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A Variant Pfcrt Isoform Can Contribute To Plasmodium Falciparum Resistance To The First-Line Partner Drug Piperaquine, Satish K. Dhingra, Devasha Redhi, Jill M. Combrinck, Tomas Yeo, John Okombo, Philipp P. Henrich, Ann N. Cowell, Purva Gupta, Matthew L. Stegman, Jonathan M. Hoke, Roland A. Cooper, Elizabeth Winzeler, Sachel Mok, Timothy J. Egan, David A. Fidock

Collected Faculty and Staff Scholarship

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisininbased combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nucleasebased gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ …

Lack Of Resistance Of Plasmodium Falciparum To Dihydroartemisinin In Uganda Based On Parasitogolgical And Molecular Assays, Roland A. Cooper, Melissa D. Conrad, Quentin D. Watson, Stephanie J. Huezo, Harriet Ninsiima, Patrick Tumwebaze, Samuel L. Nsobya, Philip J. Rosenthal

Collected Faculty and Staff Scholarship

• Artemisinin-­‐based combination therapy is now standard treatment for falciparum malaria. However, this regimen is threatened by resistance to artemisinins, manifest as delayed clearance of parasitemia after therapy, in southeast Asia.

• Artemisinin resistance in southeast Asia is associated with increased parasitemias in culture, compared to those in sensi0ve parasites, 72 hours a=er a 6 hour pulse with 700 nM dihydroartemisinin (DHA), and with propeller domain polymorphisms in the Plasmodium falciparum kelch (K13; PF3D7_1343700) gene

• Given that artemether/lumefantrine has been adopted as standard therapy for malaria within the last decade in Uganda, we characterized artemisinin sensiBvity in fresh P. falciparum isolates from …

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Mutations In The Plasmodium Falciparum Chloroquine Resistance Transporter, Pfcrt, Enlarge The Parasite's Food Vacuole And Alter Drug Sensitivities, Serena Pulcini, Henry M. Staines, Andrew H. Lee, Sarah H. Shafik, Guillaume Bouyer, Catherine M. Moore, Daniel A. Daley, Matthew J. Hoke, Lindsey M. Altenhofen, Heather J. Painter, Jianbing Mu, David J. P. Ferguson, Manuel Llinas, Rowen E. Martin, David A. Fidock, Roland Cooper, Sanjeev Krishna

Collected Faculty and Staff Scholarship

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction …

Lack Of Artemisinin Resistance In Plasmodium Falciparum In Uganda Based On Parasitological And Molecular Assays, Roland A. Cooper, Melissa D. Conrad, Quentin D. Watson, Stephanie J. Huezo, Harriet Ninsiima, Patrick Tumwebaze, Samuel L. Nsobya, Philip J. Rosenthal

Collected Faculty and Staff Scholarship

We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.

Impact Of Antimalarial Treatment And Chemoprevention On The Drug Sensitivity Of Malaria Parasites Isolated From Ugandan Children, Patrick Tumwebaze, Melissa D. Conrad, Andrew Walakira, Norbert Leclair, Oswald Byaruhanga, Christine Nakazibwe, Benjamin Kozak, Jessica Bloome, Jaffer Okiring, Abel Kakuru, Victor Bigira, James Kapisi, Jennifer Legac, Jiri Gut, Roland A. Cooper, Moses R. Kamya, Diane V. Havlir, Grant Dorsey, Bryan Greenhouse, Samuel L. Nsobya, Philip J. Rosenthal

Collected Faculty and Staff Scholarship

Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. …

Using Expression Profiling To Understand The Effects Of Chronic Cadmium Exposure On Mcf-7 Breast Cancer Cells, Zelmina Lubovac-Pilav, Daniel M. Borras, Esmeralda Ponce, Maggie Louie

Collected Faculty and Staff Scholarship

Cadmium is a metalloestrogen known to activate the estrogen receptor and promote breast cancer cell growth. Previous studies have implicated cadmium in the development of more malignant tumors; however the molecular mechanisms behind this cadmium-induced malignancy remain elusive. Using clonal cell lines derived from exposing breast cancer cells to cadmium for over 6 months (MCF-7-Cd4, -Cd6, -Cd7, -Cd8 and -Cd12), this study aims to identify gene expression signatures associated with chronic cadmium exposure. Our results demonstrate that prolonged cadmium exposure does not merely result in the deregulation of genes but actually leads to a distinctive expression profile. The genes deregulated …

Chronic Cadmium Exposure Stimulates Sdf-1 Expression In An Erα Dependent Manner, Esmeralda Ponce, Natalie B. Aquino, Maggie Louie

Collected Faculty and Staff Scholarship

Cadmium is an omnipotent environmental contaminant associated with the development of breast cancer. Studies suggest that cadmium functions as an endocrine disruptor, mimicking the actions of estrogen in breast cancer cells and activating the receptor to promote cell growth. Although acute cadmium exposure is known to promote estrogen receptor-mediated gene expression associated with growth, the consequence of chronic cadmium exposure is unclear. Since heavy metals are known to bioaccumulate, it is necessary to understand the effects of prolonged cadmium exposure. This study aims to investigate the effects of chronic cadmium exposure on breast cancer progression. A MCF7 breast cancer cell …

Mutation In The Plasmodium Falciparum Crt Protein Determines The Stereospecific Activity Of Antimalarial Cinchona Alkaloids, Carol E. Griffin, Jonathan M. Hoke, Upeka Samarakoon, Junhui Duan, Jianbing Mu, Michael T. Ferdig, David C. Warhurst, Roland Cooper

Collected Faculty and Staff Scholarship

The Cinchona alkaloids are quinoline aminoalcohols that occur as diastereomer pairs, typified by (-)-quinine and (+)-quinidine. The potency of (+)-isomers is greater than the (-)-isomers in vitro and in vivo against Plasmodium falciparum malaria parasites. They may act by the inhibition of heme crystallization within the parasite digestive vacuole in a manner similar to chloroquine. Earlier studies showed that a K76I mutation in the digestive vacuole-associated protein, PfCRT (P. falciparum chloroquine resistance transporter), reversed the normal potency order of quinine and quinidine toward P. falciparum. To further explore PfCRT-alkaloid interactions in the malaria parasite, we measured the in vitro susceptibility …

The Role Of Cadmium And Nickel In Estrogen Receptor Signaling And Breast Cancer: Metalloestrogens Or Not?, Natalie B. Aquino, Mary B. Sevigny, Jackielyn Sabangan, Maggie C. Louie

Collected Faculty and Staff Scholarship

During the last half-century, incidences of breast cancer have increased globally. Various factors —genetic and environmental— have been implicated in the initiation and progression of this disease. One potential environmental risk factor that has not received a lot of attention is the exposure to heavy metals. While several mechanisms have been put forth describing how high concentrations of heavy metals play a role in carcinogenesis, it is unclear whether chronic, lowlevel exposure to certain heavy metals (i.e. cadmium and nickel), can directly result in the development and progression of cancer. Cadmium and nickel have been hypothesized to play a role …

Chloroquine Susceptibility And Reversibility In A Plasmodium Falciparum Genetic Cross, Jigar J. Patel, Drew Thacker, Jon C. Tan, Perri Pleeter, Lisa Checkley, Joseph M. Gonzales, Bingbing Deng, Paul D. Roepe, Roland A. Cooper, Michael T. Ferdig

Collected Faculty and Staff Scholarship

Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT), are major determinants of verapamil (VP)-reversible CQ resistance (CQR). In the presence of mutant PfCRT, additional genes contribute to the wide range of CQ susceptibilities observed. It is not known if these genes influence mechanisms of chemosensitization by CQR reversal agents. Using quantitative trait locus (QTL) mapping of progeny clones from the HB3 × Dd2 cross, we show that the P. falciparum multidrug resistance gene 1 (pfmdr1) interacts with the Southeast Asiaderived mutant pfcrt haplotype to modulate CQR levels. A novel chromosome 7 locus is predicted to contribute with the …

Accumulation Of Artemisinin Trioxane Derivatives Within Neutral Lipids Of Plasmodium Falciparum Malaria Parasites Is Endoperoxide-Dependent, Carmony L. Hartwig, Andrew S. Rosenthal, John D'Angelo, Carol E. Griffin, Gary H. Posner, Roland A. Cooper

Collected Faculty and Staff Scholarship

The antimalarial trioxanes, exemplified by the naturally occurring sesquiterpene lactone artemisinin and its semi-synthetic derivatives, contain an endoperoxide pharmacophore that lends tremendous potency against Plasmodium parasites. Despite decades of research, their mechanism of action remains unresolved. A leading model of anti-plasmodial activity hypothesizes that iron-mediated cleavage of the endoperoxide bridge generates cytotoxic drug metabolites capable of damaging cellular macromolecules. To probe the malarial targets of the endoperoxide drugs, we studied the distribution of fluorescent dansyl trioxane derivatives in living, intraerythrocytic-stage Plasmodium falciparum parasites using microscopic imaging. The fluorescent trioxanes rapidly accumulated in parasitized erythrocytes, localizing within digestive vacuole-associated neutral lipid …

Genome-Wide Compensatory Changes Accompany Drug Selected Mutations In The Plasmodium Falciparum Crt Gene, Hongying Jiang, Jigar J. Patel, Ming Yi, Jianbing Mu, Jinhui Ding, Robert Stephens, Roland Cooper, Michael T. Ferdig, Xin-Zhuan Su

Collected Faculty and Staff Scholarship

Mutations in PfCRT (Plasmodium falciparum chloroquine-resistant transporter), particularly the substitution at amino acid position 76, confer chloroquine (CQ) resistance in P. falciparum. Point mutations in the homolog of the mammalian multidrug resistance gene (pfmdr1) can also modulate the levels of CQ response. Moreover, parasites with the same pfcrt and pfmdr1 alleles exhibit a wide range of drug sensitivity, suggesting that additional genes contribute to levels of CQ resistance (CQR). Reemergence of CQ sensitive parasites after cessation of CQ use indicates that changes in PfCRT are deleterious to the parasite. Some CQR parasites, however, persist in the field and grow well …

Androgen-Induced Recruitment Of Rna Polymerase Ii To A Nuclear Receptor–P160 Coactivator Complex, Maggie C. Louie, Hong Qiong Yang, Ai-Hong Ma, Wei Xu, June X. Zou, Hsing-Jien Kung, Hong-Wu Chen

Collected Faculty and Staff Scholarship

The androgen receptor, like other nuclear receptors, activates target genes by binding to hormone-responsive enhancers. Here we demonstrate that androgen induces robust recruitment of androgen receptor, members of the p160 coactivator family, and CREB-binding protein/p300 specifically at the distant enhancer of prostate-specific antigen (PSA) gene. Unexpectedly, we found that RNA polymerase II (Pol II) is directly recruited to the enhancer in a hormone-dependent manner, independent of the proximal promoter, and that the isolated PSA enhancer can mediate efficient androgen induction of transcription. Inhibition of the Pol II carboxyl-terminal domain kinase activity with low concentrations of flavopiridol blocks Pol II transfer …