Life Sciences Commons^™

Metaboanalyst 5.0: Narrowing The Gap Between Raw Spectra And Functional Insights., Zhiqiang Pang, Jasmine Chong, Guangyan Zhou, David Anderson De Lima Morais, Le Chang, Michel Barrette, Carol Gauthier, Pierre-Étienne Jacques, Shuzhao Li, Jianguo Xia

Faculty Research 2021

Since its first release over a decade ago, the MetaboAnalyst web-based platform has become widely used for comprehensive metabolomics data analysis and interpretation. Here we introduce MetaboAnalyst version 5.0, aiming to narrow the gap from raw data to functional insights for global metabolomics based on high-resolution mass spectrometry (HRMS). Three modules have been developed to help achieve this goal, including: (i) a LC-MS Spectra Processing module which offers an easy-to-use pipeline that can perform automated parameter optimization and resumable analysis to significantly lower the barriers to LC-MS1 spectra processing; (ii) a Functional Analysis module which expands the previous MS Peaks …

Network Topology Of Biological Aging And Geroscience-Guided Approaches To Covid-19., Alan Landay, Jenna Bartley, Dishary Banerjee, Geneva Hargis, Laura Haynes, Ali Keshavarzian, Chia-Ling Kuo, Oh Sung Kwon, Sheng Li, Shuzhao Li, Julia Oh, Ibrahim Tarik Ozbolat, Duygu Ucar, Ming Xu, Xudong Yao, Derya Unutmaz, George A Kuchel

Faculty Research 2021

Aging has emerged as the greatest and most prevalent risk factor for the development of severe COVID-19 infection and death following exposure to the SARS-CoV-2 virus. The presence of multiple co-existing chronic diseases and conditions of aging further enhances this risk. Biological aging not only enhances the risk of chronic diseases, but the presence of such conditions further accelerates varied biological processes or "hallmarks" implicated in aging. Given growing evidence that it is possible to slow the rate of many biological aging processes using pharmacological compounds has led to the proposal that such geroscience-guided interventions may help enhance immune resilience …

Long Non-Coding Rnas And Their Targets As Potential Biomarkers In Breast Cancer., Maryam Khalid, Rehan Zafar Paracha, Maryum Nisar, Sumaira Malik, Salma Tariq, Iqra Arshad, Amnah Siddiqa, Zamir Hussain, Jamil Ahmad, Amjad Ali

Faculty Research 2021

Breast cancer is among the lethal types of cancer with a high mortality rate, globally. Its high prevalence can be controlled through improved analysis and identification of disease-specific biomarkers. Recently, long non-coding RNAs (lncRNAs) have been reported as key contributors of carcinogenesis and regulate various cellular pathways through post-transcriptional regulatory mechanisms. The specific aim of this study was to identify the novel interactions of aberrantly expressed genetic components in breast cancer by applying integrative analysis of publicly available expression profiles of both lncRNAs and mRNAs. Differential expression patterns were identified by comparing the breast cancer expression profiles of samples with …

Rna-Seq Reveals Changes In Human Placental Metabolism, Transport And Endocrinology Across The First-Second Trimester Transition., Malwina Prater, Russell S Hamilton, Hong Wa Yung, Andrew M Sharkey, Paul Robson, N Erlyani Abd Hamid, Eric Jauniaux, D Stephen Charnock-Jones, Graham J Burton, Tereza Cindrova-Davies

Faculty Research 2021

The human placenta is exposed to major environmental changes towards the end of the first trimester associated with full onset of the maternal arterial placental circulation. Changes include a switch from histotrophic to hemotrophic nutrition, and a threefold rise in the intraplacental oxygen concentration. We evaluated their impact on trophoblast development and function using RNA-sequencing (RNA-Seq) and DNA-methylation analyses performed on the same chorionic villous samples at 7-8 (n=8) and 13-14 (n=6) weeks of gestation. Reads were adjusted for fetal sex. Most DEGs were associated with protein processing in the endoplasmic reticulum (ER), hormone secretion, transport, extracellular matrix, vasculogenesis, and …

E2f6 Initiates Stable Epigenetic Silencing Of Germline Genes During Embryonic Development., Thomas Dahlet, Matthias Truss, Ute Frede, Hala Al Adhami, Anaïs F Bardet, Michael Dumas, Judith Vallet, Johana Chicher, Philippe Hammann, Sarah Kottnik, Peter Hansen, Uschi Luz, Gonzalo Alvarez, Ghislain Auclair, Jochen Hecht, Peter N Robinson, Christian Hagemeier, Michael Weber

Faculty Research 2021

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads …

Human Kit+ Myeloid Cells Facilitate Visceral Metastasis By Melanoma., Chun I. Yu, Jan Martinek, Te-Chia Wu, Kyung In Kim, Joshy George, Elaheh Ahmadzadeh, Richard S. Maser, Florentina Marches, Patrick Metang, Pierre Authie, Vanessa K P Oliveira, Victor G Wang, Jeffrey Chuang, Paul Robson, Jacques Banchereau, Karolina Palucka

Faculty Research 2021

Metastasis of melanoma significantly worsens prognosis; thus, therapeutic interventions that prevent metastasis could improve patient outcomes. Here, we show using humanized mice that colonization of distant visceral organs with melanoma is dependent upon a human CD33+CD11b+CD117+ progenitor cell subset comprising <4% of the human CD45+ leukocytes. Metastatic tumor-infiltrating CD33+ cells from patients and humanized (h)NSG-SGM3 mice showed converging transcriptional profiles. Single-cell RNA-seq analysis identified a gene signature of a KIT/CD117-expressing CD33+ subset that correlated with decreased overall survival in a TCGA melanoma cohort. Thus, human CD33+CD11b+CD117+ myeloid cells represent a novel candidate biomarker as well as a therapeutic target for metastatic melanoma.

Endometrial Receptivity And Implantation Require Uterine Bmp Signaling Through An Acvr2a-Smad1/Smad5 Axis., Diana Monsivais, Takashi Nagashima, Renata Prunskaite-Hyyryläinen, Kaori Nozawa, Keisuke Shimada, Suni Tang, Clark Hamor, Julio E Agno, Fengju Chen, Ramya P Masand, Steven L Young, Chad J Creighton, Francesco J Demayo, Masahito Ikawa, Se-Jin Lee, Martin M Matzuk

Faculty Research 2021

During early pregnancy in the mouse, nidatory estrogen (E2) stimulates endometrial receptivity by activating a network of signaling pathways that is not yet fully characterized. Here, we report that bone morphogenetic proteins (BMPs) control endometrial receptivity via a conserved activin receptor type 2 A (ACVR2A) and SMAD1/5 signaling pathway. Mice were generated to contain single or double conditional deletion of SMAD1/5 and ACVR2A/ACVR2B receptors using progesterone receptor (PR)-cre. Female mice with SMAD1/5 deletion display endometrial defects that result in the development of cystic endometrial glands, a hyperproliferative endometrial epithelium during the window of implantation, and impaired apicobasal transformation that prevents …

Maximizing The Potential Of Aggressive Mouse Tumor Models In Preclinical Drug Testing., M Tarek Elghetany, Jia-Min Ho, Lois Hew Shi-Qi, Sekar Karthik, Jack M F Su, Qi Lin, Yuchen Du, Jianhe Shen, Wing-Yuk Chow, Ching C Lau, Adekunle Adesina, Angela Major, Anat Erdreich-Epstein, Kam-Man Hui, Xiao-Nan Li, Wan-Yee Teo

Faculty Research 2021

Atypical teratoid rhabdoid tumor (ATRT) is an aggressive embryonal brain tumor among infants and young children. Two challenges exist for preclinical testing in ATRT. First, genetically quiet, ATRT is a difficult tumor to target molecularly. Tumor cells need to divide to propagate tumor growth-intercepting the common crossroads in cell cycle progression is a feasible strategy. KIF11 is needed for bipolar spindle formation in metaphase. We identified KIF11 as a universal target of all ATRT-molecular-subtypes. Ispinesib, a KIF11-inhibitor, effectively inhibited tumor proliferation in all seven cell lines. A second challenge-a major challenge in preclinical drug testing in-vivo among aggressive tumor models, …

Modeling Seizures In The Human Phenotype Ontology According To Contemporary Ilae Concepts Makes Big Phenotypic Data Tractable., David Lewis-Smith, Peter D Galer, Ganna Balagura, Hugh Kearney, Shiva Ganesan, Mahgenn Cosico, Margaret O'Brien, Priya Vaidiswaran, Roland Krause, Colin A Ellis, Rhys H Thomas, Peter N Robinson, Ingo Helbig

Faculty Research 2021

OBJECTIVE: The clinical features of epilepsy determine how it is defined, which in turn guides management. Therefore, consideration of the fundamental clinical entities that comprise an epilepsy is essential in the study of causes, trajectories, and treatment responses. The Human Phenotype Ontology (HPO) is used widely in clinical and research genetics for concise communication and modeling of clinical features, allowing extracted data to be harmonized using logical inference. We sought to redesign the HPO seizure subontology to improve its consistency with current epileptological concepts, supporting the use of large clinical data sets in high-throughput clinical and research genomics.

METHODS: We …

Integrating Genomics And Metabolomics For Scalable Non-Ribosomal Peptide Discovery., Bahar Behsaz, Edna Bode, Alexey Gurevich, Yan-Ni Shi, Florian Grundmann, Deepa Acharya, Andrés Mauricio Caraballo-Rodríguez, Amina Bouslimani, Morgan Panitchpakdi, Annabell Linck, Changhui Guan, Julia Oh, Pieter C Dorrestein, Helge B Bode, Pavel A Pevzner, Hosein Mohimani

Faculty Research 2021

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, …

Somatostatin-Expressing Parafacial Neurons Are Co2/H+ Sensitive And Regulate Baseline Breathing., Colin M Cleary, Brenda M Milla, Fu-Shan Kuo, Shaun James, William F Flynn, Paul Robson, Daniel K Mulkey

Faculty Research 2021

Glutamatergic neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating breathing in response to tissue CO2/H+. The RTN and greater parafacial region may also function as a chemosensing network composed of CO2/H+-sensitive excitatory and inhibitory synaptic interactions. In the context of disease, we showed that loss of inhibitory neural activity in a mouse model of Dravet syndrome disinhibited RTN chemoreceptors and destabilized breathing (Kuo et al., 2019). Despite this, contributions of parafacial inhibitory neurons to control of breathing are unknown, and synaptic properties of RTN neurons have not been characterized. Here, we show the parafacial region contains …

Macrophage Mediated Recognition And Clearance Of Borrelia Burgdorferi Elicits Myd88-Dependent And -Independent Phagosomal Signals That Contribute To Phagocytosis And Inflammation., Sarah J Benjamin, Kelly L Hawley, Paola Vera-Licona, Carson J La Vake, Jorge L Cervantes, Yijun Ruan, Justin D Radolf, Juan C Salazar

Faculty Research 2021

BACKGROUND: Macrophages play prominent roles in bacteria recognition and clearance, including Borrelia burgdorferi (Bb), the Lyme disease spirochete. To elucidate mechanisms by which MyD88/TLR signaling enhances clearance of Bb by macrophages, we studied wildtype (WT) and MyD88

RESULTS: MyD88

CONCLUSION: Our findings show that MyD88 signaling enhances, but is not required, for bacterial uptake or phagosomal maturation and provide mechanistic insights into how MyD88-mediated phagosomal signaling enhances Bb uptake and clearance.

Transfer Learning-Trained Convolutional Neural Networks Identify Novel Mri Biomarkers Of Alzheimer's Disease Progression., Yi Li, Annat Haber, Christoph Preuss, Cai John, Asli Uyar, Hongtian Stanley Yang, Benjamin A Logsdon, Vivek M. Philip, Radha Krishna Murthy Karuturi, Gregory W. Carter, Alzheimer's Disease Neuroimaging Initative

Faculty Research 2021

Introduction: Genome-wide association studies (GWAS) for late onset Alzheimer's disease (AD) may miss genetic variants relevant for delineating disease stages when using clinically defined case/control as a phenotype due to its loose definition and heterogeneity.

Methods: We use a transfer learning technique to train three-dimensional convolutional neural network (CNN) models based on structural magnetic resonance imaging (MRI) from the screening stage in the Alzheimer's Disease Neuroimaging Initiative consortium to derive image features that reflect AD progression.

Results: CNN-derived image phenotypes are significantly associated with fasting metabolites related to early lipid metabolic changes as well as insulin resistance and with genetic …

A New Twist: The Combination Of Sulbactam/Avibactam Enhances Sulbactam Activity Against Carbapenem-Resistant, Fernando Pasteran, Jose Cedano, Michelle Baez, Ezequiel Albornoz, Melina Rapoport, Jose Osteria, Sabrina Montaña, Casin Le, Grace Ra, Robert A Bonomo, Marcelo E Tolmasky, Mark D Adams, Alejandra Corso, Maria Soledad Ramirez

Faculty Research 2021

An increasing number of untreatable infections are recorded every year. Many studies have focused their efforts on developing new β-lactamase inhibitors to treat multi-drug resistant (MDR) isolates. In the present study, sulbactam/avibactam and sulbactam/relebactam combination were tested against 187 multi-drug resistant (MDR) Acinetobacter clinical isolates; both sulbactam/avibactam and sulbactam/relebactam restored sulbactam activity. A decrease ≥2 dilutions in sulbactam MICs was observed in 89% of the isolates when tested in combination with avibactam. Sulbactam/relebactam was able to restore sulbactam susceptibility in 40% of the isolates. In addition, the susceptibility testing using twenty-three A. baumannii AB5075 knockout strains revealed potential sulbactam and/or …

Sarcomere Function Activates A P53-Dependent Dna Damage Response That Promotes Polyploidization And Limits In Vivo Cell Engraftment., Anthony M Pettinato, Dasom Yoo, Jennifer Vanoudenhove, Yu-Sheng Chen, Rachel Cohn, Feria A Ladha, Xiulan Yang, Ketan Thakar, Robert Romano, Nicolas Legere, Emily Meredith, Paul Robson, Michael Regnier, Justin L Cotney, Charles E Murry, J Travis Hinson

Faculty Research 2021

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or …

User Testing Of A Diagnostic Decision Support System With Machine-Assisted Chart Review To Facilitate Clinical Genomic Diagnosis., Alanna Kulchak Rahm, Nephi A Walton, Lynn K Feldman, Conner Jenkins, Troy Jenkins, Thomas N Person, Joeseph Peterson, Jonathon C Reynolds, Peter N Robinson, Makenzie A Woltz, Marc S Williams, Michael M Segal

Faculty Research 2021

OBJECTIVES: There is a need in clinical genomics for systems that assist in clinical diagnosis, analysis of genomic information and periodic reanalysis of results, and can use information from the electronic health record to do so. Such systems should be built using the concepts of human-centred design, fit within clinical workflows and provide solutions to priority problems.

METHODS: We adapted a commercially available diagnostic decision support system (DDSS) to use extracted findings from a patient record and combine them with genomic variant information in the DDSS interface. Three representative patient cases were created in a simulated clinical environment for user …

Sfrp4 Drives Invasion In Gastric Cancer And Is An Early Predictor Of Recurrence., Rita A Busuttil, Joshy George, Colin M House, Stephen Lade, Catherine Mitchell, Natasha S Di Costanzo, Sharon Pattison, Yu-Kuan Huang, Patrick Tan, Jae-Ho Cheong, Sun Young Rha, Alex Boussioutas

Faculty Research 2021

OBJECTIVE: Gastric cancer patients generally have a poor outcome, particularly those with advanced-stage disease which is defined by the increased invasion of cancer locally and is associated with higher metastatic potential. This study aimed to identify genes that were functional in the most fundamental hallmark of cancer, namely invasion. We then wanted to assess their value as biomarkers of gastric cancer progression and recurrence.

DESIGN: Data from a cohort of patients profiled on cDNA expression arrays was interrogated using K-means analysis. This genomic approach classified the data based on patterns of gene expression allowing the identification of the genes most …

Defects In Translation-Dependent Quality Control Pathways Lead To Convergent Molecular And Neurodevelopmental Pathology., Markus Terrey, Scott I Adamson, Jeffrey H Chuang, Susan L Ackerman

Faculty Research 2021

Translation-dependent quality control pathways such as no-go decay (NGD), non-stop decay (NSD), and nonsense-mediated decay (NMD) govern protein synthesis and proteostasis by resolving non-translating ribosomes and preventing the production of potentially toxic peptides derived from faulty and aberrant mRNAs. However, how translation is altered and the in vivo defects that arise in the absence of these pathways are poorly understood.

Here, we show that the NGD/NSD factors Pelo and Hbs1l are critical in mice for cerebellar neurogenesis but expendable for survival of these neurons after development. Analysis of mutant mouse embryonic fibroblasts revealed translational pauses, alteration of signaling pathways, and …

Genome-Based Targeted Sequencing As A Reproducible Microbial Community Profiling Assay., Jacquelynn Benjamino, Benjamin Leopold, Daniel S Phillips, Mark D Adams

Faculty Research 2021

Current sequencing-based methods for profiling microbial communities rely on marker gene (e.g., 16S rRNA) or metagenome shotgun sequencing (mWGS) analysis. We present an approach based on a single-primer extension reaction using a highly multiplexed oligonucleotide probe pool. This approach, termed MA-GenTA (microbial abundances from genome tagged analysis), enables quantitative, straightforward, cost-effective microbiome profiling that combines desirable features of both 16S rRNA and mWGS strategies. The use of multiple probes per target genome and rigorous probe design criteria enabled robust determination of relative abundance. To test the utility of the MA-GenTA assay, probes were designed for 830 genome sequences representing bacteria …

Sars-Cov-2 Infects Human Engineered Heart Tissues And Models Covid-19 Myocarditis., Adam L Bailey, Oleksandr Dmytrenko, Lina Greenberg, Andrea L Bredemeyer, Pan Ma, Jing Liu, Vinay Penna, Emma S Winkler, Sanja Sviben, Erin Brooks, Ajith P Nair, Kent A Heck, Aniket S Rali, Leo Simpson, Mehrdad Saririan, Dan Hobohm, W Tom Stump, James A Fitzpatrick, Xuping Xie, Xianwen Zhang, Pei-Yong Shi, J Travis Hinson, Weng-Tein Gi, Constanze Schmidt, Florian Leuschner, Chieh-Yu Lin, Michael S Diamond, Michael J Greenberg, Kory J Lavine

Faculty Research 2021

There is ongoing debate as to whether cardiac complications of coronavirus disease-2019 (COVID-19) result from myocardial viral infection or are secondary to systemic inflammation and/or thrombosis. We provide evidence that cardiomyocytes are infected in patients with COVID-19 myocarditis and are susceptible to severe acute respiratory syndrome coronavirus 2. We establish an engineered heart tissue model of COVID-19 myocardial pathology, define mechanisms of viral pathogenesis, and demonstrate that cardiomyocyte severe acute respiratory syndrome coronavirus 2 infection results in contractile deficits, cytokine production, sarcomere disassembly, and cell death. These findings implicate direct infection of cardiomyocytes in the pathogenesis of COVID-19 myocardial pathology …

Classification Of Molecular Subtypes Of High-Grade Serous Ovarian Cancer By Maldi-Imaging., Wanja Kassuhn, Oliver Klein, Silvia Darb-Esfahani, Hedwig Lammert, Sylwia Handzik, Eliane T Taube, Wolfgang D Schmitt, Carlotta Keunecke, David Horst, Felix Dreher, Joshy George, David D Bowtell, Oliver Dorigo, Michael Hummel, Jalid Sehouli, Nils Blüthgen, Hagen Kulbe, Elena I Braicu

Faculty Research 2021

Despite the correlation of clinical outcome and molecular subtypes of high-grade serous ovarian cancer (HGSOC), contemporary gene expression signatures have not been implemented in clinical practice to stratify patients for targeted therapy. Hence, we aimed to examine the potential of unsupervised matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to stratify patients who might benefit from targeted therapeutic strategies. Molecular subtyping of paraffin-embedded tissue samples from 279 HGSOC patients was performed by NanoString analysis (ground truth labeling). Next, we applied MALDI-IMS paired with machine-learning algorithms to identify distinct mass profiles on the same paraffin-embedded tissue sections and distinguish HGSOC subtypes by …

Perspective Of Mesenchymal Transformation In Glioblastoma., Yona Kim, Frederick S Varn, Sung-Hye Park, Byung Woo Yoon, Hye Ran Park, Charles Lee, Roel G W Verhaak, Sun Ha Paek

Faculty Research 2021

Despite aggressive multimodal treatment, glioblastoma (GBM), a grade IV primary brain tumor, still portends a poor prognosis with a median overall survival of 12-16 months. The complexity of GBM treatment mainly lies in the inter- and intra-tumoral heterogeneity, which largely contributes to the treatment-refractory and recurrent nature of GBM. By paving the road towards the development of personalized medicine for GBM patients, the cancer genome atlas classification scheme of GBM into distinct transcriptional subtypes has been considered an invaluable approach to overcoming this heterogeneity. Among the identified transcriptional subtypes, the mesenchymal subtype has been found associated with more aggressive, invasive, …

Cerebellar Kv3.3 Potassium Channels Activate Tank-Binding Kinase 1 To Regulate Trafficking Of The Cell Survival Protein Hax-1., Yalan Zhang, Luis Varela, Klara Szigeti-Buck, Adam Williams, Milan Stoiljkovic, Matija Šestan-Peša, Jorge Henao-Mejia, Pasquale D'Acunzo, Efrat Levy, Richard A Flavell, Tamas L Horvath, Leonard K Kaczmarek

Faculty Research 2021

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival …

Single-Cell Longitudinal Analysis Of Sars-Cov-2 Infection In Human Airway Epithelium Identifies Target Cells, Alterations In Gene Expression, And Cell State Changes., Neal G Ravindra, Mia Madel Alfajaro, Victor Gasque, Nicholas C Huston, Han Wan, Klara Szigeti-Buck, Yuki Yasumoto, Allison M Greaney, Victoria Habet, Ryan D Chow, Jennifer S Chen, Jin Wei, Renata B Filler, Bao Wang, Guilin Wang, Laura E Niklason, Ruth R Montgomery, Stephanie C Eisenbarth, Sidi Chen, Adam Williams, Akiko Iwasaki, Tamas L Horvath, Ellen F Foxman, Richard W Pierce, Anna Marie Pyle, David Van Dijk, Craig B Wilen

Faculty Research 2021

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we …

Single Cell Analysis Of Blood Mononuclear Cells Stimulated Through Either Lps Or Anti-Cd3 And Anti-Cd28., Nathan Lawlor, Djamel Nehar-Belaid, Jessica D S Grassmann, Marlon Stoeckius, Peter Smibert, Michael L. Stitzel, Virginia Pascual, Jacques Banchereau, Adam Williams, Duygu Ucar

Faculty Research 2021

Immune cell activation assays have been widely used for immune monitoring and for understanding disease mechanisms. However, these assays are typically limited in scope. A holistic study of circulating immune cell responses to different activators is lacking. Here we developed a cost-effective high-throughput multiplexed single-cell RNA-seq combined with epitope tagging (CITE-seq) to determine how classic activators of T cells (anti-CD3 coupled with anti-CD28) or monocytes (LPS) alter the cell composition and transcriptional profiles of peripheral blood mononuclear cells (PBMCs) from healthy human donors. Anti-CD3/CD28 treatment activated all classes of lymphocytes either directly (T cells) or indirectly (B and NK cells) …

Employees' Views And Ethical, Legal, And Social Implications Assessment Of Voluntary Workplace Genomic Testing, Kunal Sanghavi, W Gregory Feero, Debra Jh Mathews, Anya Er Prince, Lori Lyn Price, Edison Liu, Kyle Brothers, J Scott Roberts, Charles Lee

Faculty Research 2021

Employers have begun to offer voluntary workplace genomic testing (wGT) as part of employee wellness benefit programs, but few empirical studies have examined the ethical, legal, and social implications (ELSI) of wGT. To better understand employee perspectives on wGT, employees were surveyed at a large biomedical research institution. Survey respondents were presented with three hypothetical scenarios for accessing health-related genomic testing: via (1) their doctor; (2) their workplace; and 3) a commercial direct-to-consumer (DTC) genetic testing company. Overall, 594 employees (28%) responded to the survey. Respondents indicated a preference for genomic testing in the workplace setting (70%; 95% CI 66-74%), …

A Novel Cd4+ Ctl Subtype Characterized By Chemotaxis And Inflammation Is Involved In The Pathogenesis Of Graves' Orbitopathy., Yue Wang, Ziyi Chen, Tingjie Wang, Hui Guo, Yufeng Liu, Ningxin Dang, Shiqian Hu, Liping Wu, Chengsheng Zhang, Kai Ye, Bingyin Shi

Faculty Research 2021

Graves' orbitopathy (GO), the most severe manifestation of Graves' hyperthyroidism (GH), is an autoimmune-mediated inflammatory disorder, and treatments often exhibit a low efficacy. CD4+ T cells have been reported to play vital roles in GO progression. To explore the pathogenic CD4+ T cell types that drive GO progression, we applied single-cell RNA sequencing (scRNA-Seq), T cell receptor sequencing (TCR-Seq), flow cytometry, immunofluorescence and mixed lymphocyte reaction (MLR) assays to evaluate CD4+ T cells from GO and GH patients. scRNA-Seq revealed the novel GO-specific cell type CD4+ cytotoxic T lymphocytes (CTLs), which are characterized by chemotactic and inflammatory features. The clonal …

Targeted Rnaseq Assay Incorporating Unique Molecular Identifiers For Improved Quantification Of Gene Expression Signatures And Transcribed Mutation Fraction In Fixed Tumor Samples., Chunxiao Fu, Michal Marczyk, Michael Samuels, Alexander J Trevarton, Jiaxin Qu, Rosanna Lau, Lili Du, Todd Pappas, Bruno V Sinn, Rebekah E Gould, Lajos Pusztai, Christos Hatzis, W Fraser Symmans

Faculty Research 2021

BACKGROUND: Our objective was to assess whether modifications to a customized targeted RNA sequencing (RNAseq) assay to include unique molecular identifiers (UMIs) that collapse read counts to their source mRNA counts would improve quantification of transcripts from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples. The assay (SET4) includes signatures that measure hormone receptor and PI3-kinase related transcriptional activity (SET

METHODS: Modifications included steps to introduce eight nucleotides-long UMIs during reverse transcription (RT) in bulk solution, followed by polymerase chain reaction (PCR) of labeled cDNA in droplets, with optimization of the polymerase enzyme and reaction conditions. We used Lin's concordance correlation coefficient …

Capsule Carbohydrate Structure Determines Virulence In Acinetobacter Baumannii., Yuli Talyansky, Travis B Nielsen, Jun Yan, Ulrike Carlino-Macdonald, Gisela Di Venanzio, Somnath Chakravorty, Amber Ulhaq, Mario F Feldman, Thomas A Russo, Evgeny Vinogradov, Brian Luna, Meredith S Wright, Mark D Adams, Brad Spellberg

Faculty Research 2021

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and …

Genome-Scale Crispr Screening Identifies Cell Cycle And Protein Ubiquitination Processes As Druggable Targets For Erlotinib-Resistant Lung Cancer., Jieun Lee, Ahyoung Choi, Sung-Yup Cho, Yukyung Jun, Deukchae Na, Ahra Lee, Giyong Jang, Jee Young Kwon, Jaesang Kim, Sanghyuk Lee, Charles Lee

Faculty Research 2021

Erlotinib is highly effective in lung cancer patients with epidermal growth factor receptor (EGFR) mutations. However, despite initial favorable responses, most patients rapidly develop resistance to erlotinib soon after the initial treatment. This study aims to identify new genes and pathways associated with erlotinib resistance mechanisms in order to develop novel therapeutic strategies. Here, we induced knockout (KO) mutations in erlotinib-resistant human lung cancer cells (NCI-H820) using a genome-scale CRISPR-Cas9 sgRNA library to screen for genes involved in erlotinib susceptibility. The spectrum of sgRNAs incorporated among erlotinib-treated cells was substantially different to that of the untreated cells. Gene set analyses …