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Spring 2018 Montana ACS Meeting-in-Miniature

Friday, April 6, 2018

Montana State University Bozeman, MT

Organized by the Graduate Student Association,

Department of Chemistry and Biochemistry, Montana State University

http://montana.site.acs.org

https://www.facebook.com/Montana-ACS-275145155836505/

Mark your calendars and save the date!

Fall Meeting and Social 2018

October 12-14, 2018 at Chico Hot Springs

Selected student presentations, undergraduate posters, banquette dinner, keynote speaker.

Breakfast buffet in main dining room Local Montana ACS section board and business meeting

Meeting Schedule of Events All events will take place in Animal Biosciences Buildings

except the Plenary Talk! Friday, April 6th

8:30 – 9:30 am Arrival & Check-in in ABB 145 (equipment check, setup for all morning speakers; coffee+donuts for registered participants) Morning Parallel Sessions: I. Materials Chemistry – ABB 134 9:30 – 9:40 am Opening comments by the moderator

Aoife Casey 9:40 – 10:00 am Geoffrey Zath: High-Throughput

IdentificationofInfluenzaInterferingParticlesUsingDroplet-BasedMicrofluidics

10:00 – 10:20 am Tom LeFevre: CharacterizingtheAdhesionandRheologyofSoftMaterialsUsingCentrifugeMicroscopy

10:20 – 10:40 am Humberto Sanchez: AnalyzingViralPopulationDynamicswithMicrofluidics

10:40 – 11:00 am Ranalda Tsosie: SelectiveSolidPhaseExtractionofUraniumandArsenicUsingSilicaPolyamineCompositesandtheImplementationofTraditionalKnowledgeandChemistry.

11:00 – 11:20 am Joelle Romo: ZeoliteBeadHeterogeneousCatalystforBiomassUpgrading

11:20 – 11:40 am Reha Abbasi: PrintingChannelsinHydrogelswithStereolithography-Based3DPrinting

11:40 – noon Nada Naser: SingleCellStudyofE.coliBiomineralizationusingDropletMicrofluidics

II. Biochemistry – ABB 138 9:30 – 9:40 am Opening comments by the moderator

Casey Kennedy 9:40 – 10:00 am Jacob Remmington: MolecularDynamics

andTime-ResolvedFluorescenceStudiesofBaseStackinginDNADinucleotides

10:00 – 10:20 am Burcu Ozay: UltrasensitiveMicroRNADetectionforDiseaseDiagnosis

10:20 – 10:40 am Jenna Mattice: ABacterialFlavin-DependentOxidoreductasethatCapturesCarbonDioxideintoBiomass

10:40 – 11:00 am Luke Berry: MassSpectrometry-BasedStructuralAnalysisofMoFeandFeFeNitrogenasetoElucidatetheRoleofStructure-FunctioninNitrogenReduction

11:00 – 11:20 am Ross Hartman: ElucidatingAttachmentofaHyper-ThermophillicViruswithCryo-ElectronTomography

11:20 – 11:40 am Stella Impano: UnderstandingtheBiochemistryof[FeFe]HydrogenaseMaturaseEnzyme,HydE

11:40 – noon Nickolas Avila: QuantifyingEnhancedMassTransferofCO2intoHighAlkalinityAlgaeCultureMedium

Lunch Break: noon – 12:55 pm Lunch in ABB 145

(equipment check, setup for afternoon speakers; sandwich or wrap/cookie/soda for registered participants)

Afternoon Parallel Sessions:

III. Organic Chemistry – ABB 134 12:55 – 1:00 pm opening comments by the moderator

Arianna Celis 1:00 – 1:20 pm Samuel Bernhard: Quantifying Protein-

Carbohydrate Binding Affinity Through Time-Dependent Fluorescence

1:20 – 1:40 pm Leidy Hooker: Catalyst-Controlled Selectivity in Nickel-Catalyzed Cross-Coupling

1:40 – 2:00 pm Harrison VanKoten: ProbingtheLEC-1andLEC-10OxidativeStressPathwayinCaenorhabditiselegansUsingGalβ1-4FucDendrimers

2:00 – 2:20 pm Danica Walsh: Design,SynthesisandEvaluationofPro-DrugAntimicrobialstoControlBiofilms

2:20 – 2:40 pm Jessie Langlais: SynthesisofLactoseFunctionalizedLinearPolymersusingRing-OpeningMetathesisPolymerization

2:40 – 3:00 pm John Russell: SelectiveSuzukiCross-CouplingtoSynthesizeFunctionalizedOrganostannanes

3:00 – 3:20 pm Pierce Fix: Thermodynamicsof2-MethylthiopheneHomocoupling

IV. Biochemistry – ABB 138 12:55 – 1:00 pm opening comments by the moderator

Casey Kennedy 1:00 – 1:20 pm Genevive Coe: Deciphering the Role of

Gln185 in the Peroxide Dependent Coproheme Decarboxylation Reaction

1:20 – 1:40 pm Angela Patterson: TheRoleofAllosteryintheAssemblyoftheHBVCapsid

1:40 – 2:00 pm Elizabeth McDaniels: PFL-AEandGD-AE,aTaleofTwoRadicalSAMEnzymes

2:00 – 2:20 pm Amanda Fuchs: Quantitative1HNMRMetabolomicsAnalysisRevealsDistinctMetabolicAdaptationsinHumanMacrophagesFollowingActivation

2:20 – 2:40 pm Canberk Kayalar: TransducingProteinsintoAmplifiableDNASignalsusingAptamers

2:40 – 3:00 pm Melodie Machovina: FromPlantBiopolymertoHigh-ValueProduct:UnderstandingHowaNovelandPromiscuousCytochromeP450CatabolizesLignin

3:00 – 3:20 pm Mackenzie Fricke: UsingLactoseFunctionalizedDendrimerstoStudytheImpactofGalectin-3onCancerCellMigration

Awards and Closing Comments 3:30 – 3:45 pm announcements of travel awards

by GSA president Casey Kennedy and Robert K. Szilagyi, organizers

4:00 – 5:00 pm Keynote presentation: Regents

Professor Pat Callis "Seeking Microscopic Truth about Biomolecules" in Gaines 101

Keynote Presentation:

Regents Professor Pat Callis "Seeking Microscopic Truth about Biomolecules"

Montana State University professor Pat Callis instructs a chemistry class on Oct. 17, 2017. Callis, a photophysicist who is nearing his 50th year as MSU faculty, was named an MUS Regents Professor on Nov. 16. MSU photo by Adrian Sanchez-Gonzalez. Prof. Callis, who is nearing his 50th year in MSU’s Department of Chemistry and Biochemistry in the College of Letters and Science, brought recognition to MSU with his renowned contributions to the science of photophysics and his laudable integration of learning, discovery and engagement. “I strongly believe in the synergic balance of teaching and research-creativity, as reflected in the expectations of the College of Letters and Science at MSU,” Callis explained. “Had I not been granted a sabbatical leave in 1979 to Cornell University — during which I brought my research up to the quality of my teaching — I am sure I would have died on the vine academically decades ago. I wish to acknowledge Professor Gary Strobel for spearheading the nascent EPSCoR program at MSU and Chemistry-Biochemistry Department Head Ed Abbott for encouragement and support during that time.”

ExcerptsfromMontanaSectionACSBylaws...

BYLAWV.Officers,ExecutiveCommittee,andCouncilors

Section 1. The officers of the Section shall be members of the SOCIETY and the Sec on and shall consist of the Chair, Chair-Elect, Secretary, and Treasurer. The Secretary and Treasurer positions may be held by the same person. Section 4. The dues of the officers shall be such as usually pertain to their offices, together with those required by these bylaws and by the Constitution on and Bylaws of the SOCIETY, and such other duties as may be assigned to them from me to me by the Executive Committee. 4.a. The duties of the Chair shall be to preside at meetings of the Executive Committee, to carry into effect the decisions and recommendations of that Committee, to preside at meetings of the Section to conduct governance business, to appoint, with the approval of the Executive Committee, all committee chairs and committee members except as stated elsewhere in these bylaws, and to carry out the duties required by the Constitution and Bylaws of the SOCIETY. 4.b. The duties of the Chair-Elect shall be to assist the Chair with the direction and management of the Section. In the absence of the Chair, the duties of the office shall devolve upon the Chair-Elect. 4.c. The duties of the Secretary shall be to keep a record of the minutes of the meetings of the Sec on and of the Executive Committee, to maintain a list of members and affiliates, to send to members and affiliates such notices as the business of the Section may require, to submit a report to the Sec on at its annual meeting, and to carry out the duties required by the Constitution and Bylaws of the SOCIETY and elsewhere in these bylaws. The Secretary shall preside over meetings in the absence of both the Chair and Chair-Elect. 4.d. The Treasurer shall have charge of the funds of the Section, keep an accurate record of all receipts and disbursements, receive dues, and make those disbursements approved by the Executive Committee. The Treasurer shall render an account of all transactions and of the financial condition of the Section to the Executive Committee at times set by the Committee, and shall submit such reports as are required by the Constitution and Bylaws of the SOCIETY.

Presentation Abstracts

QUANTIFYING ENHANCED MASS TRANSFER OF CO2 INTO HIGH ALKALINITY ALGAE CULTURE MEDIUM

Agasteswar Vadlamani,1 Nickolas Avila,2 Matt Jackson,2 Mohammadmatin Hanifzadeh,1 Brahmaiah Pendyala,1 Sasidhar

Varanasi,1 Robin Gerlach,2 Sridhar Viamajala,1

1 The University of Toledo, Toledo, OH 2 Montana State University, Bozeman, MT

Email: [email protected]

High pH and high alkalinity solutions can simultaneously allow for a high rate of CO2

mass transfer from the atmosphere and provide non-limiting concentrations of

bicarbonate in algal growth media for photosynthetic fixation, especially by microalgae.

We developed a mathematical model describing this enhanced CO2 mass transfer by

relating it to the hydroxyl ion concentration, diffusion constants, total alkalinity,

equilibrium constants of the carbonate/bicarbonate equilibrium equations, and the

volumetric mass transfer coefficient (kLa). All of the constants are well known from the

literature and changes in the hydroxyl ion concentration are easily measured as pH. Thus,

kLa can be estimated by fitting the data relating pH and time to the ordinary differential

equation that describes the unsteady state mass transfer.

In this work, we determined the kLa for the transport of atmospheric CO2 into

paddlewheel-mixed alkaline open ponds by minimizing the sum of the square of the

residuals between experimentally determined pH versus time data and the model

prediction via an algorithm developed in the programming language Python. This

algorithm finds the differential equation solution via a numerical ordinary differential

equation solver in the SciPy software library of Python. Correlation coefficients between

data and solutions are between 0.6341 and 0.9987 with a median correlation coefficient

of 0.9830, using 7 sets of data measured over a period of 24 hours, having pH values

between 9.95 and 10.55. This generally good fit is providing the basis for increased algal

biomass productivity for broad applications in biotechnology.

scheduled presentation time: II. Biochemistry ABB138, 11:40-noon

- 1 -


QUANTIFYING PROTEIN-CARBOHYDRATE BINDING AFFINITY THROUGH TIME-DEPENDENT

FLUORESCENCE

Samuel Bernhard, Candace Goodman, Jon Cousin, and Mary Cloninger

Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717


In this study, intrinsic fluorescence measurements were used to investigate and quantify the binding of multiple systems: a series of carbohydrates and carbohydrate functionalized PAMAM dendrimers to human galectin-3 and galectin-1. Galectin-3 binding dissociation constants (Kd) were quantified; lactose (75 ± 6 µM), 1-methyl lactose (72 ± 8 µM), lactose functionalized PAMAM G2, 4, and 6 dendrimers (24 ± 11 µM, 91 ± 17 µM, 44 ± 10 µM respectively). Galectin-1 binding dissociation constants (Kd) were quantified: lactose (255 µM), galactose (36.2 µM). The chosen examples showcase the widespread utility this technique has for determining binding constants, including cases for which standard methods have limited utility.

scheduled presentation time: III. Organic Chemistry ABB134, 1:00-1:20 pm

- 2 -


MASS SPECTROMETRY-BASED STRUCTURAL ANALYSIS OF MoFe AND FeFe NITROGENASE TO

ELUCIDATE THE ROLE OF STRUCTURE-FUNCTION IN NITROGEN REDUCTION

Luke Berry, Monika Tokmina-Lukaszewska, Derek F. Harris, Oleg A. Zadvornyy, John W. Peters, Lance C. Seefeldt, and Brian

Bothner



Nitrogenase enzymes are a class of enzymes capable of forming reduced species of nitrogen (NH3) from nitrogen gas (N2) through a process known as nitrogen fixation. Organisms possessing these enzymes are responsible for producing 50% of the world’s reduced nitrogen supply. There are three types of nitrogenases, each characterized by a unique metal cluster at the active site (MoFe, VFe, and FeFe). Structural models have successfully been developed for the MoFe and VFe nitrogenases. However, a high resolution structural model for the Iron only class of nitrogenase (FeFe) has not been obtained despite numerous attempts. We have employed mass spectrometry-based chemical cross-linking and H/D exchange with computational modeling to generate quaternary structural models of the FeFe Nitrongenase. This study not only provided an accurate 3D structure of the FeFe nitrogenase, but preliminary HDX data also revealed differential deuterium incorporation between the FeFe and MoFe nitrogeanses. This differential exchange suggests alternative mechanisms of communication with the nitrogenase complex, which can influence the rate and efficiency of nitrogen reduction.

scheduled presentation time: II. Biochemistry ABB138 10:40-11:00

- 3 -


DECIPHERING THE ROLE OF Gln185 IN THE PEROXIDE DEPENDENT COPROHEME DECARBOXYLATION

REACTION CATALYZED BY HemQ

Genevive Coe and Jennifer DuBois



Heme (iron-protoporphyrin IX) is a cofactor involved in electron transport, cell signaling and degradation of harmful oxygen species. Recent examination of the classical pathway of heme biosynthesis has revealed that gram-positive bacteria and archaea possess a unique set of genes encoding for different proteins involved in the final steps of synthesis. In particular, the final step of heme synthesis in gram-positive bacteria consists of the decarboxylation of two propionate groups of coproheme, catalyzed by HemQ. Part of the chlorite dismutase superfamily, HemQ demonstrates novel mechanistic features as a noncanonical peroxidase. HemQ catalyzes the decarboxylation of coproheme to heme independent of cofactors, but relies on hydrogen peroxide as a co-substrate. While the mechanism of classical peroxidases is well-known, the autocatalytic peroxidase mechanism of HemQ is not well understood. The distal pocket of the active site, above the plane of the coproheme substrate, is unique as it is largely hydrophobic and non-polar, features not shared by other peroxidases or related chlorite dismutases. We sought to understand the unique mechanism of heterolytic O-O bond cleavage in the HemQ active site by probing the role of a distal pocket residue (Gln185), which is not conserved in classical peroxidases. Site-directed mutagenesis of Gln185 to Ala and Arg demonstrates the effect of size and charge of this residue in the distal pocket with respect to small neutral and charged ligand interactions. Characterization of a protein unique to gram- positive organisms is useful for understanding the mechanism of heme biosynthesis, a process critical to gram-positive pathogen virulence.

scheduled presentation time: IV. Biochemistry ABB138 1:00-1:20 pm

- 4 -


THERMODYNAMICS OF 2-METHYLTHIOPHENE HOMOCOUPLING

Pierce Fix, Ian Jacobson, Marc Malek, and John Rowley

1 Carroll College, Helena, MT


The homocoupling of 2-methylthiophene to yield 5,5’-dimethyl-2,2’-bithiophene was observed with the following chemical oxidants: hydrogen peroxide (H2O2), cerium (Ce4+), dichromate (Cr2O7

2-), and persulfate (S2O8

2-). The reaction kinetics and equilibrated system concentration for the homocoupling of 2-methylthiophene was quantified using 1H-NMR spectroscopy. Under the conditions studied the system equilibrated in under 48 hours. Using the oxidant hydrogen peroxide the equilibrium concentrations of 2-methylthiophene and 5,5’-dimethyl-2,2’-bithiophene were 0.0994 M, and 7.398x10-4 M. The equilibrium constant for the homocoupling in the presence of hydrogen peroxide was calculated to be 0.146 at 25 °C. Using this equilibrium constant, the reduction potential of 5,5’-dimethyl-2,2’-bithiophene to 2-methylthiophene was calculated to be 1.80 V.

scheduled presentation time: III. Organic Chemistry ABB134 3:00-3:20pm

- 5 -


USING LACTOSE FUNCTIONALIZED DENDRIMERS TO STUDY THE IMPACT OF GALECTIN-3 ON CANCER

CELL MIGRATION

Mackenzie Fricke and Mary J. Cloninger



Tissue invasion and metastasis is devastating process and is associated with a poor prognosis for cancer patients. Galectin-3 is a protein with varied expression in cancers which has recently made it a highly investigated protein. Galectin-3 selectively binds β-galactosides and demonstrates the ability to oligomerize through aggregation of the N-terminal domain. Based on its cellular localization, galectin-3 can be involved in many processes such as gene regulation, apoptosis and angiogenesis. In addition, when excreted to the extracellular space, galectin-3 can impact intercellular and matrix adhesions, endocytosis, tumor metastasis and angiogenesis. To investigate the role of galectin-3 in cancer cell migration in vitro, multivalently displayed lactose was used to present competitive and energetically favorable binding partners for galectin-3. These multivalent compounds significantly impacted the migration of cancer cells in the presence of exogenously added galectin-3. Further investigations through immunofluorescence revealed that galectin-3 was localized on the periphery of the migrating cells and underwent aggregation while being endocytosed. This talk describes details of the migration assays including results and conclusions that have emerged, to date, from these experiments.


- 6 -


QUANTITATIVE 1H NMR METABOLOMICS ANALYSIS REVEALS DISTINCT METABOLIC ADAPTATIONS IN HUMAN MACROPHAGES FOLLOWING ACTIVATION

Amanda L. Fuchs, Mary Cloud B. Ammons, and Valerie Copie



Macrophages are phagocytic immune cells that can be found in nearly all tissues of the

human body, where they coordinate innate and adaptive immune responses in order to

maintain homeostasis. Therefore, macrophages are capable of displaying a spectrum of

functional phenotypes as a result of microenvironmental and stress-induced stimuli.

Recently, evidence has emerged demonstrating that metabolism is not only crucial for the

generation of energy and biomolecular precursors, but also contributes to the function

and plasticity of macrophages. Here, we used 1D 1H NMR-based metabolomics to

identify metabolic pathways that are differentially modulated following macrophage

activation with inflammatory, lipopolysaccharide (LPS) and interferon-ɣ (IFN-ɣ), or anti-

inflammatory, interleukin 4 (IL-4), cytokines relative to resting macrophages. Metabolic

profiling of inflammatory macrophages indicated a substantial increase in oxidative stress

and glutamate anaplerosis as well as a decrease in mitochondrial respiration; in addition,

these metabolic profiles demonstrate compelling evidence that inflammatory

macrophages divert metabolites from de novo glycerophospholipid synthesis to inhibit

oxidative phosphorylation and produce energy. Furthermore, glycolysis and lactic acid

fermentation were found to be significantly increased in both inflammatory and anti-

inflammatory macrophages; therefore, this finding is considered to be a universal

metabolic marker of activation. Our study supports previously published results, presents

novel metabolic pathway modulation findings with regard to human macrophage

activation, and contributes to the rapidly evolving field of immunometabolism.


- 7 -


ELUCIDATING ATTACHMENT OF A HYPER-THERMOPHILLIC VIRUS WITH CRYO-ELECTRON

TOMOGRAPHY

Ross Hartman, Mark Young, and C. Martin Lawrence



Archaeal Viruses represent an understudied field. The complete replication cycle has not been determined for any of these organisms. Although Sulfolobus Turreted Icosahedral virus (STIV) is one of the most studied archaeal viruses, still nothing is known about its attachment and entry phase. The virus uses unique 7-sided lysis structures for release from the cell. Given the demanding environment 80 ºC, pH 2-3, and low cell density, the virus may also utilize novel attachment and entry strategies. Using cryo-electron tomography we have imaged the virus bound to host pili as part of its initial infection sequence. The host pilus appears to bind at a cleft in the vial turret. Further work is needed to identify the pili structure and confirm its role in attachment and entry.


- 8 -


CATALYST-CONTROLLED SELECTIVITY IN NICKEL-CATALYZED CROSS-COUPLING

Leidy Hooker, Kayla Creelman, Emily Reeves, Emily Entz, and Sharon Neufeldt



Suzuki cross-couplings reactions allow creation of carbon-carbon bonds, usually between two aryl groups, using a metal catalyst. This work utilizes nickel as the catalyst. Nickel’s nucleophilic nature increases its reactivity with a broad array of electrophiles, specifically phenol-derived electrophiles. With numerous functional groups to work with, the goal of this project is to use ligands on nickel to control selectivity for different electrophilic functional groups in Suzuki cross-coupling reactions. In particular, we have found that nickel can be induced to react selectively with a halide functional group or, alternatively, with a phenol-derived functional group simply by switching the ligand in the reaction. This presentation describes our path to discovering this divergent selectivity and our efforts to understand it. We have screened numerous variables such as ligand, base, and solvent. Nuclear magnetic resonance (NMR) studies were performed to evaluate major intermediates in the Suzuki cross-coupling catalytic cycle. Evaluation of the NMR spectra obtained from these studies allow us to paint a picture of how and when the various reagents interact with the nickel catalyst. These studies are aimed at gaining an understanding of how sterics and electronics of the ligand play a role in determining what functional group reacts with the catalyst. Although further work is needed to improve yields and selectivities, we anticipate that these results will have significant implications for organic synthesis. The ability to switch catalyst selectivity in cross coupling reactions could facilitate synthesis of compounds ranging from polymers (e.g., plastics) to pharmaceuticals (e.g., drugs to treat leukemia).

scheduled presentation time: III. Organic Chemistry ABB134 1:20-1:40 pm

- 9 -


UNDERSTANDING THE BIOCHEMISTRY OF [FeFe] HYDROGENASE MATURASE ENZYME, HydE

Stella Impano and Joan B. Broderick



[FeFe] hydrogenase enzymes catalyze the reversible reaction between protons and electrons to

generate hydrogen gas, using an organometallic center termed the H-cluster. Assembly of this

cofactor is achieved by three maturase enzymes; HydF, a GTPase scaffold protein, HydE and

HydG, two radical S-adenosyl-L-methionine (SAM) enzymes. Collectively, these enzymes

biosynthesize a uniquely decorated 2Fe subcluster, whose irons are coordinated by diatomic

ligands CO and CN- and further bridged by a dithiomethylamine (DTMA) bridge. While HydG is

known to produce the diatomic ligands through a radical mediated decomposition mechanism of

its substrate tyrosine, the substrate used and function of HydE in hydrogenase maturation are still

a mystery to be uncovered. HydE is thought to synthesize the dithiomethylamine (DTMA) bridge

of the H-cluster through a condensation reaction between a substrate presumed to contain a thiol

functionality and ammonium. For this to happen, we hypothesize that this polyatomic ion would

need to bind at a site in/near the active site, surrounded by one or more negatively charged

residues. To investigate this possibility, thallium, a monoatomic cation that was previously shown

to interact with paramagnetic centers through hyperfine coupling, is used. Spectroscopic studies

show that in the presence of thallium, the electron paramagnetic resonance (EPR) spectra of

HydE’s radical SAM cluster experiences line broadening that can be partially reverted to the

original when ammonium is added. Additionally, in 2008, Nicolet et al showed that a strictly

conserved Glu58 is required for hydrogen activation. This discovery directed our focus on trying

to investigate if this residue facilitates ammonium binding and what would happen if it was

mutated. While our results show that Glu58 is not essential for Tl+, it was uncovered that

replacing this residue with alanine perturbs SAM binding and hence reduces the amount of

hydrogen produced, by hydrogenase.

scheduled presentation time: II. Biochemistry ABB138, 11:20-11:40 am

- 10 -


TRANSDUCING PROTEINS INTO AMPLIFIABLE DNA SIGNALS USING APTAMERS

Canberk Kayalar and Stephanie McCalla

Department of Chemical and Biological Engineering, Montana State University, Bozeman, MT, 59717


Recent advances in disease diagnostic technologies improve patient care and treatment; however they are mostly laboratory – based. The required centralized laboratories and highly trained staff are crucial for these methods, but hard to access in limited resource settings. New diagnostic methods come with a steep price tag which further impairs its ability to be utilized as a go to healthcare tool. An inexpensive method that is also simple and robust would fill the large gap between current technology and clinical need. Rapid and simple methods that detect biomarkers such as DNA, RNA and proteins sacrifices robustness and reproducibility for simplicity for speed. These sacrificed parameters are especially important when conducted in limited resource settings, because they can produce erroneous results which can lead to catastrophic outcomes. We propose a new molecular detection method for disease diagnostics based on a chemical amplification switch that is similar to the behavior that commonly observed in biological systems. The proposed amplification switch will convert the target antigen/protein to a high concentration of DNA signal by using aptamers. When target molecule is bound to an aptamer, it will initiate a conformational change in the aptamer, freeing the 3’ end and hence producing an “on” state. Non target molecules will produce minimal DNA signal since they won’t be able to trigger a conformational change and switch will remain in “off” state. Our goal is to deliver a fast and robust molecular diagnostics for all patients around world.


- 11 -


SYNTHESIS OF LACTOSE FUNCTIONALIZED LINEAR POLYMERS USING RING-OPENING METATHESIS

POLYMERIZATION

Jessi Langlais and Mary Cloninger



Protein aggregation is a phenomenon that can often be initiated by multivalent protein-carbohydrate interactions. Carbohydrate-functionalized polymers can be used very effectively to nucleate the formation of protein/polymer aggregates. For example, the Cloninger group has previously demonstrated that lactose functionalized polyamidoamine (PAMAM) dendrimers direct the aggregation of galectin-3, a β-galactoside binding protein that is important for cancer progression. In this research project, linear glycopolymers will be compared to the spherical glycodendrimers in terms of protein aggregation. The size and heterogeneity of galectin-3/glycopolymer nanoparticles will be compared. This talk will describe the synthesis and characterization of the linear glycopolymers using ring-opening metathesis polymerization (ROMP). The synthesis of the monomer units and of the carbohydrate derivatives will also be explained.


- 12 -


CHARACTERIZING THE ADHESION AND RHEOLOGY OF SOFT MATERIALS USING CENTRIFUGE

MICROSCOPY

Thomas B. LeFevre and James N. Wilking

1 Montana State University, Bozeman, MT


Soft materials are materials that can be easily deformed by mechanical or thermal stress. They include gels, colloids, granules, and liquids. One important example of a soft material is a biofilm. Biofilms - communities of microbes living on a surface - cost the U.S. billions of dollars every year in industry and medicine. Their adhesion strength, and the effects of anti-biofilm treatments on that strength, are not well quantified. I am using centrifuge microscopy (CM) to study biofilm adhesion. In CM, a microscope-camera assembly is placed in a centrifuge to observe the effects of the artificial gravity on a biofilm sample. When the biofilm detaches from its surface, the camera records the speed at which the centrifuge was running, which determines the force applied. This method addresses drawbacks of previously used methods that lack quantifiable adhesion data. Another application for CM is gravitational microrheology. Generally, microrheology is the study of material properties by tracking the motion of microbeads embedded in a material. The mechanism driving the microbeads varies between methods. In this method the motion is driven by the artificial gravitational force of the centrifuge. Image tracking software tracks the beads and then characterizes the viscosity and homogeneity of the material. Gravitational microrheology is new way of doing microrheology without applying heat, light, or electromagnetic fields to the sample.

scheduled presentation time: I. Materials Chemistry ABB134 10:00-10:20 am

- 13 -


FROM PLANT BIOPOLYMER TO HIGH-VALUE PRODUCT: UNDERSTANDING HOW A NOVEL AND

PROMISCUOUS CYTOCHROME P450 CATABOLIZES LIGNIN

Melodie M. Machovina, Sam J.B. Mallinson, Rodrigo L. Silveira, Marc Garcia-Borras, Christopher W. Johnson, Ellen L. Neidle,

Kendall N. Houk, Gregg T. Beckham, John E. McGeehan, Jennifer L. DuBois



Microbial conversion of plant-derived lignin, a vast and renewable carbon storehouse, to

useful products is of high interest and need in today’s society. O-aryl-demethylation is an

essential biochemical reaction in the pathway for catabolizing lignin-derived aromatic

compounds. Recently, we characterized a promiscuous P450 aryl-O-demethylase,

consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-

domain reductase (GcoB) that together represent a new two-component P450 class.

Though originally described as demethylating guaiacol to catechol, we show that this

system efficiently demethylates both guaiacol and an unexpectedly wide variety of

lignin-relevant monomers. Structural, biochemical, and computational studies of this

novel two-component system elucidate the mechanism of its broad substrate specificity,

presenting it as a new tool for a critical step in biological lignin conversion.


- 14 -


A BACTERIAL FLAVIN-DEPENDENT OXIDOREDUCTASE THAT CAPTURES CARBON

DIOXIDE INTO BIOMASS

Jenna R. Mattice,1 Bennett R. Streit,1 Greg Prussia,1,2 John W. Peters,2 and Jennifer L. DuBois,1

1 Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717

2 Department of Biochemistry, Washington State University, Pullman, WA


Atmospheric carbon dioxide is used as a carbon source for building biomass in plants and

photosynthetic microbes. Non-photosynthetic processes that also fix carbon dioxide have more

recently been discovered. This research focuses on a microbial mechanism for coupling

acetone to CO2 to make a central metabolite, acetoacetate. The key reaction is catalyzed by

NADPH-2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), a bacterial

enzyme that is part of the flavin and cysteine-disulfide containing oxidoreductase family

(DSORs) which are best known for reducing metallic or disulfide substrates. 2-KPCC lacks a

conserved, catalytically essential acid-base histidine possessed by all other DSOR enzymes,

having instead a phenylalanine (F501) at the same position. We hypothesized that F501 is

important for the reductive half reaction – which generates the reactive, C-S bond breaking

form of the active site. In typical DSORs, this reaction generates a Cys/FAD charge transfer

species. However, we showed that 2-KPCC generates an electronically unique form of the

active site, in which the flavin is oxidized and a pair of active site histidines are reduced and

protonated. We hypothesize that this form of the active site generates the substrate-reactive

Cys in a more nucleophilic form, where it is capable of cleaving relatively strong C-S bonds.

The resulting enolacetone carbanion is an extremely potent nucleophile that is capable of

directly attacking CO2. Research on 2-KPCC and other biological CO2 fixation methods adds

to our arsenal of strategies for carbon dioxide capture and use.


- 15 -


PFL-AE AND GD-AE, A TALE OF TWO RADICAL SAM ENZYMES

Elizabeth McDaniel and Joan B. Broderick



Radical S-adenosyl-L-methionine (SAM) enzymes make up one of the largest and most diverse enzyme families with over 113,000 known members. These enzymes are characterized by their utilization of a Cx3Cx2C motif coordinating a [4Fe-4S] cluster, which binds SAM and initiates homolytic cleavage of the small molecule into methionine and a deoxyadenosyl (dAdo) radical. Within this vast superfamily lies a subclass of enzymes, glycyl radical enzyme activating enzymes (GRE-AEs), which remove a hydrogen atom from a glycine residue in a given protein substrate producing a glycyl radical. Despite structural and mechanistic similarities, radical GRE-AEs are crucial in a variety of processes including anaerobic glucose metabolism in the case of pyruvate formate-lyase activating enzyme (PFL-AE) and polyether and polyester formation assisted by glycerol dehydratase activating enzyme (GD-AE). Over the past twenty-five years, the Broderick lab has developed a variety of growth and purification methods tailored to each radical SAM (RS) protein and has employed spectroscopic techniques to investigate mechanistic similarities among these enzymes. The research presented will highlight the commonalities and contrasts of the tools used to generate functional enzyme in the cases of two RS GRE-AEs, PFL-AE and GD-AE. Additionally, by using high performance liquid chromatography (HPLC), we counter previous statements that GD-AE cleaves SAM into 5'-deoxy-5'-(methylthio)adenosine and a 3-amino-3-carboxypropyl radical instead of the typical methionine and dAdo radical by showing MTA as a side product of SAM degradation rather than a product of its cleavage.

scheduled presentation time: IV. Biochemistry ABB138, 1:40-2:00 pm

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SINGLE CELL STUDY OF E. COLI BIOMINERALIZATION USING DROPLET

MICROFLUIDICS

Nada Naser, Neerja Zambare, Robin Gerlach, and Connie Chang



The main goal of this project is to investigate the fundamental principles of

Microbiologically Induced Calcite Precipitation (MICP), crystal nucleation, and factors

that affect the size and shape of crystals on the individual cell scale. MICP is the

chemical modification of an environment by biological activity that results in

supersaturation and the precipitation of minerals. It is an important field of study for

many eng -ineered and natural systems including: geologic carbon sequestration,

radionuclide remediation, soil stabilization and permeability manipulation. Even though

MICP has been of great interest for a number of years, the specific mechanisms

concerning induction of precipitation by bacteria have not been explicitly studied. In

previous studies, it has been hypothesized that the crystal nucleation starts at the cell

surface. However, this has not yet been definitively shown.

To study MICP, Escherichia coli MJK2, a bacterial strain engineered to express

green fluorescent protein (GFP) and carry out ureolysis was used. . Single MJK2 cells

were suspended in droplets of growth medium in oil, using a microfluidic droplet maker,

and observed with real-time imaging using Confocal Laser Scanning Microscopy

(CLSM). In preliminary experiments, the droplets enabled the observation of single

bacteria growing and precipitating calcium carbonate. Using microfluidic droplets to

encapsulate single biomineralizing cells is a novel approach to this area of research and is

anticipated to provide insight into crystal formation on a microscale as well as cell-to-cell

heterogeneity in the production of calcium carbonate.

scheduled presentation time: I. Materials Chemistry ABB134 11:40-noon

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ULTRASENSITIVE MICRORNA DETECTION FOR DISEASE DIAGNOSIS

Burcu Ozay, Stephanie E. McCalla, Cara Robertus, and Jackson Negri



Early detection of diseases has a huge impact on treatment success. Recent studies show that microRNA expression (miRNA) levels change in response to disease, often in the early stages, which makes them potential biomarkers for early diagnostics. However, miRNAs have short sequences and are usually in low concentrations, such that cost-effective specific detection is a challenge. Currently, qPCR is considered the gold standard for miRNA detection. However, qPCR is a time-consuming, expensive method that is not suitable for limited resource settings. In this study, we developed and characterized a new tunable DNA amplification chemistry that mimics the switch-like characteristics of biological systems such as cell signaling and genetic regulation. This tunable switch-like chemistry allows DNA amplification to be more specific by providing a yes/no answer for the presence of the miRNA molecules. The non-linear kinetics and high output nature of the reaction will make suppressing amplification below a threshold level possible, which turns a rapid method that can have false positives into a specific method. This application can be converted into a quantitative assay by counting single amplified molecules with help of microfluidic tools to create a digital assay for miRNA detection. A switch-based assay in clinics for early diagnosis, detection of diseases, or determining disease progression will be one step closer with this new technology. Our future goal is to diagnose a variety of diseases in early stages using newly developed miRNA detection methods that are rapid, affordable and easier to apply.

scheduled presentation time: II. Biochemistry ABB138 10:00-10:20 am

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THE ROLE OF ALLOSTERY IN THE ASSEMBLY OF THE HBV CAPSID

Angela Patterson, Navid Movahed, Lisa Selzer, Adam Zlotnick, and Brian Bothner



Hepatitis B Virus (HBV) is a global health issue that affects over 240 million people worldwide. Though there is an effective vaccine for HBV, we have yet to develop a cure for this virus. Further understanding of how the capsid assembles may lead to the determination of a possible drug target for HBV. The capsid is made up of 240 copies of the capsid protein (Cp) which spontaneously dimerize through interactions between the four-helix bundles of the monomers, and further oligomerize through interactions between hydrophobic patches on the dimers. Here we explore the role of allostery in HBV capsid assembly through the comparison of the dynamics of the assembly deficient mutant (Y132A) to an assembly competent wild type Cp construct (WTCp149). The mutation, which is located distal to the dimer-dimer interaction interface, produces a dimer that is structurally similar to WTCp149 yet the two proteins show differences in chemical and thermal stability as well as dynamics (tested through the use of hydrogen deuterium exchange-mass spectrometry). The differences observed in the dynamics of the two protein dimers are propagated throughout the entire protein structure showing the potential for an allosteric network to exist within the protein. Through the comparison of the dynamics of the free WTCp149 dimer, the WTCp149 dimer within a capsid, and the Y132A dimer showed the dynamics that are necessary to allow for assembly competence and which areas of increased stability cause assembly incompetence. This information may be used to help rationally design a capsid assembly inhibitor.

scheduled presentation time: IV. Biochemistry ABB138 1:20-1:40

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MOLECULAR DYNAMICS AND TIME-RESOLVED FLUORESCENCE STUDIES OF BASE STACKING IN

DNA DINUCLEOTIDES

Jacob Remington, Martin McCullagh, Pat Callis, and Bern Kohler



2-Aminopurine (2Ap), a fluorescent isomer of adenine (A), is frequently used to probe the local conformation of DNA through site-specific replacement of A with 2Ap and steady state and/or time resolved fluorescence measurements. In picosecond time-resolved fluorescence measurements of 2Ap labeled DNA oligomers, the signals are complicated and require 3-5 discrete exponential components, or a continuous rate distribution, to fit the data. Even after 50 years, the physical mechanism that gives rise to the fluorescence decay heterogeneity is poorly understood and vaguely attributed to base stacking. Here, time-correlated single photon counting (TCSPC) and molecular dynamics simulations (MD) are employed to unravel the heterogeneity present in the simplest 2Ap labeled DNA system, a 2Ap and A containing dinucleotide. The TCSPC results are consistent with literature and require four exponentials with lifetimes ranging from 300 ps to 10 ns, the lifetime of free 2Ap. Five independent two microsecond Amber MD runs in an explicit water model demonstrate the existence of four different base stacks, two partial stacks, and an unstacked structure. Leveraging state-of-the-art methods from the protein folding literature, a Markov State Model (MSM) of the dinucleotide conformational landscape reveals multiple sub-10 ns structural relaxation time scales, consistent with the TCSPC experiment. These results expand understanding of a commonly used fluorescence probe, while simultaneously quantify the kinetics of fundamental structural transitions in short DNA oligomers.

scheduled presentation time: I. Biochemistry ABB138, 9:40-10:00 am

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ZEOLITE BEAD HETEROGENEOUS CATALYST FOR BIOMASS UPGRADING

Joelle Romo, Tara Sundsted, Ting Wu, Jolie Lucero, Stephanie Wettstein, and Moises Carreon



Due to decreasing fossil fuel reserves, synthesizing value-added products from biomass is of significant interest. However, traditional homogeneous catalysts, such as sulfuric and hydrochloric acid, can poison downstream catalysts in addition to requiring special handling and resistant materials due to their corrosiveness. In this work, we show promising yields for the synthesis of platform chemicals, like furfural, levulinic acid, and 5-hydroxymethyl furfural, using solid acid catalysts in monophasic and biphasic systems with γ-valerolactone (GVL)/water. Zeolite 5A beads layered with SAPO-34 were used for the dehydration of xylose to furfural, with preliminary results of up to 24% yield in monophasic systems. The furfural yield can be increased to approximately 41% using a biphasic GVL/water system. This current work builds upon work with powdered SAPO-34 where furfural yields of 40% from xylose were obtained. The 5A layered SAPO-34 bead catalysts have potential for easier recovery and recyclability in addition to the 5A zeolite’s chemical and thermal stability. The use of GVL, a solvent readily obtained from biomass, provides a greener chemical pathway for furfural production.


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SELECTIVE SUZUKI CROSS-COUPLING TO SYNTHESIZE FUNCTIONALIZED ORGANOSTANNANES

John Russell, Emily Entz, and Sharon Neufeldt



Current strategies to synthesize tin reagents frequently involve high temperatures or the use of organolithium or Grignard reagents, thereby limiting functional group tolerance during the synthesis of tin reagents. However, we have found that room temperature Pd-catalyzed Suzuki cross-coupling between arylboronic acids and aryl halides takes place selectively in the presence of arylstannanes. This result is remarkable because tin reagents are also known to be competent nucleophiles in Pd-catalyzed cross-coupling reactions (the Stille coupling). The observed selectivity for cross coupling of boronic acids instead of organostannanes allows the installation of aryl rings bearing sensitive functional groups onto a substrate that contains a pre-installed tin substituent. The products are functionalized organostannanes, which can then be used as reagents in a Stille cross-coupling reaction. Because of the mild reaction conditions, this chemistry offers an alternative strategy for introducing sensitive functionalities into organostannanes without needing protecting groups. This presentation will describe our optimization of this selective cross coupling and our application of the methodology to obtain several functionalized tin reagents.

scheduled presentation time: III. Organic Chemistry ABB134 2:40-3:00

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ANALYZING VIRAL POPULATION DYNAMICS WITH MICROFLUIDICS

Humberto Sanchez and Connie Chang



Current approaches to treating viral pathogens target the virus at the time of diagnosis and are static. Therapeutic interfering particles (TIPs) are a promising solution of producing co-evolving viral therapies. One route to their development is to study how defective-interfering particles (DIPs) naturally occur and evolve during in vitro experiments, so that they can be used as a basis for TIP design. To determine how these TIPs and DIPs affect viral propagation and diversity we intend to track viral lineages in single-cell serial passages over time, quantify the viral diversity that results from infections of individual cells, and determine the parentage of the viral genome segments. The basis of this technology is the encapsulation of single cells infected with influenza A virus (IAV) and individual unique barcoding hydrogels. These polyacrylamide spheres are synthesized with unique DNA primers using a split and pool approach giving each sphere a barcoded sequence. When these primers are released from the hydrogels, an IAV specific sequence will complement the viral RNA and subsequent amplification will integrate the barcode sequences. Our protocol will generate barcoded viral RNA from all segments of the IAV genome which can be sequenced using PacBio technology. We have successfully synthesized polyacrylamide spheres and performed viral passaging from single-cell infection events with droplet microfluidics. Taken together, this methodology will allow us to perform whole genome sequencing to determine viral diversity from single-cell infection events.


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SELECTIVE SOLID PHASE EXTRACTION OF URANIUM AND ARSENIC USING SILICA POLYAMINE

COMPOSITES AND THE IMPLEMENTATION OF TRADITIONAL KNOWLEDGE AND CHEMISTRY

Ranalda L. Tsosie and Edward Rosenberg

Department of Chemistry and Biochemistry, University of Montana, Missoula, MT 59812


The legacy of mine waste contamination continues to affect many Indigenous communities

throughout the U.S. Often resulting in numerous water sources that exceed established

maximum contaminate levels for uranium, arsenic and other toxic metals. These

contaminations are a direct result from the improper disposal of mine waste materials and

abandoned mines. Some of these legacy-contaminations posed significant health and

environmental impacts on the community members and livestock.

To date, there are challenges for access to safe water despite many years of research

and remediation efforts. Traditional solvent extraction methods are expensive, time consuming

and pose additional problems with the generation of waste products. The aim of this study is to

use solid phase extraction methods to remediate contaminated water sources. An example is

Silica Polyamine Composites (SPC) that are engineered materials are rigid, thermally stable,

have high porosity and can be easily modified with metal selective functional groups. SPCs

have been primarily used in industrial settings, but this is the first application of SPCs to target

well water remediation.

Given the high valent nature of uranium and the effectiveness of adsorption of metals

from wastewaters and mine leachates by SPCs, we hypothesized that functionalized SPCs will be

effective at removing uranyl and arsenic ions from contaminated water. In addition to addressing

longstanding water contamination issues, a research framework that implements Diné philosophy

and teachings has been developed to pair with the aims of the study and to assist the community

with bringing about awareness of environmental exposures and risks.


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PROBING THE LEC-1 AND LEC-10 OXIDATIVE STRESS PATHWAY IN CAENORHABDITIS ELEGANS USING

GALΒ1-4FUC DENDRIMERS

Harrison W. VanKoten, Rebecca Moore, Coleen Murphy, Mary J. Cloninger



Reactive oxygen species (ROS) have been proposed to advance the universal process of aging. Although the role of galectins in this process is not yet fully understood, galectins have been proposed to serve as protective proteins during periods of high oxidative stress. For example, lec-1 and lec-10 knockouts in the model organism C. elegans showed an increased susceptibility to oxidative stress. Herein, we describe the synthesis of Galβ1-4Fuc functionalized onto the poly(amidoamine) (PAMAM) dendrimer framework. The dendrimer provides a scaffold for the multivalent display of Galβ1-4Fuc, since this glyco-epitope has been reported to bind well to C. elegans galectins and since multivalent interactions are important in mammalian galectin/glycan interactions. The knockdowns lec-1i and lec-10i were treated with these glycodendrimers and then exposed to oxidative stress. First, the knockdowns were given hydrogen peroxide to confirm they performed similarly to the lec-1 and lec-10 knockouts. C. elegans that had been pre-treated with the dendrimers were less susceptible to oxidative stress. An Alexaflour-488 dye was appended to the glycodendrimers, confocal microscopy images of C. elegans were obtained after exposure to the fluorescently-labeled glycodendrimers. The glycodendrimers mainly appeared within the digestive tract of the worms, and uptake into the vulva and proximal gonads could also be observed in some instances. The dendrimers were also seen in the neural channels where fluorescent dyes are known to collect.


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DESIGN, SYNTHESIS AND EVALUATION OF PRODRUG ANTIMICROBIALS TO CONTROL BIOFILMS

Danica Walsh, Tom Livinghouse, and Phil Stewart



The majority of microorganisms in nature, including those responsible for hospital-acquired infections and fouling of industrial processing equipment, live in association with surfaces as biofilms. Due to the secretion of proteins, extracellular DNA and lipopolysaccharides, biofilm communities are encased in a robust matrix which reduces their susceptibility to antimicrobial agents. The long term goal of my research project is to develop efficient, prodrug antimicrobial reagents that are able to permeate the biofilm matrix, as well as the cell membrane in order to eradicate biofilm colonization. This began with the literature review and minimum inhibitory concentration (MIC) evaluation of simple phenolic compounds with antimicrobial activity, followed by functionalization to increase toxicity and lipophilicity. Ester appendages are being placed on select compounds to implement a prodrug design, as is used in cellular dyes such as Calcein AM and pharmaceuticals such as Bolmantalate. As with fluorescent cellular dyes, once inside the cell the ester appendages will be cleaved, and the resulting compound will be negatively charged and trapped within the cell. It is our hypothesize that the efficacy of antimicrobial agents towards biofilms will be efficiently restored through this strategic design and synthesis of derivatives with modulated polarity that are engineered to have high levels of cellular retention upon undergoing a cleavage event in the cell. This new class of prodrugs presents a wide array of potential applications, from the control of biologically induced corrosion to the incorporation into household cleaning products.

scheduled presentation time: III. Organic ABB134 2:00-2:20 pm

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PRINTING CHANNELS IN HYDROGELS WITH STEREOLITHOGRAPHY-BASED 3D PRINTING

James N. Wilking, Aaron D. Benjamin, and Reha Abbasi



Hydrogels are soft, water-based gels with widespread applications in personal care products, medicine and biomedical engineering. Many of these applications require structuring the hydrogel into complex three-dimensional shapes. For these applications, 3D printing offers exquisite control over material structure. Light-based 3D printing, in particular, is rapid and can be used to create high-resolution material features; however, the use of this approach for structuring hydrogels is underdeveloped. In particular, the ability to print hydrogel objects containing submillimeter channels and overhangs is limited. This is largely due to the lack of water-soluble, biocompatible photoblockers, which can be used to limit the propagation of light into the object and prevent unwanted internal curing. Here, we present the use of chlorophyllin, a hydrolyzed derivative of chlorophyll, as a water-soluble, biocompatible photoblocker for light-based 3D printing of hydrogels. We show that the attenuation provided by chlorophyllin dramatically improves resolution for printed hydrogels. By systematically varying the chlorophyllin concentration and the layer thickness, we determine optimal conditions for printing 3D hydrogel structures with high resolution and minimal bleedthrough. We demonstrate the potential of this formulation by printing hydrogels with complex, submillimeter channels.


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HIGH-THROUGHPUT IDENTIFICATION OF INFLUENZA INTERFERING PARTICLES USING DROPLET-BASED

MICROFLUIDICS

Geoffrey Zath and Connie Chang



The influenza virus infects millions every year while causing over 300,000 deaths worldwide. The current form of treatment is through vaccinations of the public; however, vaccinations are a passive approach and are not able to adapt with the virus over the course of a flu season. A proposed method for dealing with a mutating virus is to engineer Therapeutic Interfering Particles (TIPs) that co-evolve with the virus and outcompete for the same essential viral components within the host’s cells. The challenge we face is finding a TIP candidate that remains effective in fighting a virus over the course of an infection. We aim to use droplet-based microfluidics to increase the throughput of this search over a thousand-fold when compared to traditional techniques and at a fraction of the cost. Sample encapsulation into droplets provides us the unique ability to observe interactions at a single cell level in picoliter volumes. Our research plan is twofold: first, develop a droplet-based microfluidic method for the passaging of viruses and TIPs with host MDCK cells in droplets, and second, develop an in-drop qPCR method to quantify viruses and TIPs within droplets. We plan to use these devices for multiple rounds of passaging and compare our results to parallel experiments in bulk. By drastically increasing our sample size over what is possible in bulk, we are increasing the possible infection/competition events between virus and TIP and providing an extensive amount of data that will hopefully give us insight into the effectiveness of specific TIP candidates.


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