Publications by authors named "Savas Tay"

Protein activity state, rather than protein or mRNA abundance, is a biologically regulated and relevant input to many processes in signaling, differentiation, development, and diseases such as cancer. While there are numerous methods to detect and quantify mRNA and protein abundance in biological samples, there are no general approaches to detect and quantify endogenous protein activity with single-cell resolution. Here, we report the development of a chemoproteomic platform, single-cell activity-dependent proximity ligation, which uses automated, microfluidics-based single-cell capture and nanoliter volume manipulations to convert the interactions of family-wide chemical activity probes with native protein targets into multiplexed, amplifiable oligonucleotide barcodes.

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Unlabelled: Herpes simplex virus type 1 (HSV-1) is a highly prevalent human pathogen that causes a range of clinical manifestations, including oral and genital herpes, keratitis, encephalitis, and disseminated neonatal disease. Despite its significant health and economic burden, there is currently only a handful of approved antiviral drugs to treat HSV-1 infection. Acyclovir and its analogs are the first-line treatment, but resistance often arises during prolonged treatment periods, such as in immunocompromised patients.

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Complex cellular functions occur via the coordinated formation and dissociation of protein complexes. Functions such as the response to a signaling ligand can incorporate dozens of proteins and hundreds of complexes. Until recently, it has been difficult to measure multiple protein complexes at the single-cell level.

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Proximity sequencing (Prox-seq) simultaneously measures gene expression, protein expression and protein complexes on single cells. Using information from dual-antibody binding events, Prox-seq infers surface protein dimers at the single-cell level. Prox-seq provides multi-dimensional phenotyping of single cells in high throughput, and was recently used to track the formation of receptor complexes during cell signaling and discovered a novel interaction between CD9 and CD8 in naïve T cells.

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DUX4 is a germline transcription factor and a master regulator of zygotic genome activation. During early embryogenesis, DUX4 is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In adult somatic cells, DUX4 expression is silenced and its activation in adult muscle cells causes the genetic disorder Facioscapulohumeral Muscular Dystrophy (FSHD).

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Cells at the site of an infection experience numerous biochemical signals that vary in amplitude, space, and time. Despite the diversity of dynamic signals produced by pathogens and sentinel cells, information-processing pathways converge on a limited number of central signaling nodes to ultimately control cellular responses. In particular, the NF-κB pathway responds to dozens of signals from pathogens and self, and plays a vital role in processing proinflammatory inputs.

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Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types.

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Recently, a high-throughput screen of 1900 clinically used drugs identified masitinib, an orally bioavailable tyrosine kinase inhibitor, as a potential treatment for COVID-19. Masitinib acts as a broad-spectrum inhibitor for human coronaviruses, including SARS-CoV-2 and several of its variants. In this work, we rely on atomistic molecular dynamics simulations with advanced sampling methods to develop a deeper understanding of masitinib's mechanism of M inhibition.

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Proximity sequencing (Prox-seq) measures gene expression, protein expression, and protein complexes at the single cell level, using information from dual-antibody binding events and a single cell sequencing readout. Prox-seq provides multi-dimensional phenotyping of single cells and was recently used to track the formation of receptor complexes during inflammatory signaling in macrophages and to discover a new interaction between CD9/CD8 proteins on naïve T cells. The distribution of protein abundance affects identification of protein complexes in a complicated manner in dual-binding assays like Prox-seq.

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Article Synopsis
  • Stimulation of the innate immune system is essential for successful vaccinations and immunotherapies, often involving adjuvants that activate specific immune signaling pathways.
  • The study shows how small molecule "immunomodulators" can modify and enhance adjuvant activity by altering immune signaling rather than just binding to receptors.
  • These modulators improve immune responses in flu vaccines and decrease side effects in typhoid vaccines, suggesting potential uses in preventative and therapeutic applications.
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Study of spatial and temporal aspects of signaling between individual cells is essential in understanding development, the immune response, and host-pathogen interactions. We present an automated high-throughput microfluidic platform that chemically stimulates immune cells to initiate cytokine secretion, and controls the formation of signal gradients that activate neighboring cell populations. Furthermore, our system enables controlling the cell type and density based on distance, and retrieval of cells from different regions for gene expression analysis.

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We present proximity sequencing (Prox-seq) for simultaneous measurement of proteins, protein complexes and mRNAs in thousands of single cells. Prox-seq combines proximity ligation assay with single-cell sequencing to measure proteins and their complexes from all pairwise combinations of targeted proteins, providing quadratically scaled multiplexing. We validate Prox-seq and analyze a mixture of T cells and B cells to show that it accurately identifies these cell types and detects well-known protein complexes.

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Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses. However, these metrics are difficult and labor-intensive to obtain for high-throughput image datasets.

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Studying viral-host protein-protein interactions can facilitate the discovery of therapies for viral infection. We use high-throughput yeast two-hybrid experiments and mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of 739 high-confidence binary and co-complex interactions, validating 218 known SARS-CoV-2 host factors and revealing 361 novel ones. Our results show the highest overlap of interaction partners between published datasets and of genes differentially expressed in samples from COVID-19 patients.

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed to be essential for viral replication.

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Infected cells communicate through secreted signaling molecules like cytokines, which carry information about pathogens. How differences in cytokine secretion affect inflammatory signaling over space and how responding cells decode information from propagating cytokines are not understood. By computationally and experimentally studying NF-κB dynamics in cocultures of signal-sending cells (macrophages) and signal-receiving cells (fibroblasts), we find that cytokine signals are transmitted by wave-like propagation of NF-κB activity and create well-defined activation zones in responding cells.

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Many scenarios in cellular communication require cells to interpret multiple dynamic signals. It is unclear how exposure to inflammatory stimuli alters transcriptional responses to subsequent stimulus. Using high-throughput microfluidic live-cell analysis, we systematically profile the NF-κB response to different signal sequences in single cells.

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Objective: Airborne spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant risk for healthcare workers. Understanding transmission of SARS-CoV-2 in the hospital could help minimize nosocomial infection. The objective of this pilot study was to measure aerosolization of SARS-CoV-2 in the hospital rooms of COVID-19 patients.

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Unlabelled: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available, or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication.

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Physical interactions between viral and host proteins are responsible for almost all aspects of the viral life cycle and the host's immune response. Studying viral-host protein-protein interactions is thus crucial for identifying strategies for treatment and prevention of viral infection. Here, we use high-throughput yeast two-hybrid and affinity purification followed by mass spectrometry to generate a comprehensive SARS-CoV-2-human protein-protein interactome network consisting of both binary and co-complex interactions.

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Article Synopsis
  • The study investigates the effects of cannabidiol (CBD) on SARS-CoV-2 infection, revealing that CBD inhibits the virus's replication in both cells and mice while not being effective like other cannabinoids such as THC.
  • CBD works by acting after the virus enters host cells, blocking viral gene expression and positively influencing host gene responses, particularly enhancing the IRE1α RNase ER stress response and interferon signaling pathways.
  • In human patient data, a significant negative relationship was found between CBD use and positive COVID-19 test results, suggesting potential for CBD as a preventative measure; however, the authors advise against using non-medical forms of CBD for treatment at this time.
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The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity.

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Digital proximity ligation assay (PLA) detects single protein molecules with a pair of oligonucleotide-conjugated antibodies and digital PCR (dPCR) readout, which allows absolute quantitation of proteins in single cells with high sensitivity. The pipeline also allows simultaneous measurement of protein and mRNA from the same single cell. The sensitivity of the assay has been further improved with implementation of the assay on a microfluidic system, which enables quantitation of rare protein species, with expression level as low as ~3000 protein molecules per cell.

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Individual cells are heterogeneous when responding to environmental cues. Under an external signal, certain cells activate gene regulatory pathways, while others completely ignore that signal. Mechanisms underlying cellular heterogeneity are often inaccessible because experiments needed to study molecular states destroy the very states that we need to examine.

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