Schistosoma mansoni: A diamidinophenylindole probe for in vitro death of schistosomula

Linden, P. W. G. Van Der; Deelder, A. M.

doi:10.1016/0014-4894(84)90071-7

Cited by 9 publications

(6 citation statements)

References 8 publications

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“…1B). Upon induction of TbSPT2 RNAi, the cells stopped dividing over a period of 5-6 days, lost motility and began to die, as indicated by 4Ј,6Ј-diamidino-2-phenylindole hydrochloride (DAPI)-exclusion staining (Van der Linden and Deelder, 1984) (data not shown). A 16-hour treatment of the parental 29-13 procyclic cells and the 90-13 bloodstream cells with myriocina fungal inhibitor that targets SPT2 (Miyake et al, 1995) -at concentrations of 0.5-5 M and 0.05-0.5 M, respectively, also resulted in cell death as assessed by DAPI permeability of living cells.…”

Section: Identification Of Genes Encoding the Subunits Of T Brucei Smentioning

confidence: 99%

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Fridberg

Olson

Nakayasu

et al. 2008

Journal of Cell Science

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Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.

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Section: Identification Of Genes Encoding the Subunits Of T Brucei Smentioning

confidence: 99%

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Fridberg

Olson

Nakayasu

et al. 2008

Journal of Cell Science

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“…Some fluorescent compounds such as DNA intercalating dyes, ethidium bromide (EB) and Propidium Iodide (PI) [60, 61], carboxyfluorescein [61] as well as resazurin have also been used to quantify schistosome viability in low-throughput, microscopic read-out methods. In more detail, resazurin, the active ingredient of Alamar Blue (AB), a non-toxic and cell permeable compound, is blue in color and virtually non-fluorescent.…”

Section: In Vitro Drug Sensitivity Assays With Newly Transformed Schimentioning

confidence: 99%

“…Ethidium bromide has been used to differentiate dead schistosomula from live ones during microscopic examination [61], while PI has successfully been used for the same reason (as a differential stain of dead schistosomula) for both microscopic examination and flow cytometry [60]. Carboxyfluorescein in contrast to ethidium bromide and propidium iodide, has been tested as live staining for schistosomula.…”

Section: In Vitro Drug Sensitivity Assays With Newly Transformed Schimentioning

confidence: 99%

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Mechanically produced schistosomula as a higher-throughput tools for phenotypic pre-screening in drug sensitivity assays: current research and future trends

Tekwu¹,

Anyan²,

Boamah³

et al. 2016

Biomark Res

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It is crucial to develop new antischistosomal drugs since there is no vaccine and the whole world is relying on only a single drug for the treatment of schistosomiasis. One of the obstacles to the development of drugs is the absence of the high throughput objective screening methods to assess drug compounds efficacy. Thus for identification of new drug compounds candidates, fast and accurate in vitro assays are unavoidable and more research efforts in the field of drug discovery can target schistosomula. This review presents a substantial overview of the present state of in vitro drug sensitivity assays developed so far for the determination of anti-schistosomula activity of drug compounds, natural products and derivatives using newly transformed schistosomula (NTS). It highlights some of the challenges involved in in vitro compound screening using NTS and the way forward.

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“…For schistosomes, various DNA binding fluorescent probes have been used to test for viability. These include DAPI, ethidium bromide and propidium iodide (PI) which flood into dead schistosomula and bind to DNA but are excluded from live larvae . Alternatively, to specifically label live schistosome larvae, the membrane‐permanent reactive tracers carboxyfluorescein diacetate and fluorescein diacetate (FDA) have been used.…”

Section: Helminth Screening Activity Readoutsmentioning

confidence: 99%

Automated Imaging and other developments in whole‐organism anthelmintic screening

Paveley

Bickle

2013

Parasite Immunology

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Helminth infections still represent a huge public health problem throughout the developing world and in the absence of vaccines control is based on periodic mass drug administration. Poor efficacy of some anthelmintics and concerns about emergence of drug resistance has highlighted the need for new drug discovery. Most current anthelmintics were discovered through in vivo screening of selected compounds in animal models but recent approaches have shifted towards screening for activity against adult or larval stages in vitro. Larvae are normally available in greater numbers than adults, can often be produced in vitro and are small enough for microplate assays. However, the manual visualization of drug effects in vitro is subjective, laborious and slow. This can be overcome by application of automated readouts including high-content imaging. Incorporated into robotically controlled HTS platforms such methods allow the very large compound collections being made available by the pharmaceutical industry or academic organizations to be screened against helminths for the first time, invigorating the drug discovery pipeline. Here, we review the status of whole-organism screens based on in vitro activity against living worms and highlight the recent progress towards automated image-based readouts.

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Schistosoma mansoni: A diamidinophenylindole probe for in vitro death of schistosomula

Cited by 9 publications

References 8 publications

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei

Mechanically produced schistosomula as a higher-throughput tools for phenotypic pre-screening in drug sensitivity assays: current research and future trends

Automated Imaging and other developments in whole‐organism anthelmintic screening

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