and authorized 
research personnel. All data and materials collected during this study are for research purposes 
only, and the data will be kept in strict confidence. De-identified information and biospecimens 
collected for this research may be used by or distributed to investigators for other research without 
obtaining additional informed consent from you. 
 
Individuals must include their name on the MRI screening form, which includes private health 
information. This form is only visible to the study staff and the MRI technologist.  
 
4.2 Obtaining Identifiable Information About Non-Subjects 
Not applicable 
 
4.3 Number of Subjects 
 
A. Total Number of Subjects 
10 patients; 5 male and 5 female 
B. Total Number of Subjects If Multicenter Study 
N/A 
C. Feasibility 
DEEP6 identified 26 patients via EPIC system who had the diagnosis of PWS with only 5 patients 
meeting our inclusion criteria. For this reason we are expanding our diagnosis coding to include 
hyperphagia syndrome. Given hyperphagia is a hallmark feature of PWS and the particular behavior 
we are aiming to study, this expansion should improve our subject pool and allow us to enroll 10 
patients. 

 
 
 
 
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4.4 Consent Procedures 
 
A. Consent Process 
▪ 
Location of Consent Process 
The electronic consent process will take place during virtual interviews on Rutgers Zoom, a 
HIPPA compliant platform. Individuals who are willing to participate in this study will provide 
their electronic informed consent along with their guardians on DocuSign, a HIPPA compliant 
platform during the interview.  
 
▪ 
Ongoing Consent 
Not applicable 
 
▪ 
Individual Roles for Researchers Involved in Consent 
Study coordinators will conduct a consent interview to explain all aspects of the study and 
answer any questions the prospective participants may have. 
 
▪ 
Consent Discussion Duration 
The consent discussion will take approximately 30 min. 
 
▪ 
Coercion or Undue Influence 
In order to minimize the possibility of coercion or undue influence, the consent process will 
not be conducted by research personnel who have any known relationship or conflict of 
interest with the prospective participants. 
 
▪ 
Subject Understanding 
Throughout the consent process the prospective participants will be encouraged to ask any 
questions should they feel the need to do so. The consent process will be divided into a 
series of small sections. Members of the research team will summarize each section, use 
probe questions to check for sufficient understanding before proceeding to the next section.  
 
▪ 
Protecting Privacy 
Should any participants desire not to interact with or provide personal information to specific 
members of the research team, they may report such a need to the PI and the arrangement 
of the research personnel will be adjusted accordingly. Participants will not be required to 
provide a reason if they desire not to do so. Should changing of the research personnel is not 
possible, the PI will discuss with the subjects about alternative solutions.  
 
Each participant will be assigned a unique identification code. A master code identifier which 
links the identification code and personal information will be maintained separately from the 
study data. Subject information and de-identified study data are only accessible to named 
investigators in this protocol and authorized research personnel. All data and materials 
collected during this study are for research purpose only, and the data will be kept in strict 
confidence. No information will be given to anyone without permission from the subjects. 
Should an incidental finding appear on the subject’s MRI, the MRI will be shared with a 
neuroradiologist from University Radiology Group. No name will appear on the MRI to protect 
the participant’s privacy. 
 
B. Waiver or Alteration of Consent Process  
▪ 
Waiver or Alteration Details 
We are requesting an alteration of the consent process. Prospective participants will be 
provided with a copy of the consent form prior to a virtual consent interview. During this 
interview, the study coordinator will go through the contents of the consent form with the all 

 
 
 
 
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applicable parties (subject, surrogate, witness, and research team member) will provide 
signatures on the consent form if the prospective participants are willing to provide information 
for eligibility screening. This is being done to minimize in-person contact during the pandemic. 
The alteration refers to the utilization of virtual consent. 
 
▪ 
Destruction of Identifiers 
The master code identifier, which contains personal information of subjects and the participant 
code assigned to them, will be deleted from the server 6 years after study completion. 
 
▪ 
Use of Deception/Concealment 
Not applicable 
 
C. Documentation of Consent 
▪ 
Documenting Consent 
Prospective participants and the study coordinator who conducts the consent process will 
view and provide electronic signatures on the consent form in real-time through DocuSign, 
and both parties will keep a copy of the form.  
 
▪ 
Waiver of Documentation of Consent (i.e., will not obtain subject’s signature) 
Not applicable 
 
4.5 Special Consent Populations 
 
A. Enrolling Minors-Subjects Who Are Not Yet Adults  
N/A 
B. Enrolling Wards of the State 
N/A 
C. Enrolling Non-English-Speaking Subjects 
N/A 
D. Enrolling Adults Lacking Decision-Making Capacity (Surrogate Consent) 
Given the nature of PWS, all patients will lack decision-making capacity and require 
surrogate consent.  
 
Subjects will be assessed by Dr. Kunal Shah, not affiliated with the study team. He will 
assess if they are able to understand the details of the research and if she/he can make a 
decision about taking part in the study. He will document this determination in the electronic 
medical record. If the answer is yes, consent will be obtained by the individual.  
 
If the subject does not have capacity to make informed decisions, Dr. Marshall will be notified 
and will obtain their Advance Directive from the patient or surrogate who has accompanied 
them to their clinic visit.  If the advanced directive exists and indicates that the potential 
subject would not want to take part in proposed medical research, the subject will not be 
considered for the study. If the advanced directive does not exist or does not indicate that the 
subject would object to taking part of proposed medical research, The determination of 
consent will be provided to the patient and a surrogate.  
 
Surrogate will be chosen from the highest category of surrogate decision-making priority. The 
surrogate will be contacted by phone. If one or two or more available persons in the same 
order of priority expresses opposition to the participation of the subject in the study, the 
investigator must exclude the subject from the study. If two or more available persons are in 
different orders of priority, refusal to consent by a potential surrogate who is of higher priority 
controls and will not be superseded by the consent of a person who is of a lower priority.  

 
 
 
 
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Once identified, the research staff in Rutgers will contact the surrogate via a phone call to 
provide an overview of the study, requirements for research participation, and review 
eligibility for the study (Appendix A). If the surrogate provides assent to participation and 
candidates are eligible for the study, a virtual informed consent interview with the research 
team will be scheduled (Appendix B). Subjects who are able to understand the concept of 
participating in a particular study will be asked to assent to the study; those that are unable to 
understand the concept of participating in a particular study will not be asked to assent.  
For studies at RWJUH, the Chaplain is the designated witness for studies utilizing surrogate 
consent.  
If subject express dissent to participation or if a surrogate withdraws consent, subject will be 
withdrawn from the study. No further testing will take place and high-fiber supplement will be 
stopped.  
 
E. Special Consent Considerations 
N/A 
 
4.6 Economic Burden and/or Compensation for Subjects 
 
A. Expenses  
We do not expect any expenses the participants will incur by taking part in the research. The 
compensation should cover travel costs.  
 
B. Compensation/Incentives 
Compensation will be awarded via ClinCards. Participants will receive compensation for the 
following milestones: 
- 
$25.00 after the completion of sample collection and shipment at Day 0 
- 
$25.00 after the completion of sample collection and shipment at Day 28 
- 
$30.00 after the completion of questionnaire at Day 0 
- 
$30.00 after the completion of questionnaire at Day 28 
- 
$50.00 after the completion of fMRI examination at Day 0 
- 
$50.00 after the completion of fMRI examination at Day 28 
- 
$50.00 after the completion of blood draw at Day 0 
- 
$50.00 after the completion of blood draw at Day 28 
 
C Compensation Documentation 
I Compensation will be awarded via ClinCards. Compensation will be tracked utilizing an excel sheet, which 
will also be stored in an encrypted, password-protected file.  
 
. 
  
4.7 Risks of Harm/Potential for Benefits to Subjects 
 
A. Description of Risks of Harm to Subjects 
▪ 
Reasonably Foreseeable Risks of Harm 
Measuring fasting blood glucose during the study both involves a finger prick that pierces the 
skin. There may be a bruise, bleeding, or infection, at the place where the skin is pierced. 
However, infection is rare. Collecting fecal samples at home may impose inconvenience for the 
participants and their families. Participants taking NBT-BN108 may experience gastrointestinal 
symptoms (e.g. abdominal pain/discomfort, constipation, or bloating). These discomforts are 

 
 
 
 
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minor and should not last longer than a few hours. This product is low in digestible and simple 
carbohydrates and may reduce food intake.  
There are no known health risks associated with the magnetic field produced by this 3.0T 
scanner in healthy adults. The FDA has indicated that they consider MR imaging on 
machines up to 4.0T to pose no risk (https://www.fda.gov/medical-devices/cdrh-research-
programs/magnetic-resonance-imaging-mri-safety-and-effectiveness). Sammet (2016) 
provides discussion of MRI safety issues, each of which can be avoided with proper 
management. These include:1) attractive forces and torque caused by the magnet, 2) thermal 
effects caused by the radiofrequency fields, 3) peripheral nerve stimulation due to switching 
gradients, 4) hearing damage caused by the acoustic noise of the gradient system, 5) 
questions of risk for a fetus, 6) claustrophobia and other discomforts. These are briefly 
detailed below.  
Attractive Forces: Ferrous material can be pulled toward the magnet, causing injury 
to a subject. These forces can cause metallic implants to be attracted to the magnet. The 
magnet can erase credit cards and other types of swipe cards.  
Thermal Effects: The radiofrequency field can cause heating of ferrous material that 
are in the field, this is a concern for metal implants, and tattoos on the skin that specifically 
contain metal (iron).  
Peripheral Nerve Stimulation (PNS): Although none of the planned scans involve 
parameters that should cause any peripheral nerve stimulation (tingling sensation caused by 
stimulation of a sensory nerve or motor twitch caused by stimulation of a motor nerve), it is 
hypothetically possible for this to occur in a scan in which magnetic gradients are being 
rapidly switched because the gradient cause a neuron near the surface of the skin or muscle 
to “fire”.  While the gradient performance of the magnet is not expected to produce PNS 
within the operational limits defined by Siemens, they remain a possibility for a small minority 
of sequences. Note that PNS is not considered an adverse event because when it occurs, it 
is an issue only of patient comfort rather than a safety concern.  
Hearing damage: The gradient system noise often reaches 100dB. Sustained 
exposure to this volume of noise without noise reduction devices can cause hearing damage. 
Claustrophobia, and  
 
Additional Discomforts: Some participants experience anxiety due to the enclosed 
nature of the space in the scanner and head coil. Some potential minor discomforts 
associated with the procedures include back discomfort from lying in one position.  
Incidental Findings: An incidental finding could arise during scanning. Learning of an 
incidental finding could cause psychological distress. 
 
▪ 
Risk of Harm from an Intervention on a Subject with an Existing Condition 
Although 3.0 T MRI is safe for healthy individuals several existing conditions, or situations can 
increase risk, or can increase the risk of discomfort. Persons at primary risk from exposure to the 
magnet include persons with metal surgical implants, and anyone with implanted electric/magnetic 
devices (e.g. a pacemaker, aneurism clips, IUDs). Such individuals are therefore excluded from the 
study.  
 
▪ 
Other Foreseeable Risks of Harm 
N/A 
 
▪ 
Observation and Sensitive Information 
N/A 

 
 
 
 
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B. Procedures which Risk Harm to Embryo, Fetus, and/or Pregnant Subjects 
N/A 
 
C. Risks of Harm to Non-Subjects 
Fecal sample collection at home may impose inconvenience for individuals in the same 
household. 
D. Assessment of Social Behavior Considerations 
N/A 
 
E. Minimizing Risks of Harm 
Participants will be provided with oral and written instructions and supplies to collect fecal samples 
at home without direct physical contact with the specimens throughout the process (Appendix D). 
The specimens will be stored in a sealed container, a leak-proof secondary package (a sealable 
plastic bag lined with absorbent pads) and an outer package (e.g. a shipping box). Participants are 
not required to store the samples at home; they will mail the specimens to the research team using 
the shipping supplies provided. 
To minimize adverse effects due to NBT-NM108, they will also be advised to drink enough fluids 
throughout the study (approximately eight 8-ounce glasses of fluid a day including the amount of water 
taken together with NBT-NM108) to avoid constipation. Participants will be asked to monitor potential 
adverse events daily. If participants report an adverse event, a member of the study team will contact 
them to ask follow-up questions about the degree of severity (mild, moderate, or severe) and the 
frequency of the adverse events. The study physician (PI Dr. Kesavarapu) will assess the adverse 
events and, if necessary, she will instruct the participants to discontinue treatment. 
Measuring fasting blood glucose during the study both involves a finger prick that pierces the skin. 
There may be a bruise, bleeding, or infection, at the place where the skin is pierced. However, 
infection is rare. To minimize adverse effects, venipuncture will be done by a certified 
venupuncturist.  
Minimizing risks from the fMRI 
Exclusion of individuals with contra-indications for scanning: Individuals at primary risk for the 
exposure to high magnetic fields due to the presence of potentially dangerous ferrous materials in 
the body are excluded. This eliminates the risk of attractive forces or thermal heating. Participants 
are told of these exclusions during recruitment and must complete a comprehensive screen of 
any potential metal on their body. This screening form is reviewed by a member of the team with 
level 2 training, and it is confirmed by the scanner operator before the participant is allowed in the 
scanner room. For participants who possess orthopedic screws or pins that are firmly in bone, 
participants can only be scanned if the type of screw or pin is considered safe according to the 
Reference Manual for Magnetic Resonance Safety. Implants and Devices, 2020 Edition 
Participants are withdrawn from the study if it is determined there is any contra-indication for 
scanning.  
 
Avoidance of other risks from the high magnetic field: The MRI suite is coded in terms of zones. 
The entire scanner suite has controlled access, and only individuals with appropriate training (see 
staff training) are allowed to permit people into the scanner control room or MRI and only after 
they have been screened. Subjects are reminded to remove any jewelry before entering the 
scanner room and are screened with a handheld metal detector before entering the scanner 
room. Subjects are given a box/locker to place their wallet, keys and other personal belongings 
before entering the scanner room. All study personnel have undergone safety training and must 
adhere to safety rules to limit potential risks in the scanner suite. 

 
 
 
 
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Even if a participant passes screening for the presence of ferrous implants or tattoos, participants 
with orthopedic hardware are told to squeeze a ball so that they can be removed from the 
scanner in the unlikely event that they feel sensations such as warming at the site of an 
orthopedic screw or tattoo.   
 
Avoidance of claustrophobia: Participants are excluded if they report a history of claustrophobia, 
and they are warned in advance that the space is cramped and that they will have a head coil 
placed around their head.  If an individual does become claustrophobic during the scan, they can 
inform the study staff and discontinue the study at any time. Subjects will be given a ball to 
squeeze so that they can stop the scan at any time.  
 
Avoidance of physical discomfort from lying in the scanner: Individuals with a history of chronic or 
recent acute back pain are excluded from the study. Subjects are warned that they will need to lie 
on their back in a still position for up to 90 minutes. Pillows and cushions are used to keep the 
subject comfortable and the scan operator always checks that the person is comfortable before 
closing the scan room door. If the subject does need to move during the scan, they can be 
repositioned as long as it is done between scans.  
 
Avoidance of discomfort from volume of the scanner: Subjects are given earplugs and noise 
reducing headphones to limit the volume of the scanner.  
Incidental Findings: If an incidental finding is observed during a scan, it will not be initially shown 
to the subject.  After scanning, the scan data will be electronically submitted to the University 
Radiology Group imaging server, where it will be read by a neuroradiologist who will provide a 
brief written report. If the neuroradiologist determines the scan has clinical significance that 
warrants attention, the PI will inform the participant and after a release of information form is 
completed, will provide the imaging data and report to a physician of the participant’s choice.  
 
Hospitalization during study 
If patients are hospitalized during the study, they will be removed from the study and discontinue 
all data and sample collection. Subjects will need to contact the study PI, Keerthana Kesavarapu, 
D.O. at (732)235-7784 if hospitalized during the study. 
 
▪ 
Certificate of Confidentiality 
N/A 
 
▪ 
Provisions to Protect the Privacy Interests of Subjects 
Should any participants desire not to interact with or provide personal information to specific 
members of the research team, they may report such a need to the PI and the arrangement of 
the research personnel will be adjusted accordingly. Participants will not be required to provide 
a reason if they desire not to do so. If changing of research personnel is not possible, the PI 
will discuss with the subjects about alternative solutions.  
Each participant will be assigned a unique identification code. A master code identifier which 
links the identification code and personal information will be maintained separately from the 
study data. Subject information and de-identified study data are only accessible to named 
investigators in this protocol and authorized research personnel. All data and materials 
collected during this study are for research purposes only, and the data will be kept in strict 
confidence. No information will be given to anyone without permission from the subjects. 
 

 
 
 
 
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F. Potential Direct Benefits to Subjects 
There are no known direct benefits to subjects.  
 
5.0 Special Considerations 
 
5.1 Health Insurance Portability and Accountability Act (HIPAA) 
We will ask participants for their consent so that designated members of the research team can review 
his/her medical record to retrieve medical history, medications, clinical and laboratory data related to 
PWS or hyperphagia. All received health information will be stored in an encrypted password-protected 
file which is stored on the university’s secure server and will only be accessible to authorized research 
personnel. 
 
5.2 Family Educational Rights and Privacy Act (FERPA) 
N/A 
 
5.3 Code of Federal Regulations Title 45 Part 46 (Vulnerable Populations) 
 
A. Special Populations 
N/A 
 
5.4 General Data Protection Regulation (GDPR) 
N/A 
 
5.5 NJ Access to Medical Research Act (Surrogate Consent) 
N/A 
 
6.0 Data Management Plan 
 
6.1 Data Analysis 
Microbiome sequencing data will be analyzed based on the amplicon sequence variants (ASVs) [28] to 
determine the gut microbiota composition using QIIME 2 [29]. Shannon index and ASV richness will be 
used to assess alpha diversity. Principal coordinate analysis will be used to compare and visualize 
dissimilarity in gut microbiota structure between samples based on beta diversity (Weighted and 
Unweighted UniFrac distance) and statistical differences will be tested using permutational multivariate 
analysis of variance (PERMANOVA). PICRUSt 2 will be used to predict the functions of individual ASVs 
and the collective functions of the gut microbiota at the gene content and pathway levels [30]. Global 
functional profiles of the gut microbiota will be compared and visualized by principal component analysis. 
Repeat measures correlation will be used to assess the relationships between ASVs [31], followed by ASV 
clustering based on co-abundance patterns. Multivariate methods such as MaAsLin2, that allow adjustment 
for confounding variables and model the covariates as random effects, will be applied to interpret the 
relationship between ASVs / ASV co-abundance groups / functions and the clinical metadata.  
 
Difference in continuous variables of metadata, such as measurements of hormones, blood inflammatory 
markers and total caloric intake, between week 0 and week 4 will be tested by Paired t-test (two-tailed) if 
meeting normal distribution or Paired Wilcoxon Signed-Rank test (two-tailed) if not meeting normal 
distribution. Difference in the continuous variables of metadata between male and female will be tested by 
Student’s t-test (two-tailed) if meeting normal distribution or Mann-Whitney test if not meeting normal 
distribution. Fisher’s exact test will be used to compare proportions of categorical metadata, such as data 
in the appetite and physical activity questionnaires, between male and female, and between Week 0 and 
Week 4. The false discovery rate will be applied for multiple testing, where appropriate. 

 
 
 
 
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Functional MRI data will be preprocessed with fMRI/PREP and analyzed with Statistical Parametric 
Mapping. Functional connectivity in the resting state will be extracted in the feeding related networks using 
the Conn Toolbox with a specific focus on connectivity between anatomically defined ROIs in the 
hypothalamus, insula, anterior cingulate, ventromedial/orbitofrontal prefrontal cortex, ventral striatum and 
amygdala. Activation to food (vs non-food) images will be assessed for responsivity in appetitive feeding 
networks by first modelling food stimulus presentations vs. non food presentations on a voxel wise basis 
and then extracting activations in anatomically defined regions of interest. The extracted data will then be 
analyzed as dependent variables following procedures like those described above.  
 
6.2 Data Security 
The study data will include two key components: 1) a master code identifier that contains the personal 
information of the participants and the participant code assigned to them; and 2) study data files that contain 
all the de-identified data generated in this trial. All received health information will be stored in an encrypted 
password-protected file which is stored on the university’s secure server and will only be accessible to 
authorized research personnel. The data will only be accessible to authorized research personnel. 
 
The master code identifier and the data files will be kept in two separate locations. The master code 
identifier is only accessible to the Principal Investigator and research personnel who schedule appointments 
and shipments. Data in hard copies and the master code identifier will be kept for 6 years after study 
completion, then any hard copies will be shredded, and the master code identifier will be permanently 
deleted from the server. As study data will then exist in de-identified form, the electronic data files will be 
kept indefinitely for utilization in future research. Data will not be distributed to other investigators for future 
research. 
 
6.3 Data and Safety Monitoring 
A. Data/Safety Monitoring Plan 
A Data and Safety Monitoring Board (DSMB) will approve the protocol before the study is initiated, 
and after 30% of the participants complete 14 days of the intervention to review study progress 
including recruitment, retention, adherence, adverse events and study outcomes, as well as to 
ensure that participant safety is addressed adequately. Reports of adverse events will be used to 
assess the safety of the study.   
 
B. Data/Safety Monitoring Board Details 
The DSMB will include Rutgers University faculty members with expertise in medicine, 
epidemiology and/or statistics who are not directly involved with the study. The Board will meet 
before the study is initiated, and after 30% of the participants complete 14 days of the intervention 
to review study progress and provide recommendations related to continuing, modifying or 
terminating the study. Any serious adverse event that is life-threatening or results in death, that is 
possibly, probably, or related to the research protocol, will trigger an immediate suspension of the 
research. All details about the DSMB are found in Appendix F.  
 
6.4 Reporting Results 
 
A. Individual Subjects’ Results  
No individual study results will be released to participants. 
B. Aggregate Results 
No aggregate study results will be released to participants. 
C. Professional Reporting 
Preliminary results will be presented in local and international academic conferences. The final study 
findings will be published in peer-reviewed academic journals. 

 
 
 
 
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D. Clinical Trials Registration, Results Reporting and Consent Posting 
This trial needs to be registered on ClinicalTrials.gov 
 
6.5 Secondary Use of the Data    
Fecal samples, blood samples, and de-identified data collected from participants may be stored in 
Lipman Hall, Rutgers University for future research by the principal investigator, 
 
7.0 Research Repositories – Specimens and/or Data 
 
N/A 
  
8.0 Approvals/Authorizations 
 
Approval from Institutional Biosafety Committee of Rutgers University for fecal sample processing and 
microbiota profiling has been obtained under Co-PI Dr. Zhao’s program of work titled “Personalized gut 
microbiota-targeted dietary intervention for patients with type 2 diabetes” at Rutgers University (#17-051). 
9.0 Bibliography 
 
1. 
Holland AJ, Treasure J, Coskeran P, Dallow J, Milton N, Hillhouse E. Measurement of excessive 
appetite and metabolic changes in Prader-Willi syndrome. Int J Obes Relat Metab Disord. 
1993;17(9):527-32. 
2. 
Fieldstone A, Zipf WB, Sarter MF, Berntson GG. Food intake in Prader-Willi syndrome and 
controls with obesity after administration of a benzodiazepine receptor agonist. Obes Res. 1998;6(1):29-
33. 
3. 
Proffitt J, Osann K, McManus B, Kimonis VE, Heinemann J, Butler MG, et al. Contributing factors 
of mortality in Prader-Willi syndrome. Am J Med Genet A. 2019;179(2):196-205. 
4. 
Martinez Michel L, Haqq AM, Wismer WV. A review of chemosensory perceptions, food 
preferences and food-related behaviours in subjects with Prader-Willi Syndrome. Appetite. 2016;99:17-
24. 
5. 
Zhang C, Yin A, Li H, Wang R, Wu G, Shen J, et al. Dietary Modulation of Gut Microbiota 
Contributes to Alleviation of Both Genetic and Simple Obesity in Children. EBioMedicine. 2015;2(8):968-
84. 
6. 
Lam YY, Maguire S, Palacios T, Caterson ID. Are the Gut Bacteria Telling Us to Eat or Not to Eat? 
Reviewing the Role of Gut Microbiota in the Etiology, Disease Progression and Treatment of Eating 
Disorders. Nutrients. 2017;9(6). 
7. 
Purtell L, Sze L, Loughnan G, Smith E, Herzog H, Sainsbury A, et al. In adults with Prader-Willi 
syndrome, elevated ghrelin levels are more consistent with hyperphagia than high PYY and GLP-1 levels. 
Neuropeptides. 2011;45(4):301-7. 
8. 
Holsen LM, Savage CR, Martin LE, Bruce AS, Lepping RJ, Ko E, et al. Importance of reward and 
prefrontal circuitry in hunger and satiety: Prader-Willi syndrome vs simple obesity. Int J Obes (Lond). 
2012;36(5):638-47. 
9. 
Lips MA, Wijngaarden MA, van der Grond J, van Buchem MA, de Groot GH, Rombouts SA, et al. 
Resting-state functional connectivity of brain regions involved in cognitive control, motivation, and 
reward is enhanced in obese females. Am J Clin Nutr. 2014;100(2):524-31. 

 
 
 
 
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10. 
Wijngaarden MA, Veer IM, Rombouts SA, van Buchem MA, Willems van Dijk K, Pijl H, et al. 
Obesity is marked by distinct functional connectivity in brain networks involved in food reward and 
salience. Behav Brain Res. 2015;287:127-34. 
11. 
Bruce AS, Holsen LM, Chambers RJ, Martin LE, Brooks WM, Zarcone JR, et al. Obese children 
show hyperactivation to food pictures in brain networks linked to motivation, reward and cognitive 
control. Int J Obes (Lond). 2010;34(10):1494-500. 
12. 
Frank S, Laharnar N, Kullmann S, Veit R, Canova C, Hegner YL, et al. Processing of food pictures: 
influence of hunger, gender and calorie content. Brain Res. 2010;1350:159-66. 
13. 
Stice E, Spoor S, Ng J, Zald DH. Relation of obesity to consummatory and anticipatory food 
reward. Physiol Behav. 2009;97(5):551-60. 
14. 
Dykens EM, Maxwell MA, Pantino E, Kossler R, Roof E. Assessment of hyperphagia in Prader-
Willi syndrome. Obesity (Silver Spring). 2007;15(7):1816-26. 
15. 
Wang R, Li H, Yang X, Xue X, Deng L, Shen J, et al. Genetically Obese Human Gut Microbiota 
Induces Liver Steatosis in Germ-Free Mice Fed on Normal Diet. Front Microbiol. 2018;9:1602. 
16. 
Zhao L, Zhang F, Ding X, Wu G, Lam YY, Wang X, et al. Gut bacteria selectively promoted by 
dietary fibers alleviate type 2 diabetes. Science. 2018;359(6380):1151-6. 
17. 
Chimerel C, Emery E, Summers DK, Keyser U, Gribble FM, Reimann F. Bacterial metabolite indole 
modulates incretin secretion from intestinal enteroendocrine L cells. Cell Rep. 2014;9(4):1202-8. 
18. 
Bala V, Rajagopal S, Kumar DP, Nalli AD, Mahavadi S, Sanyal AJ, et al. Release of GLP-1 and PYY in 
response to the activation of G protein-coupled bile acid receptor TGR5 is mediated by Epac/PLC-epsilon 
pathway and modulated by endogenous H2S. Front Physiol. 2014;5:420.