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

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    Megakaryocyte membrane-wrapped nanoparticles for targeted cargo delivery to hematopoietic stem and progenitor cells
    (Bioengineering and Translational Medicine, 2022-11-29) Das, Samik; Harris, Jenna C.; Winter, Erica J.; Kao, Chen-Yuan; Day, Emily S.; Papoutsakis, Eleftherios Terry
    Hematopoietic stem and progenitor cells (HSPCs) are desirable targets for gene therapy but are notoriously difficult to target and transfect. Existing viral vector-based delivery methods are not effective in HSPCs due to their cytotoxicity, limited HSPC uptake and lack of target specificity (tropism). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are attractive, nontoxic carriers that can encapsulate various cargo and enable its controlled release. To engineer PLGA NP tropism for HSPCs, megakaryocyte (Mk) membranes, which possess HSPC-targeting moieties, were extracted and wrapped around PLGA NPs, producing MkNPs. In vitro, fluorophore-labeled MkNPs are internalized by HSPCs within 24 h and were selectively taken up by HSPCs versus other physiologically related cell types. Using membranes from megakaryoblastic CHRF-288 cells containing the same HSPC-targeting moieties as Mks, CHRF-wrapped NPs (CHNPs) loaded with small interfering RNA facilitated efficient RNA interference upon delivery to HSPCs in vitro. HSPC targeting was conserved in vivo, as poly(ethylene glycol)–PLGA NPs wrapped in CHRF membranes specifically targeted and were taken up by murine bone marrow HSPCs following intravenous administration. These findings suggest that MkNPs and CHNPs are effective and promising vehicles for targeted cargo delivery to HSPCs.
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    Stress deprivation of tendon explants or Tpm3.1 inhibition in tendon cells reduces F-actin to promote a tendinosis-like phenotype
    (Molecular Biology of the Cell, 2022-12-01) Inguito, Kameron L.; Schofield, Mandy M.; Faghri, Arya D.; Bloom, Ellen T.; Heino, Marissa; West, Valerie C.; Ebron, Karl Matthew M.; Elliot, Dawn M.; Parreno, Justin
    Actin is a central mediator between mechanical force and cellular phenotype. In tendons, it is speculated that mechanical stress deprivation regulates gene expression by reducing filamentous (F)-actin. However, the mechanisms regulating tenocyte F-actin remain unclear. Tropomyosins (Tpms) are master regulators of F-actin. There are more than 40 Tpm isoforms, each having the unique capability to stabilize F-actin subpopulations. We investigated F-actin polymerization in stress-deprived tendons and tested the hypothesis that stress fiber–associated Tpm(s) stabilize F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon down-regulated tenogenic and up-regulated protease (matrix metalloproteinase-3) mRNA levels. Concomitant with mRNA modulation were increases in G/F-actin, confirming reduced F-actin by tendon stress deprivation. To investigate the molecular regulation of F-actin, we identified that tail, Achilles, and plantaris tendons express three isoforms in common: Tpm1.6, 3.1, and 4.2. Tpm3.1 associates with F-actin in native and primary tenocytes. Tpm3.1 inhibition reduces F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression in mouse and human tenocytes. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin regulation in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.
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    Coupling Novel Probes with Molecular Localization Microscopy Reveals Cell Wall Homeostatic Mechanisms in Staphylococcus aureus
    (ACS Chemical Biology, 2022-11-22) Lund, Victoria A.; Gangotra, Haneesh; Zhao, Zhen; Sutton, Joshua A. F.; Wacnik, Katarzyna; DeMeester, Kristen; Liang, Hai; Santiago, Cintia; Grimes, Catherine Leimkuhler; Jones, Simon; Foster, Simon J.
    Bacterial cell wall peptidoglycan is essential for viability, and its synthesis is targeted by antibiotics, including penicillin. To determine how peptidoglycan homeostasis controls cell architecture, growth, and division, we have developed novel labeling approaches. These are compatible with super-resolution fluorescence microscopy to examine peptidoglycan synthesis, hydrolysis, and the localization of the enzymes required for its biosynthesis (penicillin binding proteins (PBPs)). Synthesis of a cephalosporin-based fluorescent probe revealed a pattern of PBPs at the septum during division, supporting a model of dispersed peptidoglycan synthesis. Metabolic and hydroxylamine-based probes respectively enabled the synthesis of glycan strands and associated reducing termini of the peptidoglycan to be mapped. Foci and arcs of reducing termini appear as a result of both synthesis of glycan strands and glucosaminidase activity of the major peptidoglycan hydrolase, SagB. Our studies provide molecular level details of how essential peptidoglycan dynamics are controlled during growth and division.
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    Sex-related external factors influence pulmonary vascular angiogenesis in a sex-dependent manner
    (American Journal of Physiology - Heart and Circulatory Physiology, 2023-01-01) Hayward-Piatkovskyi, Brielle; Gonyea, Cailin R.; Pyle, Sienna C.; Lingappan, Krithika; Gleghorn, Jason P.
    Bronchopulmonary dysplasia (BPD) is a disease with a significant sexual dimorphism where males have a disadvantage compared with their female counterparts. Although mechanisms behind this sexual dimorphism are poorly understood, sex differences in angiogenesis have been identified as one possible source of the male disadvantage in BPD. Pulmonary angiogenesis was assessed in vitro using a bead sprouting assay with pooled male or female human pulmonary microvascular endothelial cells (HPMECs, 18–19 wk gestation, canalicular stage of human lung development) in standard (sex-hormone containing) and hormone-stripped medium. We identified sex-specific phenotypes in angiogenesis where male HPMECs produce fewer but longer sprouts compared with female HPMECs. The presence of sex hormones from standard culture medium modifies the male HPMEC phenotype with shorter and fewer sprouts but does not influence the female phenotype. Using a conditioned medium model, we further characterized the influence of the sex-specific secretome. Male and female HPMECs secrete factors that increase the maximum length of sprouts in female, but not male HPMECs. The presence of sex hormones abolishes this response. The male HPMEC secretome inhibits angiogenic sprouting in male HPMECs in the absence of sex hormones. Taken together, these results demonstrate that the pulmonary endothelial cell phenotypes are influenced by sex hormones and sex-specific secreted factors in a sex-dependent manner. NEW & NOTEWORTHY We identified a sex-specific phenotype wherein male HPMECs produce fewer but longer sprouts than females. Surprisingly, the presence of sex hormones only modifies the male phenotype, resulting in shorter and even fewer sprouts. Furthermore, we found the sex-specific secretome has a sex-dependent influence on angiogenesis that is also sex-hormone sensitive. These new and surprising findings point to the unappreciated role of sex and sex-related exogenous factors in early developmental angiogenesis.
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    microRNA-124 regulates Notch and NeuroD1 to mediate transition states of neuronal development
    (Developmental Neurobiology, 2022-11-23) Konrad, Kalin D.; Song, Jia L.
    MicroRNAs regulate gene expression by destabilizing target mRNA and/or inhibiting translation in animal cells. The ability to mechanistically dissect miR-124′s function during specification, differentiation, and maturation of neurons during development within a single system has not been accomplished. Using the sea urchin embryo, we take advantage of the manipulability of the embryo and its well-documented gene regulatory networks (GRNs). We incorporated NeuroD1 as part of the sea urchin neuronal GRN and determined that miR-124 inhibition resulted in aberrant gut contractions, swimming velocity, and neuronal development. Inhibition of miR-124 resulted in an increased number of cells expressing transcription factors (TFs) associated with progenitor neurons and a concurrent decrease of mature and functional neurons. Results revealed that in the early blastula/gastrula stages, miR-124 regulates undefined factors during neuronal specification and differentiation. In the late gastrula/larval stages, miR-124 regulates Notch and NeuroD1 during the transition between neuronal differentiation and maturation. Overall, we have improved the neuronal GRN and identified miR-124 to play a prolific role in regulating various transitions of neuronal development.
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