Single-molecule Biophysics: Machine Learning for Automated Data Processing

Abstract-Single-molecule fluorescence microscopy has been greatly successful in understanding biophysics at molecular level. This technique has been advancing toward higher throughput, which creates a need for a data-analysis tool to distinguish molecule of interest from other fluorescence signals. Here, we have used supervised machine-learning approaches to filter biological events of our interest, and present three approaches applicable to different data set size

Imaging cell lineage with a synthetic digital recording system

Cell lineage plays a pivotal role in cell fate determination. Chow et al. demonstrate the use of an integrase-based synthetic barcode system called intMEMOIR, which uses the serine integrase Bxb1 to perform irreversible nucleotide edits. Inducible editing either deletes or inverts its target region, thus encoding information in three-state memory elements, or trits, and avoiding undesired recombination events. Using intMEMOIR combined with single-molecule fluorescence in situ hybridization, the authors were able to identify clonal structures as well as gene expression patterns in the fly brain, enabling both clonal analysis and expression profiling with intact spatial information. The ability to visualize cell lineage relationships directly within their native tissue context provides insights into development and disease

Biased TAS2R Bronchodilators Inhibit Airway Smooth Muscle Growth by Downregulating pERK1/2

Bitter taste receptor (TAS2R) agonists dilate airways by receptor-dependent smooth muscle relaxation. Besides coupling to relaxation, we have found that human airway smooth muscle (HASM) cell TAS2Rs activate (phosphorylate) ERK1/2, but the cellular effects are not known. Here we show in HASM cells that TAS2R agonists initially stimulate pERK1/2, but by 24 hrs cause a marked (50-70%) downregulation of pERK1/2 without a change in total ERK1/2. It was hypothesized that TAS2R agonists suppress cell growth through this pERK1/2 downregulation. Agonist-dependent inhibition of cell proliferation was indeed found in HASM cells derived from normal and asthmatic human lungs, as well as an immortalized HASM cell line. pERK1/2 downregulation was linked to downregulation of the upstream kinase MEK1/2. Various structurally diverse TAS2R agonists evoked a range of inhibition of HASM proliferation, the magnitude of which directly correlated with the downregulation of pERK1/2 (R2 = 0.86). Some TAS2R agonists were as effective in suppressing growth as pharmacological inhibitors of Raf1 and MEK1/2. siRNA silencing of TAS2Rs (subtypes 10, 14, 31) ablated the pERK1/2 and growth inhibiting effects of TAS2R agonists. These phenotypes were attenuated by inhibiting the TAS2R G-protein Gα, and by knocking-down β-arrestin1/2, indicating a dual pathway, although there may be additional mechanisms involved in this HASM TAS2R multidimensional signaling. Thus TAS2R agonist structure can be manipulated to maintain the relaxation response, and be biased towards suppression of HASM growth. The latter response is of potential therapeutic benefit in asthma, where an increase in smooth muscle mass contributes to airway obstruction

Imaging cell lineage with a synthetic digital recording system

Multicellular development depends on the differentiation of cells into specific fates with precise spatial organization. Lineage history plays a pivotal role in cell fate decisions, but is inaccessible in most contexts. Engineering cells to actively record lineage information in a format readable in situ would provide a spatially resolved view of lineage in diverse developmental processes. Here, we introduce a serine integrase-based recording system that allows in situ readout, and demonstrate its ability to reconstruct lineage relationships in cultured stem cells and flies. The system, termed intMEMOIR, employs an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. intMEMOIR accurately reconstructed lineage trees in stem cells and enabled simultaneous analysis of single cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable clonal analysis and recording in diverse systems

Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers.

Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling

Dairy Cattle, a Potential Reservoir of Human Campylobacteriosis: Epidemiological and Molecular Characterization of Campylobacter jejuni From Cattle Farms

Campylobacter jejuni is a major foodborne pathogen that is increasingly found worldwide and that is transmitted to humans through meat or dairy products. A detailed understanding of the prevalence and characteristics of C. jejuni in dairy cattle farms, which are likely to become sources of contamination, is imperative and is currently lacking. In this study, a total of 295 dairy cattle farm samples from 15 farms (24 visits) in Korea were collected. C. jejuni prevalence at the farm level was 60% (9/15) and at the animal level was 23.8% (68/266). Using the multivariable generalized estimating equation (GEE) model based on farm-environmental factors, we estimated that a high density of cattle and average environmental temperature (7 days prior to sampling) below 24°C affects the presence and survival of C. jejuni in the farm environment. Cattle isolates, together with C. jejuni from other sources (chicken and human), were genetically characterized based on analysis of 10 virulence and survival genes. A total of 19 virulence profile types were identified, with type 01 carrying eight genes (all except hcp and virB11) being the most prevalent. The prevalence of virB11 and hcp was significantly higher in isolates from cattle than in those from other sources (p < 0.05). Multilocus sequence typing (MLST) of C. jejuni isolates from three different sources mainly clustered in the CC-21 and CC-48. Within the CC-21 and CC-48 clusters, cattle isolates shared an indistinguishable pattern with human isolates according to pulsed-field gel electrophoresis (PFGE) and flaA-restriction fragment length polymorphism (RFLP) typing. This suggests that CC-21 and CC-48 C. jejuni from dairy cattle are genetically related to clinical campylobacteriosis isolates. In conclusion, the farm environment influences the presence and survival of C. jejuni, which may play an important role in cycles of cattle re-infection, and dairy cattle represent potential reservoirs of human campylobacteriosis. Thus, environmental management practices could be implemented on cattle farms to reduce the shedding of C. jejuni from cattle, subsequently reducing the potential risk of the spread of cattle-derived C. jejuni to humans through the food chain

Biased TAS2R Bronchodilators Inhibit Airway Smooth Muscle Growth by Downregulating Phosphorylated Extracellular Signal–regulated Kinase 1/2

Bitter taste receptor (TAS2R) agonists dilate airways by receptor-dependent smooth muscle relaxation. Besides their coupling to relaxation, we have found that human airway smooth muscle (HASM) cell TAS2Rs activate (phosphorylate) extracellular signal–related kinase 1/2 (ERK1/2), but the cellular effects are not known. In the present study, we show in HASM cells that TAS2R agonists initially stimulate phosphorylated ERK1/2 (pERK1/2) but by 24 hours cause a marked (50–70%) downregulation of pERK1/2 without a change in total ERK1/2. It was hypothesized that TAS2R agonists suppress cell growth through this pERK1/2 downregulation. Agonist-dependent inhibition of cell proliferation was indeed found in HASM cells derived from normal and asthmatic human lungs, as well as in an immortalized HASM cell line. pERK1/2 downregulation was linked to downregulation of the upstream kinase MEK1/2 (mitogen-activated protein kinase/extracellular signal–regulated kinase). Various structurally diverse TAS2R agonists evoked a range of inhibition of HASM proliferation, the magnitude of which directly correlated with the downregulation of pERK1/2 (R^2 = 0.86). Some TAS2R agonists were as effective as pharmacological inhibitors of Raf1 and MEK1/2 in suppressing growth. siRNA silencing of TAS2Rs (subtypes 10, 14, and 31) ablated the pERK1/2 and growth-inhibitory effects of TAS2R agonists. These phenotypes were attenuated by inhibiting the TAS2R G protein G_(αi) and by knocking down β-arrestin 1/2, indicating a dual pathway, although there may be additional mechanisms involved in this HASM TAS2R multidimensional signaling. Thus, TAS2R agonist structure can be manipulated to maintain the relaxation response and can be biased toward suppression of HASM growth. The latter response is of potential therapeutic benefit in asthma, in which an increase in smooth muscle mass contributes to airway obstruction

A standardized pathology report for gastric cancer: 2nd edition

The first edition of ‘A Standardized Pathology Report for Gastric Cancer’ was initiated by the Gastrointestinal Pathology Study Group of the Korean Society of Pathologists and published 17 years ago. Since then, significant advances have been made in the pathologic diagnosis, molecular genetics, and management of gastric cancer (GC). To reflect those changes, a committee for publishing a second edition of the report was formed within the Gastrointestinal Pathology Study Group of the Korean Society of Pathologists. This second edition consists of two parts: standard data elements and conditional data elements. The standard data elements contain the basic pathologic findings and items necessary to predict the prognosis of GC patients, and they are adequate for routine surgical pathology service. Other diagnostic and prognostic factors relevant to adjuvant therapy, including molecular biomarkers, are classified as conditional data elements to allow each pathologist to selectively choose items appropriate to the environment in their institution. We trust that the standardized pathology report will be helpful for GC diagnosis and facilitate large-scale multidisciplinary collaborative studies

The Dynamics of Culture in Entrepreneurial Ventures

This dissertation focuses on the dynamics of organizational culture in the context of entrepreneurial ventures. As ventures face a myriad of challenges to grow in uncertain environments, early-stage culture—a system of norms, values, and beliefs shared by organizational members in the early stage of ventures—can be used as valuable resources that lead to a competitive advantage for ventures. Despite the acknowledged importance of culture in the success of organizations, there is a lack of empirical evidence on the role of culture and the factors that affect culture in ventures. One possible reason behind the limited progress on this research may be that it is empirically challenging to observe culture of ventures longitudinally from their inception on a large scale. This dissertation aims to overcome this challenge and investigate the dynamics of ventures’ culture computationally. Hence, throughout this dissertation, I apply various machine learning techniques to measure organizational culture and employee values in ventures. The primary data used for my dissertation are a large sample of venture employee reviews from Glassdoor, which allows me to obtain elements of culture in ventures throughout the stages of ventures’ development. In the three studies, I investigate how culture influences strategic decisions and growth outcomes of ventures, as well as the factors that influence employee values in social ventures. Chapter 1 proposes a cultural perspective to explain how the emotional and cognitive organizational culture shaped by employees influences the type of entrepreneurial strategies a venture pursues. This study finds that ventures with emotional cultural diversity tend to choose strategies guided by exploration, while those with cognitive cultural diversity tend to select strategies guided by ambidexterity. Chapter 2 focuses on the performance implications of organizational culture and the importance of founding culture in providing a competitive advantage for ventures. This study investigates the effects of temporal transmission of founding culture in terms of emotional and cognitive dimensions, shedding new light on the importance of founding culture in ventures through the lens of emotion and cognition. Chapter 3 explores the antecedents of change in employee values, which are one of the layers of organizational culture, driven by an exogenous shock that affects some ventures more than others. I examine how the priorities in different employee values change in the aftermath of a shock. Taken together, my dissertation provides new insights into the role of early-stage organizational culture and the factors that influence employee values in entrepreneurial contexts. I hope that my dissertation will provide insight into how entrepreneurs can effectively manage and make sense of organizational culture to navigate the growth of their ventures