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Tag: Silvia Moreno

RGS10 physically and functionally interacts with STIM2 and requires store-operated calcium entry to regulate proinflammatory gene expression in microglia

Chronic activation of microglia is a driving factor in the progression of neuroinflammatory diseases, and mechanisms that regulate microglial inflammatory signaling are potential targets for novel therapeutics. Regulator of G protein Signaling 10 is the most abundant RGS protein in microglia, where it suppresses inflammatory gene expression and reduces microglia-mediated neurotoxicity. In particular, microglial RGS10 downregulates the expression of pro-inflammatory mediators including cyclooxygenase 2 (COX-2) following stimulation with lipopolysaccharide (LPS). However, the mechanism by which RGS10 affects inflammatory signaling is unknown and is independent of its canonical G protein targeted mechanism. Here, we sought to identify non-canonical RGS10 interacting partners that mediate its anti-inflammatory mechanism. Through RGS10 co-immunoprecipitation coupled with mass spectrometry, we identified STIM2, an endoplasmic reticulum (ER) localized calcium sensor and a component of the store-operated calcium entry (SOCE) machinery, as a novel RGS10 interacting protein in microglia. Direct immunoprecipitation experiments confirmed RGS10-STIM2 interaction in multiple microglia and macrophage cell lines, as well as in primary cells, with no interaction observed with the homologue STIM1. We further determined that STIM2, Orai channels, and the Ca2+--dependent phosphatase calcineurin are essential for LPS-induced COX-2 production in microglia, and this pathway is required for the inhibitory effect of RGS10 on COX-2. Additionally, our data demonstrated that RGS10 suppresses SOCE triggered by ER calcium depletion and that ER calcium depletion, which induces SOCE, amplifies proinflammatory genes. In addition to COX-2, we also show that RGS10 suppresses the expression of proinflammatory cytokines in microglia in response to thrombin and LPS stimulation, and all of these effects require SOCE. Collectively, the physical and functional links between RGS10 and STIM2 suggest a complex regulatory network connecting RGS10, SOCE, and pro-inflammatory gene expression in microglia, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.

Menbere Wendimu, Mohammed Alqinyah, Stephen Vella, Phillip Dean, Faris Almutairi, Roseanne Davila Rivera, Shima Rayatpisheh, James Wohlschlegel, Silvia Moreno, Shelley B Hooks. Cell Signal. 2021 Mar 8;109974. doi: 10.1016/j.cellsig.2021.109974

Silvia Moreno elected as American Academy of Microbiology Fellow

Silvia Moreno
Photo credit: Dorothy Kozlowski

University of Georgia researcher, a member of the Center for Tropical and Emerging Global Diseases and a Distinguished Research Professor in cellular biology, has been elected as a 2021 American Academy of Microbiology Fellow. Holding courtesy appointments in microbiology and infectious diseases, Silvia N. Moreno also serves as director of the NIH-funded Training in Tropical and Emerging Global Diseases program.

“This is an honor that represents the hard work and commitment of the members of my lab, past and present,” said Moreno.

Her research focuses on the parasite Toxoplasma gondii, which can cause encephalitis and cardiogenic shock in immunocompromised patients and can result in devastating birth defects in children born from infected pregnant women. Almost a third of the human population is infected. The parasite also infects cats, dogs and cattle.

In particular, Moreno’s laboratory is interested in discovering unique metabolic differences that can be used as targets for chemotherapy as current treatment options are for only one phase of the disease and have harmful side effects.

In 2018, she was named a corresponding member of the Latin American Academy of Sciences. Since 2015, she has been leading the Training in Tropical and Emerging Global Diseases program which is funded by an NIH T32 training grant. In the most recent competing renewal of the grant, CTEGD was awarded $1.9 million.

Under Moreno’s leadership the program has expanded to provide fellowships to seven graduate students and two post-doctoral fellows, a mini-sabbatical program for faculty members of local colleges with a higher proportion of diversity students to offer undergraduates and faculty research experience, and organize a number of professional development workshops.

Moreno joins more than 2,500 AAM fellows who are elected through a highly selective, peer-reviewed process, based on their record of scientific achievement and original contributions that have advanced the field of microbiology. Of the 150 researchers nominated this year, only 65 were elected to the 2021 Fellowship Class.

The role of potassium and host calcium signaling in Toxoplasma gondii egress

Toxoplasma gondii is an obligate intracellular parasite and replicates inside a parasitophorous vacuole (PV) within the host cell. The membrane of the PV (PVM) contains pores that permits for equilibration of ions and small molecules between the host cytosol and the PV lumen. Ca2+ signaling is universal and both T. gondii and its mammalian host cell utilize Ca2+ signals to stimulate diverse cellular functions. Egress of T. gondii from host cells is an essential step for the infection cycle of T. gondii, and a cytosolic Ca2+ increase initiates a Ca2+ signaling cascade that culminates in the stimulation of motility and egress. In this work we demonstrate that intracellular T. gondii tachyzoites are able to take up Ca2+ from the host cytoplasm during host cell signaling events. Both intracellular and extracellular Ca2+ sources are important in reaching a threshold of parasite cytosolic Ca2+ needed for successful egress. Two peaks of Ca2+ were observed in egressing single parasites with the second peak resulting from Ca2+ entry. We patched infected host cells to allow the delivery of precise concentrations of Ca2+ for the stimulation of motility and egress. Using this approach of patching infected host cells, allowed us to determine that increasing the host cytosolic Ca2+ to a specific concentration can trigger egress, which is further accelerated by diminishing the concentration of potassium (K+).

Stephen A Vella, Christina A Moore, Zhu-Hong Li, Miryam A Hortua Triana, Evgeniy Potapenko, Silvia N J Moreno. Cell Calcium. 2021 Jan 19;94:102337. doi: 10.1016/j.ceca.2020.102337

Trainee Spotlight: Melissa Sleda

Melissa Sleda


Melissa Sleda, a Ph.D. trainee is Silvia Moreno’s laboratory, is in her third year at UGA. She is originally from Sandusky, Michigan and attended Lawrence Technological University where she majored in Molecular and Cell Biology with a minor in Chemistry. At UGA, she has held positions as the Secretary for the Cell Bio Grad Student Association (2019-2020), and as Treasurer (2019-2020) and current President (2020-2021) of the CTEGD grad student association.

Melissa Sleda has been awarded a T32 Trainee Fellowship for the 2020-2021 academic year.

Why did you choose UGA?

I chose UGA because of the Integrated Life Sciences Umbrella program. As an incoming graduate student, I was not set on studying a particular organism, and I was excited for the opportunity to rotate in labs across different departments.

What is your research project?

My project seeks to characterize enzymes of the isoprenoid biosynthetic pathway in Toxoplasma gondii and to investigate these enzymes as potential chemotherapeutic targets. The current chemotherapy for Toxoplasmosis is ineffective because it does not eliminate the chronic stage of infection. My project seeks to test drugs that target enzymes of the isoprenoid pathway in both the acute and chronic forms of infection in order to find a more effective chemotherapy.

What are your future professional plans?

My future career goal is to stay in academia and become a professor at a smaller institution with a higher emphasis on teaching and leading smaller research projects. I want to help students at smaller universities gain research experience through classroom labs and one-on-one research projects.

What do you hope to do for your Capstone Experience?

For my capstone experience, I hope to be able to do research in another country to gain a wider perspective of how research is done in other countries. I hope that I am able to do research in a lab that I can learn new techniques that will translate into my research project.

What is your favorite thing about Athens?

My favorite thing about Athens is the warm weather and the great sense of community.

What advice do you have for students interested in this field?

Do things out of your comfort zone because it will help you develop as a scientist.


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

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

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.”

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Martin J. McPhillie, Ying Zhou, Mark R. Hickman, James A. Gordon, Christopher R. Weber, Qigui Li, Patty J. Lee, Kangsa Amporndanai, Rachel M. Johnson, Heather Darby, Stuart Woods, Zhu-hong Li, Richard S. Priestley, Kurt D. Ristroph, Scott B. Biering, Kamal El Bissati, Seungmin Hwang, Farida Esaa Hakim, Sarah M. Dovgin, Joseph D. Lykins, Lucy Roberts, Kerrie Hargrave, Hua Cong, Anthony P. Sinai, Stephen P. Muench, Jitender P. Dubey, Robert K. Prud’homme, Hernan A. Lorenzi, Giancarlo A. Biagini, Silvia N. Moreno, Craig W. Roberts, Svetlana V. Antonyuk, Colin W. G. Fishwick, and Rima McLeod. Front. Cell. Infect. Microbiol., 09 June 2020 |

Isolation and Characterization of Acidocalcisomes from Trypanosomatids

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

Guozhong Huang, Silvia N. J. Moreno, Roberto Docampo. Methods Mol Biol. 2020;2116:673-688. doi: 10.1007/978-1-0716-0294-2_40.

An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum endoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites.

IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.

Manuel A. Fierro, Beejan Asady, Carrie F. Brooks, David W. Cobb, Alejandra Villegas, Silvia N. J. Moreno, Vasant Muralidharan. mBio. 2020 Feb 25;11(1). pii: e03078-19. doi: 10.1128/mBio.03078-19.

Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.

The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing “tried and true” methods of our lab who pioneered the first use of GECI’s in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

Stephen A. Vella, Abigail Calixto, Beejan Asady, Zhu-Hong Li, Silvia N. J. Moreno. Methods Mol Biol. 2020;2071:187-207. doi: 10.1007/978-1-4939-9857-9_11.

Synthesis and biological evaluation of 1-alkylaminomethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii

As an extension of our project aimed at the search for new chemotherapeutic agents against Chagas disease and toxoplasmosis, several 1,1-bisphosphonates were designed, synthesized and biologically evaluated against Trypanosoma cruzi and Toxoplasma gondii, the etiologic agents of these diseases, respectively. In particular, and based on the antiparasitic activity exhibited by 2-alkylaminoethyl-1,1-bisphosphonates targeting farnesyl diphosphate synthase, a series of linear 2-alkylaminomethyl-1,1-bisphosphonic acids (compounds 2133), that is, the position of the amino group was one carbon closer to the gem-phosphonate moiety, were evaluated as growth inhibitors against the clinically more relevant dividing form (amastigotes) of T. cruzi. Although all of these compounds resulted to be devoid of antiparasitic activity, these results were valuable for a rigorous SAR study. In addition, unexpectedly, the synthetic designed 2-cycloalkylaminoethyl-1,1-bisphosphonic acids 4749 were free of antiparasitic activity. Moreover, long chain sulfur-containing 1,1-bisphosphonic acids, such as compounds 545659, turned out to be nanomolar growth inhibitors of tachyzoites of T. gondii. As many bisphosphonate-containing molecules are FDA-approved drugs for the treatment of bone resorption disorders, their potential nontoxicity makes them good candidates to control American trypanosomiasis and toxoplasmosis.

Tamila Galaka, Bruno N. Falcone, Catherine Li, Sergio H. Szajnman, Silvia N.J. Moreno, Roberto Docampo, Juan B.Rodriguez. Bioorg Med Chem. 2019 Jul 4. pii: S0968-0896(19)30740-0. doi: 10.1016/j.bmc.2019.07.004.

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