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1.  Prospective Memory Performance in Non-Psychotic First-Degree Relatives of Patients with Schizophrenia: A Controlled Study 
PLoS ONE  2014;9(11):e111562.
Objective
We aimed at investigating prospective memory and its socio-demographic and neurocognitive correlates in non-psychotic, first-degree relatives (FDRs) of patients with schizophrenia compared to patients with first episode schizophrenia (FES), and healthy controls (HCs).
Methods
Forty-seven FES patients, 50 non-psychotic FDRs (23 offspring and 27 siblings) of patients with chronic schizophrenia (unrelated to the FES group) and 51 HCs were studied. The Chinese version of the Cambridge Prospective Memory Test (C-CAMPROMPT) was used to measure time-based prospective memory (TBPM) and event-based prospective memory (EBPM) performance. Other cognitive functions (involving respective memory and executive functions) were evaluated with standardized tests.
Results
After controlling for basic demographic characteristics including age, gender and educational level, there was a significant difference between FDRs, FES and HCs with respect to both TBPM (F(2,142) = 10.4, p<0.001) and EBPM (F(2,142) = 10.8, p<0.001). Multiple linear regression analyses revealed that lower scores of the Hopkins Verbal Learning Test-Revised (HVLT-R) and the STROOP Word-Color Test (SWCT) contributed to TBPM impairment, while lower educational level and higher scores of the Color Trails Test-2 (CTT-2) contributed to EBPM deficit in FDRs.
Conclusions
FDRs share similar but attenuated prospective memory impairments with schizophrenia patients, suggesting that prospective memory deficits may represent an endophenotype of schizophrenia.
doi:10.1371/journal.pone.0111562
PMCID: PMC4218767  PMID: 25365028
2.  Inhibition of Dengue Virus Replication by a Class of Small-Molecule Compounds That Antagonize Dopamine Receptor D4 and Downstream Mitogen-Activated Protein Kinase Signaling 
Journal of Virology  2014;88(10):5533-5542.
ABSTRACT
Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds—dihydrodibenzothiepines (DHBTs), identified through high-throughput screening—with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds.
IMPORTANCE The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.
doi:10.1128/JVI.00365-14
PMCID: PMC4019099  PMID: 24599995
3.  Plasmid-based shRNA lentiviral particle production for RNAi applications 
Journal of biomolecular screening  2014;19(9):1309-1313.
Lentiviral vectors have become mainstream gene transfer vehicles for their ability to delivery and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express dsRNA molecules usually as short hairpin RNA (shRNA) enabling long-term gene silencing and when pseudotyped with a broad host glycoprotein envelope; allows a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral particles in high-titer and uniformity. Typical methods require the transfection of three or more plasmids in which essential viral elements have been encoded separated so as to remain replication deficient. These transfection procedures are of critical importance; however, methods often vary among laboratories making it difficult to assess the overall efficiency of lentiviral particle production. In this report, we focused exclusively on this step and compared the overall impact of the commercial transfection reagent FuGENE 6 to FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard ELISA assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production.
doi:10.1177/1087057114539390
PMCID: PMC4170048  PMID: 24939963
shRNA; FuGENE 6; FuGENE HD; RNAi; lentiviral particles; viral titer; plasmid; DNA
4.  A 1536-well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins 
RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10μM. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs.
PMCID: PMC4135234  PMID: 24912481
RNA-binding protein; MUSASHI; HTS; fluorescence polarization; cancer; small-molecule inhibitors
5.  Discovery of a Dicer-Independent, Cell-Type Dependent Alternate Targeting Sequence Generator: Implications in Gene Silencing & Pooled RNAi Screens 
PLoS ONE  2014;9(7):e100676.
There is an acceptance that plasmid-based delivery of interfering RNA always generates the intended targeting sequences in cells, making it as specific as its synthetic counterpart. However, recent studies have reported on cellular inefficiencies of the former, especially in light of emerging gene discordance at inter-screen level and across formats. Focusing primarily on the TRC plasmid-based shRNA hairpins, we reasoned that alleged specificities were perhaps compromised due to altered processing; resulting in a multitude of random interfering sequences. For this purpose, we opted to study the processing of hairpin TRCN#40273 targeting CTTN; which showed activity in a miRNA-21 gain-of-function shRNA screen, but inactive when used as an siRNA duplex. Using a previously described walk-through method, we identified 36 theoretical cleavage variants resulting in 78 potential siRNA duplexes targeting 53 genes. We synthesized and tested all of them. Surprisingly, six duplexes targeting ASH1L, DROSHA, GNG7, PRKCH, THEM4, and WDR92 scored as active. QRT-PCR analysis on hairpin transduced reporter cells confirmed knockdown of all six genes, besides CTTN; revealing a surprising 7 gene-signature perturbation by this one single hairpin. We expanded our qRT-PCR studies to 26 additional cell lines and observed unique knockdown profiles associated with each cell line tested; even for those lacking functional DICER1 gene suggesting no obvious dependence on dicer for shRNA hairpin processing; contrary to published models. Taken together, we report on a novel dicer independent, cell-type dependent mechanism for non-specific RNAi gene silencing we coin Alternate Targeting Sequence Generator (ATSG). In summary, ATSG adds another dimension to the already complex interpretation of RNAi screening data, and provides for the first time strong evidence in support of arrayed screening, and questions the scientific merits of performing pooled RNAi screens, where deconvolution of up to genome-scale pools is indispensable for target identification.
doi:10.1371/journal.pone.0100676
PMCID: PMC4079264  PMID: 24987961
6.  Chemical & RNAi screening at MSKCC: a collaborative platform to discover & repurpose drugs to fight disease 
Memorial Sloan-Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution’s commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator’s research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia.
PMCID: PMC4050342  PMID: 24661215
HTS; HCS; RNAi; siRNA; shRNA; miRNA; automation; robotics; small molecule; chemical; robotics; cell-based assay; target-based assay; screen data analysis; drug discovery
7.  Comparative analysis of RNAi screening technologies at genome-scale reveals an inherent processing inefficiency of the plasmid-based shRNA hairpin 
RNAi screening in combination with the genome-sequencing projects would constitute the Holy Grail of modern genetics; enabling discovery and validation towards a better understanding of fundamental biology leading to novel targets to combat disease. Hit discordance at inter-screen level together with the lack of reproducibility is emerging as the technology's main pitfalls. To examine some of the underlining factors leading to such discrepancies, we reasoned that perhaps there is an inherent difference in knockdown efficiency of the various RNAi technologies. For this purpose, we utilized the two most popular ones, chemically synthesized siRNA duplex and plasmid-based shRNA hairpin, in order to perform a head to head comparison. Using a previously developed gain-of-function assay probing modulators of the miRNA biogenesis pathway, we first executed on a siRNA screen against the Silencer Select V4.0 library (AMB) nominating 1,273, followed by an shRNA screen against the TRC1 library (TRC1) nominating 497 gene candidates. We observed a poor overlap of only 29 hits given that there are 15,068 overlapping genes between the two libraries; with DROSHA as the only common hit out of the seven known core miRNA biogenesis genes. Distinct genes interacting with the same biogenesis regulators were observed in both screens, with a dismal cross-network overlap of only 3 genes (DROSHA, TGFBR1, and DIS3). Taken together, our study demonstrates differential knockdown activities between the two technologies, possibly due to the inefficient intracellular processing and potential cell-type specificity determinants in generating intended targeting sequences for the plasmid-based shRNA hairpins; and suggests this observed inefficiency as potential culprit in addressing the lack of reproducibility.
PMCID: PMC4007059  PMID: 24433414
8.  An Arrayed Genome-Scale Lentiviral-Enabled Short Hairpin RNA Screen Identifies Lethal and Rescuer Gene Candidates 
Abstract
RNA interference technology is becoming an integral tool for target discovery and validation.; With perhaps the exception of only few studies published using arrayed short hairpin RNA (shRNA) libraries, most of the reports have been either against pooled siRNA or shRNA, or arrayed siRNA libraries. For this purpose, we have developed a workflow and performed an arrayed genome-scale shRNA lethality screen against the TRC1 library in HeLa cells. The resulting targets would be a valuable resource of candidates toward a better understanding of cellular homeostasis. Using a high-stringency hit nomination method encompassing criteria of at least three active hairpins per gene and filtered for potential off-target effects (OTEs), referred to as the Bhinder–Djaballah analysis method, we identified 1,252 lethal and 6 rescuer gene candidates, knockdown of which resulted in severe cell death or enhanced growth, respectively. Cross referencing individual hairpins with the TRC1 validated clone database, 239 of the 1,252 candidates were deemed independently validated with at least three validated clones. Through our systematic OTE analysis, we have identified 31 microRNAs (miRNAs) in lethal and 2 in rescuer genes; all having a seed heptamer mimic in the corresponding shRNA hairpins and likely cause of the OTE observed in our screen, perhaps unraveling a previously unknown plausible essentiality of these miRNAs in cellular viability. Taken together, we report on a methodology for performing large-scale arrayed shRNA screens, a comprehensive analysis method to nominate high-confidence hits, and a performance assessment of the TRC1 library highlighting the intracellular inefficiencies of shRNA processing in general.
doi:10.1089/adt.2012.475
PMCID: PMC3619155  PMID: 23198867
9.  An Arrayed RNA Interference Genome-Wide Screen Identifies Candidate Genes Involved in the MicroRNA 21 Biogenesis Pathway 
Abstract
MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder–Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function.
doi:10.1089/adt.2012.477
PMCID: PMC3619226  PMID: 23153064
10.  Flaviviruses Are Sensitive to Inhibition of Thymidine Synthesis Pathways 
Journal of Virology  2013;87(17):9411-9419.
Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.
doi:10.1128/JVI.00101-13
PMCID: PMC3754125  PMID: 23824813
11.  Modulators of the microRNA biogenesis pathway via arrayed lentiviral enabled RNAi screening for drug and biomarker discovery 
MicroRNAs (miRNAs) are small endogenous and conserved non-coding RNA molecules that regulate gene expression. Although the first miRNA was discovered well over sixteen years ago, little is known about their biogenesis and it is only recently that we have begun to understand their scope and diversity. For this purpose, we performed an RNAi screen aimed at identifying genes involved in their biogenesis pathway with a potential use as biomarkers. Using a previously developed miRNA 21 (miR-21) EGFP-based biosensor cell based assay monitoring green fluorescence enhancements, we performed an arrayed short hairpin RNA (shRNA) screen against a lentiviral particle ready TRC1 library covering 16,039 genes in 384-well plate format, and interrogating the genome one gene at a time building a panoramic view of endogenous miRNA activity. Using the BDA method for RNAi data analysis, we nominate 497 gene candidates the knockdown of which increased the EGFP fluorescence and yielding an initial hit rate of 3.09%; of which only 22, with reported validated clones, are deemed high-confidence gene candidates. An unexpected and surprising result was that only DROSHA was identified as a hit out of the seven core essential miRNA biogenesis genes; suggesting that perhaps intracellular shRNA processing into the correct duplex may be cell dependent and with differential outcome. Biological classification revealed several major control junctions among them genes involved in transport and vesicular trafficking. In summary, we report on 22 high confidence gene candidate regulators of miRNA biogenesis with potential use in drug and biomarker discovery.
PMCID: PMC3884689  PMID: 23977983
miRNA; biogenesis; shRNA; H score; BDA method; RNAi; HCS; biomarker; HCA; miRNA 21; DROSHA; biomarker; diagnostics
12.  A Novel High-Throughput 1536-well Notch1 γ-Secretase AlphaLISA Assay 
The Notch pathway plays a crucial role in cell fate decisions through controlling various cellular processes. Overactive Notch signal contributes to cancer development from leukemias to solid tumors. γ-Secretase is an intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch fragment for signaling. Therefore, γ-secretase is an attractive drug target in treating Notch-mediated cancers. However, the absence of high-throughput γ-secretase assay using Notch substrate has limited the identification and development of γ-secretase inhibitors that specifically target the Notch signaling pathway. Here, we report on the development of a 1536-well γ-secretase assay using a biotinylated recombinant Notch1 substrate. We effectively assimilated and miniaturized this newly developed Notch1 substrate with the AlphaLISA detection technology and demonstrated its robustness with a calculated Z’ score of 0.66. We further validated this optimized assay by performing a pilot screening against a chemical library consisting of ~5,600 chemicals and identified known γ-secretase inhibitors e.g. DAPT, and Calpeptin; as well as a novel γ-secretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and represents a novel high-throughput Notch1-γ-secretase assay, which provides an unprecedented opportunity to discover Notch-selective γ-secretase inhibitors that can be potentially used for the treatment of cancer and other human disorders.
PMCID: PMC3664143  PMID: 23448293
Alzheimer disease; AlphaLISA; cancer; γ-secretase; γ-secretase modulators; Notch signaling
13.  Planning Following Stroke: A Relational Complexity Approach Using the Tower of London 
Planning on the 4-disk version of the Tower of London (TOL4) was examined in stroke patients and unimpaired controls. Overall TOL4 solution scores indicated impaired planning in the frontal stroke but not non-frontal stroke patients. Consistent with the claim that processing the relations between current states, intermediate states, and goal states is a key process in planning, the domain-general relational complexity metric was a good indicator of the experienced difficulty of TOL4 problems. The relational complexity metric shared variance with task-specific metrics of moves to solution and search depth. Frontal stroke patients showed impaired planning compared to controls on problems at all three complexity levels, but at only two of the three levels of moves to solution, search depth and goal ambiguity. Non-frontal stroke patients showed impaired planning only on the most difficult quaternary-relational and high search depth problems. An independent measure of relational processing (viz., Latin square task) predicted TOL4 solution scores after controlling for stroke status and location, and executive processing (Trail Making Test). The findings suggest that planning involves a domain-general capacity for relational processing that depends on the frontal brain regions.
doi:10.3389/fnhum.2014.01032
PMCID: PMC4274981  PMID: 25566042
Tower of London; planning; moves to solution; search depth; goal ambiguity; relational complexity; stroke; frontal lobes
14.  Developmental trajectories of schizotypal personality disorder-like behavioural manifestations: a two-year longitudinal prospective study of college students 
BMC Psychiatry  2013;13:323.
Background
Previous evidence has shown that schizotypal personality disorder (SPD) is part of the schizophrenia spectrum. Few studies have examined latent classes in the developmental trajectories of SPD features over time in individuals with SPD features.
Methods
We adopted a longitudinal prospective study design to follow up a cohort of 660 college students during a two-year period. Participants’ SPD-like symptoms and psychosocial function were measured by a comprehensive set of questionnaires that covered SPD features and cognitive, emotional, and psychosocial functions. Latent class growth analysis was used to examine the trajectory classes.
Results
Three trajectory classes were identified: a low, a medium, and a high SPD features group. Participants in the low group reported few SPD features and their symptoms declined over time. The medium group students had more SPD features than the low group and these symptoms stabilized during the follow up period. Participants in the high group reported the most SPD features and their symptoms increased over time. The three groups differed in paranoid thoughts, psychological distress, neurocognition function, and emotional expression over time. Results of multivariate regression analysis suggested that paranoid thoughts, emotional experience and prospective memory were predictors of social functioning in the high SPD feature group.
Conclusions
Our findings suggest that individuals with SPD features may be delineated into different developmental subgroups and these subgroups differ significantly in psychosocial function. Delusions, emotion, and prospective memory may be important features to consider in early diagnosis and interventions for individuals predisposed to SPD and schizophrenia.
doi:10.1186/1471-244X-13-323
PMCID: PMC3875349  PMID: 24289659
Schizotypal personality disorder; Developmental trajectories; Psychosocial function; Latent class growth analysis
15.  Comparison of error-based and errorless learning for people with severe traumatic brain injury: study protocol for a randomized control trial 
Trials  2013;14:369.
Background
Poor skills generalization poses a major barrier to successful outcomes of rehabilitation after traumatic brain injury (TBI). Error-based learning (EBL) is a relatively new intervention approach that aims to promote skills generalization by teaching people internal self-regulation skills, or how to anticipate, monitor and correct their own errors. This paper describes the protocol of a study that aims to compare the efficacy of EBL and errorless learning (ELL) for improving error self-regulation, behavioral competency, awareness of deficits and long-term outcomes after TBI.
Methods/Design
This randomized, controlled trial (RCT) has two arms (EBL and ELL); each arm entails 8 × 2 h training sessions conducted within the participants’ homes. The first four sessions involve a meal preparation activity, and the final four sessions incorporate a multitasking errand activity. Based on a sample size estimate, 135 participants with severe TBI will be randomized into either the EBL or ELL condition. The primary outcome measure assesses error self-regulation skills on a task related to but distinct from training. Secondary outcomes include measures of self-monitoring and self-regulation, behavioral competency, awareness of deficits, role participation and supportive care needs. Assessments will be conducted at pre-intervention, post-intervention, and at 6-months post-intervention.
Discussion
This study seeks to determine the efficacy and long-term impact of EBL for training internal self-regulation strategies following severe TBI. In doing so, the study will advance theoretical understanding of the role of errors in task learning and skills generalization. EBL has the potential to reduce the length and costs of rehabilitation and lifestyle support because the techniques could enhance generalization success and lifelong application of strategies after TBI.
Trial registration
ACTRN12613000585729.
doi:10.1186/1745-6215-14-369
PMCID: PMC4228339  PMID: 24192067
Brain injury; Metacognition; Self-awareness; Rehabilitation; Functional activities; Randomized controlled trial
16.  A high density assay format for the detection of novel cytotoxicagents in large chemical libraries 
The Alamar Blue (AB) assay, which incorporates a redox indicator that causes a fluorescence signal enhancement in response to metabolic activity, is commonly used to assess the viability of mammalian cells. In response to the need for homogeneous, inexpensive, high throughput assays for anti-cancer drug screening, a 1536-well microtiter plate based assay which utilizes the AB fluorescent dye as a measure of cellular growth was developed and validated in 10 µL assay volume. The performance and robustness of the miniaturized assay was assessed using a human Mantle Cell Lymphoma (MCL) cell line in a pilot screen against a library of 2,000 known bioactive chemicals; with an overall Z’ value of 0.89 for assay robustness, several known cytotoxic agents were identified including and not limited to anthracyclines, cardiac glycosides, gamboges, and quinones. To further test the sensitivity of the assay, IC50 determinations were performed in both 384-well and 1536-well formats and the obtained results show a very good correlation between the two density formats. These findings demonstrate that this newly developed assay is simple to set up, robust, highly sensitive and inexpensive. The non-radiometric strategy employed in this study should also offer the potential for the rapid screening, without a wash or a lysis step, of well established and primary tumor cell lines against large chemical libraries using the 1536-well microtiter plates.
doi:10.1080/14756360701810082
PMCID: PMC3710589  PMID: 18608772
Assay; miniaturization; Alamar Blue; cytotoxicity; anthracyclines; screening; HTS; fluorescence; resazurin; cell viability; NCEB1; cancer
17.  A High-Throughput Scintillation Proximity-Based Assay for Human DNA Ligase IV 
Abstract
Ionizing radiation (IR) and certain chemotherapeutic drugs are designed to generate cytotoxic DNA double-strand breaks (DSBs) in cancer cells. Inhibition of the major DSB repair pathway, nonhomologous end joining (NHEJ), will enhance the cytotoxicity of these agents. Screening for inhibitors of the DNA ligase IV (Lig4), which mediates the final ligation step in NHEJ, offers a novel target-based drug discovery opportunity. For this purpose, we have developed an enzymatic assay to identify chemicals that block the transfer of [α-33P]-AMP from the complex Lig4-[α-33P]-AMP onto the 5′ end of a double-stranded DNA substrate and adapted it to a scintillation proximity assay (SPA). A screen was performed against a collection of 5,280 compounds. Assay statistics show an average Z′ value of 0.73, indicative of a robust assay in this SPA format. Using a threshold of >20% inhibition, 10 compounds were initially scored as positive hits. A follow-up screen confirmed four compounds with IC50 values ranging from 1 to 30 μM. Rabeprazole and U73122 were found to specifically block the adenylate transfer step and DNA rejoining; in whole live cell assays, these compounds were found to inhibit the repair of DSBs generated by IR. The ability to screen and identify Lig4 inhibitors suggests that they may have utility as chemo- and radio-sensitizers in combination therapy and provides a rationale for using this screening strategy to identify additional inhibitors.
doi:10.1089/adt.2011.0404
PMCID: PMC3374410  PMID: 22192310
18.  A High Throughput Scintillation Proximity Imaging Assay for Protein Methyltransferases 
Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z′ values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.
PMCID: PMC3553658  PMID: 22256970
protein methyl transferases; drug discovery; inhibit or; SET7/9; SET8; SETD2; EuHMTase1; SPA technology; red shifted imaging beads
19.  Social Functioning in Chinese College Students with and without Schizotypal Personality Traits: An Exploratory Study of the Chinese Version of the First Episode Social Functioning Scale 
PLoS ONE  2013;8(5):e61115.
Objectives
The First Episode Social Functioning Scale (FESFS) was designed to measure social functioning of young individuals with schizophrenia. The aim of this study was to validate a Chinese version of the FESFS in a sample of young Chinese adults.
Method
The FESFS was translated to Chinese prior to being administered to 1576 college students. The factor structure, reliability, and validity of the scale were examined.
Results
Two items were deleted after item analysis and the internal consistency of the whole scale was .89. A six-factor structure was derived by exploratory factor analysis. The factors were interpersonal, family and friends, school, living skills, intimacy, and balance. Estimates of the structural equation model supported this structure, with Goodness of Fit Chi-Square χ2 = 1097.53 (p<0.0001), the root mean square error of approximation (RMSEA) = 0.058, and the comparative fit index (CFI) = 0.93. Scale validity was supported by significant correlations between social functioning factors scores and schizophrenia personality questionnaire (SPQ) scores. Individuals with schizotypal personality features presented poorer social functioning than those without schizotypal personality features.
Conclusions
The Chinese revised version of the FESFS was found to have good psychometric properties and could be used in the future to examine social functioning in Chinese college students.
doi:10.1371/journal.pone.0061115
PMCID: PMC3653910  PMID: 23690922
20.  Structure-activity relationships of 6-(2,6-dichlorophenyl)-8-methyl-2-(phenylamino)pyrido[2,3-d]pyrimidin-7-ones: toward selective Abl inhibitors 
We report the design, synthesis and structure-activity relationship (SAR) of a series of novel pyrido[2,3-d]pyrimidin-7-one compounds as potent Abl kinase inhibitors. We evaluate their specificity profile against a panel of human recombinant kinases, as well as their biological profile toward a panel of well characterized cancer cell lines. Our study reveals that substitutions in the -3 and -4 positions of the phenylamino moiety lead to improved potency and improved selectivity both in target-based and cell based assays. Altogether, our results provide an insight into the SAR of pyrido[2,3-d]pyrimidin-7-ones for the development of drug candidates with improved potency and selectivity for the targeted treatment of CML.
doi:10.1016/j.bmcl.2009.10.085
PMCID: PMC3629380  PMID: 19889540
Pyridopyrimidines; CML; Abl kinase; inhibitor
21.  Development and Validation of a High-density Fluorescence Polarization-based Assay for the Trypanosoma RNA Triphosphatase TbCet1 
RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener™ ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high-throughput screening.
PMCID: PMC3626118  PMID: 19275531
triphosphatase; drug discovery; high-throughput screening; fluorescence polarization
22.  Neural correlates of uncertain decision making: ERP evidence from the Iowa Gambling Task 
In our daily life, it is very common to make decisions in uncertain situations. The Iowa Gambling Task (IGT) has been widely used in laboratory studies because of its good simulation of uncertainty in real life activities. The present study aimed to examine the neural correlates of uncertain decision making with the IGT. Twenty-six university students completed this study. An adapted IGT was administered to them, and the EEG data were recorded. The adapted IGT we used allowed us to analyze the choice evaluation, response selection, and feedback evaluation stages of uncertain decision making within the same paradigm. In the choice evaluation stage, the advantageous decks evoked larger P3 amplitude in the left hemisphere, while the disadvantageous decks evoked larger P3 in the right hemisphere. In the response selection stage, the response of “pass” (the card was not turned over; the participants neither won nor lost money) evoked larger negativity preceding the response compared to that of “play” (the card was turned over; the participant either won or lost money). In the feedback evaluation stage, feedback-related negativity (FRN) was only sensitive to the valence (win/loss) but not the magnitude (large/small) of the outcome, and P3 was sensitive to both the valence and the magnitude of the outcome. These results were consistent with the notion that a positive somatic state was represented in the left hemisphere and a negative somatic state was represented in the right hemisphere. There were also anticipatory ERP effects that guided the participants' responses and provided evidence for the somatic marker hypothesis with more precise timing.
doi:10.3389/fnhum.2013.00776
PMCID: PMC3828619  PMID: 24298248
uncertain decision making; Iowa Gambling Task; emotion; ERP; somatic marker hypothesis
23.  Study protocol: a randomised controlled trial of the effects of a multi-modal exercise program on cognition and physical functioning in older women 
BMC Geriatrics  2012;12:60.
Background
Intervention studies testing the efficacy of cardiorespiratory exercise have shown some promise in terms of improving cognitive function in later life. Recent developments suggest that a multi-modal exercise intervention that includes motor as well as physical training and requires sustained attention and concentration, may better elicit the actual potency of exercise to enhance cognitive performance. This study will test the effect of a multi-modal exercise program, for older women, on cognitive and physical functioning.
Methods/design
This randomised controlled trial involves community dwelling women, without cognitive impairment, aged 65–75 years. Participants are randomised to exercise intervention or non-exercise control groups, for 16 weeks. The intervention consists of twice weekly, 60 minute, exercise classes incorporating aerobic, strength, balance, flexibility, co-ordination and agility training. Primary outcomes are measures of cognitive function and secondary outcomes include physical functioning and a neurocognitive biomarker (brain derived neurotrophic factor). Measures are taken at baseline and 16 weeks later and qualitative data related to the experience and acceptability of the program are collected from a sub-sample of the intervention group.
Discussion
If this randomised controlled trial demonstrates that multimodal exercise (that includes motor fitness training) can improve cognitive performance in later life, the benefits will be two-fold. First, an inexpensive, effective strategy will have been developed that could ameliorate the increased prevalence of age-related cognitive impairment predicted to accompany population ageing. Second, more robust evidence will have been provided about the mechanisms that link exercise to cognitive improvement allowing future research to be better focused and potentially more productive.
Trial registration
Australian and New Zealand Clinical Trial Registration Number: ANZCTR12612000451808
doi:10.1186/1471-2318-12-60
PMCID: PMC3503610  PMID: 23009189
Exercise; Cognition; Aged; Multi-modal exercise; Brain derived neurotrophic factor
24.  Can exercise ameliorate treatment toxicity during the initial phase of testosterone deprivation in prostate cancer patients? Is this more effective than delayed rehabilitation? 
BMC Cancer  2012;12:432.
Background
There has been substantial increase in use of androgen deprivation therapy as adjuvant management of prostate cancer. However, this leads to a range of musculoskeletal toxicities including reduced bone mass and increased skeletal fractures compounded with rapid metabolic alterations, including increased body fat, reduced lean mass, insulin resistance and negative lipoprotein profile, increased incidence of cardiovascular and metabolic morbidity, greater distress and reduced quality of life. Numerous research studies have demonstrated certain exercise prescriptions to be effective at preventing or even reversing these treatment toxicities. However, all interventions to date have been of rehabilitative intent being implemented after a minimum of 3 months since initiation of androgen deprivation, by which time considerable physical and psychological health problems have manifested. The pressing question is whether it is more efficacious to commence exercise therapy at the same time as initiating androgen deprivation, so treatment induced adverse effects can be immediately attenuated or indeed prevented.
Methods/design
We are proposing a multi-site randomized controlled trial with partial crossover to examine the effects of timing of exercise implementation (immediate or delayed) on preserving long-term skeletal health, reversing short- and long-term metabolic and cardiovascular risk factors, and supporting mental health in men receiving androgen deprivation therapy. 124 men who are about to initiate androgen deprivation for prostate cancer will be randomized to immediate or delayed groups. Immediate will commence a 6-month exercise program within 7–10 days of their first dose. Delayed will receive usual care for 6 months and then commence the exercise program for 6 months (partial cross-over). Immediate will be free to adopt the lifestyle of their choosing following the initial 6-month intervention. Measurements for primary and secondary endpoints will take place at baseline, 6 months and 12 months.
Discussion
This project is unique as it explores a fundamental question of when exercise implementation will be of most benefit and addresses both physical and psychological consequences of androgen deprivation initiation. The final outcome may be adjunct treatment which will reduce if not prevent the toxicities of androgen deprivation, ultimately resulting in reduced morbidity and mortality for men with prostate cancer.
Trial registration
ACTRN12612000097842
doi:10.1186/1471-2407-12-432
PMCID: PMC3519595  PMID: 23013489
Prostate cancer; Androgen deprivation therapy; Exercise; Resistance training; Aerobic training; Side-effects
25.  Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase 
Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their struture activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.
doi:10.1016/j.bmcl.2011.05.129
PMCID: PMC3139024  PMID: 21719286
Human peptide deformylase; Benzofuran-4,5-diones; Structure activity relationships; Fluorescence polarization; Antiproliferative agents

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