Medical Molecular Biology Commons^™

Protocol To Identify The Core Gene Supported By An Essential Gene In E. Coli Bacteria Using A Genome-Wide Suppressor Screen, Isao Masuda, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

We describe here a genome-wide screening approach to identify the most critical core reaction among a network of many that are supported by an essential gene to establish cell viability. We describe steps for maintenance plasmid construction, knockout cell construction, and phenotype validation. We then detail isolation of suppressors, whole-genome sequencing analysis, and reconstruction of CRISPR mutants. We focus on E. coli trmD, which encodes an essential methyl transferase that synthesizes m1G37 on the 3'-side of the tRNA anticodon. For complete details on the use and execution of this protocol, please refer to Masuda et al. (2022).

Language: Its Origin And Ongoing Evolution, Ilia Markov, Kseniia Kharitonova, Elena L Grigorenko

Faculty, Staff and Students Publications

With the present paper, we sought to use research findings to illustrate the following thesis: the evolution of language follows the principles of human evolution. We argued that language does not exist for its own sake, it is one of a multitude of skills that developed to achieve a shared communicative goal, and all its features are reflective of this. Ongoing emerging language adaptations strive to better fit the present state of the human species. Theories of language have evolved from a single-modality to multimodal, from human-specific to usage-based and goal-driven. We proposed that language should be viewed as a …

Developing A Bacterial Panel For The Evaluation Of Novel Anti-Infective Compounds, Clare Euteneuer

UNO Student Research and Creative Activity Fair

Antibiotic resistance is one of the leading causes of concern for the world health community. Drugs used to stop various infections for years are now becoming easier for bacteria to resist due to mutations and plasmids conferring resistance. To combat this problem, new drug can help alleviate this concern. We developed an assay that allows us to screen novel drug-like compounds against bacteria in an effort to identify promising new anti-infective compounds. Our assay was designed using known drugs against a panel of gram positive and negative bacilli and cocci including S. epidermidis, P. mirabilis, N. mucosa, and E. …

Defects In Lipid Homeostasis Reflect The Function Of Tango2 In Phospholipid And Neutral Lipid Metabolism, Agustin Leonardo Lujan, Ombretta Foresti, Conor Sugden, Nathalie Brouwers, Alex Mateo Farre, Alessio Vignoli, Mahshid Azamian, Alicia Turner, Jose Wojnacki, Vivek Malhotra

Faculty, Staff and Students Publications

We show that TANGO2 in mammalian cells localizes predominantly to mitochondria and partially at mitochondria sites juxtaposed to lipid droplets (LDs) and the endoplasmic reticulum. HepG2 cells and fibroblasts of patients lacking TANGO2 exhibit enlarged LDs. Quantitative lipidomics revealed a marked increase in lysophosphatidic acid (LPA) and a concomitant decrease in its biosynthetic precursor phosphatidic acid (PA). These changes were exacerbated in nutrient-starved cells. Based on our data, we suggest that TANGO2 function is linked to acyl-CoA metabolism, which is necessary for the acylation of LPA to generate PA. The defect in acyl-CoA availability impacts the metabolism of many other …

Conjugation's Toolkit: The Roles Of Nonstructural Proteins In Bacterial Sex, Matthew B Cooke, Christophe Herman

Faculty, Staff and Students Publications

Bacterial conjugation, a form of horizontal gene transfer, relies on a type 4 secretion system (T4SS) and a set of nonstructural genes that are closely linked. These nonstructural genes aid in the mobile lifestyle of conjugative elements but are not part of the T4SS apparatus for conjugative transfer, such as the membrane pore and relaxosome, or the plasmid maintenance and replication machineries. While these nonstructural genes are not essential for conjugation, they assist in core conjugative functions and mitigate the cellular burden on the host. This review compiles and categorizes known functions of nonstructural genes by the stage of conjugation …

Dna Damage Response-Related Proteins Are Prognostic For Outcome In Both Adult And Pediatric Acute Myelogenous Leukemia Patients: Samples From Adults And From Children Enrolled In A Children's Oncology Group Study, Stefan E Hubner, Eduardo S De Camargo Magalhães, Fieke W Hoff, Brandon D Brown, Yihua Qiu, Terzah M Horton, Steven M Kornblau

Faculty, Staff and Student Publications

The survival of malignant leukemic cells is dependent on DNA damage repair (DDR) signaling. Reverse Phase Protein Array (RPPA) data sets were assembled using diagnostic samples from 810 adult and 500 pediatric acute myelogenous leukemia (AML) patients and were probed with 412 and 296 strictly validated antibodies, respectively, including those detecting the expression of proteins directly involved in DDR. Unbiased hierarchical clustering identified strong recurrent DDR protein expression patterns in both adult and pediatric AML. Globally, DDR expression was associated with gene mutational statuses and was prognostic for outcomes including overall survival (OS), relapse rate, and remission duration (RD). In …

Esrage-Expressing Ohsv Enhances Anti-Tumor Efficacy By Inhibition Of Endothelial Cell Activation, Jessica Swanner, Ji Seon Shim, Kimberly A Rivera-Caraballo, Karina Vázquez-Arreguín, Bangxing Hong, Alberto J Bueso-Perez, Tae Jin Lee, Yeshavanth Kumar Banasavadi-Siddegowda, Balveen Kaur, Ji Young Yoo

Faculty, Staff and Student Publications

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) molecule that plays an important role in inflammation and tumorigenesis. Receptor for advanced glycation end products (RAGE) is one of the major receptors to which extracellular HMGB1 binds to mediate its activity. RAGE is highly expressed on the endothelial cells (ECs) and regulates endothelial permeability during inflammation. Here, we introduced the endogenous secretory form of RAGE (esRAGE) as a decoy receptor for RAGE ligands into an oncolytic herpes simplex virus 1 (oHSV) (OVesRAGE), which, upon release, can function to block RAGE signaling. OVesRAGE significantly decreased phosphorylation of MEK1/2 and …

Tsks Localizes To Nuage In Spermatids And Regulates Cytoplasmic Elimination During Spermiation, Keisuke Shimada, Soojin Park, Seiya Oura, Taichi Noda, Akane Morohoshi, Martin M Matzuk, Masahito Ikawa

Faculty, Staff and Students Publications

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding …

Acute Acat1/Soat1 Blockade Increases Mam Cholesterol And Strengthens Er-Mitochondria Connectivity., Taylor C Harned, Radu V Stan, Ze Cao, Rajarshi Chakrabarti, Henry N Higgs, Catherine C Y Chang, Ta Yuan Chang

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain …

Prognostication Of Dna Damage Response Protein Expression Patterns In Chronic Lymphocytic Leukemia, Ti'ara L Griffen, Fieke W Hoff, Yihua Qiu, Jan Burger, William Wierda, Steven M Kornblau

Faculty, Staff and Student Publications

Proteomic DNA Damage Repair (DDR) expression patterns in Chronic Lymphocytic Leukemia were characterized by quantifying and clustering 24 total and phosphorylated DDR proteins. Overall, three protein expression patterns (C1-C3) were identified and were associated as an independent predictor of distinct patient overall survival outcomes. Patients within clusters C1 and C2 had poorer survival outcomes and responses to fludarabine, cyclophosphamide, and rituxan chemotherapy compared to patients within cluster C3. However, DDR protein expression patterns were not prognostic in more modern therapies with BCL2 inhibitors or a BTK/PI3K inhibitor. Individually, nine of the DDR proteins were prognostic for predicting overall survival and/or …

Reverse Phase Protein Array Profiling Identifies Recurrent Protein Expression Patterns Of Dna Damage-Related Proteins Across Acute And Chronic Leukemia: Samples From Adults And The Children's Oncology Group, Fieke W Hoff, Ti'ara L Griffen, Brandon D Brown, Terzah M Horton, Jan Burger, William Wierda, Stefan E Hubner, Yihua Qiu, Steven M Kornblau

Faculty, Staff and Student Publications

DNA damage response (DNADR) recognition and repair (DDR) pathways affect carcinogenesis and therapy responsiveness in cancers, including leukemia. We measured protein expression levels of 16 DNADR and DDR proteins using the Reverse Phase Protein Array methodology in acute myeloid (AML) (n = 1310), T-cell acute lymphoblastic leukemia (T-ALL) (n = 361) and chronic lymphocytic leukemia (CLL) (n = 795) cases. Clustering analysis identified five protein expression clusters; three were unique compared to normal CD34+ cells. Individual protein expression differed by disease for 14/16 proteins, with five highest in CLL and nine in T-ALL, and by age in …

A Gene Regulatory Network Approach Harmonizes Genetic And Epigenetic Signals And Reveals Repurposable Drug Candidates For Multiple Sclerosis, Astrid M Manuel, Yulin Dai, Peilin Jia, Leorah A Freeman, Zhongming Zhao

Faculty, Staff and Student Publications

Multiple sclerosis (MS) is a complex dysimmune disorder of the central nervous system. Genome-wide association studies (GWAS) have identified 233 genetic variations associated with MS at the genome-wide significant level. Epigenetic studies have pinpointed differentially methylated CpG sites in MS patients. However, the interplay between genetic risk factors and epigenetic regulation remains elusive. Here, we employed a network model to integrate GWAS summary statistics of 14 802 MS cases and 26 703 controls with DNA methylation profiles from 140 MS cases and 139 controls and the human interactome. We identified differentially methylated genes by aggregating additive effects of differentially methylated …

The Fly Homolog Of Supt16h, A Gene Associated With Neurodevelopmental Disorders, Is Required In A Cell-Autonomous Fashion For Cell Survival, Mengqi Ma, Xi Zhang, Yiming Zheng, Shenzhao Lu, Xueyang Pan, Xiao Mao, Hongling Pan, Hyung-Lok Chung, Hua Wang, Hong Guo, Hugo J Bellen

Faculty, Staff and Students Publications

SUPT16H encodes the large subunit of the FAcilitate Chromatin Transcription (FACT) complex, which functions as a nucleosome organizer during transcription. We identified two individuals from unrelated families carrying de novo missense variants in SUPT16H. The probands exhibit global developmental delay, intellectual disability, epilepsy, facial dysmorphism and brain structural abnormalities. We used Drosophila to characterize two variants: p.T171I and p.G808R. Loss of the fly ortholog, dre4, causes lethality at an early developmental stage. RNAi-mediated knockdown of dre4 in either glia or neurons causes severely reduced eclosion and longevity. Tissue-specific knockdown of dre4 in the eye or wing leads to the loss …

Whole-Exome Sequencing Study Identifies Four Novel Gene Loci Associated With Diabetic Kidney Disease, Yang Pan, Xiao Sun, Xuenan Mi, Zhijie Huang, Yenchih Hsu, James E Hixson, Donna Munzy, Ginger Metcalf, Nora Franceschini, Adrienne Tin, Anna Köttgen, Michael Francis, Nhlbi Trans-Omics For Precision Medicine (Topmed) Consortium Topmed Kidney Function Working Group, Jennifer A Brody, Bryan Kestenbaum, Colleen M Sitlani, Josyf C Mychaleckyj, Holly Kramer, Leslie A Lange, Xiuqing Guo, Shih-Jen Hwang, Marguerite R Irvin, Jennifer A Smith, Lisa R Yanek, Dhananjay Vaidya, Yii-Der Ida Chen, Myriam Fornage, Donald M Lloyd-Jones, Lifang Hou, Rasika A Mathias, Braxton D Mitchell, Patricia A Peyser, Sharon L R Kardia, Donna K Arnett, Adolfo Correa, Laura M Raffield, Ramachandran S Vasan, L Adrienne Cupple, Daniel Levy, Robert C Kaplan, Kari E North, Jerome I Rotter, Charles Kooperberg, Alexander P Reiner, Bruce M Psaty, Russell P Tracy, Richard A Gibbs, Alanna C Morrison, Harold Feldman, Eric Boerwinkle, Jiang He, Tanika N Kelly, Cric Study Investigators

Faculty, Staff and Students Publications

Diabetic kidney disease (DKD) is recognized as an important public health challenge. However, its genomic mechanisms are poorly understood. To identify rare variants for DKD, we conducted a whole-exome sequencing (WES) study leveraging large cohorts well-phenotyped for chronic kidney disease and diabetes. Our two-stage WES study included 4372 European and African ancestry participants from the Chronic Renal Insufficiency Cohort and Atherosclerosis Risk in Communities studies (stage 1) and 11 487 multi-ancestry Trans-Omics for Precision Medicine participants (stage 2). Generalized linear mixed models, which accounted for genetic relatedness and adjusted for age, sex and ancestry, were used to test associations between …

Assigning Pathogenicity For Tab2 Variants Using A Novel Scalable Functional Assay And Expanding Tab2 Disease Spectrum, Weiyi Xu, Andrea Graves, Monika Weisz-Hubshman, Lamees Hegazy, Christina Magyar, Zian Liu, Eleni Nasiotis, Md Abul Hassan Samee, Thomas Burris, Seema Lalani, Lilei Zhang

Faculty, Staff and Students Publications

Haploinsufficiency of TGF-beta-activated kinase 1 (MAP3K7) binding protein 2 (TAB2) has been associated with congenital heart disease and more recently multiorgan structural abnormalities. Missense variant represents a major proportion of non-synonymous TAB2 variants reported in gnomAD (295/576) and Clinvar (16/73), most of which are variants of uncertain significance (VUSs). However, interpretation of TAB2 missense variants remains challenging because of lack of functional assays. To address this issue, we established a cell-based luciferase assay that enables high-throughput screening of TAB2 variants to assess the functional consequence for predicting variant pathogenicity. Using this platform, we screened 47 TAB2 variants including five pathogenic …

Effects Of Protein-Coding Variants On Blood Metabolite Measurements And Clinical Biomarkers In The Uk Biobank, Abhishek Nag, Ryan S Dhindsa, Lawrence Middleton, Xiao Jiang, Dimitrios Vitsios, Eleanor Wigmore, Erik L Allman, Anna Reznichenko, Keren Carss, Katherine R Smith, Quanli Wang, Benjamin Challis, Dirk S Paul, Andrew R Harper, Slavé Petrovski

Faculty, Staff and Students Publications

Genome-wide association studies (GWASs) have established the contribution of common and low-frequency variants to metabolic blood measurements in the UK Biobank (UKB). To complement existing GWAS findings, we assessed the contribution of rare protein-coding variants in relation to 355 metabolic blood measurements-including 325 predominantly lipid-related nuclear magnetic resonance (NMR)-derived blood metabolite measurements (Nightingale Health Plc) and 30 clinical blood biomarkers-using 412,393 exome sequences from four genetically diverse ancestries in the UKB. Gene-level collapsing analyses were conducted to evaluate a diverse range of rare-variant architectures for the metabolic blood measurements. Altogether, we identified significant associations (p < 1 × 10

Endotrophin Neutralization Through Targeted Antibody Treatment Protects From Renal Fibrosis In A Podocyte Ablation Model, Yu A An, Wei Xiong, Shiuhwei Chen, Dawei Bu, Joseph M Rutkowski, Joel P Berger, Christine M Kusminski, Ningyan Zhang, Zhiqiang An, Philipp E Scherer

Faculty, Staff and Student Publications

OBJECTIVE: Renal fibrosis is a hallmark for chronic kidney disease (CKD), and often leads to end stage renal disease (ESRD). However, limited interventions are available clinically to ameliorate or reverse renal fibrosis.

METHODS: Herein, we evaluated whether blockade of endotrophin through neutralizing antibodies protects from renal fibrosis in the podocyte insult model (the "POD-ATTAC" mouse). We determined the therapeutic effects of endotrophin targeted antibody through assessing renal function, renal inflammation and fibrosis at histological and transcriptional levels, and podocyte regeneration.

RESULTS: We demonstrated that neutralizing endotrophin antibody treatment significantly ameliorates renal fibrosis at the transcriptional, morphological, and functional levels. In …

Epstein-Barr Virus-Associated Lymphoepithelial Carcinoma Arising In The Parotid Gland: A Case Report And Literature Review, Akinobu Kubota, Nobuyuki Bandoh, Takashi Goto, Ken-Ichi Matsumoto, Tomomi Yamaguchi-Ishochi, Yasutaka Kato, Hiroshi Nishihara, Hidehiro Takei

Faculty, Staff and Student Publications

A 60-year-old woman presented with a 3-year history of a slow-growing, painless mass in their left parotid gland. Ultrasonography revealed a well-circumscribed, lobulated, hypoechoic mass measuring 19x12x10 mm in the left parotid gland. Computed tomography revealed a well-circumscribed, solid mass with homogeneous enhancement. Fluorodeoxyglucose-positron emission tomography revealed uptake by the tumor but no uptake in other organs, including the nasopharynx. The patient underwent superficial parotidectomy with adequate safety margins and selective neck dissection followed by radiotherapy. No facial paralysis or recurrence of the tumor had been observed as of 20 months post-operation. Histologically, the tumor was composed of sheets of …

Ferroptosis In Life: To Be Or Not To Be, Ling Xu, Yu'e Liu, Xi Chen, Hua Zhong, Yi Wang

Faculty, Staff and Students Publications

Ferroptosis is a novel type of programmed cell death, characterized by a dysregulated iron metabolism and accumulation of lipid peroxides. It features the alteration of mitochondria and aberrant accumulation of excessive iron as well as loss of the cysteine-glutathione-GPX4 axis. Eventually, the accumulated lipid peroxides result in lethal damage to the cells. Ferroptosis is induced by the overloading of iron and the accumulation of ROS and can be inhibited by the activation of the GPX4 pathway, FS1-CoQ10 pathway, GCH1-BH4 pathway, and the DHODH pathway, it is also regulated by the oncogenes and tumor suppressors. Ferroptosis involves various physiological and pathological …

Bacterial Division Ring Stabilizing Zapa Versus Destabilizing Slma Modulate Ftsz Switching Between Biomolecular Condensates And Polymers, Begoña Monterroso, Miguel Ángel Robles-Ramos, Marta Sobrinos-Sanguino, Juan Román Luque-Ortega, Carlos Alfonso, William Margolin, Germán Rivas, Silvia Zorrilla

Faculty, Staff and Student Publications

Cytokinesis is a fundamental process for bacterial survival and proliferation, involving the formation of a ring by filaments of the GTPase FtsZ, spatio-temporally regulated through the coordinated action of several factors. The mechanisms of this regulation remain largely unsolved, but the inhibition of FtsZ polymerization by the nucleoid occlusion factor SlmA and filament stabilization by the widely conserved cross-linking protein ZapA are known to play key roles. It was recently described that FtsZ, SlmA and its target DNA sequences (SlmA-binding sequence (SBS)) form phase-separated biomolecular condensates, a type of structure associated with cellular compartmentalization and resistance to stress. Using biochemical …