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PubMed2002

Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection.

The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.

Anti-Inflammatory Agents↗
PubMed2026

Integrative chemical genetics platform identifies condensate modulators linked to neurological disorders.

Dysregulation of biomolecular condensates is implicated across multiple neurological disorders. However, approaches to systematically identify their modulators remain limited. Here, we expand the utility of MLF2 as a versatile condensate biomarker and develop CondenScreen, an integrated high-content screening and bioinformatics pipeline enabling identification of condensate modulators across chemical and genetic space. Screening 1760 bioactive compounds in a cellular DYT1 dystonia model, we validate the platform for condensate-targeted drug discovery, identifying drugs that prevent the accumulation of the MLF2 reporter into nuclear envelope condensates. In parallel, a genome-wide CRISPR/Cas9 screen correlates nuclear condensate abundance with genes implicated in microcephaly and over eight additional neurodevelopmental disorders. Machine learning and confocal imaging resolve distinct condensate phenotypes, with RNF26 deletion provoking nuclear envelope condensates that phenocopy hallmarks of torsin deficiency. Our study provides a scalable platform for identifying modulators of condensates and establishes a correlative connection between nuclear condensate accumulation and genes implicated in neurodevelopmental disorders.

Humans↗
PubMed2003

Identification of gap junction blockers using automated fluorescence microscopy imaging.

Gap junctions coordinate electrical signals and facilitate metabolic synchronization between cells. In this study, the authors have developed a novel assay for the identification of gap junction blockers using fluorescence microscopy imaging-based high-content screening technology. In the assay, the communication between neighboring cells through gap junctions was measured by following the redistribution of a fluorescent marker. The movement of calcein dye from dye-loaded donor cells to dye-free acceptor cells through gap junctions overexpressed on cell surface membranes was monitored using automated fluorescence microscopy imaging in a high-throughput compatible format. The fluorescence imaging technology consisted of automated focusing, image acquisition, image processing, and data mining. The authors have successfully performed a high-throughput screening of a 486,000- compound program with this assay, and they were able to identify false positives without additional experiments. Selective and pharmacologically interesting compounds were identified for further optimization.

Animals↗
PubMed2004

Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828.

A multiparametric high-content screening assay for measurement of apoptosis was developed. HeLa cells and lymphoma U-937 cells were exposed to cytotoxic drugs in flat-bottomed optical microtiter plates. After incubation, the DNA-binding dye Hoechst 33342, fluorescein-tagged probes that covalently bind active caspases and chloromethyl-X-rosamine to detect mitochondrial membrane potential (MMP) were added. Image acquisition and quantitative measurement of fluorescence in a defined number of cells per well was performed using the automated image capture and analysis instrument ArrayScan. The usefulness of the assay was tested in cells exposed to standard cytotoxic drugs as well as in experimental cytotoxic cyanoguanidine CHS 828. A time- and dose-dependent activation of caspase-3, decrease in MMP, and increase in nuclear fragmentation and condensation were observed for the standard drugs, with the ability to correlate the parameters on a single cell basis. CHS 828 induced caspase-3 activation and reduction in MMP with modest changes in nuclear morphology. The method described was considered to be a rapid and information-rich apoptosis assay suitable both for correlating morphological and biochemical apoptotic events in single cells as well as for screening and evaluation of novel substances with apoptosis-inducing capabilities.

Antineoplastic Agents↗
PubMed1991

Factors influencing determination of high-level aminoglycoside resistance in Enterococcus faecalis.

The ability of seven methods to detect high-level gentamicin (58 strains) and streptomycin resistance (56 strains) among 107 Enterococcus faecalis isolates was investigated at the University of Chicago Medical Center and the University of Nebraska Medical Center. Methods included a standard agar screen plate, high-content disk diffusion, Remel (Lenexa, Kans.) EF Synergy Quad plates, standard microdilution panels prepared in house, Pasco MIC Gram-Positive panels (Difco Laboratories, Detroit, Mich.), MicroScan MIC Type 5 dry panels (Baxter Healthcare Corp., MicroScan Div., West Sacramento, Calif.), and Vitek GPS-TA cards (Vitek Systems Inc., Hazelwood, Mo.). Results indicating false resistance were not obtained by any method, and there was 100% agreement between the results of the disk diffusion and standard agar screen methods. Prolonging incubation from 24 to 48 h increased resistance detection for both agar and microdilution screens. EF Synergy Quad plates inoculated with micropipettes detected 100% of the streptomycin- and gentamicin-resistant isolates. Resistance detection for streptomycin and gentamicin, respectively, was 93 and 96% by standard microdilution, 93 and 98% by Pasco panels, 88 and 89% by MicroScan panels, and 88 and 91% by Vitek GPS-TA cards. False susceptibility occurred more frequently with streptomycin-resistant isolates than it did with gentamicin-resistant strains and appeared to be strain related in some instances. The use of an increased inoculum size enhanced resistance detection with these strains, but it complicated interpretation of results and led to the selection of streptomycin-resistant mutants. Until results of further studies delineate optimum test conditions, a delay in the final interpretation of agar and microdilution screen results until 48 h for isolates showing no or light growth at 24 h may help to minimize the occurrence of false susceptibility reporting.

Anti-Bacterial Agents↗
PubMed2026

Field-isolate recombinant tick-borne encephalitis viruses define reporter-stability guidelines for antiviral testing in flaviviruses.

As arthropod-borne viruses continue to threaten populations globally, there is a pressing need for experimental systems that enable rapid antiviral discovery. Reverse-genetics platforms producing recombinant reporter orthoflaviviruses have been developed to address this gap. Here, we present two new recombinant tick-borne encephalitis viruses (TBEVrec) generated on a European-subtype Haselmühl Tiho1 isolate backbone. A reporter gene, either eGFP or Nluc, was inserted in the capsid-coding region of the genome downstream of the capsid RNA regulatory signal and separated from the complete viral polyprotein by a 2A self-cleaving peptide. TBEVrec was better rescued using the circular polymerase extension reaction (CPER) than with the infectious subgenomic amplicon (ISA) method. TBEVrec replicated efficiently in relevant human cell lines, with comparable replication to wild-type TBEV in a neuronal cell line and moderately reduced titers and RNA levels in immune-derived cell lines. Using either eGFP or Nluc, we illustrate how TBEVrec enabled high-content RNAi screening, highlighting Nucleolin and PRKD1 as potential TBEV host factors, and drug testing on a benchtop plate reader. Nanopore sequencing of the eGFP insert revealed that the reporter is excised without affecting flanking regions. Comparative analysis of eGFP and Nluc further shows that this instability is time- and cell type-dependent, and that Nluc is comparatively more stable. From these observations, we outline safeguards and design principles that are broadly applicable both to the rescue of existing constructs and to the design of future recombinant reporter virus platforms.

CPER↗
PubMed2025

Panduratin A Induces Autophagy Through AMPK Activation Independent of mTOR Inhibition and Restricts Mycobacterium tuberculosis in Host Macrophages.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health burden, especially with the increasing prevalence of drug-resistant strains. There is an urgent need for new therapeutics that act via alternative mechanisms. Autophagy, a vital cell-autonomous defense process, allows macrophages to degrade intracellular pathogens such as Mtb and has gained attention as a potential target for host-directed therapy. In this study, we conducted a high-content imaging screen of herb-derived compounds to identify autophagy inducers in RAW264.7 macrophages. Panduratin A (NPA), a natural compound from Boesenbergia rotunda, was found to potently induce autophagy. NPA promoted autophagic vacuole formation in a dose-dependent fashion at low micromolar levels. Its autophagy-inducing effect was validated using RFP-GFP-LC3 dual fluorescence assays and immunoblotting in the presence of bafilomycin A1. Further mechanistic analysis revealed that NPA activates autophagy through AMPK activation, independent of mTOR inhibition. Importantly, NPA significantly promoted intracellular Mtb clearance and increased colocalization of Mtb with autophagosomes and lysosomes, in a manner dependent on Beclin-1. These findings highlight NPA as a potent enhancer of macrophage antimicrobial responses via autophagy, supporting its potential as a candidate for host-directed adjunctive therapy against TB.

Autophagy↗
PubMed2003

A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells.

The PI3K/PTEN/Akt signal transduction pathway plays a key role in many tumors. Downstream targets of this pathway include the Forkhead family of transcription factors (FOXO1a, FOXO3a, FOXO4). In PTEN null cells, FOXO1a is inactivated by PI3K-dependent phosphorylation and mislocalization to the cytoplasm, yet still undergoes nucleocytoplasmic shuttling. Since forcible localization of FOXO1a to the nucleus can reverse tumorigenicity of PTEN null cells, a high-content, chemical genetic screen for inhibitors of FOXO1a nuclear export was performed. The compounds detected in the primary screen were retested in secondary assays, and structure-function relationships were identified. Novel general export inhibitors were found that react with CRM1 as well as a number of compounds that inhibit PI3K/Akt signaling, among which are included multiple antagonists of calmodulin signaling.

Active Transport, Cell Nucleus↗
PubMed2025

Monitoring Influenza A Virus Entry Using Quantitative Fluorescence Microscopy.

Influenza A virus (IAV) is a major threat to global human health and is a topic of intense research. With the continuous problem of seasonal influenza and the threat of potential pandemics due to frequent emergence of new viral strains, development of new, broad-spectrum antivirals is an urgent priority. In antiviral development against influenza, the process of host cell entry of IAV is of particular interest as inhibiting the virus at the entry step should stop infection early on, blocking the downstream infection processes including viral replication and transcription. Therefore, a detailed understanding of the IAV entry processes is essential to illuminate virus-assisting host factors that can serve as potentially valuable targets for therapeutic interventions. To accelerate the identification of novel antivirals or host-directed targets that play essential role in IAV entry, quantitative assays that can be used to monitor the virus at sequential entry steps would be important for performing high-content genetic or inhibitor screens. In this chapter, we describe how IAV entry can be monitored at the sequential entry steps, spanning from the initial attachment of the virus particle to the cell surface to the transmission of the viral genome to the nucleus, by fluorescence microscopy. Further, we provide the methods to quantify the images acquired with high-content microscope for each of the major IAV entry steps. The fluorescence microscopy-based IAV entry assays and the image quantification methods described here can be used to boost our understanding of the virus-host cell interactions and can lead to the discovery of novel host-directed prophylactic or therapeutic interventions.

Humans↗
PubMed2025

Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency.

Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12, ARIH2, and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo. CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced metabolic fitness, increased secretion of proinflammatory cytokines, and expansion of cytotoxic NK cell subsets. Through high-content genome-wide CRISPR screening in NK cells, this study reveals critical regulators of NK cell function and provides a valuable resource for engineering next-generation NK cell therapies with improved efficacy against cancer.

Humans↗
PubMed1995

Detection of aminoglycoside-penicillin synergy against Enterococcus faecium using high-content aminoglycoside disks.

Thirty-seven Enterococcus faecium strains were screened for high-level aminoglycoside resistance with an agar diffusion test using high-content aminoglycoside disks (300 micrograms of streptomycin and 120 micrograms of gentamicin, tobramycin, kanamycin or amikacin). The inhibition zones obtained were correlated with results of time-kill penicillin-aminoglycoside synergy studies. An 11 mm breakpoint differentiated strains susceptible or resistant to the synergy of streptomycin plus penicillin. Irrespective of the inhibition zones obtained with tobramycin and kanamycin disks, Enterococcus faecium strains never showed synergy with penicillin in combination with these aminoglycosides. Penicillin-amikacin synergy cannot be predicted by the amikacin disks. Nevertheless, even though kanamycin disks do not predict penicillin-kanamycin synergy, they can be used to predict penicillin-amikacin synergy. In summary, high-content streptomycin, gentamicin and kanamycin disks can be used to predict the susceptibility of Enterococcus faecium strains to the synergistic combination of penicillin plus one of the aminoglycosides (streptomycin, gentamicin or amikacin, respectively).

Aminoglycosides↗
PubMed2004

A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.

BACKGROUND: Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries. RESULTS: We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate. CONCLUSIONS: The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.

Actins↗
PubMed1998

Prevalence of high level aminoglycoside and vancomycin resistance among enterococci in Turkey.

Enterococcus spp. have become the third most common cause of nosocomial infections. High-level aminoglycoside resistance (HLAR), an important clinical concern, has been associated with some species of enterococci. The synergistic effect obtained by the combination of aminoglycosides with penicillin or vancomycin disappears in strains that show high-level resistance (HLR) to the aminoglycosides. We evaluated the agar dilution and high content disk diffusion tests for the detection of HLAR for 60 isolated enterococci. In our study 4 strains of Enterococcus faecalis (6.6%) and 8 strains of Enterococcus faecium (13.3%), totally 20% were found to be highly resistant to gentamicin; 2 strains of E. faecalis (13.3%), 8 strains of E. faecium (3.3%), totally 16.6% showed HLR to streptomycin by agar dilution method and the same rates were found by high-content disk diffusion method. 2 strains of E. faecalis (3.3%), 4 strains of E. faecium (6.6%), totally 10% showed vancomycin resistance by agar dilution method. The ratio for beta lactamase activity was found to be 26.6% for E. faecalis and 3.3% for E. faecium. Our results demonstrate that high-content disk diffusion test should be performed by using both gentamicin and streptomycin in routine screening of HLAR among enterococci and the determination of beta lactamase activity at the same time is also useful for detecting resistance to a beta lactam agent.

Anti-Bacterial Agents↗
PubMed2003

Hit and lead generation: beyond high-throughput screening.

The identification of small-molecule modulators of protein function, and the process of transforming these into high-content lead series, are key activities in modern drug discovery. The decisions taken during this process have far-reaching consequences for success later in lead optimization and even more crucially in clinical development. Recently, there has been an increased focus on these activities due to escalating downstream costs resulting from high clinical failure rates. In addition, the vast emerging opportunities from efforts in functional genomics and proteomics demands a departure from the linear process of identification, evaluation and refinement activities towards a more integrated parallel process. This calls for flexible, fast and cost-effective strategies to meet the demands of producing high-content lead series with improved prospects for clinical success.

Amino Acid Motifs↗
PubMed2004

Reliable and sensitive identification of occult tumor cells using the improved rare event imaging system.

PURPOSE: The purpose of this study was to assess the feasibility of using rare event imaging system (REIS)-assisted analysis to detect occult tumor cells (OTCs) in peripheral blood (PB). The study also sought to determine whether REIS-assisted OTC detection presents a clinically viable alternative to manual microscopic detection to establish the true significance of OTC from solid epithelial tumors. EXPERIMENTAL DESIGN: We recently demonstrated proof of concept using a fluorescence-based automated microscope system, REIS, for OTC detection from the PB. For this study, the prototype of the system was adopted for high-throughput and high-content cellular analysis. RESULTS: The performance of the improved REIS was examined using normal blood (n = 10), normal blood added to cancer cells (n = 20), and blood samples obtained from cancer patients (n = 80). Data from the screening of 80 clinical slides from breast and lung cancer patients, by manual microscopy and by the REIS, revealed that as many as 14 of 35 positive slides (40%) were missed by manual screening but positively identified by REIS. In addition, REIS-assisted scanning reliably and reproducibly quantified the total number of cells analyzed in the assay and categorized positive cells based on their marker expression profile. CONCLUSIONS: REIS-assisted analysis provides excellent sensitivity and reproducibility for OTC detection. This approach may enable an improved method for screening of PB samples and for obtaining novel information about disease staging and about risk evaluation in cancer patients.

Breast Neoplasms↗
PubMed1995

Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC 51299 for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci.

Studies were conducted to validate the use of Enterococcus faecalis ATCC 51299 (which is vancomycin resistant and resistant to high levels of gentamicin and streptomycin) and E. faecalis ATCC 29212 (which is susceptible to vancomycin and against which gentamicin or streptomycin and cell wall-active agents have synergistic kill activity) as controls in an agar screening test for vancomycin resistance and high-level streptomycin and gentamicin resistance and a broth microdilution screening test for high-level streptomycin and gentamicin resistance. Both organisms performed as expected in these tests and will serve as appropriate controls. However, E. faecalis ATCC 29212 was occasionally noted to produce light growth on the vancomycin screening plate with certain lots of agar. Quality control ranges for disk diffusion tests with disks with large amounts of streptomycin (300 micrograms) and gentamicin (120 micrograms) were established for E. faecalis ATCC 29212; zone limits are 16 to 22 mm for gentamicin and 14 to 19 mm for streptomycin. No zones for inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.

Aminoglycosides↗
PubMed1991

Detection of high-level aminoglycoside resistance in enterococci other than Enterococcus faecalis.

The ability of six screening methods to detect high-level aminoglycoside resistance in enterococcal species other than Enterococcus faecalis was investigated. The 85 Enterococcus isolates, which included 55 E. faecium, 11 E. gallinarum, 9 E. casseliflavus, 5 E. raffinosus, 4 E. avium, and 1 E. mundtii, were tested by using aminoglycoside-supplemented brain heart infusion agar (BHI), Remel EF Synergy Quad plates, high-content aminoglycoside diffusion disks, standard (prepared in-house) microdilution panels, Pasco MIC Gram Positive microdilution panels, and Vitek GPS-TA cards. When tested on BHI, 32 and 35 strains showed resistance to gentamicin and streptomycin, respectively. Resistance profiles obtained with Remel EF Synergy Quad plates were in complete agreement with those obtained on BHI. However, growth on Mueller-Hinton agar-based plates was not as heavy. Some isolates showed only weak growth and required 48 h for resistance to become evident, especially with swab inoculation of quadrants containing 2,000 micrograms of gentamicin per ml. Profiles obtained by use of the agar-based screens were used as the basis for evaluating the other methods. Disk diffusion showed complete agreement. No false resistance occurred by either microdilution method, but 48 h of incubation was needed for detection of some gentamicin-resistant isolates, and 14% of the streptomycin-resistant strains were not detected by standard microdilution. The Vitek GPS-TA card detected 81 and 100% of the gentamicin- and streptomycin-resistant isolates, respectively. In general, most methods used to detect high-level aminoglycoside resistance in E. faecalis appear to be reliable for the testing of the other enterococcal species. However, further investigations with a greater number of resistant E. raffinosus, E. avium, and E. mundtii isolates, when they are available, will be useful for establishing the full range of enterococci that can reliably be tested by the various methods.

Anti-Bacterial Agents↗
PubMed2025

Genome-wide analysis of host-encoded microRNAs modulating SARS-CoV-2 infection.

Viruses exploit cellular machinery to complete their replication cycle. Furthering our understanding of this process provides insight into the mechanism of virus replication and potential targets for antiviral therapeutics. Genome-wide CRISPR screens have identified cellular pathways important in the SARS-COV-2 infection process, including vesicular traffic, lipid homeostasis and PI3K signalling. Functional genomics-driven analysis of host-encoded microRNAs (miRNAs) impacting SARS-CoV-2 infection would provide further unbiased and discovery-driven insight into the host-pathogen interface. Here we present findings from genome-wide complementary miRNA mimic and inhibitor screens performed in a bio-safety level (BSL)-4 laboratory using a combination of high-throughput robotics, high-content imaging and novel data analysis pipelines. This dataset has identified both miRNA promoters and inhibitors of SARS-CoV-2 replication which may be used by researchers to further explore therapeutic targets against SARS-CoV-2 and the host factors influencing COVID pathogenesis.

MicroRNAs↗