Increased erythrophagocytosis induces ferroptosis in red pulp macrophages in a mouse model of transfusion.

Macrophages play important roles in recycling iron derived from the clearance of red blood cells (RBCs). They are also a critically important component of host defense, protecting against invading pathogens. However, the effects on macrophage biology of acutely ingesting large numbers of RBCs are not completely understood. To investigate this issue, we used a mouse model of RBC transfusion and clearance, which mimics the clinical setting. In this model, transfusions of refrigerator storage-damaged (ie, "old") RBCs led to increased erythrophagocytosis by splenic red pulp macrophages (RPMs). This robust erythrophagocytosis induced ferroptosis, an iron-dependent form of cell death, in RPMs. This was accompanied by increases in reactive oxygen species and lipid peroxidation in vivo, which were reduced by treatment in vitro with ferrostatin-1, a ferroptosis inhibitor. Old RBC transfusions also induced RPM-dependent chemokine expression by splenic Ly6Chi monocytes, which signaled Ly6Chi monocyte migration from bone marrow to spleen, where these cells subsequently differentiated into RPMs. The combination of cell division among remaining splenic RPMs, along with the influx of bone marrow-derived Ly6Chi monocytes, suggests that, following RPM depletion induced by robust erythrophagocytosis, there is a coordinated effort to restore homeostasis of the RPM population by local self-maintenance and contributions from circulating monocytes. In conclusion, these findings may be clinically relevant to pathological conditions that can arise as a result of increased erythrophagocytosis, such as transfusion-related immunomodulation and impaired host immunity.

Systematic, network-based characterization of therapeutic target inhibitors.

A large fraction of the proteins that are being identified as key tumor dependencies represent poor pharmacological targets or lack clinically-relevant small-molecule inhibitors. Availability of fully generalizable approaches for the systematic and efficient prioritization of tumor-context specific protein activity inhibitors would thus have significant translational value. Unfortunately, inhibitor effects on protein activity cannot be directly measured in systematic and proteome-wide fashion by conventional biochemical assays. We introduce OncoLead, a novel network based approach for the systematic prioritization of candidate inhibitors for arbitrary targets of therapeutic interest. In vitro and in vivo validation confirmed that OncoLead analysis can recapitulate known inhibitors as well as prioritize novel, context-specific inhibitors of difficult targets, such as MYC and STAT3. We used OncoLead to generate the first unbiased drug/regulator interaction map, representing compounds modulating the activity of cancer-relevant transcription factors, with potential in precision medicine.

Multiwell-based G0-PCC assay for radiation biodosimetry.

In cytogenetic biodosimetry, assessing radiation exposure typically requires over 48 hours for cells to reach mitosis, significantly delaying the administration of crucial radiation countermeasures needed within the first 24 hours post-exposure. To improve medical response times, we incorporated the G0-Premature Chromosome Condensation (G0-PCC) technique with the Rapid Automated Biodosimetry Tool-II (RABiT-II), creating a faster alternative for large-scale radiation emergencies. Our findings revealed that using a lower concentration of Calyculin A (Cal A) than recommended effectively increased the yield of highly-condensed G0-PCC cells (hPCC). However, integrating recombinant CDK1/Cyclin B kinase, vital for chromosome condensation, proved challenging due to the properties of these proteins affecting interactions with cellular membranes. Interestingly, Cal A alone was capable of inducing chromosome compaction in some G0 cells even in the absence of mitotic kinases, although these chromosomes displayed atypical morphologies. This suggests that Cal A mechanism for compacting G0 chromatin may differ from condensation driven by mitotic kinases. Additionally, we observed a correlation between radiation dose and extent of hPCC chromosome fragmentation, which allowed us to automate radiation damage quantification using a Convolutional Neural Network (CNN). Our method can address the need for a same-day cytogenetic biodosimetry test in radiation emergency situations.

Network-based elucidation of colon cancer drug resistance mechanisms by phosphoproteomic time-series analysis.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.

Network-based elucidation of colon cancer drug resistance by phosphoproteomic time-series analysis.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies, over the past decade, we developed VESPA-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases.

Validation of a High-Throughput Dicentric Chromosome Assay Using Complex Radiation Exposures.

Validation of biodosimetry assays is routinely performed using primarily orthovoltage irradiators at a conventional dose rate of approximately 1 Gy/min. However, incidental/ accidental exposures caused by nuclear weapons can be more complex. The aim of this work was to simulate the DNA damage effects mimicking those caused by the detonation of a several kilotons improvised nuclear device (IND). For this, we modeled complex exposures to: 1. a mixed (photons + IND-neutrons) field and 2. different dose rates that may come from the blast, nuclear fallout, or ground deposition of radionuclides (ground shine). Additionally, we assessed whether myeloid cytokines affect the precision of radiation dose estimation by modulating the frequency of dicentric chromosomes. To mimic different exposure scenarios, several irradiation systems were used. In a mixed field study, human blood samples were exposed to a photon field enriched with neutrons (ranging from 10% to 37%) from a source that mimics Hiroshima's A-bomb's energy spectrum (0.2-9 MeV). Using statistical analysis, we assessed whether photons and neutrons act in an additive or synergistic way to form dicentrics. For the dose rates study, human blood was exposed to photons or electrons at dose rates ranging from low (where the dose was spread over 32 h) to extremely high (where the dose was delivered in a fraction of a microsecond). Potential effects of cytokine treatment on biodosimetry dose predictions were analyzed in irradiated blood subjected to Neupogen or Neulasta for 24 or 48 h at the concentration recommended to forestall manifestation of an acute radiation syndrome in bomb survivors. All measurements were performed using a robotic station, the Rapid Automated Biodosimetry Tool II, programmed to culture lymphocytes and score dicentrics in multiwell plates (the RABiT-II DCA). In agreement with classical concepts of radiation biology, the RABiT-II DCA calibration curves suggested that the frequency of dicentrics depends on the type of radiation and is modulated by changes in the dose rate. The resulting dose-response curves suggested an intermediate dicentric yields and additive effects of photons and IND-neutrons in the mixed field. At ultra-high dose rate (600 Gy/s), affected lymphocytes exhibited significantly fewer dicentrics (P < 0.004, t test). In contrast, we did not find the dose-response modification effects of radiomitigators on the yields of dicentrics (Bonferroni corrected P > 0.006, ANOVA test). This result suggests no bias in the dose predictions should be expected after emergency cytokine treatment initiated up to 48 h prior to blood collection for dicentric analysis.

Elucidating Compound Mechanism of Action and Polypharmacology with a Large-scale Perturbational Profile Compendium.

The Mechanism of Action (MoA) of a drug is generally represented as a small, non-tissue-specific repertoire of high-affinity binding targets. Yet, drug activity and polypharmacology are increasingly associated with a broad range of off-target and tissue-specific effector proteins. To address this challenge, we have implemented an efficient integrative experimental and computational framework leveraging the systematic generation and analysis of drug perturbational profiles representing >700 FDA-approved and experimental oncology drugs, in cell lines selected as high-fidelity models of 23 aggressive tumor subtypes. Protein activity-based analyses revealed highly reproducible, drug-mediated modulation of tissue-specific targets, leading to generation of a proteome-wide polypharmacology map, characterization of MoA-related drug clusters and off-target effects, and identification and experimental validation of novel, tissue-specific inhibitors of undruggable oncoproteins. The proposed framework, which is easily extended to elucidating the MoA of novel small-molecule libraries, could help support more systematic and quantitative approaches to precision oncology.

OncoLoop: A Network-Based Precision Cancer Medicine Framework.

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide. SIGNIFICANCE: OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. This article is highlighted in the In This Issue feature, p. 247.

A Model for Network-Based Identification and Pharmacological Targeting of Aberrant, Replication-Permissive Transcriptional Programs Induced by Viral Infection.

Precise characterization and targeting of host cell transcriptional machinery hijacked by viral infection remains challenging. Here, we show that SARS-CoV-2 hijacks the host cell transcriptional machinery to induce a phenotypic state amenable to its replication. Specifically, analysis of Master Regulator (MR) proteins representing mechanistic determinants of the gene expression signature induced by SARS-CoV-2 in infected cells revealed coordinated inactivation of MRs enriched in physical interactions with SARS-CoV-2 proteins, suggesting their mechanistic role in maintaining a host cell state refractory to virus replication. To test their functional relevance, we measured SARS-CoV-2 replication in epithelial cells treated with drugs predicted to activate the entire repertoire of repressed MRs, based on their experimentally elucidated, context-specific mechanism of action. Overall, >80% of drugs predicted to be effective by this methodology induced significant reduction of SARS-CoV-2 replication, without affecting cell viability. This model for host-directed pharmacological therapy is fully generalizable and can be deployed to identify drugs targeting host cell-based MR signatures induced by virtually any pathogen.

A model for network-based identification and pharmacological targeting of aberrant, replication-permissive transcriptional programs induced by viral infection.

SARS-CoV-2 hijacks the host cell transcriptional machinery to induce a phenotypic state amenable to its replication. Here we show that analysis of Master Regulator proteins representing mechanistic determinants of the gene expression signature induced by SARS-CoV-2 in infected cells revealed coordinated inactivation of Master Regulators enriched in physical interactions with SARS-CoV-2 proteins, suggesting their mechanistic role in maintaining a host cell state refractory to virus replication. To test their functional relevance, we measured SARS-CoV-2 replication in epithelial cells treated with drugs predicted to activate the entire repertoire of repressed Master Regulators, based on their experimentally elucidated, context-specific mechanism of action. Overall, 15 of the 18 drugs predicted to be effective by this methodology induced significant reduction of SARS-CoV-2 replication, without affecting cell viability. This model for host-directed pharmacological therapy is fully generalizable and can be deployed to identify drugs targeting host cell-based Master Regulator signatures induced by virtually any pathogen.

The use of a centrifuge-free RABiT-II system for high-throughput micronucleus analysis.

The cytokinesis-block micronucleus (CBMN) assay is considered to be the most suitable biodosimetry method for automation. Previously, we automated this assay on a commercial robotic biotech high-throughput system (RABiT-II) adopting both a traditional and an accelerated micronucleus protocol, using centrifugation steps for both lymphocyte harvesting and washing, after whole blood culturing. Here we describe further development of our accelerated CBMN assay protocol for use on high-throughput/high content screening (HTS/HCS) robotic systems without a centrifuge. This opens the way for implementation of the CBMN assay on a wider range of commercial automated HTS/HCS systems and thus increases the potential capacity for dose estimates following a mass-casualty radiological event.

The RABiT-II DCA in the Rhesus Macaque Model.

An automated platform for cytogenetic biodosimetry, the "Rapid Automated Biodosimetry Tool II (RABiT-II)," adapts the dicentric chromosome assay (DCA) for high-throughput mass-screening of the population after a large-scale radiological event. To validate this test, the U.S. Federal Drug Administration (FDA) recommends demonstrating that the high-throughput biodosimetric assay in question correctly reports the dose in an in vivo model. Here we describe the use of rhesus macaques (Macaca mulatta) to augment human studies and validate the accuracy of the high-throughput version of the DCA. To perform analysis, we developed the 17/22-mer peptide nucleic acid (PNA) probes that bind to the rhesus macaque's centromeres. To our knowledge, these are the first custom PNA probes with high specificity that can be used for chromosome analysis in M. mulatta. The accuracy of fully-automated chromosome analysis was improved by optimizing a low-temperature telomere PNA FISH staining in multiwell plates and adding the telomere detection feature to our custom chromosome detection software, FluorQuantDic V4. The dicentric frequencies estimated from in vitro irradiated rhesus macaque samples were compared to human blood samples of individuals subjected to the same ex vivo irradiation conditions. The results of the RABiT-II DCA analysis suggest that, in the lymphocyte system, the dose responses to gamma radiation in the rhesus macaques were similar to those in humans, with small but statistically significant differences between these two model systems.

The Host Cell ViroCheckpoint: Identification and Pharmacologic Targeting of Novel Mechanistic Determinants of Coronavirus-Mediated Hijacked Cell States.

Most antiviral agents are designed to target virus-specific proteins and mechanisms rather than the host cell proteins that are critically dysregulated following virus-mediated reprogramming of the host cell transcriptional state. To overcome these limitations, we propose that elucidation and pharmacologic targeting of host cell Master Regulator proteins-whose aberrant activities govern the reprogramed state of coronavirus-infected cells-presents unique opportunities to develop novel mechanism-based therapeutic approaches to antiviral therapy, either as monotherapy or as a complement to established treatments. Specifically, we propose that a small module of host cell Master Regulator proteins (ViroCheckpoint) is hijacked by the virus to support its efficient replication and release. Conventional methodologies are not well suited to elucidate these potentially targetable proteins. By using the VIPER network-based algorithm, we successfully interrogated 12h, 24h, and 48h signatures from Calu-3 lung adenocarcinoma cells infected with SARS-CoV, to elucidate the time-dependent reprogramming of host cells and associated Master Regulator proteins. We used the NYS CLIA-certified Darwin OncoTreat algorithm, with an existing database of RNASeq profiles following cell perturbation with 133 FDA-approved and 195 late-stage experimental compounds, to identify drugs capable of virtually abrogating the virus-induced Master Regulator signature. This approach to drug prioritization and repurposing can be trivially extended to other viral pathogens, including SARS-CoV-2, as soon as the relevant infection signature becomes available.

SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila.

Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes sigma(S), does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that sigma(S) affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that sigma(S) affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between sigma(S) and the posttranscriptional regulator CsrA. We analyzed the network of sigma(S)-controlled genes by mutational analysis of transcriptional regulators affected by sigma(S). One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that sigma(S) is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.

RABiT-II: A Fully-Automated Micronucleus Assay System with Shortened Time to Result.

In this work, we describe a fully automated cytokinesis-block micronucleus (CBMN) assay with a significantly shortened time to result, motivated by the need for rapid high-throughput biodosimetric estimation of radiation doses from small-volume human blood samples. The Rapid Automated Biodosimetry Tool (RABiT-II) currently consists of two commercial automated systems: a PerkinElmer cell::explorer Workstation and a GE Healthcare IN Cell Analyzer 2000 Imager. Blood samples (30 µl) from eight healthy volunteers were gamma-ray irradiated ex vivo with 0 (control), 0.5, 1.5, 2.5, 3.5 or 4.5 Gy and processed with full automation in 96-well plates on the RABiT-II system. The total cell culture time was 54 h and total assay time was 3 days. DAPI-stained fixed samples were imaged on an IN Cell Analyzer 2000 with fully-automated image analysis using the GE Healthcare IN Cell Developer Toolbox version 1.9. A CBMN dose-response calibration curve was established, after which the capability of the system to predict known doses was assessed. Various radiation doses for irradiated samples from two donors were estimated within 20% of the true dose (±0.5 Gy below 2 Gy) in 97% of the samples, with the doses in some 5 Gy irradiated samples being underestimated by up to 25%. In summary, the findings from this work demonstrate that the accelerated CBMN assay can be automated in a high-throughput format, using commercial biotech robotic systems, in 96-well plates, providing a rapid and reliable bioassay for radiation exposure.

RABiT-II-DCA: A Fully-automated Dicentric Chromosome Assay in Multiwell Plates.

We developed a fully-automated dicentric chromosome assay (DCA) in multiwell plates. All operations, from sample loading to chromosome scoring, are performed, without human intervention, by the second-generation Rapid Automated Biodosimetry Tool II (RABiT-II) robotic system, a plate imager and custom software, FluorQuantDic. The system requires small volumes of blood (30 µl per individual) to determine radiation dose received as a result of a radiation accident or terrorist attack. To visualize dicentrics in multiwell plates, we implemented a non-classical protocol for centromere FISH staining at 37°C. The RABiT-II performs rapid analysis of chromosomes after extracting them from metaphase cells. With the use of multiwell plates, many samples can be screened at the same time. Thus, the RABiT-II DCA provides an advantage during triage when risk-based stratification and medical management are required for a large population exposed to unknown levels of ionizing radiation.

Automated Triage Radiation Biodosimetry: Integrating Imaging Flow Cytometry with High-Throughput Robotics to Perform the Cytokinesis-Block Micronucleus Assay.

The cytokinesis-block micronucleus (CBMN) assay has become a fully-validated and standardized method for radiation biodosimetry. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a large-scale radiological/nuclear event. Imaging flow cytometry (IFC), which combines the statistical power of traditional flow cytometry with the sensitivity and specificity of microscopy, has been recently used to perform the CBMN assay. Since this technology is capable of automated sample acquisition and multi-file analysis, we have integrated IFC into our Rapid Automated Biodosimetry Technology (RABiT-II). Assay development and optimization studies were designed to increase the yield of binucleated cells (BNCs), and improve data acquisition and analysis templates to increase the speed and accuracy of image analysis. Human peripheral blood samples were exposed ex vivo with up to 4 Gy of c rays at a dose rate of 0.73 Gy/min. After irradiation, samples were transferred to microtubes (total volume of 1 ml including blood and media) and organized into a standard 8 × 12 plate format. Sample processing methods were modified by increasing the blood-to-media ratio, adding hypotonic solution prior to cell fixation and optimizing nuclear DRAQ5 staining, leading to an increase of 81% in BNC yield. Modification of the imaging processing algorithms within IFC software also improved BNC and MN identification, and reduced the average time of image analysis by 78%. Finally, 50 ll of irradiated whole blood was cultured with 200 ll of media in 96-well plates. All sample processing steps were performed automatically using the RABiT-II cell: :explorer robotic system adopting the optimized IFC-CBMN assay protocol. The results presented here detail a novel, high-throughput RABiT-IFC CBMN assay that possesses the potential to increase capacity for triage biodosimetry during a large-scale radiological/nuclear event.

Identification of N-(quinolin-8-yl)benzenesulfonamides as agents capable of down-regulating NFkappaB activity within two separate high-throughput screens of NFkappaB activation.

We describe here a series of N-(quinolin-8-yl)benzenesulfonamides capable of suppressing the NFkappaB pathway identified from two high-throughput screens run at two centers of the NIH Molecular Libraries Initiative. These small molecules were confirmed in both primary and secondary assays of NFkappaB activation and expanded upon through analogue synthesis. The series exhibited potencies in the cell-based assays at as low as 0.6 microM, and several indications suggest that the targeted activity lies within a common region of the NFkappaB pathway.

Guaiacol as a drug candidate for treating adult polyglucosan body disease.

Adult polyglucosan body disease (APBD) is a late-onset disease caused by intracellular accumulation of polyglucosan bodies, formed due to glycogen-branching enzyme (GBE) deficiency. To find a treatment for APBD, we screened 1,700 FDA-approved compounds in fibroblasts derived from APBD-modeling GBE1-knockin mice. Capitalizing on fluorescent periodic acid-Schiff reagent, which interacts with polyglucosans in the cell, this screen discovered that the flavoring agent guaiacol can lower polyglucosans, a result also confirmed in APBD patient fibroblasts. Biochemical assays showed that guaiacol lowers basal and glucose 6-phosphate-stimulated glycogen synthase (GYS) activity. Guaiacol also increased inactivating GYS1 phosphorylation and phosphorylation of the master activator of catabolism, AMP-dependent protein kinase. Guaiacol treatment in the APBD mouse model rescued grip strength and shorter lifespan. These treatments had no adverse effects except making the mice slightly hyperglycemic, possibly due to the reduced liver glycogen levels. In addition, treatment corrected penile prolapse in aged GBE1-knockin mice. Guaiacol's curative effects can be explained by its reduction of polyglucosans in peripheral nerve, liver, and heart, despite a short half-life of up to 60 minutes in most tissues. Our results form the basis to use guaiacol as a treatment and prepare for the clinical trials in APBD.

Identification of Fluorescent Small Molecule Compounds for Synaptic Labeling by Image-Based, High-Content Screening.

Few tools are available for noninvasive imaging of synapses in the living mammalian brain. Current paradigms require the use of genetically modified mice or viral delivery of genetic material to the brain. To develop an alternative chemical approach, utilizing the recognition of synaptic components by organic small molecules, we designed an imaging-based, high-content screen in cultured cortical neurons to identify molecules based on their colocalization with fluorescently tagged synaptic proteins. We used this approach to screen a library of ∼7000 novel fluorescent dyes, and identified a series of compounds in the xanthone family that exhibited consistent synaptic labeling. Follow-up studies with one of these compounds, CX-G3, demonstrated its ability to label acidic organelles and in particular synaptic vesicles at glutamatergic synapses in cultured neurons and murine brain tissue, indicating the potential of this screening approach to identify promising lead compounds for use as synaptic markers, sensors, and targeting devices.