Johanna sets a date for her master’s thesis defense!

Prevention of Inflammatory Bowel Diseases by Broccoli Sourced and Microbially Produced Bioactives.

Presented by Johanna Holman in fulfillment of her Master’s of Science in Nutrition degree at the University of Maine. Jul 25, 2022 09:30 – 10:30 AM Eastern Time (US and Canada)

Register in advance to attend this presentation over Zoom, which will also be held in person in 206 Rogers Hall at the University of Maine (no RSVP required). After registering, you will receive a confirmation email containing information about joining the meeting.

About Johanna

I met Johanna in the fall of 2019, when I was just establishing myself as a new Assistant Professor at UMaine and she was looking for an advisor for a graduate degree.  Right away, she impressed me with her background and enthusiasm for research.  I learned that Johanna began her undergraduate study as an art student before transitioning fluidly to science.  The ability to design visual aids and graphical representations of data is hugely important to science and sadly, not always a skill that scientists are trained to do, and Johanna has made some incredible art for her research.  

Once she became a science student during her undergraduate study, she worked in the laboratories of Drs. Yanyan Li, previously an Associate Professor (of nutrition) in the College of Science and Humanities, and Tao Zhang, Assistant Professor of Basic Pharmaceutical Sciences, both of Husson University in Bangor.  There, she performed nutritional biochemistry, worked with mouse models, and developed an idea of what she wanted to study in graduate school and pursue as a career.  Johanna continues to work closely with both researchers, especially now that Dr. Li has taken a position at UMaine. 

Johanna and I continued to plan her graduate work and career goals, she officially joined my lab as a Master’s Student of Nutrition at UMaine in fall 2020, and immediately got to work.  Not only did she begin preparations for the massive undertaking that is part of her project, but she began mentoring several undergraduates on and off campus, and started as a first time teaching assistant for the Chemistry department, which required navigating virtual labs.  She served as a chemistry TA for academic year 20/21 and 21/22, with up to 60 students per semester. For the last year and a half, she has been coordinating a large-scale research project with investigators at 4 different institutions and undergraduate researchers from 3 different institutions, involving hundreds of samples – while being a masters student, a graduate teaching assistant, and mentoring undergrads in the lab, and all during a pandemic!  She managed that all so well, that despite being a first-year graduate student, she was awarded a 2020-2021 University of Maine Graduate Student Employee of the Year award, and the 2022 Norris Charles Clements Graduate Student Award from the College of Natural Sciences, Forestry, and Agriculture. 

Johanna’s project focuses on whether consumption of specific broccoli sprout preparations will elicit changes in the gut microbiota, to the effect of improving the production of microbiota-specific bioactives that have local anti-inflammatory effects, and promoting intestinal homeostasis by reducing dysbiosis. Broccoli sprouts represent an effective, and accessible way to add dietary intervention to existing treatment and prevention strategies for IBD patients. This project is a continuation of previous research on bioactive compounds in broccoli, completed in the labs of Drs. Yanyan Li and Tao Zhang at Husson University in Bangor.  While some of the work may be similar, the skill set she has gained in her graduate work is entirely new.  For the 2020/2021 winter break, Johanna was managing a 40-mouse study looking at DSS-treatment and different preparations of a broccoli sprout diet for 5 weeks, which resulted in hundreds of samples collected, hundreds of data time points, and enough follow-up laboratory and analysis work to keep her occupied for an entire year.  She has learned how to culture bacteria in an anaerobic chamber, which is a notoriously fussy machine that requires regular attention, as well as to grow them under different conditions for biochemical analysis and enzyme activity.  She is currently learning additional histology skills, DNA extraction, DNA sequencing library preparation, DNA sequence analysis, and more. Recently, she has participated in a pilot study to develop an immunological model of IBD, using IL-10 knockout mice. While IL-10 mice have been used to study IBD, they have never been applied in this way to study the interaction of diet, microbes, and disease.

She has presented this work at the American Society for Microbiology annual meeting, and at the UC Davis Research Experience for Undergraduates (REU) symposium, and has several conference presentations planned for 2022. Johanna has an author on a paper in early 2022 for work she contributed to as an undergraduate, and is preparing 3 manuscripts generated from her masters work which will be submitted for peer review at a scientific journal in 2022.

Johanna has been accepted to the Nutrition PhD program at UMaine, and will continue working with Dr. Li and I, as well as the full research team. Based on those preliminary results, Johanna’s doctoral work will focus on developing that new model, synthesizing information from both models, and using those results to develop diet intervention trials in human patients. After her PhD, Johanna intends to conduct research at an institution here in Maine, and to continue her work connecting the biochemistry of nutrition with gut microbiology and human health.

Student point of view on researching microbes, flying squirrels, and mice around farms in Maine

Five women taking a photo together at a farm.  They are standing a few feet apart from each other, and standing in front of a cow feedlot with two cows eating.

This summer, a collaborative project was launched by the Ishaq Lab, Danielle Levesque, and Pauline Kamath at UMaine Orono and Jason Johnston at UMaine Presque Isle; “Climate Change Effects on Wild Mammal Ranges and Infectious Disease Exposure Risk at Maine Farms.”

Funded by the University of Maine Rural Health and Wellbeing Grand Challenge Grant Program, this project assesses pathogen carriage by mice and flying squirrels on or near farms in several locations in Maine. We live-capture mice and flying squirrels in traps, collect the poop they’ve left in the trap, and conduct a few other health screening tests in the field before releasing them. To maximize the information we collect while minimizing stress and interference to the animals, information is being collected for other projects in the Levesque Lab at the same time. We will be collecting samples for another few weeks, and then working on the samples we collected in the lab over the fall and winter.

One of the major goals of the funding program, and this project, is to engage students in research. After a few months on the project, some of our students describe their role and their experiences so far…


A close-up of a deer mouse sitting in a live capture trap in the forest.  In the background is one of the researchers kneeling on the ground.

Marissa Edwards

Undergraduate in Biology

Levesque Lab

Hi! My name is Marissa Edwards and I am an undergraduate research assistant with Danielle Levesque. This summer, my role has been to set traps, handle small mammals, and collect fecal and tissue samples from deer mice.

A pine marten sitting in a live capture trap in a forest.

One of the skills I’ve learned this summer is how to properly ear tag a mouse. To catch mice, we set traps across UMaine’s campus as well as other parts of Maine, including Moosehead Lake, Flagstaff Lake, and Presque Isle.

During our trip to Moosehead Lake, I saw a marten for the first time (it was in one of our traps). I did not know martens existed and initially thought it was a fisher cat. It was both a cool and terrifying experience!


Northern flying squirrel sitting on a net with a forest in the background.

Elise Gudde

Master’s Student of Ecology and Environmental Sciences

Levesque Lab

Hello, my name is Elise Gudde, and I am currently a master’s student at the University of Maine in the Ecology and Environmental Sciences program. I work in Dr. Danielle Levesque’s lab studying small mammal physiology in Maine.

Northern flying squirrel sitting on a net with a forest in the background.

This summer, as a part of the squirrel project, I work to trap small mammal species in Maine, such as white footed mice, deer mice, and flying squirrels in order to determine which species have shifted their range distributions as a result of climate change. Being a part of the research team, this summer has brought me all over Maine! I have been able to travel to Orono, Greenville, New Portland, and Aroostook County to study many interesting mammals. I even got to handle an Eastern chipmunk for the first time! As a member of the animal-handling side of the research team, I also collect fecal and tissue samples from the animals. These samples are then handed off for other members of the team to research in the lab!


Rebecca French wearing a white laboratory coat, a fabric face mask, and beige latex gloves while using a yellow plastic loop tool to spread bactrial cultures on fresh agar media plates to look for growth.  Rebecca is sitting at a biosafety cabinet with the glass window slide down between her and what she is working on.  Assorted scientific materials can be seen in the background.

Rebecca French

Undergraduate in Animal and Veterinary Sciences

Ishaq Lab

In the beginning of this project, I had no idea what I was getting myself into when I began researching flying squirrels and mice. I came into it with almost no in-person lab experience, so I had a lot to learn.

So far, I have been focusing on making media on petri dishes for culturing bacterial growth and after plating fecal bacteria on said plates; discerning what that growth can be identified as.

We are using media with specific nutrients, and colored dyes, and certain bacteria we are interested in will be able to survive or produce a color change. I have also been performing fecal flotations and viewing possible eggs and parasites under a microscope. What I’ve found most fun about this project is putting into practice what I have learned only in a classroom setting thus far. It is also very satisfying to be a part of every step of the project; from catching mice, to making media, to using that media to yield results and then to be able to have a large cache of information to turn it all into a full fledged project.


Joe Beale, posing for a photo in an open office space.

Joe Beale

Undergraduate in Animal and Veterinary Sciences

Kamath Lab

Hello! My name is Joseph Beale, and I am an undergraduate at the University of Maine working on the squirrel project as a part of my capstone requirement for graduation. My primary responsibility in this project is the molecular testing of samples obtained from the field. Primarily I will be working with ear punch samples taken from flying squirrels and field mice. DNA extracts from these field samples will be run via qPCR. The results of this qPCR will tell us if these squirrels are carrying any pathogens. 

The pathogens we will be testing for are those found in Ixodes ticks. The qPCR panel which we will be running the extracted DNA from the ear punches on tests for Borrelia burgdorferi, the causative agent of Lyme disease, Anaplasma phagocytophilum, the causative agent of anaplasmosis, and Babesia microti, the causative agent of Babesiosis. These pathogens and respective diseases discussed are all transmitted through Ixodes ticks. Deer ticks are the most common and famous of the Ixodes genus. The Ixodes genus encapsulates hard-bodied ticks. Along with deer ticks, Ixodes ticks found in Maine include: woodchuck ticks, squirrel ticks, mouse ticks, seabird ticks, and more. Mice and squirrel are ideal hosts for these Ixodes ticks, therefore becoming prime reservoirs for these diseases. In our research, we are interested in determining the prevalence of these diseases in squirrels and mice as these hosts can spread these diseases to humans and other animals in high tick areas. 

qPCR, quantitative polymerase chain reaction, allows for the quantification of amplified DNA in samples.  This will help tell us if these pathogens are present in samples and in what capacity. In qPCR provided DNA strands are added to the reaction. These strands match with the genome of the intended pathogens. If the pathogens are present in our samples, the provided DNA strands will bind to the present pathogen DNA. PCR will then work to manufacture billions of copies of this present pathogen DNA. 

When not working on this project, I also work in the University of Maine Cooperative Extension Diagnostic Research Laboratory as a part of the Tick Lab. In this position I have honed the molecular biology skills that I will in turn use for the squirrel project. 


Yvonne Booker

Undergraduate, Tuskeegee University

Levesque Lab

Microbes and the Mammalian Mystery“, reblogged from the University of Maine REU program.

Hello everyone! My name is Yvonne Booker and I am a rising senior, animal and poultry science major at Tuskegee University in Tuskegee, Alabama. I am interested in animal health research, with a particular focus in veterinary medicine. I’ve always wanted to be a veterinarian, but as I progressed throughout  college, I became interested in learning more about animal health and how I might help animals on a much larger and impactful scale–which led me to the REU ANEW program. Currently climate change is causing an increase in global temperatures, putting pressure on animals’ ability to interact and survive within their environment. Consequently, scientists are now attempting to understand not just how to prevent climate change, but how these creatures are adapting to this emerging challenge.

My research experience this summer is geared toward addressing this global issue. I am currently working in Dr. Danielle Levesque’s Lab, which aims to study the evolutionary and ecological physiology of mammals in relation to climate. My project involves conducting a literature review of the microbiome of mammals, to learn more about how their microbial community plays a role in how they adapt in a heat-stressed environment.

Our knowledge of vertebrate-microbe interactions derives partly from research on ectotherms. While this research paves the path for a better understanding of how organisms react to temperature changes, fewer studies have focused on how mammals deal with these extreme temperature shifts—specifically, the abrupt surge in climate change. The ability of endotherms to  thermoregulate alters our knowledge of (1) how mammals create heat tolerance against these environmental challenges and (2) how this internal process alters mammals’ adaptability and physiology over time. We suggest that the microbiome plays an essential part in understanding mammals’ heat tolerance and that this microbial community can help researchers further understand the various processes that allow mammals to survive extreme temperatures.

As a student of the REU ANEW program my goal was to go out of my comfort zone and study animals in an applied fashion that would impact animal health on an environmental and ecological scale; and this program was just that! My mentor, Dr. Levesque was wonderful in guiding me through conducting this research, while giving me the independence to create my own voice. The program directors, Dr. Anne Lichtenwalner and Dr. Kristina Cammen, have also  been extremely supportive throughout this entire program equipping students with the tools they need to succeed as researchers. Although research was my primary focus this summer, some of my favorite memories involved building community with the students and the staff. From weekly check-ins on zoom to virtual game nights of complete smiles and laughter, this program has been one for the books! The One Health and the Environment approach to this Research Experience for Undergraduate students has encouraged me to build on my curiosity within the field of science, and I’m looking forward to applying what I’ve learned to my career in the future.

A study I contributed to was published!

A study was recently published, led by Dr. Huawei Zeng, USDA Animal Research Station, on gut health, nutrition, and gut microbiota! I contributed analysis and interpretation for the gut community data, and though I appear as last author on this publication, it is truly because I contributed the least and not because I was administrative lead or the lead PI. I have worked with Dr. Zeng for several years, although we have never met in person,

Dr. Zeng’s presentation of this project can be found here: Adequacy of calcium and vitamin D enriches probiotic bacteria and reduces dysbiotic Parasutterela bacteria and inflammation in the colon of C57BL/6 mice fed a Western-style diet


Zeng, H., Safratowich, B.D., Liu, Z., Bukowski, , M.R., Ishaq, S.L. 2021. Adequacy of calcium and vitamin D reduces inflammation, β-catenin signaling, and dysbiotic Parasutterella bacteria in the colon of C57BL/6 mice fed a Western-style diet. Journal of Nutritional Biochemistry. In press.

Abstract

Adoption of an obesogenic diet low in calcium and vitamin D (CaD) leads to increased obesity, colonic inflammation, and cancer. However, the underlying mechanisms remain to be elucidated. We tested the hypothesis that CaD supplementation (from inadequacy to adequacy) may reduce colonic inflammation, oncogenic signaling, and dysbiosis in the colon of C57BL/6 mice fed a Western diet. Male C57/BL6 mice (4-week old) were assigned to 3 dietary groups for 36 weeks: (1) AIN76A as a control diet (AIN); (2) a defined rodent “new Western diet” (NWD); or (3) NWD with CaD supplementation (NWD/CaD). Compared to the AIN, mice receiving the NWD or NWD/CaD exhibited more than 0.2-fold increase in the levels of plasma leptin, tumor necrosis factor α (TNF-α) and body weight. The levels of plasma interleukin 6 (IL-6), inflammatory cell infiltration, and β-catenin/Ki67 protein (oncogenic signaling) were increased more than 0.8-fold in the NWD (but not NWD/CaD) group compared to the AIN group. Consistent with the inflammatory phenotype, colonic secondary bile acid (BA, inflammatory bacterial metabolite) levels increased more than 0.4-fold in the NWD group compared to the NWD/CaD and AIN groups. Furthermore, the abundance of colonic Proteobacteria (e.g., Parasutterela), considered signatures of dysbiosis, was increased more than 4-fold; and the α diversity of colonic bacterial species, indicative of health, was decreased by 30% in the NWD group compared to the AIN and NWD/CaD groups. Collectively, CaD adequacy reduces colonic inflammation, β-catenin oncogenic signaling, secondary BAs, and bacterial dysbiosis in mice fed with a Western diet.


This is part of a multi-year collaboration, with previous publications:

  • Zeng, H., Ishaq, S.L., Liu, Z., Bukowski, M.R. 2017. Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet. Journal of Nutritional Biochemistry 54:18-27. Impact 4.418. Article.
  • Zeng, H., Ishaq, S.L., Zhao, F-Q., Wright, A-D.G. 2016. Colonic inflammation accompanies an increase of b-catenin signaling Lachnospiraceae/Streptococcaceae in the hind-gut of high-fat diet-fed mice. Journal of Nutritional Biochemistry 25:30-36. Impact 4.518. Article
A very close-up image of a small, dark brown mouse perched on the arm of a graduate researcher wearing a surgical gown.

The first mouse study involving the Ishaq Lab begins!

Mice have arrived for a collaborative project on diet, gut microbes, and health in conjunction with researchers at Husson University! This is the first mouse project for the Ishaq Lab, and also my first hands-on mouse project (in my previous publications with mice, I received datasets but the mouse work was performed solely by my collaborators).

This is one of my first new collaborations at the University of Maine, which began in September 2019 as I was just finding my way around campus. An established researcher at Husson University, Dr. Yanyan Li, reached out to welcome me and talk about overlap between our work. Yanyan, her husband Dr. Tao Zhang, also a researcher at Husson University, and collaborator Dr. Grace Chen at Michigan State University, had been working on beneficial compounds found in broccoli using mice as an experimental model for Inflammatory Bowel Disease (IBD). Over the past year, in consultation with IBD experts Drs. Gary Mawe and Peter Moses (who I worked with previously while at UVM!), we have written several proposals for funding to expand the project.

Johanna Holman worked for several years with Yanyan and Tao, as an undergraduate researcher and then as a research assistant. She joined the Ishaq Lab this fall to continue her work as a graduate student and add gut microbiology to her skill repertoire. This experiment will form the base of her graduate thesis, and Johanna is taking a lead role in managing the project as well as several undergraduate researchers, including Dorien Baudewyns, assisting with the mice and lab work. As an early career researcher, and new to mice, I’m extremely lucky to be able to learn from an experienced team of researchers!

A study to which I contributed got published!

 

I’m pleased to announce that one of my collaborators,  Dr. Huawei Zeng of the USDA Agricultural Research Service, recently published another study of his, to which I contributed some analysis of bacterial communities from mice.  Several years ago, during my Ph.D. at the University of Vermont, I provided wet-lab and DNA sequence analysis work for a previous project of Dr. Zeng, investigating the health effects of a low or high fat diet on mice, which can be found here.

 

Colonic aberrant crypt formation accompanies an increase of opportunistic pathogenic bacteria in C57BL/6 mice fed a high-fat diet.

Zeng, H., Ishaq, S.L., Liu, Z., Bukowski, M.R. 2017. Journal of Nutritional Biochemistry. In press, doi.org/10.1016/j.jnutbio.2017.11.001.

Abstract

The increasing worldwide incidence of colon cancer has been linked to obesity and consumption of a high-fat western diet. To test the hypothesis that a high fat diet (HFD) promotes colonic aberrant crypt (AC) formation in a manner associated with gut bacterial dysbiosis, we examined the susceptibility to azoxymethane (AOM)-induced colonic AC and microbiome composition in C57/BL6 mice fed a modified AIN93G diet (AIN, 16% fat, energy) or a HFD (45% fat, energy) for 14 weeks. Mice receiving the HFD exhibited increased plasma leptin, body weight, body fat composition and inflammatory cell infiltration in the ileum compared with those in the AIN group. Consistent with the gut inflammatory phenotype, we observed an increase in colonic AC, plasma interleukin 6 (IL6), tumor necrosis factor α (TNF α), monocyte chemoattractant protein 1 (MCP1), and inducible nitric oxide synthase (iNOS) in the ileum of the HFD-AOM group compared with the AIN-AOM group. Although the HFD and AIN groups did not differ in bacterial species number, the HFD and AIN diets resulted in different bacterial community structures in the colon. The abundance of certain short chain fatty acid (SCFA) producing bacteria (e.g., Barnesiella) and fecal SCFA (e.g., acetic acid) content were lower in the HFD-AOM group compared with the AIN and AIN-AOM groups. Furthermore, we identified a high abundance of Anaeroplasma bacteria, an opportunistic pathogen in the HFD-AOM group. Collectively, we demonstrate that a HFD promotes AC formation concurrent with an increase of opportunistic pathogenic bacteria in the colon of C57BL/6 mice.

Manuscript published on the effect of low/high fat diets on health and intestinal bacteria

Several years ago, during my Ph.D. at the University of Vermont, I provided wet-lab and DNA sequence analysis work for a project investigating the health effects of a low or high fat diet on mice with Dr. Huawei Zeng of the USDA Agricultural Research Service.  It was just recently published in the Journal of Nutritional Biochemistry!

Abstract

Consumption of an obesigenic/high-fat diet (HFD) is associated with a high colon cancer risk and may alter the gut microbiota. To test the hypothesis that long-term high-fat (HF) feeding accelerates inflammatory process and changes gut microbiome composition, C57BL/6 mice were fed HFD (45% energy) or a low-fat (LF) diet (10% energy) for 36 weeks. At the end of the study, body weights in the HF group were 35% greater than those in the LF group. These changes were associated with dramatic increases in body fat composition, inflammatory cell infiltration, inducible nitric oxide synthase protein concentration and cell proliferation marker (Ki67) in ileum and colon. Similarly, β-catenin expression was increased in colon (but not ileum). Consistent with gut inflammation phenotype, we also found that plasma leptin, interleukin 6 and tumor necrosis factor α concentrations were also elevated in mice fed the HFD, indicative of chronic inflammation. Fecal DNA was extracted and the V1–V3 hypervariable region of the microbial 16S rRNA gene was amplified using primers suitable for 454 pyrosequencing. Compared to the LF group, the HF group had high proportions of bacteria from the family Lachnospiraceae/Streptococcaceae, which is known to be involved in the development of metabolic disorders, diabetes and colon cancer. Taken together, our data demonstrate, for the first time, that long-term HF consumption not only increases inflammatory status but also accompanies an increase of colonic β-catenin signaling and Lachnospiraceae/Streptococcaceae bacteria in the hind gut of C57BL/6 mice.