scholarly journals Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Roberto R. Moraes Barros ◽  
Kittisak Thawnashom ◽  
Tyler J. Gibson ◽  
Jennifer S. Armistead ◽  
Ramoncito L. Caleon ◽  
...  
Keyword(s):  
Life Cycle ◽  
Plasmodium Vivax ◽  
Rhesus Macaque ◽  
Plasmodium Knowlesi ◽  
Luciferase Expression ◽  
Parasite Life Cycle ◽  
Anopheles Dirus ◽  
Exogenous Dna ◽  
The Stability

Abstract Background Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. Methods In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5′ UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. Results Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. Conclusion All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

scholarly journals Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle

2021 ◽  
Author(s):  
Roberto R Moraes Barros ◽  
Kittisak Thawnashom ◽  
Tyler J Gibson ◽  
Jennifer S Armistead ◽  
Ramoncito L Caleon ◽  
...  
Keyword(s):  
Life Cycle ◽  
Plasmodium Vivax ◽  
Rhesus Macaque ◽  
Plasmodium Knowlesi ◽  
Luciferase Expression ◽  
Parasite Life Cycle ◽  
Exogenous Dna ◽  
Ic50 Values ◽  
The Stability

Background: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. Methods: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated P. vivax or Plasmodium falciparum 5′ UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. Results: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, antimalarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. Conclusion: All three P. vivax promoters tested in this study functioned in P. knowlesi whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new antimalarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

scholarly journals Monoclonal Antibodies to Intracellular Stages ofCryptosporidium parvumDefine Life Cycle ProgressionIn Vitro

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Georgia Wilke ◽  
Soumya Ravindran ◽  
Lisa Funkhouser-Jones ◽  
Jennifer Barks ◽  
Qiuling Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Life Cycle ◽  
Gastrointestinal Disease ◽  
Epithelial Cell Line ◽  
Type I ◽  
Parasite Life Cycle ◽  
Major Barrier ◽  
Content Type ◽  
Life Cycle Stages

ABSTRACTAmong the obstacles hinderingCryptosporidiumresearch is the lack of anin vitroculture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti-Cryptosporidiumantibodies and a lack of markers for staging developmental progression. Previously developed antibodies againstCryptosporidiumwere raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supportedC. parvumgrowth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for theCryptosporidiumresearch community and will facilitate future investigation into parasite biology.IMPORTANCECryptosporidiumis a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages ofCryptosporidium. We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation intoCryptosporidiumbiology and help illuminate growth differences between various culture platforms.

scholarly journals In Vitro Cultivation of Cryptosporidium Species

2002 ◽  
Vol 15 (3) ◽  
pp. 390-400 ◽  
Author(s):  
Michael J. Arrowood
Keyword(s):  
Life Cycle ◽  
Life Cycle Stage ◽  
In Vitro Cultivation ◽  
Parasite Life Cycle ◽  
Disinfection Methods ◽  
Intracellular Parasites ◽  
Development And Differentiation ◽  
Stage Development ◽  
Life Cycle Development

SUMMARY The in vitro cultivation of protozoan parasites of the genus Cryptosporidium has advanced significantly in recent years. These obligate, intracellular parasites colonize the epithelium of the digestive and respiratory tracts, are often difficult to obtain in significant numbers, produce durable oocysts that defy conventional chemical disinfection methods, and are persistently infectious when stored at refrigerated temperatures (4 to 8°C). While continuous culture and efficient life cycle completion (oocyst production) have not yet been achieved in vitro, routine methods for parasite preparation and cell culture infection and assays for parasite life cycle development have been established. Parasite yields may be limited, but in vitro growth is sufficient to support a variety of research studies, including assessing potential drug therapies, evaluating oocyst disinfection methods, and characterizing life cycle stage development and differentiation.

scholarly journals Small-Molecule Xenomycins Inhibit All Stages of the Plasmodium Life Cycle

2014 ◽  
Vol 59 (3) ◽  
pp. 1427-1434 ◽  
Author(s):  
Jessey Erath ◽  
Julio Gallego-Delgado ◽  
Wenyue Xu ◽  
Grasiella Andriani ◽  
Scott Tanghe ◽  
...  
Keyword(s):  
Life Cycle ◽  
Small Molecule ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Parasite Life Cycle ◽  
Content Type ◽  
Effective Inhibition ◽  
Potential Activity ◽  
Sexual Stages

ABSTRACTWidespread resistance to most antimalaria drugs in use has prompted the search for novel candidate compounds with activity againstPlasmodiumasexual blood stages to be developed for treatment. In addition, the current malaria eradication programs require the development of drugs that are effective against all stages of the parasite life cycle. We have analyzed the antimalarial properties of xenomycins, a novel subclass of small molecule compounds initially isolated for anticancer activity and similarity to quinacrine in biological effects on mammalian cells.In vitrostudies show potent activity of Xenomycins againstPlasmodium falciparum. Oral administration of xenomycins in mouse models result in effective clearance of liver and blood asexual and sexual stages, as well as effective inhibition of transmission to mosquitoes. These characteristics position xenomycins as antimalarial candidates with potential activity in prevention, treatment and elimination of this disease.

scholarly journals Plasmodium vivax infection compromises reticulocyte stability

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Martha A. Clark ◽  
Usheer Kanjee ◽  
Gabriel W. Rangel ◽  
Laura Chery ◽  
Anjali Mascarenhas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Cell ◽  
Plasmodium Vivax ◽  
Malaria Infection ◽  
Structural Integrity ◽  
Osmotic Stability ◽  
Reticulocyte Maturation ◽  
The Stability ◽  
New Permeability Pathways

AbstractThe structural integrity of the host red blood cell (RBC) is crucial for propagation of Plasmodium spp. during the disease-causing blood stage of malaria infection. To assess the stability of Plasmodium vivax-infected reticulocytes, we developed a flow cytometry-based assay to measure osmotic stability within characteristically heterogeneous reticulocyte and P. vivax-infected samples. We find that erythroid osmotic stability decreases during erythropoiesis and reticulocyte maturation. Of enucleated RBCs, young reticulocytes which are preferentially infected by P. vivax, are the most osmotically stable. P. vivax infection however decreases reticulocyte stability to levels close to those of RBC disorders that cause hemolytic anemia, and to a significantly greater degree than P. falciparum destabilizes normocytes. Finally, we find that P. vivax new permeability pathways contribute to the decreased osmotic stability of infected-reticulocytes. These results reveal a vulnerability of P. vivax-infected reticulocytes that could be manipulated to allow in vitro culture and develop novel therapeutics.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Reverse torque values of abutment screws after dynamic loading: The effects of sealant agents and the taper of conical connections

2020 ◽  
Author(s):  
Arda Ozdiler ◽  
suleyman dayan ◽  
Burc Gencel ◽  
Gulbahar Isık-Ozkol
Keyword(s):  
Dynamic Loading ◽  
Antibacterial Agent ◽  
In Vitro Study ◽  
Control Group ◽  
Silicone Sealant ◽  
Reverse Torque ◽  
The Stability ◽  
Torque Values ◽  
Chlorhexidine Gel

This in vitro study evaluated the influence of taper angles on the internal conical connections of implant systems and of the application of chlorhexidine gel as an antibacterial agent or a polyvinyl siloxane (PVS) sealant on the reverse torque values of abutment screws after dynamic loading. The current study tested four implant systems with different taper angles (5.4°, 12°, 45°, and 60°). Specimens were divided into three groups: control (neither chlorhexidine gel filled nor silicone sealed), 2% chlorhexidine gel-filled or silicone-sealed group, and group subjected to a dynamic load of 50 N at 1 Hz for 500,000 cycles prior to reverse torque measurements. Quantitative positive correlation was observed between the taper angle degree and the percentage of tightening torque loss. However, this correlation was significant only for the 60° connection groups except in the group in which a sealant was applied ( p = 0.013 for the control group, p = 0.007 for the chlorhexidine group). Percentages of decrease in the torque values of the specimens with silicone sealant application were significantly higher compared with both the control and chlorhexidine groups ( p = 0.001, p = 0.002, p = 0.001, and p = 0.002, respectively, according to the increasing taper angles); the percentage of decrease in torque values due to chlorhexidine application was statistically insignificant when compared with the control group. The application of gel-form chlorhexidine as an antibacterial agent does not significantly affect the stability of the implant–abutment connection under dynamic loads. PVS sealants may cause screw loosening under functional loads.

Using FRET to Measure the Time it Takes for a Cell to Destroy a Virus

2019 ◽  
Author(s):  
Candace E. Benjamin ◽  
Zhuo Chen ◽  
Olivia Brohlin ◽  
Hamilton Lee ◽  
Stefanie Boyd ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Rhodamine B ◽  
Virus Like Particle ◽  
A Cell ◽  
Viral Nanoparticles ◽  
The Stability ◽  
Open Question ◽  
Viral Surface ◽  
Fret Pair

<div><div><div><p>The emergence of viral nanotechnology over the preceding two decades has created a number of intellectually captivating possible translational applications; however, the in vitro fate of the viral nanoparticles in cells remains an open question. Herein, we investigate the stability and lifetime of virus-like particle (VLP) Qβ - a representative and popular VLP for several applications - following cellular uptake. By exploiting the available functional handles on the viral surface, we have orthogonally installed the known FRET pair, FITC and Rhodamine B, to gain insight of the particle’s behavior in vitro. Based on these data, we believe VLPs undergo aggregation in addition to the anticipated proteolysis within a few hours of cellular uptake.</p></div></div></div>

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