Browsing by Author "Alves, Nathan J."

Now showing 1 - 10 of 26
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    Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant
    (Elsevier, 2017-02) Alves, Nathan J.; Turner, Kendrick B.; DiVito, Kyle A.; Daniele, Michael A.; Walper, Scott A.; Department of Emergency Medicine, School of Medicine
    To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.
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    Antibody Conjugation and Formulation
    (Oxford, 2019) Alves, Nathan J.; Emergency Medicine, School of Medicine
    In an era where ultra-high antibody concentrations, high viscosities, low volumes, auto-injectors, and long storage requirements are already complex problems with the current unconjugated monoclonal antibodies on the market the formulation demands for antibody-drug conjugates (ADCs) are significant. Antibodies have historically been administered at relatively low concentrations through intravenous (IV) infusion due to their large size and the inability to formulate for oral delivery. Due to the high demands associated with IV infusion and the development of novel antibody targets and unique antibody conjugates more accessible routes of administration such as intramuscular (IM), and subcutaneous (SC) are being explored. This review will summarize various site-specific and non-site-specific antibody conjugation techniques in the context of antibody-drug conjugates (ADCs) and the demands of formulation for high concentration clinical implementation.
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    Comparison of isoflurane and α-chloralose in an anesthetized swine model of acute pulmonary embolism producing right ventricular dysfunction
    (American Association for Laboratory Animal Science, 2015-02) Beam, Daren M.; Neto-Neves, Evandro M.; Stubblefield, William B.; Alves, Nathan J.; Tune, Johnathan D.; Kline, Jeffrey A.; Department of Emergency Medicine, IU School of Medicine
    Pulmonary embolism (PE) is a leading cause of sudden cardiac death, and a model is needed for testing potential treatments. In developing a model, we compared the hemodynamic effects of isoflurane and α-chloralose in an acute swine model of PE because the choice of anesthesia will likely affect the cardiovascular responses of an animal to PE. At baseline, swine that received α-chloralose (n = 6) had a lower heart rate and cardiac output and higher SpO2, end-tidal CO2, and mean arterial pressure than did those given isoflurane (n = 9). After PE induction, swine given α-chloralose compared with isoflurane exhibited a lower heart rate (63 ± 10 compared with 116 ± 15 bpm) and peripheral arterial pressure (52 ± 12 compared with 61 ± 12 mm Hg); higher SpO2 (98% ± 3% compared with 95% ± 1%), end-tidal CO2 (35 ± 4 compared with 32 ± 5), and systolic blood pressure (121 ± 8 compared with 104 ± 20 mm Hg); and equivalent right ventricular:left ventricular ratios (1.32 ± 0.50 compared with 1.23 ± 0.19) and troponin I mean values (0.09 ± 0.07 ng/mL compared with 0.09 ± 0.06 ng/mL). Isoflurane was associated with widely variable fibrinogen and activated partial thromboplastin time. Intraexperiment mortality was 0 of 6 animals for α-chloralose and 2 of 9 swine for isoflurane. All swine anesthetized with α-chloralose survived with sustained pulmonary hypertension, RV-dilation-associated cardiac injury without the confounding vasodilatory or coagulatory effects of isoflurane. These data demonstrate the physiologic advantages of α-chloralose over isoflurane for anesthesia in a swine model of severe submassive PE.
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    Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
    (2016) Alves, Nathan J.; Turner, Kendrick B.; Walper, Scott A.; Department of Emergency Medicine, School of Medicine
    A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.
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    Disinhibiting neurons in the dorsomedial hypothalamus delays the onset of exertional fatigue and exhaustion in rats exercising in a warm environment
    (Elsevier, 2018-06) Zaretsky, Dmitry V.; Kline, Hannah; Zaretskaia, Maria V.; Brown, Mary Beth; Durant, Pamela J.; Alves, Nathan J.; Rusyniak, Daniel E.; Emergency Medicine, School of Medicine
    Stimulants cause hyperthermia, in part, by increasing heat generation through exercise. Stimulants also delay the onset of fatigue and exhaustion allowing animals to exercise longer. If used in a warm environment, this combination (increased exercise and decreased fatigue) can cause heat stroke. The dorsomedial hypothalamus (DMH) is involved in mediating locomotion from stimulants. Furthermore, inhibiting the DMH decreases locomotion and prevents hyperthermia in rats given stimulants in a warm environment. Whether the DMH is involved in mediating exercise-induced fatigue and exhaustion is not known. We hypothesized that disinhibiting neurons in the dorsomedial hypothalamus (DMH) would delay the onset of fatigue and exhaustion in animals exercising in a warm environment. To test this hypothesis, we used automated video tracking software to measure fatigue and exhaustion. In rats, using wearable mini-pumps, we demonstrated that disinhibiting the DMH, via bicuculline perfusion (5 µM), increased the duration of exercise in a warm environment as compared to control animals (25 ± 3 min vs 15 ± 2 min). Bicuculline-perfused animals also had higher temperatures at exhaustion (41.4 ± 0.2 °C vs 40.0 ± 0.4 °C). Disinhibiting neurons in the DMH also increased the time to fatigue. Our data show that the same region of the hypothalamus that is involved in mediating locomotion to stimulants, is also involved in controlling exhaustion and fatigue. These findings have implications for understanding the cause and treatment of stimulant-induced-hyperthermia.
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    Disinhibiting neurons in the dorsomedial hypothalamus delays the onset of exertional fatigue and exhaustion in rats exercising in a warm environment
    (Elsevier, 2018-06-15) Zaretsky, Dmitry V.; Kline, Hannah; Zaretskaia, Maria V.; Brown, Mary Beth; Durant, Pamela J.; Alves, Nathan J.; Rusyniak, Daniel E.; Emergency Medicine, School of Medicine
    Stimulants cause hyperthermia, in part, by increasing heat generation through exercise. Stimulants also delay the onset of fatigue and exhaustion allowing animals to exercise longer. If used in a warm environment, this combination (increased exercise and decreased fatigue) can cause heat stroke. The dorsomedial hypothalamus (DMH) is involved in mediating locomotion from stimulants. Furthermore, inhibiting the DMH decreases locomotion and prevents hyperthermia in rats given stimulants in a warm environment. Whether the DMH is involved in mediating exercise-induced fatigue and exhaustion is not known. We hypothesized that disinhibiting neurons in the dorsomedial hypothalamus (DMH) would delay the onset of fatigue and exhaustion in animals exercising in a warm environment. To test this hypothesis, we used automated video tracking software to measure fatigue and exhaustion. In rats, using wearable mini-pumps, we demonstrated that disinhibiting the DMH, via bicuculline perfusion (5 µM), increased the duration of exercise in a warm environment as compared to control animals (25 ± 3 min vs 15 ± 2 min). Bicuculline-perfused animals also had higher temperatures at exhaustion (41.4 ± 0.2 °C vs 40.0 ± 0.4 °C). Disinhibiting neurons in the DMH also increased the time to fatigue. Our data show that the same region of the hypothalamus that is involved in mediating locomotion to stimulants, is also involved in controlling exhaustion and fatigue. These findings have implications for understanding the cause and treatment of stimulant-induced-hyperthermia.
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    Effect of Chandler loop shear and tubing size on thrombus architecture
    (Springer, 2023-05-12) Zeng, Ziqian; Chakravarthula, Tanmaye Nallan; Christodoulides, Alexei; Hall, Abigail; Alves, Nathan J.; Emergency Medicine, School of Medicine
    Thrombosis can lead to a wide variety of life-threatening circumstances. As current thrombolytic drug screening models often poorly predict drug profiles, leading to failure of thrombolytic therapy or clinical translation, more representative clot substrates are necessary for drug evaluation. Utilizing a Chandler loop device to form clot analogs at high shear has gained popularity in stroke societies. However, shear-dependent clot microstructure has not been fully addressed and low shear conditions are often overlooked. We herein characterized the impact of wall shear rate (126 to 951 s-1) on clot properties in the Chandler loop. Different revolutions (20-60) per minute and tubing sizes (3.2 to 7.9 mm) were employed to create different sized clots to mimic various thrombosis applications. Increased shear resulted in decreased RBC counts (76.9 ± 4.3% to 17.6 ± 0.9%) and increased fibrin (10 to 60%) based on clot histology. Increased fibrin sheet morphology and platelet aggregates were observed at higher shear under scanning electron microscope. These results show the significant impact of shear and tubing size on resulting clot properties and demonstrate the capability of forming a variety of reproducible in-vivo-like clot analogs in the Chandler loop device controlling for simple parameters to tune clot characteristics.
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    Emerging Therapeutic Delivery Capabilities and Challenges Utilizing Enzyme/Protein Packaged Bacterial Vesicles
    (Future Science, 2015-07) Alves, Nathan J.; Turner, Kendrick B.; Medintz, Igor L.; Walker, Scott A.; Emergency Medicine, School of Medicine
    Nanoparticle-based therapeutics are poised to play a critical role in treating disease. These complex multifunctional drug delivery vehicles provide for the passive and active targeted delivery of numerous small molecule, peptide and protein-derived pharmaceuticals. This article will first discuss some of the current state of the art nanoparticle classes (dendrimers, lipid-based, polymeric and inorganic), highlighting benefits/drawbacks associated with their implementation. We will then discuss an emerging class of nanoparticle therapeutics, bacterial outer membrane vesicles, that can provide many of the nanoparticle benefits while simplifying assembly. Through molecular biology techniques; outer membrane vesicle hijacking potentially allows for stringent control over nanoparticle production allowing for targeted protein packaged nanoparticles to be fully synthesized by bacteria.
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    Environmental Decontamination of a Chemical Warfare Simulant Utilizing a Membrane Vesicle-Encapsulated Phosphotriesterase
    (ACS, 2018) Alves, Nathan J.; Moore, Martin; Johnson, Brandy J.; Dean, Scott N.; Turner, Kendrick B.; Medintz, Igor L.; Walper, Scott A.; Emergency Medicine, School of Medicine
    While technologies for the remediation of chemical contaminants continue to emerge, growing interest in green technologies has led researchers to explore natural catalytic mechanisms derived from microbial species. One such method, enzymatic degradation, offers an alternative to harsh chemical catalysts and resins. Recombinant enzymes, however, are often too labile or show limited activity when challenged with nonideal environmental conditions that may vary in salinity, pH, or other physical properties. Here, we demonstrate how phosphotriesterase encapsulated in a bacterial outer membrane vesicle can be used to degrade the organophosphate chemical warfare agent (CWA) simulant paraoxon in environmental water samples. We also carried out remediation assays on solid surfaces, including glass, painted metal, and fabric, that were selected as representative materials, which could potentially be contaminated with a CWA.
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    Environmental Microplastic and Nanoplastic: Exposure Routes and Effects on Coagulation and the Cardiovascular System
    (Elsevier, 2021) Lett, Zachary; Hall, Abigail; Skidmore, Shelby; Alves, Nathan J.; Emergency Medicine, School of Medicine
    Plastic pollution has been a growing concern in recent decades due to the proliferation and ease of manufacturing of single use plastic products and inadequate waste and recycling management. Microplastic, and even smaller nanoplastic, particles are persistent pollutants in aquatic and terrestrial systems and are the subject of active and urgent research. This review will explore the current research on how exposure to plastic particles occurs and the risks associated from different exposure routes: ingestion, inhalation, and dermal exposure. The effects of microplastics on the cardiovascular system are of particular importance due to its sensitivity and ability to transport particles to other organ systems. The effects of microplastics and nanoplastics on the heart, platelet aggregation, and thrombus formation will all be explored with focus on how the particle characteristics modulate their effect. Plastic particle interactions are highly dependent on both their size and their surface chemistry and interesting research is being done with the interaction of particle characteristics and effect on thrombosis and the cardiovascular system. There is significant uncertainty surrounding some of the findings in this field as research in this area is still maturing. There are undoubtedly more physiological consequences than we are currently aware of resulting from environmental plastic exposure and more studies need to be conducted to reveal the full extent of pathologies caused by the various routes of microplastic exposure, with particular emphasis on longitudinal exposure effects. Further research will allow us to recognize the full extent of physiological impact and begin developing viable solutions to reduce plastic pollution and potentially design interventions to mitigate in-vivo plastic effects following significant or prolonged exposure.