Biochemistry, Biophysics, and Structural Biology Commons^™

Gut Microbiota-Derived Short-Chain Fatty Acids Promote Poststroke Recovery In Aged Mice, Juneyoung Lee, John D'Aigle, Louise Atadja, Victoria Quaicoe, Pedram Honarpisheh, Bhanu P Ganesh, Ahmad Hassan, Joerg Graf, Joseph Petrosino, Nagireddy Putluri, Liang Zhu, David J Durgan, Robert M Bryan, Louise D Mccullough, Venugopal Reddy Venna

Faculty, Staff and Students Publications

RATIONALE: The elderly experience profound systemic responses after stroke, which contribute to higher mortality and more severe long-term disability. Recent studies have revealed that stroke outcomes can be influenced by the composition of gut microbiome. However, the potential benefits of manipulating the gut microbiome after injury is unknown.

OBJECTIVE: To determine if restoring youthful gut microbiota after stroke aids in recovery in aged subjects, we altered the gut microbiome through young fecal transplant gavage in aged mice after experimental stroke. Further, the effect of direct enrichment of selective bacteria producing short-chain fatty acids (SCFAs) was tested as a more targeted …

Nuclear Localization Of A Novel Calpain-2 Mediated Junctophilin-2 C-Terminal Cleavage Peptide Promotes Cardiomyocyte Remodeling, Satadru K Lahiri, Ann P Quick, Benoit Samson-Couterie, Mohit Hulsurkar, Ies Elzenaar, Ralph J Van Oort, Xander H T Wehrens

Faculty, Staff and Students Publications

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Patients with HF exhibit a loss of junctophilin-2 (JPH2), a structural protein critical in forming junctional membrane complexes in which excitation-contraction takes place. Several mechanisms have been proposed to mediate the loss of JPH2, one being cleavage by the calcium-dependent protease calpain. The downstream mechanisms underlying HF progression after JPH2 cleavage are presently poorly understood. In this study, we used Labcas to bioinformatically predict putative calpain cleavage sites on JPH2. We identified a cleavage site that produces a novel C-terminal JPH2 peptide (JPH2-CTP) using several domain-specific antibodies. Western …

Acute Tubular Injury In A Patient On A Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitor, June K Pickett, Maulin Shah, Michael Gillette, Peter Jones, Salim Virani, Christie Ballantyne, Vijay Nambi

Faculty, Staff and Students Publications

A 72-year-old man with coronary artery disease, statin intolerance, and chronic kidney disease stage IIIa was initiated on alirocumab, a proprotein convertase subtilisin/kexin type 9 inhibitor, and developed acute kidney injury. A kidney biopsy was performed and suggested acute tubular injury. The serum creatinine returned to baseline after discontinuation of alirocumab. (Level of Difficulty: Intermediate.)

Aav-Crispr Gene Editing Is Negated By Pre-Existing Immunity To Cas9, Ang Li, Mark R Tanner, Ciaran M Lee, Ayrea E Hurley, Marco De Giorgi, Kelsey E Jarrett, Timothy H Davis, Alexandria M Doerfler, Gang Bao, Christine Beeton, William R Lagor

Faculty, Staff and Students Publications

Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo. However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by …

Age-Dependent Involvement Of Gut Mast Cells And Histamine In Post-Stroke Inflammation, Maria Pilar Blasco, Anjali Chauhan, Pedram Honarpisheh, Hilda Ahnstedt, John D'Aigle, Arunkumar Ganesan, Sriram Ayyaswamy, Frank Blixt, Susan Venable, Angela Major, David Durgan, Anthony Haag, Julia Kofler, Robert Bryan, Louise D Mccullough, Bhanu Priya Ganesh

Faculty, Staff and Students Publications

BACKGROUND: Risk of stroke-related morbidity and mortality increases significantly with age. Aging is associated with chronic, low-grade inflammation, which is thought to contribute to the poorer outcomes after stroke seen in the elderly. Histamine (HA) is a major molecular mediator of inflammation, and mast cells residing in the gut are a primary source of histamine.

METHODS: Stroke was induced in male C57BL/6 J mice at 3 months (young) and 20 months (aged) of age. Role of histamine after stroke was examined using young (Yg) and aged (Ag) mice; mice underwent MCAO surgery and were euthanized at 6 h, 24 h, …

Tfeb Regulates Murine Liver Cell Fate During Development And Regeneration, Nunzia Pastore, Tuong Huynh, Niculin J Herz, Alessia Calcagni', Tiemo J Klisch, Lorenzo Brunetti, Kangho Ho Kim, Marco De Giorgi, Ayrea Hurley, Annamaria Carissimo, Margherita Mutarelli, Niya Aleksieva, Luca D'Orsi, William R Lagor, David D Moore, Carmine Settembre, Milton J Finegold, Stuart J Forbes, Andrea Ballabio

Faculty, Staff and Students Publications

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that …

Proteolysis-Targeting Chimera (Protac) For Targeted Protein Degradation And Cancer Therapy, Xin Li, Yongcheng Song

Faculty, Staff and Students Publications

Proteolysis-targeting chimera (PROTAC) has been developed to be a useful technology for targeted protein degradation. A bifunctional PROTAC molecule consists of a ligand (mostly small-molecule inhibitor) of the protein of interest (POI) and a covalently linked ligand of an E3 ubiquitin ligase (E3). Upon binding to the POI, the PROTAC can recruit E3 for POI ubiquitination, which is subjected to proteasome-mediated degradation. PROTAC complements nucleic acid-based gene knockdown/out technologies for targeted protein reduction and could mimic pharmacological protein inhibition. To date, PROTACs targeting ~ 50 proteins, many of which are clinically validated drug targets, have been successfully developed with several …

Integrating Mouse And Human Genetic Data To Move Beyond Gwas And Identify Causal Genes In Cholesterol Metabolism, Zhonggang Li, James A Votava, Gregory J M Zajac, Jenny N Nguyen, Fernanda B Leyva Jaimes, Sophia M Ly, Jacqueline A Brinkman, Marco De Giorgi, Sushma Kaul, Cara L Green, Samantha L St Clair, Sabrina L Belisle, Julia M Rios, David W Nelson, Mary G Sorci-Thomas, William R Lagor, Dudley W Lamming, Chi-Liang Eric Yen, Brian W Parks

Faculty, Staff and Students Publications

Identifying the causal gene(s) that connects genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, we develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we identified 48 genes showing replication in mice and associated with plasma lipid traits in humans and six genes on the X chromosome. Among these 54 genes, 25 have no previously identified role in lipid metabolism. Based on functional studies and integration with additional human lipid GWAS datasets, we pinpoint Sestrin1 as …

An In Vivo Genome-Wide Crispr Screen Identifies The Rna-Binding Protein Staufen2 As A Key Regulator Of Myeloid Leukemia, Jeevisha Bajaj, Michael Hamilton, Yutaka Shima, Kendall Chambers, Kyle Spinler, Eric L Van Nostrand, Brian A Yee, Steven M Blue, Michael Chen, David Rizzeri, Charles Chuah, Vivian G Oehler, H Elizabeth Broome, Roman Sasik, James Scott-Browne, Anjana Rao, Gene W Yeo, Tannishtha Reya

Faculty, Staff and Students Publications

Aggressive myeloid leukemias such as blast crisis chronic myeloid leukemia and acute myeloid leukemia remain highly lethal. Here we report a genome-wide in vivo CRISPR screen to identify new dependencies in this disease. Among these, RNA-binding proteins (RBPs) in general, and the double-stranded RBP Staufen2 (Stau2) in particular, emerged as critical regulators of myeloid leukemia. In a newly developed knockout mouse, loss of Stau2 led to a profound decrease in leukemia growth and improved survival in mouse models of the disease. Further, Stau2 was required for growth of primary human blast crisis chronic myeloid leukemia and acute myeloid leukemia. Finally, …

Nr2f1 Heterozygous Knockout Mice Recapitulate Neurological Phenotypes Of Bosch-Boonstra-Schaaf Optic Atrophy Syndrome And Show Impaired Hippocampal Synaptic Plasticity, Chun-An Chen, Wei Wang, Steen E Pedersen, Ayush Raman, Michelle L Seymour, Fernanda R Ruiz, Anping Xia, Meike E Van Der Heijden, Li Wang, Jiani Yin, Joanna Lopez, Megan E Rech, Richard A Lewis, Samuel M Wu, Zhandong Liu, Fred A Pereira, Robia G Pautler, Huda Y Zoghbi, Christian P Schaaf

Faculty, Staff and Students Publications

Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) has been identified as an autosomal-dominant disorder characterized by a complex neurological phenotype, with high prevalence of intellectual disability and optic nerve atrophy/hypoplasia. The syndrome is caused by loss-of-function mutations in NR2F1, which encodes a highly conserved nuclear receptor that serves as a transcriptional regulator. Previous investigations to understand the protein's role in neurodevelopment have mostly used mouse models with constitutive and tissue-specific homozygous knockout of Nr2f1. In order to represent the human disease more accurately, which is caused by heterozygous NR2F1 mutations, we investigated a heterozygous knockout mouse model and found that this model …

Discovery Of A Bacterial Peptide As A Modulator Of Glp-1 And Metabolic Disease, Catherine Tomaro-Duchesneau, Stephanie L Levalley, Daniel Roeth, Liang Sun, Frank T Horrigan, Markus Kalkum, Joseph M Hyser, Robert A Britton

Faculty, Staff and Students Publications

Early work in rodents highlighted the gut microbiota's importance in metabolic disease, including Type II Diabetes Mellitus (T2DM) and obesity. Glucagon-like peptide-1 (GLP-1), an incretin secreted by L-cells lining the gastrointestinal epithelium, has important functions: promoting insulin secretion, insulin sensitivity, and β-cell mass, while inhibiting gastric emptying and appetite. We set out to identify microbial strains with GLP-1 stimulatory activity as potential metabolic disease therapeutics. Over 1500 human-derived strains were isolated from healthy individuals and screened for GLP-1 modulation by incubating bacterial cell-free supernatants with NCI H716 L-cells. Approximately 45 strains capable of increasing GLP-1 were discovered. All GLP-1 positive …

Cell Type Composition And Circuit Organization Of Clonally Related Excitatory Neurons In The Juvenile Mouse Neocortex, Cathryn R Cadwell, Federico Scala, Paul G Fahey, Dmitry Kobak, Shalaka Mulherkar, Fabian H Sinz, Stelios Papadopoulos, Zheng H Tan, Per Johnsson, Leonard Hartmanis, Shuang Li, Ronald J Cotton, Kimberley F Tolias, Rickard Sandberg, Philipp Berens, Xiaolong Jiang, Andreas Savas Tolias

Faculty, Staff and Students Publications

Clones of excitatory neurons derived from a common progenitor have been proposed to serve as elementary information processing modules in the neocortex. To characterize the cell types and circuit diagram of clonally related excitatory neurons, we performed multi-cell patch clamp recordings and Patch-seq on neurons derived from Nestin-positive progenitors labeled by tamoxifen induction at embryonic day 10.5. The resulting clones are derived from two radial glia on average, span cortical layers 2–6, and are composed of a random sampling of transcriptomic cell types. We find an interaction between shared lineage and connection type: related neurons are more likely to …

Losartan Rescues Inflammation-Related Mucociliary Dysfunction In Relevant Models Of Cystic Fibrosis, Michael D Kim, Nathalie Baumlin, Makoto Yoshida, Deepika Polineni, Sebastian F Salathe, Joseph K David, Charles A Peloquin, Adam Wanner, John S Dennis, Juliette Sailland, Philip Whitney, Frank T Horrigan, Juan R Sabater, William M Abraham, Matthias Salathe

Faculty, Staff and Students Publications

Rationale: Despite therapeutic progress in treating cystic fibrosis (CF) airway disease, airway inflammation with associated mucociliary dysfunction remains largely unaddressed. Inflammation reduces the activity of apically expressed large-conductance Ca²⁺-activated and voltage-dependent K⁺ (BK) channels, critical for mucociliary function in the absence of CFTR (CF transmembrane conductance regulator).

Objectives: To test losartan as an antiinflammatory therapy in CF using CF human bronchial epithelial cells and an ovine model of CF-like airway disease.

Methods: Losartan’s antiinflammatory effectiveness to rescue BK activity and thus mucociliary function was tested in vitro using primary, fully redifferentiated human airway epithelial cells homozygous for …

Rhoa-Rock Signaling As A Therapeutic Target In Traumatic Brain Injury, Shalaka Mulherkar, Kimberley F Tolias

Faculty, Staff and Students Publications

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBIs, which range in severity from mild to severe, occur when a traumatic event, such as a fall, a traffic accident, or a blow, causes the brain to move rapidly within the skull, resulting in damage. Long-term consequences of TBI can include motor and cognitive deficits and emotional disturbances that result in a reduced quality of life and work productivity. Recovery from TBI can be challenging due to a lack of effective treatment options for repairing TBI-induced neural damage and alleviating functional impairments. Central nervous system (CNS) …

The Vimentin Rod Domain Blocks P-Selectin-P-Selectin Glycoprotein Ligand 1 Interactions To Attenuate Leukocyte Adhesion To Inflamed Endothelium, Fong Wilson Lam, Cameron August Brown, Christian Valladolid, Dabel Cynthia Emebo, Timothy Gerald Palzkill, Miguel Angel Cruz

Faculty, Staff and Students Publications

Acute inflammation begins with leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin on inflamed endothelium and platelets. In pathologic conditions, this process may contribute to secondary organ damage, like sepsis-induced liver injury. Therefore, developing novel therapies to attenuate inflammation may be beneficial. We previously reported that recombinant human vimentin (rhVim) binds P-selectin to block leukocyte adhesion to endothelium and platelets. In this study, we used SPOT-peptide arrays to identify the rod domain as the active region within rhVim that interacts with P-selectin. Indeed, recombinant human rod domain of vimentin (rhRod) binds to P-selectin with high affinity, with in silico modeling …

Ultrafast Two-Photon Imaging Of A High-Gain Voltage Indicator In Awake Behaving Mice, Vincent Villette, Mariya Chavarha, Ivan K Dimov, Jonathan Bradley, Lagnajeet Pradhan, Benjamin Mathieu, Stephen W Evans, Simon Chamberland, Dongqing Shi, Renzhi Yang, Benjamin B Kim, Annick Ayon, Abdelali Jalil, François St-Pierre, Mark J Schnitzer, Guoqiang Bi, Katalin Toth, Jun Ding, Stéphane Dieudonné, Michael Z Lin

Faculty, Staff and Students Publications

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the …

Defining The Layers Of A Sensory Cilium With Storm And Cryoelectron Nanoscopy, Michael A Robichaux, Valencia L Potter, Zhixian Zhang, Feng He, Jun Liu, Michael F Schmid, Theodore G Wensel

Faculty, Staff and Students Publications

Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular …

Advances In Gene Ontology Utilization Improve Statistical Power Of Annotation Enrichment, Eugene Waverly Hinderer Iii, Robert M. Flight, Rashmi Dubey, James N. Macleod, Hunter N. B. Moseley

Maxwell H. Gluck Equine Research Center Faculty Publications

Gene-annotation enrichment is a common method for utilizing ontology-based annotations in gene and gene-product centric knowledgebases. Effective utilization of these annotations requires inferring semantic linkages by tracing paths through edges in the ontological graph, referred to as relations. However, some relations are semantically problematic with respect to scope, necessitating their omission or modification lest erroneous term mappings occur. To address these issues, we created the Gene Ontology Categorization Suite, or GOcats—a novel tool that organizes the Gene Ontology into subgraphs representing user-defined concepts, while ensuring that all appropriate relations are congruent with respect to scoping semantics. Here, we demonstrate the …

Extracellular Vesicles As Biological Shuttles For Targeted Therapies., Stefania Raimondo, Gianluca Giavaresi, Aurelio Lorico, Riccardo Alessandro

College of Osteopathic Medicine (TUN) Publications and Research

The development of effective nanosystems for drug delivery represents a key challenge for the improvement of most current anticancer therapies. Recent progress in the understanding of structure and function of extracellular vesicles (EVs)-specialized membrane-bound nanocarriers for intercellular communication-suggests that they might also serve as optimal delivery systems of therapeutics. In addition to carrying proteins, lipids, DNA and different forms of RNAs, EVs can be engineered to deliver specific bioactive molecules to target cells. Exploitation of their molecular composition and physical properties, together with improvement in bio-techniques to modify their content are critical issues to target them to specific cells/tissues/organs. Here, …

Stress-Induced Epinephrine Enhances Lactate Dehydrogenase A And Promotes Breast Cancer Stem-Like Cells, Bai Cui, Yuanyuan Luo, Pengfei Tian, Fei Peng, Jinxin Lu, Yongliang Yang, Qitong Su, Bing Liu, Jiachuan Yu, Xi Luo, Liu Yin, Wei Cheng, Fan An, Bin He, Dapeng Liang, Sijin Wu, Peng Chu, Luyao Song, Xinyu Liu, Huandong Luo, Binhua P. Zhou

Molecular and Cellular Biochemistry Faculty Publications

Chronic stress triggers activation of the sympathetic nervous system and drives malignancy. Using an immunodeficient murine system, we showed that chronic stress–induced epinephrine promoted breast cancer stem-like properties via lactate dehydrogenase A–dependent (LDHA-dependent) metabolic rewiring. Chronic stress–induced epinephrine activated LDHA to generate lactate, and the adjusted pH directed USP28-mediated deubiquitination and stabilization of MYC. The SLUG promoter was then activated by MYC, which promoted development of breast cancer stem-like traits. Using a drug screen that targeted LDHA, we found that a chronic stress–induced cancer stem-like phenotype could be reversed by vitamin C. These findings demonstrated the critical importance of psychological …

Immunization Of Alpacas (Lama Pacos) With Protein Antigens And Production Of Antigen-Specific Single Domain Antibodies, K. Martin Chow, Sidney W. Whiteheart, Jeffrey R. Smiley, Savita Sharma, Kathy Boaz, Meggie J. Coleman, Alvina Maynard, Louis B. Hersh, Craig W. Vander Kooi

Molecular and Cellular Biochemistry Faculty Publications

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies …

Antisense Oligonucleotides Targeting Angiotensinogen: Insights From Animal Studies, Chia-Hua Wu, Ya Wang, Murong Ma, Adam E. Mullick, Rosanne M. Crooke, Mark J. Graham, Alan Daugherty, Hong S. Lu

Saha Cardiovascular Research Center Faculty Publications

Angiotensinogen (AGT) is the unique substrate of all angiotensin peptides. We review the recent preclinical research of AGT antisense oligonucleotides (ASOs), a rapidly evolving therapeutic approach. The scope of the research findings not only opens doors for potentially new therapeutics of hypertension and many other diseases, but also provides insights into understanding critical physiological and pathophysiological roles mediated by AGT.

Developmental Expression Of Monocarboxylate Transporter 1 And 4 In Rat Liver, Michael Ng, Justin Louie, Jieyun Cao, Melanie A. Felmlee

School of Pharmacy Faculty Articles

PURPOSE: Monocarboxylate transporters (MCT) are proton-coupled integral membrane proteins that control the influx and efflux of endogenous monocarboxylates such as lactate, acetate and pyruvate. They also transport and mediate the clearance of drugs such as valproate and gamma-hydroxybutyrate. CD147 functions as ancillary protein that chaperones MCT1 and MCT4 to the cell membrane. There is limited data on the maturation of MCT and CD147 expression in tissues related to drug distribution and clearance. The objective of the present study was to quantify hepatic MCT1, MCT4, and CD147 mRNA, whole cell and membrane protein expression from birth to sexual maturity.

METHODS: Liver …

Targeting The Brd4/Foxo3a/Cdk6 Axis Sensitizes Akt Inhibition In Luminal Breast Cancer, Jingyi Liu, Weijie Guo, Zhibing Duan, Lei Zeng, Yadi Wu, Yule Chen, Fang Tai, Yifan Wang, Yiwei Lin, Qiang Zhang, Yanling He, Jiong Deng, Rachel L. Stewart, Chi Wang, Pengnian Charles Lin, Saghi Ghaffari, B. Mark Evers, Suling Liu, Ming-Ming Zhou, Binhua P. Zhou, Jian Shi

Molecular and Cellular Biochemistry Faculty Publications

BRD4 assembles transcriptional machinery at gene super-enhancer regions and governs the expression of genes that are critical for cancer progression. However, it remains unclear whether BRD4-mediated gene transcription is required for tumor cells to develop drug resistance. Our data show that prolonged treatment of luminal breast cancer cells with AKT inhibitors induces FOXO3a dephosphorylation, nuclear translocation, and disrupts its association with SirT6, eventually leading to FOXO3a acetylation as well as BRD4 recognition. Acetylated FOXO3a recognizes the BD2 domain of BRD4, recruits the BRD4/RNAPII complex to the CDK6 gene promoter, and induces its transcription. Pharmacological inhibition of either BRD4/FOXO3a association or …

A Xenopus Oocyte Model System To Study Action Potentials, Aaron Corbin-Leftwich, Hannah E Small, Helen H Robinson, Carlos A. Villalba-Galea, Linda M Boland

School of Pharmacy Faculty Articles

Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na+- and K+-selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the …

An Expanded Toolkit For Gene Tagging Based On Mimic And Scarless Crispr Tagging In, David Li-Kroeger, Oguz Kanca, Pei-Tseng Lee, Sierra Cowan, Michael T Lee, Manish Jaiswal, Jose Luis Salazar, Yuchun He, Zhongyuan Zuo, Hugo J Bellen

Faculty, Staff and Students Publications

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced …

Structure Of The Mouse Trpc4 Ion Channel, Jingjing Duan, Jian Li, Bo Zeng, Gui-Lan Chen, Xiaogang Peng, Yixing Zhang, Jianbin Wang, David E. Clapham, Zongli Li, Jin Zhang

Molecular and Cellular Biochemistry Faculty Publications

Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP …

Transcriptional Correlates Of Proximal-Distal Identify And Regeneration Timing In Axolotl Limbs, S. Randal Voss, David Murrugarra, Tyler B. Jensen, James R Monaghan

Neuroscience Faculty Publications

Cells within salamander limbs retain memories that inform the correct replacement of amputated tissues at different positions along the length of the arm, with proximal and distal amputations completing regeneration at similar times. We investigated the possibility that positional memory is associated with variation in transcript abundances along the proximal-distal limb axis. Transcripts were deeply sampled from Ambystoma mexicanum limbs at the time they were administered fore arm vs upper arm amputations, and at 19 post-amputation time points. After amputation and prior to regenerative outgrowth, genes typically expressed by differentiated muscle cells declined more rapidly in upper arms while cell …

Expression, Purification, And Inhibition Profile Of Dihydrofolate Reductase From The Filarial Nematode Wuchereria Bancrofti, Andrew M. Tobias, Dea Toska, Keith Lange, Tyler Eck, Rohit Bhat, Cheryl A. Janson, David P. Rotella, Ueli Gubler, Nina M. Goodey

Department of Chemistry and Biochemistry Faculty Scholarship and Creative Works

Filariasis is a tropical disease caused by the parasitic nematodes Wuchereria bancrofti and Brugia malayi. Known inhibitors of dihydrofolate reductase (DHFR) have been previously shown to kill Brugia malayi nematodes and to inhibit Brugia malayi DHFR (BmDHFR) at nanomolar concentrations. These data suggest that BmDHFR is a potential target for the treatment of filariasis. Here, protocols for cloning, expression and purification of Wuchereria bancrofti DHFR (WbDHFR) were developed. The Uniprot entry J9F199-1 predicts a 172 amino acid protein for WbDHFR but alignment of this sequence to the previously described BmDHFR shows that this WbDHFR sequence lacks a crucial, conserved 13 …

Amylin And Diabetic Cardiomyopathy – Amylin-Induced Sarcolemmal Ca2+ Leak Is Independent Of Diabetic Remodeling Of Myocardium, Miao Liu, Amanda Hoskins, Nirmal Verma, Donald M. Bers, Sanda Despa, Florin Despa

Pharmacology and Nutritional Sciences Faculty Publications

Amylin is a pancreatic β-cell hormone co-secreted with insulin, plays a role in normal glucose homeostasis, and forms amyloid in the pancreatic islets of individuals with type-2 diabetes. Aggregated amylin is also found in blood and extra-pancreatic tissues, including myocardium. Myocardial amylin accumulation is associated with myocyte Ca²⁺ dysregulation in diabetic rats expressing human amylin. Whether deposition of amylin in the heart is a consequence of or a contributor to diabetic cardiomyopathy remains unknown. We used amylin knockout (AKO) mice intravenously infused with either human amylin (i.e, the aggregated form) or non-amyloidogenic (i.e., monomeric) rodent amylin to test the …