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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.Item The Significance of Aldehyde Dehydrogenase 1 in Cancers(International Journal of Molecular Sciences, 2024-12-30) Nguyen, Anh L.; Facey, Caroline O. B.; Boman, Bruce M.The goal of this paper is to discuss the role of ALDH isozymes in different cancers, review advances in ALDH1-targeting cancer therapies, and explore a mechanism that explains how ALDH expression becomes elevated during cancer development. ALDH is often overexpressed in cancer, and each isoform has a unique expression pattern and a distinct role in different cancers. The abnormal expression of ALDHs in different cancer types (breast, colorectal, lung, gastric, cervical, melanoma, prostate, and renal) is presented and correlated with patient prognosis. ALDH plays a significant role in various cellular functions, such as metabolism, oxidative stress response, detoxification, and cellular differentiation. Among the ALDH families, ALDH1 has gained considerable attention as a cancer stem cell (CSC) marker due to its significant role in the maintenance of stemness and the differentiation of stem cells (SCs), along with its involvement in tumorigenesis. A description of the cellular mechanisms and physiology of ALDH1 that underlies cancer development is provided. Moreover, current advances in ALDH1-targeting cancer therapies are discussed.Item The Meq Genes of Nigerian Marek’s Disease Virus (MDV) Field Isolates Contain Mutations Common to Both European and US High Virulence Strains(Viruses, 2024-12-31) Patria, Joseph N.; Jwander, Luka; Mbachu, Ifeoma; Parcells, Levi; Ladman, Brian; Trimpert, Jakob; Kaufer, Benedikt B.; Tavlarides-Hontz, Phaedra; Parcells, Mark S.Background: Marek’s disease (MD) is a pathology affecting chickens caused by Marek’s disease virus (MDV), an acute transforming alphaherpesvirus of the genus Mardivirus. MD is characterized by paralysis, immune suppression, and the rapid formation of T-cell (primarily CD4+) lymphomas. Over the last 50 years, losses due to MDV infection have been controlled worldwide through vaccination; however, these live-attenuated vaccines are non-sterilizing and potentially contributed to the virulence evolution of MDV field strains. Mutations common to field strains that can overcome vaccine protection were identified in the C-terminal proline-rich repeats of the oncoprotein Meq (Marek’s EcoRI-Q-encoded protein). These mutations in meq have been found to be distinct to their region of origin, with high virulence strains obtained in Europe differing from those having evolved in the US. The present work reports on meq mutations identified in MDV field strains in Nigeria, arising at farms employing different vaccination practices. Materials and Methods: DNA was isolated from FTA cards obtained at 12 farms affected by increased MD in the Plateau State, Nigeria. These sequences included partial whole genomes as well as targeted sequences of the meq oncogenes from these strains. Several of the meq genes were cloned for expression and their localization ability to interact with the chicken NF-IL3 protein, a putative Meq dimerization partner, were assessed. Results: Sequence analysis of the meq genes from these Nigerian field strains revealed an RB1B-like lineage co-circulating with a European Polen5-like lineage, as well as recombinants harboring a combination of these mutations. In a number of these isolates, Meq mutations accumulated in both N-terminal and C-terminal domains. Discussion: Our data, suggest a direct effect of the vaccine strategy on the selection of Meq mutations. Moreover, we posit the evolution of the next higher level of virulence MDVs, a very virulent plus plus pathotype (vv++).Item Soybean Bradyrhizobium spp. Spontaneously Produce Abundant and Diverse Temperate Phages in Culture(Viruses, 2024-11-07) Richards, Vanessa A.; Ferrell, Barbra D.; Polson, Shawn W.; Wommack, K. Eric; Fuhrmann, Jeffry J.Soybean bradyrhizobia (Bradyrhizobium spp.) are symbiotic root-nodulating bacteria that fix atmospheric nitrogen for the host plant. The University of Delaware Bradyrhizobium Culture Collection (UDBCC; 353 accessions) was created to study the diversity and ecology of soybean bradyrhizobia. Some UDBCC accessions produce temperate (lysogenic) bacteriophages spontaneously under routine culture conditions without chemical or other apparent inducing agents. Spontaneous phage production may promote horizontal gene transfer and shape bacterial genomes and associated phenotypes. A diverse subset (n = 98) of the UDBCC was examined for spontaneously produced virus-like particles (VLPs) using epifluorescent microscopy, with a majority (69%) producing detectable VLPs (>1 × 107 mL−1) in laboratory culture. Phages from the higher-producing accessions (>2.0 × 108 VLP mL−1; n = 44) were examined using transmission electron microscopy. Diverse morphologies were observed, including various tail types and lengths, capsid sizes and shapes, and the presence of collars or baseplates. In many instances, putative extracellular vesicles of a size similar to virions were also observed. Three of the four species examined (B. japonicum, B. elkanii, and B. diazoefficiens) produced apparently tailless phages. All species except B. ottawaense also produced siphovirus-like phages, while all but B. diazoefficiens additionally produced podovirus-like phages. Myovirus-like phages were restricted to B. japonicum and B. elkanii. At least three strains were polylysogens, producing up to three distinct morphotypes. These observations suggest spontaneously produced phages may play a significant role in the ecology and evolution of soybean bradyrhizobia.Item A long-term high-fat diet induces differential gene expression changes in spatially distinct adipose tissue of male mice(Physiological Genomics, 2024-11-11) Alradi, Malak; Askari, Hassan; Shaw, Mark; Bhavsar, Jaysheel D.; Kingham, Brewster F.; Polson, Shawn W.; Fancher, Ibra S.The accumulation of visceral adipose tissue (VAT) is strongly associated with cardiovascular disease and diabetes. In contrast, individuals with increased subcutaneous adipose tissue (SAT) without corresponding increases in VAT are associated with a metabolic healthy obese phenotype. These observations implicate dysfunctional VAT as a driver of disease processes, warranting investigation into obesity-induced alterations of distinct adipose depots. To determine the effects of obesity on adipose gene expression, male mice (n = 4) were fed a high-fat diet to induce obesity or a normal laboratory diet (lean controls) for 12–14 mo. Mesenteric VAT and inguinal SAT were isolated for bulk RNA sequencing. AT from lean controls served as a reference to obesity-induced changes. The long-term high-fat diet induced the expression of 169 and 814 unique genes in SAT and VAT, respectively. SAT from obese mice exhibited 308 differentially expressed genes (164 upregulated and 144 downregulated). VAT from obese mice exhibited 690 differentially expressed genes (262 genes upregulated and 428 downregulated). KEGG pathway and GO analyses revealed that metabolic pathways were upregulated in SAT versus downregulated in VAT while inflammatory signaling was upregulated in VAT. We next determined common genes that were differentially regulated between SAT and VAT in response to obesity and identified four genes that exhibited this profile: elovl6 and kcnj15 were upregulated in SAT/downregulated in VAT while trdn and hspb7 were downregulated in SAT/upregulated in VAT. We propose that these genes in particular should be further pursued to determine their roles in SAT versus VAT with respect to obesity. NEW & NOTEWORTHY A long-term high-fat diet induced the expression of more than 980 unique genes across subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). The high-fat diet also induced the differential expression of nearly 1,000 AT genes. We identified four genes that were oppositely expressed in SAT versus VAT in response to the high-fat diet and propose that these genes in particular may serve as promising targets aimed at resolving VAT dysfunction in obesity.Item Engineered and hybrid human megakaryocytic extracellular vesicles for targeted non-viral cargo delivery to hematopoietic (blood) stem and progenitor cells(Frontiers in Bioengineering and Biotechnology, 2024-09-24) Das, Samik; Thompson, Will; Papoutsakis, Eleftherios TerryNative and engineered extracellular vesicles generated from human megakaryocytes (huMkEVs) or from the human megakaryocytic cell line CHRF (CHEVs) interact with tropism delivering their cargo to both human and murine hematopoietic stem and progenitor cells (HSPCs). To develop non-viral delivery vectors to HSPCs based on MkEVs, we first confirmed, using NOD-scid IL2Rγnull (NSG™) mice, the targeting potential of the large EVs, enriched in microparticles (huMkMPs), chosen for their large cargo capacity. 24 h post intravenous infusion into NSG mice, huMkEVs induced a nearly 50% increase in murine platelet counts. PKH26-labeled huMkEVs or CHEVs localized to the HSPC-rich bone marrow preferentially interacting with murine HSPCs, thus confirming their receptor-mediated tropism for NSG HSPCs, and their potential to treat thromobocytopenias. We explored this tropism to functionally deliver synthetic cargo, notably plasmid DNA coding for a fluorescent reporter, to NSG HSPCs both in vitro and in vivo. We loaded huMkEVs with plasmid DNA either through electroporation or by generating hybrid particles with preloaded liposomes. Both methods facilitated successful functional targeted delivery of pDNA, as tissue weight-normalized fluorescence intensity of the expressed fluorescent reporter was significantly higher in bone marrow than other tissues. Furthermore, the fraction of fluorescent CD117+ HSPCs was nearly 19-fold higher than other cell types within the bone marrow 72-h following administration of the hybrid particles, further supporting that HSPC tropism is retained when using hybrid particles. These data demonstrate the potential of these EVs as a non-viral, HSPC-specific cargo vehicle for gene therapy applications to treat hematological diseases. Graphical abstract available at: https://doi.org/10.3389/fbioe.2024.1435228Item Helicobacter pylori and Campylobacter jejuni bacterial holocytochrome c synthase structure-function analysis reveals conservation of heme binding(Communications Biology, 2024-08-13) Yeasmin, Tania; Carroll, Susan C.; Hawtof, David J.; Sutherland, Molly C.Heme trafficking is essential for cellular function, yet mechanisms of transport and/or heme interaction are not well defined. The System I and System II bacterial cytochrome c biogenesis pathways are developing into model systems for heme trafficking due to their functions in heme transport, heme stereospecific positioning, and mediation of heme attachment to apocytochrome c. Here we focus on the System II pathway, CcsBA, that is proposed to be a bi-functional heme transporter and holocytochrome c synthase. An extensive structure-function analysis of recombinantly expressed Helicobacter pylori and Campylobacter jejuni CcsBAs revealed key residues required for heme interaction and holocytochrome c synthase activity. Homologous residues were previously identified to be required for heme interaction in Helicobacter hepaticus CcsBA. This study provides direct, biochemical evidence that mechanisms of heme interaction are conserved, leading to the proposal that the CcsBA WWD heme-handling domain represents a novel target for therapeutics.Item Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior(Journal of Visualized Experiments, 2023-05-26) Pastorino, Nicole G.; Tomatsu, Saori; Lin, Amy; Doerr, Jackson; Waterman, Zachary; Sershen, Krisztina; Ray, Pulak; Rodriguez, Analiz; Galileo, Deni S.Summary Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis. Abstract The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 µm) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 µm) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.Item Mutations in tyrosyl-DNA phosphodiesterase 2 suppress top-2 induced chromosome segregation defects during Caenorhabditis elegans spermatogenesis(Journal of Biological Chemistry, 2024-06-28) Kwah, Ji Kent; Bhandari, Nirajan; Rourke, Christine; Gassaway, Gabriella; Jaramillo-Lambert, AimeeMeiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5′-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.Item Development of an efficient, effective, and economical technology for proteome analysis(Cell Reports: Methods, 2024-06-11) Martin, Katherine R.; Le, Ha T.; Abdelgawad, Ahmed; Yang, Canyuan; Lu, Guotao; Keffer, Jessica L.; Zhang, Xiaohui; Zhuang, Zhihao; Asare-Okai, Papa Nii; Chan, Clara S.; Batish, Mona; Yu, YanbaoHighlights • Rapid, robust, and cost-effective alternative to proteomics sample preparation • Versatile filter devices can meet a wide range of proteomics analysis needs • On-filter in-cell digestion facilitates low-input proteomics • Ready-to-go E3 and E4 filter devices are available Motivation Conventional proteomics sample processing methods often have high technical barriers to broad biomedical scientists, leading to difficulties for quick adoption and standardization. Existing protocols are also typically associated with costly reagents and accessories, making them less feasible for resource-limited settings as well as for clinical proteomics and/or core facilities where large numbers of samples are usually processed. Thus, there is a strong unmet need for an easy-to-use, reliable, and low-cost approach for general proteomics sample preparation. Summary We present an efficient, effective, and economical approach, named E3technology, for proteomics sample preparation. By immobilizing silica microparticles into the polytetrafluoroethylene matrix, we develop a robust membrane medium, which could serve as a reliable platform to generate proteomics-friendly samples in a rapid and low-cost fashion. We benchmark its performance using different formats and demonstrate them with a variety of sample types of varied complexity, quantity, and volume. Our data suggest that E3technology provides proteome-wide identification and quantitation performance equivalent or superior to many existing methods. We further propose an enhanced single-vessel approach, named E4technology, which performs on-filter in-cell digestion with minimal sample loss and high sensitivity, enabling low-input and low-cell proteomics. Lastly, we utilized the above technologies to investigate RNA-binding proteins and profile the intact bacterial cell proteome. Graphical abstract available at: https://doi.org/10.1016/j.crmeth.2024.100796Item Chicken intestinal organoids: a novel method to measure the mode of action of feed additives(Frontiers in Immunology, 2024-05-20) Mitchell, Jordan; Sutton, Kate; Elango, Jeyashree Nathan; Borowska, Dominika; Perry, Famatta; Lahaye, Ludovic; Santin, Elizabeth; Arsenault, Ryan J.; Vervelde, LonnekeThere is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or “mini-guts” has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.Item Shared genetic risk between major orofacial cleft phenotypes in an African population(Genetic Epidemiology, 2024-04-18) Alade, Azeez; Peter, Tabitha; Busch, Tamara; Awotoye, Waheed; Anand, Deepti; Abimbola, Oladayo; Aladenika, Emmanuel; Olujitan, Mojisola; Rysavy, Oscar; Nguyen, Phuong Fawng; Naicker, Thirona; Mossey, Peter A.; Gowans, Lord J. J.; Eshete, Mekonen A.; Adeyemo, Wasiu L.; Zeng, Erliang; Van Otterloo, Eric; O'Rorke, Michael; Adeyemo, Adebowale; Murray, Jeffrey C.; Lachke, Salil A.; Romitti, Paul A.; Butali, AzeezNonsyndromic orofacial clefts (NSOFCs) represent a large proportion (70%–80%) of all OFCs. They can be broadly categorized into nonsyndromic cleft lip with or without cleft palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO). Although NSCL/P and NSCPO are considered etiologically distinct, recent evidence suggests the presence of shared genetic risks. Thus, we investigated the genetic overlap between NSCL/P and NSCPO using African genome-wide association study (GWAS) data on NSOFCs. These data consist of 814 NSCL/P, 205 NSCPO cases, and 2159 unrelated controls. We generated common single-nucleotide variants (SNVs) association summary statistics separately for each phenotype (NSCL/P and NSCPO) under an additive genetic model. Subsequently, we employed the pleiotropic analysis under the composite null (PLACO) method to test for genetic overlap. Our analysis identified two loci with genome-wide significance (rs181737795 [p = 2.58E−08] and rs2221169 [p = 4.5E−08]) and one locus with marginal significance (rs187523265 [p = 5.22E−08]). Using mouse transcriptomics data and information from genetic phenotype databases, we identified MDN1, MAP3k7, KMT2A, ARCN1, and VADC2 as top candidate genes for the associated SNVs. These findings enhance our understanding of genetic variants associated with NSOFCs and identify potential candidate genes for further exploration.Item Modeling Cataract Surgery in Mice(Journal of Visualized Experiments, 2023-12-01) O'Neill, Leah M.; Wang, Yan; Duncan, Melinda K.Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.Item Recessive TMOD1 mutation causes childhood cardiomyopathy(Communications Biology, 2024-01-02) Vasilescu, Catalina; Colpan, Mert; Ojala, Tiina H.; Manninen, Tuula; Mutka, Aino; Ylänen, Kaisa; Rahkonen, Otto; Poutanen, Tuija; Martelius, Laura; Kumari, Reena; Hinterding, Helena; Brilhante, Virginia; Ojanen, Simo; Lappalainen, Pekka; Koskenvuo, Juha; Carroll, Christopher J.; Fowler, Velia M.; Gregorio, Carol C.; Suomalainen, AnuFamilial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant’s effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.Item Structure-function analysis of the heme-binding WWD domain in the bacterial holocytochrome c synthase, CcmFH(mBio, 2023-11-06) Grunow, Amber L.; Carroll, Susan C.; Kreiman, Alicia N.; Sutherland, Molly C.Heme trafficking is a fundamental biological process, yet its direct study has been hampered due to heme’s tight intracellular regulation, heme cytotoxicity, and the transient nature of trafficking. The bacterial System I and System II cytochrome c biogenesis pathways are developing into models to interrogate heme trafficking mechanisms, as they function to transport heme from inside to outside the cell for attachment to apocytochrome c. Cytochromes c require heme for folding and to function in the context of electron transport chains for critical cellular functions, such as respiration. We focus on System I, comprised of eight membrane proteins, CcmABCDEFGH, proposed to function in two steps: CcmABCD mediates the transfer of heme and attachment to CcmE. HoloCcmE chaperones heme to CcmFH for attachment to apocytochrome c. While CcmFH is known to be the holocytochrome c synthase, the mechanism of heme interaction and positioning for attachment to apocytochrome c remains to be elucidated. A comprehensive structure-function analysis of the conserved WWD domain in CcmF was undertaken utilizing alanine-scanning and cysteine-scanning, revealing residues critical for CcmF’s synthase function and residues required for interaction with the 2- and 4-vinyls of heme. This analysis demonstrates for the first time that the CcmF WWD domain directly interacts with heme and that heme interactions within this domain are required for attachment to apocytochrome c. This in-depth interrogation of heme binding now allows for comparison across cytochrome c biogenesis proteins CcmF, CcmC, and CcsBA, revealing common mechanisms of heme interaction in these heme trafficking pathways. IMPORTANCE Heme is an essential co-factor for proteins involved with critical cellular functions, such as energy production and oxygen transport. Thus, understanding how heme interacts with proteins and is moved through cells is a fundamental biological question. This work studies the System I cytochrome c biogenesis pathway, which in some species (including Escherichia coli) is composed of eight integral membrane or membrane-associated proteins called CcmA-H that are proposed to function in two steps to transport and attach heme to apocytochrome c. Cytochrome c requires this heme attachment to function in electron transport chains to generate cellular energy. A conserved WWD heme-handling domain in CcmFH is analyzed and residues critical for heme interaction and holocytochrome c synthase activity are identified. CcmFH is the third member of the WWD domain-containing heme-handling protein family to undergo a comprehensive structure-function analysis, allowing for comparison of heme interaction across this protein family.Item Fascination with Fluctuation: Luria and Delbrück’s Legacy(Axioms, 2023-03-07) Robeva, Raina S.; Jungck, John R.While Luria and Delbrück’s seminal work has found its way to some college biology textbooks, it is now largely absent from those in mathematics. This is a significant omission, and we consider it a missed opportunity to present a celebrated conceptual model that provides an authentic and, in many ways, intuitive example of the quantifiable nature of stochasticity. We argue that it is an important topic that could enrich the educational literature in mathematics, from the introductory to advanced levels, opening many doors to undergraduate research. The paper has two main parts. First, we present in detail the mathematical theory behind the Luria–Delbrück model and make suggestions for further readings from the literature. We also give ideas for inclusion in various mathematics courses and for projects that can be used in regular courses, independent projects, or as starting points for student research. Second, we briefly review available hands-on activities as pedagogical ways to facilitate problem posing, problem-based learning, and investigative case-based learning and to expose students to experiments leading to Poisson distributions. These help students with even limited mathematics backgrounds understand the significance of Luria–Delbrück’s work for determining mutation rates and its impact on many fields, including cancer chemotherapy, antibiotic resistance, radiation, and environmental screening for mutagens and teratogens.