extfieldsofstudy: Medicine Biology

text:
Assay development and high-throughput antiviral drug screening against Bluetongue virus

Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5000 cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z′-values ≥0.70, Coefficient of Variations ≥5.68 and signal-to-background ratio ≥7.10. This assay was further validated using a 9532 compound library. The fully validated assay was then used to screen the 194 950 compound collection, which identified 693 compounds with >30% CPE inhibition. The 10-concentration dose response assay identified 185 structures with IC50 ≤ 100 μM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease.

Introduction
Bluetongue virus (BTV) is a non-enveloped, multi-layered, double-stranded RNA virus in the genus Orbivirus, family Reoviridae, one of the largest families of viruses including major human pathogens (e.g., rotavirus) and other vertebrate, plant and insect pathogens. BTV is transmitted by biting midges of Culicoides, including C. variipennis and C. imocola (Mellor, 2000). BTV replicates in both vertebrate and insect hosts, causing diseases in wild ruminants and economic important animals including sheep, goat, cattle and other domestic animals around the world (Mellor, 2000;Mellor and Wittmann, 2002), with $3 billion/year loss worldwide (MacLachlan and Osburn, 2006;Mellor et al., 2008). Due to its economic significance, BTV has been the subject of extensive molecular, genetic and structural studies and now represents one of the well-characterized viruses.
There are no antiviral drugs and no effective control measures developed against BTV. Currently, vector control and vaccination using BTV virus-like particles are two main prevention strategies being implemented to prevent the possible BTV epidemics (Blair et al., 2000;Travanty et al., 2004). However, such strategies have been demonstrated to be insufficient due to the changes of vector habitats and new virus strains. The 1998-2001 outbreak of BTV in the Mediterranean Basin was the greatest epizootic of the disease on record, showing that BTV had extended its range northwards into areas of Europe that were never affected before and has persisted in many of these locations (Meiswinkel et al., 2008). This epidemic has been linked to the recent distribution extensions of its major vector, C. imicola, the involvement of novel Culicoides vector(s), and an apparent ability of the virus to overwinter in the absence of adult vectors Mellor and Wittmann, 2002). Thus, it is urgent to develop effective antiviral drugs against BTV infection to avoid possible epidemics.
Developing robust and reliable biological assay(s) is critical for antiviral drug discovery. Traditional antiviral drug discovery assays include cytopathic effects (CPE)-based assay, genomic and/or subgenomic replicon-based assay, and plaque forming assay (Puig-Basagoiti et al., 2005;Severson et al., 2007Severson et al., , 2008. CPE is evident in BTV infected vertebrate cells, such as HeLa, BSR, and HEK 293T, including both the biochemical and morphological hallmarks of apoptosis (caspase-3 activation, DNA fragmentation, membrane blebbing and cellular shrinkage) (Li et al., 2007). BTV-induced CPE and/or apoptosis could be monitored using different CPE and apoptosis detection methods, which make the development of a robust and reliable assay possible. Here, we present the development of a robust and reliable CPE-based high-throughput screening (HTS) assay for antiviral drug discovery against BTV infection, and the identification of novel antiviral compounds that protect host cells from BTV infection.

Methods and materials
2.1. Cell culture BSR cell line, a derivative cell line from baby hamster kidney (BHK) cells, was maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 5% fetal calf serum (FCS), 100 U/ml of penicillin, and 100 g/ml of streptomycin (Sigma-Aldrich Chemical Co., St. Louis, MO). Unless otherwise noted, cells were harvested and re-suspended in assay medium, DMEM with 100 g/ml streptomycin, 100 IU penicillin/l and 1% FBS. After plating and/or compound treatment, cells were incubated at 37 • C, 5% CO 2 and 80-95% humidity for the time period as noted.

BTV culture
The BTV-10 stocks were propagated in BSR cells as previously described (Li et al., 2007). Briefly, BSR cells were grown in T75 flasks to 90% confluent, and then infected with BTV-10 virus at multiplicity of infection (MOI) of 0.01. After 72 h incubation, cells were harvested and centrifuged. The cell pellet was re-suspended in 20 ml media, and sonicated. The cell debris were then centrifuged at 1500 × g for 10 min, and the supernatants were aliquoted (1 ml each tube) and saved at −80 • C. The BTV stocks from the cells were titrated in BSR cells using the TCID 50 assay. The final titer was at 1 × 10 8 TCID 50 /ml.

Virus titration using the TCID 50 assay
Virus titers were determined in BSR cell monolayers grown in 96-well plates. BSR cells were seeded at 15 000/well in a 96-well plate, and incubated for 2 h at 37 • C, 5% CO 2 and 80-95% humidity. A volume of 100 l of 10-fold serially diluted virus was inoculated into each well in quadruplicate format. Plates were further incubated at 37 • C, 5% CO 2 and 80-95% humidity for 72 h, after which, CPE was observed microscopically at 40× magnification. Virus titers were expressed as TCID 50 unit per ml of solution.

Control and compound preparation
Carrier control consisted of dimethyl sulfoxide (DMSO) diluted in complete assay media to 0.6% and 5 l of which was dispensed to both cells and virus control wells of 384-well black clear-bottom tissue culture treated plates (Corning). Test compounds were diluted in media to 60 M with a DMSO concentration of 0.6%. A total volume of 5 l was dispensed to each well in the assay plate with a final assay volume of 30 l resulting in final concentrations of 0.1% DMSO and 10 M test compounds. Compound dilution and addition were performed with a Beckman Coulter FX liquid handler. After brief centrifugation, assay plates were transferred to the cell plating facility. For 10-concentration dose response assay, the compounds were added to assay media by a two-fold serial dilution, ranging from 20 M to 0.04 M.

Cell plating
For the validation assay and large scale HTS assay, cells were plated in 384-well plates at a cell density of 5000 cells/well (or oth-erwise noted) in 15 l assay media using a WellMate bulk dispenser (Matrix, Inc.). Plates were then incubated for 2-4 h at 37 • C, 5.0% CO 2 and 80-95% humidity incubators (Thermo-Scientific).

Virus addition
Cells were infected with BTV at MOI of 0.01, or otherwise noted in the text. The BTV stock (1 × 10 8 TCID 50 /ml) was first diluted in assay media containing 5000 TCID 50 /ml of the virus. The diluted virus (10 l) was added to the compound wells and the virus only control wells. Media only (mock infection) was added to the cell control wells. All additions were carried out using a Matrix Well-Mate housed in a class II Biosafety Cabinet within the Bio-Safety Level 2 laboratory. The plates were incubated for another 72 h, at 37 • C, 5% CO 2 and 80-95% humidity.

CPE assay and endpoint read
The CellTiter-Glo buffer and the lyophilized CellTiter-Glo substrate (Promega Inc., Madison, WI) were thawed and equilibrated to room temperature prior to use. The homogeneous CellTiter-Glo reagent solution was reconstituted after mixing the lyophilized enzyme/substrate and the buffer reagent according to the manufacturer's instructions. Meanwhile, the assay plates were also equilibrated to room temperature for 15 min. An equal volume (30 l) of CellTiter-Glo reagent was added to each well. Plates were further incubated for 15 min at room temperature to stabilize luminescent signals. Luminescence was measured using a Perkin Elmer Envision multi-label reader with an integration time of 0.1 s.

Compound clustering
In order to identify common structural elements within the identified actives hits, the compound sets were clustered using cheminformatics tools available in LeadScope (LeadScope, Inc., Columbus, OH). A hierarchical cluster analysis was performed using a Tanimoto index threshold of 0.7, applying the complete linkage method. For each cluster, a representative substructure was identified as a common motif shared by all members of a cluster. Enrichment ratios were computed for the cluster substructures in order to predict whether the substructure may be considered privileged or not.

Data analysis
During the HTS campaign, 32 control wells containing cells only and 32 wells containing cells and virus were included on each assay plate. These controls were used to calculate Z -value for each plate and to normalize the data on a per plate basis. All raw data were imported to Activity Base Data Management software (IDBS, Alameda, CA) for the determination of Z -value, signal-tobackground (S/B, fold decrease) ratio and percent inhibition for assayed compounds. Results were expressed as percent inhibition of CPE where 100% inhibition of CPE was equal to the mean of the cell only control. Compounds showing greater than average CPE inhibition plus three times standard deviation of % inhibition of viral-induced CPE of all compounds were considered "hits" and tested in 10-concentration dose response assay. Statistical calculations of Z -values were made as follows: Here Mean S is the mean luminescent signals from cell control, StdDev S is the standard deviation of the luminescent signals from cell control, Mean B is the mean luminescent signals from BTV infection control, and StdDev B is the standard deviation of the luminescent signals from virus infection control. S/B = Mean S /Mean B (Ghosh et al., 2005;Zhang et al., 1999). Each assay validation step was performed in triplicate with the exception of the validation screening of 9532 compounds which was performed in duplicate. The screening of NIH Molecular Libraries Small Molecule Repository (MLSMR) with 194 950 compounds and dose response assay were carried out once.

BTV infection induces apoptosis and CPE in BSR cells
BTV infection induces apoptosis and/or CPE in a number of mammalian cells, including Vero, HeLa, BHK/BSR (Li et al., 2007;Mortola et al., 2004;Nagaleekar et al., 2007;Schwartz-Cornil et al., 2008). Characteristics of apoptosis and CPE, including cell shrinkage, membrane blebbing, and DNA fragmentation, have been shown and the apoptotic pathway has been elucidated (Li et al., 2007;Mortola et al., 2004;Nagaleekar et al., 2007). The quantitative analysis of the CPE induced by BTV infection was shown here using 96-well plate. BSR cells were seeded with a density of 15 000 cells/well, and infected with BTV at different MOIs, 0.5, 1.0 and 10, respectively. At different hours post-infection (h.p.i.), BTV-induced CPE was determined using the CellTiter-Glo Luminescent Cell Viability kit, a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The single step "add-mix-measure" format resulting in cell lysis and the generation of luminescent signals is also ideal for future large scale HTS of various compound libraries. While there was no significant decrease of cell viability in BTV infected cell at 24 h.p.i., a two-fold decrease of cell viability in BTV infected cells was observed in all three MOIs at 48 h.p.i. The decrease of cell viability was much more apparent at 72 h.p.i., with 9.7-, 12.0-and 11.7-fold decreases, i.e. S/B ratio, for MOI of 0.5, 1.0 and 10, respectively ( Fig. 1). This result showed that BTV infection induced CPE can be quantitated, especially at 72 h.p.i. with remarkable high S/B ratios. Meanwhile, this single step CPE-based assay will be suitable for future HTS assays, although further optimizations were necessary to improve this CPEbased assay into a primary HTS assay.

DMSO sensitivity analysis
For large scale HTS, all compounds to be tested will be dissolved in DMSO solution. It has been shown that high DMSO concentra-  tions (>1%) have dramatics effect on membrane permeability, cell viability and virus infection in MDCK cells (Irvine et al., 1999;Noah et al., 2007). Thus, it is necessary to evaluate the effect of DMSO on cell growth and cell viability of BSR cells. After being seeded in 96well plates at 15 000 cells/well, BSR cells were treated with DMSO at different concentrations, ranging from 0.02% up to 2% (Fig. 2). After 72 h incubation, cell viability was determined using the CellTiter-Glo reagent. The effect of DMSO on cell viability was compared between the cell viability in DMSO treated cells relative to cell viability in cells that were not treated with DMSO. When BSR cells were treated with DMSO at 1% or lower concentration, there were no decreases on the cell viability. In contrast, there was a slight increase of cell viability, ranging from 5% to 12%, when cells were treated with DMSO concentration below 1%. However, when cells were treated with 2% DMSO, there was 28.9% decrease of cell viability, a considerable decrease comparing with cells without DMSO treatment (Fig. 2). This result supported that the designated 0.6% DMSO concentration for our HTS campaign will be suitable for the screening. Meanwhile, there should be no more than 1% DMSO added to the cell culture for the entire screening when using this assay.

Optimization of cell density
The seeding cell density is important to avoid possible cell overgrowth, to obtain a maximum and consistent luminescent signal strength and good S/B ratio (minimum > 5) in each well. To optimize the seeding cell density, BSR cells were seeded in 384-well plates at different density-2500, 5000, 7500 and 10 000 cells/well, respectively. Cell viability were evaluated and analyzed at different time post-seeding using CellTiter-Glo reagent (Fig. 3a). At cell density of 7500 and 10 000 cell/wells, cell growth reached a plateau at 48 h post-seeding and luminescent signals decreased at 72 h postseeding. At cell density of 2500 and 5000 cells/well, cell growth did not reach a plateau at 72 h post-seeding and luminescent signals were increased from 48 to 72 h, and the 5000 cells/well density showed the maximal luminescent signals, i.e. cell viability, and was chosen as the optimal cell density for all subsequent experiments.

Optimization of FBS concentration
Cell growth and/or viability are also affected by the FBS concentration in the cell culture media. Traditional cell culture medium contains 5% or higher FBS which keeps the cells grow rapidly, however, the risk of overgrowth, which may lead to the decrease of cell viability, is high. Also, some compounds and viruses are known to bind to FBS and therefore would show up as false negatives in a screen. Two different FBS concentrations, 5% and 1% in DMEM media, were evaluated at different seeding cell densities. At both concentrations, seeding density at 7500 and 10 000 cells/well showed similar pattern and the decreases of cell viability were observed at 72 h post-seeding. At 5000 cells/well seeding density, although cells were still multiplying at 72 h for both 1% and 5% FBS, the cell growth at 1% FBS concentration was more active when compared with cell growth at 5% FBS. Thus, we chose 1% FBS concentration and 5000 cells/well cell density as the optimal cell culture conditions for all subsequent experiments.

Optimization of BTV infection
The optimization of BTV infection was carried out to achieve the relative highest luminescent signals at the lowest MOI. In the previous experiment, at MOI of 0.5, the luminescent signals were decreased to a level similar to that obtained with an MOI of 1.0 and 10. Further observation showed that at even lower MOIs, CPE was also obvious (results not shown). Thus, we chose five low MOIs, 0.0025, 0.005, 0.01, 0.02 and 0.04 to infect BSR cells seeded at optimized cell density (5000 cells/well). After cells were incubated for 48 and 72 h, cell viabilities were evaluated using the CellTiter-Glo  (Fig. 4). The luminescent signals at MOI of 0.01 was comparable to that at MOI of 0.02 and 0.04 with good S/B ratio (11.4), which fit our standard to achieve relative high signal difference with lowest MOI. Thus, MOI of 0.01 was selected for all subsequent experiments including the primary HTS assay.

Z -Value determination
The Z -value analysis is designed to evaluate if an assay is robust enough for screening on an HTS platform. The Z -value evaluates the assay's signal dynamic range, the data variation associated with the sample measurement, and the data variation associated with the reference control measurement. To determine the Z -value, the CPE-based assay in 384-well plates was run at three different days and three different assays, at the optimized conditions including cell density at 5000 cells/well, 1% FBS media, BTV infection at MOI of 0.01 and final luminescent signals read at 72 h.p.i. The 384-well plate was divided into four quadrants, with the BTV infected cells as positive controls (S) in Quadrants I and IV, and the mock infected cells as negative controls (B) in Quadrants II and III on the plate. The Z -value analysis was performed repeatedly to demonstrate reproducibility. We obtained an average Z -value of 0.70 ± 0.01 under the optimized assay condition using the formula in Section 2.9 (Zhang et al., 1999). This demonstrated that the assay was robust and suitable as a primary HTS assay. Furthermore, according to our experience in HTS setting, the Z -value would be higher once this assay was fully automated.

HTS assay validation
After the CPE-based assay was optimized and Z -value determined in the laboratory, the assay was transferred to and adapted by the HTS Center at Southern Research Institute. The assay was first repeated in 384-well plates using automated liquid handling equipment which was also used for the screening of large scale compound library in Section 3.5. Integration of the assay onto a HTS platform was evaluated at each individual step of the procedure, The antiviral assay against BTV was validated twice using the 10K compound library. The primary HTS against BTV was carried out once via the screening of a 194 950 compounds from NIH MLSMR. Results from the validations and primary screening were summarized and compared in the table. Number of hits and hit rates were expressed in two data sets. One set was expressed as the mean inhibition of CPE plus three times the standard deviation, which varied from assay to assay (>21.7% for the 10K screenings I and II, and 30% for the 200K screening). Another set was expressed as the 50% inhibition of the virus induced CPE. N: number of compound screened; Z : average Z -value from each plate screened; CV: coefficient of variation; S/B ratio: signal-to-background ratio.
from loading the plates with cells to the final data analysis. The examination of intra-and inter-run variations, including well to well, plate to plate, and day to day variability, were carefully evaluated by running three Z plates on three separate days for a total of nine plates, determined that the assay was sufficiently robust to screen a large compound library. Our results showed that the average Z -value from these repeats were 0.75 ± 0.05, average coefficient of variations (CV) were 5.68 ± 0.62% for the cell control and S/B ratio of 7.10 ± 1.72. This results demonstrated that the quality control parameters for the assay met the requirements, including CV < 10%, Z -values > 0.5, S/B > 5, and inter-run reproducibility.
The final validation step was the screening of a small compound set of 9532 compounds on two different time and statistically comparing the results for reproducibility. The 9532 compounds were screened at 10 M concentration in two duplicate screens at different time. Quality control was assessed at several different points within the assay process. The values for the cell control and the BTV infection control wells were reviewed to determine the viability of each plate of compound data. Values from the duplicate screening were also compared to determine consistency. The average Z -value from each plate, CV, S/B, and hit rates for compounds that inhibited CPE above 21.7% and 50%, were showed in Table 1. The 21.7% inhibition of CPE equaled to average CPE inhibition plus three times standard deviation of % inhibition of all compounds from the duplicated screenings. The reproducibility and signal window robustness of the duplicate screenings were demonstrated with similar Z values (0.70 vs 0.71), CVs from both cell controls (5.94 vs 4.52) and virus controls (19.87 vs 13.70), and S/B ratios that averaged from each plate (7.56 vs 4.49) (Table 1). Overall, we observed an average hit rate of 0.2-1.4% and 0.05-0.1% for compounds that inhibited 21.7% and 50% of BTV-induced CPE, respectively. The Pearson's product moment correlations were high when comparing the duplicate screens, with a correlation coefficient of 0.80 with a P value of 0.23 indicating that there was no difference between the duplicate screenings.

Primary screening MLSMR compound library
After the reproducibility and robustness of the CPE-based assay were demonstrated and validated, 194 950 compounds from MLSMR were screened. Compounds from the MLSMR collection are generically grouped into the following four categories (a) specialty sets (SS), such as known drugs and toxins, (b) targeted libraries (TL) such as kinase or GPCR inhibitors, (c) natural products (NP), and (d) diversity compounds (DC), which represents general chemical diversity. These compounds were selected based on their physicochemical properties with cutoffs applied, including MW < 1000, cLogP < 5, HBA < 20 and HBD < 10. All compounds were screened once at 10 M using the validated CPE-based assay in six sepa-rate runs under the exact optimized experimental conditions. The hits identified were based on a 30.04% inhibition of virus induced CPE which equaled to average inhibition plus three times standard deviation of % inhibition of all compounds from the entire MLSMR screen. The assay robustness, including the average Z -value and S/B ratio were graphically illustrated in Fig. 5. The average Z -value for the entire screening was 0.70 ± 0.06 (Fig. 5a), which matched to the Z -values obtained from the duplicated validation screenings. The average CVs were 4.63 ± 1.31 for cells only and 10.50 ± 3.54 for virus infection only (results not shown). The S/B ratio was 3.88 ± 0.56 (Fig. 5b) for the entire screening including the cell control, virus infection control and the virus infection plus drug treatments. The percentage of inhibition of virus induced CPE from each compound in the screening was plotted in Fig. 6. Out of the 194 950 compounds screened, we identified 693 hits with over 30% CPE inhibition, with Scheme 1. Scaffolds representative of clusters identified from clustering the compounds confirmed in dose response assay. # actives are the total number of active compounds containing the given substructure. # SD set is the total number of compounds containing the given substructure within the entire single dose screened library. Enrich.: meant the computed enrichment ratio. a hit rate of 0.03%. There were 147 compounds identified with over 50% inhibition of virus induced CPE (Table 1 and Fig. 6).
Six hundred and fifty-eight of these compounds were available for further testing using a 10-concentration dose response assay. Out of the 658 dose response tested hits, an 'active set' of 185 compounds possessing IC 50 values less than 100 M was selected for the purpose of structural analysis. In order to identify common structural elements within the 185 actives, this set was clustered. For each cluster, a representative substructure was identified as a common motif shared by all members of the cluster. The enrichment ratio characterizing a particular substructure was defined here as the ratio between the percentage of compounds that contain this substructure within the active set (185 compounds) and the same percentage within the entire library screened (194 942 compounds). Privileged scaffolds contributing to the activity of hits were typically present in higher percentages among active compounds compared to their distribution within the entire screened library. We selected scaffolds representative of hit clusters that showed enrichment ratios greater than 10 and these were listed in decreasing order of cluster sizes in Scheme 1. Privileged scaffolds were likely to be present among non-clustered actives or in Fig. 6. The percentage of inhibition of virus induced CPE from each compound in the HTS against MLSMR compound library. Each dot represents one compound from the library, and total of six batches were plotted here. The higher line at a 30.04% inhibition of virus induced CPE was calculated as the average inhibition (0.35%, the lower line) plus three times standard deviation of % inhibition of all compounds from the entire MLSMR screen. small clusters (with members less than 3) as well. Therefore selection of scaffolds further pursued will include clustered hits as well as non-clustered, singleton active structures.

Discussions
CPE is a general term used to describe the adverse effect on the cells post-viral infection. CPE involves at least two distinct processes: necrosis and apoptosis. Various assays are available to distinguish these two mechanisms through measuring a variety of different markers that indicate the number of dead cells (cytotoxicity), the number of live cells (viability), and the mechanism of cell death (apoptosis). Most conventional cell-based antiviral drug screening assays were developed based on the cell viability assay (ATPase, kinase and cytotoxicity). The introduction of CPE-based assay using ATP/luminescence readout, such as CellTiter-Glo reagent, has been recently reported, which made the HTS against large compound library possible in several different antiviral assays, including the antiviral HTS assays against influenza virus (Noah et al., 2007;Severson et al., 2008), SARS-CoV , Yellow fever virus (Gong et al., 2008), Crimean-Congo hemorrhagic fever virus (Paragas et al., 2004). Our CPE-based antiviral assay against BTV also showed its feasibility with the "mix and measure" one step protocol, and flexibility with stable luminescent signals. Meanwhile, toxic compounds that reduced the cell viability were also excluded in this CPE-based assay. This CPE-based assay was also easy to be adapted and validated in HTS Center, making it possible to screen large compound libraries, such as MLSMR collection. This assay may also be used for assays based on CPE induced by other reoviruses and arboviruses.
Our assay also demonstrated its robustness with a high degree of reproducibility and low deviation including a reproducible Zvalue over 0.7, high sensitivity and low background with S/B ratios ranging from 3.88 to 7.56. Typically, a S/B ratio greater than 5 is an adequate detection window, and the Z -value >0.5 are considered robust enough for a HTS assay (Zhang et al., 1999). The duplicated validation screening of the CPE-based assay using the 9532 testing compound library, and the primary HTS assay against MLSMR further demonstrated the assay was sufficiently robust and reproducible for a HTS platform with very similar statistical assay performance values, including Z -value between 0.70 and 0.71, CV for the cell control between 4.52% and 5.94%, and S/B between 4.49 and 7.56 (Table 1). CV from the virus infected control was higher than 10%, which is natural for the virus infected cells due to the complexity nature when virus infection process. The S/B ratio form one of the validation screenings was 4.49, lower than 5.0 as requirement, however, the consistent Z -value from the validation screenings indicated that this assay was robust for using in HTS. Indeed, similar Z -values, CV and S/B ratio were also obtained for these assay parameters in our primary HTS, which further demonstrated that the assay is reproducible and reliable.
All three screenings showed similar hit rates, between 0.05% and 0.10% for the hits that inhibited CPE by >50% (Table 1). Similar hit rates were observed in a number of HTS assays against various viruses, including 0.08% and 0.022% hit rates in two assays for identification of influenza inhibitors, respectively (Noah et al., 2007;Severson et al., 2008), and 0.01% for SARS-CoV HTS . Although it is possible to increase the hit rate by manipulating the assay conditions, including dosing at a higher compound concentration (i.e. >10 M) and/or reducing the assay length from 72 to 48 h, we were very confident that these hits identified through the primary HTS were accurate, evidenced by the corresponding hit correlation in the duplicated 10K screening and confirmed by the dose response assay.
Hit compounds with IC 50 less than 100 M were clustered to identify common substructures. Among these, Scheme 1 lists substructures with highest enrichment among active hits comparing to their distribution in the entire library screened. The clusters identified include mononuclear heterocylic compounds such as the thiophene and thiazole derivatives 1 and 3, bicyclic compounds such as the pyrimidines 2, 4 and 5 fused to a 1,3,4-thiadiazole, 1,2-4-thiadiazole and pyrazole ring systems. Scaffolds 2 and 4 have very similar core scaffolds. The thienooxazinones 6 can be considered as cyclic derivatives of the ortho sustituted thiophene derivatives represented by 1. Note, scaffold 3 may be considered a small group of closely related analog series where nearly all members in the series were active.
The development of a CPE-based assay against BTV infection and the HTS of the MLSMR library provided a unique model system and identified a number of novel antiviral compounds that protect host cells from BTV infection. BTV is the prototype virus in the genus Orbivirus within the family Reoviridae. BTV is considered to be one of the most important diseases of domestic livestock, and has been a subject of intense molecular study for the last three decades and is now one of the best understood viruses at the molecular and structural levels Roy, 2008). As a member of reoviruses and arboviruses, BTV must be capable and should be sufficiently flexible to allow its replication in evolutionarily distant hosts, which also applied to other reoviruses and arboviruses including members in the families of Togaviridae, Flaviviridae, Bunyaviridae, Rhabdoviridae, and Reoviridae. Since antiviral compounds identified here could protect host cells from BTV-induced CPE responses, these compounds might also protect host cells from other reoviruses and arboviruses infections. Meanwhile, any compound identified using this assay could be used as a chemical probe to further study the virus-host interactions. Thus, our developed HTS assay will be of direct benefit to the drug development and drug design to counter reoviruses and arboviruses diseases, thereby have a positive impact on the widespread burden of these diseases.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Differential requirement for MEK Partner 1 in DU145 prostate cancer cell migration

ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1) is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells. We find that MP1 is required for motility on fibronectin, but not for motility stimulated by serum or EGF. Surprisingly, MP1 appears not to function through its known binding partners MEK1 or PAK1, suggesting the existence of a novel pathway by which MP1 can regulate motility on fibronectin. MP1 may function by regulating the stability or expression of paxillin, a key regulator of motility.

Introduction
Prostate cancer is the most commonly diagnosed cancer in men. While in situ disease usually responds well to treatment, progression to an androgen-hypersensitive, metastatic state is often fatal [1]. Aberrant signaling through the ERK pathway correlates with progression in some models [2], although oncogenic mutations in Ras or Raf occur infrequently in prostate cancer [3,4]. Rather, prostate cancer commonly displays misregulation of cellmatrix adhesion signaling, including alterations in integrin expression and upregulation of Focal Adhesion Kinase (FAK) and p21-Activated Kinase (PAK) [5,6]. We have previously shown that PAK1 can activate MEK1 independently of Raf in model systems [7] suggesting that this alternative pathway to ERK activation may function in tumor cells lacking oncogenic mutations in Ras and Raf. Indeed, active PAK1 has been shown to stimulate ERKdependent proliferation of breast cancer cells [8], and is constitutively active in DU145 prostate cancer cells [9].
MEK Partner 1 (MP1) is a putative scaffolding protein that binds MEK1 and ERK [10]. MP1 also binds to the late endosomal protein p14 [11], and the p14-MP1-MEK1 complex regulates trafficking and degradation of the EGFR [12]. Thus MP1/p14 provides a means of colocalizing the endocytosed EGFR and its downstream effectors during proliferation stimulated by EGF. MP1 and p14 also play important roles in cell morphogenesis and migration. MP1 and/or p14 are required for efficient activation of MEK and ERK, and control focal adhesion and actin dynamics during adhesion to fibronectin [13]. Since ERK is known to regulate focal adhesion disassembly [14,15], these observations implicate MP1-scaffolded ERK in focal adhesion dynamics. However, MP1 also binds active PAK1 [13], itself a regulator of adhesion disassembly [13,16,17], consistent with alternative mechanisms for its involvement in the regulation of cytoskeletal and adhesion dynamics [13]. Elevated PAK activity is associated with altered growth and migratory properties in a number of tumor types, including prostate cancer [18]. We therefore investigated whether MP1 influences ERK and/or PAK signaling during migration of DU145 prostate cancer cells.

Materials and methods
Reagents EGF (Invitrogen) and UO126 were from Calbiochem. Gridded coverslips were from Bellco; glass-bottomed 35 mm microwell dishes were from MatTek Cultureware. Both were coated with fibronectin as described [19].

Adhesion/EGF stimulation assay
Cells were plated on fibronectin essentially as described [13]. For EGF stimulation, cells were washed in serum-free (SF) medium then fed SF medium for 2 hours prior to stimulation.

Migration assays
Cells were trypsinized and collected in SF medium containing 1 mg/mL soybean trypsin inhibitor (Sigma), pelleted, suspended in SF medium and incubated at 37°C for two hours. 2 × 10 6 cells were allowed to form confluent monolayers on fibronectin-coated grid-etched coverslips for 5-7 hours at 37°C. Cells were treated with 1 μg/mL Mitomycin C (Sigma) and either 25 μM UO126 or DMSO vehicle for an additional 3 hours. The medium was aspirated and the coverslip was scratched 4 times with a 200 μL tip to form a # wound pattern. Loose cells were removed with SF medium; cells were fed fresh SF medium containing 1 μg/mL of Mitomycin C and either 25 μM UO126 or the equivalent volume of DMSO, with or without 1% FBS or 10 ng/mL EGF as indicated. Wounds were photographed in 6 different locations at zero-hour time point, and then incubated at 37°C for 5 hours (transfected cells) or 10 hours (untransfected cells) at which time the same locations were photographed. All photographs were taken with an Olympus IX81 microscope equipped with Slidebook software (Intelligent Imaging Innovations) and the area covered by migrating cells was calculated using ImageJ software (NIH). Parallel cultures were processed for immunoblotting.

Immunofluorescence
Immunofluorescence was carried out essentially as described [13] using a Leica DMRA2 microscope equipped with Slidebook software. The number and size of vinculin-positive focal adhesions at the wound edge was calculated using Slidebook software. Kymography 2 × 10 6 cells were plated on 35 mm FN-coated glass-bottomed Petri dishes in SF medium with 1 μg/mL Mitomycin C and allowed to adhere for 5-7 hours. Confluent monolayers were wounded and imaged starting 10-20 min post wounding and again after 2 hours of migration. All cells were imaged for 5 minutes at 1 frame per second on an Olympus IX81 microscope equipped with Slidebook software. Kymographs were generated using ImageJ software.

Statistical Analysis
Wound-healing assays were repeated in triplicate and statistical analysis was performed on the pooled results. A general linear model was constructed and linear contrast was used to perform pairwise comparisons of the means for the combined data set. Tukey's Honestly Significant test was used for post-hoc pairwise comparisons of migration under experimental conditions to that of migration under SF conditions and to each other. ANOVA on pooled data indicated significant differences within the data set.

ERK activity is required for growth factor-, but not fibronectin-stimulated, DU145 migration
We developed a quantitative wound-healing assay that makes use of gridded coverslips to allow for accurate measurement of migration into a wounded monolayer over time ( Fig. 1B; Materials and Methods). To distinguish between wound closure due to cell movement and proliferation, mitosis in these assays was inhibited by the inclusion of 1 μg/mL mitomycin C; this concentration was determined as the minimal dose needed to prevent DU145 proliferation without inducing cell death under serum-free conditions (data not shown). Additionally, to distinguish between cell spreading and motility, cells were tracked in real time over the course of the assay (data not shown), and movies were analyzed for directionality and distance migrated. From these assays we were able to determine that DU145 are migrating into the wound in the monolayer over the timecourse of the assay. DU145 cells migrated 65 ± 3.5 μm 2 on fibronectin in serum-free conditions, and pre-treatment with 25 μM UO126 for 3 hr had no effect (Fig. 1A). Migration on fibronectin was increased to 115 ± 4.9 and 100 ± 5.3 μm 2 in the presence of 1% serum or 10 ng/mL EGF, respectively, and these increases were significantly inhibited by UO126 (Fig. 1A). Thus, MEK signaling is required for growth-factorinduced, but not for fibronectin-stimulated, migration of DU145 cells.

MP1/P14 is required for migration of DU145 cells on fibronectin
Since fibronectin-stimulated motility of DU145 cells is insensitive to MEK inhibitor (Fig. 1A), we predicted that ERK activation is not required for DU145 migration on FN, but is required for migration on FN induced by serum . Migration in response to fibronectin alone was not affected by UO126. Serum and EGF stimulated significant (p < 0.0001) increases in migration, that were significantly inhibited (p < 0.0001 for serum, p = 0.0004 for EGF) by UO126 treatment. B) Sample images from wound healing assay; for each condition, six grid squares were imaged at 0 and 10 h post-wounding, and wound area (outlined in white) was measured using ImageJ software (NIH). MP1 depletion would be similarly without effect on fibronectin-stimulated DU145 migration. However, since MEK activity is required for growth-factor stimulated DU145 motility (Fig. 1A), and MP1 is required for EGFand serum-stimulated MEK/ERK activation in fibroblasts [13,20], we predicted that MP1 depletion would inhibit growth-factor stimulated motility of DU145 cells. Surprisingly, we found that fibronectin-stimulated motility was inhibited by ~50% following transfection with MP1 siRNA (Fig. 2A, white bars). In contrast, the component of motility stimulated by 1% serum (above and beyond that stimulated by fibronectin alone) was unaffected by transfection of MP1 siRNA, but was substantially inhibited by UO126 ( Fig. 2A) as in naïve cultures (Fig. 1A). Consistent with these observations, depletion of MP1/p14 did not substantially inhibit levels of active ERK or active MEK in cells on fibronectin, whether or not they were exposed to growth factor (Fig. 2B), whereas UO126 essentially eliminated ERK activity in both control and MP1 siRNA-transfected cells (Fig. 2B). Note that knock-down of MP1 is difficult to measure accurately due to the presence of closely migrating proteins that cross react with our antibody. However, siRNAs targeting MP1 have been shown to cause a simultaneous reduction in p14 expression, and vice versa [13,21], most probably because the MP1-p14 heterodimer is more stable than the individual subunits. Since our p14 antiserum blots more cleanly, we used p14 levels as a surrogate measure for MP1 knockdown. p14 levels were reduced on average by 82% (Fig 2B; data not shown). Together, these data indicate that MP1/p14 but not MEK signaling is required for fibronectin-stimulated motility of DU145 cells, whereas MEK signaling but not MP1/p14 is required for motility stimulated by serum growth factors. Thus MP1/p14 and MEK play separable roles in DU145 migration.

MP1 depletion decreases membrane activity and peripheral focal adhesions in migrating cells
We next asked whether membrane protrusion or focal adhesions were altered in DU145 cells lacking MP1/p14. Kymograph analysis allows visualization of the membrane boundary over time; the y-value is determined by the position of the membrane edge, and the x-axis represents time [21,22]. Cells in MP1 siRNA-transfected cultures displayed much less protrusive and ruffling activity at the wound edge relative to control siRNA-transfected cells (Fig. 3A).
To examine focal adhesions in these assays, a monolayer was wounded and fixed after cells had migrated for 5 hrs. Control-and MP1 siRNA-transfected cells display adhesions of similar total number and size distribution (Fig.  3B, C). However, MP1 siRNA-transfected cells have ~50% fewer adhesions at the wound edge ( Fig. 3B; defined as adhesions occurring between the cell periphery and the cortical actin ring, not shown; vinculin spots occurring at sites of cell-cell contact were not counted as focal adhesions). This suggests that early nucleation events required for adhesion formation at the wound edge occur at a lower frequency in cells depleted of MP1/p14, or that turnover of this population of adhesions is more pronounced in cells lacking MP1/p14.

Depletion of MP1/p14 modestly inhibits PAK activity
Membrane protrusion and ruffling are governed in part by localized Rac/PI3-Kinase signaling, while membrane extensions are stabilized by focal complexes that also require PAK activity [16,17]. Focal adhesion maturation and disassembly are complex processes governed by GTPases (including Rac and Rho), kinases (including PAK, FAK, and ERK) and molecular organizing components including paxillin. Since fibronectin-stimulated DU145 motility was insensitive to MEK inhibitor but sensitive to knock-down of MP1/p14, we reasoned that MP1 functions through a MEK activity-independent pathway to regulate membrane and adhesion dynamics. One candidate molecule is PAK1, to which MP1 binds [13]. Group I PAK kinases phosphorylate a number of cellular targets, including MEK1 [7,19,23], paxillin [24], and themselves [25,26]. Depletion of MP1/p14 slightly delays autophosphorylation of PAK1-3 on S141 following adhesion to fibronectin, with phosphorylation recovering around 1 hr and persisting throughout the 5 hr time course. Maximal phosphorylation of MEK1 on S298 is similarly delayed in MP1 siRNA-transfected cells (Fig. 4A). While we cannot exclude that these subtle differences in kinetics of phosphorylation in the population as a whole represent significant quantitative differences at the wound edge, our data suggest that depletion of MP1/p14 has minor effects on the overall activity of the PAKs phosphorylating these substrates.

Paxillin is largely absent in DU145 cells depleted of MP1/ p14
To extend these observations, we asked whether PAK phosphorylation of the cytoskeletal linker/adapter paxillin was regulated by MP1/p14. Paxillin has been shown to play essential roles in focal adhesion dynamics, membrane protrusion and motility [14,[27][28][29]. PAK phosphorylates paxillin on S273, and phosphorylation of this site is required for stabilizing membrane protrusion [24]. Surprisingly, the abundance of paxillin was substantially reduced in cells depleted of MP1/p14 (~60% reduction, Fig. 4B), suggesting that paxillin is poorly expressed or unstable in cells lacking MP1/p14, or that it resides in a compartment not extracted by our lysis buffer. To exclude the latter possibility, we prepared extracts in SDS-PAGE sample buffer which is expected to solubilize all cellular proteins. The abundance of paxillin was similarly reduced (by ~60%) in SDS-PAGE buffer extracts of DU145 cells   transfected with MP1 siRNA, while expression of other focal adhesion markers vinculin, talin and zyxin was unaffected by knock-down of MP1/p14 (Fig. 4B). Reduction of paxillin levels is unlikely to result from off-target effects of the MP1 siRNA since similar results were obtained with an independent p14 siRNA (Fig. 4C). These data indicate that the MP1 and/or p14 are required to maintain normal paxillin protein levels.

MP1/p14 regulated signaling events
MP1/p14 is essential for ERK activation during adhesion of fibroblasts to fibronectin, and in this context, depletion of MP1/p14 causes an apparent stabilization of focal adhesions [13]. We therefore predicted that depletion of MP1/p14 would cause inhibition of fibronectin-stimulated ERK activation and stabilization of focal adhesions in DU145 cells, and thus inhibit cell motility. To our surprise, we found that migration of DU145 cells on fibronectin does not require acute MEK activity but does require MP1/p14. Conversely, migration stimulated by growth factors requires acute MEK activity, but does not require MP1/p14. Furthermore, MP1/p14 depletion does not significantly inhibit ERK phosphorylation in response to fibronectin or growth factor stimulation. These data indicate that there are separable MEK activity-and MP1/ p14-dependent pathways leading to DU145 motility in response to serum growth factors and fibronectin respectively.
Because Group I PAKs have multiple roles in cell motility [30] and PAK1 binds MP1 [13], we reasoned that MP1 might function through PAK to regulate fibronectin-stimulated DU145 migration. However, depletion of MP1 caused only modest differences in PAK autophosphorylation or PAK phosphorylation of MEK1; phosphorylation of the PAK-dependent substrate cofilin was also unaffected by depletion of MP1 (data not shown). These data suggest that PAK is not a major effector for MP1 in DU145 cell motility.

MP1/p14 is required for maintenance of paxillin levels
Surprisingly, we found that paxillin was largely absent from DU145 cells depleted of MP1/P14 while expression of other focal adhesion proteins was unaffected. Future experiments will determine whether this loss of paxillin results from decreased mRNA or protein synthesis, or increased turnover of paxillin protein.

Depletion of MP1/p14 influences membrane dynamics and focal adhesion abundance
We have found that depletion of MP1/p14 results in a loss of paxillin, decreases membrane protrusion, and reduces the number of focal adhesions at the wound edge. Paxillin serves as a signaling nexus at focal adhesions and in the leading edge of migrating cells. In particular, the GIT1-PIX-PAK complex is stabilized by PAK phosphorylation of paxillin, leading to increased localized Rac activation, membrane protrusion, and migration [24]. This complex localizes to a population of dynamic focal complexes at the leading edge [14,24], suggesting that it may be important for stabilizing membrane protrusion. Alternatively, paxillin may liberate p190A RhoGAP from p120 RasGAP at the leading edge of motile cells [31]. Locally released p190A RhoGAP is free to stimulate hydrolysis of GTP on Rho allowing membrane protrusion. Interestingly, while the loss of paxillin correlates with a failure of DU145 to migrate on fibronectin, it does not preclude serum-stimulated motility.
Our data are the first to indicate that MP1/P14 has important functional roles outside the ERK pathway. These observations suggest that MP1/p14-and paxillin-dependent and -independent pathways for motility may be operational in DU145 cells in a context-specific manner.


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Do apicomplexan parasite-encoded proteins act as both ligands and receptors during host cell invasion?

Apicomplexan parasites are responsible for a wide range of diseases in animals, including humans, in whom Plasmodium species cause the devastating disease malaria. Several recent discoveries now indicate that these intracellular parasites may use a conserved mechanism to infect their host cells by using parasite-encoded proteins as both parasite ligands and receptors anchored to the host cells.

Introduction and context
Apicomplexa are a phylum of unicellular eukaryotic organisms that often parasitise multiple animal hosts during their life cycles. This group of obligate intracellular parasites are so called because the invasive zoite stages possess a characteristic cell apex containing several secretory organelles and associated cytoskeletal machinery specialised for penetrating host cells and tissues. The diseases associated with apicomplexan infections are of great health, social, and economic importance. Leading them is malaria caused by Plasmodium species, the most deadly form of which is P. falciparum [1]. Other apicomplexans that infect humans include Toxoplasma gondii and Cryptosporidium, but they usually cause less severe diseaseexcept in the developing foetus and immunocompromised individuals. Understanding how parasites enter their hosts' cells is of great interest because this offers an attractive target for the development of novel therapeutics. Some apicomplexans such as T. gondii are able to invade a wide variety of host cells despite the enormous variety of host surface proteins between tissues and species. Other apicomplexans such as Plasmodium species are highly selective for their target species and can even bias their infectivity toward cells of a particular level of maturation. In addition, some plasmodia, in particular the red blood cell invasive stage of P. falciparum, are able to switch invasion pathways to use different parasite ligands [members of the erythrocyte-binding antigen (EBA) family and reticulocyte-binding protein homologue (Rh) family] to mediate interaction with different host cell receptors [2][3][4][5][6]. For example, an individual parasite clone can adapt from using host-cell glycophorin A as a key receptor to relying on a completely different host cell receptor for entry.

Major recent advances
How are these parasites able to have so much adaptability in the receptors and ligands they use to determine host specificity yet apparently maintain similar invasion efficiency? Recently, Besteiro et al. [7] provided evidence for how efficiency might remain constant; not only do apicomplexans carry their own invasion ligands but they may provide their own invasion receptors, which they 'plug' into their hosts' cells and which serve as anchoring points for host cell penetration.
In Figure 1, we have illustrated the basic steps of invasion, using Toxoplasma tachyzoites as an example. After initial attachment and recognition, presumably involving the parasite genus-specific ligands and host cell-encoded receptors referred to above, the apical end of the parasite becomes juxtaposed to the host cell surface and a strong ring of attachment, or tight junction, is formed (Figure 1). Parasite actin/myosin motor proteins are engaged and the parasite pulls itself through the tight junction and enters the host cell [8][9][10][11][12][13]. The parasite concomitantly secretes proteins and lipids from its apical secretory organelles that help form a parasitophorous vacuole into which the parasite enters ( Figure 1). The parasite ligand relevant to the work of Besteiro et al. [7] is known as apical membrane antigen 1 (AMA1), a type 1 integral membrane protein with a large ectodomain and small cytoplasmic region [14,15]. AMA1 is stored in secretory microneme organelles and released onto the plasma membrane prior to host cell attachment ( Figure 1) [16,17]. Prior to internalisation, a subset of AMA1 molecules become restricted to the tight junction and appear to interact with a complex of three or four rhoptry neck proteins (RONs) [18][19][20]. These interactions have been known for some time, but Besteiro and colleagues suggest that, rather than residing on the parasite side of the junction, the RON complex might reside on the host side of the junction, with one or more RON components penetrating the host plasma membrane making contact with the host cytoskeleton ( Figure 1). Many apicomplexans possess homologues of AMA1 and the RONs, raising the possibility that this invasion mechanism is commonly used within the phylum.
Although the interaction between AMA1 and the RONs has been studied by other groups, similar conclusions about the orientation and host cell localisation of the RON complex have not been reached previously [18][19][20]. If this observation is substantiated by follow-up studies, it will be of tremendous significance for the field. Some support for the findings of Besteiro et al. [7] has been provided by another study that indicates that a ring of

invasion of their host cells apicomplexan zoites may transfer ligands into their hosts which they then bind to during invasion
Apicomplexan parasites may insert their own ligands into their host cells to provide an adhesive point for invasion, as illustrated by a Toxoplasma tachyzoite invading an animal cell. After host cell contact the parasite apically reorientates. Apical membrane antigen 1 (AMA1) is secreted from micronemes onto the tachyzoite surface, where a small amount interacts with the rhoptry neck proteins (RONs) to form the tight junction. How this transfer is mediated is unknown but could involve the release of RON-containing evacuoles or lipoprotein complexes. Concurrent with the formation of the AMA1-RON tight junction is the creation of an F-actin ring below the host cell surface which possibly helps anchor the tight junction. Internalisation of the parasites ensues after activation of the parasite's actin/myosin motor. The parasite migrates into a vacuole formed from host and secreted parasite lipids and proteins.
host cell F-actin rapidly forms beneath the tight junction in Toxoplasma tachyzoites and P. berghei sporozoites invading tissue-cultured cells [21]. F-actin ring formation greatly assists parasite invasion by presumably acting as a solid anchoring point, and inhibition of its formation with actin-destabilising drugs significantly reduces invasion efficiency. Actin polymerisation appears to be stimulated by recruitment of F-actin nucleating factors such as the Arp2/3 complex and cortactin. The proteins that ultimately recruit the F-actin nucleators are unknown, but the possibility of parasite effector proteins being involved has been raised [21]. These may well be members of the RON complex, and examples of direct parasite protein-mediated stimulation of host cell F-actin formation are known from bacterial pathogens [22].
If a common mechanism of tight junction formation and internalisation is used for apicomplexan invasion, then the preceding molecular interactions presumably provide the necessary specificity between the parasite and host. Initial contact between parasite and host is seemingly mediated by low-affinity long-range interactions between parasite surface coat proteins and the host surface. These may trigger the release of high-specificity EBA and Rh family proteins from apical secretory microneme organelles in Plasmodium parasites that bind blood cell surface proteins such as the glycophorins. It will be interesting to discover whether the apical concentration of these parasite receptors aids reorientating of the parasite and/or triggers a signal cascade that results in the release of the RON complex and formation of the tight junction. In Toxoplasma, the microneme (MIC) proteins perform this role, with MIC8 acting upstream of AMA1 and regulating the release of the RONs [23].

Future directions
Although early contact events between host cells and apicomplexan parasites require specific parasite-host cell recognition events, the process of tight junction formation and host cell penetration may involve a core of conserved parasite-encoded proteins acting as both ligands and receptors. AMA1 is already a leading vaccine candidate, and understanding its conserved interaction with the RON complex offers the promise of novel intervention approaches with a possible broad effect for multiple parasite species. Furthermore, the involvement of intracellular host cell proteins during invasion presents new targets for intervention that centre on the functions of host cell components.


Neurotensin receptor 1 immunoreactivity in the peripheral ganglia and carotid body

In the present study we investigated, through immunohistochemistry, the presence and location of neurotensin receptor 1 (NTR1) in the peripheral ganglia and carotid body of 16 humans and 5 rats. In both humans and rats, NTR1 immunostained ganglion cells were found in superior cervical ganglia (57.4±11.6% and 72.4±11.4%, respectively, p<0.05), enteric ganglia (51.9±10.4% and 64.6±6.1%, p<0.05), sensory ganglia (69.2±10.7% and 73.0±13.1%, p>0.05) and parasympathetic ganglia (52.1±14.1% and 59.4±14.0%, p>0.05), supporting a modulatory role for NT in these ganglia. Positivity was also detected in 45.6±9.2% and 50.8±6.8% of human and rat type I glomic cells, respectively, whereas type II cells were negative. Our findings suggest that NT produced by type I cells acts in an autocrine or paracrine way on the same cell type, playing a modulatory role on chemoception.

©2009 European Journal of Histochemistry
In the present study we investigated, through immunohistochemistry, the presence and location of neurotensin receptor 1 (NTR1) in the peripheral ganglia and carotid body of 16 humans and 5 rats. In both humans and rats, NTR1 immunostained ganglion cells were found in superior cervical ganglia (57.4±11.6% and 72.4±11.4%, respectively, p<0.05), enteric ganglia (51.9±10.4% and 64.6±6.1%, p<0.05), sensory ganglia (69.2±10.7% and 73.0±13.1%, p>0.05) and parasympathetic ganglia (52.1±14.1% and 59.4±14.0%, p>0.05), supporting a modulatory role for NT in these ganglia. Positivity was also detected in 45.6±9.2% and 50.8±6.8% of human and rat type I glomic cells, respectively, whereas type II cells were negative. Our findings suggest that NT produced by type I cells acts in an autocrine or paracrine way on the same cell type, playing a modulatory role on chemoception. N eurotensin (NT) is a tridecapeptide which was first isolated from bovine hypothalamus (Carraway and Leeman, 1973) and is widely distributed in the nervous system and intestine. In the nervous system, neurotensin acts as a neurotransmitter and neuromodulator (Dobner, 2006); in the periphery, as a paracrine or endocrine factor (Mazzocchi et al., 1997;Malendowicz, 1998). It also acts as a growth factor on various cell types (Malendowicz, 1993;Markowska et al., 1994aMarkowska et al., , 1994bEvers, 2006). Three different NT receptors, termed NTR1, NTR2 and NTR3, have been identified and cloned to date. NTR1 and NTR2 are, respectively, high-and low-affinity seven transmembrane domain G protein-coupled receptors. NTR3 is a high-affinity single trans-membrane domain type 1 receptor, with 100% homology with the sorting protein, gp95/sortilin (Kitabgi, 2006;Mazella et al., 1998). NTR3 can also form heterodimers with NTR1 in the plasma membrane (Martin et al., 2002). Nuclear internalization of the NTR1 has been reported and has been suggested to play a role in the production of long-term genomic effects (Feldberg et al., 1998;Laduron, 1992). It has also been reported that NTR2, but not NTR1, returns to the plasma membrane after NT-induced sequestration (Mazella and Vincent, 2006).
In the peripheral nervous system, pregangliar fibers containing NT have been found in sympathetic, parasympathetic and enteric ganglia, and functional studies also suggest the expression of NTRs in ganglion cells. However, direct evidence of NTR1 protein expression in the different cell types of the ganglia has not yet been provided for human and rat. Only in rat dorsal root ganglia has evidence of NTR1 expression been given through hybridization in situ (Zhang et al., 1995), but there are no data on protein location or internalization.
The carotid body is an arterial chemoreceptor, sensitive to reductions in partial blood oxygen pres-sure and pH and to increases in partial CO2 pressure, the stimulation of which induces increases in ventilatory frequency and volume.The carotid body is situated at the carotid bifurcation, and is composed of parenchymal lobules separated by connective tissue, in which afferent fibers of the glossopharyngeal nerve, arising from the petrosal ganglion, occur (Porzionato et al., 2005).Two different cell populations are present in the carotid body: type I cells, in turn separated into light, dark and pyknotic, and type II cells, at the edges of the clusters. Post-ganglionic sympathetic nerve fibers from the superior cervical ganglion are present, innervating blood vessels and type I cells, and preganglionic parasympathetic and sympathetic fibers reaching ganglion cells near the glomic cells. NT has been detected in glomic cells (Heath et al., 1988;Heym and Kummer, 1989;Smith et al., 1990) but the presence of the corresponding receptors in the various glomic cell types has not yet been investigated.
Thus, the aim of the present study was to investigate, through immunohistochemistry, the presence and location of NTR1 in the peripheral ganglia and carotid body of both human and rat, with particular reference to the different cell types.

Materials and Methods
Materials consisted of dorsal root ganglia, trigeminal ganglia, superior cervical ganglia, enteric (both myenteric and submucous) ganglia, and carotid bodies obtained at autopsy from 16 adult subjects (10 males, 6 females; mean age 44.3 years, Standard Deviation (SD) ± 3.4), clinically negative for chronic pulmonary or cardiovascular diseases, and from 5 adult Wistar rats weighing 200-250 g. Samples for evaluation of enteric ganglia were taken at the level of the first jejunal loop. Further materials consisted in ciliary ganglia sampled from the above rats and pelvic ganglia sampled from the above humans. Autopsies were performed between 24 and 30 hours after death. Sampling from rats was performed soon after sacrifice.
Samples were fixed in neutral 10% formalin and embedded in paraffin wax. Immunohistochemical examination was carried out on 3 µm thick sections. Sections were incubated in 0.3% hydrogen peroxide for 10 min at room temperature, to remove endogenous peroxidase activity, and then in blocking serum (0.04% bovine serum albumin (A2153, Sigma-Aldrich, Milan, Italy) and 0.5% normal goat serum (X0907, Dako Corporation, Carpinteria, CA, USA) in PBS) for 30 min. NTR1 immunoreactivity was detected with a rabbit polyclonal anti-NTR1 antibody at 1:600 dilution in blocking serum for 1 hour at room temperature. This antibody was raised against amino acids 1-28 and 50-69 of rat NTR1 and also detected human NTR1 (Brun et al., 2005). Sections were then washed twice for 5 min in phosphate buffered saline (PBS), revealed with anti-rabbit serum for 20 min (DAKO ® EnVision + TM Peroxidase, Rabbit, Dako Corporation, Carpinteria, CA, USA), and developed in 3,3'-diaminobenzidine (DAB, Sigma-Aldrich, Milan, Italy). Lastly, sections were counterstained with hematoxylin. Negative controls were performed by omission of primary antibody. NTR1 immunoreactions detected in rat and human substantia nigra were used as positive controls.
Immunohistochemistry anti-tyrosine hydroxylase (TH) was used to distinguish between sympathetic (TH-positive) and parasympathetic (TH-negative) human pelvic ganglia (Keast et al., 1995).To block endogenous peroxidase activity, rehydrated sections were incubated in 0.3% hydrogen peroxide for 10 min at room temperature and then predigested with 0.1% trypsin for antigen retrieval. After 2 rinses in PBS, sections were incubated for 30 min in blocking serum at room temperature to suppress nonspecific binding. Thereafter, sections were incubated in monoclonal mouse anti-human TH (DiaSorin, Stillwater, MN, USA, 1:1000, raised to TH purified from rat PC12 cells) overnight at 4C°and in biotinylated rabbit anti-mouse antibody (Elite Vecta Stain ABC Kit, Vector Laboratories, CA, 1:200) for 30 min. Lastly, sections were developed in 3,3'-diaminobenzidine (DAB, Sigma-Aldrich, Milan, Italy).
In the peripheral ganglia and carotid bodies, percentages of ganglion and glomic (types I and II) NTR1-positive cells, respectively, were evaluated at a magnification of 40X, and 5 sections and 3 fields per section were examined. Mean percentages were calculated for each case and for the entire series. For each human case, an average of 100 neurons were counted for sympathetic, parasympathetic and sensory ganglia, 40 neurons for enteric ganglia, 200 type I cells for carotid body. For each rat, an average of 200 neurons were counted for sympathetic and sensory ganglia, 150 neurons for parasympathetic ganglia, 50 neurons for enteric ganglia, 300 type I cells for carotid body. Counterstaining with hematoxylin permitted differentiation between the light, dark and pyknotic cells of the carotid body, showing both positive and negative immunoreactions, according to the morphological characteristics and staining properties of the cells.The percentages of dark, light and pyknotic cells showing NTR1 immunoreactivity were evaluated at a magnification of 40X, and 5 sections and 3 fields per section were examined. The mean percentages of positive dark, light and pyknotic cells were then calculated for each case and for the entire series.
The Mann-Whitney U-test was performed to verify any differences in NTR1 immunoreactivity between human and rat samples and different types of carotid body cells and of sensory, enteric, sympathetic and parasympathetic ganglionic cells. In order to reveal differences between glomic type I cells and sensory (dorsal root and trigeminal ganglia), sympathetic (cervical superior ganglia), parasympathetic (ciliary and pelvic parasympathetic ganglia) and enteric ganglionic cells, statistical analysis was performed in both human and rat samples by the Kruskal-Wallis test and Dunn's multiple comparison test. A p of 0.05 was considered significant.
In order to verify the immunohistochemical specificity of the reaction, absorption tests were also performed through incubation with the N-terminal peptides used to generate the antiserum for 2 hours at room temperature.

Results
In both humans ( Figure 1) and rats (Figure 2), NTR1 immunostaining was found in all the peripheral ganglia and carotid bodies examined. Ganglion and glomic cell immunostaining was eliminated when antiserum, preabsorbed with its peptide antigen, was used ( Figures 1B, 1G, 2B, 2G). As regards the subcellular location of NTR1 immunostaining, in human superior cervical ganglia the percentage of nuclear positivity on the total was 34.2±16.3%. In rat superior cervical ganglia and in sensory, parasympathetic and enteric ganglia of both humans and rats, NTR1 positivity was almost only found in the nuclei of ganglion cells. In sensory ganglia, weak/moderate nuclear immunostaining was also found in some satellite cells. Positivity involved both A-and B-cells of the sensory ganglia, without statistically significant differences.
In both humans and rats, immunohistochemistry revealed the coexistence of both NTR1-positive and -negative type I cells in all specimens examined. When present, NTR1 immunoreactivity was intense, and distributed in the cytoplasm or nucleus. The percentage of nuclear NTR1 immunostaining on the total was higher in rats (65.4±12.9%) than in humans (13.9±11.1%; p<0.01). A preliminary analysis of subpopulations of type I cells did not reveal any statistically significant differences between dark, light or pyknotic cells (p>0.05). Immunostained cells were distributed in both the center and periphery of the lobules.Type II cells did not show immunostaining.

Discussion
NT has been found in the preganglionic axon terminals of the cat sympathetic paravertebral and prevertebral ganglia, but no NT immunoreactivity was detected in principal ganglion cells (Heym et al., 1984;Caverson et al., 1989;Maher et al., 1994;Zetina et al., 1999). Functional studies reveal release of NT from preganglionic axon terminals after stimulation (Caverson et al., 1989; negative controls (B, G), performed through absorption with the N-terminal peptides used to generate the antiserum. Scale bars = 30 µ µm. negative controls (B, G) performed through absorption with the N-terminal peptides used to generate the antiserum. Scale bars = 30 µ µm. Maher et al., 1994) and excitation of sympathetic ganglion cells by exogen neurotensin (Bachoo and Polosa, 1988). Moreover, anti-NT immunohistochemistry shows NT internalization in ganglion cells after stimulation of preganglionic fibers, and this phenomenon is partially blocked by the NT antagonist SR48692 (Zetina et al., 1999). NTimmunoreactive fibers have also been found in mammalian parasympathetic intracardiac ganglia (Weihe and Reinecke, 1981;Reinecke et al., 1982), and sections from rat nodose ganglia incubated with 125I-NT exhibit dense radioautographic labeling of 15% of ganglion cells, with nuclear sparing (Kessler and Beaudet, 1989). NT-positive fibers have also been detected in canine submucous and myenteric ganglia (Buchan and Barber, 1987) and NT binding sites have been found, through autoradiography, in porcine (Seybold et al., 1990) and human (Azriel and Burcher, 2001) submucous and myenteric ganglia. Although the above studies strongly suggest the presence of high-affinity NT receptors on sympathetic, parasympathetic and enteric ganglion cells, this is the first work which identifies NTR1 in rat and human ganglia by immunohistochemistry. Nuclear location of NTR1 has also been reported in other cell types (Boudin et al., 1998;Dana et al., 1989;Feldberg et al., 1998;Toy-Miou-Leong et al., 2004). For instance, electron microscopy analysis of the distribution of NTR1 in rat substantia nigra by immunohistochemistry (Boudin et al., 1998;Toy-Miou-Leong et al., 2004) and autoradiography (Boudin et al., 1998) has revealed the strongest NTR1 immunoreactivity and the highest number of 125I-NT binding sites in the nucleus with respect to other somatic structures. Mast cells have also been found to show particularly intense anti-NTR1 nuclear immunostaining (Feldberg et al., 1998). In the literature, nuclear location of NTR1 has mainly been ascribed to internalization of the receptor (Boudin et al., 1998;Feldberg et al., 1998;Toy-Miou-Leong et al., 2004). NTR contains a strong nuclear targeting signal (Feldberg et al., 1998), and it has also been proposed that nuclear internalization of NT ligated to its receptor plays a role in the production of long-term genomic effects (Laduron, 1992). Nuclear localization has also been described for other G-protein-coupled receptors, such as angiotensin type 1, VIP, opioid, prostaglandin, and muscarinic receptors. However, it has also been alternatively hypothesized that nuclear location of NTR1 could be ascribed to the presence of neosynthesized receptors, because the nuclear envelope has long been considered a major site of protein synthesis (Boudin et al., 1998). In the present work, suppression of nuclear immunostaining after absorption tests (Figures 1 and 2) confirms the specificity of our results.

Figure 2. Anti-NTR1 immunohistochemistry in rats -Sections of superior cervical (A-B), trigeminal (C), ciliary parasympathetic (D), enteric (E) ganglia and carotid bodies (F-G), showing selective positivity of ganglionic cells (A, C-E) and glomic type I cells (F), while immunostaining is largely eliminated in
Dorsal root ganglia are known to express different neurotransmitter receptors, such as nicotinic, muscarinic, glutamate and opioid receptors. NT immunoreactivity has also been found in the neurons of cat, dog, primate, sloth and rat dorsal root ganglia (Yaksh et al., 1988;Zhang et al., 1996). Electrophysiological studies on extirpated rat dorsal root ganglia after NT exposure showed outward and inward currents in C-and A-types neurons, respectively, suggesting the expression of different types of receptors (Xu et al., 1997). Inward currents in response to NT were also found in guineapig dorsal root ganglia (Kawarada et al., 2000). In situ hybridization revealed expression of NTR1 mainly in small sensory neurons of rat dorsal root ganglia (Zhang et al., 1995), and immunofluorescence analysis revealed the expression of NTR2 in both small and large cells (Sarret et al., 2005). We also confirmed by immunohistochemistry NTR1 expression in sensory ganglion cells, and nuclear NTR1 location supports internalization, also in this structure. In addition, in both humans and rats it 140 A. Porzionato, et al. must be stressed that the sensory ganglia showed the highest NTR1 immunostaining among the different structures examined.
Many neurotransmitters and neuromodulators have been identified in glomic cells, such as dopamine, serotonin, noradrenaline, adrenaline, acetylcholine, adenosine, met-and leu-enkephalins, neuropeptide Y, CGRP, galanin, endothelins, bombesin, NO and adrenomedullin (Bairam and Carroll, 2005;Porzionato et al., 2006Porzionato et al., , 2008aPorzionato et al., , 2008b. NT occurrence and distribution has been studied by radioimmunoassay (Heath et al., 1988), immunohistochemistry (Heym and Kummer, 1989) and immunogold techniques (Smith et al., 1990). In human carotid bodies, radioimmunoassay showed higher levels of NT expression than VIP and substance P, and lower levels of bombesin, met-and leu-enkephalin (Heath et al., 1988). The immunogold technique revealed the weakest immunoreactivity for neurotensin with respect to the other neuropeptides (Smith et al., 1990). A species-specific pattern of NT expression was also reported, with NT immunolabeling only in beagle glomus cells among various animal species (Heym and Kummer, 1989). The location of NTR1 in the carotid body has never been investigated before, so that our study demonstrates for the first time the selective expression of NTR1 in human and rat type I cells.
In some cases, immunoreaction was cytoplasmic and in others nuclear, thus also confirming NT internalization in the carotid body. The higher cytoplasmic NTR1 positivity in humans than in rats may be explained on the basis of the different kinetics of internalization, or longer post-mortem intervals. Thus, NT produced by type I cells may act in an autocrine or paracrine way on the same cell type. It is possible that NT acting on type I cells also derives from NT-positive sympathetic or parasympathetic fibers innervating type I cells, although there is no direct evidence of this in the literature. NT probably plays a modulatory role on chemoception by type I cells, as the neuromodulatory effects of NT have already been proposed in ganglion transmission (Bachoo and Polosa, 1988). Further functional studies will be necessary to understand the effect of NT in glomic cells. Binding of NT to NTR1 stimulates phosphorylation of ERK and AKT (Hassan et al., 2004) and probably contributes to the activation of ERK and AKT signalling pathways in the carotid body and peripheral ganglia (Porzionato et al., In press). Cell line PC12, which shows oxygen-sensing properties and has been considered as a model for carotid body function, cannot be used for investigation of NT action, as these cells express and release neurotensin (Tischler et al., 1991) but seem not to show functional NTR (Caillaud et al., 1995). However, it is intriguing to note that NT attenuates dopamine-induced inhibition of midbrain dopamine neurons (Seutin et al., 1989;Shi et al., 1992), as dopamine is the main glomic neurotransmitter. It may be hypothesized that, also in the carotid body, NT reduces the inhibitory effect of dopamine on chemoception.


text:
Regulation of Bestrophins by Ca2+: A Theoretical and Experimental Study

Bestrophins are a recently discovered family of Cl− channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BKCa). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl− current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl− current in agreement with modeling results.

Text S1
Test of the accuracy of the force field used in the MD simulation.
Structures and energetic of Ca 2+ -binding proteins are difficult to reproduce with current classical force fields [1,2]. Factors, which limit the accuracy, include the lack of polarizability and charge transfer effects [3]. Here we tested the accuracy of three commonly used force fields: the Aqvist [4], the Bradbrook [5] and the AMBER [6,7] force fields. We performed 20-ns MD calculations of TSP-1 T3 6 for which the X-ray structure is available [8] and compared X-ray and MD structures.
TSP-1 T3 6 is a 39 amino acids long loop resembling EF hands motif in the spacing of acidic side chains [9,10]. It binds five Ca 2+ ions by eleven Asp and two Asn residues (See Figure). Here we considered a model in which all five (Ca 1-5 ) calcium ions were present. The TSP-1 T3 6 domain was inserted in a box of edges 46, 51 and 49 Å filled with ~3700 water molecules and 2 Cl − counterions.
Computational details for the relaxation and unrestrained MD simulations were the same as reported in the main text of the paper.
In the three simulations, the final structure turned out to be rather similar to the X-ray structure: the RMSD values calculated onto the N-terminal and C-terminal halves are indeed equal to 3.2 Å and 2.4 Å, respectively (see table below) and Ca 1-4 remained bound. However, the total backbone RMSD from the X-ray structure was relatively large because of conformational changes of Ca 5binding groups (D880 and D887) upon Ca 5 solvation after few ns (~2 ns for Aqvist-and Bradbrookbased MD, ~1.3 ns for the AMBER-based MD). The loss of the Ca 2+ ion might be caused by several factors, including the limitations of the accuracy of the force field describing the Ca 2+ ions [2] and/or the absence of electrostatic packing forces in the simulation with respect to the crystal, along with a difference in temperature (the simulation was run at 300 K, the crystal structure was solved at 100 K). Force-matching methods, which were already applied by us for other metalcontaining systems [11], might be used to further address this issue, which is however beyond the scope of the present paper and the severe computational resources that would be needed prevent the application of these methods to the present problem. In all of the other calculations presented in this work we have indeed used the AMBER force field.

Figure.
Crystal Because of its limitations, we used molecular modeling to identify possible Ca 2+ -binding conformations of the Asp-rich domain and alanine mutations, which could affect Ca 2+ binding. No attempts were made to predict the structure of disease-linked mutants [12] and/or mutants for which electrophysiological experiments have been previously carried out [13] .
N-term corresponds to residues from A865 to H878, whereas C-term corresponds to residues from D879 to N897.

Table.
TSP-1 T3 6 backbone RMSD values relative to the X-ray structure after 20-ns MD simulations based on three different force fields.