Traditional Screening Approaches Research Articles

Selection of DNA-encoded chemical libraries against endogenous membrane proteins on live cells.

Membrane proteins on the cell surface perform a myriad of biological functions; however, ligand discovery for membrane proteins is highly challenging, because a natural cellular environment is often necessary to maintain protein structure and function. DNA-encoded chemical libraries (DELs) have emerged as a powerful technology for ligand discovery, but they are mainly limited to purified proteins. Here we report a method that can specifically label membrane proteins with a DNA tag, and thereby enable target-specific DEL selections against endogenous membrane proteins on live cells without overexpression or any other genetic manipulation. We demonstrate the generality and performance of this method by screening a 30.42-million-compound DEL against the folate receptor, carbonic anhydrase 12 and the epidermal growth factor receptor on live cells, and identify and validate a series of novel ligands for these targets. Given the high therapeutic significance of membrane proteins and their intractability to traditional high-throughput screening approaches, this method has the potential to facilitate membrane-protein-based drug discovery by harnessing the power of DEL.

Connectivity Enhanced Structure Activity Relationship (ceSAR): A Novel Approach to Increase Accuracy and Speed of Virtual Drug Discovery

Experimental drug discovery and repurposing is a costly and time‐consuming process. Here, a novel approach, dubbed connectivity enhanced structure activity relationship (ceSAR) is introduced. ceSAR combines traditional virtual screening methodologies with cheminformatics and Library of Integrated Network‐Based Cellular Signatures (LINCS) gene expression signatures of genetic and chemical perturbations. This methodology combines structural similarity of small molecules with transcriptional connectivity and virtual screening‐predicted specific interacting molecules for the enhancement of structure activity relationship (SAR) and lead compound discovery. The transcriptional connectivity aspect of ceSAR is implemented as an R Shiny package, titled Sig2Lead, that employs connectivity between chemical and genetic signatures included in the LINCS library to identify small molecule inhibitors of user‐targeted pathways. Chemical libraries are reduced with ceSAR by first measuring concordance of transcriptomic signatures between genetic and chemical perturbation, identifying putative pathway inhibitors, followed by traditional docking approaches to re‐score the remaining library to identify compounds likely to interact with the target of interest. This combination identifies compounds that are both pathway inhibitors and have shape complementarity to the target of interest, making the most likely scenario that the molecules are directly interacting with the target protein of interest to modulate the pathway. Through integration of these orthogonal approaches, both an increased accuracy and speed can be observed, with the speed increase being several orders of magnitude.Support or Funding InformationThis work was supported in part by the National Institutes of Health grants U54 HL127624ceSAR combines transcriptional connectivity and virtual screening to identify novel inhibitors from a screened chemical library. The library is first reduced using transcriptional connectivity to a knockdown of the target gene to restrict the search to putative pathway inhibitors. These pathway inhibitors are then reduced further using traditional virtual screening approaches to find compounds with shape complementarity to the protein of interest.Figure 1Comparison of results of an early drug screen using a combination of docking with AutoDock4.2.6 and Sig2Lead shows a dramatic reduction in compounds without losing the best‐performing compounds. If used in this case, the ceSAR method would have excluded 51 of the 147 tested compounds, of which 45 had no observable IC50 via a Fluorescence Polarization assay, thus driving library enrichment. This reduction would have saved time and reagents through preventing testing of as many spurious compounds.Figure 2

Discovery of Small-Molecule Stabilizers of 14-3-3 Protein-Protein Interactions via Dynamic Combinatorial Chemistry.

Protein-protein interactions (PPIs) play an important role in numerous biological processes such as cell-cycle regulation and multiple diseases. The family of 14-3-3 proteins is an attractive target as they serve as binding partner to various proteins and are therefore capable of regulating their biological activities. Discovering small-molecule modulators, in particular stabilizers, of such complexes via traditional screening approaches is a challenging task. Herein, we pioneered the first application of dynamic combinatorial chemistry (DCC) to a PPI target, to find modulators of 14-3-3 proteins. Evaluation of the amplified hits from the DCC experiments for their binding affinity via surface plasmon resonance (SPR), revealed that the low-micromolar (K D 15-16 μM) acylhydrazones are 14-3-3/synaptopodin PPI stabilizers. Thus, DCC appears to be ideally suited for the discovery of not only modulators but even the more elusive stabilizers of notoriously challenging PPIs.

A drug discovery platform to identify compounds that inhibit EGFR triple mutants.

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

Competitive profiling for enzyme inhibitors using chemical probes.

Enzyme inhibitors are central tools for chemical biology. In this chapter we will discuss the application of chemical probes for competitive profiling of inhibitors of the quinolone biosynthesis enzyme PqsD of Pseudomonas aeruginosa. The human pathogen P. aeruginosa produces a large diversity of 2-alkyl-4(1H)-quinolones and their derivatives as metabolites with major roles in quorum sensing, virulence, and interspecies competition. PqsD is a central enzyme in the biosynthesis of all of these quinolones and hence an interesting target for inhibitor discovery. Activity-based probes with an electrophilic warhead bind covalently to active site nucleophiles like cysteine or serine. An α-chloroacetamide probe with terminal alkyne tag allowed to selectively label the active site cysteine of PqsD and was demonstrated to be a useful tool for inhibitor discovery using competition experiments. Potent inhibitors bind to the active site and thereby prevent labeling of the enzyme by the probe. Labeling intensity is quantified on polyacrylamide gels by the fluorescence of a reporter tag appended by bioorthogonal click chemistry. The competitive inhibitor profiling strategy has many advantages over traditional screening approaches and is applicable in vitro as well as in live cells. Here we describe the synthesis of an activity-based probe and provide our detailed protocols for target enzyme labeling as well as its application for the screening for potent enzyme inhibitors of PqsD by a competitive profiling strategy.

Can Machine Learning Improve Screening for Targeted Delinquency Prevention Programs?

The cost-effectiveness of targeted delinquency prevention programs for children depends on the accuracy of the screening process. Screening accuracy is often poor, resulting in wasted resources and missed opportunities to avert negative outcomes. This study examined whether screening approaches based on logistic regression or machine learning algorithms could improve accuracy relative to traditional sum-score approaches when identifying boys in the 5th grade (N = 1012) who would be repeatedly arrested for violent and serious crimes from ages 13 to 30. Screening algorithms were developed that incorporated facets of teacher-reported externalizing problems and other known risk factors (e.g., peer rejection). The predictive performance of these algorithms was evaluated and compared in holdout (i.e., test) data using the area under the receiver operating curve (AUROC) and Brier score. Both the logistic and machine learning methods yielded AUROC superior to traditional sum-score screening approaches when a broad set of risk factors for future delinquency was considered. However, this improvement was modest and was not present when using item-level information from a composite scale assessing externalizing problems. Contrary to expectations, machine learning algorithms performed no better than simple logistic models. There was a large apparent advantage of machine learning that disappeared after appropriate cross-validation, underscoring the importance of careful evaluation of these methods. Results suggest that screening using logistic regression could improve the cost-effectiveness of targeted delinquency prevention programs in some cases, but screening using machine learning would confer no marginal benefit under currently realistic conditions.

Evolution-based screening enables genome-wide prioritization and discovery of DNA repair genes

DNA repair is critical for genome stability and is maintained through conserved pathways. Traditional genome-wide mammalian screens are both expensive and laborious. However, computational approaches circumvent these limitations and are a powerful tool to identify new DNA repair factors. By analyzing the evolutionary relationships between genes in the major DNA repair pathways, we uncovered functional relationships between individual genes and identified partners. Here we ranked 17,487 mammalian genes for coevolution with 6 distinct DNA repair pathways. Direct comparison to genetic screens for homologous recombination or Fanconi anemia factors indicates that our evolution-based screen is comparable, if not superior, to traditional screening approaches. Demonstrating the utility of our strategy, we identify a role for the DNA damage-induced apoptosis suppressor (DDIAS) gene in double-strand break repair based on its coevolution with homologous recombination. DDIAS knockdown results in DNA double-strand breaks, indicated by ATM kinase activation and 53BP1 foci induction. Additionally, DDIAS-depleted cells are deficient for homologous recombination. Our results reveal that evolutionary analysis is a powerful tool to uncover novel factors and functional relationships in DNA repair.

Clinical application of noninvasive prenatal screening for sex chromosome aneuploidies in 50,301 pregnancies: initial experience in a Chinese hospital.

To evaluate the clinical performance of noninvasive prenatal screening (NIPS) for fetal sex chromosome aneuploidies (SCAs), pregnant women were recruited in this retrospective observational study. The NIPS test was undertaken using high-throughput gene sequencing. In total,50,301 pregnant women were analysed for demographic characteristics and medical history. Of them, 308 women (0.61%) had high risk for fetal SCAs, including 138 for 45,X, 111 for 47,XXY, 42 for 47,XXX, and 17 for 47,XYY. After the pre-test counselling, 182 participants chose to undergo invasive prenatal diagnosis, confirming 59 positive cases. The combined positive predictive value of NIPS was 32.42% (59/182), 18.39% (16/87), 44.4% (12/27), 39.29% (22/56), and 75% (9/12) for detecting SCAs, 45,X, 47,XXX, 47,XXY, and 47,XYY, respectively. NIPS can be a useful method to detect the fetal SCAs using high-throughput gene sequencing, though accuracy can still be improved, especially for 45,X. Although the value of NIPS compare favorably with those seen in traditional screening approaches for SCAs, it is important to highlight the limitations of NIPS while educating clinicians and patients.

Identification of novel antimicrobial-producing bacteria from an ancient water source by Oxford Nanopore Whole Genome Sequencing and Natural Product Chemistry

Microbially-derived antimicrobial compounds are a rich source of clinical antibiotic leads. However, discovery rates have declined over the past 40 years due, in part, to high rediscovery rates of known compounds from traditional soil-based screening approaches. In this study, an ancient hot-spring water source was tested for the presence of antimicrobial-producing bacteria using culture techniques which led to isolation of two organisms capable of inhibiting the growth of multiple bacterial species. Oxford Nanopore whole genome sequencing was used to identify these two isolates as being in one of two genera; Streptomyces and Paenibacillus. Bioinformatic analysis revealed both isolates to have multiple novel secondary metabolite gene clusters. Investigations of the Streptomyces sp. by natural product chemistry techniques showed the organism to produce multiple antimicrobial compounds, these were effective methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. This study underlines the value of investigating non-traditional habitats in the search for novel antibiotic-producing organisms.

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Positive predictive value estimates for cell-free noninvasive prenatal screening from data of a large referral genetic diagnostic laboratory

Since its debut in 2011, cell-free fetal DNA screening has undergone rapid expansion with respect to both utilization and coverage. However, conclusive data regarding the clinical validity and utility of this screening tool, both for the originally included common autosomal and sex-chromosomal aneuploidies as well as the more recently added chromosomal microdeletion syndromes, have lagged behind. Thus, there is a continued need to educate clinicians and patients about the current benefits and limitations of this screening tool to inform pre- and posttest counseling, pre/perinatal decision making, and medical risk assessment/management. The objective of this study was to determine the positive predictive value and false-positive rates for different chromosomal abnormalities identified by cell-free fetal DNA screening using a large data set of diagnostic testing results on invasive samples submitted to the laboratory for confirmatory studies. We tested 712 patient samples sent to our laboratory to confirm a cell-free fetal DNA screening result, indicating high risk for a chromosome abnormality. We compiled data from all cases in which the indication for confirmatory testing was a positive cell-free fetal DNA screen, including the common trisomies, sex chromosomal aneuploidies, microdeletion syndromes, and other large genome-wide copy number abnormalities. Testing modalities included fluorescence in situ hybridization, G-banded karyotype, and/or chromosomal microarray analysis performed on chorionic villus samples, amniotic fluid, or postnatally obtained blood samples. Positive predictive values and false-positive rates were calculated from tabulated data. The positive predictive values for trisomy 13, 18, and 21 were consistent with previous reports at 45%, 76%, and 84%, respectively. For the microdeletion syndrome regions, positive predictive values ranged from 0% for detection of Cri-du-Chat syndrome and Prader-Willi/Angelman syndrome to 14% for 1p36 deletion syndrome and 21% for 22q11.2 deletion syndrome. Detection of sex chromosomal aneuploidies had positive predictive values of 26% for monosomy X, 50% for 47,XXX, and 86% for 47,XXY. The positive predictive values for detection of common autosomal and sex chromosomal aneuploidies by cell-free fetal DNA screening were comparable with other studies. Identification of microdeletions was associated with lower positive predictive values and higher false-positive rates, likely because of the low prevalence of the individual targeted microdeletion syndromes in the general population. Although the obtained positive predictive values compare favorably with those seen in traditional screening approaches for common aneuploidies, they highlight the importance of educating clinicians and patients on the limitations of cell-free fetal DNA screening tests. Improvement of the cell-free fetal DNA screening technology and continued monitoring of its performance after introduction into clinical practice will be important to fully establish its clinical utility. Nonetheless, our data provide valuable information that may aid result interpretation, patient counseling, and clinical decision making/management.

Cost savings from a teledentistry model for school dental screening: an Australian health system perspective.

Objective The aim of the present study was to compare the costs of teledentistry and traditional dental screening approaches in Australian school children. Methods A cost-minimisation analysis was performed from the perspective of the oral health system, comparing the cost of dental screening in school children using a traditional visual examination approach with the cost of mid-level dental practitioners (MLDPs), such as dental therapists, screening the same cohort of children remotely using teledentistry. A model was developed to simulate the costs (over a 12-month period) of the two models of dental screening for all school children (2.7million children) aged 5-14 years across all Australian states and territories. The fixed costs and the variable costs, including staff salary, travel and accommodation costs, and cost of supply were calculated. All costs are given in Australian dollars. Results The total estimated cost of the teledentistry model was $50million. The fixed cost of teledentistry was $1million and that of staff salaries (tele-assistants, charters and their supervisors, as well as information technology support was estimated to be $49million. The estimated staff salary saved with the teledentistry model was $56million, and the estimated travel allowance and supply expenses avoided were $16million and $14million respectively; an annual reduction of $85million in total. Conclusions The present study shows that the teledentistry model of dental screening can minimise costs. The estimated savings were due primarily to the low salaries of dental therapists and the avoidance of travel and accommodation costs. Such savings could be redistributed to improve infrastructure and oral health services in rural or other underserved areas. What is known about the topic? Caries is a preventable disease, which, if it remains untreated, can cause significant morbidity requiring costly treatment. Regular dental screening and oral health education have the great potential to improve oral health and save significant resources. The use of role substitution, such as using MLDPs to provide oral care has been well acknowledged worldwide because of their ability to provide safe and effective care. The teledentistry approach for dental screening offers a comparable diagnostic performance to the traditional visual approach. What does this paper add? The results of the present study suggest that teledentistry is a practical and economically viable approach for mass dental screening not only for isolated communities, but also for underserved urban communities. The costs of the teledentistry model were substantially lower than the costs associated with a conventional, face-to-face approach to dental screening in both remote and urban areas. The primary driver of net savings is the low salary of MLDPs and avoidance of travel and overnight accommodation by MLDPs. What are the implications for practitioners? The use of lower-cost MLDPs and a teledentistry model for dental screening has the potential to save significant economic and human resources that can be redirected to improve infrastructure and oral care services in underserved regions. In the absence of evidence of the economic usefulness of teledentistry, studies such as the present one can increase the acceptance of this technology among dental care providers and guide future decisions on whether or not to implement teledentistry services.

Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.

Abstract 4189: Cancer drugs on the fly: whole-animal chemical screening in Drosophila identifies unexpected role for EGFR in RAF intestinal stem cell tumors

Abstract A major challenge in cancer therapeutics is to identify drugs that target cancer stem cells. This presents a problem for traditional cell-based screening approaches because the complex cellular interactions underlying stem cell biology are difficult to recapitulate in vitro. Conversely, mice, which are arguably among the best animal models for cancer—are not amenable to high-throughput screening. However, many aspects of stem cell initiated tumorigenesis can be modeled in the fruit fly Drosophila melanogaster, which due to its small size is well suited for large-scale screens. The Drosophila intestine, in particular, presents a compelling model to study stem-cell derived intestinal tumors because Drosophila intestinal stem cells share many features with their mammalian counterparts including a propensity to form tumors in response to mutations in WNT, NOTCH, or EGFR signaling. We therefore developed an in vivo screening pipeline using adult Drosophila, to identify compounds that can suppress the growth of stem cell initiated tumors. In previous work we showed that ectopically expressing a constitutively active human c-RAF transgene, RAF(gof) in Drosophila intestinal stem cells results in fast growing tumors that are sensitive to several FDA approved chemotherapeutics. These results establish the clinical relevance of using Drosophila to model stem cell cancers. Here we present results from a screen of known bioactives, that reveal an unexpected role of the EGF receptor, EGFR, in RAF(gof) intestinal stem cell tumors. Based on the linear order of genes in the canonical RAF pathway—EGFR→RAS→RAF→MEK→ERK—we expected that drugs that inhibit genes acting downstream of RAF would score as hits in the screen, whereas genes that act upstream of RAF would not. Indeed, that same logic is applied when assessing whether to give human patients certain pathway specific drugs: patients with activating mutations in RAS or RAF are typically not given drugs that target the upstream EGFR. Consistent with our expectation, we found that drugs that inhibit genes downstream of RAF, effectively stopped Raf(gof) tumor growth. However, to our surprise, we also found that inhibitors of EGFR blocked the growth of RAF(gof) tumors. Using RNAi against EGFR, we genetically validated the chemical results. Our preliminary evidence suggests that EGFR is required in a parallel pathway, upstream of AKT. These findings are significant because they suggest that in some cases, human patients with RAF activated tumors may benefit from drug therapies that target EGFR. Importantly, our results demonstrate that taking an unbiased approach in assessing which drug therapies to apply to a given cancer, may yield unexpectedly good results. Citation Format: Michele Markstein, Hannah Dayton, Samantha Dettorre. Cancer drugs on the fly: whole-animal chemical screening in Drosophila identifies unexpected role for EGFR in RAF intestinal stem cell tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4189.

Unexpected Challenges in Noninvasive Prenatal Testing.

The clinical paradigm for prenatal screening for fetal chromosome aneuploidies has been transformed by the introduction of noninvasive prenatal testing (NIPT). NIPT sequences cell-free DNA from maternal plasma, which contains a mixture of small maternal DNA fragments as well as placental DNA fragments that serve as a fetal surrogate. The relative proportion of cell-free fetal DNA increases with gestational age and can reliably be detected by 9–10 weeks of gestation. As sequencing technologies have improved and become less expensive, there has been a rapid adoption of NIPT since its commercial launch in late 2011. NIPT is a screening test, similar to traditional prenatal screening approaches that combine maternal serum screening plus ultrasound. A principal advantage of NIPT is that the false-positive rate is <0.2% compared to an average of 5% for traditional screening methods. Confirmatory …

Next-generation sequencing approach for connecting secondary metabolites to biosynthetic gene clusters in fungi.

Genomics has revolutionized the research on fungal secondary metabolite (SM) biosynthesis. To elucidate the molecular and enzymatic mechanisms underlying the biosynthesis of a specific SM compound, the important first step is often to find the genes that responsible for its synthesis. The accessibility to fungal genome sequences allows the bypass of the cumbersome traditional library construction and screening approach. The advance in next-generation sequencing (NGS) technologies have further improved the speed and reduced the cost of microbial genome sequencing in the past few years, which has accelerated the research in this field. Here, we will present an example work flow for identifying the gene cluster encoding the biosynthesis of SMs of interest using an NGS approach. We will also review the different strategies that can be employed to pinpoint the targeted gene clusters rapidly by giving several examples stemming from our work.

Noninvasive Prenatal Testing: An Update on Prenatal Screening and Testing Options

The landmark discovery that cell-free fetal DNA was present in maternal plasma paved the way for significant changes in prenatal screening and diagnosis. Although traditional screening approaches relied on measuring maternal serum markers and ultrasound findings, analyzing cell-free fetal DNA held the promise of increasing the sensitivity and specificity of prenatal screening. Nearly 15 years after the initial discovery, this testing finally became a reality with the publication of several large clinical trials that validated its use for aneuploidy screening. We review several approaches for noninvasive prenatal testing that are currently available, including whole genome massively parallel shotgun sequencing, targeted sequencing, and analysis of single nucleotide polymorphisms. Although noninvasive prenatal testing holds great promise, it is important that patients receive adequate counseling of its benefits and limitations. It is anticipated that this field will continue to experience rapid growth, and the ability to test for smaller genomic changes and single gene disorders by using this noninvasive testing will likely follow.

Molecular detection of bacterial contamination in gnotobiotic rodent units

Gnotobiotic rodents provide an important technique to study the functional roles of commensal bacteria in host physiology and pathophysiology. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches of culturing and Gram staining feces have inherent limitations, as many bacteria are uncultivable and fecal Gram stains are difficult to interpret. Thus, we developed and validated molecular methods to definitively detect and identify contamination in germ-free (GF) and selectively colonized animals. Fresh fecal pellets were collected from rodents housed in GF isolators, spontaneously contaminated ex-GF isolators, selectively colonized isolators and specific pathogen-free (SPF) conditions. DNA isolated from mouse and rat fecal samples was amplified by polymerase chain reaction (PCR) and subjected to quantitative PCR (qPCR) using universal primers that amplify the 16S rRNA gene from all bacterial groups. PCR products were sequenced to identify contaminating bacterial species. Random amplification of polymorphic DNA (RAPD) PCR profiles verified bacterial inoculation of selectively colonized animals. These PCR techniques more accurately detected and identified GF isolator contamination than current standard approaches. These molecular techniques can be utilized to more definitively screen GF and selectively colonized animals for bacterial contamination when Gram stain and/or culture results are un-interpretable or inconsistent.

A Microfluidic Platform for Evaporation-based Salt Screening of Pharmaceutical Parent compounds

We describe a microfluidic platform to screen for salt forms of pharmaceutical compounds (PCs) via controlled evaporation. The platform enables on-chip combinatorial mixing of PC and salt former solutions in a 24-well array (~200 nL/well), which is a drastic reduction in the amount of PC needed per condition screened compared to traditional screening approaches that require ~100 μL/well. The reduced sample needs enable salt screening at a much earlier stage in the drug development process, when only limited quantities of PCs are available. Compatibility with (i) solvents commonly used in the pharmaceutical industry, and (ii) Raman spectroscopy for solid form identification was ensured by using a hybrid microfluidic platform. A thin layer of elastomeric PDMS was utilized to retain pneumatic valving capabilities. This layer is sandwiched between layers of cyclic-olefin copolymer, a material with low air and solvent permeability and low Raman background to yield a physically rigid and Raman compatible chip. A solvent-impermeable thiolene layer patterned with evaporation channels permits control over the rate of solvent evaporation. Control over the rate of solvent evaporation (2-15 nL h(-1)) results in consistent, known rates of increase in the supersaturation levels attained on-chip, and increases the probability for crystalline solids to form. The modular nature of the platform enables on-chip Raman and birefringence analysis of the solid forms. Model compounds, tamoxifen and ephedrine, were used to validate the platform's ability to screen for salts. On-chip Raman analysis helped to identify six different salts each of tamoxifen and ephedrine.

Non-Traditional Antibacterial Screening Approaches for the Identification of Novel Inhibitors of the Glyoxylate Shunt in Gram-Negative Pathogens

Antibacterial compounds that affect bacterial viability have traditionally been identified, confirmed, and characterized in standard laboratory media. The historical success of identifying new antibiotics via this route has justifiably established a traditional means of screening for new antimicrobials. The emergence of multi-drug-resistant (MDR) bacterial pathogens has expedited the need for new antibiotics, though many in the industry have questioned the source(s) of these new compounds. As many pharmaceutical companies' chemical libraries have been exhaustively screened via the traditional route, we have concluded that all compounds with any antibacterial potential have been identified. While new compound libraries and platforms are being pursued, it also seems prudent to screen the libraries we currently have in hand using alternative screening approaches. One strategy involves screening under conditions that better reflect the environment pathogens experience during an infection, and identifying in vivo essential targets and pathways that are dispensable for growth in standard laboratory media in vitro. Here we describe a novel screening strategy for identifying compounds that inhibit the glyoxylate shunt in Pseudomonas aeruginosa, a pathway that is required for bacterial survival in the pulmonary environment. We demonstrate that these compounds, which were not previously identified using traditional screening approaches, have broad-spectrum antibacterial activity when they are tested under in vivo-relevant conditions. We also show that these compounds have potent activity on both enzymes that comprise the glyoxylate shunt, a feature that was supported by computational homology modeling. By dual-targeting both enzymes in this pathway, we would expect to see a reduced propensity for resistance development to these compounds. Taken together, these data suggest that understanding the in vivo environment that bacterial pathogens must tolerate, and adjusting the antibacterial screening paradigm to reflect those conditions, could identify novel antibiotics for the treatment of serious MDR pathogens.