In vitro and in vivo activity of cinnamaldehyde against Eimeria kofoidi in chukar partridge (Alectoris chukar).

One hundred and twenty one-day-old chukar partridges were randomly divided into eight groups which received diets with different supplementations. There were four unchallenged groups. One group received salinomycin (50 ppm), two groups received cinnamaldehyde (CINN) (100 and 200 mg/kg of diet), and another one received only the basal diet from the 1st to the 31st day. There were also four corresponding groups orally challenged by 3 × 105Eimeria kofoidi sporulated oocysts at the 21st day. Three samplings were done at the 24th, 26th, and 31st days of rearing for pathological and biochemical assessments. Fecal samples were daily taken to check the pattern of oocyst shedding from the 26th to 31st day. The body weight of birds was measured at 21st and 31st days. Along with the in vivo experiment, an in vitro sporulation inhibition test was carried out. The in vitro results showed that CINN decreased sporulation rate at 1 and 0.5 mg/ml. In vivo, it was found that CINN did not prevent the oocyst shedding. Furthermore, the histopathological findings revealed that CINN and salinomycin had no effect on infection establishment. However, our findings showed that CINN (200 mg/kg of diet) could enhance the body weight and improve antioxidant status. Although our results did not support the in vivo anticoccidial activity of CINN, it had a promising potential to improve antioxidant status and body weight in the chukar partridge.

Role of a Concentration Gradient in Malaria Drug Resistance Evolution: A Combined within- and between-Hosts Modelling Approach.

Resistance to antimalarial drugs is currently a growing public health problem, resulting in more cases with treatment failure. Although previous studies suggested that a concentration gradient facilitates the antibiotic resistance evolution in bacteria, no attempt has been made to investigate the roles of a concentration gradient in malaria drug resistance. Unlike the person-to-person mode of transmission of bacteria, the malaria parasites need to switch back and forth between the human and mosquito hosts to complete the life cycle and to spread the resistant alleles. Here we developed a stochastic combined within- and between-hosts evolutionary dynamics model specific to malaria parasites in order to investigate the influence of an antimalarial concentration gradient on the evolutionary dynamics of malaria drug resistance. Every stage of malaria development in both human and mosquito hosts are individually modelled using the tau-leaping algorithm. We found that the concentration gradient can accelerate antimalarial resistance evolution. The gain in resistance evolution was improved by the increase in the parasite mutation rate and the mosquito biting rate. In addition, even though the rate of resistance evolution is not sensitive to the changes in parasite reduction ratios (PRRs) of antimalarial drugs, the probability of finding the antimalarial drug resistant parasites decreases when the PRR increases.

Activation of artemisinin and heme degradation in Leishmania tarentolae promastigotes: A possible link.

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.

Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells.

BACKGROUND: The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. RESULTS: Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. CONCLUSIONS: Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

High-Throughput Screening of Entamoeba Identifies Compounds Which Target Both Life Cycle Stages and Which Are Effective Against Metronidazole Resistant Parasites.

Neglected tropical diseases, especially those caused by parasites, are significantly underserved by current drug development efforts, mostly due to the high costs and low economic returns. One method for lowering the costs of drug discovery and development for these diseases is to repurpose drugs developed for other indications. Here, we present the results of a screen of five repurposed drug libraries to identify potential new lead compounds to treat amebiasis, a disease that affects tens of millions of people and causes ~100,000 deaths annually. E. histolytica, the causative agent of amebiasis, has two major life cycle stages, the trophozoite and the cyst. The current primary treatment for amebiasis, nitroimidazole compounds, do not eliminate parasites from the colonic lumen, necessitating a multi-drug treatment regimen. We aimed to address this problem by screening against both life stages, with the aim of identifying a single drug that targets both. We successfully identified eleven compounds with activity against both cysts and trophozoites, as well as multiple compounds that killed trophozoites with improved efficacy over existing drugs. Two lead compounds (anisomycin and prodigiosin) were further characterized for activity against metronidazole (MNZ) resistant parasites and mature cysts. Anisomycin and prodigiosin were both able to kill MNZ resistant parasites while prodigiosin and its analog obatoclax were active against mature cysts. This work confirms the feasibility of identifying drugs that target both Entamoeba trophozoites and cysts, and is an important step toward developing improved treatment regimens for Entamoeba infection.

Effect of antiprotozoal molecules on hypnospores of Perkinsus spp. parasite.

Perkinsus protozoan parasites have been associated with high mortality of bivalves worldwide, including Brazil. The use of antiproliferative drugs to treat the Perkinsosis is an unusual prophylactic strategy. However, because of their environment impact it could be used to control parasite proliferation in closed system, such as hatchery. This study evaluated the anti-Perkinsus activity potential of synthesized and commercial compounds. Viability of hypnospores of Perkinsus spp. was assessed in vitro. Cells were incubated with three 2-amino-thiophene (6AMD, 6CN, 5CN) and one acylhydrazone derivatives (AMZ-DCL), at the concentrations of 31.25; 62.5; 125; 250 and 500 µM and one commercial chlorinated phenoxy phenol derivative, triclosan (2, 5, 10 and 20 µM), for 24-48 h. Two synthetic molecules (6CN and AMZ-DCL) caused a significant decline (38 and 39%, respectively) in hypnospores viability, at the highest concentration (500 µM), after 48 h. Triclosan was the most cytotoxic compound, causing 100% of mortality at 20 µM after 24 h and at 10 µM after 48 h. Cytotoxic effects of the compounds 6CN, AMZ-DCL, and triclosan were investigated by measuring parasite's zoosporulation, morphological changes and metabolic activities (esterase activity, production of reactive oxygen species and lipid content). Results showed that zoosporulation occurred in few cell. Triclosan caused changes in the morphology of hypnospores. The 6CN and AMZ-DCL did not alter the metabolic activities studied whilst Triclosan significantly increased the production of reactive oxygen species and changed the amount and distribution of lipids in the hypnospores. These results suggest that three compounds had potential to be used as antiprotozoal drugs, although further investigation of their mechanism of action must be enlightened.

Combating Acanthamoeba spp. cysts: what are the options?

Acanthamoeba spp. are protist pathogens and causative agents of serious infections including keratitis and granulomatous amoebic encephalitis. Its ability to convert into dormant and highly resistant cysts form limits effectiveness of available therapeutic agents and presents a pivotal challenge for drug development. During the cyst stage, Acanthamoeba is protected by the presence of hardy cyst walls, comprised primarily of carbohydrates and cyst-specific proteins, hence synthesis inhibition and/or degradation of cyst walls is of major interest. This review focuses on targeting of Acanthamoeba cysts by identifying viable therapeutic targets.

Metabolomes of Potato Root Exudates: Compounds That Stimulate Resting Spore Germination of the Soil-Borne Pathogen Spongospora subterranea.

Root exudation has importance in soil chemical ecology influencing rhizosphere microbiota. Prior studies reported root exudates from host and nonhost plants stimulated resting spore germination of Spongospora subterranea, the powdery scab pathogen of potato, but the identities of stimulatory compounds were unknown. This study showed that potato root exudates stimulated S. subterranea resting spore germination, releasing more zoospores at an earlier time than the control. We detected 24 low molecular weight organic compounds within potato root exudates and identified specific amino acids, sugars, organic acids, and other compounds that were stimulatory to S. subterranea resting spore germination. Given that several stimulatory compounds are commonly found in exudates of diverse plant species, we support observations of nonhost-specific stimulation. We provide knowledge of S. subterranea resting spore biology and chemical ecology that may be useful in formulating new disease management strategies.

Sentinel cells, symbiotic bacteria and toxin resistance in the social amoeba Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is unusual among eukaryotes in having both unicellular and multicellular stages. In the multicellular stage, some cells, called sentinels, ingest toxins, waste and bacteria. The sentinel cells ultimately fall away from the back of the migrating slug, thus removing these substances from the slug. However, some D. discoideum clones (called farmers) carry commensal bacteria through the multicellular stage, while others (called non-farmers) do not. Farmers profit from their beneficial bacteria. To prevent the loss of these bacteria, we hypothesize that sentinel cell numbers may be reduced in farmers, and thus farmers may have a diminished capacity to respond to pathogenic bacteria or toxins. In support, we found that farmers have fewer sentinel cells compared with non-farmers. However, farmers produced no fewer viable spores when challenged with a toxin. These results are consistent with the beneficial bacteria Burkholderia providing protection against toxins. The farmers did not vary in spore production with and without a toxin challenge the way the non-farmers did, which suggests the costs of Burkholderia may be fixed while sentinel cells may be inducible. Therefore, the costs for non-farmers are only paid in the presence of the toxin. When the farmers were cured of their symbiotic bacteria with antibiotics, they behaved just like non-farmers in response to a toxin challenge. Thus, the advantages farmers gain from carrying bacteria include not just food and protection against competitors, but also protection against toxins.

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.

Exposure to synthetic gray water inhibits amoeba encystation and alters expression of Legionella pneumophila virulence genes.

Water conservation efforts have focused on gray water (GW) usage, especially for applications that do not require potable water quality. However, there is a need to better understand environmental pathogens and their free-living amoeba (FLA) hosts within GW, given their growth potential in stored gray water. Using synthetic gray water (sGW) we examined three strains of the water-based pathogen Legionella pneumophila and its FLA hosts Acanthamoeba polyphaga, A. castellanii, and Vermamoeba vermiformis. Exposure to sGW for 72 h resulted in significant inhibition (P < 0.0001) of amoebal encystation versus control-treated cells, with the following percentages of cysts in sGW versus controls: A. polyphaga (0.6 versus 6%), A. castellanii (2 versus 62%), and V. vermiformis (1 versus 92%), suggesting sGW induced maintenance of the actively feeding trophozoite form. During sGW exposure, L. pneumophila culturability decreased as early as 5 h (1.3 to 2.9 log10 CFU, P < 0.001) compared to controls (Δ0 to 0.1 log10 CFU) with flow cytometric analysis revealing immediate changes in membrane permeability. Furthermore, reverse transcription-quantitative PCR was performed on total RNA isolated from L. pneumophila cells at 0 to 48 h after sGW incubation, and genes associated with virulence (gacA, lirR, csrA, pla, and sidF), the type IV secretion system (lvrB and lvrE), and metabolism (ccmF and lolA) were all shown to be differentially expressed. These results suggest that conditions within GW may promote interactions between water-based pathogens and FLA hosts, through amoebal encystment inhibition and alteration of bacterial gene expression, thus warranting further exploration into FLA and L. pneumophila behavior in GW systems.

Suppression of Eimeria tenella sporulation by disinfectants.

The disinfectant effects (DEs) of 10 types of chemicals, defined by their ability to destroy or inhibit oocysts and consequently prevent sporulation of Eimeria tenella field isolate, were evaluated in vitro. Correct species assignments and sample purities were confirmed by the singular internal transcribed spacer (ITS)-PCR analysis. A total of 18 treatments were performed, and the disinfection suppression levels were 75.9% for 39% benzene + 22% xylene (1:10 dilution), 85.5% for 30% cresol soup (1:1 dilution), and 91.7% for 99.9% acetic acid (1:2 dilution) group. The results indicate that acetic acid, cresol soup, and benzene+xylene are good candidates for suppression of E. tenella oocyst sporulation.

Effect of ingested human antibodies induced by RTS, S/AS01 malaria vaccination in children on Plasmodium falciparum oocyst formation and sporogony in mosquitoes.

BACKGROUND: The circumsporozoite protein (CS protein) on the malaria parasites in mosquitoes plays an important role in sporogony in mosquitoes. The RTS,S/AS01 malaria vaccine candidate, which has shown significant efficacy against clinical malaria in a large Phase 3 trial, targets the Plasmodium falciparum CS protein, but the ability of serum from vaccinated individuals to inhibit sporogony in mosquitoes has not been evaluated. METHODS: Previously a double-blind, randomized trial of RTS,S/AS01 vaccine, as compared with rabies vaccine, in five- to 17-month old children in Tanzania was conducted. In this study, polyclonal human antibodies were purified from the pools of sera taken one month after the third vaccination. IgGs were purified from four pools of sera from 25 RTS,S/AS01 vaccinated children each, and two pools of sera from 25 children vaccinated with rabies vaccine each. The ability of antibodies to inhibit P. falciparum oocyst formation and/or sporogony in the mosquito host was evaluated by a standard membrane-feeding assay. The test antibodies were fed on day 0 (at the same time as the gametocyte feed), or on days 3 or 6 (serial-feed experiments). The oocyst and sporozoite counts were performed on days 8 and 16, respectively. In addition, two human anti-CS monoclonal antibodies (mAb) and a control mAb were also evaluated. RESULTS: Polyclonal anti-CS IgG preparations from RTS,S-vaccinated children tested at concentrations of 149-210 ELISA units (EU)/ml did not show significant inhibition in oocyst and sporozoite formation when the antibodies were fed with gametocytes at the same time, or later (serial-feed experiments). Similarly, anti-CS mAbs tested at 6,421 or 7,122 EU/ml did not show reduction in oocyst and sporozoite formation. CONCLUSIONS: This study does not support the concept that anti-CS antibodies induced by the RTS,S/AS01 vaccines in humans noticeably reduce malaria transmission by blocking P. falciparum sporozoite development or salivary gland invasion in mosquitoes when taken up during feeding.

Oxygen stress reduces zoospore survival of Phytophthora species in a simulated aquatic system.

BACKGROUND: The genus Phytophthora includes a group of agriculturally important pathogens and they are commonly regarded as water molds. They produce motile zoospores that can move via water currents and on their own locomotion in aquatic environments. However, zoosporic response to dissolved oxygen, an important water quality parameter, is not known. Like other water quality parameters, dissolved oxygen concentration in irrigation reservoirs fluctuates dramatically over time. The aim of this study was to determine whether and how zoospore survival may be affected by elevated and low concentrations of dissolved oxygen in water to better understand the aquatic biology of these pathogens in irrigation reservoirs. RESULTS: Zoospores of P. megasperma, P. nicotianae, P. pini and P. tropicalis were assessed for survival in 10% Hoagland's solution at a range of dissolved concentrations from 0.9 to 20.1 mg L(-1) for up to seven exposure times from 0 to 72 h. Zoospore survival was measured by resultant colony counts per ml. Zoospores of these species survived the best in control Hoagland's solution at dissolved oxygen concentrations of 5.3 to 5.6 mg L(-1). Zoospore survival rates decreased with increasing and decreasing concentration of dissolved oxygen, depending upon Phytophthora species and exposure time. Overall, P. megasperma and P. pini are less sensitive than P. nicotianae and P. tropicalis to hyperoxia and hypoxia conditions. CONCLUSION: Zoospores in the control solution declined over time and this natural decline process was enhanced under hyperoxia and hypoxia conditions. These findings suggest that dramatic fluctuations of dissolved oxygen in irrigation reservoirs contribute to the population decline of Phytophthora species along the water path in the same reservoirs. These findings advanced our understanding of the aquatic ecology of these pathogens in irrigation reservoirs. They also provided a basis for pathogen risk mitigation by prolonging the turnover time of runoff water in recycling irrigation systems via better system designs.

The in vitro efficacy of antimicrobial agents against the pathogenic free-living amoeba Balamuthia mandrillaris.

The free-living amoeba Balamuthia mandrillaris causes usually fatal encephalitis in humans and animals. Only limited studies have investigated the efficacy of antimicrobial agents against the organism. Assay methods were developed to assess antimicrobial efficacy against both the trophozoite and cyst stage of B. mandrillaris (ATCC 50209). Amphotericin B, ciclopirox olamine, miltefosine, natamycin, paromomycin, pentamidine isethionate, protriptyline, spiramycin, sulconazole and telithromycin had limited activity with amoebacidal levels of > 135-500 µM. However, diminazene aceturate (Berenil(®) ) was amoebacidal at 7.8 µM and 31.3-61.5 µM for trophozoites and cysts, respectively. Assays for antimicrobial testing may improve the prognosis for infection and aid in the development of primary selective culture isolation media.

Occurrence of organic chlorinated pesticides and their ecological effects on soil protozoa in the agricultural soils of North Western Beijing, China.

The occurrence of ∑HCHs, ∑DDTs, protozoa abundance and their community structure in surface soils of orchards, vegetable lands, and barren lands in northern west outskirts of Beijing were detected in order to investigate the protozoa responses to low dose organic chlorinated Pesticides (OCPs) after long-term field-based exposure. Significant differences in total concentrations of HCHs and DDTs were found among the three general groups ranking in decreasing order of concentration from orchard>vegetable lands >barren lands. Ciliate was the rare group in surface soils of all the sampling groups. The abundance of flagellate, ciliate, and amoebae in vegetable soils were significantly higher than those in orchard soils. The abundance of all the taxa of protozoa was strongly negative correlated with the residue level of ∑HCHs and ∑DDTs (P<0.05) in agricultural soils. However, no negative correlation between the residue levels of OCPs and protozoa abundance was shown in both the orchard and the barren soils. This field study demonstrated a considerable long-term impact of the OCPs residue on the abundance of protozoa in soils, and that the abundance of soil protozoa was much more influenced by land use type in association with different soil properties.

An image analysis algorithm for malaria parasite stage classification and viability quantification.

With more than 40% of the world's population at risk, 200-300 million infections each year, and an estimated 1.2 million deaths annually, malaria remains one of the most important public health problems of mankind today. With the propensity of malaria parasites to rapidly develop resistance to newly developed therapies, and the recent failures of artemisinin-based drugs in Southeast Asia, there is an urgent need for new antimalarial compounds with novel mechanisms of action to be developed against multidrug resistant malaria. We present here a novel image analysis algorithm for the quantitative detection and classification of Plasmodium lifecycle stages in culture as well as discriminating between viable and dead parasites in drug-treated samples. This new algorithm reliably estimates the number of red blood cells (isolated or clustered) per fluorescence image field, and accurately identifies parasitized erythrocytes on the basis of high intensity DAPI-stained parasite nuclei spots and Mitotracker-stained mitochondrial in viable parasites. We validated the performance of the algorithm by manual counting of the infected and non-infected red blood cells in multiple image fields, and the quantitative analyses of the different parasite stages (early rings, rings, trophozoites, schizonts) at various time-point post-merozoite invasion, in tightly synchronized cultures. Additionally, the developed algorithm provided parasitological effective concentration 50 (EC50) values for both chloroquine and artemisinin, that were similar to known growth inhibitory EC50 values for these compounds as determined using conventional SYBR Green I and lactate dehydrogenase-based assays.

A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species.

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species.