scholarly journals Evaluating 5-Nitrofurans as Trypanocidal Agents

2013 ◽  
Vol 57 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Christopher Bot ◽  
Belinda S. Hall ◽  
Guzmán Álvarez ◽  
Rossanna Di Maio ◽  
Mercedes González ◽  
...  
Keyword(s):  
Human African Trypanosomiasis ◽  
Bloodstream Form ◽  
Type I ◽  
Biochemical Screening ◽  
African Trypanosomiasis ◽  
Open Chain ◽  
Content Type ◽  
Growth Inhibitory ◽  
Initial Activation

ABSTRACTThe nitroheterocycle nifurtimox, as part of a nifurtimox-eflornithine combination therapy, represents one of a limited number of treatments targetingTrypanosoma brucei, the causative agent of human African trypanosomiasis. The mode of action of this prodrug involves an initial activation reaction catalyzed by a type I nitroreductase (NTR), an enzyme found predominantly in prokaryotes, leading to the formation of a cytotoxic unsaturated open-chain nitrile metabolite. Here, we evaluate the trypanocidal activities of a library of other 5-nitrofurans against the bloodstream form ofT. bruceias a preliminary step in the identification of additional nitroaromatic compounds that can potentially partner with eflornithine. Biochemical screening against the purified enzyme revealed that all 5-nitrofurans were effective substrates forT. bruceiNTR (TbNTR), with the preferred compounds having apparentkcat/Kmvalues approximately 50-fold greater than those of nifurtimox. For several compounds,in vitroreduction by this nitroreductase yielded products characterized by mass spectrometry as either unsaturated or saturated open-chain nitriles. When tested against the bloodstream form ofT. brucei, many of the derivatives displayed significant growth-inhibitory properties, with the most potent compounds generating 50% inhibitory concentrations (IC50s) around 200 nM. The antiparasitic activities of the most potent agents were demonstrated to be NTR dependent, as parasites having reduced levels of the enzyme displayed resistance to the compounds, while parasites overexpressing TbNTR showed hypersensitivity. We conclude that other members of the 5-nitrofuran class of nitroheterocycles have the potential to treat human African trypanosomiasis, perhaps as an alternative partner prodrug to nifurtimox, in the next generation of eflornithine-based combinational therapies.

scholarly journals Evaluating 5-Nitrothiazoles as Trypanocidal Agents

2015 ◽  
Vol 60 (2) ◽  
pp. 1137-1140 ◽  
Author(s):  
Ivan P. O'Shea ◽  
Mohammed Shahed ◽  
Benjamín Aguilera-Venegas ◽  
Shane R. Wilkinson
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Cytotoxic Effect ◽  
Human African Trypanosomiasis ◽  
Type I ◽  
Antiparasitic Activity ◽  
Content Type ◽  
Growth Inhibitory ◽  
Activity Dependent ◽  
Related Compounds

ABSTRACTThe growth-inhibitory properties of a 5-nitrothiazole series were evaluated againstTrypanosoma brucei. A subset of related compounds displayed the greatest potency toward the parasite while exhibiting little cytotoxic effect on mammalian cells, with this antiparasitic activity dependent on expression of a type I nitroreductase by the trypanosome. We conclude that the 5-nitrothiazole class of nitroheterocyclic drugs may represent a new lead in the treatment of human African trypanosomiasis.

scholarly journals Characterization of a Melamino Nitroheterocycle as a Potential Lead for the Treatment of Human African Trypanosomiasis

2014 ◽  
Vol 58 (10) ◽  
pp. 5747-5757 ◽  
Author(s):  
Federica Giordani ◽  
Annamaria Buschini ◽  
Alessandro Baliani ◽  
Marcel Kaiser ◽  
Reto Brun ◽  
...  
Keyword(s):  
Human African Trypanosomiasis ◽  
Micronucleus Assay ◽  
Oral Route ◽  
Drug Candidate ◽  
African Trypanosomiasis ◽  
Content Type ◽  
Pharmacokinetic Properties ◽  
Toxic Potential ◽  
Biopharmaceutical Properties

ABSTRACTThis paper reports an evaluation of a melamino nitroheterocycle, a potential lead for further development as an agent against human African trypanosomiasis (HAT). Studies on its efficacy, physicochemical and biopharmaceutical properties, and potential for toxicity are described. The compound previously had been shown to possess exceptional activity againstTrypanosoma bruceiinin vitroassays comparable to that of melarsoprol. Here, we demonstrate that the compound also was curative in the stringent acute mouse modelT. brucei rhodesienseSTIB 900 when given intraperitoneally at 40 mg/kg of body weight. Nevertheless, activity was only moderate when the oral route was used, and no cure was obtained when the compound was tested in a stage 2 rodent model of infection. Genotoxic profiling revealed that the compound induces DNA damage by a mechanism apparently independent from nitroreduction and involving the introduction of base pair substitutions (Ames test), possibly caused by oxidative damage of the DNA (comet test). No significant genotoxicity was observed at the chromosome level (micronucleus assay). The lack of suitable properties for oral and central nervous system uptake and the genotoxic liabilities prevent the progression of this melamine nitroheterocycle as a drug candidate for HAT. Further modification of the compound is required to improve the pharmacokinetic properties of the molecule and to separate the trypanocidal activity from the toxic potential.

scholarly journals New Chemical Scaffolds for Human African Trypanosomiasis Lead Discovery from a Screen of Tyrosine Kinase Inhibitor Drugs

2014 ◽  
Vol 58 (4) ◽  
pp. 2202-2210 ◽  
Author(s):  
Ranjan Behera ◽  
Sarah M. Thomas ◽  
Kojo Mensa-Wilmot
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Human African Trypanosomiasis ◽  
Kinase Inhibitor ◽  
New Drugs ◽  
Proof Of Concept ◽  
African Trypanosomiasis ◽  
Lead Discovery ◽  
Content Type ◽  
Growth Inhibitory

ABSTRACTHuman African trypanosomiasis (HAT) is caused by the protozoanTrypanosoma brucei. New drugs are needed to treat HAT because of undesirable side effects and difficulties in the administration of the antiquated drugs that are currently used. In human proliferative diseases, protein tyrosine kinase (PTK) inhibitors (PTKIs) have been developed into drugs (e.g., lapatinib and erlotinib) by optimization of a 4-anilinoquinazoline scaffold. Two sets of facts raise a possibility that drugs targeted against human PTKs could be “hits” for antitrypanosomal lead discoveries. First, trypanosome protein kinases bind some drugs, namely, lapatinib, CI-1033, and AEE788. Second, the pan-PTK inhibitor tyrphostin A47 blocks the endocytosis of transferrin and inhibits trypanosome replication. Following up on these concepts, we performed a focused screen of various PTKI drugs as possible antitrypanosomal hits. Lapatinib, CI-1033, erlotinib, axitinib, sunitinib, PKI-166, and AEE788 inhibited the replication of bloodstreamT. brucei, with a 50% growth inhibitory concentration (GI50) between 1.3 μM and 2.5 μM. Imatinib had no effect (i.e., GI50> 10 μM). To discover leads among the drugs, a mouse model of HAT was used in a proof-of-concept study. Orally administered lapatinib reduced parasitemia, extended the survival of all treated mice, and cured the trypanosomal infection in 25% of the mice. CI-1033 and AEE788 reduced parasitemia and extended the survival of the infected mice. On the strength of these data and noting their oral bioavailabilities, we propose that the 4-anilinoquinazoline and pyrrolopyrimidine scaffolds of lapatinib, CI-1033, and AEE788 are worth optimizing againstT. bruceiin medicinal chemistry campaigns (i.e., scaffold repurposing) to discover new drugs against HAT.

scholarly journals Pharmacokinetics, Trypanosoma brucei gambiense Efficacy, and Time of Drug Action of DB829, a Preclinical Candidate for Treatment of Second-Stage Human African Trypanosomiasis

2013 ◽  
Vol 57 (11) ◽  
pp. 5330-5343 ◽  
Author(s):  
Tanja Wenzler ◽  
Sihyung Yang ◽  
Olivier Braissant ◽  
David W. Boykin ◽  
Reto Brun ◽  
...  
Keyword(s):  
Single Dose ◽  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Drug Action ◽  
African Trypanosomiasis ◽  
Content Type ◽  
Second Stage ◽  
Trypanosoma Brucei Gambiense

ABSTRACTHuman African trypanosomiasis (HAT, also called sleeping sickness), a neglected tropical disease endemic to sub-Saharan Africa, is caused by the parasitesTrypanosoma brucei gambienseandT. brucei rhodesiense. Current drugs against this disease have significant limitations, including toxicity, increasing resistance, and/or a complicated parenteral treatment regimen. DB829 is a novel aza-diamidine that demonstrated excellent efficacy in mice infected withT. b. rhodesienseorT. b. bruceiparasites. The current study examined the pharmacokinetics,in vitroandin vivoactivity againstT. b. gambiense, and time of drug action of DB829 in comparison to pentamidine. DB829 showed outstandingin vivoefficacy in mice infected with parasites ofT. b. gambiensestrains, despite having higherin vitro50% inhibitory concentrations (IC50s) than againstT. b. rhodesiensestrain STIB900. A single dose of DB829 administered intraperitoneally (5 mg/kg of body weight) cured all mice infected with differentT. b. gambiensestrains. No cross-resistance was observed between DB829 and pentamidine inT. b. gambiensestrains isolated from melarsoprol-refractory patients. Compared to pentamidine, DB829 showed a greater systemic exposure when administered intraperitoneally, partially contributing to its improved efficacy. Isothermal microcalorimetry andin vivotime-to-kill studies revealed that DB829 is a slower-acting trypanocidal compound than pentamidine. A single dose of DB829 (20 mg/kg) administered intraperitoneally clears parasites from mouse blood within 2 to 5 days. In summary, DB829 is a promising preclinical candidate for the treatment of first- and second-stage HAT caused by bothTrypanosoma bruceisubspecies.

scholarly journals Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

2010 ◽  
Vol 54 (7) ◽  
pp. 2893-2900 ◽  
Author(s):  
Antoaneta Y. Sokolova ◽  
Susan Wyllie ◽  
Stephen Patterson ◽  
Sandra L. Oza ◽  
Kevin D. Read ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Parent Drug ◽  
Cross Resistance ◽  
Pharmacokinetic Studies ◽  
African Trypanosomiasis ◽  
Trypanosoma Brucei Brucei ◽  
Resistant Lines

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

scholarly journals Plasmodium falciparum Line-Dependent Association ofIn VitroGrowth-Inhibitory Activity and Risk of Malaria

2012 ◽  
Vol 80 (5) ◽  
pp. 1900-1908 ◽  
Author(s):  
Josea Rono ◽  
Anna Färnert ◽  
Daniel Olsson ◽  
Faith Osier ◽  
Ingegerd Rooth ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Inhibitory Activity ◽  
Content Type ◽  
Phenotypic Differences ◽  
Erythrocyte Invasion ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity ◽  
Control Study

ABSTRACTPlasmodium falciparum's ability to invade erythrocytes is essential for its survival within the human host. Immune mechanisms that impair this ability are therefore expected to contribute to immunity against the parasite. Plasma of humans who are naturally exposed to malaria has been shown to have growth-inhibitory activity (GIA)in vitro. However, the importance of GIA in relation to protection from malaria has been unclear. In a case-control study nested within a longitudinally followed population in Tanzania, plasma samples collected at baseline from 171 individuals (55 cases and 116 age-matched controls) were assayed for GIA using threeP. falciparumlines (3D7, K1, and W2mef) chosen based on their erythrocyte invasion phenotypes. Distribution of GIA differed between the lines, with most samples inhibiting the growth of 3D7 and K1 and enhancing the growth of W2mef. GIA to 3D7 was associated with a reduced risk of malaria within 40 weeks of follow-up (odds ratio, 0.45; 95% confidence interval [CI], 0.21 to 0.96;P= 0.04), whereas GIA to K1 and W2mef was not. These results show that GIA, as well as its association with protection from malaria, is dependent on theP. falciparumline and can be explained by differences in erythrocyte invasion phenotypes between parasite lines. Our study contributes knowledge on the biological importance of growth inhibition and the potential influence ofP. falciparumerythrocyte invasion phenotypic differences on its relationship to protective immunity against malaria.

scholarly journals A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

2014 ◽  
Vol 59 (1) ◽  
pp. 356-364 ◽  
Author(s):  
Wesley Wu ◽  
Zachary Herrera ◽  
Danny Ebert ◽  
Katie Baska ◽  
Seok H. Cho ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Drug Targets ◽  
Specific Activity ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Rescue ◽  
Growth Inhibitory ◽  
Parasite Populations

ABSTRACTThe apicoplast is an essential plastid organelle found inPlasmodiumparasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stagePlasmodium falciparumgrowth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations inP. falciparumIspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activityin vitrowith a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

scholarly journals Bacterial Enzymes Catalyzing the Synthesis of 1,8-Dihydroxynaphthalene, a Key Precursor of Dihydroxynaphthalene Melanin, fromSorangium cellulosum

2018 ◽  
Vol 84 (9) ◽  
Author(s):  
Yusuke Sone ◽  
Shuto Nakamura ◽  
Makoto Sasaki ◽  
Fumihito Hasebe ◽  
Seung-Young Kim ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Optical Purity ◽  
Brown Pigment ◽  
Type I ◽  
Enzymatic System ◽  
Type Iii Polyketide Synthase ◽  
Content Type ◽  
Type Iii ◽  
Bacterial Origin

ABSTRACT1,8-Dihydroxynaphthalene (1,8-DHN) is a key intermediate in the biosynthesis of DHN melanin, which is specific to fungi. In this study, we characterized the enzymatic properties of the gene products of an operon consisting ofsoceCHS1,bdsA, andbdsBfrom the Gram-negative bacteriumSorangium cellulosum. Heterologous expression ofsoceCHS1,bdsA, andbdsBinStreptomyces coelicolorcaused secretion of a dark-brown pigment into the broth. High-performance liquid chromatography (HPLC) analysis of the broth revealed that the recombinant strain produced 1,8-DHN, indicating that the operon encoded a novel enzymatic system for the synthesis of 1,8-DHN. Simultaneous incubation of the recombinant SoceCHS1, BdsA, and BdsB with malonyl-coenzyme A (malonyl-CoA) and NADPH resulted in the synthesis of 1,8-DHN. SoceCHS1, a type III polyketide synthase (PKS), catalyzed the synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN)in vitro. T4HN was in turn converted to 1,8-DHN by successive steps of reduction and dehydration, which were catalyzed by BdsA and BdsB. BdsA, which is a member of the aldo-keto reductase (AKR) superfamily, catalyzed the reduction of T4HN and 1,3,8-tetrahydroxynaphthalene (T3HN) to scytalone and vermelone, respectively. The stereoselectivity of T4HN reduction by BdsA occurred on thesi-face to give (R)-scytalone with more than 99% optical purity. BdsB, a SnoaL2-like protein, catalyzed the dehydration of scytalone and vermelone to T3HN and 1,8-DHN, respectively. The fungal pathway for the synthesis of 1,8-DHN is composed of a type I PKS, naphthol reductases of the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (SD). These findings demonstrated 1,8-DHN synthesis by novel enzymes of bacterial origin.IMPORTANCEAlthough the DHN biosynthetic pathway was thought to be specific to fungi, we discovered novel DHN synthesis enzymes of bacterial origin. The biosynthesis of bacterial DHN utilized a type III PKS for polyketide synthesis, an AKR superfamily for reduction, and a SnoaL2-like NTF2 superfamily for dehydration, whereas the biosynthesis of fungal DHN utilized a type I PKS, SDR superfamily enzyme, and SD-like NTF2 superfamily. Surprisingly, the enzyme systems comprising the pathway were significantly different from each other, suggesting independent, parallel evolution leading to the same biosynthesis. DHN melanin plays roles in host invasion and adaptation to stress in pathogenic fungi and is therefore important to study. However, it is unclear whether DHN biosynthesis occurs in bacteria. Importantly, we did find that bacterial DHN biosynthetic enzymes were conserved among pathogenic bacteria.

scholarly journals SSP3 Is a Novel Plasmodium yoelii Sporozoite Surface Protein with a Role in Gliding Motility

2014 ◽  
Vol 82 (11) ◽  
pp. 4643-4653 ◽  
Author(s):  
Anke Harupa ◽  
Brandon K. Sack ◽  
Viswanathan Lakshmanan ◽  
Nadia Arang ◽  
Alyse N. Douglass ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Biological Activities ◽  
Surface Protein ◽  
Plasmodium Yoelii ◽  
Gliding Motility ◽  
Surface Proteins ◽  
Type I ◽  
Content Type ◽  
Mosquito Midgut

ABSTRACTPlasmodiumsporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasitePlasmodium yoelii. SSP3 is a putative type I transmembrane protein unique toPlasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion ofSSP3adversely affectedin vitrosporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasionin vitro, nor infectivityin vivo. Together, these data reveal a previously unappreciated complexity of thePlasmodiumsporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites.

scholarly journals A Tale of Three Species: Adaptation ofSodalis glossinidiusto Tsetse Biology,WigglesworthiaMetabolism, and Host Diet

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Rebecca J. Hall ◽  
Lindsey A. Flanagan ◽  
Michael J. Bottery ◽  
Vicki Springthorpe ◽  
Stephen Thorpe ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Strong Dependence ◽  
Tsetse Fly ◽  
Disease Spread ◽  
Growth Media ◽  
Insect Vector ◽  
African Trypanosomiasis ◽  
Content Type ◽  
Sodalis Glossinidius

ABSTRACTThe tsetse fly is the insect vector for theTrypanosoma bruceiparasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium,Sodalis glossinidius. The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model ofS. glossinidiusmetabolism. The model enabled the design and experimental verification of a defined medium that supportsS. glossinidiusgrowthex vivo. This has been used subsequently to analyzein vitroaspects ofS. glossinidiusmetabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomerN-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence onl-glutamate as a source of carbon and nitrogen and by the ability to rescue a predictedl-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiontWigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution ofS. glossinidiusto exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibitS. glossinidiusgrowth and aid the reduction of trypanosomal transmission.IMPORTANCEHuman African trypanosomiasis is caused by theTrypanosoma bruceiparasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential forT. bruceilife cycle progression and transmission. The tsetse’s mutualistic endosymbiontSodalis glossinidiushas a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model ofS. glossinidiusmetabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects ofS. glossinidiusmetabolism. We presentS. glossinidiusas uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally,S. glossinidiushas adapted to the tsetse’s obligate symbiontWigglesworthia glossinidiaby scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission.

Sign in / Sign up

Export Citation Format

Share Document