bluuebunny/crossref_metadata_2025 huggingface dataset:
HISTOCHEMICAL ANALYSIS OF STOMACH OF GRASSCUTTER (Thryonomys swinderianus)
Authors: Alawa Judith Nkiruka , Barnabas Kas K , Ibegbu Austin O , Alawa Clement Barikuma
Published: June 2021

The Grasscutter (Thryonomys swinderianus), is a wild herbivorous rodent peculiar to Africa. It is widely eaten in Nigerian rural communities as a protein source hence its domestication as microlivestock. There is paucity of data on the histochemical studies. This study was aimed at histochemically analyzing the stomach in order to determine if it exhibits regional differences in its qualitative expression of acid, neutral and carboxylated mucin. Seven animals obtained from the wild and domesticated in the animal house of the Department of Human Anatomy, Ahmadu Bello University, Zaria were used. Histologically, stomach of the Grasscutter was simple, glandular and composed of three regions: cardia, fundic‐body and pyloric. Cardia region showed enlongated tubular glands, coiled at the lower end with an opening continuous with the long conspicuous gastric pits. Fundic‐body region showed numerous gastric glands which open into fewer shorter gastric pits while the pyloric region showed simple tubular glands which are highly branched as such obscures the depth of the gastric pits. Histochemically, stomach of the Grasscutter exhibits regional polymorphism in the mucin content with the glands showing weak alcianophilic reactions. Cardia and pyloric regions demonstrated neutral periodic acid Schiff (PAS) reaction characterized by purple coloration thus indicating the presence of neutral mucins. Fundic‐body region exhibited strong PAS positive reaction particularly at the apical cytoplasm of the oxynticopeptic cells indicating the presence of carboxylated mucins. Results demonstrate that regional differences in the expression of mucins occur in the stomach of African Grasscutters, thus this may provide basic information for further nutritional and conservative studies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Iodine Staining Results in Significant Shrinkage of Sarcomere Lengths in Macaque Biceps Brachii Muscle
Authors: Green Katherine D. , Orsbon Courtney P. , Ross Callum F. , Taylor Andrea B.
Published: June 2021

Sarcomere lengths are widely used to calculate scaled sarcomere models of whole muscle length‐tension curves. Previous studies have shown that scaled sarcomere models of fresh tissues accurately predict experimental whole muscle length‐tension curves. Diffusible iodine‐based contrast enhanced CT (diceCT) imaging employs iodine staining with Lugol's solution (I2KI) to improve visualization of muscle tissue to the level of individual fascicles, which holds great promise to allow length‐tension modeling of multiple muscles. However, staining skeletal muscles with I2KI causes significant volumetric shrinkage of the muscle, even at levels as low as 2% I2/4% KI, which may affect the validity of length‐tension curves calculated from these tissues. Here we test the hypothesis that iodine staining distorts sarcomere length measurements. We evaluated the impact of I2KI on sarcomere lengths measured from muscle samples of the right biceps brachii from one formalin‐fixed rhesus macaque (Macaca mulatta). We sectioned the muscle into a primary control (no I2KI) and two experimental groups: one treated with 20% (w/v) I2KI and one pretreated with a sucrose prewash (20% w/v) prior to iodine staining. The muscle sections then were chemically digested in 70% nitric acid in saline and mounted on slides. A minimum of three sarcomere length measurements was taken per fiber using laser diffraction and the averages used for ANOVA and post hoc t‐tests. The results show that iodine staining significantly shrinks sarcomere lengths (2.08 ± 0.22 μm) compared with primary controls (2.45 ± 0.25 μm) (p < 0.001). The sucrose prewash group also experienced significant shrinkage (2.13 ± 0.23 μm). These data demonstrate the need for methods which correct for this pronounced sarcomere shrinkage before iodine stained skeletal muscle samples can be used to calculate whole muscle length‐tension curves.

Support or Funding Information

National Science Foundation Grant, Division of Behavioral and Cognitive Science, DDRI Bio Anthro, Award #1732175

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mesterolone Increases Satellite Cell Number without Inducing Hypertrophy in Type I Oxidative Muscle Fibers
Authors: Allouh Mohd (Mohammed) Zohair
Published: June 2021

Mesterolone is a synthetic oral anabolic androgenic steroid used to treat hypogonadism. Limited information is available about how this drug exerts its effects on skeletal muscle. Satellite cells (SCs) are mononuclear myogenic stem cells that contribute to postnatal skeletal muscle growth and maintenance. As SCs' activation and subsequent differentiation to new myonuclei is a major event during skeletal muscle hypertrophy, this study investigated the influence of Mesterolone on SC distribution within type I oxidative fibers of the adult avian skeletal muscle. Immunocytochemical techniques and morphometric analyses were used to calculate the fiber size, and numbers of SCs and myonuclei within the anterior latissimus dorsi (ALD) muscle of the chicken. The ALD is a slow (tonic) contracting muscle that is made up exclusively of slow‐twitch type I oxidative red fibers. The number of SCs was significantly (P<0.01) increased by very high doses (12 and 16 mg/kg) of Mesterolone administration. Similarly, the number of myonuclei was increased by administering the high doses of Mesterolone. However, this increase did not reach significant levels except at the Mesterolone dose of 12 mg/kg as the number of myonuclei increased significantly (P<0.05) by 32% compared to the number of myonuclei in the control birds. In addition, no significant (P>0.05) differences were detected in the fiber area and the myonuclear domain size between control and Mesterolone‐treated birds. This study indicates that Mesterolone treatment can increase the numbers of SCs and myonuclei without inducing hypertrophy in type I fibers of the adult avian skeletal muscle. This is because SCs in type I fibers are, most probably, required for fiber maintenance rather than hypertrophy since these fibers have a higher rate of protein breakdown because they are the ones that mostly used in daily routine activities.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intrinsic Cardiac Ganglionic Neurons Projecting to the SA node in the Rat and Pig Hearts: Retrograde Labeling and Neurolucida 3‐D Reconstruction
Authors: Chen Jin , Hanna Peter , Ardell Jeffrey L , Hoover Donald B , Vadigepalli Rajanikanth , Schwaber James , Shivkumar Kalyanam , Cheng Zixi J
Published: June 2021

Intrinsic cardiac ganglionic neurons (ICNs) within the heart are essential for regulation of cardiac function. ICNs include afferent, efferent neurons and interneurons and constitute a local integration center. In rat and pig hearts, there are multiple cardiac ganglia. Among these ganglia, the largest ganglionated plexus in the rat heart and the right atrial ganglionic plexus (RAGP) in the pig heart are near the sinoatrial node (SAN). However, it is not clear whether both plexi project to the SAN. In this study, retrograde tracer Fast DiI was injected into the SAN in rats (n=5) and a pig. SAN cells were identified by immunostaining for pacemaker channel HCN4. Fluorogold (FG) was injected (i.p.) to label all of the ICNs in rats. FG‐ and DiI‐labeled ICNs were identified and analyzed using a confocal microscopic system and a Neurolucida Tracing and Digitization System. Sixteen days after Fast DiI injection in rats, the animals were perfused with paraformaldehyde, and the whole‐mount atria were prepared. There were multiple ganglionated plexi and the DiI‐labeled neurons were mainly found in the largest ganglionated plexus near the SAN in all these rats. Twenty‐one days after Fast DiI injection in the pig, the animal was sacrificed, and the RAGP was removed and sectioned at 40 μm. Numerous DiI‐labeled neurons were found in the RAGP of this pig. Interestingly, these DiI‐labeled neurons were organized in five spatially‐separated columns perpendicular to the epicardial surface. Our data suggest that the largest ganglionated plexus in rat indeed projects mainly to the SAN, and the pig RAGP projects very heavily to the SAN. The functional significance of this large ganglionated plexus in rat and the five spatially‐separated columns of RAGP ICN projecting neurons in the pig will be elucidated in the future.

Support or Funding Information

Supported by NIH SPARC grant OT2OD023848, NIH R15HL137143‐01A1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Periocular Mesenchyme (POM) Contributes to Anterior Eye Structures
Authors: Svoboda Kathy K H , Thomson Robert , Serrano Maria Juliana , Zhao Hu , Petroll Matthew
Published: June 2021

Purpose

Establish a new glaucoma animal model. The objective of this project is to determine if Gli1 positive cells contribute to the POM and anterior eye structures by using inducible Gli1‐CreERT2; tdTomatoflox (Gli1‐tdTomato) mouse model to serve as a probe for anterior eye development. Pitx2, FOXC1, and FOXC2 are expressed in the periocular mesenchyme, and are critical for anterior eye development. Mutations in these genes are associated with a broad spectrum of abnormalities in these tissues. A second objective is to establish that FOXC1, FOXC2, and Pitx2 are expressed in the Gli1 positive cells.

Methods

Gli1‐tdTomato expressing mice were induced at different stages with tamoxifen. Eyes were examined that were induced on embryonic (E13.5) and postnatal days: P3, P7, P30 and sacrificed several days to weeks later. Mouse tissues were fixed and prepared for frozen or paraffin sectioning. Tissue antigen retrieval was done using citric acid buffer. Indirect immunohistochemistry for FOXC1, FOXC2, and Pitx2 was completed. Some samples were counterstained with phalloidin. Sections were analyzed with confocal microscopy for spatial distribution of the Gli1+ labeled cells and FOXC1, FOXC2, and Pitx2.

Results

In Gli1‐tdTomato mice induced on P3, P7, and P30, the POM and anterior angle of the eye, eyelids and optic nerve were labeled several days to 3 weeks later. In contrast, the cornea, lens and sclera did not have positive cells for Gli1. Expression levels for FOXC1, FOXC2, and Pitx2 were generally higher in mice harvested at younger postnatal ages. After postnatal day 30, the expression decreased with little to no expression of FOXC1 and FOXC2 while Pitx2 expression remained relatively constant in the anterior angle tissues and was expressed in some Gli1 positive cells.

Conclusion

This inducible Gli1‐tdTomato mouse model may be an excellent system for determining the role of POM in anterior eye development. The elegance of this model is it can be activated at specific developmental stages and Gli1+ cell derivatives can be tracked and other detection probes (phalloidin, nuclear stains, Immunohistochemistry etc), can be incorporated to determine cell characteristics or over‐all tissue structure. Our results provide evidence that POM cells are contributing to the development of the anterior angle chamber that forms between P3‐P10 in mice.

Support or Funding Information

Biomedical Sciences Department Seed Grant (K. Svoboda), NIH K08DE025090 (H. Zhao)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Emergent Properties of Facial Morphogenesis Regulated by Fgf Signaling
Authors: Marcucio Ralph , Hu Diane , Hallgrimsson Benedikt , Young Nathan
Published: June 2021

Craniosynostosis is a severe disorder that may be caused by activating mutations in Fibroblast growth factor receptors (FgfRs) that affects development of the skull and is characterized by premature fusion of the cranial sutures. In addition to dysmorphology of the skull, affected individuals also have facial dysmorphology. We have previously used an avian model to explore the role that a disease‐causing allele of FgfR2 (FgfR2C278F) plays in producing malformations of the middle and upper face at early stages of development. In that work we observed a wider midface, which resembles what is observed in human patients, and this was directly related to decreased cell proliferation and a disrupted net polarization of the mesenchymal cells in the growing facial primordia. We have extended this work by examining the role of the Map kinase, PLC‐gamma, and PI3K pathways downstream of Fgf signaling in producing facial dysmorphology, as well as directly blocking FgfR activation. We implanted beads soaked in small molecular inhibitors (MEK1/2: U0126, PLC‐gamma: U‐73122, PI3K: LY 294002, FgfR activation: SU5402) into the right side of the developing avian face at HH22 and examined morphological and cellular outcomes at various times of development. First, we determined that each inhibitor substantially and specifically down‐regulated its respective pathway using immunohistochemistry to assess expression of down‐stream targets of activation of each pathway. We then determined that each pathway significantly reduced cell proliferation and altered cell polarization in the mesenchyme. Blocking each pathway also created severe craniofacial malformations that were observed at early and late time points. There are many inputs to each of these pathways, and our data suggest that each participate in regulation of a similar set of cellular processes that contribute to the emergence of morphogenetic processes in facial development.

Support or Funding Information

NIH: R01DE019638, R01DE018234, R21DE028198

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of alpha‐amylase during murine palatine gland development: A light microscopic immunocytochemical study
Authors: Kalfut Mahasin , Alhazmi Ahlam , Alhakami Khalid , Hakami Zaki
Published: June 2021

Introduction

Salivary alpha‐amylase is one of the major enzymes in the oral cavity. Beyond its primary function of initiation of digestion in the oral cavity, it is involved in defense against bacteria. Alpha‐amylase, a product of serous cells, is the most abundant protein of parotid saliva and it is also secreted by sublingual glands. However, its presence in the minor glands, including palatine glands, has been contradictory. Therefore, this study was conducted to observe the expression of alpha‐amylase at different developmental stages of rat palatine glands using light microscopic immunohistochemistry.

Materials and Methods

An avidin‐biotin technique was used to detect the binding of alpha‐amylase on sagittal frozen sections of palatine glands of pre‐ and post‐natal rats.

Results

Positive reaction of alpha‐amylase was observed at the basolateral and perinuclear membranes of the developing acinar cells at the embryonic day 20 (E20). At birth, in addition to the basolateral membranes, weak cytoplasmic reaction labelling a few mucous cells was observed. Amylase reactivity increased with maturation and reached peak reactivity at postnatal day (PN) 10 and 14, thereafter, decreased into adulthood. Serous cells expressed amylase starting from PN7. Whereas ducts showed variable reaction on the luminal membrane starting from PN3. In adult rats, heterogeneous distribution was revealed between the anterior and posterior portions of palatine glands, as amylase showed more reactivity in the posterior portion.

Conclusion

The expression and distribution of alpha‐amylase showed significant modifications during cytodifferentiation and maturation of the palatine glands.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cold‐Inducible RNA Binding Protein (CIRP) Decreases Inflammation and Breast Tumor Growth
Authors: Lujan Daniel Albino , Hathaway Helen J , Kusewitt Donna F , Hartley Rebecca S
Published: June 2021

RNA binding proteins (RBPs) post‐transcriptionally regulate gene expression by associating with the regulatory sequences in the untranslated regions of mRNAs. RBPs are often disrupted in cancer, but current knowledge of their role in regulating cancer‐associated genes is largely based on in vitro studies. Cold‐inducible RBP (CIRP) is a stress‐induced RBP that was recently shown to modulate inflammation in response to cellular stress. CIRP expression is altered in several cancers, including breast cancer. The effect of CIRP on inflammation in breast cancer is not known. This study tested the hypothesis, based on our preliminary data and published studies that CIRP changes the inflammatory profile of breast tumors from pro‐ to anti‐tumorigenic, and that CIRP expression and this altered inflammatory milieu are associated with lower tumorgenicity. To test this hypothesis a transgenic mouse overexpressing CIRP in the mammary epithelium was crossed with the PyMT mouse model of breast cancer. Effects on both early (7‐week‐old mice) and late (14‐week‐old mice) tumorigenesis and inflammation were assessed in CIRP/PyMT and PyMT mice. Mammary glands from 7‐week‐old CIRP/PyMT mice had 50% fewer mammary intraepithelial neoplasias than PyMT mice while 14 week‐old CIRP/PyMT mice had slower tumor growth kinetics, smaller tumors, decreased tumor burden and decreased pulmonary metastasis compared to PyMT mice. Importantly, CIRP protein level correlated with decreased tumorigenesis in CIRP/PyMT mice. To assess effects on the inflammatory environment, mammary glands from 7 and 14‐week‐old mice were incubated in medium overnight and Luminex cytokine assays were performed on the medium. Luminex assays were also performed on sera from 14‐wk mice to assess effects on systemic inflammation. There was a decrease in cytokines that promote inflammation, angiogenesis and metastasis at both timepoints for mammary glands, and in 14‐week sera. Overall, these data support a role for CIRP as a potent anti‐tumor molecule that suppresses local and systemic pro‐tumorigenic inflammation. To begin to probe whether CIRP's effects on cytokine levels were cell‐autonomous, we performed in silico analysis of CIRPs binding affinity for individual targets using the web‐based beRBP tool (Yu et al.). CIRP was not predicted to bind the cytokine mRNAs so may be acting in a non‐cell autonomous fashion.

Support or Funding Information

National Institutes of Health Grant/Award Number: F31‐CA213933

American Association of Anatomists, Fellows Grant Award Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of a GDF5 Associated Enhancer During Limb Development
Authors: Cooper Allen M. , Shankel Matthew , Haro Endika , Pira Charmaine U. , Oberg Kerby C.
Published: June 2021

Growth differentiation factor 5 (GDF5) is an anabolic growth factor that plays a critical role in the formation, maintenance and repair of bones and joints. Reduced levels of GDF5 are associated with accelerated degradation of joint cartilage, a disease called osteoarthritis (OA). Identification of factors involved in GDF5 regulation may offer therapeutic targets for treating or preventing OA. A GDF5 Associated Regulatory Region (GARR) has been previously identified as a 900 bp enhancer located 78 kb downstream of the GDF5 locus. GARR contains a number of potential transcription factor binding sites, including sites for odd‐skipped related (Osr) zinc‐finger and Sry box (Sox) transcription factors. As Osr1/2 and Sox11 are important for normal Gdf5 expression, and their expression patterns overlap that of Gdf5, we hypothesized that mutation of OSR and SOX binding sites within GARR would disrupt enhancer activity.

To identify whether putative OSR and SOX transcription factor binding sites contribute to GARR activity, we performed site‐directed mutagenesis on both potential OSR binding sites (GARRmutOSR), or on both potential SOX binding sites (GARRmutSOX) in the GARR‐tk‐EGFP reporter construct (GARRcontrol). We used targeted regional electroporation (TREP) to introduce control and mutated reporter constructs into Hamburger‐Hamilton stage 23 chicken limb buds near the developing elbow joint. Transfection efficiency was determined by co‐transfection with a β‐actin promoter‐driven RFP construct. We determined GARR activity by fluorescence microscopy, following 48 hours of incubation after TREP.

Comparison of GARRcontrol and GARRmutOSR demonstrated similar GFP fluorescence intensity localized to the elbow joint. In contrast, fluorescence of GARRmutSOX was markedly reduced relative to GARRcontrol, although similarly restricted to the elbow. Though Osr transcription factors are associated with Gdf5 expression, mutation of the OSR binding sites in GARR was insufficient to substantially alter the level of GARR activity. Mutation of SOX binding sites, however, dramatically reduced GARR activity, implicating SOX transcription factors as critical regulators of GDF5 expression.

Support or Funding Information

The research was funded in part by a grant from the LLU Pathology Research Endowment.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circularization of linear chromosomes and telomerase RNA gain‐of‐function mutations in Saccharomyces cerevisiae
Authors: Mefford Melissa Anne
Published: June 2021

Telomeres are repeated DNA sequences located at the ends of linear eukaryotic chromosomes. While they act as protective caps, they cannot be fully copied by the DNA replication machinery. To overcome this end‐replication problem, most eukaryotic organisms express the ribonucleoprotein enzyme complex telomerase. I will present preliminary results investigating basic, broad questions of telomere and telomerase evolution in the budding yeast, Saccharomyces cerevisiae. First, we are genetically engineering strains with circularized versions of each of the 16 chromosomes to assess the viability and potential fitness effects on the organism. Second, we are screening for gain‐of‐function mutations in telomerase RNA to gain further insights into how this component contributes to enzyme function.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Roles of Nap1, Taz2, and H1 on Nucleosome Assembly
Authors: Blackwell Virginia Katherine , Sarkar Prithwijit , D'Arcy Sheena
Published: June 2021

In eukaryotic cells, DNA is packaged into the nucleus by extensive coiling, the most fundamental unit of which is the nucleosome. Nucleosomes are comprised of 146 base pairs of DNA wrapped around an octameric histone core, along with linker histone H1, controlling access to the genome. Proteins such as nucleosome assembly proteins (Naps) and p300 interact to shuttle histones to and from the nucleosome, regulating gene expression. In this project, we investigated the molecular details of interactions between Nap1 and the Taz2 domain of p300, their interactions with H1, and studied the role of these protein complexes on nucleosome dynamics. We purified Nap1, Taz2, and H1 using column chromatography, then assayed interactions between Nap1‐Taz2 and Nap1‐H1. We also performed experiments investigating the formation of a ternary complex of Nap1‐Taz2‐H1. Electrophoretic mobility shift assay (EMSA) and size exclusion chromatography with multi‐angle light scattering (SEC‐MALS) were used to study binding and determine the stoichiometry of these protein‐protein interactions. With these methods, we observed that Nap1 and Taz2 interact in vitro at an equimolar stoichiometry. We performed nucleosome assembly/disassembly assays in the presence of these protein complexes to study their effects on the nucleosome. Since Nap1, p300, and histones are a few of many ‘gatekeepers’ to our genetic code, it is critical that we determine how they function and cooperate to protect DNA. It is known that both Nap1 as well as p300 mutations contribute to cancer. While there is still much to unravel, p300 and interacting proteins are crucial to proper cell growth and division. This study furthers our understanding of nucleosome dynamics.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rexinoid signaling promotes myogenic differentiation through a direct regulation of MyoD
Authors: Hamed Munerah , Khilji Saadia , Li Qiao
Published: June 2021

Readout of epigenetic modification has provided us with plentiful information to thoroughly understand the regulation of genes during myogenic differentiation. While skeletal myogenesis is coordinated by sequential expression of myogenic regulatory factors including MyoD and myogenin, chromatin modifications have emerged as vital mechanisms of myogenic regulation. We have recently found that bexarotene, a retinoid X receptor (RXR)‐selective agonist, promotes the specification and differentiation of muscle lineage; however, the genome wide impact of rexinoid action on myogenic expression has not yet been investigated. Through genome wide association studies, we here determined rexinoid‐responsive residue‐specific histone acetylation at a distinct chromatin state associated with MyoD and myogenin. We also define a new mechanism of rexinoid action which is mediated by the receptor and largely reconciled through a direct regulation of MyoD expression. Thus, we provide novel molecular insights into the interplay between retinoid X receptor signaling and chromatin states associated with myogenic programs in early differentiation

Support or Funding Information

NSERC

The Cancer Research society

IRSC

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of lncRNA HOTAIR in the regulation of glucose metabolism
Authors: Obaid Monira , Mandal Subhrangsu S
Published: June 2021

Glucose is a fuel for immune cells and glucose transporters have influential roles to supply glucose for immune cell functional responses. Recently we have reported that long noncoding RNA (lncRNA) HOTAIR is involved in the regulation of inflammatory and immune response. Our studies demonstrate that HOTAIR expression is induced in immune cells (macrophages) upon treatment with lipopolysaccharide (LPS). Glucose transporters have been found to be upregulated in immune cells. Our present study has correlated HOTAIR with the upregulation of the glucose transporters in the presence of LPS. Currently, we are investigating the detail mechanism how HOTAIR induces expression of glucose transporters and metabolic reprogramming. Thus, we hypothesize that HOTAIR is involved in metabolic reprogramming via increased glucose availability and modulate the macrophage inflammatory response. Understanding the role of HOTAIR in metabolic reprogramming will identify signaling pathways that would serve as novel therapeutic targets in the treatment of diabetes and obesity.

Support or Funding Information

Subhrangsu S. Mandal

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered dosage of noncoding RNAs expressed from the Dlk1‐Dio3 locus impairs skeletal muscle differentiation
Authors: Petty Christopher Anthony , Naya Francisco J
Published: June 2021

Noncoding RNAs (ncRNAs) have emerged as key components of gene regulatory networks, yet their role in skeletal muscle diseases is still poorly understood. The maternally imprinted Dlk1‐Dio3 ncRNA locus has been shown to play an essential role in skeletal muscle development, regeneration, and metabolism. The diverse ncRNAs expressed from this locus, including over 50 microRNAs (miRNAs), several long noncoding RNAs (lncRNAs), and a cluster of C/D box snoRNAs, are thought to be transcribed as a ~200 kb polycistron initiating from the proximal promoter immediately upstream of the Gtl2 lncRNA, the most 5′ ncRNA in the locus. This project aimed to understand the importance of proper dosage of the Dlk1‐Dio3 ncRNAs in skeletal muscle differentiation. Toward this end, we utilized CRISPR‐Cas9 gene editing to generate deletions in the Gtl2 proximal promoter and the overlapping differentially methylated region (DMR) with the goal of modulating expression of the maternally expressed ncRNAs in C2C12 myoblasts, an established muscle cell line. This gene editing strategy yielded a clonal population of C2C12 myoblasts that harbored a deletion spanning a portion of the Gtl2 proximal promoter while leaving the Gtl2 promoter's TATA box intact. This CRISPR‐edited cell line showed a dramatic increase in the Dlk1‐Dio3 ncRNAs. As a result of Dlk1‐Dio3 ncRNA upregulation, this C2C12 cell line displays significantly impaired myogenic differentiation. The continued characterization of this novel C2C12 cell line will help elucidate the importance of maintaining proper dosage of the Dlk1‐Dio3 locus in skeletal muscle, which could serve as a therapeutic target for treating muscle diseases.

Support or Funding Information

This project was supported with funds from the Arnold & Mabel Beckman Foundation and the Boston University Undergraduate Research Opportunities Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glucose‐induced dysregulation of lncRNAs in a model of chronic kidney disease
Authors: Tsotakos Nikolaos , Caldwell Eric Adam , Cerny Garret , Hartman Hannah L , Saade Namir
Published: June 2021

Diabetic nephropathy (DN) is the leading cause for end‐stage renal disease worldwide. Despite the extensive research that has been done to identify biomarkers and novel therapeutic targets for DN, long non‐coding RNAs (lncRNAs) have not been very well studied. This study sought to identify lncRNAs that are differentially expressed in diabetic podocytes compared to normal podocytes. To do so, Human Glomerular Epithelial Cells (HGEC) were cultured in either normal or high glucose environments (5 mM and 25 mM, respectively). Differential expression of lncRNAs was detected using qPCR. HGEC cultured in high glucose levels demonstrated upregulation of MEG3, MEG8, and H19 compared to cells cultured in normal glucose. Additionally, a modest upregulation of Xist was exhibited by HGEC cultured in high glucose. No differences in MALAT1, TUG1, HOTAIR, or NEAT1 were detected. Podocalyxin, a marker of podocyte health, was used as a control. The results indicate that some lncRNAs, specifically MEG3, MEG8, Xist, and H19, are upregulated in diabetic podocytes. Further research is needed to elucidate the mechanism by which these lncRNAs contribute to the progression of DN.

Support or Funding Information

This work was supported by the Research and Development Committee of Dickinson College and by the School of Science, Engineering, and Technology of Penn State Harrisburg.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Complex Formation on Motion in DNA Binding Domains of ARFs
Authors: George Natalie A , Carroll Margaret C , Gonzalez Daniel , Ellis Jamie P
Published: June 2021

The auxin response factor (ARF) family is a set of DNA binding transcription factors found in the model plant Arabidopsis thaliana in addition to many other food and fuel crops. Along with two other frequently conserved domains, all ARFs contain an N‐terminus DNA binding domain (DBD) that includes a B3 DNA binding motif and a dimerization subdomain. While the structure of example DBDs have been solved, motion within this domain is poorly understood. The interplay of these domains may provide selectivity in binding to variants of target DNA constructs. Our work examines the DBDs of ARF1 and ARF5, a transcription repressor and transcription activator respectively, as ARF5 is known to be more permissive than ARF1 to a larger spacing between binding sites on DNA. Our work directly tests the hypothesis that the ability to bind to a broader range of DNA targets relates to observable differences in structural flexibility. We combine spectroscopic and chromatographic methods of resolving both complex formation and molecular motion for different combinations of target DNA and DBD. We utilize fluorescence spectroscopy as it resolves protein motions and associations that occur on the same timescale as the fluorescent lifetimes (~ns). Labeled proteins can also be studied at low physiological concentrations and within an aqueous buffer. We optimized the expression and purification of the protein as well as two alternative fluorophore conjugation methods to compare thiol‐reactive fluorophore labeling to a newer technique that targets the N‐terminus. Under conditions of known dissociated versus associated states, we apply both steady‐state and time‐resolved fluorescence anisotropy to sense rotational correlation times of these proteins and distinguish subdomain motions in the presence and absence of DNA. Our work demonstrates that the N‐terminal probes are more sensitive to molecular changes likely due to its placement at the hinge between the DNA binding domain and the dimerization domain. Consistent with our hypothesis, ARF5 shifts from a highly dynamic state to a more rigid state upon complex formation while ARF1 remains more unperturbed. These differences in the available structural ensemble provide valuable insights into the structural basis of target specificity as well as the interplay of these transcriptional on‐ and off‐switches.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation Impact on the Behavior of Substrate‐Bound Tau
Authors: Harries Lukas , Donhauser Zachary
Published: June 2021

Tau proteins are commonly found in neural cells and provide a variety of functions, the most notable being the regulation of microtubule stability and spacing. Tau aggregation has been implicated in several neurodegenerative diseases, namely dementia and Alzheimer's, making tau‐based therapeutics an area of high research interest. Site‐specific phosphorylation of tau has been observed to direct its behavior but can also result in a lower binding affinity of tau to microtubules, increased propensity of tau to aggregate, and cell death. As the dynamics of tau aggregation are poorly understood, more investigation is needed to elucidate tau aggregation and the effects of phosphorylation. Most of the research in this area has been centered on tau's unbound state, requiring more information regarding the dynamics of its oligomerization upon microtubule binding and cause of disassociation leading to aggregation. To study tau‐tau and tau‐microtubule interactions, we utilized a pseudohyperphosphorylated tau containing seven pseudophosphorylation changes and mimicked the surface of negatively‐charged microtubules with mica. Atomic force microscopy was used to study tau bound to mica, quantify tau‐tau intermolecular interactions, and to assess its bound structure. Changes to solution conditions, such as ionic strength or presence of aggregation inducers, were employed to alter tau's structure and its ability to oligomerize. Comparisons of resulting intermolecular interactions and bound‐tau structures between wild‐type tau and pseudohyperphosphorylated mutants are described, in an effort to address the mechanism linking phosphorylation and tau malfunction.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Studies of GMP Reductase from Escherichia coli
Authors: Harris Darnell , Torelli Andrew T
Published: June 2021

Guanosine Monophosphate Reductase (GMPR) is an NADPH‐dependent enzyme that catalyzes the reductive deamination of guanosine (G) monophosphate nucleotides into their corresponding inosine (I) monophosphate derivatives. GMPR plays an important role in purine salvage, re‐utilization of free intracellular bases, and contributes overall to the intracellular balance of G and adenine (A) nucleotides. As with many other enzymes in the purine salvage or biosynthesis pathways, GMPR is widespread in nature across eukaryotic and prokaryotic taxa. Given the interest in purine metabolism as a potential target for drug design, GMPR has been characterized both structurally and kinetically in different studies using GMPR derived from several species. We report progress towards solving the crystal structure of GMPR from Escherichia coli in order to extend the insight from existing studies of the E. coli GMPR kinetic and thermodynamic characteristics. Additionally, lessons learned from integrating related research activities into an undergraduate laboratory course will be presented in order to promote adoption of similar course‐based research practices at other primarily‐undergraduate institutions.

Support or Funding Information

Ithaca College Department of Chemistry

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular mechanism for accurate dephosphorylation of RNA polymerase II during eukaryotic transcription
Authors: Irani Seema , Zhang Yan
Published: June 2021

The phosphorylation states of the C‐terminal domain of RNA polymerase II (CTD) coordinate eukaryotic transcription by recruiting protein regulators to Pol II. The precise placement and removal of phosphate groups on the specific residues of the heptad repeat of CTD is critical for the fidelity and effectiveness of transcription. In spite of highly similar flanking residues, Ser5 dephosphorylation is carried out efficiently during transcription elongation when Ser2 phosphorylation accumulates, while Ser2 dephosphorylation occurs predominantly at the end of transcription. To understand the molecular mechanisms of how the CTD phosphatases Ssu72 and Fcp1 specifically remove CTD phosphorylation marks in the context of other phosphorylated CTD residues, we used a combination of high resolution MALDI‐TOF mass spectrometry, X‐ray crystallography, and protein engineering to analyze CTD phosphoregulation. Biochemical and bioanalytical characterization revealed that Ssu72 dephosphorylates Ser5 effectively but with much less activity towards Ser7 and Ser2 and no activity against Tyr1 and Thr4. Structural analysis demonstrated that the requirement for cis‐proline substrates of Ssu72 inhibits binding of bulky flanking residues like Tyr1 next to Ser2, which illustrates why Ser5 is the preferred substrate for Ssu72. Phosphorylation of Tyr1 even further prohibits binding of pSer2 by Ssu72, preventing the untimely dephosphorylation of Ser2 at elongation by Ssu72. Finally, we show that Fcp1 not only dephosphorylates phosphorylated Ser2 and Ser5 but also phosphorylated Tyr1. General clearance of phosphorylation marks on the CTD by Fcp1 supports its reported role in recycling Pol II for the next round of transcription. Our results reveal novel roles for Tyr1 in preventing inappropriate dephosphorylation of Ser2 by Ssu72 during transcriptional elongation and for Fcp1 in removing Tyr1 phosphorylation marks at the end of transcription.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Phenotypic Consequences of EmrE Expression Under Various Environmental Stressors
Authors: Beeninga Will , Spreacker Peyton , Henzler‐Wildman Katherine
Published: June 2021

EmrE is a multidrug resistance transporter found in the inner membrane of E. coli. EmrE has been shown to efflux many different toxic compounds, most notably quaternary ammonium compounds (QACs), such as ethidium bromide (EtBr), and other polyaromatic cations. While this protein has been extensively studied, the native substrate and function of EmrE is unknown. To understand the range of EmrE activity under various environmental conditions, we tested ethidium efflux under different salt and pH conditions. In these screens, we also noted phenotypic differences arise between different knockout strains of E. coli. Additionally, recent literature suggests that variation in carbon sources may also affect expression and/or function of multidrug efflux pumps, such as EmrE. We have screened EmrE efflux activity in E. coli grown on different carbon sources to determine the phenotypic effect for this particular pump.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural mechanism of corepressor‐selective inverse agonism of the nuclear receptor PPARγ
Authors: Kojetin Douglas , Shang Jinsai , Brust Richard , Bass Jared
Published: June 2021

The structural mechanisms affording ligand‐induced activation of nuclear receptor transcription factor peroxisome proliferator‐activated receptor gamma (PPARγ) are well understood; however, it remains poorly understood how to design transcriptionally repressive PPARγ inverse agonists. We discovered that a simple phenyl‐to‐pyridyl substitution between two common covalent orthosteric ligands targeting PPARγ converts a transcriptionally neutral antagonist into a corepressor‐selective inverse agonist. Using X‐ray crystallography, molecular dynamics simulations, NMR spectroscopy, and mutagenesis coupled to activity assays, we discovered that hydrogen bond formation with Arg288 is essential for corepressor‐selective inverse agonism. For one corepressor‐selective inverse agonist, NMR spectroscopy revealed that PPARγ exchanges between two long‐lived conformations, including a conformation that prepopulates a corepressor‐bound state that primes PPARγ for high affinity corepressor binding. We also determined the crystal structure of corepressor‐bound PPARγ, which reveals several unique conformational features compared to the well studied active conformation. Our findings define new structural features for repressing PPARγ transcription with ligands.

Support or Funding Information

This work was supported by National Institutes of Health (NIH) grants R01DK101871, F32DK108442; and American Heart Association (AHA) award 16POST27780018 (RB)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calmodulin Induced Changes in the Active Site of Nitric Oxide Synthase
Authors: Sanchez Austin Johnny , Esquerra Raymond , Refuerzo Ruszel
Published: June 2021

Nitric oxide is a transient signaling molecule in mammalian physiology, known as a vasodilating neurotransmitter and effector during innate immune responses. As such, studies about the family of nitric oxide synthases (NOS) responsible for the rapid conversion of amino acid L‐arginine into nitric oxide have been of great interest. The heme domain within the NOS active site critically binds molecular oxygen which combines with L‐arginine in a series of redox reactions. NOS activity is regulated by a number of cofactors, most notably the ubiquitous regulatory protein calmodulin (CaM) which, in the presence of calcium, binds NOS and promotes nitric oxide production. Though this relationship is well‐documented, the molecular mechanism is not understood. Previously, time resolved spectroscopy has been used to probe the active sites of heme proteins and observe differences in absorption on a nanosecond time frame during changes in the catalytic pockets. Our investigation studies the kinetic effects of CaM binding NOS during the photolysis of carbon monoxide from its heme domain. CaM has been shown to directly influence the active site, promoting availability to substrates and rebinding of ligands to the NOS heme, thus enhancing enzymatic activity. A major facet of our project involves further investigation of the various domains of NOS to determine their significance in CaM‐regulated binding. To this end, we prepared a number of knockout NOS variants which, through assaying via time‐resolved spectroscopy, further elucidate the molecular mechanism of active site regulation.

Support or Funding Information

NIH MARC: T34‐GM008574

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The inflammasome adapter ASC and truncated constructs containing only its Death Domains self‐assemble into different macrostructures
Authors: Sandin Suzanne , Nambayan Reinard Jeffery , Quint David , Satyadi David , de Alba Eva
Published: June 2021

ASC (apoptosis‐associated speck‐like protein containing a CARD) is an adaptor protein that mediates in inflammasome assembly. The inflammasome is a multiprotein complex that triggers the inflammatory response. Multiple copies of a sensor protein, procaspase‐1 and ASC acting as a molecular bridge between them, self‐associate and oligomerize to form the inflammasome particle, a micrometer‐size filamentous ring in the cell cytosol. Thus, ASC self‐assembly properties and interacting capabilities via its two Death Domains (PYD and CARD) are critical for inflammasome formation. Our Transmission Electron Microscopy (TEM) studies show that full length ASC, and the truncated constructs ASCPYD and ASCCARD, form macrostructures with significantly different characteristics. In addition, we have analyzed in detail the ASCCARD–ASCCARD interaction by solution NMR and TEM, which is of particular relevance due to the current lack of information. Our analysis includes CARD mutants designed based on the NMR data.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms of G Protein‐Selectivity in Muscarinic Acetylcholine Receptor Family
Authors: Santiago Luis Jaimes , Abrol Ravinder
Published: June 2021

Muscarinic acetylcholine receptors are integral membrane proteins that regulate physiological activity. Acetylcholine is a neurotransmitter that binds to the extracellular side of these G protein coupled receptors (GPCRs) activating the Gα subunit of the trimeric G protein inside the cell. There are 5 different muscarinic receptor subtypes (M1–M5), where the same ligand acetylcholine causes M2 and M4 receptors to preferentially couple to the Gαi subunit and the receptors M1, M3, and M5 to preferentially couple to the Gαq subunit. This selectivity of Gα subunits mediates different secondary messenger pathways in the cell: Gαi being responsible for deactivating adenyl cyclase activity and Gαq being responsible for activating phospholipase C. We are using computational biophysical methods to probe the mechanisms of G protein selectivity of this muscarinic receptor family. We have built structural models for the M1 and M2 receptors coupled to Gαi or Gαq proteins and used molecular dynamics simulations to obtain relaxed structures of these complexes, so that the thermodynamic analysis of the receptor G protein interactions can be conducted. The analyses are beginning to show that thermodynamically, some complexes are more stable than others in agreement with corresponding biochemical experiments. In addition, some receptor ‐ G protein interactions may be kinetically controlled. Overall, these studies are beginning to provide us with the missing mechanistic insight into the G protein selectivity of the muscarinic receptors.

Support or Funding Information

We acknowledge the CSUPERB New Investigator Grant. We thank the RISE (GM063787) program for their student support.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.