Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new antifungal drugs targeted to fungal virulence proteins using C. albicans as a model fungal pathogen.
Automated syndrome classification aims to aid near real-time syndromic
surveillance to serve as an early warning system for disease outbreaks,
using Emergency Department (ED) data. We present a system that improves the
automatic classification of an ED record with triage note into one or more
syndrome categories using the vector space model coupled with a
‘learning’ module that employs a pseudo-relevance feedback
mechanism. Materials and Methods: Terms from standard syndrome
definitions are used to construct an initial reference dictionary for
generating the syndrome and triage note vectors. Based on cosine similarity
between the vectors, each record is classified into a syndrome category. We
then take terms from the top-ranked records that belong to the syndrome of
interest as feedback. These terms are added to the reference dictionary and
the process is repeated to determine the final classification. The system
was tested on two different datasets for each of three syndromes:
Gastro-Intestinal (GI), Respiratory (Resp) and Fever-Rash (FR). Performance
was measured in terms of sensitivity (Se) and specificity (Sp).
Results: The use of relevance feedback produced high values
of sensitivity and specificity for all three syndromes in both test sets:
GI: 90% and 71%, Resp: 97% and 73%, FR: 100% and 87%, respectively, in test
set 1, and GI: 88% and 69%, Resp: 87% and 61%, FR: 97% and 71%,
respectively, in test set 2. Conclusions: The new system for
pre-processing and syndromic classification of ED records with triage notes
achieved improvements in Se and Sp. Our results also demonstrate that the
system can be tuned to achieve different levels of performance based on user
Disease outbreaks; electronic health records/classification; machine learning; natural language processing; public health informatics; public health surveillance/methods
Electronic laboratory reporting (ELR) reduces the time between communicable disease diagnosis and case reporting to local health departments (LHDs). However, it also imposes burdens on public health agencies, such as increases in the number of unique and duplicate case reports. We assessed how ELR affects the timeliness and accuracy of case report processing within public health agencies.
Using data from May–August 2010 and January–March 2012, we assessed timeliness by calculating the time between receiving a case at the LHD and reporting the case to the state (first stage of reporting) and between submitting the report to the state and submitting it to the Centers for Disease Control and Prevention (second stage of reporting). We assessed accuracy by calculating the proportion of cases returned to the LHD for changes or additional information. We compared timeliness and accuracy for ELR and non-ELR cases.
ELR was associated with decreases in case processing time (median = 40 days for ELR cases vs. 52 days for non-ELR cases in 2010; median = 20 days for ELR cases vs. 25 days for non-ELR cases in 2012; both p<0.001). ELR also allowed time to reduce the backlog of unreported cases. Finally, ELR was associated with higher case reporting accuracy (in 2010, 2% of ELR case reports vs. 8% of non-ELR case reports were returned; in 2012, 2% of ELR case reports vs. 6% of non-ELR case reports were returned; both p<0.001).
The overall impact of increased ELR is more efficient case processing at both local and state levels.
ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anti-cancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.
Efflux inhibition; ABCB1; ABCC1; ABCG2; Fluorescent substrate; Flow cytometry
Background: Air pollution epidemiologic studies, often conducted in large metropolitan areas because of proximity to regulatory monitors, are limited in their ability to examine potential associations between air pollution exposures and health effects in rural locations.
Methods: Using a time-stratified case-crossover framework, we examined associations between asthma emergency department (ED) visits in North Carolina (2006–2008), collected by a surveillance system, and short-term ozone (O3) exposures using predicted concentrations from the Community Multiscale Air Quality (CMAQ) model. We estimated associations by county groupings based on four urbanicity classifications (representative of county size and urban proximity) and county health.
Results: O3 was associated with asthma ED visits in all-year and warm season (April–October) analyses [odds ratio (OR) = 1.019; 95% CI: 0.998, 1.040; OR = 1.020; 95% CI: 0.997, 1.044, respectively, for a 20-ppb increase in lag 0–2 days O3]. The association was strongest in Less Urbanized counties, with no evidence of a positive association in Rural counties. Associations were similar when adjusted for fine particulate matter in copollutant models. Associations were stronger for children (5–17 years of age) compared with other age groups, and for individuals living in counties identified with poorer health status compared with counties that had the highest health rankings, although estimated associations for these subgroups had larger uncertainty.
Conclusions: Associations between short-term O3 exposures and asthma ED visits differed by overall county health and urbanicity, with stronger associations in Less Urbanized counties, and no positive association in Rural counties. Results also suggest that children are at increased risk of O3-related respiratory effects.
Citation: Sacks JD, Rappold AG, Davis JA Jr, Richardson DB, Waller AE, Luben TJ. 2014. Influence of urbanicity and county characteristics on the association between ozone and asthma emergency department visits in North Carolina. Environ Health Perspect 122:506–512; http://dx.doi.org/10.1289/ehp.1306940
Drug overdose; Public health surveillance; Emergency department data
Chemotherapeutics tumor resistance is a principal reason for treatment failure and clinical and experimental data indicate that multidrug transporters such as ATP-binding Cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate we identified a piperazine substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused SAR-driven chemistry effort we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2 over-expressing tumor model. At least two analogs significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.
Multi-drug resistance; ABC Transporter; ABCG2; ABCB1; Efflux inhibition
When using emergency department (ED) data sets for public health surveillance, a standard approach is needed to define visits attributable to asthma. Asthma can be the first (primary) or a subsequent (2nd through 11th) diagnosis. Our study objective was to develop a definition of ED visits attributable to asthma for public health surveillance. We evaluated the effect of including visits with an asthma diagnosis in primary-only versus subsequent positions.
The study was a cross-sectional analysis of population-level ED surveillance data. Of the 114 North Carolina EDs eligible to participate in a statewide surveillance system in 2008–2009, we used data from the 111 (97%) that participated during those years. Included were all ED visits with an ICD-9-CM diagnosis code for asthma in any diagnosis position (1 through 11). We formed 11 strata based on the diagnosis position of asthma and described common chief complaint and primary diagnosis categories for each. Prevalence ratios compared each category’s proportion of visits that received either asthma- or cardiac-related procedure codes.
Respiratory diagnoses were most common in records of ED visits in which asthma was the first or second diagnosis, while primary diagnoses of injury and heart disease were more common when asthma appeared in positions 3–11. Asthma-related chief complaints and procedures were most common when asthma was the first or second diagnosis, whereas cardiac procedures were more common in records with asthma in positions 3–11.
ED visits should be defined as asthma-related when asthma is in the first or second diagnosis position.
Public health officials use syndromic surveillance systems to facilitate early detection and response to infectious disease outbreaks. Emergency department clinical notes are becoming more available for surveillance but present the challenge of accurately extracting concepts from these text data. The purpose of this study was to implement a new system, Emergency Medical Text Classifier (EMT-C), into daily production for syndromic surveillance and evaluate system performance and user satisfaction. The system was designed to meet user preferences for a syndromic classifier that maximized positive predictive value and minimized false positives in order to provide a manageable workload. EMT-C performed better than the baseline system on all metrics and users were slightly more satisfied with it. It is vital to obtain user input and test new systems in the production environment.
Emergency department (ED) visits are made by cancer patients for symptom management, treatment effects, oncologic emergencies, or end of life care. While most patients prefer to die at home, many die in health care institutions. The purpose of this study is to describe visit characteristics of cancer patients who died in the ED and their most common chief complaints using 2008 ED visit data from the North Carolina Disease Event Tracking and Epidemiologic Collection Tool (NC DETECT). Of the 37,760 cancer-related ED visits, 283 resulted in death. For lung cancer patients, 104 died in the ED with 70.9% dying on their first ED visit. Research on factors precipitating ED visits by cancer patients is needed to address end of life care needs.
Keywords: cancer; emergency department; death; dying; end of life; symptom management
A real-time surveillance method is developed with emphasis on rapid and accurate detection of emerging outbreaks. We develop a model with relatively weak assumptions regarding the latent processes generating the observed data, ensuring a robust prediction of the spatiotemporal incidence surface. Estimation occurs via a local linear fitting combined with day-of-week effects, where spatial smoothing is handled by a novel distance metric that adjusts for population density. Detection of emerging outbreaks is carried out via residual analysis. Both daily residuals and AR model-based de-trended residuals are used for detecting abnormalities in the data given that either a large daily residual or an increasing temporal trend in the residuals signals a potential outbreak, with the threshold for statistical significance determined using a resampling approach.
Disease surveillance; Local linear estimation; Residual analysis; Lattice Data; Time series modeling
Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (Ki) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase as well as serving as a model for other small molecular weight GTPase inhibitors.
Rab, Rho, Rac, Cdc42 and Ras GTPases; chemical biology; drug discovery; therapeutics; fluorescent GTP and GDP; enzyme kinetics
Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt® high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of ~25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells.
HyperCyt® high-throughput flow cytometry; small molecule screening; intracellular granularity; prostate cancer
We sought to describe the integration of syndromic surveillance data into daily surveillance practice at local health departments (LHDs) and make recommendations for the effective integration of syndromic and reportable disease data for public health use.
Structured interviews were conducted with local health directors and communicable disease nursing staff from a stratified random sample of LHDs from May through September 2009. Interviews captured information on direct access to the North Carolina syndromic surveillance system and on the use of syndromic surveillance information for outbreak management, program management, and the creation of reports. We analyzed syndromic surveillance system data to assess the number of signals resulting in a public health response.
Syndromic surveillance data were used for outbreak investigation (19% of respondents) and program management and report writing (43% of respondents); a minority reported use of both syndromic and reportable disease data for these purposes (15% and 23%, respectively). Receiving data from frequent system users was associated with using data for these purposes (p=0.016 and p=0.033, respectively, for syndromic and reportable disease data). A small proportion of signals (<25%) resulted in a public health response.
Use of syndromic surveillance data by North Carolina local public health authorities resulted in meaningful public health action, including both case investigation and program management. While useful, the syndromic surveillance data system was oriented toward sensitivity rather than efficiency. Successful incorporation of new surveillance data is likely to require systems that are oriented toward efficiency.
TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.
To describe a collaboration with the Johns Hopkins Applied Physics Laboratory (JHU APL), the North Carolina Division of Public Health (NC DPH), and the UNC Department of Emergency Medicine Carolina Center for Health Informatics (CCHI) to implement time-of-arrival analysis (TOA) for hospital emergency department (ED) data in NC DETECT to identify clusters of ED visits for which there is no pre-defined syndrome or sub-syndrome.
TOA identifies clusters of patients arriving to a hospital ED within a short temporal interval. Past implementations have been restricted to records of patients with a specific type of complaint. The Florida Department of Health uses TOA at the county level for multiple sub-syndromes (1). In 2011, NC DPH, CCHI and CDC collaborated to enhance and evaluate this capability for NC DETECT, using NC DETECT data in BioSense 1.0 (2). After this successful evaluation based on exposure complaints, discussions were held to determine the best approach to implement this new algorithm into the production environment for NC DETECT. NC DPH was particularly interested in determining if TOA could be used for identifying clusters of ED visits not filtered by any syndrome or sub-syndrome. In other words, can TOA detect a cluster of ED visits relating to a public health event, even if symptoms from that event are not characterized by a predefined syndrome grouping? Syndromes are continuously added to NC DETECT but a syndrome cannot be created for every potential event of public health concern. This TOA approach is the first attempt to address this issue in NC DETECT. The initial goal is to identify clusters of related ED visits whose keywords, signs and/or symptoms are NOT all expressed by a traditional syndrome, e.g. rash, gastrointestinal, and flu-like illnesses. The goal instead is to identify clusters resulting from specific events or exposures regardless of how patients present – event concepts that are too numerous to pre-classify.
In late 2011, NC DPH and JHU APL signed a Software License Agreement and soon thereafter CCHI received the TOA software package. In May 2012, the TOA controller was adapted and set up to run against ED visit data for all NC DETECT hospitals. The TOA looks for clusters in all ED visits by hospital based solely on arrival time in both 30-minute and 60-minute intervals. There is no pre-classification of the chief complaints or triage notes into syndromes. TOA alerts are viewable on the NC DETECT Web application and, as of August 2012, users are able to document any actions taken on these alerts.
From April 15, 2012 to July 31, 2012, TOA generated 173 alerts across all 115 hospitals reporting to NC DETECT. The TOA identified a group of scabies-related ED visits that was not captured in another syndrome. The TOA also identified clusters identified by hospitals as disaster-related which included misspellings that had not been previously identified, e.g. “diaster” and “disater,” as well as events involving out-of-town groups that will not be identified spatially (Table 1). This preliminary review of TOA alerts did not evaluate TOA for false negatives.
Our preliminary review of TOA shows that this algorithm approach can be helpful for identifying clusters of ED visits that are not captured by existing syndromes and can be used to identify hospital coding schemes for disaster events. The TOA will continue to be monitored in our production environment and evaluated for additional effectiveness. We will also explore tools that will display counts of terms within a TOA alert to assist in signal investigation.
Cluster detection; Time-of-arrival analysis; Syndrome classification
To investigate hospital admissions and short-term return visits resulting from chronic obstructive pulmonary disease (COPD)-related emergency department (ED) visits.
COPD is a prevalent chronic disease among older adults; exacerbations often result in ED visits and subsequent hospital admissions. [1,2] A portion of such patients return to the ED within a few days or weeks.  In this study, we investigated patterns of hospital admissions and short-term return visits resulting from COPD-related ED visits.
We performed a population-based study of ED visits for COPD using state-wide surveillance data from NC DETECT, including all ED visits made by NC residents aged ≥45 years in 2008–2009. Visits were considered COPD-related if the first- or second-listed discharge diagnoses contained one of the following ICD-9-CM codes: 491.*, 492.*, 493.2*, 494.*, or 496.*. Hospital admissions were captured by ED disposition codes. If a patient had made another COPD-related ED visit within the prior 3 or 30 days, we defined the current visit as a 3-day or 30-day return visit. We compared the prevalence of hospitalization and 3- and 30-day return visits by age, sex, and payment method. We also described the disposition patterns for return visit pairs.
There were 97,511 COPD-related ED visits made by adults age 45 and older in NC in 2008–2009, made by 64,568 individuals. HOSPITAL ADMISSIONS: Nearly half (46.3%) of all COPD-related ED visits resulted in hospital admission. Hospitalization prevalence increased with age, but there were no differences by gender. ED visits that were non-insured (self-pay) or paid by Medicare or Medicaid were less likely to lead to hospitalization than those with private insurance. RETURN VISITS: 1.6% (1607) of the COPD-related ED visits were categorized as 3-day return visits and 11.2% (10922) were considered 30-day return visits. There were no statistical differences by gender for 3-day returns, while 30-day returns were more likely to be made by men. Prevalence of return visits for both intervals initially increased with age compared to the 45–49 years age group, then decreased steadily after age 65. Visits that were non-insured or paid by Medicare or Medicaid were statistically more likely to be 3-day or 30-day returns than those paid by private insurance. DISPOSITION PATTERNS: We also examined the permutations of 1st and 2nd ED visit dispositions that make up these return visit pairs. While many return visits were discharged at both visits in the return visit pair, a substantial proportion were admitted at one or both visits. Surprisingly, in 8% of the 3-day return visit pairs, the patient was hospitalized at the 1st ED visit but yet still returned to the ED within 3 days; for the 30-day visit pairs, 37% returned despite the patient being admitted at the 1st visit.
This population-based study describes the short-term outcomes of a large number of COPD-related ED visits using a unique state-wide surveillance system. We found a high prevalence of hospital admissions and return ED visits, including many repeat hospitalizations. This study also demonstrates how surveillance data can be used for research on “acute on chronic” disease epidemiology.
Chronic obstructive pulmonary disease; Chronic disease surveillance; Emergency department data; Hospitalizations; Return visits
Our objective was to describe changes in use following syndromic surveillance system modifications and assess the effectiveness of these modifications.
Syndromic surveillance systems offer richer understanding of population health. However, because of their complexity, they are less used at small public health agencies, such as many local health departments (LHDs). The evolution of these systems has included modifying user interfaces for more efficient and effective use at the local level. The North Carolina Preparedness and Emergency Response Research Center previously evaluated use of syndromic surveillance information at LHDs in North Carolina. Since this time, both the NC DETECT system and distribution of syndromic surveillance information by the state public health agency have changed. This work describes use following these changes.
Data from NC DETECT were used to assess the number of users and usage time. Staff from 14 NC LHDs in 2009 and from 39 LHDs in 2012 were surveyed (May–August of 2009 and June of 2012) to gather information on the mode of access to syndromic surveillance information and how this information was used. Data were analyzed to assess the link between the mode of access and use of syndromic surveillance data.
System changes made between 2009 and 2012 included the creation of “dashboards” (Figure 1) which present users with LHD-specific charts and graphs upon login and increases in the distribution of syndromic surveillance information by the state public health agency. The number of LHD-based NC DETECT system users increased from 99 in 2009 to 175 in 2012. Sixty-two of 72 respondents completed the 2012 survey (86%). Syndromic surveillance information was used in 28/40 LHDs (70%) for key public health tasks. Among 20 NC EDSS leads reporting an outbreak in the past year, 25% reported using data from NC DETECT for outbreak response, compared to 23% in 2009 (Figure 2). Among 30 responding NC EDSS leads, 57% reported using data from NC DETECT to respond to seasonal events such as heat-related illness or influenza, compared to 46% in 2009. NC DETECT data were reported to have been used for program management by 30% (compared to 25% in 2009), and to have been used in reports by 33% (compared to 23% in 2009).
Changes in how syndromic surveillance information was distributed supported modest increases in use in LHDs. Because use of syndromic surveillance data at smaller LHDs is rare, these modest increases are important indicators of effective modification of the NC syndromic surveillance system.
evaluation; public health practice; syndromic surveillance; surveillance; local health department
Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases.
Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays.
Conclusion: The identified compound is a highly Cdc42-selective inhibitor.
Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research.
Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.
Cdc42; Cytoskeleton; GTPase; Integrin; Migration
Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small molecule inhibitors. We describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different GST-tagged small GTPases were bound to glutathione beads each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This novel multiplex assay allowed us to screen a library of almost 200,000 compounds and identify over 1,200 positive compounds, which were further verified by dose response analyses, using 6 to 8-plex assays. After the elimination of false positive and negative compounds, several small molecule families with opposing effects on GTP-binding activity were identified. Here we detail the characterization of MLS000532223, a general inhibitor that prevents GTP-binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high throughput screening (HTS) via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.
Ras; Rab and Rho GTPases; actin cytoskeleton; bead-based multiplex assay; flow cytometry; fluorescent GTP binding
ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.
Emergency departments (EDs) in the United States are used by patients with cancer for disease or treatment-related problems and unrelated issues. The North Carolina Disease Event Tracking and Epidemiologic Collection Tool (NC DETECT) collects information about ED visits through a statewide database.
Patients and Methods
After approval by the institutional review board, 2008 NC DETECT ED visit data were acquired and cancer-related visits were identified. Descriptive statistics and logistic regressions were performed. Of 4,190,911 ED visits in 2008, there were 37,760 ED visits by 27,644 patients with cancer.
Among patients, 77.2% had only one ED visit in 2008, the mean age was 64 years, and there were slightly more men than women. Among visits, the payor was Medicare for 52.4% and Medicaid for 12.1%. More than half the visits by patients with cancer occurred on weekends or evenings, and 44.9% occurred during normal hours. The top three chief complaints were related to pain, respiratory distress, and GI issues. Lung, breast, prostate, and colorectal cancers were identified in 26.9%, 6.3%, 6%, and 7.7% of visits, respectively, with diagnosis. A total of 63.2% of visits resulted in hospital admittance. When controlling for sex, age, time of day, day of week, insurance, and diagnosis position, patients with lung cancer were more likely to be admitted than patients with other types of cancer.
To the best of our knowledge, this is the first study to provide a population-based snapshot of ED visits by patients with cancer in North Carolina. Efforts that target clinical problems and specific populations may improve delivery of quality cancer care and avoid ED visits.
Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the “valley of death” by bridging basic to clinical sciences.
Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by Gαi-coupled G protein-coupled receptors (GPCRs) and deactivated by Gαs-coupled GPCRs. The effects of Gαs-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific α I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step.
G Protein-coupled Receptors (GPCR); Integrin; Kinetics; Leukocyte; Receptor Regulation; Binding; LFA-1; Ligand; Receptor