Quantifying pH in Malaria Using pHluorin and Flow Cytometry.

Intracellular pH (pHi) plays a critical role in the regulation of numerous biological functions where specific pH ranges are required for optimal operation within cells. Slight pH changes can impact the regulation of diverse molecular processes, including enzymatic activities, ion channels, and transporters, which all play a role in cell functions. Methods for quantifying pHi continue to evolve and include various optical methods using fluorescent pH indicators. Here, we provide a protocol to measure pHi in the cytosol of Plasmodium falciparum blood stage parasites by means of flow cytometry and using pHluorin2, a pH-sensitive fluorescent protein that has been introduced into the genome of the parasite.

Protein Profiling of Malaria-Derived Extracellular Vesicles Reveals Distinct Subtypes.

Malaria is caused by obligate intracellular parasites belonging to the genus Plasmodium. Red blood cells (RBCs) infected with different stages of Plasmodium spp. release extracellular vesicles (EVs). Extensive studies have recently shown that these EVs are involved in key aspects of the parasite's biology and disease pathogenesis. However, they are yet to be fully characterized. The blood stages of Plasmodium spp., namely the rings, trophozoites and schizonts, are phenotypically distinct, hence, may induce the release of characteristically different EVs from infected RBCs. To gain insights into the biology and biogenesis of malaria EVs, it is important to characterize their biophysical and biochemical properties. By differential centrifugation, we isolated EVs from in vitro cultures of RBCs infected with different stages of Plasmodium falciparum. We performed a preliminary characterization of these EVs and observed that important EV markers were differentially expressed in EVs with different sedimentation properties as well as across EVs released from ring-, trophozoite- or schizont-infected RBCs. Our findings show that RBCs infected with different stages of malaria parasites release EVs with distinct protein expression profiles.

OPD (Online Plasmodium Diagnosis): An ALA-PpIX based functional assay to predict active malaria.

BACKGROUND: Malaria kills one child every 30 seconds reaching up to 3000 children a day. The mosquito borne malarial parasite invades the blood stream and hijacks red blood cells (RBCs). One of the medical successes of the 20th century was development of malaria diagnostic tests. However, poor specificity and sensitivity along with the inability of these assays to distinguish active malarial infections has put the management scheme in jeopardy. AIM: To develop an in-vitro functional assay to predict active malarial infections. METHODS: Plasmodium falciparum (3D7) parasites were incubated with delta-aminolevulinic acid (ALA) for 7 h and imaged using a confocal microscope for protoporphyrin IX (PpIX) fluorescence. Similarly, PpIX was extracted, and fluorescence was estimated by fluorimetry. RESULTS: Imaging showed that the falciparum-infected RBCs fluoresced while the non-infected cells did not. Moreover, fluorimetry showed fluorescent peaks only in actively infected RBCs. CONCLUSIONS: ALA was only taken up by infected RBCs. When the parasites were loaded with ALA, they fluoresced. These proof-of-concept results demonstrate the first functional assay to detect/diagnose active malaria.

Ability of Vital and Fluorescent Staining in the Differentiation of Schistosoma haematobium Live and Dead Eggs.

This study reports (for the first time) the staining ability of vital (0.4% trypan blue and 1% neutral red) and fluorescent (Hoechst 33258) dyes to differentiate between live and dead Schistosoma haematobium (S. haematobium) eggs in human urine samples. Since S. haematobium egg is important in disease pathology, diagnosis, transmission, and drug development research, it is essential to be able to easily distinguish live eggs from dead ones. Staining is considered a way of enhancing the identification of live and dead eggs. Urine samples from school children were examined for the presence of S. haematobium eggs. Vital and fluorescent dyes were used to stain the samples that contained S. haematobium eggs, after which they were observed using light and fluorescent microscopes, respectively. The Hoechst 33258 provided a good staining outcome for differentiation between live and dead eggs, followed by 0.4% Trypan blue. Regarding the 1% neutral red stain, even though it provided some evidence of which egg was alive or dead, the distinction was not very clear; therefore, it could be useful when used in combination with other stains for egg viability determination. The benefits of this study will include assessing the effect of drugs on S. haematobium eggs in Schistosomiasis research.

In vitro anti-Leishmania activity of tetracyclic iridoids from Morinda lucida, benth.

Leishmaniasis is an infectious disease transmitted by the sand fly. It is caused by over 20 different species of Leishmania and has affected over 14 million people worldwide. One of the main forms of control of leishmaniasis is chemotherapy, but this is limited by the high cost and/or toxicity of available drugs. We previously found three novel compounds with an iridoid tetracyclic skeleton to have activity against trypanosome parasites. In this study, we determined the activity of the three anti-trypanosome compounds against Leishmania using field strain, 010, and the lab strain Leishmania hertigi. The minimum inhibitory concentration (MIC) of the compounds against 010 was determined by microscopy while the IC50 of compounds against L. hertigi was determined by fluorescence-activated cell sorting with Guava viacount analysis. We found two of the three compounds, molucidin and ML-F52, to have anti-Leishmania activity against both strains. The fluor-microscope observation with DAPI stain revealed that both Molucidin and ML-F52 induced abnormal parasites with two sets of nucleus and kinetoplast in a cell, suggesting that compounds might inhibit cytokinesis in Leishmania parasites. Molucidin and ML-F52 might be good lead compounds for the development of new anti-Leishmania chemotherapy.

Ovipositional Behavior of Anopheles gambiae Mosquitoes.

Mosquito eggs laid within two hours are necessary for transgenic (injection) studies, because mosquito eggs become hard after that period. Thus, in order to have eggs available within this two-hour window, it is important to understand the ovipositional behavior of Anopheles gambiae s.s.. In the present study, the ovipositional behavior of An. gambiae s.s. (Kisumu) was investigated in several different conditions: age of mosquitoes, time post blood meal to access oviposition substrate, and light conditions. Two groups of mosquitoes, 3-5 days old and 9-11 days old were blood-fed. For those mosquito groups, an oviposition dish was set either at 48 hours or 72 hours after the blood meal either in a light condition or in an artificial dark condition. The number of laid eggs was compared among the different conditions. The 3-5 day-old mosquitoes apparently produced a higher number of eggs than 9-11 day-old mosquitoes, while there was no significant difference between the two groups. The number of laid eggs per one surviving blood-fed mosquito in the dark condition was significantly higher than that in the light condition (p = 0.03). Providing an oviposition dish at 72 hours after blood meal resulted in a significantly higher number of laid eggs per one surviving blood-fed mosquito than at 48 hours after blood meal (p = 0.03). In conclusion, the optimal condition to have readily available egg supply for transgenic analysis was as follows: 3-5 day-old mosquitoes with an oviposition dish placed at 72 hours after the blood meal in a dark environment.