Shantanu Singh

Neuronal synapses contain hundreds of different protein species important for regulating signal transmission. Characterizing differential expression profiles of proteins within synapses in distinct regions of the brain has revealed a high degree of synaptic diversity defined by unique molecular organization. Multiplexed imaging of in vitro neuronal culture models at single synapse resolution offers new opportunities for exploring synaptic reorganization in response to chemical and genetic perturbations. Here, we combine 12-color multiplexed fluorescence imaging with quantitative image analysis and machine learning to identify novel synaptic subtypes within excitatory and inhibitory synapses based on the expression profiles of major synaptic components. We characterize differences in the correlated expression of proteins within these subtypes and we examine how the distribution of these synapses is modified following induction of synaptic plasticity. Under chronic suppression of neur...

A key to the pathogenic success of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the capacity to survive within host macrophages. Although several factors required for this survival have been identified, a comprehensive knowledge of such factors and how they work together to manipulate the host environment to benefit bacterial survival are not well understood. To systematically identify Mtb factors required for intracellular growth, we screened an arrayed, non-redundant Mtb transposon mutant library by high-content imaging to characterize the mutant-macrophage interaction. Based on a combination of imaging features, we identified mutants impaired for intracellular survival. We then characterized the phenotype of infection with each mutant by profiling the induced macrophage cytokine response. Taking a systems-level approach to understanding the biology of identified mutants, we performed a multiparametric analysis combining pathogen and host phenotypes to...

Large-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications. This microscopy data set includes 919,874 five-channel fields of view representing 30,616 tested compounds, available at 'The Cell Image Library' repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments a...

We hypothesized that human genes and disease-associated alleles might be systematically functionally annotated using morphological profiling of cDNA constructs, via a microscopy-based Cell Painting assay. Indeed, 50% of the 220 tested genes yielded detectable morphological profiles, which grouped into biologically meaningful gene clusters consistent with known functional annotation (e.g., the RAS-RAF-MEK-ERK cascade). We used novel subpopulation-based visualization methods to interpret the morphological changes for specific clusters. This unbiased morphologic map of gene function revealed TRAF2/c-REL negative regulation of YAP1/WWTR1-responsive pathways. We confirmed this discovery of functional connectivity between the NF-κB pathway and Hippo pathway effectors at the transcriptional level, thereby expanding knowledge of these two signaling pathways that critically regulate tumor initiation and progression. We make the images and raw data publicly available, providing an initial mor...

Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer.

Quantitative microscopy has proven a versatile and powerful phenotypic screening technique. Recently, image-based profiling has shown promise as a means for broadly characterizing molecules’ effects on cells in several drug-discovery applications, including target-agnostic screening and predicting a compound’s mechanism of action (MOA). Several profiling methods have been proposed, but little is known about their comparative performance, impeding the wider adoption and further development of image-based profiling. We compared these methods by applying them to a widely applicable assay of cultured cells and measuring the ability of each method to predict the MOA of a compendium of drugs. A very simple method that is based on population means performed as well as methods designed to take advantage of the measurements of individual cells. This is surprising because many treatments induced a heterogeneous phenotypic response across the cell population in each sample. Another simple meth...

The elasto-plastic analysis of reinforced concrete square columns was introduced to study the relationship between lateral force and curvature by using the open source program OpenSees. A pseudo static test on the inverted "T" shape reinforced concrete square column was conducted for the comparative analysis. The results indicated that the lateral force calculated by program agree with experimental data with an axial load ratio of 0.33. The calculated yield lateral force was 29.7 kN, the error was lower than 7% compared with experimental results. In addition, the cross-section curvature were obtained, which were difficult to obtain through the traditional experimental study, the calculated yield curvature was 1.825×10-5. The calculated results with different axial load ratios were presented in this paper, which showed that both the yield lateral force and curvature of reinforced concrete square columns were increased with low axial load ratio, and at the same time it could...