Calumenin, a Ca2+ Binding Protein, Is Required for Dauer Formation in Caenorhabditis elegans.

Caenorhabditis elegans can adapt and survive in dynamically changing environments by the smart and delicate switching of molecular plasticity. C. elegans dauer diapause is a form of phenotypic and developmental plasticity that induces reversible developmental arrest upon environmental cues. An ER (endoplasmic reticulum)-resident Ca2+ binding protein, calumenin has been reported to function in a variety of malignant diseases in vertebrates and in the process of muscle contraction-relaxation. In C. elegans, CALU-1 is known to function in Ca2+-regulated behaviors (pharyngeal pumping and defecation) and cuticle formation. The cuticles of dauer larvae are morphologically distinct from those of larvae that develop in favorable conditions. The structure of the dauer cuticle is thicker and more highly reinforced than that of other larval stages to protect dauer larvae from various environmental insults. Since the calu-1(tm1783) mutant exhibited abnormal cuticle structures such as highly deformed annuli and alae, we investigated whether CALU-1 is involved in dauer formation or not. Ascaroside pheromone (ascr#2) and crude daumone were used under starvation conditions to analyze the rate of dauer formation in the calu-1(tm1783) mutant. Surprisingly, the dauer ratio of the calu-1(tm1783) mutant was extremely low compared to that of the wild type. In fact, the calu-1(tm1783) mutants were mostly unable to enter diapause. We also found that calu-1 is expressed in body-wall muscle and AIA interneurons at the dauer stage. Taken together, our results suggest that CALU-1 is required for normal entry into diapause in C. elegans.

BAM15 Relieves Neurodegeneration in Aged Caenorhabditis elegans and Extends Lifespan.

BAM15 was recently screened as a protonophore uncoupler specifically for the mitochondrial membrane but not the plasma membrane. It is equally as potent as FCCP, but less toxic. Previously, mitochondrial uncoupling via DNP alleviates neurodegeneration in the nematode Caenorhabditis elegans during aging. Therefore, we investigated whether BAM15 uncouplers could phenotypically and functionally reduce neuronal defects in aged nematodes. We observed green fluorescence protein-tagged mechanosensory neurons and performed touch and chemotaxis assays during aging. Wild-type animals treated with both 50 µM BAM15 and 10 µM DNP showed reduced mechanosensory neuronal defects during aging, which correlates with the maintenance of touch responses and short-term memory during aging. Uncoupler mutant ucp-4 also responded the same way as the wild-type, reducing neurodegeneration in 50 µM BAM15 and 10 µM DNP-treated animals compared to the DMSO control. These results suggest that 50 µM BAM15 alleviates neurodegeneration phenotypically and functionally in C. elegans during aging, potentially through mitochondrial uncoupling. In accordance with the preserved neuronal shape and function in aged C. elegans, 50 µM BAM15 extended the mean lifespan of both wild-type and ucp-4 mutants.

Prediction and Feature Importance Analysis for Severity of COVID-19 in South Korea Using Artificial Intelligence: Model Development and Validation.

BACKGROUND: The number of deaths from COVID-19 continues to surge worldwide. In particular, if a patient's condition is sufficiently severe to require invasive ventilation, it is more likely to lead to death than to recovery. OBJECTIVE: The goal of our study was to analyze the factors related to COVID-19 severity in patients and to develop an artificial intelligence (AI) model to predict the severity of COVID-19 at an early stage. METHODS: We developed an AI model that predicts severity based on data from 5601 COVID-19 patients from all national and regional hospitals across South Korea as of April 2020. The clinical severity of COVID-19 was divided into two categories: low and high severity. The condition of patients in the low-severity group corresponded to no limit of activity, oxygen support with nasal prong or facial mask, and noninvasive ventilation. The condition of patients in the high-severity group corresponded to invasive ventilation, multi-organ failure with extracorporeal membrane oxygenation required, and death. For the AI model input, we used 37 variables from the medical records, including basic patient information, a physical index, initial examination findings, clinical findings, comorbid diseases, and general blood test results at an early stage. Feature importance analysis was performed with AdaBoost, random forest, and eXtreme Gradient Boosting (XGBoost); the AI model for predicting COVID-19 severity among patients was developed with a 5-layer deep neural network (DNN) with the 20 most important features, which were selected based on ranked feature importance analysis of 37 features from the comprehensive data set. The selection procedure was performed using sensitivity, specificity, accuracy, balanced accuracy, and area under the curve (AUC). RESULTS: We found that age was the most important factor for predicting disease severity, followed by lymphocyte level, platelet count, and shortness of breath or dyspnea. Our proposed 5-layer DNN with the 20 most important features provided high sensitivity (90.2%), specificity (90.4%), accuracy (90.4%), balanced accuracy (90.3%), and AUC (0.96). CONCLUSIONS: Our proposed AI model with the selected features was able to predict the severity of COVID-19 accurately. We also made a web application so that anyone can access the model. We believe that sharing the AI model with the public will be helpful in validating and improving its performance.

Flavonoids mitigate neurodegeneration in aged Caenorhabditis elegans by mitochondrial uncoupling.

Dietary supplementation of flavonoids has been shown to reduce the severity of neurodegenerative disorders such as dementia, Parkinson's disease, and Alzheimer's disease by their antioxidant effects. However, their low bioavailabilityin vivo raises the question of how much their antioxidant capacity actually contributes to the mitigating effects. The physicochemical properties of flavonoids suggest they could function as mitochondrial uncouplers. Moreover, mitochondrial uncoupling alleviated neurodegeneration in Caenorhabditis elegans during aging in previous research. Therefore, we investigated whether various flavonoids (fisetin, quercetin, apigenin, chrysin, catechin, and naringenin) could reduce neuronal defects by mitochondrial uncoupling in C. elegans. Both neuronal defects and mitochondrial membrane potential were reduced in aged worms in nearly all of the flavonoid treatments suggesting that flavonoids may reduce neurodegeneration in C. elegans. However, there was no significant reduction of neuronal defects in mitophagy-deficient pink-1/pdr-1 double mutants under flavonoid treatments. These results suggest that flavonoids could function as mitochondrial uncouplers to mitigate neurodegeneration in aged C. elegans, possibly via a PINK1/Parkin mitophagy process.

Novel Findings of Anti-Filarial Drug Target and Structure-Based Virtual Screening for Drug Discovery.

Lymphatic filariasis and onchocerciasis caused by filarial nematodes are important diseases leading to considerable morbidity throughout tropical countries. Diethylcarbamazine (DEC), albendazole (ALB), and ivermectin (IVM) used in massive drug administration are not highly effective in killing the long-lived adult worms, and there is demand for the development of novel macrofilaricidal drugs affecting new molecular targets. A Ca2+ binding protein, calumenin, was identified as a novel and nematode-specific drug target for filariasis, due to its involvement in fertility and cuticle development in nematodes. As sterilizing and killing effects of the adult worms are considered to be ideal profiles of new drugs, calumenin could be an eligible drug target. Indeed, the Caenorhabditis elegans mutant model of calumenin exhibited enhanced drug acceptability to both microfilaricidal drugs (ALB and IVM) even at the adult stage, proving the roles of the nematode cuticle in efficient drug entry. Molecular modeling revealed that structural features of calumenin were only conserved among nematodes (C. elegans, Brugia malayi, and Onchocerca volvulus). Structural conservation and the specificity of nematode calumenins enabled the development of drugs with good target selectivity between parasites and human hosts. Structure-based virtual screening resulted in the discovery of itraconazole (ITC), an inhibitor of sterol biosynthesis, as a nematode calumenin-targeting ligand. The inhibitory potential of ITC was tested using a nematode mutant model of calumenin.

Mitochondrial Uncoupling Attenuates Age-Dependent Neurodegeneration in C. elegans.

The uncoupling protein 4 (ucp-4) gene is involved in age-dependent neurodegeneration in C. elegans. Therefore, we aimed to investigate the mechanism underlying the association between mitochondrial uncoupling and neurodegeneration by examining the effects of uncoupling agents and ucp-4 overexpression in C. elegans. Treatment with either DNP or CCCP improved neuronal defects in wild type during aging. Uncoupling agents also restored neuronal phenotypes of ucp-4 mutants to those exhibited by wild type, while ucp-4 overexpression attenuated the severity of age-dependent neurodegeneration. Neuronal improvements were further associated with reductions in mitochondrial membrane potentials. However, these age-dependent neuroprotective effects were limited in mitophagy-deficient mutant, pink-1, background. These results suggest that membrane uncoupling can attenuate age-dependent neurodegeneration by stimulating mitophagy.

A Novel Malaria Pf/Pv Ab Rapid Diagnostic Test Using a Differential Diagnostic Marker Identified by Network Biology.

Rapid diagnostic tests (RDTs) can detect anti-malaria antibodies in human blood. As they can detect parasite infection at the low parasite density, they are useful in endemic areas where light infection and/or re-infection of parasites are common. Thus, malaria antibody tests can be used for screening bloods in blood banks to prevent transfusion-transmitted malaria (TTM), an emerging problem in malaria endemic areas. However, only a few malaria antibody tests are available in the microwell-based assay format and these are not suitable for field application. A novel malaria antibody (Ab)-based RDT using a differential diagnostic marker for falciparum and vivax malaria was developed as a suitable high-throughput assay that is sensitive and practical for blood screening. The marker, merozoite surface protein 1 (MSP1) was discovered by generation of a Plasmodium-specific network and the hierarchical organization of modularity in the network. Clinical evaluation revealed that the novel Malaria Pf/Pv Ab RDT shows improved sensitivity (98%) and specificity (99.7%) compared with the performance of a commercial kit, SD BioLine Malaria P.f/P.v (95.1% sensitivity and 99.1% specificity). The novel Malaria Pf/Pv Ab RDT has potential for use as a cost-effective blood-screening tool for malaria and in turn, reduces TTM risk in endemic areas.

Enhanced performance of an innovative dengue IgG/IgM rapid diagnostic test using an anti-dengue EDI monoclonal antibody and dengue virus antigen.

High levels of anti-dengue IgM or IgG can be detected using numerous rapid diagnostic tests (RDTs). However, the sensitivity and specificity of these tests are reduced by changes in envelope glycoprotein antigenicity that inevitably occur in limited expression systems. A novel RDT was designed to enhance diagnostic sensitivity. Dengue viruses cultured in animal cells were used as antigens to retain the native viral coat protein. Monoclonal antibodies (mAbs) were then developed, for the first time, against domain I of envelope glycoprotein (EDI). The anti-dengue EDI mAb was employed as a capturer, and EDII and EDIII, which are mainly involved in the induction of neutralizing antibodies in patients, were fully available to bind to anti-dengue IgM or IgG in patients. A one-way automatic blood separation device prevented reverse migration of plasma and maximize the capture of anti-dengue antibodies at the test lines. A clinical evaluation in the field proved that the novel RDT (sensitivities of 96.5% and 96.7% for anti-dengue IgM and IgG) is more effective in detecting anti-dengue antibodies than two major commercial tests (sensitivities of 54.8% and 82% for SD BIOLINE; 50.4% and 75.3% for PanBio). The innovative format of RDT can be applied to other infectious viral diseases.

Development and clinical evaluation of a highly accurate dengue NS1 rapid test: from the preparation of a soluble NS1 antigen to the construction of an RDT.

Early diagnosis of dengue virus (DENV) is important. There are numerous products on the market claiming to detect DENV NS1, but these are not always reliable. In this study, a highly sensitive and accurate rapid diagnostic test (RDT) was developed using anti-dengue NS1 monoclonal antibodies. A recombinant NS1 protein was produced with high antigenicity and purity. Monoclonal antibodies were raised against this purified NS1 antigen. The RDT was constructed using a capturing (4A6A10, Kd=7.512±0.419×10(-9)) and a conjugating antibody (3E12E6, Kd=7.032±0.322×10(-9)). The diagnostic performance was evaluated with NS1-positive clinical samples collected from various dengue endemic countries and compared to SD BioLine Dengue NS1 Ag kit. The constructed RDT exhibited higher sensitivity (92.9%) with more obvious diagnostic performance than the commercial kit (83.3%). The specificity of constructed RDT was 100%. The constructed RDT could offer a reliable point-of-care testing tool for the early detection of dengue infections in remote areas and contribute to the control of dengue-related diseases.

Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene.

BACKGROUND: Dengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy. OBJECTIVES: This study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches. STUDY DESIGN: Serotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens. RESULTS: The LOD of the assay was 3.0 log10 RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8-47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection. CONCLUSIONS: The assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.

Evaluation of anti-coccidial effects of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one and identification of its potential target proteins in Toxoplasma gondii.

Coccidiosis affects many vertebrates worldwide, but treatment with known anti-coccidial drugs causes several adverse side effects. There is a critical need for the development and evaluation of new drugs. The anti-coccidial effect of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one (NPPP), a synthetic compound, was studied in vitro and in vivo. Treatment with NPPP showed anti-Toxoplasma activity in vitro with a lower EC50 value than pyrimethamine. In ICR mice infected with Toxoplasma gondii, oral administration of NPPP for 4 days showed statistically significant anti-Toxoplasma activity with lower numbers of tachyzoite than those of the negative control (p < 0.01). NPPP also exhibited strong anti-Eimeria activity in Eimeria tenella-infected chickens when treated for 4 days with orally administered NPPP at a dose of 100 mg/kg. Potential target proteins of NPPP were analyzed by proteomic profiles of T. gondii tachyzoites. Two hypothetical proteins were identified as possible targets of NPPP, a putative ortholog of vacuolar ATP synthase subunit C and a class I S-adenosylmethionine-dependent methyltransferase. Our data show that the NPPP might be an anti-coccidial drug candidate for clinical application against coccidial infections. Future investigations will focus on identifying the function of proteins regulated by NPPP.

6-trifluoromethyl-2-thiouracil possesses anti-Toxoplasma gondii effect in vitro and in vivo with low hepatotoxicity.

Since pyrimethamine, the general therapeutic drug for toxoplasmosis, presents several adverse side effects, the need to develop and evaluate new drugs for the condition is critical. In this study, anti-Toxoplasma gondii activities of 3-[{2-((E)-furan-2-ylmethylene)hydrazinyl}methylene]-1,3-dihydroindol-2-one (ATT-5126) and 6-trifluoromethyl-2-thiouracil (KH-0562) were evaluated in vitro using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and in vivo by measuring amount of the tachyzoites in mice ascites. Biochemical parameters such as lipid peroxidation (LPO), glutathione (GSH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were also evaluated in livers of mice at 4 days post-infection. As a result, the ATT-5126 and KH-0562 showed anti-T. gondii activity in vitro. Treatment of ATT-5126 or KH-0562 decreased the amount of tachyzoites in T. gondii infected ICR mice. The relative weight of liver and spleen increased by T. gondii infection were decreased by treatment of ATT-5126 or KH-0562. The levels of LPO, ALT and AST, which are biochemical parameters involved in liver injury, were also significantly recovered by treatment of ATT-5126 or KH-0562 (p<0.05). In particular, the recovered levels by KH-0562 were similar to those of pyrimethamine-treated group (p<0.05). However, the level of GSH, which is an antioxidant indicator, showed insignificant statistics. The results suggest that KH-0562 show anti-T. gondii activities in vitro and in vivo with low hepatotoxicity. Therefore, KH-0562 may be a useful candidate for treating T. gondii infection.

Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development.

BACKGROUND: With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified. METHODS: Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa) was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety. RESULTS: Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn) formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity. CONCLUSION: The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite. In addition, the monomeric form of enzyme may contribute to relatively simple synthesis of selective inhibitors.

Anti-malarial activity of new N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) derivatives against Plasmodium falciparum.

Malaria is the most common of the parasitic diseases in tropical and subtropical regions. Adverse side effects of anti-malarial drugs have precluded them as a potential clinical drug. In this study, novel derivatives of N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) based on a variety of dipeptidyl α,ß-unsaturated amides containing lysine as a part were synthesized and evaluated. Lower toxicity was achieved by reducing or eliminating the tendency of forming chemically reactive and toxic intermediates and metabolites. The synthesized compounds were evaluated for anti-malarial efficacy against Plasmodium falciparum and cytotoxicity in human epitheloid carcinoma cervix (HeLa cells) by estimating the therapeutic index (TI). N-Methyl amide with N'-Boc protection among them exhibited strong anti-malarial activity and N-methyl amide with N'-m-methylbenzyl amide showed excellent anti-malarial activity with much lower toxicity than the ALLN. Therefore, the two chemicals, as well as the underlying design rationale, could be useful in the discovery and development of new anti-malarial drugs.

Development of a novel fluorophore for real-time biomonitoring system.

Rapid in-field diagnosis is very important to prevent the outbreak of various infectious and contagious diseases. Highly sensitive and quantitative detection of diseases can be performed using fluorescent immunochemical assay with specific antigen-antibody binding and a good quality fluorophore. This can lead to the development of a small, portable, quantitative biosensor to transmit diagnostic results to a control center in order to systematically prevent disease outbreaks. In this study, we developed a novel fluorophore, coumarin-derived dendrimer, with high emission intensity, strong signal brightness, and high photostability. It is easily coupled with biomolecules and emits strong and stable fluorescence at 590 nm with excitation at 455 nm. Application to fluorescent immunochromatographic test (FICT) showed that the novel coumarin-derived dendrimer bioconjugate could detect antigens at amount as low as 0.1 ng. The clinical results and the spectral characteristics of the novel coumarin-derived dendrimer open, for the first time, the possibility of developing a cost/energy efficient LED-based portable quantitative biosensor for point-of-care (POC) disease diagnosis, which can permit real time monitoring (U-healthcare system) by a disease control center.

Ethyl gallate induces apoptosis of HL-60 cells by promoting the expression of caspases-8, -9, -3, apoptosis-inducing factor and endonuclease G.

Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.

Production of monoclonal antibodies for Plasmodium vivax lactate dehydrogenase and patient sera screening using sandwich ELISA.

Malaria is a worldwide infectious disease. There are many diagnostic kits to detect malaria infection. However, the sensitivity of these diagnostic kits remains a problem. To develop a diagnostic kit for malaria that has high sensitivity, it is necessary to produce monoclonal antibodies (McAbs) with high affinity. The present study was undertaken to produce hybridoma cells that can be used to generate McAbs with high affinity and specificity against Plasmodium vivax lactate dehydrogenase (pvLDH). In this study, BALB/c mice were immunized with purified recombinant polypeptides that encode pvLDH. McAbs against pvLDH were produced according to the protocol of hybridoma technique using myeloma cells (SP2/0 cell lines). The McAbs were characterized by isotyping and by Western blot analysis. Two McAbs (D2H and D7E) against pvLDH antigen were obtained. The isotypes of D2H and D7E were IgG2b. They recognize 33 kDa proteins that were defined as pvLDH by Western blot analysis. In the affinity test, D2H and D7E showed positively optical density value until each McAbs were serially diluted at concentrations of 0.156 and 0.078 µg/ml, respectively. To evaluate sensitivity and specificity against clinical specimens of P. vivax, purified McAbs were tested with alkaline phosphatase-conjugated monoclonal antibodies and blood samples (n = 180) of P. vivax patients using the sandwich enzyme-linked immunosorbent assay, showing the 98% sensitivity. We suggest that McAbs produced in this study may be used for developing efficient and rapid diagnostic kits.

Synthesis and evaluation of peptidyl α,ß-unsaturated carbonyl derivatives as anti-malarial calpain inhibitors.

Malarial calpain is a cysteine protease believed to be a central mediator essential for parasitic activities. N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN), a calpain inhibitor, showed an excellent inhibitory effect on the erythrocytic stages of Plasmodium falciparum. However the aldehyde group of ALLN makes it susceptible to metabolism. Therefore, we designed α,ß-unsaturated carbonyl peptides that could serve as electrophiles for cysteine residues in calpain. Among the synthetic analogs based on the structure of ALLN, peptidyl esters 7, 8 and 9 showed the most potent anti-malarial effects, with the same IC50 values of 5.0 µM. Also they showed the high selective toxicity for the malaria versus Hela cell with 40.6, 69.2 and 24.3 fold for 7, 8 and 9, respectively. Dipeptidyl α,ß-unsaturated carbonyl derivatives consisting of two amino acids gave better anti-malarial effects than those consisting with one amino acid. The fluctuation in anti-malarial activity with small changes in chemical structure indicates the possibilities of improving synthetic analogs.

Simple and efficient model systems of screening anti-Toxoplasma drugs in vitro.

INTRODUCTION: A lot of in vitro technologies have been developed to screen drugs for toxoplasmosis, which is caused by Toxoplasma gondii and is one of the most serious infectious diseases in the world. However, developed screening methods still have limitation such as inaccuracy, labor-intensive and time-consuming procedure. Therefore, the development of simpler, more efficient and accurate high-throughput screening assay is needed. AREAS COVERED: The present review gives the overview of in vitro screening technologies described in literatures so far including morphological assay, incorporation of [(3)H]uracil assay, enzyme-linked immunosorbent assay (ELISA), colorimetric microtiter assay (ß-galactosidase assay), flow cytometric quantification assay, yellow fluorescent protein assay and cell viability assay. The authors discuss how these methods are efficient and/or limited for screening anti-T. gondii drugs. The authors further suggest brand-new technologies which are faster, simpler, more effective and available for high-throughput screening. EXPERT OPINION: Options for clinical treatment of toxoplasmosis are currently very limited. Thus, more accurate in vitro screening methods must be established to identify the most effective anti-T. gondii drugs from random screening of compounds. At the same time, based on genome information, combination of an appropriate screening technology, combinatorial chemistry and computational biology may increase the efficiency of target-based drug discovery against T. gondii.

CNP-1 (ARRD-17), a novel substrate of calcineurin, is critical for modulation of egg-laying and locomotion in response to food and lysine sensation in Caenorhabditis elegans.

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase involved in calcium signaling pathways. In Caenorhabditis elegans, the loss of calcineurin activity causes pleiotropic defects including hyperadaptation of sensory neurons, hypersensation to thermal difference and hyper-egg-laying when worms are refed after starvation. In this study, we report on arrd-17 as calcineurin-interacting protein-1 (cnp-1), which is a novel molecular target of calcineurin. CNP-1 interacts with the catalytic domain of the C. elegans calcineurin A subunit, TAX-6, in a yeast two-hybrid assay and is dephosphorylated by TAX-6 in vitro. cnp-1 is expressed in ASK, ADL, ASH and ASJ sensory neurons as TAX-6. It acts downstream of tax-6 in regulation of locomotion and egg-laying after starvation, ASH sensory neuron adaptation and lysine chemotaxis, that is known to be mediated by ASK neurons. Altogether, our biochemical and genetic evidence indicates that CNP-1 is a direct target of calcineurin and required in stimulated egg-laying and locomotion after starvation, adaptation to hyperosmolarity and attraction to lysine, which is modulated by calcineurin. We suggest that the phosphorylation status of CNP-1 plays an important role in regulation of refed stimulating behaviors after starvation and attraction to amino acid, which provides valuable nutritious information.