Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations

The rapid development of parasite drug resistance as well as the lack of medications targeting both the asexual and the sexual blood stages of the malaria parasite necessitate the search for novel antimalarial compounds. Eleven organoarsenic compounds were synthesized and tested for their effect on the asexual blood stages and sexual transmission stages of the malaria parasite Plasmodium falciparum using in vitro assays. The inhibitory potential of the compounds on blood stage viability was tested on the chloroquine (CQ)-sensitive 3D7 and the CQ-resistant Dd2 strain using the Malstat assay. The most effective compounds were subsequently investigated for their effect on impairing gametocyte development and gametogenesis, using the gametocyte-producing NF54 strain in respective cell-based assays. Their potential toxicity was investigated on leukemia cell line Nalm-6 and non-infected erythrocytes. Five out of the 11 compounds showed antiplasmodial activities against 3D7, with half-maximal inhibitory concentration (IC50) values ranging between 1.52 and 8.64 µM. Three of the compounds also acted against Dd2, with the most active compound As-8 exhibiting an IC50 of 0.35 µM. The five compounds also showed significant inhibitory effects on the parasite sexual stages at both IC50 and IC90 concentrations with As-8 displaying the best gametocytocidal activity. No hemolytic and cytotoxic effect was observed for any of the compounds. The organoarsenic compound As-8 may represent a good lead for the design of novel organoarsenic drugs with combined antimalarial and transmission blocking activities.

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Reversal of Mefloquine and Quinine Resistance in Plasmodium falciparum with NP30

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.8.2393-2396.2003 ◽

2003 ◽

Vol 47 (8) ◽

pp. 2393-2396 ◽

Cited By ~ 15

Author(s):

Michelle Ciach ◽

Kathleen Zong ◽

Kevin C. Kain ◽

Ian Crandall

Keyword(s):

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Genetic Analysis ◽

Resistance Genes ◽

Mammalian Cells ◽

Point Mutations ◽

In Vitro Assays ◽

Drug Efflux ◽

P Glycoprotein

ABSTRACT Quinoline resistance in malaria is frequently compared with P-glycoprotein-mediated multidrug resistance (mdr) in mammalian cells. We have previously reported that nonylphenolethoxylates, such as NP30, are potential Plasmodium falciparum P-glycoprotein substrates and drug efflux inhibitors. We used in vitro assays to compare the ability of verapamil and NP30 to sensitize two parasite isolates to four quinolines: chloroquine (CQ), mefloquine (MF), quinine (QN), and quinidine (QD). NP30 was able to sensitize (reversal, >80%) P. falciparum to MF, QN, QD, and, to a lesser extent, CQ. The presence of 2 μM verapamil had no effect on mefloquine resistance; however, the presence of verapamil modulated the activities of QN and QD in a manner parallel to that observed for CQ. Genetic analysis of putative quinoline resistance genes did not suggest an association between known point mutations in pfcrt and pfmdr1 and NP30 sensitization activity. We conclude that the sensitization action of NP30 is distinct both phenotypically and genotypically from that of verapamil.

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Plasmodium falciparum K13 mutations in Africa and Asia present varying degrees of artemisinin resistance and an elevated fitness cost in African parasites

10.1101/2021.01.05.425445 ◽

2021 ◽

Author(s):

Barbara H. Stokes ◽

Kelly Rubiano ◽

Satish K. Dhingra ◽

Sachel Mok ◽

Judith Straimer ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Southeast Asia ◽

Survival Rates ◽

Point Mutations ◽

Artemisinin Resistance ◽

Parasite Clearance ◽

Definitive Evidence ◽

The Impact

AbstractThe emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites has led to increasing rates of treatment failure with first-line ART-based combination therapies (ACTs) in Southeast Asia. In this region, select mutations in K13 can result in delayed parasite clearance rates in vivo and enhanced survival in the ring-stage survival assay (RSA) in vitro. Our genotyping of 3,299 P. falciparum isolates across 11 sub-Saharan countries reveals the continuing dominance of wild-type K13 and confirms the emergence of a K13 R561H variant in Rwanda. Using gene editing, we provide definitive evidence that this mutation, along with M579I and C580Y, can confer variable degrees of in vitro ART resistance in African P. falciparum strains. C580Y and M579I were both associated with substantial fitness costs in African parasites, which may counter-select against their dissemination in high-transmission settings. We also report the impact of multiple K13 mutations, including the predominant variant C580Y, on RSA survival rates and fitness in multiple Southeast Asian strains. No change in ART susceptibility was observed upon editing point mutations in ferrodoxin or mdr2, earlier associated with ART resistance in Southeast Asia. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

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97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab97 ◽

2005 ◽

Vol 17 (2) ◽

pp. 199 ◽

Cited By ~ 3

Author(s):

B. Peachey ◽

K. Hartwich ◽

K. Cockrem ◽

A. Marsh ◽

A. Pugh ◽

...

Keyword(s):

Ethylene Glycol ◽

Survival Rates ◽

Slow Freezing ◽

High Survival ◽

Bovine Embryos ◽

Embryo Survival ◽

Frozen Embryos ◽

Morula Stage ◽

Fresh Embryos

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

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Genotypes and phenotypes of resistance in Ecuadorian Plasmodium falciparum

Malaria Journal ◽

10.1186/s12936-019-3044-z ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 1

Author(s):

Gabriela Valenzuela ◽

L. Enrique Castro ◽

Julio Valencia-Zamora ◽

Claudia A. Vera-Arias ◽

Petra Rohrbach ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Live Cell Imaging ◽

Cell Imaging ◽

Chloroquine Resistance ◽

In Vitro Assays ◽

Wild Type ◽

The Status ◽

Transport Assay ◽

Resistance Markers

Abstract Background Malaria continues to be endemic in the coast and Amazon regions of Ecuador. Clarifying current Plasmodium falciparum resistance in the country will support malaria elimination efforts. In this study, Ecuadorian P. falciparum parasites were analysed to determine their drug resistance genotypes and phenotypes. Methods Molecular analyses were performed to search for mutations in known resistance markers (Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, k13). Pfmdr1 copy number was determined by qPCR. PFMDR1 transporter activity was characterized in live parasites using live cell imaging in combination with the Fluo-4 transport assay. Chloroquine, quinine, lumefantrine, mefloquine, dihydroartemisinin, and artemether sensitivities were measured by in vitro assays. Results The majority of samples from this study presented the CVMNT genotype for Pfcrt (72–26), NEDF SDFD mutations in Pfmdr1 and wild type genotypes for Pfdhfr, Pfdhps and k13. The Ecuadorian P. falciparum strain ESM-2013 showed in vitro resistance to chloroquine, but sensitivity to quinine, lumefantrine, mefloquine, dihydroartemisinin and artemether. In addition, transport of the fluorochrome Fluo-4 from the cytosol into the digestive vacuole (DV) of the ESM-2013 strain was minimally detected in the DV. All analysed samples revealed one copy of Pfmdr1. Conclusion This study indicates that Ecuadorian parasites presented the genotype and phenotype for chloroquine resistance and were found to be sensitive to SP, artemether-lumefantrine, quinine, mefloquine, and dihydroartemisinin. The results suggest that the current malaria treatment employed in the country remains effective. This study clarifies the status of anti-malarial resistance in Ecuador and informs the P. falciparum elimination campaigns in the country.

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High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-019-0390-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Sergio Ledda ◽

Jen M. Kelly ◽

Stefano Nieddu ◽

Daniela Bebbere ◽

Federica Ariu ◽

...

Keyword(s):

Survival Rate ◽

Survival Rates ◽

Blastocyst Stage ◽

Hatching Rate ◽

High Survival ◽

Apoptotic Cells ◽

Embryo Survival ◽

New Device ◽

In Vitro Survival

Abstract Background To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 195), using a new device named “E.Vit”, composed by a 0.25-mL straw with a 50-μm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos (n = 102) were maintained in in vitro culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay. Results Blastocyst re-expansion (2 h) after warming was higher (P < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control. Conclusions A high survival rate of IVP embryos can be achieved by the new “E.Vit” device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

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Generation of UCiPSCs-Derived Neurospheres for Cell Therapy and its Application in Cell Shipment at Ambient Temperature

10.21203/rs.3.rs-47992/v1 ◽

2020 ◽

Author(s):

Shuai Li ◽

Huifang Zhao ◽

Xiaobo Han ◽

Lang He ◽

Omar Mukama ◽

...

Keyword(s):

Stem Cells ◽

Cell Therapy ◽

Neural Stem Cells ◽

Ambient Temperature ◽

Low Cost ◽

Survival Rates ◽

High Survival ◽

Simple Method

Abstract BackgroundNeural stem cells（NSCs）therapy remains one of the most potential approaches for neurological disorders treatment. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized our technique to cell therapy. Meanwhile, it is often required that NSCs are stored and transported long distances for research or treatment. Although high survival rates could be maintained, conventional methods of cell transport (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore，the establishment of a safe, affordable, and frequent obtained hiPSCs and hiNSCs, with characteristics that match fetal hNSCs and a simple, low-cost way to store and transport, are incredibly urgent. MethodsWe reprogrammed human urinary cells to iPSCs using a virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis and in vitro and in vivo differentiation capabilities, etc).ResultsHere, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique and their differentiation into neural networks. Moreover，hiNSCs survived longer as neurospheres at ambient temperature than those cultured in a monolayer. Approximately 7 days, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to ambient temperature that were placed under standard culture conditions (37 ℃, 5% CO2) recovered their typical morphology, and retained their ability to proliferate and differentiate. ConclusionsIn this study, we provided a simple method for the storage of NSCs as neurospheres at ambient temperature as an alternative to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at ambient temperature and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

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Characterization of PRMT1 from Plasmodium falciparum

Biochemical Journal ◽

10.1042/bj20090185 ◽

2009 ◽

Vol 421 (1) ◽

pp. 107-118 ◽

Cited By ~ 29

Author(s):

Qi Fan ◽

Jun Miao ◽

Long Cui ◽

Liwang Cui

Keyword(s):

Plasmodium Falciparum ◽

Fluorescent Protein ◽

Arginine Methylation ◽

In Vitro Assays ◽

Parasite Development ◽

Type I ◽

Histone H4 ◽

Turnover Rates ◽

Post Translational Modification

Arginine methylation is a post-translational modification that affects many cellular processes in eukaryotes. The malaria parasite Plasmodium falciparum encodes three conserved PRMTs (protein arginine N-methyltransferases). We have determined that PfPRMT1 (P. falciparum PRMT1) has authentic type I PRMT activity to form monomethylarginines and asymmetric dimethylarginines. Compared with mammalian PRMT1s, PfPRMT1 possesses a distinctive N-terminal sequence that is ∼50 amino acids longer and is essential for enzyme activity. Recombinant PfPRMT1 methylated histones H4 and H2A and several conserved substrates involved in RNA metabolism, including fibrillarin, poly(A)-binding protein II, ribosomal protein S2 and a putative splicing factor. Using synthetic peptides and MS, we determined target arginines in several substrates and studied the enzyme kinetics. Whereas the kinetic parameters of recombinant PfPRMT1 on an H4 peptide and S-adenosylmethionine were similar to those of mammalian PRMT1s, PfPRMT1 had much higher substrate-turnover rates. In the histone H4 N-terminus, PfPRMT1 could methylate only Arg3, a mark for transcription activation. Western blotting detected dynamic dimethylation of H4-Arg3 during parasite development, suggesting that histone-arginine methylation may play a conserved role in chromatin-mediated gene regulation. Consistent with the presence of potential substrates in both the cytoplasm and nucleus, green fluorescent protein-tagged PfPRMT1 and untagged PfPRMT1 were localized in both cellular compartments, with the majority in the cytoplasm. in vitro assays showed that PfPRMT1 could be inhibited by several small-molecule inhibitors, with IC50-values in the sub-micromolar range. Most of these compounds also effectively inhibited parasite growth, suggesting that parasite PRMTs are promising targets for developing antiparasitic drugs.

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The G9a Histone Methyltransferase Inhibitor BIX-01294 Modulates Gene Expression during Plasmodium falciparum Gametocyte Development and Transmission

International Journal of Molecular Sciences ◽

10.3390/ijms20205087 ◽

2019 ◽

Vol 20 (20) ◽

pp. 5087 ◽

Cited By ~ 5

Author(s):

Che Julius Ngwa ◽

Meike Jutta Kiesow ◽

Lindsey Marie Orchard ◽

Afia Farrukh ◽

Manuel Llinás ◽

...

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Histone Methylation ◽

Blood Feeding ◽

In Vitro Assays ◽

Multi Stage ◽

Parasite Plasmodium Falciparum ◽

Stress Signals

Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is initiated by specialized sexual cells, the gametocytes. In the human, gametocytes are formed in response to stress signals and following uptake by a blood-feeding Anopheles mosquito initiate sexual reproduction. Gametocytes need to fine-tune their gene expression in order to develop inside the mosquito to continue life-cycle progression. Previously, we showed that post-translational histone acetylation controls gene expression during gametocyte development and transmission. However, the role of histone methylation remains poorly understood. We here use the histone G9a methyltransferase inhibitor BIX-01294 to investigate the role of histone methylation in regulating gene expression in gametocytes. In vitro assays demonstrated that BIX-01294 inhibits intraerythrocytic replication with a half maximal inhibitory concentration (IC50) of 13.0 nM. Furthermore, BIX-01294 significantly impairs gametocyte maturation and reduces the formation of gametes and zygotes. Comparative transcriptomics between BIX-01294-treated and untreated immature, mature and activated gametocytes demonstrated greater than 1.5-fold deregulation of approximately 359 genes. The majority of these genes are transcriptionally downregulated in the activated gametocytes and could be assigned to transcription, translation, and signaling, indicating a contribution of histone methylations in mediating gametogenesis. Our combined data show that inhibitors of histone methylation may serve as a multi-stage antimalarial.

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Clonal propagation of Pelargonium x hortorum through tissue culture: Effects of salt dilution and growth regulator concentration

Canadian Journal of Plant Science ◽

10.4141/cjps93-113 ◽

1993 ◽

Vol 73 (3) ◽

pp. 871-878 ◽

Cited By ~ 7

Author(s):

Hélène Desilets ◽

Yves Desjardins ◽

Richard R. Bélanger

Keyword(s):

Growth Regulators ◽

Doubling Time ◽

Culture Media ◽

Survival Rates ◽

Basal Medium ◽

Multiple Shoots ◽

Naphthaleneacetic Acid ◽

High Survival ◽

Half Strength

Different culture media were compared at the initiation and multiplication steps to develop a rapid production system for geranium (Pelargonium × hortorum) in vitro. Different salt dilutions of the Murashige and Skoog (MS) (1962) mineral medium were used in combination with different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) in order to optimize initiation of shoots of four geranium cultivars. The use of a MS basal medium with half-strength macrosalts supplemented with 0.11 μM NAA and 0.89 μM BA gave the best results in initiation. More than 40% of the apices initiated on this medium produced multiple shoots within a month. Subsequently, the effect of different concentrations of growth regulators was quantified by the mean of "shoot doubling time" evaluation. The shortest time recorded was 10.5 d for a theoretical production of 1 × 109 plantlets/apex/year. This is the first quantitative evaluation of geranium production in vitro. Geranium plantlets rooted easily on a half-strength MS medium without growth regulators. Acclimatization of geranium plantlets was characterized by high survival rates (94%) and the plants thus produced were phenotypically comparable to seed-derived plants. Key words: Geranium, micropropagation, shoot doubling time, in vitro

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