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Author: Donna Huber

p53 Hinders CRISPR/Cas9-Mediated Targeted Gene Disruption in Memory CD8 T Cells In Vivo

Donna Huber on September 4, 2020

CRISPR/Cas9 technology has revolutionized rapid and reliable gene editing in cells. Although many cell types have been subjected to CRISPR/Cas9-mediated gene editing, there is no evidence of success in genetic alteration of Ag-experienced memory CD8 T cells. In this study, we show that CRISPR/Cas9-mediated gene editing in memory CD8 T cells precludes their proliferation after Ag re-encounter in vivo. This defect is mediated by the proapoptotic transcription factor p53, a sensor of DNA damage. Temporarily inhibiting p53 function offers a window of opportunity for the memory CD8 T cells to repair the DNA damage, facilitating robust recall responses on Ag re-encounter. We demonstrate this by functionally altering memory CD8 T cells using CRISPR/Cas9-mediated targeted gene disruption under the aegis of p53siRNA in the mouse model. Our approach thus adapts the CRISPR/Cas9 technology for memory CD8 T cells to undertake gene editing in vivo, for the first time, to our knowledge.

Samarchith P. Kurup, Steven J. Moioffer, Lecia L. Pewe and John T. Harty. J Immunol. 2020 Sep 4;ji2000654. doi: 10.4049/jimmunol.2000654.

IP3 receptor-mediated Ca2+ release from acidocalcisomes regulates mitochondrial bioenergetics and prevents autophagy in Trypanosoma cruzi

Donna Huber on September 2, 2020

In contrast to animal cells, the inositol 1,4,5-trisphosphate receptor of Trypanosoma cruzi (TcIP3R) localizes to acidocalcisomes instead of the endoplasmic reticulum. Here, we present evidence that TcIP3R is a Ca2+ release channel gated by IP3 when expressed in DT40 cells knockout for all vertebrate IP3 receptors, and is required for Ca2+ uptake by T. cruzi mitochondria, regulating pyruvate dehydrogenase dephosphorylation and mitochondrial O2 consumption, and preventing autophagy. Localization studies revealed its co-localization with an acidocalcisome marker in all life cycle stages of the parasite. Ablation of TcIP3R by CRISPR/Cas9 genome editing caused: a) a reduction in O2 consumption rate and citrate synthase activity; b) decreased mitochondrial Ca2+ transport without affecting the membrane potential; c) increased ammonia production and AMP/ATP ratio; d) stimulation of autophagosome formation, and e) marked defects in growth of culture forms (epimastigotes) and invasion of host cells by infective stages (trypomastigotes). Moreover, TcIP3R overexpressing parasites showed decreased metacyclogenesis, trypomastigote host cell invasion and intracellular amastigote replication. In conclusion, the results suggest a modulatory activity of TcIP3R-mediated acidocalcisome Ca2+ release on cell bioenergetics in T. cruzi.

Miguel A. Chiurillo, Noelia Lander, Anibal E. Vercesi, Roberto Docampo, 2020. Cell Calcium; 92:102284, https://doi.org/10.1016/j.ceca.2020.102284.

Fine-scale heterogeneity in Schistosoma mansoni force of infection measured through antibody response

Donna Huber on August 31, 2020

Schistosomiasis is among the most common parasitic diseases in the world, with over 142 million people infected in low- and middle-income countries. Measuring population-level transmission is centrally important in guiding schistosomiasis control programs. Traditionally, human Schistosoma mansoni infections have been detected using stool microscopy, which is logistically difficult at program scale and has low sensitivity when people have low infection burdens. We compared serological measures of transmission based on antibody response to S. mansoni soluble egg antigen (SEA) with stool-based measures of infection among 3,663 preschool-age children in an area endemic for S. mansoni in western Kenya. We estimated force of infection among children using the seroconversion rate and examined how it varied geographically and by age. At the community level, serological measures of transmission aligned with stool-based measures of infection (ρ = 0.94), and serological measures provided more resolution for between-community differences at lower levels of infection. Force of infection showed a clear gradient of transmission with distance from Lake Victoria, with 94% of infections and 93% of seropositive children in communities <1.5 km from the lake. Force of infection increased through age 3 y, by which time 65% (95% CI: 53%, 75%) of children were SEA positive in high-transmission communities—2 y before they would be reached by school-based deworming programs. Our results show that serologic surveillance platforms represent an important opportunity to guide and monitor schistosomiasis control programs, and that in high-transmission settings preschool-age children represent a key population missed by school-based deworming programs.

Benjamin F. Arnold, Henry Kanyi, Sammy M. Njenga, Fredrick O. Rawago, Jeffrey W. Priest, W. Evan Secor, Patrick J. Lammie, Kimberly Y. Won, and Maurice R. Odiere. Proc Natl Acad Sci U S A. 2020 Aug 31;202008951. doi: 10.1073/pnas.2008951117.

Noncoding RNAs in Apicomplexan Parasites: An Update

Donna Huber on August 20, 2020

Illustration of Long Noncoding RNA (lncRNA) Functions in Apicomplexan Parasites.

Recent breakthroughs in high-throughput technologies, transcriptomics, and advances in our understanding of gene regulatory networks have enhanced our perspective on the complex interplay between parasite and host. Noncoding RNA molecules have been implicated in critical roles covering a broad range of biological processes in the Apicomplexa. Processes that are affected range from parasite development to host–parasite interactions and include interactions with epigenetic machinery and other regulatory factors. Here we review recent progress involving noncoding RNAs and their functions in the Apicomplexa, with a focus on three parasites: Plasmodium, Toxoplasma, and Cryptosporidium. We discuss the limitations and challenges of current methods applied to apicomplexan noncoding RNA study and discuss future directions in this exciting field.

 

 

 

Yiran Li, Rodrigo P. Baptista, Jessica C. Kissinger. Trends Parasitol. 2020 Aug 19;S1471-4922(20)30189-6. https://doi.org/10.1016/j.pt.2020.07.006

Trainee Spotlight: Alona Botnar

Donna Huber on August 18, 2020

Alona Botnar

T32 trainee Alona Botnar is entering her fifth year as a Ph.D. candidate in Dr. Dennis Kyle’s laboratory. She is from Doylestown, Pennsylvania and completed her B.S. in Chemistry with a minor in Biochemistry at the University of Georgia in 2015. During her undergraduate career, she also worked at Janssen, a pharmaceutical company of Johnson & Johnson as a Biologics R&D co-op.

As a graduate student, she was able to spend three semesters teaching Anatomy and Physiology labs here at UGA, and in 2019, she was awarded the Outstanding Teaching Assistant Award sponsored by the Office of the Vice President for Instruction. She also received a graduate school travel award to attend the 2018 annual meeting of the American Society of Tropical Medicine and Hygiene in New Orleans, Louisiana, and an Office for Vice President and Research travel award to attend the 2020 Molecular Approaches to Malaria meeting held in Lorne, Victoria, Australia.

Why did you choose UGA?

Having been at UGA for my undergraduate degree, I was already in love with UGA and Athens and all that they have to offer. I was attracted to the Integrated Life Sciences program because it gives incoming graduate students the freedom to explore a wide range of research topics among 14 different departments before choosing the lab they would ultimately like to join. Furthermore, I found the interdisciplinary approach of the program appealing. I love the Center for Tropical and Emerging Global Diseases because of all the resources available to us as scientists. Not only do we have state of the art microscopy and flow cytometry cores, but we also have very collaborative labs that are happy to share equipment, supplies, and expertise.

What is your research focus?

My research is focused on malaria and addressing significant problems at the stages of development at which the malaria parasite enters a drug-induced dormant period and evades the antimalarial. The mechanism by which the parasite enters drug-induced dormancy and later recrudesces to continue development is currently unknown.

Half the world’s population is at risk of malaria with about half a million people dying each year from it. A majority of these deaths are in children under the age of 5, located mainly in sub-Saharan Africa. While there are 5 species that can infect humans, Plasmodium falciparum is the most lethal and is responsible for a majority of the deaths. Our current arsenal of malaria drugs is failing at an alarming rate as drug-resistant strains of the parasite continue to emerge.

Thus I chose this research project because it is vital that we respond to the challenge of antimalarial drug resistance by not only developing novel drugs but also by understanding the mechanisms the parasite is using to evade the drugs.

What are your future career goals?

I plan on continuing my work in the field of infectious diseases. I am leaning towards industry research but I’m keeping an open mind. Lately, I have been interested in alternative careers available to life science Ph.D.’s such as consulting and being a medical science liaison.

What is your favorite thing about UGA?

UGA football. There’s nothing quite like a fall Saturday in Athens between the hedges. GO DAWGS!

Do you have any advice for a student interested in this field?

It’s never too early or too late to get into the field. Don’t be afraid to send those emails and get involved in research. And always ask questions.

 

Support trainees like Alona by giving today to the Center for Tropical & Emerging Global Diseases.

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Age influences thermal tolerance in Asian malaria mosquito

Donna Huber on August 11, 2020

Environmental portrait of Courtney Murdock in her laboratory at UGA’s School of Veterinary Medicine (Dorothy Kozlowski)

Malaria disease transmission models are important tools for controlling and eliminating disease spread. However, a model is only as good as the assumptions about the various variables. Dr. Courtney Murdock, a member of the UGA’s Center for Tropical and Emerging Global Diseases and professor at Cornell University, has been studying how various biological and environmental factors influence mosquito survival. In a study recently published in the Proceedings of the Royal Society B led by graduate student Kerri Miazgowicz, Murdock and her colleagues examined several life traits, such as biting, feeding, and egg production, over the course of the life span of the mosquito Anopheles stephensi in hopes of providing better data for the models.

“Due to a lack of high-quality entomological data in general, researchers are often forced to input data from multiple disease systems to inform models in a given system or use approximations of key model components,” said Murdock.

An. stephensi is the primary mosquito species that transmits malaria in India. While most of the focus on malaria is most often associated with sub-Saharan Africa, it is widespread in the Indian subcontinent and throughout southeast Asia. Several Plasmodium species cause malaria, but P. falciparum is the deadliest of them. It has also shown drug resistance to current treatments. Control of the mosquito population is an important component in malaria control and elimination programs. Researchers need to be able to more accurately predict where mosquito populations will occur as climate changes and current territories become unsuitable living and breeding grounds. Program managers need to be prepared to incorporate more northern regions in their control efforts.

trainee field work
Kerri Miazgowicz, a graduate student in the Murdock Laboratory at the University of Georgia, led the study on the effects of age on thermal tolerances.

Current models rely on data that are only snapshots in time and often from multiple mosquito species, particularly the African mosquitoes, which are vectors for different malaria species. Miazgowicz, Murdock, and colleagues wanted to determine if the data from a single species of mosquito and parasite, over the course of its entire lifespan, significantly influenced current models in determining disease transmission in hopes of creating more accurate models.

The single most important factor driving current models is temperature. Mosquitoes are cold-blooded animals and therefore rely on their environment to regulate their body temperature. However, temperature is not the only factor influencing life traits. Currently, data are only available as snapshots in time. These incomplete data do not take into account for changes in mosquito behavior and life traits that occur over the course of the mosquito’s life. Murdock and her colleagues have recorded changes in biological function as the mosquito ages. Just as people slow down biologically as they age – metabolism slows, reproduction ability declines, etc. – the same is true for mosquitos. They also found that various traits peak at different times depending on temperature. Importantly they found that temperature and age significantly affected the number of females taking a blood meal (this is the means in which malaria parasites are transmitted to humans) on a given day, average daily egg production, and ultimately survival.

The findings in this study indicated that the addition of An. stephensi data yielded qualitatively different temperature-transmission suitability relationships compared to models that included multiple malaria vectors. With An. stephensi data, the model predicted a broader geographical range of temperature suitability.

“Accounting for these age and species effects in models of transmission potential alters how much of South Asia is predicted to be suitable for malaria relative to models that do not account for these factors,” said Murdock.

These findings can lead to improved malaria transmission models. However, more study outside the laboratory is needed to truly understand the impact mosquito age has on life traits and thermal tolerance.

“This study highlights a critical need for more research in natural settings characterizing the effects of age on mosquito biology to improve predictions of current and future risk,” concluded Murdock.

 

NIH awards CTEGD $1.9 million to support training in tropical and emerging global diseases

Donna Huber on August 6, 2020

UGA’s Center for Tropical and Emerging Global Diseases has been awarded $1.9 million from the National Institutes of Health to continue its pre- and post-doctoral training program for the next five years. First funded in 2004, CTEGD has received nearly $2 million from NIH to train the next generation of scientists in the fight against neglected tropical diseases. The new funding will support additional fellowships and new training initiatives.

Every year, parasitic diseases are responsible for more than a million deaths and leave hundreds of millions more with chronic infections lasting years. However, there are few U.S. programs that specifically train researchers in this field.

CTEGD is recognized as a pre-eminent research center that brings together the largest number of laboratories in the U.S. to study the full gamut of parasitic diseases, which are highly prevalent in sub-Saharan Africa, South America and Asia. Often, these diseases are the consequence and cause of poverty, and they are increasingly emerging (or re-emerging) in industrialized nations. Most of these diseases have no cure and few treatment options, and growing drug resistance complicates the fight against them. Now more than ever, researchers are needed in the field of parasitology and tropical diseases in order to improve global health.

NIH recognizes the need for researchers trained in parasitology and tropical disease, as well as UGA’s and CTEGD’s ability to fill it, by providing funding for two additional pre-doctoral fellowships. CTEGD will now be able to award five pre-doctoral fellowships and two post-doctoral fellowships each year

“This is incredibly exciting,” said Dr. Silvia Moreno, program director of CTEGD’s Training in Tropical and Emerging Global Diseases (TTEGD) and Distinguished Research Professor in cellular biology. “It’s a very competitive program and having additional pre-doc positions funded is even more exciting. Our program offers a great environment for training in parasitology which is the result of the hard work of the CTEGD community plus great UGA institutional support”.

The support of the university community

UGA’s support of the program has been instrumental to its continuing funding by NIH. The state-of-the-art equipment and facilities available to trainees allow them to learn cutting-edge techniques and technologies.

Under the direction of Vice President David Lee, the Office of Research has committed significant support over the next five years to fund the capstone experience, which allows each fellow to conduct research in an international setting, and two additional pre-doctoral assistantships.

“The Center for Tropical and Emerging Global Diseases is one of the key components of UGA’s strength in the wide-ranging fight against human disease,” Lee said. “The exceptional training that students and posdocs receive at CTEGD is a point of significant pride for the university, and I am committed to supporting the further growth of the Center and the TTEGD program.”

The Office of Postdoctoral Affairs will continue to partner with CTEGD to provide grant-writing workshops and career development opportunities for post-doctoral fellows. Last year, CTEGD organized a workshop on NIH’s K-Award grants that are available to post-doctoral fellows.

Franklin College of Arts & Sciences has also committed over the next 5 years to match funds from CTEGD to provide for a mini-sabbatical program a faculty member from a local college or university, along with up to two undergraduate students from that school, to spend a semester working in a CTEGD laboratory. The goal of this program is to increase awareness of post-undergraduate opportunities and spark interest in neglected tropical disease research.

Committed to training the next generation of scientists

To date, 37 pre- and post-doctoral fellows have received training support through the NIH T32 Training Grant. The fellowships awarded by CTEGD include a stipend, travel award, health insurance, and some financial support for research supplies. Pre-doctoral fellows also receive a tuition waiver. In the past 15 years, graduate students have assisted in field studies in Haiti, Tanzania, Argentina, Thailand, Ecuador, and Kenya as part of their capstone experience.

“My trip to Ecuador was unimaginably enriching,” said Dr. Manuel Fierro, a former T32 fellow who completed his Ph.D. this year. “I was presented with the way great research is done in a setting containing fewer resources than in the US and it gave me important connections for my professional development. More importantly, it gave me hope of returning to my home country and continue applying my parasitology training.”

Trainees have gone on to positions in academia, government and industry. They have secured positions as post-doctoral fellows, staff scientists, and assistant professors at such institutions as the Centers for Disease Control and Prevention, Food and Drug Administration, the National Institutes of Health, University of California, Emory University School of Medicine, Tufts University’s Cummings Veterinary Medical School, and Wellcome Centre for Anti-Infective Research in Scotland.

Embarking on its 16th year this fall, CTEGD is committed as ever to increasing rigor, diversity, and career development in its training program. New requirements for post-doctoral trainees, more rigorous training for new trainers, strategic recruitment initiatives to target underrepresented minorities, and expansion of training to include large data mining and computer science are just a few of the changes the program will undertake over the next five years.

“The T32 training grant represents the core of CTEGD’s effort to prepare the next generation of scientists to tackle the scourge of the neglected parasitic diseases of mankind,” said Dr. Dennis Kyle, director of CTEGD. “The renewal of this award allows us to expand our efforts and is recognition of the excellence of our students, postdocs, staff, and faculty.”

Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum

Donna Huber on August 6, 2020

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite’s development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.

Flavia M Zimbres, Ana Lisa Valenciano, Emilio F Merino, Anat Florentin, Nicole R Holderman, Guijuan He, Katarzyna Gawarecka, Karolina Skorupinska-Tudek, Maria L Fernández-Murga, Ewa Swiezewska, Xiaofeng Wang, Vasant Muralidharan, Maria Belen Cassera. Sci Rep. 2020 Aug 6;10(1):13264. doi: 10.1038/s41598-020-70246-0.

Trainee Spotlight: Emma Troth

Donna Huber on July 23, 2020

Emma Troth

Emma Troth, a Ph.D. trainee in Dennis Kyle‘s laboratory, is entering her fourth year at UGA. She is originally from Eureka, Illinois, and attended Bradley University where she majored in Biology with a minor in Ethics. While at UGA, Emma has served as president of the CTEGD Graduate Student Association (2019-2020) and is currently the CTEGD Graduate Student Association representative.

How did you get interested in neglected tropical diseases?

As an undergraduate, I participated in a Research Experience for Undergraduates program at the University of Notre Dame. At Notre Dame, I worked on a project characterizing malaria vectors in the Solomon Islands and Indonesia. My summer at Notre Dame sparked my interest in neglected tropical diseases.

Why did you choose UGA?

I chose UGA because of the diversity of research. Coming into graduate school, I knew I was interested in infectious diseases but did not have my heart set on a particular organism to work on. UGA works on the biggest selection of infectious organisms. With the Integrated Life Sciences Program, I had the opportunity to experience multiple labs working on different organisms, regardless of department, to help me identify where I would like to complete my doctoral degree.

What is your research focus? Why are you interested in this topic?

My project focuses on drug discovery for Naegleria fowleri, the brain-eating amoebae. My main project focuses on structure-based drug design to develop novel drug targets against N. fowleri. Additionally, I am working to develop phenotypic drug screening assays to complement our high-throughput drug discovery. I am fascinated by N. fowleri because it is such a mysterious, deadly organism. Though infections may not be as common as other parasitic diseases, nearly all cases of primary amebic meningoencephalitis (PAM) are fatal. This amoeba is grossly understudied; very few labs in the world research this organism. It is both a privilege and a challenge to be able to work on this neglected parasite.

Have you done any fieldwork or is there a collaborator/field site that you would like to visit in order to enhance your training?

I hope to complete an internship with the Task Force for Global Health. This internship will ideally include fieldwork with one of their neglected tropical disease programs.

What are your future professional plans?

Going forward, I would like to continue my career in neglected tropical diseases with an emphasis on global health. I am particularly interested in a career involving field research. Ultimately, I hope to work for a government agency like the Centers for Disease Control and Prevention (CDC) or a non-profit organization focused on neglected tropical diseases.

What is your favorite thing about Athens?

My favorite thing about Athens is the food! There is such a variety of local restaurants and new restaurants are continually opening. Coming from a small undergraduate institution, I really enjoy the atmosphere of a large university in a small college town. Athens is a very easy city to feel “at home”.

Any advice for a student interested in this field?

Never be afraid to reach out for help, wherever you need it! Coming into graduate school can be intimidating and at times, isolating. There are so many people eager to help you on your graduate school journey and ultimately want to see you succeed. Particularly within the CTEGD, I have always been met with kind and willing responses. All it takes is for you to take the step and reach out!

Support trainees like Emma by giving today to the Center for Tropical & Emerging Global Diseases.

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Age influences the thermal suitability of Plasmodium falciparum transmission in the Asian malaria vector Anopheles stephensi

Donna Huber on July 22, 2020

Models predicting disease transmission are vital tools for long-term planning of malaria reduction efforts, particularly for mitigating impacts of climate change. We compared temperature-dependent malaria transmission models when mosquito life-history traits were estimated from a truncated portion of the lifespan (a common practice) versus traits measured across the full lifespan. We conducted an experiment on adult female Anopheles stephensi, the Asian urban malaria mosquito, to generate daily per capita values for mortality, egg production and biting rate at six constant temperatures. Both temperature and age significantly affected trait values. Further, we found quantitative and qualitative differences between temperature–trait relationships estimated from truncated data versus observed lifetime values. Incorporating these temperature–trait relationships into an expression governing the thermal suitability of transmission, relative R0(T), resulted in minor differences in the breadth of suitable temperatures for Plasmodium falciparum transmission between the two models constructed from only An. stephensi trait data. However, we found a substantial increase in thermal niche breadth compared with a previously published model consisting of trait data from multiple Anopheles mosquito species. Overall, this work highlights the importance of considering how mosquito trait values vary with mosquito age and mosquito species when generating temperature-based suitability predictions of transmission.

K. L. Miazgowicz, M. S. Shocket, S. J. Ryan, O. C. Villena, R. J. Hall, J. Owen, T. Adanlawo, K. Balaji, L. R. Johnson, E. A. Mordecai and C. C. Murdock. Proc Biol Sci. 2020 Jul 29;287(1931):20201093. doi: 10.1098/rspb.2020.1093.