Digital Commons Network^™

Early Growth Response 1 Transcription Factor Is Essential For The Pathogenic Properties Of Human Endometriotic Epithelial Cells, Vineet K Maurya, Maria M Szwarc, Rodrigo Fernandez-Valdivia, David M Lonard, Song Yong, Niraj Joshi, Asgerally T Fazleabas, John P Lydon

Faculty, Staff and Students Publications

Although a non-malignant gynecological disorder, endometriosis displays some pathogenic features of malignancy, such as cell proliferation, migration, invasion and adaptation to hypoxia. Current treatments of endometriosis include pharmacotherapy and/or surgery, which are of limited efficacy and often associated with adverse side effects. Therefore, to develop more effective therapies to treat this disease, a broader understanding of the underlying molecular mechanisms that underpin endometriosis needs to be attained. Using immortalized human endometriotic epithelial and stromal cell lines, we demonstrate that the early growth response 1 (EGR1) transcription factor is essential for cell proliferation, migration and invasion, which represent some of the …

Salivary Biomarker Evaluation Of Chronic Pancreatitis Patients Reveals Alterations In Human Proteins, Cytokines, Prostaglandin E2 Levels, And Bacterial Diversity, Richard T Waldron, Elaina K Jones, Vincent I Anani, Jolaine M Hines, Jing Zhao, Aurelia Lugea, Marcio A Diniz, Sungjin Kim, Aida Habtezion, Kristi L Hoffman, Joseph F Petrosino, William E Fisher, Liang Li, Ryan J Lennon, Ravinder Jit Singh, Santhi Swaroop Vege, Stephen J Pandol, Mark D Topazian

Faculty, Staff and Students Publications

OBJECTIVES: Chronic pancreatitis (CP) is a chronic fibroinflammatory condition of the pancreas difficult to diagnose in early stages. Novel biomarkers useful to facilitate early diagnosis or treatment responses may be found in biofluids. Although saliva can be easily and noninvasively collected from patients, useful salivary biomarkers from CP patients have not yet been identified.

METHODS: Here, we analyzed the proteome by quantitative proteomics, cytokine/chemokine levels by Luminex analysis, prostaglandin E2 (PGE2) levels by a mass spectrometry-based assay, and bacterial species diversity by 16S ribosomal ribonucleic acid sequencing in saliva samples from confirmed CP patients and healthy controls.

RESULTS: Our results …

Maladaptive Positive Feedback Production Of Chrebpβ Underlies Glucotoxic Β-Cell Failure, Liora S Katz, Gabriel Brill, Pili Zhang, Anil Kumar, Sharon Baumel-Alterzon, Lee B Honig, Nicolás Gómez-Banoy, Esra Karakose, Marius Tanase, Ludivine Doridot, Alexandra Alvarsson, Bennett Davenport, Peng Wang, Luca Lambertini, Sarah A Stanley, Dirk Homann, Andrew F Stewart, James C Lo, Mark A Herman, Adolfo Garcia-Ocaña, Donald K Scott

Faculty, Staff and Students Publications

Preservation and expansion of β-cell mass is a therapeutic goal for diabetes. Here we show that the hyperactive isoform of carbohydrate response-element binding protein (ChREBPβ) is a nuclear effector of hyperglycemic stress occurring in β-cells in response to prolonged glucose exposure, high-fat diet, and diabetes. We show that transient positive feedback induction of ChREBPβ is necessary for adaptive β-cell expansion in response to metabolic challenges. Conversely, chronic excessive β-cell-specific overexpression of ChREBPβ results in loss of β-cell identity, apoptosis, loss of β-cell mass, and diabetes. Furthermore, β-cell "glucolipotoxicity" can be prevented by deletion of ChREBPβ. Moreover, ChREBPβ-mediated cell death is …

Genomic And Functional Characterization Of Vancomycin-Resistant Enterococci-Specific Bacteriophages In The Galleria Mellonella Wax Moth Larvae Model, Lynn El Haddad, Georgios Angelidakis, Justin R Clark, Jesus F Mendoza, Austen L Terwilliger, Christopher P Chaftari, Mark Duna, Serena T Yusuf, Cynthia P Harb, Mark Stibich, Anthony Maresso, Roy F Chemaly

Faculty, Staff and Students Publications

Phages are naturally occurring viruses that selectively kill bacterial species without disturbing the individual’s normal flora, averting the collateral damage of antimicrobial usage. The safety and the effectiveness of phages have been mainly confirmed in the food industry as well as in animal models. In this study, we report on the successful isolation of phages specific to Vancomycin-resistant Enterococci, including Enterococcus faecium (VREfm) and Enterococcus faecalis from sewage samples, and demonstrate their efficacy and safety for VREfm infection in the greater wax moth Galleria mellonella model. No virulence-associated genes, antibiotic resistance genes or integrases were detected in …

Holarctic Species In The Pluteus Romellii Clade Five New Species Described And Old Names Reassessed, Hana Ševčíková, Ekaterina Malysheva, Giuliano Ferisin, Francesco Dovana, Egon Horak, Jacob Kalichman, Oğuzhan Kaygusuz, Renée Lebeuf, Guillermo Muñoz González, Andrew M Minnis, Stephen D Russell, Michal Sochor, Bálint Dima, Vladimír Antonín, Alfredo Justo

Faculty, Staff and Students Publications

We studied the taxonomy of Pluteus romellii, and morphologically similar Holarctic species in the/romellii clade of section Celluloderma, using morphological and molecular data (nrITS, TEF1-α). Pluteus romellii is lectotypified and epitypified and accepted as an exclusively Eurasian species. Pluteus lutescens and P. pallescens are considered synonyms of P. romellii. Pluteus fulvibadius is accepted as a related, but separate, North American species. Five species in the/romellii clade are described as new to science: two from North America (P. austrofulvus and P. parvisporus), one from Asia (P. parvicarpus), one from Europe (P. siccus …

A Phospholipid Mimetic Targeting Lrh-1 Ameliorates Colitis, Suzanne G Mays, Emma H D'Agostino, Autumn R Flynn, Xiangsheng Huang, Guohui Wang, Xu Liu, Elizabeth J Millings, C Denise Okafor, Anamika Patel, Michael L Cato, Jeffery L Cornelison, Diana Melchers, René Houtman, David D Moore, John W Calvert, Nathan T Jui, Eric A Ortlund

Faculty, Staff and Students Publications

Phospholipids are ligands for nuclear hormone receptors (NRs) that regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions is a robust strategy to improve agonists of liver receptor homolog-1 (LRH-1), a therapeutic target for colitis. Conventional LRH-1 modulators only partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. We show that the phospholipid-mimicking groups interact with the targeted residues in crystal structures and improve binding affinity, LRH-1 transcriptional activity, …

Paclitaxel And Urolithin A Prevent Histamine-Induced Neurovascular Breakdown Alike, In An, Srijayaprakash Uppada, Dianxiong Zou, Erin M Scott, Gladys Ko, Stephen Pflugfelder, M N V Ravi Kumar, Raghu Ganugula

Faculty, Staff and Students Publications

Neurovascular eye problems are better prevented than managed or treated. Despite growing concern of occurrence in aging populations and development secondary to diseases such as diabetes and hypertension, we currently have very few options to tackle this global problem. Creating effective and high-throughput screening strategies is as important as the intervention itself. Here, we present for the first time a robust ex vivo rat eye model of histamine-induced vascular damage for investigating the therapeutic potential of paclitaxel (PTX) and urolithin A (UA) as alternatives to dexamethasone for preventing vascular damage in the retina. Extensive loss of vascularization and apoptosis were …

Hippo-Taz Signaling Is The Master Regulator Of The Onset Of Triple-Negative Basal-Like Breast Cancers, Hirotoshi Soyama, Miki Nishio, Junji Otani, Toshiko Sakuma, Shintaro Takao, Shigeo Hara, Takaaki Masuda, Koshi Mimori, Shinya Toyokuni, John P Lydon, Kazuwa Nakao, Hiroshi Nishina, Takumi Fukumoto, Tomohiko Maehama, Akira Suzuki

Faculty, Staff and Students Publications

A universal oncogenic driver of basal-like breast cancer (BLBC) has resisted identification. We show that continuous transcriptional coactivator with PDZ-binding motif (TAZ) activation in precancerous murine luminal cells generates luminal cancers that later become BLBCs. Subsequent TP53 alteration, a feature of invasive human BLBCs, accelerates tumor progression. Because BLBC development is inhibited by TAZ inactivation in vivo, our work provides a sound rationale for targeting Hippo-TAZ signaling as therapy for human BLBC. Our mouse model of BLBC represents a powerful tool for evaluating such drugs.

The Testis-Specific E3 Ubiquitin Ligase Rnf133 Is Required For Fecundity In Mice, Kaori Nozawa, Yoshitaka Fujihara, Darius J Devlin, Ricardo E Deras, Katarzyna Kent, Irina V Larina, Kohei Umezu, Zhifeng Yu, Courtney M Sutton, Qiuji Ye, Laura K Dean, Chihiro Emori, Masahito Ikawa, Thomas X Garcia, Martin M Matzuk

Faculty, Staff and Students Publications

BACKGROUND: Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin-proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated …

Internal Standard Triggered-Parallel Reaction Monitoring Mass Spectrometry Enables Multiplexed Quantification Of Candidate Biomarkers In Plasma, Jacob J Kennedy, Jeffrey R Whiteaker, Richard G Ivey, Aura Burian, Shrabanti Chowdhury, Chia-Feng Tsai, Tao Liu, Chenwei Lin, Oscar D Murillo, Rachel A Lundeen, Lisa A Jones, Philip R Gafken, Gary Longton, Karin D Rodland, Steven J Skates, John Landua, Pei Wang, Michael T Lewis, Amanda G Paulovich

Faculty, Staff and Students Publications

Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Previously, mass spectrometry-based approaches have been used to fill this gap but feature poor quantitative performance and were generally limited to hundreds of proteins. Here, we demonstrate the capability of an internal standard triggered-parallel reaction monitoring (IS-PRM) assay to greatly expand the numbers of candidates that can be …

Extracellular Domain Of Pept1 Interacts With Tm1 To Facilitate Substrate Transport, Jiemin Shen, Miaohui Hu, Xiao Fan, Zhenning Ren, Corinne Portioli, Xiuwen Yan, Mingqiang Rong, Ming Zhou

Faculty, Staff and Students Publications

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has twelve transmembrane (TM) helices that fold into an N-terminal domain (NTD, TM1–6) and a C-terminal domain (CTD, TM7–12), and has a large extracellular domain (ECD) between TM9–10. It is well-recognized that peptide transport requires movements of the NTD and CTD, but the role of the ECD in PepT1 remains unclear. Here we report the structure of horse PepT1 encircled in lipid nanodiscs and captured in the inward-open apo …

Maternal Adipocyte Connexin43 Gap Junctions Affect Breastmilk Lactose Levels And Neonate Growth In Mice, Mingyang Huang, Anying Song, Xi Chen, Sarah Ishtiaq, Chunmei Wang, Darryl L Hadsell, Qiong A Wang, Yi Zhu

Faculty, Staff and Students Publications

Simple Summary

Breastfeeding offers many health benefits for both mothers and infants. However, overnutrition and a steady increase in obesity in the U.S. has made it harder for many mothers to produce and express breastmilk. Moreover, the quality of breastmilk from obese mothers is frequently compromised in that it contains fewer nutrients and more inflammatory components. In this study, we used mice to model this phenomenon. We found that short-term high-fat feeding at the start of breeding reduces litter size and pups’ body weight. It also impairs adipocyte remodeling during lactation. Connexin43 is the primary building block for gap junctions …

Mir-181a Promotes Multiple Protumorigenic Functions By Targeting Tgfβr3, Vida Chitsazzadeh, Tran N Nguyen, Alvaro De Mingo Pulido, Bruna B Bittencourt, Lili Du, Charles H Adelmann, Ivannie Ortiz Rivera, Kimberly A Nguyen, Leah D Guerra, Andrew Davis, Marco Napoli, Wencai Ma, Richard Eric Davis, Kimal Rajapakshe, Cristian Coarfa, Elsa R Flores, Kenneth Y Tsai

Faculty, Staff and Students Publications

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFβ signaling, TGFβR3. miR-181a and TGFβR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFβR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal …

Monkeypox: A Contemporary Review For Healthcare Professionals, Boghuma K Titanji, Bryan Tegomoh, Saman Nematollahi, Michael Konomos, Prathit A Kulkarni

Faculty, Staff and Students Publications

The ongoing 2022 multicountry outbreak of monkeypox is the largest in history to occur outside of Africa. Monkeypox is an emerging zoonotic disease that for decades has been viewed as an infectious disease with significant epidemic potential because of the increasing occurrence of human outbreaks in recent years. As public health entities work to contain the current outbreak, healthcare professionals globally are aiming to become familiar with the various clinical presentations and management of this infection. We present in this review an updated overview of monkeypox for healthcare professionals in the context of the ongoing outbreaks around the world.

Activating Connexin43 Gap Junctions Primes Adipose Tissue For Therapeutic Intervention, Yi Zhu, Na Li, Mingyang Huang, Xi Chen, Yu A An, Jianping Li, Shangang Zhao, Jan-Bernd Funcke, Jianhong Cao, Zhenyan He, Qingzhang Zhu, Zhuzhen Zhang, Zhao V Wang, Lin Xu, Kevin W Williams, Chien Li, Kevin Grove, Philipp E Scherer

Faculty, Staff and Students Publications

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic …

Spatial Analysis Of Drug Absorption, Distribution, Metabolism, And Toxicology Using Mass Spectrometry Imaging, Michelle L Spruill, Mirjana Maletic-Savatic, Howard Martin, Feng Li, Xinli Liu

Faculty, Staff and Students Publications

Mass spectrometry imaging (MSI) is emerging as a powerful analytical tool for detection, quantification, and simultaneous spatial molecular imaging of endogenous and exogenous molecules via in situ mass spectrometry analysis of thin tissue sections without the requirement of chemical labeling. The MSI generates chemically specific and spatially resolved ion distribution information for administered drugs and metabolites, which allows numerous applications for studies involving various stages of drug absorption, distribution, metabolism, excretion, and toxicity (ADMET). MSI-based pharmacokinetic imaging analysis provides a histological context and cellular environment regarding dynamic drug distribution and metabolism processes, and facilitates the understanding of the spatial pharmacokinetics …

A Role For Insulin-Like Growth Factor 1 In The Generation Of Epileptic Spasms In A Murine Model, Carlos J Ballester-Rosado, John T Le, Trang T Lam, Carrie A Mohila, Sandi Lam, Anne E Anderson, James D Frost, John W Swann

Faculty, Staff and Students Publications

Objective: Infantile spasms are associated with a wide variety of clinical conditions, including perinatal brain injuries. We have created a model in which prolonged infusion of tetrodotoxin (TTX) into the neocortex, beginning in infancy, produces a localized lesion and reproduces the behavioral spasms, electroencephalogram (EEG) abnormalities, and drug responsiveness seen clinically. Here, we undertook experiments to explore the possibility that the growth factor IGF-1 plays a role in generating epileptic spasms.

Methods: We combined long-term video EEG recordings with quantitative immunohistochemical and biochemical analyses to unravel IGF-1's role in spasm generation. Immunohistochemistry was undertaken in surgically resected tissue from infantile …

Cholestasis Alters Brain Lipid And Bile Acid Composition And Compromises Motor Function In Neonatal Piglets, Nicole Lind Henriksen, Svend Høime Hansen, Matthew Domenic Lycas, Xiaoyu Pan, Thomas Eriksen, Lars Søndergaard Johansen, Richard R Sprenger, Christer Stenby Ejsing, Douglas G Burrin, Kerstin Skovgaard, Vibeke Brix Christensen, Thomas Thymann, Stanislava Pankratova

Faculty, Staff and Students Publications

Infants with neonatal cholestasis are prone to neurodevelopmental deficits, however, the underlying pathogenesis is unclear. Lipid malabsorption and accumulation of potentially neurotoxic molecules in the blood such as bile acids are important yet relatively unexplored pathways. Here, we developed a translational piglet model to understand how the molecular bile acid and lipid composition of the brain is affected by this disease and relates to motor function. Piglets (8-days old) had bile duct ligation or sham surgery and were fed a formula diet for 3 weeks. Alongside sensory-motor deficits observed in bile duct-ligated animals, we found a shift toward a more …

Chronic Lung Allograft Dysfunction, Anupam Kumar, Puneet Singh Garcha

Faculty, Staff and Students Publications

Lung transplantation remains the only cure for selected patients with advanced irreversible lung diseases. More than 4000 lung transplants are performed worldwide annually. In the last two decades, significant advances have been made in this arena, the most impactful being a modest but improved survival of the recipients. Unfortunately majority of recipients still succumb to chronic lung allograft dysfunction (CLAD), and it remains the most common cause of death after the first year of transplantation. Below is a concise review of the current definition of CLAD, including the various phenotypes, and a brief discussion of the tools available for its …

Regulation Of Pgc1Α Downstream Of The Insulin Signaling Pathway Plays A Role In The Hepatic Proteotoxicity Of Mutant Α1-Antitrypsin Deficiency Variant Z, David A Rudnick, Jiansheng Huang, Tunda Hidvegi, Andrew S Chu, Pamela Hale, Admire Munanairi, Dennis J Dietzen, Paul F Cliften, Eric Tycksen, Andrew J Lutkewitte, Brian N Finck, Stephen C Pak, Gary A Silverman, David H Perlmutter

Faculty, Staff and Students Publications

BACKGROUND & AIMS: Insulin signaling is known to regulate essential proteostasis mechanisms.

METHODS: The analyses here examined effects of insulin signaling in the PiZ mouse model of α1-antitrypsin deficiency in which hepatocellular accumulation and proteotoxicity of the misfolded α1-antitrypsin Z variant (ATZ) causes liver fibrosis and cancer.

RESULTS: We first studied the effects of breeding PiZ mice to liver-insulin-receptor knockout (LIRKO) mice (with hepatocyte-specific insulin-receptor gene disruption). The results showed decreased hepatic ATZ accumulation and liver fibrosis in PiZ x LIRKO vs PiZ mice, with reversal of those effects when we bred PiZ x LIRKO mice onto a FOXO1-deficient background. …

Deletion Of The Lmna Gene In Fibroblasts Causes Senescence-Associated Dilated Cardiomyopathy By Activating The Double-Stranded Dna Damage Response And Induction Of Senescence-Associated Secretory Phenotype, Leila Rouhi, Gaelle Auguste, Qiong Zhou, Raffaella Lombardi, Melis Olcum, Kimia Pourebrahim, Sirisha M Cheedipudi, Saman Asghar, Kui Hong, Matthew J Robertson, Cristian Coarfa, Priyatansh Gurha, Ali J Marian

Faculty, Staff and Students Publications

INTRODUCTION: Mutations in the LMNA gene, encoding Lamin A/C (LMNA), are established causes of dilated cardiomyopathy (DCM). The phenotype is typically characterized by progressive cardiac conduction defects, arrhythmias, heart failure, and premature death. DCM is primarily considered a disease of cardiac myocytes. However, LMNA is also expressed in other cardiac cell types, including fibroblasts.

AIM: The purpose of the study was to determine the contribution of the fibroblasts to DCM caused by LMNA deficiency.

METHODS AND RESULTS: The Lmna gene was deleted by crossing the platelet-derived growth factor receptor α-Cre recombinase (Pdgfra-Cre) and floxed Lmna (LmnaF/F) …

The Evolution And Structure/Function Of Bhlh-Pas Transcription Factor Family, Hailey E Edwards, Daniel A Gorelick

Faculty, Staff and Students Publications

Proteins that contain basic helix-loop-helix (bHLH) and Per-Arnt-Sim motifs (PAS) function as transcription factors. bHLH-PAS proteins exhibit essential and diverse functions throughout the body, from cell specification and differentiation in embryonic development to the proper function of organs like the brain and liver in adulthood. bHLH-PAS proteins are divided into two classes, which form heterodimers to regulate transcription. Class I bHLH-PAS proteins are typically activated in response to specific stimuli, while class II proteins are expressed more ubiquitously. Here, we discuss the general structure and functions of bHLH-PAS proteins throughout the animal kingdom, including family members that do not fit …

High-Throughput Profiling Of Histone Post-Translational Modifications And Chromatin Modifying Proteins By Reverse Phase Protein Array, Xuan Wang, Zhongcheng Shi, Hsin-Yi Lu, Jean J Kim, Wen Bu, Jose A Villalobos, Dimuthu N Perera, Sung Yun Jung, Tao Wang, Sandra L Grimm, Bethany C Taylor, Kimal Rajapakshe, Hyekyung Park, Julia Wulfkuhle, Nicolas L Young, Yi Li, Cristian Coarfa, Dean P Edwards, Shixia Huang

Faculty, Staff and Students Publications

Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by …

Dietary Fatty Acid Intake And The Colonic Gut Microbiota In Humans, Anthony A Xu, Luke K Kennedy, Kristi Hoffman, Donna L White, Fasiha Kanwal, Hashem B El-Serag, Joseph F Petrosino, Li Jiao

Faculty, Staff and Students Publications

A high-fat diet has been associated with systemic diseases in humans and alterations in gut microbiota in animal studies. However, the influence of dietary fatty acid intake on gut microbiota in humans has not been well studied. In this cross-sectional study, we examined the association between intake of total fatty acids (TFAs), saturated fatty acids (SFAs), trans fatty acids (TrFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), n3-FAs, and n6-FAs, and the community composition and structure of the adherent colonic gut microbiota. We obtained 97 colonic biopsies from 34 participants with endoscopically normal colons. Microbial DNA was used to …

Mycn-Driven Fatty Acid Uptake Is A Metabolic Vulnerability In Neuroblastoma, Ling Tao, Mahmoud A Mohammad, Giorgio Milazzo, Myrthala Moreno-Smith, Tajhal D Patel, Barry Zorman, Andrew Badachhape, Blanca E Hernandez, Amber B Wolf, Zihua Zeng, Jennifer H Foster, Sara Aloisi, Pavel Sumazin, Youli Zu, John Hicks, Ketan B Ghaghada, Nagireddy Putluri, Giovanni Perini, Cristian Coarfa, Eveline Barbieri

Faculty, Staff and Students Publications

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and …

Gain-Of-Signal Assays For Probing Inhibition Of Sars-Cov-2 Mpro/3clpro In Living Cells, Seyed Arad Moghadasi, Morgan A Esler, Yuka Otsuka, Jordan T Becker, Sofia N Moraes, Constance B Anderson, Srinivas Chamakuri, Christopher Belica, Chloe Wick, Daniel A Harki, Damian W Young, Louis Scampavia, Timothy P Spicer, Ke Shi, Hideki Aihara, William L Brown, Reuben S Harris

Faculty, Staff and Students Publications

The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that …

Expanding Anti-Cd38 Immunotherapy For Lymphoid Malignancies, Xu Wang, Xinfang Yu, Wei Li, Praveen Neeli, Ming Liu, Ling Li, Mingzhi Zhang, Xiaosheng Fang, Ken H Young, Yong Li

Faculty, Staff and Students Publications

BACKGROUND: Lymphoid neoplasms, including multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and NK/T cell neoplasms, are a major cause of blood cancer morbidity and mortality. CD38 (cyclic ADP ribose hydrolase) is a transmembrane glycoprotein expressed on the surface of plasma cells and MM cells. The high expression of CD38 across MM and other lymphoid malignancies and its restricted expression in normal tissues make CD38 an attractive target for immunotherapy. CD38-targeting antibodies, like daratumumab, have been approved for the treatment of MM and tested against lymphoma and leukemia in multiple clinical trials.

METHODS: We generated chimeric antigen receptor (CAR) T cells targeting …

Crispr/Cas13 Effectors Have Differing Extents Of Off-Target Effects That Limit Their Utility In Eukaryotic Cells, Yuxi Ai, Dongming Liang, Jeremy E Wilusz

Faculty, Staff and Students Publications

CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as …

Evaluation Of Long-Chain Fatty Acid Respiration In Neonatal Mouse Cardiomyocytes Using Seahorse Instrument, Aude Angelini, Xinchun Pi, Liang Xie

Faculty, Staff and Students Publications

Metabolic switches play a critical role in the pathophysiology of cardiac diseases, including heart failure. Here, we describe an assay for long-chain fatty acid oxidation in neonatal mouse cardiomyocytes by using a SeaHorse Flux Analyzer (Agilent). This protocol is a simplified but robust adaptation of the standard protocol that enables metabolic measurements in cells isolated from transgenic mouse models, which can be timesaving and informative. Cell isolation and culture represent a critical point that may require bench optimization.

For complete details on the use and execution of this protocol, please refer to Angelini et al. (2021).

Variants In Asph Cause Exertional Heat Illness And Are Associated With Malignant Hyperthermia Susceptibility, Yukari Endo, Linda Groom, Alper Celik, Natalia Kraeva, Chang Seok Lee, Sung Yun Jung, Lois Gardner, Marie-Anne Shaw, Susan L Hamilton, Philip M Hopkins, Robert T Dirksen, Sheila Riazi, James J Dowling

Faculty, Staff and Students Publications

Exertional heat illness (EHI) and malignant hyperthermia (MH) are life threatening conditions associated with muscle breakdown in the setting of triggering factors including volatile anesthetics, exercise, and high environmental temperature. To identify new genetic variants that predispose to EHI and/or MH, we performed genomic sequencing on a cohort with EHI/MH and/or abnormal caffeine-halothane contracture test. In five individuals, we identified rare, pathogenic heterozygous variants in ASPH, a gene encoding junctin, a regulator of excitation-contraction coupling. We validated the pathogenicity of these variants using orthogonal pre-clinical models, CRISPR-edited C2C12 myotubes and transgenic zebrafish. In total, we demonstrate that ASPH variants represent …