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Book ; Online: Chapter 48 In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei

Ritchie, Ryan / Barrett, Michael / Mottram, Jeremy / Myburgh, Elmarie

2020

Keywords	Biology, life sciences ; Firefly luciferase, Trypanosoma brucei brucei, In vivo imaging, Bioluminescence
Size	1 electronic resource (17 pages)
Publisher	Springer Nature
Document type	Book ; Online
Note	English ; Open Access
HBZ-ID	HT021044641
ISBN	9781071602966 ; 1071602969
Database	ZB MED Catalogue: Medicine, Health, Nutrition, Environment, Agriculture

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Article ; Online: In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei.

Ritchie, Ryan / Barrett, Michael P / Mottram, Jeremy C / Myburgh, Elmarie

Methods in molecular biology (Clifton, N.J.)

2020 Volume 2116, Page(s) 801–817

Abstract: Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment ... ...

Abstract	Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment before surviving parasites become detectable in the blood. To expedite compound screening, we have used a Trypanosoma brucei brucei GVR35 strain expressing red-shifted firefly luciferase to monitor parasite distribution in infected mice through noninvasive whole-body bioluminescence imaging. This protocol describes the infection and in vivo bioluminescence imaging of mice to assess compound efficacy against T. brucei during the two characteristic stages of disease, the hemolymphatic phase (stage 1) and the encephalitic or central nervous system phase (stage 2).
MeSH term(s)	Animals ; Disease Models, Animal ; Female ; Genes, Reporter/genetics ; Humans ; Luciferases, Firefly/chemistry ; Luciferases, Firefly/genetics ; Luminescent Agents/chemistry ; Luminescent Measurements/instrumentation ; Luminescent Measurements/methods ; Mice ; Optical Imaging/methods ; Parasitic Sensitivity Tests/instrumentation ; Parasitic Sensitivity Tests/methods ; Trypanocidal Agents/pharmacology ; Trypanocidal Agents/therapeutic use ; Trypanosoma brucei brucei/genetics ; Trypanosoma brucei brucei/isolation & purification ; Trypanosomiasis, African/diagnosis ; Trypanosomiasis, African/drug therapy ; Trypanosomiasis, African/parasitology
Chemical Substances	Luminescent Agents ; Trypanocidal Agents ; Luciferases, Firefly (EC 1.13.12.7)
Language	English
Publishing date	2020-03-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-0294-2_48
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Alpizar-Sosa, Edubiel A / Ithnin, Nur Raihana Binti / Wei, Wenbin / Pountain, Andrew W / Weidt, Stefan K / Donachie, Anne M / Ritchie, Ryan / Dickie, Emily A / Burchmore, Richard J S / Denny, Paul W / Barrett, Michael P

PLoS neglected tropical diseases

2022 Volume 16, Issue 9, Page(s) e0010779

Abstract: Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. ... ...

Abstract	Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.
MeSH term(s)	Mice ; Animals ; Amphotericin B/pharmacology ; Leishmania mexicana/metabolism ; Nystatin ; Serum Albumin, Bovine/metabolism ; Sterols ; Oxidative Stress ; Polyenes ; Transferases/metabolism ; Glucose ; Fatty Acid Desaturases/metabolism ; Antiprotozoal Agents/pharmacology
Chemical Substances	Amphotericin B (7XU7A7DROE) ; Nystatin (1400-61-9) ; Serum Albumin, Bovine (27432CM55Q) ; Sterols ; Polyenes ; Transferases (EC 2.-) ; Glucose (IY9XDZ35W2) ; Fatty Acid Desaturases (EC 1.14.19.-) ; Antiprotozoal Agents
Language	English
Publishing date	2022-09-28
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2429704-5
ISSN	1935-2735 ; 1935-2735
ISSN (online)	1935-2735
ISSN	1935-2735
DOI	10.1371/journal.pntd.0010779
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: IL-17 can be protective or deleterious in murine pneumococcal pneumonia.

Ritchie, Neil D / Ritchie, Ryan / Bayes, Hannah K / Mitchell, Tim J / Evans, Tom J

PLoS pathogens

2018 Volume 14, Issue 5, Page(s) e1007099

Abstract: Streptococcus pneumoniae is the major bacterial cause of community-acquired pneumonia, and the leading agent of childhood pneumonia deaths worldwide. Nasal colonization is an essential step prior to infection. The cytokine IL-17 protects against such ... ...

Abstract	Streptococcus pneumoniae is the major bacterial cause of community-acquired pneumonia, and the leading agent of childhood pneumonia deaths worldwide. Nasal colonization is an essential step prior to infection. The cytokine IL-17 protects against such colonization and vaccines that enhance IL-17 responses to pneumococcal colonization are being developed. The role of IL-17 in host defence against pneumonia is not known. To address this issue, we have utilized a murine model of pneumococcal pneumonia in which the gene for the IL-17 cytokine family receptor, Il17ra, has been inactivated. Using this model, we show that IL-17 produced predominantly from γδ T cells protects mice against death from the invasive TIGR4 strain (serotype 4) which expresses a relatively thin capsule. However, in pneumonia produced by two heavily encapsulated strains with low invasive potential (serotypes 3 and 6B), IL-17 significantly enhanced mortality. Neutrophil uptake and killing of the serotype 3 strain was significantly impaired compared to the serotype 4 strain and depletion of neutrophils with antibody enhanced survival of mice infected with the highly encapsulated SRL1 strain. These data strongly suggest that IL-17 mediated neutrophil recruitment to the lungs clears infection from the invasive TIGR4 strain but that lung neutrophils exacerbate disease caused by the highly encapsulated pneumococcal strains. Thus, whilst augmenting IL-17 immune responses against pneumococci may decrease nasal colonization, this may worsen outcome during pneumonia caused by some strains.
MeSH term(s)	Animals ; Bacteremia/immunology ; Bacteremia/microbiology ; Bacterial Capsules/immunology ; Bacterial Capsules/ultrastructure ; Bronchoalveolar Lavage Fluid/cytology ; Bronchoalveolar Lavage Fluid/microbiology ; Disease Models, Animal ; Interleukin-17/immunology ; Lung/cytology ; Lung/enzymology ; Lung/immunology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Nasopharynx/microbiology ; Neutrophils/cytology ; Neutrophils/immunology ; Peroxidase/metabolism ; Phagocytosis ; Pneumonia, Pneumococcal/immunology ; Pneumonia, Pneumococcal/mortality ; Pneumonia, Pneumococcal/prevention & control ; Receptors, Antigen, T-Cell, gamma-delta/genetics ; Receptors, Antigen, T-Cell, gamma-delta/immunology ; Receptors, Interleukin-17/genetics ; Specific Pathogen-Free Organisms ; Streptococcus pneumoniae/immunology ; Streptococcus pneumoniae/ultrastructure
Chemical Substances	Il17ra protein, mouse ; Interleukin-17 ; Receptors, Antigen, T-Cell, gamma-delta ; Receptors, Interleukin-17 ; Peroxidase (EC 1.11.1.7)
Language	English
Publishing date	2018-05-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1007099
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Article ; Online: Attempts to Image the Early Inflammatory Response during Infection with the Lymphatic Filarial Nematode Brugia pahangi in a Mouse Model.

Myburgh, Elmarie / Ritchie, Ryan / Goundry, Amy / O'Neill, Kerry / Marchesi, Francesco / Devaney, Eileen

PloS one

2016 Volume 11, Issue 12, Page(s) e0168602

Abstract: Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A ... ...

Abstract	Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals.
MeSH term(s)	Animals ; Antigens, Helminth/immunology ; Brugia pahangi/immunology ; Cytokines/metabolism ; Disease Models, Animal ; Elephantiasis, Filarial/immunology ; Luminescent Agents/metabolism ; Luminescent Measurements ; Luminol/metabolism ; Lymphatic System/immunology ; Male ; Mice
Chemical Substances	Antigens, Helminth ; Cytokines ; Luminescent Agents ; Luminol (5EXP385Q4F)
Language	English
Publishing date	2016-12-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0168602
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence.

Herbert, Jenny A / Mitchell, Andrea M / Ritchie, Ryan / Ma, Jiangtao / Ross-Hutchinson, Kirsty / Mitchell, Timothy J

PloS one

2018 Volume 13, Issue 1, Page(s) e0189426

Abstract: Bioluminescence has been harnessed for use in bacterial reporter systems and for in vivo imaging of infection in animal models. Strain Xen35, a bioluminescent derivative of Streptococcus pneumoniae serotype 4 strain TIGR4 was previously constructed for ... ...

Abstract	Bioluminescence has been harnessed for use in bacterial reporter systems and for in vivo imaging of infection in animal models. Strain Xen35, a bioluminescent derivative of Streptococcus pneumoniae serotype 4 strain TIGR4 was previously constructed for use for in vivo imaging of infections in animal models. We have shown that strain Xen35 is less virulent than its parent TIGR4 and that this is associated with the expression of the genes for bioluminescence. The expression of the luxA-E genes in the pneumococcus reduces virulence and down regulates the expression of the pneumococcal pilus.
MeSH term(s)	Animals ; Blotting, Western ; Fimbriae, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Luminescence ; Mice ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Reverse Transcriptase Polymerase Chain Reaction ; Streptococcus pneumoniae/genetics ; Streptococcus pneumoniae/pathogenicity ; Virulence/genetics
Language	English
Publishing date	2018
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0189426
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Article ; Online: Anti-Trypanosomal Proteasome Inhibitors Cure Hemolymphatic and Meningoencephalic Murine Infection Models of African Trypanosomiasis.

Rao, Srinivasa P S / Lakshminarayana, Suresh B / Jiricek, Jan / Kaiser, Marcel / Ritchie, Ryan / Myburgh, Elmarie / Supek, Frantisek / Tuntland, Tove / Nagle, Advait / Molteni, Valentina / Mäser, Pascal / Mottram, Jeremy C / Barrett, Michael P / Diagana, Thierry T

Tropical medicine and infectious disease

2020 Volume 5, Issue 1

Abstract: Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and 'easy to use' oral drugs. Previously, we reported the discovery of the ... ...

Abstract	Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and 'easy to use' oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against
Language	English
Publishing date	2020-02-17
Publishing country	Switzerland
Document type	Journal Article
ISSN	2414-6366
ISSN (online)	2414-6366
DOI	10.3390/tropicalmed5010028
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Article ; Online: Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition.

Steketee, Pieter C / Dickie, Emily A / Iremonger, James / Crouch, Kathryn / Paxton, Edith / Jayaraman, Siddharth / Alfituri, Omar A / Awuah-Mensah, Georgina / Ritchie, Ryan / Schnaufer, Achim / Rowan, Tim / de Koning, Harry P / Gadelha, Catarina / Wickstead, Bill / Barrett, Michael P / Morrison, Liam J

PLoS pathogens

2021 Volume 17, Issue 7, Page(s) e1009734

Abstract: Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of ...

Abstract	Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.
MeSH term(s)	Animals ; Lipid Regulating Agents/pharmacology ; Mice ; Trypanosoma brucei brucei/drug effects ; Trypanosoma brucei brucei/metabolism ; Trypanosoma congolense/drug effects ; Trypanosoma congolense/metabolism ; Trypanosomiasis, African
Chemical Substances	Lipid Regulating Agents
Language	English
Publishing date	2021-07-26
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2205412-1
ISSN	1553-7374 ; 1553-7374
ISSN (online)	1553-7374
ISSN	1553-7374
DOI	10.1371/journal.ppat.1009734
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Article ; Online: RNAi screening identifies Trypanosoma brucei stress response protein kinases required for survival in the mouse.

Fernandez-Cortes, Fernando / Serafim, Tiago D / Wilkes, Jonathan M / Jones, Nathaniel G / Ritchie, Ryan / McCulloch, Richard / Mottram, Jeremy C

Scientific reports

2017 Volume 7, Issue 1, Page(s) 6156

Abstract: Protein kinases (PKs) are a class of druggable targets in Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (sleeping sickness), yet little is known about which PKs are essential for survival in mammals. A recent kinome-wide RNAi ... ...

Abstract	Protein kinases (PKs) are a class of druggable targets in Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (sleeping sickness), yet little is known about which PKs are essential for survival in mammals. A recent kinome-wide RNAi screen with 176 individual bloodstream form Trypanosoma brucei lines identified PKs required for proliferation in culture. In order to assess which PKs are also potential virulence factors essential in vivo, lines were pooled, inoculated into mice, and screened for loss of fitness after 48 h RNAi. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to growth in culture. We identified 49 PKs with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. Nine PKs had a more pronounced growth defect in vivo, than in vitro. Amongst these PKs were several with putative functions related to stress responses mediated through the PI3K/TOR or MAPK signaling cascades, which act to protect the parasite from complement-mediated and osmotic lysis. Identification of these virulence-associated PKs provides new insights into T. brucei-host interaction and reveals novel potential protein kinase drug targets.
MeSH term(s)	Animals ; Genes, Essential ; Mice ; Protein Kinases/genetics ; Protozoan Proteins/genetics ; RNA Interference ; Sequence Analysis, RNA/methods ; Signal Transduction ; Trypanosoma brucei brucei/genetics ; Trypanosoma brucei brucei/pathogenicity ; Trypanosomiasis, African/genetics ; Trypanosomiasis, African/parasitology ; Trypanosomiasis, African/veterinary ; Virulence Factors/genetics
Chemical Substances	Protozoan Proteins ; Virulence Factors ; Protein Kinases (EC 2.7.-)
Language	English
Publishing date	2017-07-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-017-06501-8
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Article ; Online: Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

Nature communications

2021 Volume 12, Issue 1, Page(s) 1052

Abstract: The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ...

Abstract	The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.
MeSH term(s)	Acute Disease ; Allosteric Regulation/drug effects ; Animals ; Glycolysis/drug effects ; Hep G2 Cells ; Humans ; Inhibitory Concentration 50 ; Kaplan-Meier Estimate ; Mice ; Parasites/drug effects ; Phosphofructokinases/antagonists & inhibitors ; Phosphofructokinases/chemistry ; Phosphofructokinases/metabolism ; Protein Binding/drug effects ; Protein Kinase Inhibitors/chemistry ; Protein Kinase Inhibitors/pharmacokinetics ; Protein Kinase Inhibitors/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Protein Multimerization ; Structure-Activity Relationship ; Trypanosoma/drug effects ; Trypanosoma/enzymology ; Trypanosomiasis, African/drug therapy ; Trypanosomiasis, African/metabolism ; Trypanosomiasis, African/parasitology
Chemical Substances	Protein Kinase Inhibitors ; Phosphofructokinases (EC 2.7.1 -)
Language	English
Publishing date	2021-02-16
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-21273-6
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