Tag: mice

Paper published on “Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease”

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The Ishaq and Li labs at UMaine are delighted to announce that our paper on “Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease.” has been published in mSystems!! ASM was kind enough to write a press release about study, found here.

The complete author list, Abstract, and Ackowledgements/Funders portions of the paper can be found at the end of this post. This paper is part of a larger Broccoli project, in which we are evaluating the use of broccoli sprouts in the diet to enlist gut microbes to produce anti-inflammatories as a way to resolve symptoms of Inflammatory Bowel Disease.

The Premise

Broccoli sprouts are very high in a compound called glucoraphanin, which is in-active for humans. When glucoraphanin comes in contact with the myrosinase enzyme, also found in the sprouts, it is transformed into sulforaphane, which drives away insect pests but acts as an anti-inflammatory in people!

If you eat raw sprouts, most of this conversion happens when you cut or chew the sprouts, and that anti-inflammatory will get absorbed in your stomach. If you steam or cook the sprouts, you can inactivate the enzyme and leave the glucoraphanin compound alone. Some of your gut microbes are able to use glucoraphanin, and produce the anti-inflammatory sulforaphane right in your gut! We are trying to understand how and when this works, so we can use it to reduce symptoms of Inflammatory Bowel Disease.

A diagram with two panels, and a cartoon mouse in the middle. The cartoon mouse is eating broccoli, and a cartoon of the digestive tract is overlaid on the mouse's abdomen. Lines emanating from the broccoli point to the left panel, and show the compound glucoraphanin being converted into sulforaphane by the myrosinase enzyme. Lines emanating from the colon of the mouse point to the panel on the right, showing the same biochemical conversion by gut microbes.
A cartoon of a woman eating broccoli, with the digestive tract shown on her shirt, and smiling microbes in the background.

The mice in this trial are used to mimic Crohn’s Disease, which is one of the main ways that Inflammatory Bowel Diseases may be classified. Crohn’s Disease is complictaed, and involves an over-active immune response to gut microbes. This is replicated in mice that are bred to lack the genes in the DNA to make interleukin-10 (IL-10). IL-10 is an immune factor that can be used to calm the immune system and tolerate microbes which are not causing harm. Without IL-10, these mice over-react to the presence of bacteria, even those which are not causing harm, and this creates symptoms similar to Crohn’s in people.

We used two age groups of mice, and in each group, half ate a mouse chow (control) diet and half ate the mouse chow with 10% of the chow replaced by raw broccoli sprouts. Crohn’s often develops in childhood and adolescence, so our two age groups of mice reflect the juvenile stage (4-5 weeks old) and the adolescence stage (5-6 weeks old) of symptom onset. After wo weeks of symptoms, we sacrificed the mice and collected as much information as we could.

Figure 1 from the paper mentioned in this post. It shows an experimental design.

The Team

The mice, their care during the experiment, and sample collection for this project was graciously provided by University of Vermont researchers Gary Mawe and Brigitte Lavoie, and then-grad-student-now-medical-student Molly Hurd, in 2021. The SUNY Bingamton team, Tao Zhang and Allesandra Stratigakis, processed metabolite and cytokine samples and analyzed those data. The UMaine team (pictured below and led by Sue Ishaq and Yanyan Li) processed and analyzed data from different locations of gut tissue for histolgy and sequencing of bacterial communities, as well as analyzing those data, and took the lead on writing the paper.

Portrait of Lola Holcomb, wearing a block sweater on a beach at sunset
Lola standing in front of her poster which displays the results of her research on gut microbes.
Louisa Colucci. Young woman with dark hair tied in a bun is sitting in a kayak with her paddle raised. She is wearing a black top and a red life-vest over it, in a light blue, single-person kayak. The kayak is in the water, most likely a lake, with the forested shoreline in the background.

The Health Benefits were most obvious in the younger mice

The mice that were eating the broccoli sprouts in their chow and did much better than the control group who ate only mouse chow when symptoms of Crohn’s Disease were induced — and we found something really interesting… The diet worked really well in the younger mice and reduced their symtpoms of inflammation and illness for almost every metric we studied. The older, adolecent mice got some benefit from eating the raw broccoli sprouts, but not nearly as much as the younger mice! Those graphs are shown in the paper.

The Gut Microbes were most changed in the younger mice

Bacterial richness (the number of different types of bacteria present) was increased, but only in younger mice consuming a 10% raw sprout diet, which is useful because pediatric Crohn’s patients usually have fewer types of bacteria present in their gut.

Younger mice consuming broccoli sprouts also had more types of bacteria that are known to convert glucoraphanin into sulforophane, and they had more of the genes needed to do it. Crohn’s patients usually have fewer of these types of bacteria, which are also known to provide other health benefits.

The Next Steps

We are currently working on replicating and expanding this project to include more age groups, so we can understand how different diet preparations of broccoli sprouts impact immune systems and gut microbiota at different developmental periods of life. We are also really interested in understanding how sex in mice, and gender in humans, plays a role in how immune systems and microbial communities develop during a critical phase of life. We have some initial data to suggest that male and female mice respond to different diets and at differnt ages, but we aren’t sure why yet.

We hope to expand our work with people to study how these diets work in the real world, and how we can tailor diet and cooking preparations of sprouts to best meet the needs of people of different ages, health statuses, and tastes.

The paper

Early life exposure to broccoli sprouts confers stronger protection against enterocolitis development in an immunological mouse model of inflammatory bowel disease. 

Lola Holcomb1$, Johanna M. Holman2$, Molly Hurd3, Brigitte Lavoie3, Louisa Colucci4, Benjamin Hunt5, Timothy Hunt5, Marissa Kinney2, Jahnavi Pathak1, Gary M. Mawe3,Peter L. Moses3,6, Emma Perry7, Allesandra Stratigakis8, Tao Zhang8, Grace Chen9, Suzanne L. Ishaq1*, Yanyan Li1*

1 Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA 04469. 2 School of Food and Agriculture, University of Maine, Orono, Maine, USA 04469. 3 Larner College of Medicine, University of Vermont, Burlington, Vermont, USA 05401. 4 Department of Biology, Husson University, Bangor, Maine, USA 04401. 5 Department of Biology, University of Maine, Orono, Maine, USA 04469. 6 Finch Therapeutics, Somerville, Massachusetts, USA 02143. 7 Electron Microscopy Laboratory, University of Maine, Orono, Maine, USA 04469. 8 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, Johnson City, New York, USA 13790. 9 Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 48109

$ these authors contributed equally.

Keywords: Crohn’s Disease, cruciferous vegetables, sulforaphane, glucoraphanin, gut microbiota, dietary bioactives, 16S rDNA, interleukin-10 knockout 

Abstract

Crohn’s Disease (CD) is a presentation of Inflammatory Bowel Disease (IBD) that manifests in childhood and adolescence, and involves chronic and severe enterocolitis, immune and gut microbial dysregulation, and other complications. Diet and gut-microbiota-produced metabolites are sources of anti-inflammatories which could ameliorate symptoms. However, questions remain on how IBD influences biogeographic patterns of microbial location and function in the gut, how early life transitional gut communities are affected by IBD and diet interventions, and how disruption to biogeography alters disease mediation by diet components or microbial metabolites. Many studies on diet and IBD use a chemically induced ulcerative colitis model, despite the availability of an immune-modulated CD model. Interleukin-10-knockout (IL-10-KO) mice on a C57BL/6 background, beginning at age 4 or 7 weeks, were fed a control diet or one containing 10% (w/w) raw broccoli sprouts, which was high in the sprout-sourced anti-inflammatory sulforaphane. Diets began 7 days prior to, and for 2 weeks after inoculation with Helicobacter hepaticus, which triggers Crohn’s-like symptoms in these immune-impaired mice. The broccoli sprout diet increased sulforaphane in plasma; decreased weight stagnation, fecal blood, and diarrhea associated; and increased microbiota richness in the gut, especially in younger mice. Sprout diets resulted in some anatomically specific bacteria in younger mice, and reduced the prevalence and abundance of pathobiont bacteria which trigger inflammation in the IL-10-KO mouse, e.g., Escherichia coli and Helicobacter. Overall, the IL-10-KO mouse model is responsive to a raw broccoli sprout diet and represents an opportunity for more diet-host-microbiome research.

Importance

To our knowledge, IL-10-KO mice have not previously been used to investigate the interactions of host, microbiota, and broccoli, broccoli sprouts, or broccoli bioactives in resolving symptoms of CD. We showed that a diet containing 10% raw broccoli sprouts increased the plasma concentration of the anti-inflammatory compound sulforaphane, and protected mice to varying degrees against disease symptoms, including weight loss or stagnation, fecal blood, and diarrhea. Younger mice responded more strongly to the diet, further reducing symptoms, as well as increased gut bacterial richness, increased bacterial community similarity to each other, and more location-specific communities than older mice on the diet intervention. Crohn’s Disease disrupts the lives of patients, and requires people to alter dietary and lifestyle habits to manage symptoms. The current medical treatment is expensive with significant side effects, and a dietary intervention represents an affordable, accessible, and simple strategy to reduce the burden of symptoms.

Acknowledgements: This project was supported by the USDA National Institute of Food and Agriculture through the Maine Agricultural & Forest Experiment Station: Hatch Project Numbers ME022102 and ME022329 (Ishaq) and ME022303 (Li); the USDA-NIFA-AFRI Foundational Program [Li and Chen; USDA/NIFA 2018-67017-27520/2018-67017-36797]; and the National Institute of Health [Li and Ishaq; NIH/NIDDK 1R15DK133826-01] which supported Marissa Kinney, Timothy Hunt, and Benjamin Hunt. Johanna Holman was supported by ME0-22303 (Li), and Lola Holcomb was supported by US National Science Foundation One Health and the Environment (OG&E): Convergence of Social and Biological Sciences NRT program grant DGE-1922560, and the UMaine Graduate School of Biomedical Science and Engineering.

Paper published on “Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice”!

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The Ishaq and Li labs at UMaine are delighted to announce that our paper on “Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice” has been published in mSystems!! The complete author list, Abstract, and Ackowledgements/Funders portions of the paper can be found at the end of this post.

This paper is part of a larger Broccoli project, in which we are evaluating the use of broccoli sprouts in the diet to enlist gut microbes to produce anti-inflammatories. You can read about the whole project here, with links to other resources.

The Premise

A cartoon of a bacteria eating broccoli and pooping out sulforaphane
Designed by Johanna Holman
A diagram with two panels, and a cartoon mouse in the middle. The cartoon mouse is eating broccoli, and a cartoon of the digestive tract is overlaid on the mouse's abdomen. Lines emanating from the broccoli point to the left panel, and show the compound glucoraphanin being converted into sulforaphane by the myrosinase enzyme. Lines emanating from the colon of the mouse point to the panel on the right, showing the same biochemical conversion by gut microbes.

Broccoli sprouts are very high in a compound called glucoraphanin. When glucoraphanin comes in contact with the myrosinase enzyme, also found in the sprouts, it is transformed into a compound that acts an an anti-inflammatory in people!

If you eat raw sprouts, this conversion happens when you cut or chew the sprouts, and that anti-inflammatory will get absorbed in your stomach. If you steam or cook the sprouts, you can inactivate the enzyme and leave the glucoraphanin compound alone. Some of your gut microbes are able to use the compound, and produce the anti-inflammatory right in your gut! We are trying to understand how and when this works, so we can use it to reduce symptoms of Inflammatory Bowel Disease.

The Mouse work

In the winter of 2020-2021, we ran a 40-day study with 40 mice housed at UMaine. The mice were divided into 4 groups: “control” which ate the mouse chow, “control+DSS” which ate the mouse chow and had colitis induced by adding DSS (a salt laxative) to their drinking water, “broccoli” which ate the mouse chow with steamed broccoli sprouts mixed in, and “broccoli+DSS” which ate the mouse chow/steamed broccoli sprouts diet and had colitis induced by adding DSS (salt laxative) to their drinking water. This work was led by Johanna Holman, who was a master’s student at the time; Lousia Colicci, who was an undergrad at Husson University at the time and is applying to medical schools now; Dorein Baudewyns, who was an undergrad at Husson University at the time and is completing a graduate program in Psychology at UMaine; and Joe Balkan, who was completing his senior year of high school at the time and has since begin an undergrad degree in Biology at Tufts University where he is preparing for medical school.

Person in a research facility holding up their arm with a mouse on it. Person is wearing a hairnet, nitrile gloves, surgical mask, and a surgical gown. They are holding their left arm up to the camera to show off a mouse with dark brown fur sitting on their arm. In the background is a metal shelf with containers of research materials.
Johanna Holman, graduate researcher in the Ishaq Lab
Louisa Colucci. Young woman with dark hair tied in a bun is sitting in a kayak with her paddle raised. She is wearing a black top and a red life-vest over it, in a light blue, single-person kayak. The kayak is in the water, most likely a lake, with the forested shoreline in the background.
Louisa Colucci.
A person holding a mouse in a research facility. The person is wearing a hairnet, nitrile gloves, a surgical mask, and a surgical gown. The mouse with dark brown fur is sitting on the top of the person's hand. In the background, there are metal shelves with bins and containers of research supplies.
Dorien Baudewyns, undergraduate researcher at Husson University
Joe Balkan, performing culturing of bacteria at the anaerobic chamber. Joe is wearing a face mask, and is holding up a culture plate. There are stacks of petri dishes inside the chamber.
Joe Balkan, undergraduate researcher at Tufts University, performing culturing of bacteria at the anaerobic chamber.
Close-up image of a small, dark brown mouse perched on the arm of a graduate researcher. The person is wearing a face mask and surgical gown.

The mice were weighed regularly and fecal samples assessed for blood (signs of colitis). At the end of the study, the mice were euthanized so we could study the bacteria in parts of the intestines that we can’t access in humans. We used as few mice as possible, and got as much information from this study as possible, to do as much good as we can with their sacrifice.

The Health Benefits

As we’d hoped, the broccoli+DSS mice that were eating the broccoli sprouts that were given colitis did much better than the control+DSS group who ate mouse chow during their colitis. The broccoli+DSS mice were able to keep gaining weight as they grew, had better consistency of their stool, and had lower amounts of proteins and other metabolities in their blood which indicate inflammation (lower cytokines and lipocalin). Those graphs are shown in the paper.

The Gut Microbes

We found a lot of interesting things with the microbial communities that were living in different parts of the intestines, but the most exciting was that broccoli sprouts in the diet helped microbial communities stay alive in their original gut locations even during colitis! Certain microbes like to live in particular places in our intestines based on where different ingredients in our diet get processed, or the local environment (like how acidic the intestinal neighborhood is), and this is called biogeography.

In the graph below, our control group mice (eating chow) or the broccoli group (eating chow plus sprouts), we see that microbial communites in the small intestines clustered away from the microbial communities in the large intestines.

The DSS salt laxative, and ulcerative colitis, wreak havoc on gut microbes because they cause physical damage to the lining of the intestine, which where many microbes that can be useful to us live on or near. When we induced colitis in mice that were eating mouse chow (control+DSS group), the damage to the intestines caused a loss to some of the microbes living in different places. The remaining microbes that could survive these tough conditions were basically the same ones regardless of where we we looked in the intestines.

But, if mice had colitis and were eating broccoli sprouts (broccoli+DSS), the microbes were able to survive in their original locations and preserved biogeography! This is important because where microbes live in the gut may determine if the beneficial things they make can help resolve IBD symptoms in specific locations in the gut.

Image by Johanna Holman, graph from the paper.

The Spatial Location of GLR-digesting-genes

Bejamin and Timothy Hunt are undergraduates in Biology who have been working on bioinformatics in the Ishaq Lab since December 2022 after completing Sue’s DNA Sequencing Data Analysis Class. They joined the DSS project to provide in-depth analysis on some of the sequences which matched bacteria that are known to convert GLR into the anti-inflammatory SFN, as well as analyze data comparing numbers of genes known to be involved in the process.

A cartoon of the intestines with bacteria of interest in the jejunum, ceculm and colon,
Cropped figure from the paper, made by Benjamin and Timothy.
Benjamin Hunt

The study of the bioproduction of SFN and its mucosal and luminal activity benefited from the biogeographical analysis of this study. It was interesting to note the extreme dominance of a Bacteroides species in the broccoli treatments. B. thetaiotaomicron was indicated based on BLASTN analysis and an evaluation of matching species but was not directly suggested by the dada-Silva taxonomy assignment. The indication of B. thetaiotaomicron suggested analyzing the presence of the operon BT2159-BT2156, which was generally minimally present (<100) but at relatively high counts (>100,000) in some samples. Significantly, the operon was found at locations where no Bacteroides were identified. We continue to reflect on the similarities and differences in the biogeography of bacterial abundance and operon presence highlighted in the different treatments of this study.

Benjamin and Timothy Hunt

The Next Steps

As part of this project, we cultured hundreds of bacteria from the intestines of mice to try and isolate some of the ones that turn glucroraphanin into sulforaphane. We have a large team of students and researchers participating on the culturing work, some of whom are pictured here. We’ll be providing plenty of updates on that project as we continue to process the bacteria this fall!

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The Paper

Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice.

Johanna M. Holman1, Louisa Colucci2, Dorien Baudewyns3, Joe Balkan4, Timothy Hunt5, Benjamin Hunt5, Marissa Kinney1, Lola Holcomb6, Allesandra Stratigakis7, Grace Chen8, Peter L. Moses9,10, Gary M. Mawe9, Tao Zhang7, Yanyan Li1*, Suzanne L. Ishaq1*

1 School of Food and Agriculture, University of Maine, Orono, Maine, USA 04469 2 Department of Biology, Husson University, Bangor, Maine, USA 04401 3 Department of Psychology, University of Maine, Orono, USA 04469 4 Department of Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, USA 02155 5 Department of Biology, University of Maine, Orono, Maine, USA 04469 6 Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA 04469 7 School of Pharmacy and Pharmaceutical Sciences, SUNY Binghamton University, Johnson City, New York, USA 13790 8Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA 48109 9Departments of Neurological Sciences and of Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont, USA 0540110 Finch Therapeutics, Somerville, Massachusetts, USA 02143

Abstract: Inflammatory Bowel Diseases (IBD) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective, and affordable, for managing symptoms. Glucosinolate compounds are highly concentrated in broccoli sprouts, especially glucoraphanin, and can be metabolized by certain mammalian gut bacteria into anti-inflammatory isothiocyanates, such as sulforaphane. Gut microbiota exhibit biogeographic patterns, but it is unknown if colitis alters these or whether the location of glucoraphanin-metabolizing bacteria affects anti-inflammatory benefits. We fed specific pathogen free C57BL/6 mice either a control diet or a 10% steamed broccoli sprout diet, and gave a three-cycle regimen of 2.5% dextran sodium sulfate (DSS) in drinking water over a 34-day experiment to simulate chronic, relapsing ulcerative colitis. We monitored body weight, fecal characteristics, lipocalin, serum cytokines, and bacterial communities from the luminal- and mucosa-associated populations in the jejunum, cecum, and colon. Mice fed the broccoli sprout diet with DSS treatment performed better than mice fed the control diet with DSS, including significantly more weight gain, lower Disease Activity Indexes, lower plasma lipocalin and proinflammatory cytokines, and higher bacterial richness in all gut locations. Bacterial communities were assorted by gut location, but were more homogenous across locations in the control diet + DSS mice. Importantly, our results showed that broccoli sprout feeding abrogated the effects of DSS on gut microbiota, as bacterial richness and biogeography were similar between mice receiving broccoli sprouts with and without DSS. Collectively, this supports the protective effect of steamed broccoli sprouts against dysbiosis and colitis induced by DSS.


Importance: Evaluating bacterial communities across different locations in the gut provides a greater insight than fecal samples alone, and provides an additional metric by which to evaluate beneficial host-microbe interactions. Here, we show that 10% steamed broccoli sprouts in the diet protects mice from the negative effects of dextran sodium sulfate induced colitis, that colitis erases biogeographical patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. Mice fed the broccoli sprout diet during colitis performed better than mice fed the control diet while receiving DSS. The identification of accessible dietary components and concentrations that help maintain and correct the gut microbiome may provide universal and equitable approaches to IBD prevention and recovery, and broccoli sprouts represent a promising strategy.

Acknowledgements: All authors have read and approved the final manuscript. The authors thank Jess Majors, University of Maine, for her kind and detailed care of the mice during the trial, and for Ellie Pelletier for her informal review of the manuscript. This project was supported by the USDA National Institute of Food and Agriculture through the Maine Agricultural & Forest Experiment Station: Hatch Project Numbers ME022102 and ME022329 (Ishaq) and ME022303 (Li) which supported Johanna Holman; the USDA-NIFA-AFRI Foundational Program [Li and Chen; USDA/NIFA 2018-67017-27520/2018-67017-36797]; and the National Institute of Health [Li and Ishaq; NIH/NIDDK 1R15DK133826-01] which supported Marissa Kinney, Timothy Hunt, and Benjamin Hunt. Lola Holcomb was supported by US National Science Foundation One Health and the Environment (OG&E): Convergence of Social and Biological Sciences NRT program grant DGE-1922560, and through the UMaine Graduate School of Biomedical Sciences and Engineering. 

The Ishaq Lab welcomes a new grad student, Marissa Kinney!

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The Ishaq Lab is pleased to welcome Marissa Kinney as a Master’s of Science student in Microbiology, beginning in January 2023! She’ll be joining ‘Team Broccoli‘ to investigate the 806 bacteria we isolated from the digestive tracts of mice eating a broccoli sprout diet, in a previous experiment on broccoli sprouts, microbes, and resolving colitis.

Marissa is a recent graduate of the UMaine Microbiology bachelor’s program, where she was part of an interdisciplinary research group and was the first author on a scientific publication this year: Suppression of Methicillin-Resistant Staphylococcus aureus and Reduction of Other Bacteria by Black Soldier Fly Larvae Reared on Potato Substrate.

Marissa Kinney

Marissa Kinney 

Master of Science student, Microbiology and Animal and Veterinary Sciences

Blurb: Marissa is a Masters student who loves learning and bench microbiology. She completed her undergraduate at the University of Maine in 2021, earning a BS in Microbiology and a BS in Cellular/Molecular Biology. She devoted a large portion of her time in undergrad to research in the laboratories of Dr. Julie Gosse and Dr. Edward Bernard. Since graduating, she worked in the field of public health at UMaine’s Margaret Chase Smith Policy Center, collecting and processing data about violent and drug-related deaths in Maine. While her role at the Center was one she loved dearly, she feels a big pull towards laboratory work and academic research. She recently joined the Ishaq lab and is excited by the new opportunities this position brings. 

Johanna defends her master’s thesis defense!

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Johanna Holman passed her Master’s of Science these defense (we knew she would succeed)!! Johanna has worked incredibly hard over the last two years to broaden her research skills and conduct several experiments, and her defense presentation was a wonderful way to see that progression all at once. She has also earned the designation of “first Ishaq Lab grad student to defend”. The defense was attended by her thesis committee, students in the Ishaq lab, collaborators on this project, and friends and family (who brought her a flower and broccoli bouquet that can be seen in the picture below). She will officially pass after a few revisions to her thesis and a formal acceptance by the committee members, which is standard for graduate defenses.

Johanna Holman standing at a wooden podium with a white board and a projection screen behind her. Johanna is presenting her master's thesis, and the title slide of the presentation is showing on the screen.
Johanna Holman beginning her master’s defense presentation.

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 mouse 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.

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

  • Johanna Holman at her ASM Microbe 2022 poster
    Johanna Holman at her ASM Microbe 2022 poster
  • Person in a research facility holding up their arm with a mouse on it. Person is wearing a hairnet, nitrile gloves, surgical mask, and a surgical gown. They are holding their left arm up to the camera to show off a mouse with dark brown fur sitting on their arm. In the background is a metal shelf with containers of research materials.
    Johanna Holman, graduate researcher in the Ishaq Lab

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

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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.

Person in a research facility holding up their arm with a mouse on it. Person is wearing a hairnet, nitrile gloves, surgical mask, and a surgical gown. They are holding their left arm up to the camera to show off a mouse with dark brown fur sitting on their arm. In the background is a metal shelf with containers of research materials.

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

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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.

Some of the media used to culture bacteria from the feces of mice and squirrels.

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!

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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!

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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).

A person holding a mouse in a research facility. The person is wearing a hairnet, nitrile gloves, a surgical mask, and a surgical gown. The mouse with dark brown fur is sitting on the top of the person's hand. In the background, there are metal shelves with bins and containers of research supplies.
Person in a research facility holding up their arm with a mouse on it. Person is wearing a hairnet, nitrile gloves, surgical mask, and a surgical gown. They are holding their left arm up to the camera to show off a mouse with dark brown fur sitting on their arm. In the background is a metal shelf with containers of research materials.

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!

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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

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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.