Scholarly Works, Mechanical Engineering
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Browsing Scholarly Works, Mechanical Engineering by Department "Biological Sciences"
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- Dynamical Localization of DivL and PleC in the Asymmetric Division Cycle of Caulobacter crescentus: A Theoretical Investigation of Alternative ModelsSubramanian, Kartik; Paul, Mark R.; Tyson, John J. (PLOS, 2015-07-01)Cell-fate asymmetry in the predivisional cell of Caulobacter crescentus requires that the regulatory protein DivL localizes to the new pole of the cell where it up-regulates CckA kinase, resulting in a gradient of CtrA~P across the cell. In the preceding stage of the cell cycle (the “stalked” cell), DivL is localized uniformly along the cell membrane and maintained in an inactive form by DivK~P. It is unclear how DivL overcomes inhibition by DivK~P in the predivisional cell simply by changing its location to the new pole. It has been suggested that co-localization of DivL with PleC phosphatase at the new pole is essential to DivL’s activity there. However, there are contrasting views on whether the bifunctional enzyme, PleC, acts as a kinase or phosphatase at the new pole. To explore these ambiguities, we formulated a mathematical model of the spatiotemporal distributions of DivL, PleC and associated proteins (DivJ, DivK, CckA, and CtrA) during the asymmetric division cycle of a Caulobacter cell. By varying localization profiles of DivL and PleC in our model, we show how the physiologically observed spatial distributions of these proteins are essential for the transition from a stalked cell to a predivisional cell. Our simulations suggest that PleC is a kinase in predivisional cells, and that, by sequestering DivK~P, the kinase form of PleC enables DivL to be reactivated at the new pole. Hence, co-localization of PleC kinase and DivL is essential to establishing cellular asymmetry. Our simulations reproduce the experimentally observed spatial distribution and phosphorylation status of CtrA in wild-type and mutant cells. Based on the model, we explore novel combinations of mutant alleles, making predictions that can be tested experimentally.
- Heparin-based hydrogel scaffolding alters the transcriptomic profile and increases the chemoresistance of MDA-MB-231 triple-negative breast cancer cellsMenon, Nidhi; Dang, Ha X.; Datla, Udaya Sree; Moarefian, Maryam; Lawrence, Christopher B.; Maher, Christopher A.; Jones, Caroline N. (2020-05-21)The tumor microenvironment plays a critical role in the proliferation and chemoresistance of cancer cells. Growth factors (GFs) are known to interact with the extracellular matrix (ECM) via heparin binding sites, and these associations influence cell behavior. In the present study, we demonstrate the ability to define signals presented by the scaffold by pre-mixing growth factors, such as epidermal growth factor, into the heparin-based (HP-B) hydrogel prior to gelation. In the 3D biomimetic microenvironment, breast cancer cells formed spheroids within 24 hours of initial seeding. Despite higher number of proliferating cells in 2D cultures, 3D spheroids exhibited a higher degree of chemoresistance after 72 hours. Further, our RNA sequencing results highlighted the phenotypic changes influenced by solid-phase GF presentation. Wnt/beta-catenin and TGF-beta signaling were upregulated in the cells grown in the hydrogel, while apoptosis, IL2-STAT5 and PI3K-AKT-mTOR signaling were downregulated. With emerging technologies for precision medicine in cancer, this nature of fine-tuning the microenvironment is paramount for cultivation and downstream characterization of primary cancer cells and rare circulating tumor cells (CTCs), and effective screening of chemotherapeutic agents.
- Microfluidic reactors for advancing the MS analysis of fast biological responsesLazar, Iuliana M.; Deng, J.; Stremler, Mark A.; Ahuja, Shreya (Nature Publishing Group, 2019-02-11)The response of cells to physical or chemical stimuli is complex, unfolding on time-scales from seconds to days, with or without de novo protein synthesis, and involving signaling processes that are transient or sustained. By combining the technology of microfluidics that supports fast and precise execution of a variety of cell handling operations, with that of mass spectrometry detection that facilitates an accurate and complex characterization of the protein complement of cells, in this work, we developed a platform that supports (near) real-time sampling and proteome-level capturing of cellular responses to a perturbation such as treatment with mitogens. The geometric design of the chip supports three critical features: (a) capture of a sufficient number of cells to meet the detection limit requirements of mass spectrometry instrumentation, (b) fluid delivery for uniform stimulation of the resident cells, and (c) fast cell recovery, lysis and processing for accurate sampling of time-sensitive cellular responses to a stimulus. COMSOL simulations and microscopy were used to predict and evaluate the flow behavior inside the microfluidic device. Proteomic analysis of the cellular extracts generated by the chip experiments revealed that the identified proteins were representative of all cellular locations, exosomes, and major biological processes related to proliferation and signaling, demonstrating that the device holds promising potential for integration into complex lab-on-chip work-flows that address systems biology questions. The applicability of the chips to study time-sensitive cellular responses is discussed in terms of technological challenges and biological relevance. © 2019, The Author(s).
- A model of yeast cell-cycle regulation based on multisite phosphorylationBarik, Debashis; Baumann, William T.; Paul, Mark R.; Novak, Bela; Tyson, John J. (Nature Publishing Group, 2010-08-01)In order for the cell’s genome to be passed intact from one generation to the next, the events of the cell cycle (DNA replication, mitosis, cell division) must be executed in the correct order, despite the considerable molecular noise inherent in any protein-based regulatory system residing in the small confines of a eukaryotic cell. To assess the effects of molecular fluctuations on cell-cycle progression in budding yeast cells, we have constructed a new model of the regulation of Cln- and Clb-dependent kinases, based on multisite phosphorylation of their target proteins and on positive and negative feedback loops involving the kinases themselves. To account for the significant role of noise in the transcription and translation steps of gene expression, the model includes mRNAs as well as proteins. The model equations are simulated deterministically and stochastically to reveal the bistable switching behavior on which proper cell-cycle progression depends and to show that this behavior is robust to the level of molecular noise expected in yeast-sized cells (B50 fL volume). The model gives a quantitatively accurate account of the variability observed in the G1-S transition in budding yeast, which is governed by an underlying sizer + timer control system.
- Modeling iontophoretic drug delivery in a microfluidic deviceMoarefian, Maryam; Davalos, Rafael V.; Tafti, Danesh K.; Achenie, Luke E. K.; Jones, Caroline N. (2020-09-21)Iontophoresis employs low-intensity electrical voltage and continuous constant current to direct a charged drug into a tissue. Iontophoretic drug delivery has recently been used as a novel method for cancer treatment in vivo. There is an urgent need to precisely model the low-intensity electric fields in cell culture systems to optimize iontophoretic drug delivery to tumors. Here, we present an iontophoresis-on-chip (IOC) platform to precisely quantify carboplatin drug delivery and its corresponding anti-cancer efficacy under various voltages and currents. In this study, we use an in vitro heparin-based hydrogel microfluidic device to model the movement of a charged drug across an extracellular matrix (ECM) and in MDA-MB231 triple-negative breast cancer (TNBC) cells. Transport of the drug through the hydrogel was modeled based on diffusion and electrophoresis of charged drug molecules in the direction of an oppositely charged electrode. The drug concentration in the tumor extracellular matrix was computed using finite element modeling of transient drug transport in the heparin-based hydrogel. The model predictions were then validated using the IOC platform by comparing the predicted concentration of a fluorescent cationic dye (Alexa Fluor 594 (R)) to the actual concentration in the microfluidic device. Alexa Fluor 594 (R) was used because it has a molecular weight close to paclitaxel, the gold standard drug for treating TNBC, and carboplatin. Our results demonstrated that a 50 mV DC electric field and a 3 mA electrical current significantly increased drug delivery and tumor cell death by 48.12% +/- 14.33 and 39.13% +/- 12.86, respectively (n = 3, p-value <0.05). The IOC platform and mathematical drug delivery model of iontophoresis are promising tools for precise delivery of chemotherapeutic drugs into solid tumors. Further improvements to the IOC platform can be made by adding a layer of epidermal cells to model the skin.
- Optimizing the restored chemotactic behavior of anticancer agent Salmonella enterica serovar Typhimurium VNP20009Broadway, Katherine M.; Suh, SeungBeum; Behkam, Bahareh; Scharf, Birgit E. (Elsevier, 2017-06-10)Bacteria, including strains of Salmonella, have been researched and applied as therapeutic cancer agents for centuries. Salmonella are particularly of interest due to their facultative anaerobic nature, facilitating colonization of differentially oxygenated tumor regions. Additionally, Salmonella can be manipulated with relative ease, resulting in the ability to attenuate the pathogen or engineer vectors for drug delivery. It was recently discovered that the anti-cancer Salmonella enterica serovar Typhimurium strain VNP20009 is lacking in chemotactic ability, due to a non-synonymous single nucleotide polymorphism in cheY. Replacing the mutated copy of cheY with the wild-type sequence restored chemotaxis to 70% of the parental strain. We aimed to investigate further if chemotaxis of VNP20009 can be optimized. By restoring the gene msbB in VNP20009 cheY+, which confers attenuation by lipid A modification, we observed a 9% increase in swimming speed, 13% increase in swim plate performance, 19% increase in microfluidic device partitioning towards the attractant at the optimum concentration gradient, and mitigation of a non-motile cell subpopulation. We conclude that chemotaxis can be enhanced further but at the cost of changing one defining characteristic of VNP20009. A less compromised strain might be needed to employ for investigating bacterial chemotaxis in tumor interactions.
- Potential Role of a Bistable Histidine Kinase Switch in the Asymmetric Division Cycle of Caulobacter crescentusSubramanian, Kartik; Paul, Mark R.; Tyson, John J. (PLOS, 2013-09-01)The free-living aquatic bacterium, Caulobacter crescentus, exhibits two different morphologies during its life cycle. The morphological change from swarmer cell to stalked cell is a result of changes of function of two bi-functional histidine kinases, PleC and CckA. Here, we describe a detailed molecular mechanism by which the function of PleC changes between phosphatase and kinase state. By mathematical modeling of our proposed molecular interactions, we derive conditions under which PleC, CckA and its response regulators exhibit bistable behavior, thus providing a scenario for robust switching between swarmer and stalked states. Our simulations are in reasonable agreement with in vitro and in vivo experimental observations of wild type and mutant phenotypes. According to our model, the kinase form of PleC is essential for the swarmer-to-stalked transition and to prevent premature development of the swarmer pole. Based on our results, we reconcile some published experimental observations and suggest novel mutants to test our predictions.
- Quaternary Ammonium Compound Disinfectants Reduce Lupus-Associated Splenomegaly by Targeting Neutrophil Migration and T-Cell FateAbdelhamid, Leila; Cabana-Puig, Xavier; Mu, Qinghui; Moarefian, Maryam; Swartwout, Brianna K.; Eden, Kristin; Das, Prerna; Seguin, Ryan P.; Xu, Libin; Lowen, Sarah; Lavani, Mital; Hrubec, Terry C.; Jones, Caroline N.; Luo, Xin M. (2020-10-21)Hypersensitivity reactions and immune dysregulation have been reported with the use of quaternary ammonium compound disinfectants (QACs). We hypothesized that QAC exposure would exacerbate autoimmunity associated with systemic lupus erythematosus (lupus). Surprisingly, however, we found that compared to QAC-free mice, ambient exposure of lupus-prone mice to QACs led to smaller spleens with no change in circulating autoantibodies or the severity of glomerulonephritis. This suggests that QACs may have immunosuppressive effects on lupus. Using a microfluidic device, we showed that ambient exposure to QACs reduced directional migration of bone marrow-derived neutrophils toward an inflammatory chemoattractant ex vivo. Consistent with this, we found decreased infiltration of neutrophils into the spleen. While bone marrow-derived neutrophils appeared to exhibit a pro-inflammatory profile, upregulated expression of PD-L1 was observed on neutrophils that infiltrated the spleen, which in turn interacted with PD-1 on T cells and modulated their fate. Specifically, QAC exposure hindered activation of splenic T cells and increased apoptosis of effector T-cell populations. Collectively, these results suggest that ambient QAC exposure decreases lupus-associated splenomegaly likely through neutrophil-mediated toning of T-cell activation and/or apoptosis. However, our findings also indicate that even ambient exposure could alter immune cell phenotypes, functions, and their fate. Further investigations on how QACs affect immunity under steady-state conditions are warranted.
- Stochastic simulation of enzyme-catalyzed reactions with disparate timescalesBarik, Debashis; Paul, Mark R.; Baumann, William T.; Cao, Yang; Tyson, John J. (Cell Press, 2008-10-01)Many physiological characteristics of living cells are regulated by protein interaction networks. Because the total numbers of these protein species can be small, molecular noise can have significant effects on the dynamical properties of a regulatory network. Computing these stochastic effects is made difficult by the large timescale separations typical of protein interactions (e. g., complex formation may occur in fractions of a second, whereas catalytic conversions may take minutes). Exact stochastic simulation may be very inefficient under these circumstances, and methods for speeding up the simulation without sacrificing accuracy have been widely studied. We show that the "total quasi-steady-state approximation'' for enzyme-catalyzed reactions provides a useful framework for efficient and accurate stochastic simulations. The method is applied to three examples: a simple enzyme-catalyzed reaction where enzyme and substrate have comparable abundances, a Goldbeter-Koshland switch, where a kinase and phosphatase regulate the phosphorylation state of a common substrate, and coupled Goldbeter-Koshland switches that exhibit bistability. Simulations based on the total quasi-steady-state approximation accurately capture the steady-state probability distributions of all components of these reaction networks. In many respects, the approximation also faithfully reproduces time-dependent aspects of the fluctuations. The method is accurate even under conditions of poor timescale separation.