Perturbation-Specific Transcriptional Mapping for unbiased target elucidation of antibiotics.

The rising prevalence of antibiotic resistance threatens human health. While more sophisticated strategies for antibiotic discovery are being developed, target elucidation of new chemical entities remains challenging. In the post-genomic era, expression profiling can play an important role in mechanism-of-action (MOA) prediction by reporting on the cellular response to perturbation. However, the broad application of transcriptomics has yet to fulfill its promise of transforming target elucidation due to challenges in identifying the most relevant, direct responses to target inhibition. We developed an unbiased strategy for MOA prediction, called Perturbation-Specific Transcriptional Mapping (PerSpecTM), in which large-throughput expression profiling of wildtype or hypomorphic mutants, depleted for essential targets, enables a computational strategy to address this challenge. We applied PerSpecTM to perform reference-based MOA prediction based on the principle that similar perturbations, whether chemical or genetic, will elicit similar transcriptional responses. Using this approach, we elucidated the MOAs of three new molecules with activity against Pseudomonas aeruginosa by comparing their expression profiles to those of a reference set of antimicrobial compounds with known MOAs. We also show that transcriptional responses to small molecule inhibition resemble those resulting from genetic depletion of essential targets by CRISPRi by PerSpecTM, demonstrating proof-of-concept that correlations between expression profiles of small molecule and genetic perturbations can facilitate MOA prediction when no chemical entities exist to serve as a reference. Empowered by PerSpecTM, this work lays the foundation for an unbiased, readily scalable, systematic reference-based strategy for MOA elucidation that could transform antibiotic discovery efforts.

"Multiplexed screen identifies a Pseudomonas aeruginosa -specific small molecule targeting the outer membrane protein OprH and its interaction with LPS".

The surge of antimicrobial resistance threatens efficacy of current antibiotics, particularly against Pseudomonas aeruginosa , a highly resistant gram-negative pathogen. The asymmetric outer membrane (OM) of P. aeruginosa combined with its array of efflux pumps provide a barrier to xenobiotic accumulation, thus making antibiotic discovery challenging. We adapted PROSPECT 1 , a target-based, whole-cell screening strategy, to discover small molecule probes that kill P. aeruginosa mutants depleted for essential proteins localized at the OM. We identified BRD1401, a small molecule that has specific activity against a P. aeruginosa mutant depleted for the essential lipoprotein, OprL. Genetic and chemical biological studies identified that BRD1401 acts by targeting the OM ß-barrel protein OprH to disrupt its interaction with LPS and increase membrane fluidity. Studies with BRD1401 also revealed an interaction between OprL and OprH, directly linking the OM with peptidoglycan. Thus, a whole-cell, multiplexed screen can identify species-specific chemical probes to reveal novel pathogen biology.

High-Throughput Neutralization and Serology Assays Reveal Correlated but Highly Variable Humoral Immune Responses in a Large Population of Individuals Infected with SARS-CoV-2 in the US between March and August 2020.

The ability to measure neutralizing antibodies on large scale can be important for understanding features of the natural history and epidemiology of infection, as well as an aid in determining the efficacy of interventions, particularly in outbreaks such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Because of the assay's rapid scalability and high efficiency, serology measurements that quantify the presence rather than function of serum antibodies often serve as proxies of immune protection. Here, we report the development of a high-throughput, automated fluorescence-based neutralization assay using SARS-CoV-2 virus to quantify neutralizing antibody activity in patient specimens. We performed large-scale testing of over 19,000 COVID-19 convalescent plasma (CCP) samples from patients who had been infected with SARS-CoV-2 between March and August 2020 across the United States. The neutralization capacity of the samples was moderately correlated with serological measurements of anti-receptor-binding domain (RBD) IgG levels. The neutralizing antibody levels within these convalescent-phase serum samples were highly variable against the original USA-WA1/2020 strain with almost 10% of individuals who had had PCR-confirmed SARS-CoV-2 infection having no detectable antibodies either by serology or neutralization, and ~1/3 having no or low neutralizing activity. Discordance between neutralization and serology measurements was mainly due to the presence of non-IgG RBD isotypes. Meanwhile, natural infection with the earliest SARS-CoV-2 strain USA-WA1/2020 resulted in weaker neutralization of subsequent B.1.1.7 (alpha) and the B.1.351 (beta) variants, with 88% of samples having no activity against the BA.1 (omicron) variant. IMPORTANCE The ability to directly measure neutralizing antibodies on live SARS-CoV-2 virus in individuals can play an important role in understanding the efficacy of therapeutic interventions or vaccines. In contrast to functional neutralization assays, serological assays only quantify the presence of antibodies as a proxy of immune protection. Here, we have developed a high-throughput, automated neutralization assay for SARS-CoV-2 and measured the neutralizing activity of ~19,000 COVID-19 convalescent plasma (CCP) samples collected across the United States between March and August of 2020. These data were used to support the FDA's interpretation of CCP efficacy in patients with SARS-CoV-2 infection and their issuance of emergency use authorization of CCP in 2020.

At-home Testing and Risk Factors for Acquisition of SARS-CoV-2 Infection in a Major US Metropolitan Area.

Background: Unbiased assessment of the risks associated with acquisition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to informing mitigation efforts during pandemics. The objective of our study was to understand the risk factors for acquiring coronavirus disease 2019 (COVID-19) in a large prospective cohort of adult residents in a large US metropolitan area. Methods: We designed a fully remote longitudinal cohort study involving monthly at-home SARS-CoV-2 polymerase chain reaction (PCR) and serology self-testing and monthly surveys. Results: Between October 2020 and January 2021, we enrolled 10 289 adults reflective of the Boston metropolitan area census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared with Whites, Black non-Hispanic participants had a 2.2-fold greater risk of acquiring COVID-19 (hazard ratio [HR], 2.19; 95% CI, 1.91-2.50; P < .001), and Hispanics had a 1.5-fold greater risk (HR, 1.52; 95% CI, 1.32-1.71; P < .016). Individuals aged 18-29, those who worked outside the home, and those living with other adults and children were at an increased risk. Individuals in the second and third lowest disadvantaged neighborhood communities were associated with an increased risk of acquiring COVID-19. Individuals with medical risk factors for severe disease were at a decreased risk of SARS-CoV-2 acquisition. Conclusions: These results demonstrate that race/ethnicity and socioeconomic status are the biggest determinants of acquisition of infection. This disparity is significantly underestimated if based on PCR data alone, as noted by the discrepancy in serology vs PCR detection for non-White participants, and points to persistent disparity in access to testing. Medical conditions and advanced age, which increase the risk for severity of SARS-CoV-2 disease, were associated with a lower risk of COVID-19 acquisition, suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics.

At-home Testing and Risk Factors for Acquisition of SARS-CoV-2 Infection in a Major US Metropolitan Area.

IMPORTANCE: Unbiased assessment of risks associated with acquisition of SARS-CoV-2 is critical to informing mitigation efforts during pandemics. OBJECTIVE: Understand risk factors for acquiring COVID-19 in a large, prospective cohort of adult residents recruited to be representative of a large US metropolitan area. DESIGN: Fully remote longitudinal cohort study launched in October 2020 and ongoing; Study data reported through June 15, 2021. SETTING: Brigham and Women’s Hospital, Boston MA. PARTICIPANTS: Adults within 45 miles of Boston, MA. INTERVENTION: Monthly at-home SARS-CoV-2 viral and antibody testing. MAIN OUTCOMES: Between October 2020 and January 2021, we enrolled 10,289 adults reflective of Massachusetts census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared to whites, Black non-Hispanic participants had a 2.2 fold greater risk of acquiring COVID-19 (HR 2.19, 95% CI 1.91-2.50; p=<0.001) and Hispanics had a 1.5 fold greater risk (HR 1.52, 95% CI 1.32-1.71; p=<0.016). Individuals aged 18-29, those who worked outside the home, and those living with other adults and children were at an increased risk. Individuals in the second and third lowest disadvantaged neighborhood communities, as measured by the area deprivation index as a marker for socioeconomic status by census block group, were associated with an increased risk in developing COVID-19. Individuals with medical risk factors for severe COVID-19 disease were at a decreased risk of SARS-CoV-2 acquisition. CONCLUSIONS: These results demonstrate that race/ethnicity and socioeconomic status are not only risk factors for severity of disease but are also the biggest determinants of acquisition of infection. Importantly, this disparity is significantly underestimated if based on PCR data alone as noted by the discrepancy in serology vs. PCR detection for non-white participants, and points to persistent disparity in access to testing. Meanwhile, medical conditions and advanced age that increase the risk for severity of SARS-CoV-2 disease were associated with a lower risk of acquisition of COVID-19 suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics. TRIAL REGISTRATION: N/A. QUESTION: What population and occupational groups in the United States are at increased risk for acquiring COVID-19? FINDINGS: In this remote, longitudinal cohort study involving monthly PCR and serology self-testing of 10,289 adult residents of the Boston metropolitan area, 9257 (90.0%) of TestBoston participants acquired evidence of immunity to SARS-CoV-2 through vaccination, infection, or both as of June 15, 2021. Residents identifying as Black, Hispanic/Latinx had an increased risk of acquisition of COVID-19. Healthcare workers were not at increased risk of SARS-CoV-2 acquisition. Individuals with medical risk factors for severe COVID-19 disease were at a decreased risk of SARS-CoV-2 acquisition. MEANING: These results demonstrate that race/ethnicity and socioeconomic status are not only risk factors for severity of disease but also are the biggest determinants of acquisition of infection. These findings highlight the need to address the consequences of structural racism during the development of mitigation programs for current and future pandemics.

A comparison of microfluidic methods for high-throughput cell deformability measurements.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.

Optofluidic real-time cell sorter for longitudinal CTC studies in mouse models of cancer.

Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.

Microfluidic platform for characterizing TCR-pMHC interactions.

The physical characteristics of the T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) interaction are known to play a central role in determining T cell function in the initial stages of the adaptive immune response. State-of-the-art assays can probe the kinetics of this interaction with single-molecular-bond resolution, but this precision typically comes at the cost of low throughput, since the complexity of these measurements largely precludes "scaling up." Here, we explore the feasibility of detecting specific TCR-pMHC interactions by flowing T cells past immobilized pMHC and measuring the reduction in cell speed due to the mechanical force of the receptor-ligand interaction. To test this new fluidic measurement modality, we fabricated a microfluidic device in which pMHC-coated beads are immobilized in hydrodynamic traps along the length of a serpentine channel. As T cells flow past the immobilized beads, their change in speed is tracked via microscopy. We validated this approach using two model systems: primary CD8+ T cells from an OT-1 TCR transgenic mouse with beads conjugated with H-2Kb:SIINFEKL, and Jurkat T cells with beads conjugated with anti-CD3 and anti-CD28 antibodies.

Deformability-based cell selection with downstream immunofluorescence analysis.

Mechanical properties of single cells have been shown to relate to cell phenotype and malignancy. However, until recently, it has been difficult to directly correlate each cell's biophysical characteristics to its molecular traits. Here, we present a cell sorting technique for use with a suspended microchannel resonator (SMR), which can measure biophysical characteristics of a single cell based on the sensor's record of its buoyant mass as well as its precise position while it traverses through a constricted microfluidic channel. The measurement provides information regarding the amount of time a cell takes to pass through a constriction (passage time), as related to the cell's deformability and surface friction, as well as the particular manner in which it passes through. In the method presented here, cells of interest are determined based on passage time, and are collected off-chip for downstream immunofluorescence imaging. The biophysical single-cell SMR measurement can then be correlated to the molecular expression of the collected cell. This proof-of-principle is demonstrated by sorting and collecting tumor cells from cell line-spiked blood samples as well as a metastatic prostate cancer patient blood sample, identifying them by their surface protein expression and relating them to distinct SMR signal trajectories.

Deformability of Tumor Cells versus Blood Cells.

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.