Open Access Publications - Department of Biological Sciences

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Open access publications by faculty, postdocs, and graduate students in the Department of Biological Sciences

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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.
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.
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.
Osteocyte Dendrites: How Do They Grow, Mature, and Degenerate in Mineralized Bone?
(Cytoskeleton, 2024-12-09) Guerra, Rosa M.; Fowler, Velia M.; Wang, Liyun
Osteocytes, 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.
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.
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.
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.
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, Justin
The 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.
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.
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, Aditya
Summary 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.
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, Aditya
The 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.
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, Aditya
Understanding 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.
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.
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++).
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.
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.
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 Terry
Native 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.2024.1435228
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.
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.
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, Aimee
Meiosis 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.

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