Browsing by Author "Ray, Bruce D."

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    1H NMR of Deep Eutectic Solvents
    (Office of the Vice Chancellor for Research, 2014-04-11) Wallis, J. Lincoln; Sapir, Liel; Harries, Daniel; Petrache, Horia I.; Ray, Bruce D.
    Deep Eutectic Solvents (DESs) form between a variety of quaternary ammonium or phosphonium salts and hydrogen-bond donors. Over the past decade, DESs have been studied as green solvents with potential applications in industrial processes, chemical extractions, and pharmaceuticals. The recent suggestion that many plants produce natural deep eutectic solvents (NADES) from primary metabolites led to investigation of the potential uses of DESs in biophysics research. This study examined the 1H NMR spectra of the choline chloride:urea 1:2, and choline chloride:ethylene glycol 1:3 molar ratio DES. Spectra of the choline chloride:urea 1:2 with various solutes were acquired to see what effect these solutes had on the DESs NMR spectrum. For both DESs tested, the NMR spectra were a superposition of the spectra of the components. DES-solute spectra showed that interaction between components persisted, indicating the solvent properties of the DESs were not lost upon addition of solutes.
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    Detecting Counterfeit Pharmaceuticals through UV Spectrophotometry
    (Office of the Vice Chancellor for Research, 2015-04-17) Figueroa, Gabriela; Palacio, Luis A.; Ray, Bruce D.; Petrache, Horia I.; Lopez-Yunez, Alfredo
    According to the World Health Organization between 10%-30% of medicines, in Africa, Asia and South America, are counterfeit or sub-standard, affecting the health of millions of people. Currently, there is no effective way to check the quality of a medicine at the point of care, leaving many with treatable diseases at risk. The goal of this study is to identify UV-Vis (240nm - 500nm) absorbance patterns that would indicate if a drug is sub-standard or counterfeit. UV-Vis spectroscopy was selected as the method for testing due to the maturity and availability of the technology. Pure Acetaminophen and Tylenol were used as controls for proof of concept. Samples were prepared by dissolving different combinations of the pure active ingredient and adulterants such as cement, rice flour, vitamin C and lactose in three different types of solvents (H2O, 0.1 M HCl, 0.1 NaOH). Various concentrations (ranging from 0.01mg/ml to 0.04mg/ml) and mixing ratios were analyzed using a UV-Vis Spectrophotometer. It was found that adulterants significantly decrease the absorption of acetaminophen at 245nm by interacting with its benzene ring, while showing a slight increase in other parts of the spectrum. UV-Vis scans show that the amount of change in absorbance at specific wavelengths, coupled with characteristic wavelength shifts produced by different solvents, can be used for detection of counterfeit drugs. The methods presented here could be used for quality control of medicines at or near the point of care in parts of the world at higher risk of encountering defective pharmaceuticals.
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    Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy
    (Elsevier, 2016-04) Matam, Yashasvi; Ray, Bruce D.; Petrache, Horia I.; Department of Physics, School of Science
    Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain’s reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells.
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    DOMAIN STRUCTURE OF THE MAJOR ALLERGEN OVOMUCOID BY SOLUTION NMR
    (Office of the Vice Chancellor for Research, 2011-04-08) Stenzoski, Natalie E.; Gebriel, Gugsa W; Ray, Bruce D.; Petrache, Horia I.
    The interest in the ovomucoid protein is twofold. First it is a protein of interest for medical studies due to its potent allergen activity. Second, as a special variety of glycosylated protein (Kazal family), it allows us to explore the role of protein glycosylation in protein-membrane interactions for a particular, model case. The nature, location, and orientation of the glycosyl groups are determining factors in proteinmembrane interactions and therefore are critical to biological processes involving glycosylated proteins. We have found that as opposed to other glycosylated proteins, ovomucoid does not induce ionic currents across lipid membranes. This behavior likely has a structural cause, yet very little overall structural data is available. In this study, we use solution NMR spectroscopy to determine the structure of the chicken ovomucoid protein, taking advantage of the division of its structure into three stable domains of 55-65 amino acids each. We present results on the protein purification steps and isolation of separate domains suitable for solution NMR spectroscopy. We then present NMR results acquired on a 500 MHz spectrometer, and we show atomic models of individual domains and of overall protein structure from analysis of NMR spectra.
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    The Effect of Free Amino Acids on Fibrin Formation
    (Office of the Vice Chancellor for Research, 2014-04-11) Dow, Anna M.; Johnson, Merrell A.; Rosen, Elliot D.; Ray, Bruce D.; Petrache, Horia I.
    Fibrinogen, a plasma protein, is a main component of blood clot formation. In the event of an injury, blood loss is hindered through a process that forms a thrombus by conversion of fibrinogen to fibrin induced by activation of the enzyme thrombin. The fibrin network structure depends on the concentration of thrombin, as expected, but is also highly influenced by its environment during formation. In particular, we find that fibrin formation is altered in the presence of zwitterions. Zwitterions are dipolar molecules, typically highly polarizable, exhibiting both a positive and a negative charge depending on the pH of the solution. Amino acids are highly abundant zwitterions in biological materials. In this study we measured by visible/UV-spectroscopy the effects of various types of amino acids on the rate of fibrin network formation. We show that the electrical charge and type of amino acid, such as lysine, glycine and arginine, inhibits or promotes formation of fibrin networks. Such an ability to decrease or increase the rate of coagulation can be valuable in the treatment of patients suffering from hemostatic and thrombotic disorders.
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    Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures
    (American Physiological Society, 2015-07-15) Schweitzer, Kelly S.; Chen, Steven X.; Law, Sarah; Van Demark, Mary; Poirier, Christophe; Justice, Matthew J.; Hubbard, Walter C.; Kim, Elena S.; Lai, Xianyin; Wang, Mu; Kranz, William D.; Carroll, Clinton J.; Ray, Bruce D.; Bittman, Robert; Goodpaster, John V.; Petrache, Irina; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.
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    Finding Active Ingredients in Pharmaceuticals by UV Spectrophotometry
    (Office of the Vice Chancellor for Research, 2014-04-11) Figueroa, Gabriela; Palacio, Luis A.; Ray, Bruce D.; Petrache, Horia I.; Lopez-Yunez, Alfredo
    The active ingredient in any pharmaceutical is the chemical that will ultimately deliver the desired effect on a patient. Knowing the quality and the quantity of the active ingredient in a pill right before ingestion is of paramount importance for the patient’s health and the desired results. Unfortunately drugs only undergo quality control testing at the manufacturing plant but not at the point of sale. Moreover, to an untrained eye, one pill may not appear different from another and if the wrong pill or the wrong dose is taken, adverse health effects may arise. Indeed, manufacturers of counterfeit drugs rely on these two points of appearance and testing. In this study we examine whether ultra violet (UV) spectrophotometry absorbance can be used to separate an active ingredient’s UV peaks from the combination of peaks generated by the inactive ingredients of the tablet. A well-understood active ingredient, acetaminophen, was used for this study. Samples were prepared by crushing Tylenol tablets, dissolving the powder in different solvents (0.1M HCl, 0.1M NaOH, and H2O) at various concentrations and mixed by vortex. After preparation, the samples were measured by UV Spectroscopy. Experimental results were compared to standard UV curves for the pure active ingredient to correlate the observed changes in absorbance within the relevant UV wavelength range. We observe that more than one solvent is needed to identify the active ingredient. Development of a simple method to accurately identify the quality of the active ingredient will provide an additional safeguard to consumers, particularly in regions where counterfeit drugs are prevalent.
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    Fluorescence Measurements of Aromatic Amino Acids in the Presence of Lipid Membranes
    (Office of the Vice Chancellor for Research, 2014-04-11) Khelifi, Sirine; Johnson, Merrell A.; Ray, Bruce D.; Petrache, Horia I.
    Amphiphilic peptides are capable of finding their way to, and occasionally through, cellular membranes using a mechanism that includes specific amino acid sequences. Physical measurements of amino acid-lipid interactions are of interest for a quantitative description of peptide affinities to biological membranes. In this study, we investigate small peptide-lipid interactions using the fluorescence of the aromatic amino acids tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe). Reference spectra in deuterated isopropanol solutions are obtained to mimic hydrophobic environments and are used to quantify the interaction of Lys-Tyr-Lys, Trp-Gly, and Gly-Phe with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and palmitoyl-oleoyl phosphatidylserine (POPS) lipid membranes. These fluorescence data complement previously reported UV absorption data and have the advantage of eliminating background and scatter from solution. Together with NMR data, these results can be used to more fully characterize lipid-aromatic amino residue interactions.
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    Fluorescence Measurements Of Phototrophic Sulfur Bacteria For Applications In Water Column Profiling
    (Office of the Vice Chancellor for Research, 2015-04-17) Gurdasani, Simran S.; Harper, Lynsie A.; Petrache, Horia I.; Ray, Bruce D.; Gilhooly, William P., III; Johnson, Merrell A.
    Unlike plants that produce oxygen during photosynthesis, phototrophic sulfur bacteria use sulfide and sunlight to produce carbohydrates and elemental sulfur. These bacteria require a unique aquatic environment to thrive: one that is anoxic (depleted of oxygen) and rich in hydrogen sulfide. Such conditions are found in a number of stratified lakes around the world including several in Northern Indiana. Studying the ecology and geochemical conditions that promote habitable conditions for phototrophic bacteria in lakes provides insight into the Early Earth (thought to be anoxic), ocean anoxic events of the Mesozoic (70-250 million years ago) and modern low oxygen conditions of coastal environments such as the Dead Zone of the Gulf of Mexico. However, locating and directly sampling these bacterial populations in vast bodies of water is not an easy task. In this project, we investigate fluorescent properties of purple sulfur bacteria in order to develop a dependable sensor that can be deployed in the water column. We report a number of measurements of purple sulfur bacterium fluorescence in the near infrared region when excited at discrete wavelengths in the UV range. We use these bench-top measurements to design a water-proof apparatus equipped with an absorption and luminescent detector for localization of bacteria in lake water. This device will be deployed in anoxic lakes of Northern Indiana to find the in situ water column position of phototrophic bacteria.
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    Hydrodynamic Hunters
    (Cell Press, 2017-03-28) Jashnsaz, Hossein; Al Juboori, Mohammed; Weistuch, Corey; Miller, Nicholas; Nguyen, Tyler; Meyerhoff, Viktoria; McCoy, Bryan; Perkins, Stephanie; Wallgren, Ross; Ray, Bruce D.; Tsekouras, Konstantinos; Anderson, Gregory G.; Pressé, Steve; Physics, School of Science
    The Gram-negative Bdellovibrio bacteriovorus (BV) is a model bacterial predator that hunts other bacteria and may serve as a living antibiotic. Despite over 50 years since its discovery, it is suggested that BV probably collides into its prey at random. It remains unclear to what degree, if any, BV uses chemical cues to target its prey. The targeted search problem by the predator for its prey in three dimensions is a difficult problem: it requires the predator to sensitively detect prey and forecast its mobile prey’s future position on the basis of previously detected signal. Here instead we find that rather than chemically detecting prey, hydrodynamics forces BV into regions high in prey density, thereby improving its odds of a chance collision with prey and ultimately reducing BV’s search space for prey. We do so by showing that BV’s dynamics are strongly influenced by self-generated hydrodynamic flow fields forcing BV onto surfaces and, for large enough defects on surfaces, forcing BV in orbital motion around these defects. Key experimental controls and calculations recapitulate the hydrodynamic origin of these behaviors. While BV’s prey (Escherichia coli) are too small to trap BV in hydrodynamic orbit, the prey are also susceptible to their own hydrodynamic fields, substantially confining them to surfaces and defects where mobile predator and prey density is now dramatically enhanced. Colocalization, driven by hydrodynamics, ultimately reduces BV’s search space for prey from three to two dimensions (on surfaces) even down to a single dimension (around defects). We conclude that BV’s search for individual prey remains random, as suggested in the literature, but confined, however—by generic hydrodynamic forces—to reduced dimensionality.