Open Access Publications - Department of Biological Sciences
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Item Inflammatory Breast Cancer: What’s in a Name?(Medical Research Archives, 2025-09-25) Van Golen, Kenneth Louis; Aquino, Hilary C.One of the most unique and highly aggressive types of cancer is known to be Inflammatory Breast Cancer. IBC is a distinct locally advanced breast cancer with a highly virulent course and extremely low 5- and 10-year survival rates. Although Inflammatory Breast Cancer only accounts for 1-3% of breast cancers it is estimated to account for 10% of breast cancer deaths annually in the United States. The accuracy of diagnosis and classification of this unique cancer is a major concern within patient and medical communities. This in turn has led to a strong advocacy movement. Historically, Inflammatory Breast Cancer was thought to be many different diseases but 100 years ago was defined as a unified disease. Over the past three decades several molecular studies have identified unique gene and protein signatures that make Inflammatory Breast Cancer a truly distinct entity. Despite this, the potential for inaccurate diagnosis and misclassification in cases of Inflammatory Breast Cancer is increased by many factors including its physical presentation.Item Whole Exome Sequencing Study Uncovers Novel Candidate Genes and Protein-Coding Variants for Cataract(Investigative Ophthalmology & Visual Science, 2025-08-13) Chaar, Dima L.; Jiang, Chen; Coomson, Sarah Y.; Matthieu, Duot,; Sangani, Poorab; Hoffmann, Thomas J.; Jorgenson, Eric; Hufnagel, Robert B.; Hysi, Pirro; Lachke, Salil A.; Choquet, HélènePurpose: To identify novel candidates for cataract and evaluate the contribution of protein-coding variants to cataract susceptibility. Methods: We first leveraged a publicly-available browser, Genebass, to extract significant gene-based and single-variant association results for cataract in UK Biobank exomes (30,550 cataract cases and 364,291 controls). We then validated findings using genome-wide association study (GWAS) summary statistics from the Genetic Epidemiology Research in Adult Health and Aging (GERA) cohort (28,092 cataract cases and 50,487 controls). Finally, we examined the expression of the prioritized genes in lens tissue using the iSyTE database. Results: Gene-based association testing identified four genes (KDM5B, COL2A1, MIP and CRYBB2) that were associated with cataract (P < 2.50 × 10−6), of which one (KDM5B) was neither previously reported to be associated with congenital cataract nor reported in GWAS. Single-variant association testing identified seven variants within six genes (BFSP2, ZNF800, MIP, HERC2, TSPAN10 and CPAMD8) that were associated with cataract (P < 1.00 × 10−8). Among the identified cataract variants, we found four missense, one synonymous, one frameshift, and one stop-gained variant. Associations at COL2A1, HERC2, and ZNF800 were validated in GERA. Importantly, majority of prioritized cataract genes were robustly expressed in iSyTE lens data and were enriched in structural constituent of eye lens, lens development in camera-type eye, visual perception, and collagen type II trimer pathways. Conclusions: Our results demonstrate the value of gene-based and single-variant association testing for understanding cataract etiology and uncovering novel genetic risk factors. Our findings also show that cataract-associated genes are significantly expressed in lens tissues and lens-related biological pathways.Item LIRTS Viewer: A Web-Based Resource to View the Transcriptional Response of Lens Epithelial Cells to Injury(Investigative Ophthalmology & Visual Science (IOVS), 2025-07-18) Gorai, Suhotro; Faranda, Adam P.; Shihan, Mahbubul H.; Wang, Yan; Duncan, Melinda K.Purpose: Residual lens epithelial cells (LECs) respond to injury after cataract surgery, leading to posterior capsular opacification (PCO). Transcriptomic profiling of lens capsule–associated cells (CACs) post-cataract surgery (PCS) revealed that LECs quickly alter their transcriptome, producing numerous pro-inflammatory cytokines within a few hours PCS. In contrast, the significant activation of TGFβ signaling and fibrotic extracellular matrix deposition related to PCO only begins 1 to 3 days later. However, the global changes in gene expression in CACs, following the establishment of robust TGFβ signaling, remain unknown. Methods: Lens fiber cells were removed from wild-type mice, and CACs were isolated at 0, 72, or 120 hours PCS to perform bulk RNA sequencing (RNA-seq) to obtain estimates of RNA abundance. These data were combined with existing RNA-seq datasets to create a web-based visualization resource to explore the expression dynamics of most protein coding genes in CACs. Results: At 72 hours PCS, CACs differentially express genes consistent with a surge in proliferation and changes in actin filament organization while also robustly expressing fibrotic marker genes by 120 hours PCS. We developed a data visualization resource, the Lens Injury Response Time Series (LIRTS) Viewer, which integrates all data to gather valuable insights from gene expression in CACs over the first 5 days PCS. Conclusions: The LIRTS Viewer is useful for generating hypotheses related to PCO pathogenesis, as it reveals CAC gene expression dynamics, gene correlations, and biological pathways during the first 5 days following lens injury in an in vivo cataract surgery model.Item Targeting Misconceptions in the Central Dogma by Examining Viral Infection(CourseSource, 2021-10-22) DeVito, Stefanie R.Understanding the central dogma and how changes in gene expression can impact cell function requires integration of several topics in molecular biology. Students often do not make the necessary connections between DNA structure, transcription, translation and how these processes work together to impact cell function. This lesson seeks to tie together these concepts through the use of data from primary literature, in the context of viral infection. This lesson asks students to think like scientists as they design experiments, make predictions and interpret and evaluate data from primary literature on how changes in the expression of a glucose transporter gene can alter the function of a cell through changes to glucose uptake and metabolism. This lesson incorporates the Vision and Change core concept of information flow and the core competency of quantitative reasoning. It also addresses The Genetics Society of America learning framework goal of Gene Expression and Regulation (How can gene activity be altered in the absence of DNA changes?). This lesson was taught in three sections of a small-enrollment undergraduate class and assessed summatively using a pre/post test and formatively using in class via personal response systems. This lesson describes the design, implementation and results of student assessment, and offers suggestions on how to adapt the materials to a variety of contexts including different class sizes, different units of introductory biology, and upper-level classes.Item Implementing a Podcast Project to Introduce Introductory STEM Students toScience Communication(Journal of College Science Teaching, 2024-06-18) DeVito, Stefanie; McDuell, Anne; McCollom, Amanda; Sykes, JenniferScience communication skills are increasingly emphasized as an important skill for undergraduate STEM students. In our science communication podcast project, students use the fundamentals of science communication to research a topic related to their inquiry-based lab project. Groups then create a podcast that presents the current science to a general audience. This project has been run in an integrated introductory biology and chemistry course each semester since 2018. Student course evaluations indicate that they enjoy having autonomy over the topic and style of their podcast. Students’ confidence in their science communication skills increased after completion of this multiweek scaffolded project.Item Biochemical mapping reveals a conserved heme transport mechanism via CcmCD in System I bacterial cytochrome c biogenesis(mBio, 2025-04-01) Kreiman, Alicia N.; Garner, Sarah E.; Carroll, Susan C.; Sutherland, Molly C.Heme is a redox-active cofactor for essential processes across all domains of life. Heme’s redox capabilities are responsible for its biological significance but also make it highly cytotoxic, requiring tight intracellular regulation. Thus, the mechanisms of heme trafficking are still not well understood. To address this, the bacterial cytochrome c biogenesis pathways are being developed into model systems to elucidate mechanisms of heme trafficking. These pathways function to attach heme to apocytochrome c, which requires the transport of heme from inside to outside of the cell. Here, we focus on the System I pathway (CcmABCDEFGH) which is proposed to function in two steps: CcmABCD transports heme across the membrane and attaches it to CcmE. HoloCcmE then transports heme to the holocytochrome c synthase, CcmFH, for attachment to apocytochrome c. To interrogate heme transport across the membrane, we focus on CcmCD, which can form holoCcmE independent of CcmAB, leading to the hypothesis that CcmCD is a heme transporter. A structure-function analysis via cysteine/heme crosslinking identified a heme acceptance domain and heme transport channel in CcmCD. Bioinformatic analysis and structural predictions across prokaryotic organisms determined that the heme acceptance domains are structurally variable, potentially to interact with diverse heme delivery proteins. In contrast, the CcmC transmembrane heme channel is structurally conserved, indicating a common mechanism for transmembrane heme transport. We provide direct biochemical evidence mapping the CcmCD heme channel and providing insights into general mechanisms of heme trafficking by other putative heme transporters. IMPORTANCE Heme is a biologically important cofactor for proteins involved with essential cellular functions, such as oxygen transport and energy production. Heme can also be toxic to cells and thus requires tight regulation and specific trafficking pathways. As a result, much effort has been devoted to understanding how this important, yet cytotoxic, molecule is transported. While several heme transporters/importers/exporters have been identified, the biochemical mechanisms of transport are not well understood, representing a major knowledge gap. Here, the bacterial cytochrome c biogenesis pathway, System I (CcmABCDEFGH), is used to elucidate the transmembrane transport of heme via CcmCD. We utilize a cysteine/heme crosslinking approach, which can trap endogenous heme in specific domains, to biochemically map the heme transport channel in CcmCD, demonstrating that CcmCD is a heme transporter. These results suggest a model for heme trafficking by other heme transporters in both prokaryotes and eukaryotes.Item Transcriptome Meta-Analysis Uncovers Cell-Specific Regulatory Relationships in Embryonic, Juvenile, Adult, and Aged Mouse Lens Epithelium and Fibers(Investigative Ophthalmology & Visual Science, 2025-04-16) Duot, Matthieu; Coomson, Sarah Y.; Shrestha, Sanjaya K.; Nagulla, M. V. Murali Krishna; Audic, Yann; Barve, Ruteja A.; Huang, Hongzhan; Gautier-Courteille, Carole; Paillard, Luc; Lachke, Salil A.Purpose: The lens transcriptome has been examined using microarrays and RNA-sequencing (RNA-seq). These omics data are the basis of the bioinformatics web-resource iSyTE that has identified new genes involved in lens development and cataract. The lens predominantly contains epithelial and fiber cells, and yet, presently, iSyTE is based on whole lens data. To gain cell-specific regulatory insights, we meta-analyzed isolated epithelium and fiber transcriptomes from embryonic/postnatal, adult and aged lenses. Methods: Mouse lens epithelium and fiber transcriptome public datasets at embryonic (E) and postnatal (P) stages E12.5, E14.5, E16.5, E18.5, P0.5, P0, P5, P13, and age one month, three months, six months, and two years were analyzed. Microarray or RNA-seq data were analyzed by appropriate methods and compared to other resources (e.g., Cat-Map, CompBio). Results: Across all RNA-seq datasets examined, 2466 genes are differentially expressed between epithelium and fibers, of which 106 are cataract-linked. Gene ontology enrichment validates epithelial and fiber expression, corroborating the meta-analysis. Whole embryonic-body–in silico subtraction and other analyses identify several new high-priority epithelial- and/or fiber-enriched genes (e.g., Casz1, Ell2). Furthermore, new insights into cell-specific regulatory processes at distinct stages are identified (e.g., ribonucleoprotein regulation in E12.5 epithelium). Finally, this data is made accessible at iSyTE (https://research.bioinformatics.udel.edu/iSyTE/). Conclusions: This spatiotemporal transcriptome meta-analysis comprehensively informs on epithelium- and fiber-specific regulatory processes in developing, adult and aged lenses. Notably, it includes the first description of an embryonic stage (i.e., E12.5) representing early primary fiber differentiation, thus informing on the initial transcriptome changes as lens cell-types are readily distinguishable.Item Lens Development: Bringing signaling complexity into focus(eLife, 2025-04-01) Coomson, Sarah Y.; Lachke, Salil A.Ask yourself, what is necessary for you to read this article? And how are you able to discern between individual words or letters? Evolution has solved these challenges by developing a complex organ, the eye, that is similar in many ways to a camera. It has a lens that focuses light onto a light-sensitive tissue, the retina, which captures this information and relays it to the brain. For the lens to be effective, it needs to be transparent, and complex genetic pathways have emerged to ensure that this happens during development. In vertebrates, the majority of the cells in the lens are fiber cells. Although the need for cross-talk between the lens and the retina during lens development was first noticed in 1901 (Spemann, 1901), the details of the impact of this cross-talk on the differentiation of lens cells did not become clear until the early 1960s. Reversing the orientation of a chick embryonic lens so that its anterior region faced the retina (rather than looking outwards) led to a dramatic cellular reorganization: the lens epithelium facing the retina differentiated into fiber cells, suggesting that a key signal from the retina drives this differentiation of lens cells (Coulombre and Coulombre, 1963). This signal was later revealed to be a protein called fibroblast growth factor (or FGF for short; Chamberlain and McAvoy, 1987), which, depending on its dosage, can induce epithelial cell proliferation or their differentiation into fiber cells in the lens (McAvoy and Chamberlain, 1989).Item Comparative analysis of rodent lens morphometrics and biomechanical properties(Frontiers in Ophthalmology, 2025-04-03) Cheheltani, Sepideh; Islam, Sadia T.; Malino, Heather; Abera, Kalekidan; Aryal, Sandeep; Forbes, Karen; Parreno, Justin; Fowler, Velia M.Introduction: Proper ocular lens function requires biomechanical flexibility, which is reduced during aging. As increasing lens size has been shown to correlate with lens biomechanical stiffness in aging, we tested the hypothesis that whole lens size determines gross biomechanical stiffness by comparing lenses of varying sizes from three rodent species (mice, rats, and guinea pigs). Methods: Coverslip compression assay was performed to measure whole lens biomechanics. Whole mount staining on fixed lenses, followed by confocal microscopy, was conducted to measure lens microstructures. Results: Among the three species, guinea pig lenses are the largest, rat lenses are smaller than guinea pig lenses, and mouse lenses are the smallest of the three. We found that rat and guinea pig lenses are stiffer than the much smaller mouse lenses. However, despite guinea pig lenses being larger than rat lenses, whole lens stiffness between guinea pigs and rats is not different. This refutes our hypothesis and indicates that lens size does not solely determine lens stiffness. We next compared lens microstructures, including nuclear size, capsule thickness, epithelial cell area, fiber cell widths, and suture organization between mice, rats, and guinea pigs. The lens nucleus is the largest in guinea pigs, followed by rats, and mice. However, the rat nucleus occupies a larger fraction of the lens. Both lens capsule thickness and fiber cell widths are the largest in guinea pigs, followed by mice and then rats. Epithelial cells are the largest in guinea pigs, and there are no differences between mice and rats. In addition, the lens suture shape appears similar across all three species. Discussion: Overall, our data indicates that whole lens size and microstructure morphometrics do not correlate with lens stiffness, indicating that factors contributing to lens biomechanics are complex and likely multifactorial.Item Actin Filament Pointed Ends: Assays for Regulation of Assembly and Disassembly by Tropomodulin and Tropomyosin(Cytoskeleton, 2025-02-24) Yamashiro, Sawako; Shekhar, Shashank; Novak, Stefanie M.; Biswas, Sudipta; Gregorio, Carol C.; Fowler, Velia M.Actin filaments are dynamic polymers whose length depends on regulated monomer association and dissociation at their ends. Actin barbed-end dynamics are relatively better understood, primarily due to the approximately tenfold faster subunit on/off rates at barbed versus pointed ends. We present experimental approaches to selectively assay actin pointed-end regulation using bulk biochemistry, single filament imaging, and live cell microscopy with an emphasis on tropomodulins (Tmods), a conserved family of eukaryotic proteins that specifically cap pointed ends. Average pointed-end assembly/disassembly rates are measured in bulk solution using pyrene-labeled actin and barbed end-capping protein CapZ. Direct rate measurements of individual pointed ends are performed via microfluidic-assisted total internal reflection fluorescence microscopy (mf-TIRF). Actin pointed-end dynamics in living cells are examined in striated muscle cells expressing fluorescent actin, where the regular arrays of 1- to 2-μm-long actin filaments in sarcomeres enable visualization of filament pointed and barbed ends. These assays will also help advance our understanding of other pointed end regulators, including cyclase-associated protein and leiomodins, which have been implicated in filament stabilization, disassembly, and elongation. This work is relevant to the musculoskeletal field, where precise regulation of filament lengths is particularly critical for sarcomere organization and striated muscle contraction.Item The Complexity and Significance of Fibroblast Growth Factor (FGF) Signaling for FGF-Targeted Cancer Therapies(Cancers, 2024-12-30) Nguyen, Anh L.; Facey, Caroline O. B.; Boman, Bruce M.Simple Summary Fibroblast growth factors (FGFs) and their receptors (FGFRs) play a crucial role in cancer development. However, due to their diverse cellular functions, the mechanisms by which they drive cancer are complex. In this review, we discuss the mechanisms of action of FGFs and FGFRs, as well as how the dysregulation of FGF signaling contributes to cancer in various tumor types. The complexity of FGF signaling is partly explained by the large number of FGF isoforms and FGFR receptor types. We provide a classification of FGF ligands based on their signaling modes and binding specificity to FGFRs. Additionally, we explore promising therapeutic strategies being developed to target FGF signaling in oncology, including small molecules, ligand traps, and monoclonal antibody-based inhibitors. Abstract Fibroblast growth factors (FGFs) have diverse functions in the regulation of cell proliferation and differentiation in development, tissue maintenance, wound repair, and angiogenesis. The goal of this review paper is to (i) deliberate on the role of FGFs and FGF receptors (FGFRs) in different cancers, (ii) present advances in FGF-targeted cancer therapies, and (iii) explore cell signaling mechanisms that explain how FGF expression becomes dysregulated during cancer development. FGF is often mutated and overexpressed in cancer and the different FGF and FGFR isoforms have unique expression patterns and distinct roles in different cancers. Among the FGF members, the FGF 15/19 subfamily is particularly interesting because of its unique protein structure and role in endocrine function. The abnormal expression of FGFs in different cancer types (breast, colorectal, hepatobiliary, bronchogenic, and others) is examined and correlated with patient prognosis. The classification of FGF ligands based on their mode of action, whether autocrine, paracrine, endocrine, or intracrine, is illustrated, and an analysis of the binding specificity of FGFs to FGFRs is also provided. Moreover, the latest advances in cancer therapeutic strategies involving small molecules, ligand traps, and monoclonal antibody-based FGF inhibitors are presented. Lastly, we discuss how the dysregulation of FGF and FGFR expression affects FGF signaling and its role in cancer development.Item Osteocyte Dendrites: How Do They Grow, Mature, and Degenerate in Mineralized Bone?(Cytoskeleton, 2024-12-09) Guerra, Rosa M.; Fowler, Velia M.; Wang, LiyunOsteocytes, the most abundant bone cells, form an extensive cellular network via interconnecting dendrites. Like neurons in the brain, the long-lived osteocytes perceive mechanical and biological inputs and signal to other effector cells, leading to the homeostasis and turnover of bone tissues. Despite the appreciation of osteocytes' vital roles in bone biology, the initiation, growth, maintenance, and eventual degradation of osteocyte dendrites are poorly understood due to their full encasement by mineralized matrix. With the advancement of imaging modalities and genetic models, the architectural organization and molecular composition of the osteocyte dendrites, as well as their morphological changes with aging and diseases, have begun to be revealed. However, several long-standing mysteries remain unsolved, including (1) how the dendrites are initiated and elongated when a surface osteoblast becomes embedded as an osteocyte; (2) how the dendrites maintain a relatively stable morphology during their decades-long life span; (3) what biological processes control the dendrite morphology, connectivity, and stability; and (4) if these processes are influenced by age, sex, hormones, and mechanical loading. Our review of long, thin actin filament (F-actin)-containing processes extending from other cells leads to a working model that serves as a starting point to investigate the formation and maintenance of osteocyte dendrites and their degradation with aging and diseases.Item Depth and Strain-Dependent Structural Responses of Mouse Lens Fiber Cells During Whole Lens Shape Changes(Investigative Ophthalmology & Visual Science, 2025-02-20) Cheheltani, Sepideh; Shihan, Mahbubul H.; Parreno, Justin; Biswas, Sondip K.; Lo, Woo-Kuen; Fowler, Velia M.Purpose: To study the relationship between whole lens shape changes and fiber cell responses to externally applied loads. Methods: Freshly dissected mouse lenses were compressed by applying glass coverslips to the lens anterior, followed by fixation to preserve lens shape, and preparation for scanning electron microscopy (SEM). SEM images were collected from the outer cortex to the nucleus, and fiber cell end-to-end curvature and membrane paddle dimensions were measured using ImageJ. Results: At 23% and 29% axial strain, cortical fiber bundle curvature increased significantly compared to control uncompressed lenses, whereas nuclear fiber bundle curvature was unaffected. Outer cortical fiber cell membrane paddles and protrusions were dramatically distorted in a radial direction, with loss of paddle-associated small protrusions in compressed lenses compared to controls, but nuclear fiber cell morphologies were unchanged. The compression-induced increases in cortical fiber cell curvature and distortion of membrane paddles were reversible, with fiber cell morphologies returning to those of control lenses after the release of load. Conclusions: Whole lens shape changes due to an increase in axial strain result in increased fiber cell curvature and distorted membrane morphologies in cortical but not nuclear fiber cells, indicating that mechanical strain dissipates with depth. The recovery of normal cortical fiber cell curvature and membrane morphologies after the removal of load and lens rounding back to its original shape suggests elastic properties of the young and mature fiber cells and their membrane paddles.Item Transcriptome Analysis of Post-Hatch Breast Muscle in Legacy and Modern Broiler Chickens Reveals Enrichment of Several Regulators of Myogenic Growth(PLoS ONE, 2015-03-30) Davis, Richard V. N.; Lamont, Susan J.; Rothschild, Max F.; Persia, Michael E.; Ashwell, Chris M.; Schmidt, Carl J.Agriculture provides excellent model systems for understanding how selective pressure, as applied by humans, can affect the genomes of plants and animals. One such system is modern poultry breeding in which intensive genetic selection has been applied for meat production in the domesticated chicken. As a result, modern meat-type chickens (broilers) exhibit enhanced growth, especially of the skeletal muscle, relative to their legacy counterparts. Comparative studies of modern and legacy broiler chickens provide an opportunity to identify genes and pathways affected by this human-directed evolution. This study used RNA-seq to compare the transcriptomes of a modern and a legacy broiler line to identify differentially enriched genes in the breast muscle at days 6 and 21 post-hatch. Among the 15,945 genes analyzed, 10,841 were expressed at greater than 0.1 RPKM. At day 6 post-hatch 189 genes, including several regulators of myogenic growth and development, were differentially enriched between the two lines. The transcriptional profiles between lines at day 21 post-hatch identify 193 genes differentially enriched and still include genes associated with myogenic growth. This study identified differentially enriched genes that regulate myogenic growth and differentiation between the modern and legacy broiler lines. Specifically, differences in the ratios of several positive (IGF1, IGF1R, WFIKKN2) and negative (MSTN, ACE) myogenic growth regulators may help explain the differences underlying the enhanced growth characteristics of the modern broilers.Item ClpS Directs Degradation of N-Degron Substrates With Primary Destabilizing Residues in Mycolicibacterium smegmatis(Molecular Microbiology, 2024-12-03) Presloid, Christopher J.; Jiang, Jialiu; Kandel, Pratistha; Anderson, Henry R.; Beardslee, Patrick C.; Swayne, Thomas M.; Schmitz, Karl R.Drug-resistant tuberculosis infections are a major threat to global public health. The essential mycobacterial ClpC1P1P2 protease has received attention as a prospective target for novel antibacterial therapeutics. However, efforts to probe its function in cells are constrained by our limited knowledge of its physiological proteolytic repertoire. Here, we interrogate the role of mycobacterial ClpS in directing N-degron pathway proteolysis by ClpC1P1P2 in Mycolicibacterium smegmatis. Binding assays demonstrate that mycobacterial ClpS binds canonical primary destabilizing residues (Leu, Phe, Tyr, Trp) with moderate affinity. N-degron binding restricts the conformational flexibility of a loop adjacent to the ClpS N-degron binding pocket and strengthens ClpS•ClpC1 binding affinity ~30-fold, providing a mechanism for cells to prioritize N-degron proteolysis when substrates are abundant. Proteolytic reporter assays in M. smegmatis confirm degradation of substrates bearing primary N-degrons, but suggest that secondary N-degrons are absent in mycobacteria. This work expands our understanding of the mycobacterial N-degron pathway and identifies ClpS as a critical component for substrate specificity, providing insights that may support the development of improved Clp protease inhibitors. Graphical Abstract available at: https://doi.org/10.1111/mmi.15334 Many bacteria possess an N-degron pathway that links the proteolytic stability of proteins to the identity of their N-terminal residue. Here, we report that Mycolicibacterium smegmatis possesses a physiological N-degron pathway that is less complex than those described in other bacteria.Item Actin Polymerization Status Regulates Tenocyte Homeostasis Through Myocardin-Related Transcription Factor-A(Cytoskeleton, 2024-11-27) West, Valerie C.; Owen, Kaelyn E.; Inguito, Kameron L.; Ebron, Karl Matthew M.; Reiner, Tori N.; Mirack, Chloe E.; Le, Christian H.; Marqueti, Rita de Cassia; Snipes, Steven; Mousavizadeh, Rouhollah; King, Rylee E.; Elliott, Dawn M.; Parreno, JustinThe actin cytoskeleton is a potent regulator of tenocyte homeostasis. However, the mechanisms by which actin regulates tendon homeostasis are not entirely known. This study examined the regulation of tenocyte molecule expression by actin polymerization via the globular (G-) actin-binding transcription factor, myocardin-related transcription factor-a (MRTF). We determined that decreasing the proportion of G-actin in tenocytes by treatment with TGFβ1 increases nuclear MRTF. These alterations in actin polymerization and MRTF localization coincided with favorable alterations to tenocyte gene expression. In contrast, latrunculin A increases the proportion of G-actin in tenocytes and reduces nuclear MRTF, causing cells to acquire a tendinosis-like phenotype. To parse out the effects of F-actin depolymerization from regulation by MRTF, we treated tenocytes with cytochalasin D. Exposure of cells to cytochalasin D increases the proportion of G-actin in tenocytes. However, as compared to latrunculin A, cytochalasin D has a differential effect on MRTF localization by increasing nuclear MRTF. This led to an opposing effect on the regulation of a subset of genes. The differential regulation of genes by latrunculin A and cytochalasin D suggests that actin signals through MRTF to regulate a specific subset of genes. By targeting the deactivation of MRTF through the inhibitor CCG1423, we verify that MRTF regulates Type I Collagen, Tenascin C, Scleraxis, and α-smooth muscle actin in tenocytes. Actin polymerization status is a potent regulator of tenocyte homeostasis through the modulation of several downstream pathways, including MRTF. Understanding the regulation of tenocyte homeostasis by actin may lead to new therapeutic interventions against tendinopathies, such as tendinosis.Item YAP/TAZ-associated cell signaling – at the crossroads of cancer and neurodevelopmental disorders(Frontiers in Cell and Developmental Biology, 2025-01-28) Ajongbolo, Aderonke O.; Langhans, Sigrid A.YAP/TAZ (Yes-associated protein/paralog transcriptional co-activator with PDZ-binding domain) are transcriptional cofactors that are the key and major downstream effectors of the Hippo signaling pathway. Both are known to play a crucial role in defining cellular outcomes, including cell differentiation, cell proliferation, and apoptosis. Aside from the canonical Hippo signaling cascade with the key components MST1/2 (mammalian STE20-like kinase 1/2), SAV1 (Salvador homologue 1), MOB1A/B (Mps one binder kinase activator 1A/B) and LATS1/2 (large tumor suppressor kinase 1/2) upstream of YAP/TAZ, YAP/TAZ activation is also influenced by numerous other signaling pathways. Such non-canonical regulation of YAP/TAZ includes well-known growth factor signaling pathways such as the epidermal growth factor receptor (EGFR)/ErbB family, Notch, and Wnt signaling as well as cell-cell adhesion, cell-matrix interactions and mechanical cues from a cell’s microenvironment. This puts YAP/TAZ at the center of a complex signaling network capable of regulating developmental processes and tissue regeneration. On the other hand, dysregulation of YAP/TAZ signaling has been implicated in numerous diseases including various cancers and neurodevelopmental disorders. Indeed, in recent years, parallels between cancer development and neurodevelopmental disorders have become apparent with YAP/TAZ signaling being one of these pathways. This review discusses the role of YAP/TAZ in brain development, cancer and neurodevelopmental disorders with a special focus on the interconnection in the role of YAP/TAZ in these different conditions.Item Separation of Avian Preovulatory Follicle Granulosa and Theca Cell Layers for Downstream Applications(Journal of Visualized Experiments, 2024-10-24) Kramer, Ashley E.; Ellwood, Kathryn M.; Brannick, Erin M.; Dutta, AdityaSummary Here, we describe a protocol for separating yolk, granulosa cells, and theca cells in avian preovulatory follicles. This precision handling enables critical investigations into the role of these layers in reproductive function, aiding the understanding of follicular development, hormonal regulation, and disease research for enhanced agricultural yield and biomedical insights. Abstract Layer hens (egg-laying chickens) and broiler breeders (breeding stock for meat-producing chickens) are crucial to the world's food supply as a reliable source of protein. They are also an emerging animal model for the study of human reproductive disease. As the field of poultry research develops, the health and function of the layer hen and broiler breeder ovary will be an important point of study for both agricultural and biomedical researchers. One of the challenges presented by this emerging interest is the need for replicable techniques that all researchers can employ in ovarian specimen collection. In particular, a detailed visual process must be established to define the proper separation of the specialized granulosa and theca cell layers from hen follicles to achieve agreement and consistency among researchers. This study describes the extraction of preovulatory follicles and ovary tissue in white leghorn hens of prime reproductive age. The separation of these follicles is performed under cold, liquid conditions to congeal the yolk for easier manipulation and to prevent the follicle's own weight from tearing apart cell layers during the separation process. Once the separation is complete, the desired cell layers can be further digested for tissue culture approaches or can be cryopreserved for genomic and proteomic analyses.Item Cross-species regulatory network analysis identifies FOXO1 as a driver of ovarian follicular recruitment(Scientific Reports, 2024-12-28) Kramer, Ashley E.; Berral-González, Alberto; Ellwood, Kathryn M.; Ding, Shanshan; De Las Rivas, Javier; Dutta, AdityaThe transcriptional regulation of gene expression in the latter stages of follicular development in laying hen ovarian follicles is not well understood. Although differentially expressed genes (DEGs) have been identified in pre-recruitment and pre-ovulatory stages, the master regulators driving these DEGs remain unknown. This study addresses this knowledge gap by utilizing Master Regulator Analysis (MRA) combined with the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) for the first time in laying hen research to identify master regulators that are controlling DEGs in pre-recruitment and pre-ovulatory phases. The constructed ARACNe network included 10,466 nodes and 292,391 edges. The ARACNe network was then used in conjunction with the Virtual Inference of Protein-activity by Enriched Regulon (VIPER) for the MRA to identify top up- and down-regulated master regulators. VIPER analysis revealed FOXO1 as a master regulator, influencing 275 DEGs and impacting pathways related to apoptosis, proliferation, and hormonal regulation. Additionally, CLOCK, known as a crucial regulator of circadian rhythm, emerged as an upregulated master regulator in the pre-ovulatory stage. These findings provide new insights into the transcriptional landscape of laying hen ovarian follicles, offering a foundation for further exploration of follicle development and enhancing reproductive efficiency in avian species.Item Transcriptomic data reveals MYC as an upstream regulator in laying hen follicular recruitment(Poultry Science, 2024-11-24) Kramer, Ashley E.; Ellwood, Kathryn M.; Guarino, Nicole; Li, Cong-Jun; Dutta, AdityaUnderstanding the mechanisms of follicular recruitment is essential for improving laying hen and broiler breeder productivity, as it directly influences egg production. Despite advancements in poultry breeding for enhanced egg production, the factors driving successful ovarian follicle maturation remain inadequately understood. This study investigates the genetic drivers mediating the transition of pre-recruitment follicles to the pre-ovulatory phase, a crucial stage before ovulation. Using RNA sequencing and bioinformatics approaches such as a differential gene expression analysis, we compared pre-recruitment follicles with the recently recruited F5 pre-ovulatory follicle to identify key genes and upstream regulators involved in this transition. Further validation through qRT-PCR confirmed these findings. Using Qiagen's Ingenuity Pathway Analysis we identified MYC proto-oncogene (C-Myc) as a pivotal upstream regulator, controlling genes essential for cell proliferation and differentiation. Additionally, TGFβ1 emerged as a key regulator, influencing pathways involving SMAD3, TNF, and TP53. The study highlights the intricate regulatory network involving MYC and other transcription factors such as CTNNB1, crucial for follicular development. These findings provide valuable insights into the molecular mechanisms governing follicular selection and maturation, which are essential for enhancing egg production efficiency. Future research should explore the roles of MYC, CTNNB1, and other driver genes in follicular development to further understand and improve reproductive efficiency in poultry.