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Author: Donna Huber

Celebrate the 10th Anniversary of SEFCIG

SECFIG annual meeting

Celebrate the 10th anniversary of the Southeast Flow Cytometry Interest Group (SEFCIG) with 4 days of educational training and scientific talks. SEFCIG was founded by Julie Nelson, the director of UGA’s CTEGD Cytometry Shared Resource Laboratory. The 10th Annual Meeting is being hosted on the University of Georgia campus in Athens, GA.

10th Annual Meeting March 5 – 8, 2019

March 5 – 6         ExCyte – Flow Cytometry Boot Camp

Register here: https://excyteatuga.eventbrite.com/

Special discounts are available to the UGA community and those attending SEFCIG, please email Tim Bushnell (timbushnell@expertcytometry.com) for details.

March 7 – TechnoFlow

Location: Paul D. Coverdell Building Room S175

11:00 – 12:00

Forensic Flow – Join us to test your skills at detecting bad flow data
Jodi Kroeger, Moffitt Cancer Institute

12:00 – 1:00

 Luncheon – Coverdell Rotunda

1:00 – 1:45

Multi-Dimensional Functional Profiling of Human Rhinovirus and Allergen- specific T-Cells By Means Of Spectral Flow Cytometry
Liesbeth Paul and Joanne Lannigan, University of Virginia

1:45 – 2:30

 Imaging and Spectral Cytometry go Viral!
Joanne Lannigan, University of Virginia

2:30 – 3:15

High Dimensional Mass Cytometry Data Analysis
Deon Bryant, Emory University

3:15 – 4:00

Next Generation Cell Sorting: New Technologies and Strategies
Joe Trotter, Becton Dickinson

6:00 – 9:00

Opening Reception – Pecan Tree Galleria

March 8 – General Session

Location: Masters Hall, Georgia Center for Continuing Education

Flow in the South

9:00 – 9:10

Welcome and Introductions

9:10 – 9:55

Canine Breast Cancer Immunotherapy as a Model of Human Disease
Curtis Bird, Auburn University

9:55 – 10:40

Single cell analyses of human B cell responses: Lessons from infectious disease and autoimmunity
Jens Wrammert, Emory University

10:40 – 11:25

Using Flow Cytometry to Catch Parasites Sleeping
Dennis Kyle, University of Georgia

11:25 – 12:00

Exhibits

Vendor Break Out Session with Box Lunches

12:00 – 2:45

Vendor Talks and Exhibits

Flow in the World

2:45 – 3:30

Extracellular Vesicle-Biome Analysis by Nanoscale High Resolution Flow Cytometry
Terry Morgan, Oregon School of Health

3:30 – 4:15

Optimizing and validating FC-based EV measurements
John Nolan, Scintillon Institute

4:15 – 5:00

Developing Flow Cytometry Assays to Support Clinical Trials
Jake Jacobberger, Case Western

Register and make hotel reservations at www.sefcig.org

Focus on Faculty: Courtney Murdock

Courtney Murdock in her laboratory at the School of Veterinary Medicine (Photo by Dorothy Kozlowski/UGA)

Courtney Murdock, an assistant professor with a joint appointment in the College of Veterinary Medicine, the Odum School of Ecology and CTEGD, studies the transmission of mosquito-borne diseases to inform predictions about disease patterns and interventions to disrupt transmission.

Where did you earn degrees and what are your current responsibilities at UGA?

I earned my Bachelor of Science degree in biology with a minor in Spanish literature at the University of Michigan, where I also earned my Ph.D. in the School of Natural Resources and the Environment. I was a postdoctoral researcher in the departments of biology and entomology at Pennsylvania State University and am currently an assistant professor with a joint appointment in the department of infectious diseases in the UGA College of Veterinary Medicine and the Odum School of Ecology.

When did you come to UGA and what brought you here?

I began my current position at UGA in 2014. I was excited to join the faculty here due to the growing expertise in infectious diseases across campus, having access to excellent colleagues in the College of Veterinary Medicine and the world-renowned Odum School of Ecology, and the plethora of resources available concerning facilities, expertise and support for graduate students.

What are your favorite courses and why?

My favorite courses that I took as an undergraduate and graduate student, and to teach as a professor, are ecology courses. Ecology is a modern science that is the study of the interactions among organisms and the environment. This field of study provides key insights into how the environment shapes interactions among organisms, their abundances, where they live, and our overall impact. Ecological knowledge is crucial for understanding and mitigating some of the biggest problems we will have to contend with in the future—some of which include global climate change, natural catastrophes, food and water scarcity, the evolution of antibiotic resistance, and emerging infectious diseases.

What are some highlights of your career at UGA?

My research on mosquito-borne diseases has been well-supported by agencies such as the National Institutes of Health and National Science Foundation, with total funding exceeding $1.2 million since 2014. These funds have supported laboratory research, as well as fieldwork in the U.S. and the Caribbean. The results of my research have been published in high-quality scientific journals of international standing, and my research findings have been cited nearly 1,000 times.

I also mentor 17 undergraduate students, one D.V.M. student, five Ph.D. students, and two postdoctoral researchers. All of my mentees gain hands-on experience working in an infectious disease system in the lab or field, as well as exposure to a diversity of host-parasite/pathogen systems and projects that are both basic and applied in nature. My students have a strong record of success, with two NSF Graduate Research Fellowships, four travel awards to attend international conferences to present their work, and two awards for presenting research at local venues.

How do you describe the scope and impact of your research or scholarship to people outside of your field?

I am interested in understanding what drives the transmission of mosquito-borne diseases. The mosquito is the deadliest organism on this planet because of the harmful organisms it transmits to humans, wildlife and domestic animals. Many of these diseases cannot be treated with drugs or prevented with vaccines. Thus, only through an understanding of the transmission process will we be able understand when we are at most risk to contract these diseases, predict how current disease distributions might change in the future, and develop interventions that efficiently disrupt transmission.

How does your research or scholarship inspire your teaching, and vice versa?

For me there is quite a bit of cross-talk between my research knowledge and experiences and my teaching. One important goal as an instructor in the sciences is to impart a solid understanding of the scientific process. Many students who take my courses do not necessarily want a career in science. I believe that to be informed citizens, however, they need to be able to think critically about science and its contributions to society. The best way I have found outside of lab sections to impart this knowledge is from drawing on my own research experiences. I also have found that my teaching informs the direction of my research program because it encourages me to think about my research from the perspective of foundational concepts in ecology.

What do you hope students gain from their classroom experience with you?

In general, the learning objectives for my courses include understanding the conceptual foundations of ecology, becoming comfortable understanding and working with scientific data, being familiar with the scientific method, and being able to engage in discussion and make informed decisions about ecological and environmental issues.

On the less concrete side, I want them to wonder at how amazing the natural world is, be curious about it, understand our part and overall impact, and to be more informed, science-literate citizens.

Describe your ideal student.

Here are some characteristics I value in both undergraduate and graduate students that I work with (this is not ranked in any particular order):

  • Curiosity—always questioning why and how.
  • Self-starter—only you can advocate for your interests and education.
  • Life learner—there is no rubric for life; college and graduate school is the perfect place to begin learning how to teach yourself the material you need to know to pass the test, complete course objectives, fulfill job expectations, answer your own questions, etc.
  • Positive—this shapes everything, your outlook on life and work, general happiness, interactions with co-workers.
  • Hardworking – willing to do what is needed to get the task at hand done.
  • Creative – ability to think outside of the box, willingness to explore and adopt concepts from other fields in order to innovate or solve existing problems.
  • Team member – working effectively with people with different backgrounds, knowledge, working styles and personalities is a life skill that is beneficial across a diversity of situations and careers.
  • Fearless – failure is an opportunity to learn and grow.
  • Responsible – this goes beyond just being reliable and detail oriented. It involves taking ownership over success and failure as well as both positive and negative interactions with others.
  • Human – have outside interests, be respectful to others, empathize with others.
Courtney Murdock works with postdoc Christine Reitmeyer in the insectary, where they conduct research on mosquito-borne diseases. (Photo by Dorothy Kozlowski/UGA)

Favorite place to be/thing to near campus is…

… eating lunch at Cali N Tito’s with colleagues or members of my lab.

Beyond the UGA campus, I like to…

In addition to being a scientist and a professor, I am a mother of two kids, a wife, a daughter, a sister, and a friend. I spend as much time as I can manage with my family and friends outside of work. This involves simple things like going swimming with the kids at the YMCA, going to the local library, going to museums or Lego Land in Atlanta, playing at local parks when the weather is nice, hiking at Fort Yargo State Park or Sandy Creek Nature Center, and occasionally camping in the mountains of Georgia. We also spend a lot of our vacation visiting family in Chicago, Illinois, and Traverse City, Michigan.

Favorite book/movie (and why)?

Favorite nonfiction: “Devil in the White City.” I grew up in the suburbs of Chicago, so it was really insightful and fun to read about how much of the city and world was shaped by the World Fair of 1893. There is also a side story involving a serial killer, which is totally gripping.

Favorite fiction: The “Outlander” series by Diana Gabaldon. These novels are historical fiction, mixed with fantasy and a pinch of romance. The characters are well developed, complex, and the history well researched, so is a perfect storm for losing oneself completely.

The one UGA experience I will always remember will be…

Every year my lab picks a themed costume and dresses up for Halloween. To me this is special, as it is an opportunity for our group to do something fun, wacky and together. Current pictures are on our website: https://www.themurdocklab.com/people.

Is there anything else you’d like to add?

I was an NCAA Division 1 scholar-athlete. I walked on to the University of Michigan softball team as a freshman during my undergraduate career. While I never started, I learned a lot of life skills from this experience that translate to my perspective on life, challenges, teamwork and leadership. I feel like there are stereotypes associated with student-athletes that are oftentimes unwarranted concerning their scholarship, and we should be mindful of this in our interactions with student-athletes in the classroom. I feel that they bring a lot of underappreciated assets to the table.

First published at UGA Today.

Mitochondrial genome sequence variation as a useful marker for assessing genetic heterogeneity among Cyclospora cayetanensis isolates and source-tracking

Abstract

BACKGROUND: Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks.

METHODS: In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products.

RESULTS: All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking.

CONCLUSIONS: The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.

KEYWORDS: Cyclospora cayetanensis; Genotyping; Mitochondrion; Source-tracking; qPCR

Yaqiong Guo, Yuanfei Wang, Xiaolan Wang, Longxian Zhang, Ynes Ortega, and Yaoyu Feng. Parasite Vectors 2019 Jan 21; 12(1):47. doi.1186/s13071-019-3294-1

Trypanosoma cruzi 13C-labeled O-Glycan standards for mass spectrometry

Abstract

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a debilitating condition that affects over 10 million humans in the American continents. In addition to its traditional mode of human entry via the ‘kissing bug’ in endemic areas, the infection can also be spread in non-endemic countries through blood transfusion, organ transplantation, eating food contaminated with the parasites, and from mother to fetus. Previous NMR-based studies established that the parasite expresses a variety of strain-specific and developmentally-regulated O-glycans that may contribute to virulence. In this report, we describe five synthetic O-glycan analytical standards and show their potential to enable a more facile analysis of native O-glycan isomers based on mass spectrometry.

M. Osman Sheikh, Elisabet Gas-Pascual, John N. Glushka, Juan M. Bustamante, Lance Wells, Christopher M. West. 2019. Glycobiology. https://doi.org/10.1093/glycob/cwy111

Kinetoplast Division Factors in a Trypanosome

Trypanosoma brucei

Highlights

  • Kinetoplasts (mitochondrial genome nucleoids) are important in bloodstream trypanosomes for the establishment of mitochondrial membrane potential.
  • Many proteins involved in segregation of kinetoplasts have been identified.
  • A region between a kinetoplast and basal bodies is described as a tripartite attachment complex (TAC).
  • A set of TAC-associated proteins (TACAPs) has been proposed as the machinery for kinetoplast segregation.
  • Subcomplexes of TACAPs that form in vivo have been described.
  • Several proteins that do not associate with TAC are involved in the maintenance of the kinetoplast.
  • New kinetoplast-associated proteins have been identified.
  • We are approaching an exciting period in the field when a molecular understanding of how all aspects of kinetoplast biogenesis are executed seems achievable.

Kojo Mensa-Wilmot, Benjamin Hoffman, Justin Wiedman, Catherine Sullenberger, Amrita Sharma. 2019. Trends in Parasitology. https://doi.org/10.1016/j.pt.2018.11.002

Survival and Internalization of Salmonella and Escherichia coli O157:H7 Sprayed onto Different Cabbage Cultivars during Cultivation in Growth Chambers

cabbage

ABSTRACT

BACKGROUND: Cabbage may become contaminated with enteric pathogens during cultivation. Using multiple cabbage cultivars at two maturity stages (small plants or plants with small heads) in growth chamber studies, the fate (internalization or surface survival) of Salmonella and Escherichia coli O157:H7 (0157) were examined in conjunction with any potential relationships to the plant’s antimicrobial content.

RESULTS: Internalized Salmonella was detected in cabbage within 24 hours with prevalence ranging from 62% (16 of 26) for the ‘Super Red 80’ cultivar to 92% (24 of 26) for the ‘Red Dynasty’ cultivar. The fate of Salmonella and O157 on small cabbage plants over nine days was significantly affected by cultivar with both these pathogens surviving the least and most on the ‘Capture’ and ‘Farao’ cultivars, respectively (P < 0.05). Survival of O157 was slightly higher on cabbage heads for O157 than small plants suggesting that the maturity stage may affect this pathogen’s fate. An inverse relationship existed between antimicrobial levels and a pathogen’s fate on cabbage heads (P < 0.05).

CONCLUSIONS: The fate of pathogens varied with the cabbage cultivar in growth chamber studies highlighting the potential to explore cultivar in field studies to reduce the risk of microbiological contamination in this crop.

Marilyn C. Erickson, Jye-Yin Liao, Alison S. Payton, Peter W. Cook, Ynes R. Ortega. 2019. Journal of the Science of Food and Agriculture.
https://doi.org/10.1002/jsfa.9573

Researchers receive $2M NIH instrumentation grant

by Alan Flurry

The National Institutes of Health has awarded University of Georgia researchers $1.956 million for a high-resolution mass spectrometer that will enhance capabilities for scientists in many fields across campus.

The award by the NIH High End Instrumentation program, which provides grants in the range of $600,000 to $2 million for a variety of expensive instrumentation, including MRI imagers, electron microscopes, DNA sequencers, and mass spectrometers, was one of 30 awards made in the program, and one of only six mass spectrometer requests funded in the 2018 cycle.

The grant funded a 12 Tesla Bruker Solarix FTMS, a high-resolution mass spectrometer capable of measuring molecular weights with precision accuracy that can be applied to molecules ranging in size from small metabolic products to intact proteins and protein complexes. It can also provide molecular structure through a multidimensional analysis method known as tandem mass spectrometry. The instrument will be used to support research in metabolomics and glycomics, the analysis of genetic, physiologic and pathologic aspects of sugar molecules involved in all biological process from modulating cell function to determining cancer development.

“This instrument will enhance the research capabilities for a number of scientists in chemistry, the biological sciences and biomedical research, and will help foster interdisciplinary research projects between groups in a number of departments and colleges at the university,” said Jon Amster, professor and head of the department of chemistry and principal investigator on the grant.

Over a dozen researchers will be major users of this instrument, which will be housed in the Amster laboratory in the department of chemistry.

“The new 12T FT-ICR instrument will greatly improve our ability to perform metabolomics analysis, especially regarding to the identification of unknown metabolites, since this instrument has higher accuracy and resolving power than the current instruments at UGA,” said Belen Cassera, associate professor of biochemistry and molecular biology, member of the Center for Tropical and Emerging Global Diseases, and co-principal investigator on the grant. “This type of grant can be particularly difficult to obtain and it is a privilege for me to be part of an amazing team of investigators that put together this application.”

“Virtually every metabolomics project we have going right now will benefit from this new instrumentation grant,” said Art Edison, GRA Eminent Scholar, professor of biochemistry and molecular biology, and a co-principal investigator on the grant. “High resolution mass spectrometry is a very important tool for the analysis of complex biological mixtures and unknown metabolite identification in applications ranging from human disease to carbon cycling in the ocean to model organisms for pathway analysis.”

Of the 104 NIH shared instrumentation grants made this year during 2018, only 10 were in the range of $1.9 million to $2 million.

Thirty-Day Daily Comparisons of Kato-Katz and CCA Assays of 45 Egyptian Children in Areas with Very Low Prevalence of Schistosoma mansoni

Egyptian children

Forty-five Schistosoma mansoni egg–negative/circulating cathodic antigen (CCA) low (Trace-1+) positive children in areas of very low prevalence were followed up daily for 30 days. Stool and urine specimens were collected and examined each day from each child. At the midpoint of the study, three egg-positive control persons with light intensity infection were included in the protocol. Stool samples were examined by the Kato–Katz (four slides/stool sample) technique and all S. mansoni egg–negative stools were further tested by the “miracidia hatching test” (MHT). Urine samples were examined by the point-of-care CCA assay (POC-CCA). Over 30 days, only one of 1,338 consecutive stool samples from study subjects was S. mansoni egg and MHT positive (0.07%). Egg counts fluctuated daily in stools from positive controls and S. mansoni miracidia were detected in all but two samples by the MHT. Point-of-care–circulating cathodic antigen bands were scored from G1 to G10 and then translated to standard Trace, 1+, 2+, 3+ banding patterns. In two districts, the POC-CCA assays were Trace or 1+ for both the study children and the positive controls. In the third district, the POC-CCA assays were Trace or 1+ for the study children and 1+ or 2+ for the positive control. We conclude that in areas with extremely low prevalence S. mansoni egg–negative and CCA-Trace or 1+ children are unlikely to pose substantial risks to continued transmission of schistosomiasis. In this setting, POC-CCA Trace or 1+ readings are likely to be false positives or perhaps represent low-level single-sex schistosome infections.

Ayat A. Haggag, Amal Rabiee, Khaled M. Abd Elaziz, Carl H. Campbell Jr., Daniel G. Colley, and Reda M. R. Ramzy. 2019 The American Journal of Tropical Medicine and Hygiene. https://doi.org/10.4269/ajtmh.18-0829

Efficacy and side effects of doxycycline versus minocycline in the three-dose melarsomine canine adulticidal heartworm treatment protocol

Abstract

Background: The American Heartworm Society currently recommends the use of a monthly macrocyclic lactone, a 28-day course of 10 mg/kg doxycycline BID, and the 3-dose protocol of melarsomine dihydrochloride for the treatment of canine heartworm disease. Doxycycline is necessary for the reduction of the bacterium Wolbachia, found in all heartworm life-stages. Previous price increases and decreasing availability prompted us to evaluate alternative tetracycline antibiotics, i.e. minocycline, for the reduction of Wolbachia during canine heartworm treatment.

Methods: Thirty-two heartworm-positive dogs were randomized to receive 10 mg/kg or 5 mg/kg of either doxycycline or minocycline for 28 days BID, for a total of 8 dogs per experimental group. All dogs received 6 months of Heartgard Plus® (ivermectin/pyrantel) and the 3-dose protocol of 2.5 mg/kg melarsomine dihydrochloride. Blood samples were collected prior to the initiation of treatment, every 7 days throughout tetracycline treatment, and then monthly thereafter until the dog tested negative for the presence of heartworm antigen. DNA was isolated from circulating microfilarial samples and qPCR was performed on each sample.

Results: A greater number of dogs in the 10 mg/kg doxycycline and minocycline treated groups experienced gastrointestinal side effects as compared to the 5 mg/kg doxycycline and minocycline treated groups. All eight dogs in the 10 mg/kg doxycycline-treated group tested negative for the presence of Wolbachia DNA by 28 days post-tetracycline treatment. A total of two dogs in both the 5 mg/kg doxycycline- and 10 mg/kg minocycline-treated groups and three dogs in the 5 mg/kg minocycline-treated group remained positive for the presence of Wolbachia DNA by the end of tetracycline treatment.

Conclusions: No lung pathology was assessed in this clinical trial, therefore the clinical effect of the remaining Wolbachia DNA in the 10 mg/kg minocycline-, 5 mg/kg doxycycline- and 5 mg/kg minocycline-treated groups cannot be determined. Owner compliance in the proper administration of these tetracyclines may be impacted by the increased severe gastrointestinal side effects reported for the 10 mg/kg doxycycline- and minocycline-treated groups. We recommend that veterinarians prescribe the recommended 10 mg/kg doxycycline for canine heartworm treatment and reduce the dosage to 5 mg/kg in cases of severe gastrointestinal side effects in order to improve owner compliance in administration of medications.

Molly D. Savadelis, Katherine M. Day, Jenna L. Bradner, Adrian J. Wolstenholme, Michael T. Dzimianski, and Andrew R. Moorhead. 2018. Parasites & Vectors; 11:671. https://doi.org/10.1186/s13071-018-3264-z