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New zebrafish model should speed research on parasite that causes toxoplasmosis

New zebrafish model should speed research on parasite that causes toxoplasmosis

Researchers at Oregon State University have found a method to speed the search for new therapies to treat toxoplasmosis – by successfully infecting zebrafish with Toxoplasma gondii.

The findings were just published in the Journal of Fish Diseases, in work supported by the Tartar Foundation and the National Institutes of Health.

  1. gondii, a protozoan parasite, can infect a wide range of hosts, and is one of the most prevalent parasites in the world. It has been estimated to infect about one-third of the human race. Treatment can be difficult because parasites often have biologic similarities to the hosts they infect.

Zebrafish have been found in recent years to be an excellent model for biomedical research because they reproduce rapidly, bear many similarities to human genetics and biological systems, and can be used in “high throughput” technologies to literally test hundreds of compounds in a fairly short period of time.

“This advance may provide a very efficient tool for the discovery of new therapies for this parasitic infection,” said Justin Sanders, an OSU postdoctoral fellow and lead author on the study. “With it we should be able to more easily screen a large library of compounds, at much less expense, and look at things that are unknown or have never been considered as a possible treatment.”

Although it infects many animals, T. gondii infection has never been observed prior to this in fish. But the OSU researchers found that by raising the temperature of the water in which zebrafish lived to a warmer-than-normal 98.6 degrees, or the temperature of a human body, they could become infected with the parasite but also survive.

T. gondii affects a wide range of mammals and birds, and cats are actually one of the most routine hosts,” said Michael Kent, a professor of microbiology in the OSU College of Science. “It can cause congenital defects, which is one reason that pregnant women are told not to clean the catbox. Many people become infected for life. These chronic infections can cause serious eye disease and can be fatal to people with weakened immune systems.

“New therapies would clearly be of value, and now we have a better way to find them,” he said.

This work was done in collaboration with researchers from the University of Chicago, Albert Einstein College of Medicine, and the U.S. Department of Agriculture.

The study this story is based on is available online: http://onlinelibrary.wiley.com/doi/10.1111/jfd.12393/full

Original article: http://oregonstate.edu/ua/ncs/archives/2015/may/new-zebrafish-model-should-speed-research-parasite-causes-toxoplasmosis

Murcia University honours Dr. Leonard Zon

Murcia University honours Dr. Leonard Zon

Jorge Galindo-Villegas, Murcia University, Spain

On April 21st, 2015, Dr. Len Zon, Director of the Stem Cell Program at Children’s Hospital Boston at Harvard Medical School (USA), was made Doctor Honoris Causa by Murcia University, Spain. The academic ceremony was presided over by the Honorable Rector Magnificus José Orihuela Calatayud and several distinguished members of the University community. The ceremony took place in the auditorium “Hermenegildo Lumeras de Castro” located beneath the Faculty of Chemistry which faces the Faculty of Biology, on the Espinardo campus. The ceremony, which was broadcast live, included the performance of different pieces by the chamber orchestra of Murcia University. Len’s promoter was Dr. Victoriano Mulero, Professor of the Department of Cell Biology and Histology, who gave the Laudatio speech.

The ceremony began with the entrance of the academic authorities to the sound of Andante, which was followed by the opening speech given by the Rector Orihuela Calatayud. The General Secretary of the University, Santiago M. Alvarez Carreño proceeded to read out the agreement made by the Board of Governors to the proposal made by the Department of Cell Biology and Histology, Faculty of Biology, to confer the Honoris Causa Degree on Dr. Zon. This agreement recognizes Len’s brilliant teaching, outstanding research track record and his renown as a leading pediatrician. He is one of the world leading figures in the field of stem cells transplantation, a researcher specialized in blood diseases and, quite importantly, founder of a new avenue of hematological research and drug screening to cure cancers, among many other diseases, using the zebrafish as a vertebrate animal model. After the agreement was pronounced, the Rector kindly asked for the presence of the Laureate. Len entered accompanied by his promoter Dr. Mulero and the Dean of the Faculty of Biology, Dr. Jose Meseguer, to the tune of Concerto for Two Trumpets in C Major (RV537). After everyone took their seat, Professor Mulero gave his Laudatio of Len in which he offered an extensive overview of his outstanding and brilliant academic career. Then, the Rector awarded Len with a Doctor Honoris Causa, thus becoming a member of the University’s Senate of Doctors. The last part of the act included the new Doctor’s speech of acceptance, following the rendering.

Len acknowledged the award, by immediately telling a funny phrase which made everyone in the audience lose the solemnity of the act with a big laugh: “I have had the opportunity of being twice in each of the three most important cities in Spain, twice in Madrid, twice in Barcelona and of course, twice in Murcia” (Murcia is a quite small town without touristic recognition). He continued by speaking a bit about his daily work. He presented himself as a hematologist by training, a medical doctor who takes care of children presenting blood diseases or cancer, and a researcher keen to learn and decipher the biology of stem cells to produce effective drugs to treat mortal human diseases.

In his speech, he stressed the several advantages displayed by the zebrafish as a vertebrate animal model. He then introduced his particular and interesting research focus by using a graphical video which he produced. The video showed a transgenic zebrafish embryo expressing fluorescence in the blood cells and he described how it was used to dissect the formation of these important cells in vertebrates. The impressive video came to a climax when he introduced how blood stem cells go into the circulation and eventually, to the intermediate cell mass at the tail where the blood cells are formed, following the process known as homing, which is accomplished through the interaction with endothelial and stromal cells in the vascular hematopoietic niche. After dividing again, they then go back into circulation and eventually will colonize the kidney. Some will go further to the thymus, allowing the animal to have blood for its entire lifetime. Len highlighted the functional importance of this process, where he transplanted stem blood cells allowing sick people to live an entire life-time. Proud of his findings he mentioned “I’ve done this procedure in a broad number of patients. Although we know how to make bone marrow transplantation, we don’t really understand how it works”.

To end his acceptance speech, Len mentioned that now his laboratory is interested in knowing more about the mechanism and pathways, using the zebrafish as a feasible live vertebrate model. So far, using thousands of small zebrafish embryos and a high-throughput chemical genetic screening, he has identified prostaglandins as stimulators of blood stem cell production both during embryogenesis and in adulthood. His studies may support a deeper comprehension of how human blood stem cells home to the marrow, engraft and self-renew, and suggest new therapeutic approaches for hematological disorders.

Duke Study Uncovers Foundations of Heart Regeneration

Duke Study Uncovers Foundations of Heart Regeneration

duke

The outer layer of the zebrafish heart (shown in green) is regenerated rapidly after damage, covering the heart like a wave from the base of one chamber to the tip of the other. Researchers have discovered properties of this mysterious outer layer — known as the epicardium — that could help explain the aquarium denizen’s remarkable ability to regrow cardiac tissue.
Photo credit: Jingli Cao

While the human heart can’t heal itself, the zebrafish heart can easily replace cells lost by damage or disease. Now, researchers have discovered properties of a mysterious outer layer of the heart known as the epicardium that could help explain the fish’s remarkable ability to regrow cardiac tissue.

After an injury, the cells in the zebrafish epicardium dive into action — generating new cells to cover the wound, secreting chemicals that prompt muscle cells to grow and divide, and supporting the production of blood vessels to carry oxygen to new tissues.

A study appearing May 4 in the journal Nature found that when this critical layer of the heart is damaged, the whole repair process is delayed as the epicardium undergoes a round of self-healing before tending to the rest of the heart. The new research showed that the process requires signaling through a protein called sonic hedgehog, and demonstrated that adding this molecule to the surface of the heart can drive the epicardial response to injury.

The finding points to a possible target for repairing the damage caused by a heart attack, a major cause of death and disability in the United States. More than five million Americans are currently experiencing heart failure, and over 900,000 suffer from a heart attack each year.

“The best way to understand how an organ regenerates is to deconstruct it. So for the heart, the muscle usually gets all the attention because it seems to do all the work,” said Kenneth D. Poss, Ph.D., senior author of the study and professor of cell biology at Duke University School of Medicine. “But we also need to look at the other components and study how they respond to injury. Clearly, there is something special about the epicardium in zebrafish that makes it possible for them to regenerate so easily.”

Poss has been studying heart regeneration in zebrafish for the last 13 years. As a postdoctoral fellow he was the first to show that the puny, striped fish could regrow severed pieces of heart tissue, like a lizard growing back a pinched tail. Since then, his group has found that this regeneration involves the input of the epicardium, a thin layer of cells that cover the surface of the heart.

“The epicardium is underappreciated, but we think it is important because similar tissues wrap up most of our organs and line our organ cavities,” Poss said. “Some people think of it as a stem cell because it can make more of its own, and can contribute all different cell types and factors when there is an injury. The truth is we know surprisingly little about this single layer of cells or how it works. It is a mystery.”

In this study, Poss and his colleagues were determined to identify the properties of the epicardium that make it such a regenerative powerhouse. First, Duke postdoctoral fellow Jinhu Wang performed open-heart surgery on live zebrafish, removing approximately one fifth of the vital organ. Afterwards he used a set of sophisticated genetic tools to kill 90 percent of the epicardial cells and then measured how well the heart healed at various time points. He found that removing this outer layer created a clear lag in regeneration, but that eventually the healing process caught up to that of zebrafish with an intact epicardium.

The results suggested that the 10 percent of epicardial cells left behind were able to rebuild the epicardial layer before moving on to heart muscle. Intrigued by the finding, Poss decided to focus the next series of experiments on the epicardium and its ability to regenerate itself. Jingli Cao, another postdoctoral fellow in his laboratory, figured out a way to remove hearts from zebrafish and grow them in dishes in the laboratory, where the tiny two-chambered organs continued to beat and behave as if they were still tucked inside the organism.

As they had before, the researchers destroyed most of the heart’s epicardial layer, but this time they put the “explanted” organs under the microscope every day to capture the regeneration in action. They showed that the epicardium regenerated rapidly, covering the heart like a wave from the base of one chamber to the tip of the other in just a week or two.

The researchers then used this model to search for small molecule compounds or drugs that would affect the ability to regenerate. They screened molecules known to be involved in development of embryos, like fibroblast growth factors and sonic hedgehog, and found that the latter was critical for the regeneration process. The researchers now plan to perform larger screens for molecules that could enhance heart repair in zebrafish, and perhaps one day provide a new treatment for humans with heart conditions.

In a second paper appearing April 1, 2015, in the journal eLife, Poss and colleagues found that the epicardium produces a molecule called neuregulin1 that makes heart muscle cells divide in response to injury. When they artificially boosted levels of neuregulin1, even without injury, the heart started building more and more muscle cells. The finding further underscores the role of this tissue in heart health.

“Studies of the epicardium in various organisms have shown that this tissue is strikingly similar between fish and mammals, indicating that what we learn in zebrafish models has great potential to reveal methods to stimulate heart regeneration in humans,” said Poss.

The Nature study was supported by postdoctoral fellowships from the American Heart Association and grants from the National Institutes of Health (HL081674) and the American Federation for Aging Research.

Read original article here https://today.duke.edu/2015/05/heartlayer

A new role for zebrafish: Larger scale gene function studies

A new role for zebrafish: Larger scale gene function studies

new role

NHGRI scientists are homing in on specific genes in zebrafish to help them better understand the function of genes in people.

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A relatively new method of targeting specific DNA sequences in zebrafish could dramatically accelerate the discovery of gene function and the identification of disease genes in humans, according to scientists at the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health.
In a study posted online on June 5, 2015, and to be published in the July 2015 issue of Genome Research, the researchers reported that the gene-editing technology known as CRISPR/Cas9 is six times more effective than other techniques at homing in on target genes and inserting or deleting specific sequences. The study also demonstrated that the CRISPR/Cas9 method can be used in a “multiplexed” fashion – that is, targeting and mutating multiple genes at the same time to determine their functions.
It was shown about a year ago that CRISPR can knock out a gene quickly,” said Shawn Burgess, Ph.D., a senior investigator with NHGRI’s Translational and Functional Genomics Branch and head of the Developmental Genomics Section. “What we have done is to establish an entire pipeline for knocking out many genes and testing their function quickly in a vertebrate model.” Researchers often try to determine the role of a gene by knocking it out — turning it off or removing it — and watching the potential effects on an organism lacking it.
Such larger scale — termed high-throughput — gene targeting in an animal model could be particularly useful for human genomic research. Only 10 to 20 percent of recognized human genes have been subjected to such rigorous testing, Dr. Burgess said. The functions of many other genes have been inferred based on analyzing proteins or have been identified as possible disease genes, but the functions of those genes have not been confirmed by knocking them out in animal models and seeing what happens.
This is a way to do that on a more cost-efficient and large scale,” Dr. Burgess said.
The study of zebrafish has already led to advances in our understanding of cancer and other human diseases,” said NHGRI Director Eric Green, M.D., Ph.D. “We anticipate that the techniques developed by NHGRI researchers will accelerate understanding the biological function of specific genes and the role they play in human genetic diseases.”
The CRISPR/Cas9 method of gene editing is one of the two essential components in the NHGRI team’s high-throughput method. Modeled on a defense mechanism evolved by bacteria against viruses, CRISPR/Cas9 activity was first described in 2012. Since then, its use has spread quickly in genomic research labs in the United States and abroad.
The acronym CRISPR stands for “clustered, regularly interspaced, short palindromic repeat,” referring to a pattern of DNA sequences that appears frequently in bacterial DNA. Scientists believe the CRISPR sequences reflect evolutionary responses to past viral attacks.

 

“We’ve shown that with relatively moderate resources, you can analyze hundreds of genes”
—Dr. Shawn Burgess
Senior Investigator, Translational and Functional Genomics Branch, NHGRI

The Cas9 protein is a nuclease, an enzyme that snips a stretch of DNA in two places, in effect cutting out a piece. Bound together, CRISPR/Cas9 becomes a powerful research tool that permits researchers to target and delete a particular sequence or to insert a new sequence into the DNA of animal-model embryos.
The other essential component of the NHGRI team’s method is the zebrafish. The zebrafish and the mouse are the most commonly studied vertebrate laboratory animals whose genomes have been completely sequenced. The zebrafish is better suited to larger scale gene editing because about 70 percent of zebrafish genes appear to have human counterparts and the fish are far less costly to maintain than are mice. They multiply astonishingly quickly; a female may produce as many as 200 eggs at one time. And the embryos are fertilized externally and are transparent, making them readily accessible to researchers.
To demonstrate the feasibility of high throughput editing, the researchers targeted 162 locations in 83 zebrafish genes – about 50 of which are similar to human genes involved in deafness. (Hearing is one of the other interests of Dr. Burgess’s lab.) This produced mutations in 82 of the 83 genes.
In screening embryos by fluorescent polymerase chain reaction (a technology that allows researchers to produce millions of copies of a specific DNA sequence) and high-throughput DNA sequencing, the researchers determined that overall, mutations were passed on to the next generation in 28 percent of cases. The transmission rate was higher for some genes than for others, but in most cases, screening offspring from parent fish should be enough to spot most mutations, the researchers reported.
The results demonstrated that using the CRISPR/Cas9 technique in zebrafish will make it possible to both generate mutants for all genes in the zebrafish genome and carry out large-scale phenotyping, they noted in the Genome Research paper.
The CRISPR/Cas9 methodology works in mice, too, but it is more costly and takes far longer. Although mice actually reach sexual maturity earlier than zebrafish, they produce far fewer offspring.
Ultimately, Dr. Burgess hopes that his lab will use the new method to knock out about 10 percent of the zebrafish’s roughly 25,000 genes, and he would like to see an even broader effort. “We’ve shown that with relatively moderate resources, you can analyze hundreds of genes,” Dr. Burgess said. “On the scale of big science, you could target every gene in the genome with what would be a relatively modest scientific investment in the low tens of millions of dollars.”
Coauthors of the Genome Research paper with Dr. Burgess were: Gaurav Varshney, Ph.D., Wuhong Pei, Ph.D., Matthew LaFave, Ph.D., Lisha Xu, M.S., Viviana Gallardo Mendieta, Ph.D., Blake Carrington, M.S., Kevin Bishop, M.S, Mary Pat Jones, M.S, Ursula Harper, M.S, and Raman Sood, Ph.D, all of NHGRI; Mingyu Li , Ph.D, and Wenbiao Chen, Ph.D, both of Vanderbilt University School of Medicine in Nashville; Sunny Huang, B.S, formerly of NHGRI, now of the University of Iowa in Iowa City; Jennifer Idol, M.S., formerly of NHGRI, now of the Jackson Laboratory in Bar Harbor, Maine; and Johan Ledin, Ph.D., of Uppsala University in Uppsala, Sweden.

How Blood Stem Cells Take Root in the Body

Live imaging captures how blood stem cells take root in the body

Fliesler, N. (2015) Live imaging captures how blood stem cells take root in the body. Vector – Boston Children’s Hospital.

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For years, the lab of Leonard Zon, MD, director of the Stem Cell Research Program at Boston Children’s Hospital, has sought ways to enhance bone marrow transplants for patients with cancer, serious immune deficiencies and blood disorders. Using zebrafish as a drug-screening platform, the lab has found a number of promising compounds, including one called ProHema that is now in clinical trials.

But truthfully, until now, Zon and his colleagues have largely been flying blind.

“Stem cell and bone marrow transplants are still very much a black box: cells are introduced into a patient and later on we can measure recovery of their blood system, but what happens in between can’t be seen,” says Owen Tamplin, PhD, in the Zon Lab. “Now we have a system where we can actually watch that middle step.”

The above animation, based on live imaging of naturally transparent zebrafish, reveals a surprisingly dynamic system in which newborn blood stem cells travel through the blood, exit into a “niche” where they get “cuddled” and nurtured, and then proceed to their final blood-making home. Their journey, also described in the January 15 issue of Cell, offers several clues for helping bone marrow transplants “take.”

“The same process occurs during a bone marrow transplant as occurs in the body naturally,” says Zon. “Our direct visualization gives us a series of steps to target, and in theory we can look for drugs that affect every step of that process.”

Read more in the Harvard Gazette.  A more technical version of the video can be viewed on Cell’s website.

Spotlight on Zebrafish

Spotlight on Zebrafish: Translational Impact

Amatruda, J.F. Dhillon, P., Patton, E. and Ramakrishnan L. (2014) Spotlight on Zebrafish: Translational Impact. Dis. Model. Mech. 7, 731733

fish

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In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue’s contents, highlighting the diversity of zebrafish disease models and their clinical applications.

Zebrafish (Danio rerio) have fast made their way from pet stores and home aquaria into research laboratories worldwide. Their weekly matings produce 100 to 200 embryos that rapidly and synchronously march through embryonic development, so that within 5 days of fertilization, they are mature, feeding larvae. Zebrafish are small and inexpensive to maintain in high numbers, facilitating large-scale experimentation and cheap in vivo drug screens. Famously, the fish are transparent during early larval stages, allowing investigators to directly observe internal development and making the fish a favorite of developmental biologists since the 1960s. But in recent years, the utility of zebrafish has been proven beyond developmental fields, and they are now being found in more and more laboratories studying behavior, diabetes, heart disease, regeneration, stem cell biology—and cancer.

Critically, zebrafish can be used to identify the important pathways and processes that cause cancer in people. Common organ systems and cell types are shared between human and zebrafish, and whether induced by transgenesis or carcinogens, cancers arising from the blood (leukemia and lymphoma), pigmented cells of the skin (melanoma), and the cells that line the bile ducts (cholangiocarcinoma) have microscopic features that are essentially indistinguishable between humans and zebrafish.

One aim of a Disease Models & Mechanisms (DMM) ‘Special Issue’ is to highlight how emerging disease models can lead to exceptional growth in particular areas of translational research. This is especially true for this issue, Spotlight on Zebrafish: Translational Impact. The zebrafish has traditionally been used to study developmental biology. Its optical transparency for the first few weeks, high fecundity and ex vivo fertilization have meant that the fundamental processes and mechanisms of vertebrate embryo development from a single cell through to a swimming fish can be studied in exquisite detail. Over the past decade these same features have enabled the zebrafish to become a preeminent disease model and tool for studying disease mechanisms. Importantly, discoveries in zebrafish disease models are leading to new perspectives on human disease and new drugs that are entering the clinic in diverse areas from cancer to tuberculosis.

We are delighted to present an issue packed with reviews, research and resource articles from researchers at the cutting-edge of their respective disease area of interest. The issue also includes a compelling interview with Len Zon, pioneer in the zebrafish disease models community, and a unique poster representation of the translational applications of zebrafish research. Here, we summarize the contents of the issue, and give our views on what makes each article special.

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Unfolded Protein Response

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish

Vacaru A.M., Di Narzo, A.F., Howarth, D.L., Tsedensodnom, O., Imrie, D., Cinaroglu, A., Amin, S., Hao, K., and Sadler, K.C. (2014) Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish. Dis. Model. Mech. 7, 823-835

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The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.

 

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Going Viral

Going Viral

University of Maine. Biology & Biomedical Sciences – Going Viral. UMaine

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Virus Study at UMaine’s Zebrafish Facility

“You can see the bacteria?”

“Do you want to say the quality value of biofilm formation?”

“Why would isolating them give you a different value?”

“Are there any other explanations?”

“How do you have 1.8 fish?”

Things can get pretty intense in the hallway outside Carol Kim’s microbiology lab in Hitchner Hall at the University of Maine. For the casual observer — especially one without a science background — the barrage of questions is overwhelming, like listening to an auctioneer calling in a foreign language.

But the three undergraduates standing there explaining their senior research projects, pointing to printouts of data and microscopic images of zebrafish taped to the walls, are completely unfazed. They answer Kim’s questions almost as quickly as she asks them. When she challenges them, they challenge back. They are confident. Eloquent. They know their stuff.

Which is exactly the way Kim, a professor of biochemistry, microbiology and molecular biology who directs UMaine’s Graduate School of Biomedical Sciences, likes it.

“I want to set up an environment where students feel comfortable and nurtured,” Kim says. “They have to know I’m going to ask tough questions and they have to be prepared. It’s going to be a lot nicer for me to ask them than to have them present in front of five professors cold. If I can be tough on them and they can answer the questions, they’ll have confidence.”

She wants to maintain rigorous and demanding standards, without the fear factor that can sometimes overwhelm students during their training in the sciences.

Kim is emblematic of a major push on campus to involve undergrads in research, and scenes like the one outside her lab play out across campus every day, especially in the weeks leading up to graduation. At UMaine, hundreds of science, humanities and engineering majors are involved in research, and close collaborations with faculty members are common.

Increasingly, UMaine has become a destination for top students interested in pre-med and biomedical studies, in large part because of the mentoring and rigorous preparation that Kim and her colleagues provide. As a result, many undergraduates are working at a graduate level long before they earn their bachelor’s degree. Like the students in the hall, they know the answers. But more important, they know which questions to ask.

hat curiosity is what drives Kim’s own career, both as an educator and a researcher. And the students who cross paths with her during their time at UMaine have a strong, demanding role model.

Kim has been fascinated by the craftiness of viruses — their resourcefulness and resilience — since grade school. But she’s equally crafty. And her research has moved the entire field of virology forward.

She conducts disease studies with zebrafish, a model organism, to better understand the human innate immune response to infection. She’s the driving force behind UMaine’s Zebrafish Facility, and since she arrived at UMaine in 1998, Kim has received continuous funding for her zebrafish research — more than $4 million in federal grants, primarily from the National Institutes of Health. Among her landmark discoveries is a zebrafish gene that produces interferon, which can inhibit the growth of a virus. She and colleague Rob Wheeler recently received a $60,000 NASA planning grant to study the effects of radiation on innate immune response and the progression of cancer.

Her current research focus provides a better understanding of how bacteria infect and cause inflammation in cystic fibrosis patients. Kim’s studies shed light on the connection between the cystic fibrosis transmembrane conductance regulator, or CFTR, and the innate immune response. That connection may someday be used as the basis for therapeutics that combat bacterial infections in cystic fibrosis.

“I love doing the brainstorming, working with a student to figure out the best question to ask.”
Carol Kim

“Clinical researchers are trying to develop therapeutics for the immediate needs of these patients, and as a result, we’ve seen significant increases in their quality of life. We’re on the other end, with basic research, trying to figure out what’s happening at the molecular and cellular level with the hope of developing those therapies. This project will be ongoing until CF is completely cured, until it’s no longer a problem. It’s going to take a while.”
Another recent collaboration with UMaine physicist Sam Hess and GSBS student Kristin Gabor focuses on immune response to viral infection — not necessarily in CF patients. By using super-resolution microscopy, the researchers are the first ever to view the single-molecule cellular interactions involving antiviral signaling in caveolae, which are flask-like invaginations in the cell membrane. While previous research has shown that viruses exploit caveolae to enter host cells, Kim took it a step further by demonstrating that viruses can evade host cell defenses by disrupting clusters of signaling molecules within the caveolae. Through a combination of fluorescent tagging and super-resolution imaging of viruses and zebrafish cells, Hess’ FPALM (Fluorescence Photoactivation Localization Microscopy) system has allowed Kim and her team to see how individual molecules and clusters move during a viral infection.

“No one has ever really looked at this,” Kim says. “No one has been able to see it the way we’ve been able to see it.”

To the uninitiated, these may seem like disparate projects, but they all have two things in common: zebrafish and the innate immune response, the body’s first line of defense against infection. Innate immunity deals with how the body reacts immediately after it comes into contact with a pathogen. This happens daily, almost constantly, and it’s why healthy people don’t get sick every time they encounter a new pathogen. It’s why your skin swells when you get a splinter or a paper cut. This is not to be confused with adaptive immunity, which is acquired through vaccination or prior infection. Zebrafish are the ideal model for this research for several reasons, including the fact that they develop rapidly and their embryos are clear, allowing researchers to see the infection as it happens.

Innate immunity is pivotal to understanding how the body defends itself against infection, how viruses and bacteria adapt to the body’s defenses, and how more effective treatments might be developed.

Even one of these accomplishments would be noteworthy. Together, they’re huge. But when asked if there is a single moment that has defined her time at UMaine, she doesn’t miss a beat.

“Every year, when students in our department get into the top graduate schools, the top medical schools, the top dental schools, when they get great jobs it makes me think, ‘Wow, that’s why we’re here.’ UMaine is only the first step, but we hope we had an impact on their lives. We’re very proud of our students”

Working in Kim’s lab has been the highlight of Walter Mowel’s four years at UMaine. Mowel is a Biology major and Pre-Med student from Montpelier, Vt., whose scientific interest lies in infectious diseases. He spent his final semester researching a virus that hasn’t been studied in zebrafish before. And when he stood up for Kim’s hallway inquisition, his enthusiasm for the project outweighed any fear.

“Before this, I spent a lot of time working on an experiment that didn’t work out, but with this project, I was able to get a lot of data that has me really excited,” Mowel says. “It’s so much fun to essentially look at something for the first time. A lot of times, when you set out to conduct a study, you’ll go to a scientific journal or you’ll go online to see what other scientists have done, but in this case, we couldn’t. We’re basically finding something new and that’s been outstanding.”

Aaron Perreault, a Biochemistry major and Honors student from Northfield, N.H., spent his junior and senior years in Kim’s lab studying Pseudomonas aeruginosa, a bacterium that regularly infects cystic fibrosis patients — one of many pathogens that the innate immune response normally quells.

“When children with cystic fibrosis are younger, they get infections with pathogens that make sense, such as Haemophilius influenzae, staph infections, more common lung pathogens. But by the time they’re in their teens, patients all have Pseudomonas infections, which are very rare in healthy individuals,” Kim says. “Why Pseudomonas rather than a more common respiratory pathogen?”

The answer to that question may someday be used to create therapeutics, and Perreault, one of five UMaine students to gain admission to Tufts University School of Medicine as a sophomore through the Tufts Maine Track Early Assurance program since 2009, may someday prescribe those therapeutics.

Though Perreault admits that the biggest lesson he’s learned is that his lab skills aren’t particularly strong, the experience has also taught him the value of analytical thinking and keeping an open mind. His work in Kim’s lab was the basis for his Honors thesis, and in the weeks leading up to his defense, he was no stranger to Kim’s hallway inquisitions.

“It can be intimidating at first, but once you figure out that she’s grilling you to make you a better student, it makes you work harder,” Perreault says.

Kim will do whatever it takes to instill confidence in her students. Sometimes that means an informal inquisition like the one in the hall. Other times, it means meeting on nights or weekends to make sure that her students are prepared to consider every angle when defending their theses or dissertations. And sometimes, it just means handing over the reins.

When Steve Altman was at UMaine — he earned a bachelor’s in microbiology in 2002 and a master’s in molecular biology in 2003 — he worked on basic immunology in zebrafish. Kim gave him a lot of wiggle room with his experiments, but she also challenged him to try things that might be outside his comfort level, and that continues to influence the way he does science.

“Some of my friends worked in labs where the principal investigator told them what to do,” recalls Altman, who now conducts Alzheimer’s research for Amgen in Cambridge, Mass. “With Carol, it was a little bit more open-ended. She gave me guidance, but she also allowed me to make decisions on my own. That stuck with me.”

Inspiring the next generation of doctors and researchers is what gets Kim out of bed in the morning. She wants them to get jazzed about how crafty bacteria and viruses are. She wants them to experience the thrill of seeing something for the first time. And the best way to do that is through research.

“I love doing the brainstorming, working with a student to figure out the best question to ask,” Kim says. “Seeing them come in to check on their fish, to see what their results are, that’s exciting to me.”

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Developing Muscles

Biology & Biomedical Sciences – Developing Muscles

Munson, D. Biology & Biomedical Sciences – Developing Muscles. UMaine

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Basic biological research on zebrafish may lead to better treatment of human diseases and injuries

“Tendons are incredibly important structures, but exceedingly little is known about tendon development. It is very understudied, but it has implications in the treatment of a variety of tendon afflictions, from tendinitis to disorders caused by antibiotics or cancer treatments,” Henry says. “Traditionally, tendons have been thought of as uniform, with the same protein structure throughout. We have found that that is decidedly not the case. Tendon structure is spatially and temporally dynamic. We’re very excited about looking further at how the type and location of tendon proteins change over time.”

Staring into the crystal-clear plastic boxes inside the University of Maine’s zebrafish research facility, it can be difficult to focus on the movements of an individual fish. Both its stripes and its size make it hard to distinguish from its brethren in a population that is now more than 40,000 strong. The fish, on the other hand, seem to be very good at tracking the movements of an individual human, darting away in a flurry of fins and sinew at the slightest agitation.

At first glance, it seems as if the frightened fish simply bullet through the water, like tiny torpedoes propelled by some hidden, turbocharged motor. But closer observation reveals the true power behind their movements: a fluttering undulation of the body and tail, dependent, of course, on muscle.

With every flip of its fins, tiny bundles of skeletal muscle extend and contract in precisely coordinated synchrony, leveraging their movements against the fish’s miniature frame.

UMaine researcher Clarissa Henry has always been fascinated by the dynamic processes that shape the complex machinery of movement, and has pioneered a unique new system for studying how muscles and tendons develop inside the zebrafish. With a $1.28 million grant from the National Institutes of Health’s National Institute of Child Health and Human Development, Henry is shining a microscope-mounted light into the darkest corners of developmental biology, revealing new truths about embryonic processes that may lead to better treatment methods for conditions ranging from muscular dystrophy to tendinitis.

Henry’s current research, aimed at developing a better understanding of tendon formation and attachment in the embryo, is the next step in her pioneering efforts to describe the complexities of early development in vertebrates using zebrafish. Her previous research, funded by the Muscular Dystrophy Association, looked at how embryonic muscle cells transform from relatively stubby globs of cytoplasm into the long, multinucleated fibers of skeletal muscle in fully developed fry.

Skeletal muscles–from the orbicularis oculi to the gluteus maximus–are primarily responsible for movement in vertebrates, and abnormalities that arise during their formation can have dire consequences. For example, muscular dystrophy, one of the most common genetic diseases in humans, is characterized by a loss in muscular function that can manifest in many ways.

“One of the critical questions related to the treatment of muscular dystrophy is: How do humans make muscle during embryonic development?” says Henry. “We were able to make a significant step forward in this area because we were able to use an in vivo model. Prior to our work, no one was able to look into a live vertebrate embryo to see how muscle cells form at high resolution. We were able to do that with the zebrafish, thanks to the MDA.”

The strength of the preliminary data was one of the reasons NIH reviewers expressed such strong support for Henry’s latest project, pointing to her well-established methodology and the work’s potential benefits in the treatment of human disease. In addition to her obvious enthusiasm for the research, Henry has a technological advantage as well, utilizing cutting-edge equipment like a Zeiss ApoTome fluorescence microscope to peer inside the living embryo.

Like their plastic tanks, the developing eggs of the zebrafish are largely transparent, allowing researchers to observe and record changes in the cells as they happen, which is difficult or impossible in other vertebrate research models, such as mice or chickens. The zebrafish model has advantages over cell culture techniques as well, revealing important nuances in growth and development that can only be seen when cells form under the influence and constraints of a living organism.

With the help of the tiny zebrafish, Henry’s early work uncovered “a phenomenal amount of data” regarding muscle cell development, laying the foundation for further research related to tendon attachment and other processes. The new research path has already led Henry and her team to some important discoveries.

“There’s a lot of basic science in this area that we just don’t understand,” says Henry. “We don’t know how these structures grow, how they increase in mass or how the attachment between the tendon and the skeleton is maintained. There’s a real opportunity here to do pioneering work.”

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Battle Lines

Battle Lines

Staples, B. (2013) Battle Lines. UMaine Today

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A UMaine biomedical lab looks for answers in the transformation of a peaceful yeast to fatal fungus

“We’re using zebrafish to ask really specific questions that cannot be
answered another way,” Wheeler says. “These questions have been inaccessible
for a long time. We hope to be able to better utilize existing therapies and be
able to develop better therapies.”

LIFE-AND-DEATH battles rage in Robert Wheeler’s lab at the University of Maine. The combatants — zebrafish and Candida albicans— fight to the bitter end in glass-bottom microplates. Similar perilous battles are being fought inside humans. The C. albicans fungus is a leading cause of hospital-acquired infection that annually kills several thousand patients nationwide. During the staged scuffles in Wheeler’s lab in Hitchner Hall, anesthetized zebrafish are injected with Candida and placed in a gelatinous material called agarose.
A laser microscope captures and magnifies the struggles inside the zebrafish blood vessels in real time in high-definition color detail. The microplate clashes provide the assistant professor of microbiology with the ability to view how immune cells fight the microbe, identify genes involved in virulence, test new drugs and learn how gene perturbations affect hostpathogen interaction.

In March 2012, Wheeler received a three-year, more than $421,600 grant from the National Institutes of Health to ask and answer these questions in the project: “Genetics & Visualization of Innate Host Response to Candida albicans Infection In Vivo.” The goal is that the resulting answers will save human lives.

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