The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown, to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan autofluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes.

Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns

Fast scan cyclic voltammetry (FSCV) and carbon-fiber microelectrodes (CFMEs) have been utilized used to detect several important neurochemicals in vivo. However, this method is limited due to the ability to discriminate dopamine from several of its metabolites. Carbon nanotube and polymer modified microelectrodes will be utilized to detect physiologically low levels of neurotransmitters that also resist surface fouling and have high temporal resolution to detect fast changes of neurotransmitters. Furthermore, novel electrode coatings and waveforms will also be utilized to detect several neurotransmitter metabolites such as dopamine, norepinephrine, normetanephrine, 3-methoxytyramine (3-MT), homovanillic acid (HVA), 3,4 dihydroxyphenylacetic acid (DOPAC), and other metabolites. Currently, dopamine is thought to be an important neurotransmitter concerning several disease states such Parkinson’s disease, drug abuse (amphetamine, cocaine, etc.), and even for gambling and sex-disorders. However, dopamine is metabolized on a subsecond timescale, and studies have pointed to the importance of neurotransmitter metabolites in these disease states apart from dopamine. Presently, there is no method to selectively co-detect these neurotransmitter metabolites of dopamine utilizing FSCV. Through several waveform modifications and polymer electrode coatings, we develop a novel method to tune the detection of dopamine and said metabolites, which will help differentiate dopamine and respective metabolites through the shapes and positions of their respective cyclic voltammograms. Preliminary measurements have also been made in zebrafish whole brain ex vivo showing the application of this technique in biological tissue. Discriminating the detection of dopamine from its metabolites will have many implications in better understanding complex disease, behavioral, and pharmacological states.

Differentiation and detection of dopamine monoamine metabolites with carbon fiber-microelectrodes and fast scan cyclic voltammetry

Flash chromatography, a staple component of medicinal chemistry workflow, consumes a lot of organic solvent (upwards of ½ million liters annually just in North America). Most, if not all, of this organic waste is incinerated off-site, liberating CO2 into the atmosphere. Because of this environmental impact, many pharma companies are instituting requirements to reduce organic solvent waste but leaving the implementation up to their chemistry departments. In this poster, we describe several proven ways to reduce organic solvent use without sacrificing purification efficiency.

Green flash chromatography

Protein arginine methyltransferase 5 (PRMT5) is an important epigenetic enzyme involved in cancer development. It carries out symmetric dimethylation on histone proteins, especially on H4R3, utilizing S-adenosyl-L-methionine (SAM) as a cofactor. PRMT5 is overexpressed in a variety of cancers, especially MTAP-deleted cancers, making it a compelling target for anticancer therapeutics. Here, we report an apo structure of the human PRMT5:MEP50 complex determined at a resolution of 3.4 Å by single-particle cryo-EM. The intrinsic flexibility of the loop region (residues 292–329) at the active site of apo-PRMT5:MEP50 suggests a huge conformational change induced by the binding of a nucleoside-containing molecule at the cofactor-binding site. Based on the structural feature of apo-PRMT5:MEP50, we have optimized our carbazole-based lead compound CMP5 into peptide-competitive inhibitors (e.g. 11-1F, 11-2F and 11-9F) that bind to the substrate-binding site in the presence of a nucleoside-containing molecule, such as 5’-methylthioadenosine (MTA), binding at the cofactor-binding site. The enzyme inhibition IC50 of compound 11-1F was determined in MTase-Glo assays at 1.3 μM, and its mechanism of inhibition was confirmed as SAM-uncompetitive and peptide-competitive. The cryo-EM structure of PRMT5:MEP50 bound with MTA and 11-2F (IC50 = 0.9 μM) was determined at a resolution of 3.3 Å. By structure-based drug design and SAR analysis, we have identified compound 11-9F as a potent and selective inhibitor with an IC50 value of 183 nM. Here we describe the design and characterization of 11-9F as a peptide-competitive, MTA- synergistic PRMT5 inhibitor with anti-proliferative effects in several breast cancer cell lines. Protein-inhibitor binding experiments were performed by Surface Plasmon Resonance (SPR) to analyze the binding preference of 11-9F towards apo and SAM/SAH/MTA-bound forms of PRMT5:MEP50 and the binding affinity KD values were determined. 11-9F showed stabilization of PRMT5 in cellular thermal shift (CETSA) target engagement assay and the melting temperature was shifted by ~2 °C. Treatment of 11-9F with or without MTA in breast cancer cell lines led to inhibition of symmetric dimethylation and cell death with IC50 values in the single-digit micromolar range. The in vivo evaluations are currently underway.

Structural and biochemical study of human PRMT5 and its peptide–competitive MTA–synergistic small molecule inhibitors for cancer therapeutics

Surfactant protein B (SP-B), a component of lung surfactant (LS), plays an essential role in lowering the surface tension of alveoli, and in trafficking lipids to the air-fluid interface. Challenges in expressing native SP-B, have resulted in a number of synthetic peptide analogs being used to mimic its functionality for mechanistic studies and surfactant replacement therapies. Specifically, the terminal ends of SP-B, SP-Bc and SP-Bn, have retained certain functional properties of the protein and can be more easily studied. SP-Bc, is examined in comparison to its synthetic mimic, KL4, which is the main ingredient of a surfactant replacement known as Surfaxin . In our studies, we utilize fluorescently labeled lipid probes, NBD-PE and Rhodamine-PE, to monitor membrane fusion events in DPPC:POPG and POPC:POPG liposomes using Fluorescence Resonance Energy Transfer (FRET). A decrease in FRET among the studied peptides will indicate increased rates of membrane fusion and may reveal a selective interaction with certain LS lipids. Current data displays a decrease in FRET within the DPPC:POPG SP-Bc system which supports previous NMR studies that showed the peptide selectively interacts with DPPC-rich liposomes and may indicate that SP-Bc facilitates membrane fusion events, which is a proposed mechanism of lipid trafficking at the air-fluid interface.

Using FRET to elucidate the lipid trafficking mechanism of SP-Bc and N terminal peptides in comparison with KL4

Coordination compounds with 1,2-ethylenediamine (en) ligands have played vital roles in the development of inorganic chemistry and stereochemistry. Nobel laureate Alfred Werner synthesized and resolved both enantiomers of [Co(en)3]3+ into their respective Λ and Δ forms over a century ago. Despite this rich background, these complexes have remained dormant in terms of synthetic utility until recently. This is largely due to the strong field amine ligands that are difficult to displace and their low spin d6 electron configuration, rendering these octahedral complexes kinetically substitution inert. Conversely, these attributes, in addition to the quantity and quality of NH units, lend Werner complexes to serve as effective chiral H-bond donating catalysts, featuring potential applications in the synthesis of pharmaceuticals and agrichemicals. Central to this goal are the anion metathesis reactions conducted for the substitution of commonly paired halide anions with lipophilic anions. These reactions enhance the solubility of the complexes in organic solvents and decrease the competition between anions and substrates for NH donor sites. Amongst the latest discoveries, lipophilic cobalt cage complexes have led us to deduce that high enantioselectivity is predicated on the substrates’ ability to hydrogen bond with the C3 faces of the catalysts. Additionally, the variation of [M(en)3]n+ to include Mn+ = Cr3+, Rh3+, Ir3+, and Pt4+ has furnished complexes with variable thermal stabilities. Finally, the ligands have been further decorated with functionalized phenyl rings via simple, stereospecific synthetic routes resulting in catalysts with a breadth of steric bulk, NH unit acidities, and solubility profiles.

Syntheses of Werner complexes: Applications as chiral hydrogen bond donors in enantioselective catalysis

Cometary ices have remained largely unprocessed since they accreted, so their study provides unique information on the earliest history of our Solar System, as well as providing insight into the chemical regents available for delivery to primitive planetary surfaces. Cometary ice abundances are derived primarily through remote observations of their atmospheres (comae). However, through observations of spatially extended distributions, several commonly observed species (such as CN, C2, HNC and H2CO) are known to be produced as a result of coma photochemistry/thermal breakdown of dust, and may not be native to the nucleus. The assumption that complex organic molecules (such as HC3N, NH2CHO, and CH3OCHO), commonly observed using single-dish radio spectroscopy are present in cometary ice is largely unproven. In fact, our new chemical/hydrodynamic coma model shows that some of these organics could instead be produced in the coma via gas-phase chemistry. Here we present chemical model calculations for HC3N and NH2CHO, which reveal a coma origin for these species via simple neutral-neutral reactions involving known coma species. This theory can be tested by mapping the distributions of these molecules using ALMA.

Chemical origins of complex organic molecules in cometary comae

Almost all chemical and biological processes can be viewed as complex networks of discrete chemical states connected by dynamic transitions. It is widely believed that to fully understand the mechanisms of these complex processes one should determine the structure of underlying networks. A new model-free theoretical method of determining mechanisms and dynamic properties of complex chemical and biological processes, which utilizes a first-passage analysis, is developed. It is stimulated by single-molecule experimental measurements on dwell times distributions of events that correspond to transitions between specific chemical states. Our approach is based on the idea that full temporal distributions of events between two arbitrary states contain full information on the dynamics of the system. Using a separation of time scales, it is shown that important aspects of the mechanisms of chemical and biological processes can be easily determined by analyzing the processes at early times and at long times. Our theoretical approach is illustrated on several biological systems.

Determining mechanisms of complex chemical and biological processes using networks analysis

Microbial biofilms are ubiquitous in nature, and they pose a serious threat to public health. Staphylococcus epidermidis and Staphylococcus aureus are the most common clinical isolates from healthcare- and medical device-related biofilm infections. No antibiotic currently on the market can eradicate pathogenic biofilms, which contain complex defense mechanisms composed of slime-like extracellular polymeric substances (EPS). Understanding the need to develop alternative approaches, we examine 600-Da branched polyethylenimine (BPEI) against methicillin-resistant S. epidermidis and S. aureus (MRSE/MRSA) biofilms. In this study, a microtiter biofilm model is used to test the synergistic effects between the two components of our combination treatment: BPEI and β-lactam antibiotics. Electron microscopy was used to confirm the growth of MRSE biofilms from the model. Minimum biofilm eradication concentration (MBEC) assays, crystal violet assays, and biofilm kill curves suggest that BPEI exhibits antibiofilm activity and can potentiate β-lactams to eradicate MRSE and MRSA biofilms.

Antibiofilm synergy of β-lactams and branched polyethylenimine against MRSE and MRSA

In the last decade, polydopamine (PDA) has become a subject of considerable scientific interest due to its universal adhesive properties. Dip coating, the traditional method of adhesion, does not allow for investigation into the adhesiveness of PDA at any given time point as the substrate and PDA solution are in continuous contact until a desired thickness of PDA is achieved. By utilizing a spin-coating  method, we aim to determine how PDA adhesiveness changes over time. This would inform both dip coating and other adhesion methods, such as spray coating, as researchers could allow a PDA solution to age to its optimal reactivity level before introducing it to substrates of interest. As PDA is a universal adhesive, we analyzed three different substrates, silicon dioxide, polydimethylsiloxane, and aminopropylmethylsiloxane-dimethylsiloxane copolymer to warrant that conclusions drawn are relevant to substrates with varying hydrophilicity and interactions with PDA. We also designed a modified dip-coating method which mimics the timing and DA concentration used in the spin-coating protocol to ensure that our results are widely applicable and not inherent to the spin-coating method itself.

Optimization of polydopamine adhesiveness during thin film fabrication

The metastability of a thin film refers to the co-existence of stable and unstable states as a function of film thickness. The film is unstable at low film thicknesses, whereas at large thicknesses, the film is stable unless heterogeneous nucleation takes place. Poly(vinyl alcohol) (PVOH) thin films were chosen in this research as PVOH is a water-soluble, thermally stable, atactic yet semi-crystalline polymer. Polydimethylsiloxane (PDMS) polymer was chosen as the substrate material because it has an extremely low glass transition temperature and can modulate the extent of the instability that it exerts on the PVOH thin films. Film-preparation methods included both static adsorption and dynamic spin coating. As-prepared and thermally annealed thin films were analyzed using contact angle goniometry, ellipsometry, optical microscopy, and atomic force microscopy. PDMS molecular weight, PVOH solution concentration, spin rate, and thermal annealing allowed the control of the PVOH film thickness and morphology. Among the parameters probed, PDMS molecular weight plays a substantial role in controlling the stability of the PVOH thin films. Metastability was observed when PDMS molecular weights between ~200 Da and ~2000 Da were employed.

Elucidation of conditions for metastable poly(vinyl alcohol) thin films on polydimethylsiloxane substrates

Over the last decade, nanoparticles have become particles of great research interest because of their inherent chemical compositions such as size, shape, ability to be coated by other molecules and control over their magnetic properties. Assembly of these particles into a monolayer allows their properties to be harnessed and applied in various fields of studies. One method that has been adopted to enable this monolayer formation is the annealing of nanoparticles. The goal of this experiment was to anneal decanoic acid coated iron oxide nanocubes at different temperatures and time intervals, to find the optimal conditions for the formation of a smooth monolayer. Using the spin casting method, the magnetic iron oxide nanocubes were spun onto a Transmission Electron Microscopy (TEM) grid, where they were subjected to different conditions of plasma cleaning, temperatures and annealing times. The grids were then imaged under TEM at different magnifications. Using the program, Image J, it was found that the nanocubes maintained their shape and the mean cubic length of 15.5 nm at temperatures ranging from 100 – 150 oC. The results so far indicates that plasma cleaning of TEM grids can hinder the formation of a uniform monolayer. This could be due to the unfavorable interaction between hydrophobic decanoic acid coating of nanocubes and the hydrophilic TEM grid.

Magnetic iron oxide nanocubes: Effect of annealing on the formation of a monolayer

An understanding of film stability is useful in crafting predictable film morphologies, which have a wide range of applications extending from self-assembled nanostructures to coatings for engineered materials. The stability of a thin film on a solid support is affected by the nature of the thin film and the underlying substrate as well as their interactions. Thin films are commonly prepared by depositing organic solutions of nonpolar materials onto silicon wafer (SiO2); aqueous solutions are rarely used due to the destabilizing polar interactions during solvent evaporation leading to dewetting. In this study, hydrophilic and semi-crystalline poly(vinyl alcohol) (PVOH) thin films from aqueous solution were fabricated on SiO2 via spin casting. Experimental parameters including PVOH concentration, PVOH degree of hydrolysis, spin rate, and thermal annealing were probed. The PVOH thin films appeared to be continuous under all the conditions examined, demonstrating the stable nature of the PVOH-SiO2 system. The extensive hydrogen bonding between the films and the substrate as well as the crystallization within the thin films appear to play more dominant roles than the destabilizing polar interactions during solvent evaporation in dictating thin film stability.

Preparation of stable poly(vinyl alcohol) thin films from aqueous solution on silicon wafer

The separation of C2H2 from CO2 in mixtures containing these gases is an important application to produce high-purity C2H2. Experimental and theoretical C2H2 and CO2 adsorption studies were performed in MPM-1-Cl and MPM-1-TIFSIX, two robust molecular porous materials (MPMs) with the empirical formula [Cu2(adenine)4Cl2]Cl2 and [Cu2(adenine)4(TiF6)2], respectively. Both MPMs consist of a hydrogen-bonding network that is self-assembled by dinuclear copper–adenine paddlewheel complexes. The difference between the two structures is simply the axial ligand. Experimental measurements revealed that MPM-1-Cl displays higher low-pressure uptake, isosteric heat of adsorption (Qst), and selectivity for C2H2 than CO2, whereas the opposite is true for MPM-1-TIFSIX. Dynamic breakthrough experiments and modeling studies confirmed that MPM-1-Cl is more selective toward C2H2, while MPM-1-TIFSIX is more selective toward CO2. Molecular simulations revealed that both MPMs display a greater preference for C2H2 within their large channels. However, MPM-1-TIFSIX contains a small accessible channel in the structure that is not present in MPM-1-Cl; this channel represents the primary binding site in the material. Calculation of the classical MPM–adsorbate interaction energies and diffusion barriers about the primary binding site in MPM-1-TIFSIX revealed that this site is more favorable for CO2 than C2H2, thus explaining why the selectivity in this MPM is inverted relative to MPM-1-Cl.

Tuning the selectivity between C2H2 and CO2 in molecular porous materials

Nanoscale phenomena are under increasingly intense investigation both in academia and industry. The unique physical and chemical properties stemming from their high surface area and confined space lead to properties that are between atomic and bulk. Students need experience in nanoscience to enter this growing workforce, and begin nanoscience research. This laboratory experiment, implemented as a two part module, introduces the optical properties of noble metal nanoparticles and probes the effect that the metal composition has on these properties. An aqueous synthesis is performed, which allows for control of the alloy composition of Au/Ag nanoparticles, while minimizing costs and hazardous waste. The composition of the synthesized particles is verified by mass analysis. Extinction spectra are recorded experimentally and determined in silico. The experimental and theoretical spectra are compared, and students identify causes for discrepancies based on mass and particle size analysis. Students actively engage in identifying how the dielectric function of a nanoparticle affects the optical properties of their synthesized nanoparticles. Students gain practical knowledge of the synthesis and characterization of metal nanoparticles. Students are also introduced to elements of theoretical chemistry. These experiments as a module have been reproducible; they utilize primarily non-hazardous, non-toxic, green materials, and they can be performed with minimal added costs to normal budgets for lab courses.

Optical properties of alloyed noble metal nanoparticles: Nanotechnology experiment for chemistry and engineering students

Process skills such as information processing, critical thinking, teamwork, and communication are key learning outcomes for undergraduate STEM programs including chemistry. For example, information processing is a skill that is necessary to evaluate all the information provided in a chemistry problem and then to be able to transform the information into another form or representation. In organic chemistry, students use information processing to convert between multiple representations such as wedge-dash forms and Newman projections. Likewise, critical thinking skills are necessary to synthesize various aspects of chemical knowledge to determine the products of a reaction. Over multiple years, process skills were assessed in a large-enrollment organic chemistry course using feedback rubrics developed by the ELIPSS Project (Enhancing Learning by Improving Process Skills in STEM). Undergraduate teaching assistants used the rubrics to assess the students’ skills during active learning activities and group quizzes and reflected on their facilitation. Students assessed their own skills and wrote reflections based on the TA rubric scores. This presentation will focus on the development of process skills in a large-enrollment organic chemistry course by discussing how the TAs assessed and provided feedback to the students, the accuracy of the student self-reflections based on the TA rubric scores, and how process skills can be connected to course content.

Using rubrics to assess process skills in a large-enrollment organic chemistry course

Improvement of sludge dewaterability is greatly hindered by the presence of large amount of water molecules trapped in sludge as a result of its strong hydrophilic characteristics. This study investigates the influence of linear and branched structures of polyacrylamide (PAM) with different charge density (CD) on the electro-kinetics, dewatering and volume reduction of highly stable industrial membrane bioreactor (MBR) sludge. The presence of PAM has induced intensive impact on the flocculation behaviour of the sludge as revealed by supernatant turbidity, zeta potential, capillary suction time (CST), flocs size and settleability. Enhancement of the flocculation and dewatering of sludge is correlated to the surface charge neutralization and bridging mechanisms. The highest optimum dose is obtained as 70 mg.L-1 for linear PAM of 40% CD whereas the lowest value is determined as 30 mg.L-1 for branched PAMs. In all cases, turbidity removal of 99.9% and CST reduction of 51-64% are attained where the linear PAMs revealed the highest CST reduction. As per the flocs formation, higher PAM dose results in bigger flocs until the saturation point, beyond which the surface saturation occurs. The sludge percent volume reduction is measured and correlated to the flocs size and PAM dose. At an optimum PAM’s dose, bigger flocs resulted in higher percent volume reduction, however, at a unified PAM’s dose, branched structure of PAM achieved higher percentage volume reduction having smaller flocs size. Overall, the branched structure of polyacrylamide was more efficient in flocculation of MBR sludge at lower dose of 30 mg.L-1 for both 40 and 60% CD and attaining the highest percentage volume reduction of 58%.

Influence of polyelectrolyte architecture on the electro-kinetics and dewaterability of industrial MBR activated sludge

A simple extraction and purification of bacterial flagella was developed, with the concentration of 83.54 mg/ml. Au nanowires were synthesized using bacterial flagella as templates with uniform aspect ratio homogeneous along the whole Au nanowires more than 60. The nanowires were scarcely clustered as straight as nanorods with no fold-tendency under transmission electron microscopy (TEM). Olive leaf extract has been shown to inhibit the growth of Listeria monocytogenes, an important foodborne pathogen. Verbascoside and Oleuropein are two major components in olive leaf extract. The synthesized Au nanowires were highly active at the concentration of 128μg/ml for electrocatalytic inhibition of Verbascoside, Oleuropein and Olive leaf extract towards L. monocytogenes F2365 which significantly reduced the Minimal Inhibitory Concentration (MIC) concentrations compared with no addition of Au nanowires. Results from Ames assays indicate that synthesized Au nanowires had no mutagenic activities at the above catalytic concentration. The AU nanowires we developed have the potential to be used as anti-microbial food packaging materials to enhance food safety.

Natural-flagella-templated Au nanowires and their neo-function as antimicrobial adjuvants

This study investigates the influence of hybrid coagulation-flocculation system on the flocculation and dewaterability efficiency of a real highly stable membrane bioreactor (MBR) sludge. Two organic coagulants, polyDADMAC (FL 4440) and polyamine (FL 2949) coupled with different structured polyacrylamides (PAMs) are used. Residual turbidity, zeta potential (ζ), flocs size and capillary suction time (CST) are used to assess the impact of the hybrid system on the degree of flocculation and dewaterability of MBR sludge. Addition of FL 4440 and FL 2949 prior to PAM has greatly reduced the turbidity and ζ of the sludge suspension by decreasing the required doses of PAMs 67% to 80% depending on the type of the PAMs used. Polyamine coagulant was more efficient in coagulation and hybrid coagulation-flocculation for MBR activated sludge. The impact of FL 2949 and FL 4440 was more significant on the linear structured PAMs with 71 and 80% reduction in the required PAM’s dose for 40% CD (FO 4490 SSH) and 60% CD (FO 4690 SSH), respectively followed by slightly and highly branched structured PAMs. Similar observations were observed for flocs size and dewaterability (CST) measurements of the flocculated MBR sludge. The results showed a maximum reduction of 58% in the flocs size for sludge conditioned with PAMs in the presence of FL 2949 and FL 4440. Nevertheless, comparable results were obtained for the dewaterability of the flocs produced by PAMs individually and hybrid system. Overall, this study successfully determines the effect of hybrid coagulation-flocculation on the flocculation performance of the MBR sludge.

Enhancement of flocculation and dewaterability of MBR activated sludge using a hybrid system

A course intended to ease the transition from Organic Chemistry I to II was designed for a two-week January term that features sharply focused topics courses. The goal is to facilitate student success in the inherently cumulative yearlong Organic Chemistry sequence. Key topics from Organic Chemistry I are revisited (e.g. spectroscopy, substitution/elimination) to ensure that students have the proper foundation to proceed. Then, a selection of Organic Chemistry II material is introduced. The course focuses heavily on interactive problem solving and utilizes an open-access text written by the instructor. YouTube videos created by the instructor also support student learning.

Bridge course between organic chemistry I and II

This project reports the development or modification of protocols used to synthesize and characterize methyl 8-(2’5’-hexylthienyl) octanoate (23%), epithiostearate (27%), cross-linked disulfide (10%), crosslinked sulfide (7.5%) and epidithiosterate (55%). Our research team is interested in these molecules because a combination of distillation, extraction, and analytical techniques (GC-FID, GC-PFPD, and GC-MS) have identified them as possible sulfur (S)-bearing contaminants in biodiesel derived from brown grease lipids (BGL). BGLs are the primary components of dewatered grease trap waste and sewage scum grease. It has recently been used as a low-value feedstock for biodiesel production; however, the S content of the resultant biodiesel is too high to meet industry specifications. The S-bearing compounds described herein do not exist in the MS library. Therefore, this project was devised to generate the analytical data necessary to verify the existence of the S-bearing compounds presumed to exist in BGL-biodiesel. The identification of S-bearing compounds in BGL-derived biodiesel is necessary to devise effective desulfurization protocols needed to reduce the concentration of S-bearing impurities in biodiesel to &lt; 15 ppm, as specified by ASTM.

Verification of sulfonated compounds found in biodiesel produced from brown grease lipids (BGL)

Our objective is to design, build and demonstrate a miniaturized instruments for analysis of organic and inorganic compounds in planetary materials with high sensitivity (parts-per-billion/trillion depending on target species) and accuracy, with a particular focus on detecting biomarkers extracted (or direct entry) from gases, rocks, soils, liquids, and ices. The ultimate goal of this research is to develop a miniaturized instruments for both robotic and human missions. We have been using Micro-Electro-Mechanical Systems (MEMS) technology to reduce the size, mass and power of the three classical chromatographic technologies: gas chromatography (GC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Both CE and HPLC are liquid chromatographic (LC) technologies. The Planetary Surface Instruments Group at JPL is drawing upon its experience in successfully developing and constructing MS flight instruments for the MEMS GC, CE, and LC systems. Our group has been responsible for the original research and flight development of several different miniature mass spectrometers including a Paul quadrupole ion trap (QIT) MS that was used in the Vehicle Cabin Atmosphere Monitor (VCAM) and will be used in the Spacecraft Atmosphere Monitor (S.A.M.). Our instrumentation is cross-cutting with the human program and, in fact, we have already flown a GC system to the International Space Station for environmental monitoring (VCAM) and will fly a MEMS-GC in 2020 (S.A.M.). HEOMD goals include (1) experiments on ISS (2), astronaut health monitoring, (3) environmental monitoring, (4) field laboratory.

MEMS liquid and gas chromatography for human and robotic missions

APOBEC3H (A3H) is a cytidine deaminase most well known for its involvement in innate human immunity to HIV. It exists primarily in an RNA-mediated dimer and acts preferentially upon a single-stranded DNA substrate containing a TCA consensus sequence. The Cisneros group recently reported a lung-cancer associated single nucleotide polymorphism on A3H that results in the A3H K121E missense variant (DNA Rep.,70, 2018, 10). Here, we characterize several aspects of A3H including substrate binding orientation and selectivity, RNA mediated interface features, and the impact on the structure/function of the cancer mutation. Our results are based on extensive molecular dynamics (MD) calculations totaling 11.25 μs of simulation time using the pmemd.cuda engine in the AMBER software suite and OpenMM on a combination of Nvidia K80 and V100 GPUs. Our calculations comprise eighteen monomer A3H systems (34,000 atoms) to investigate substrate binding and selectivity and four A3H dimer systems (260,000 atoms) to determine the impact of the cancer mutation. Based on our simulations, we have obtained a possible preferred substrate orientation, uncover residues responsible for the preferred A3H consensus sequence, and observed that the A3H K121E exhibits diminished stability at the dimer interface compared to the wild type A3H. More importantly, our simulations allowed us to predict a rescue mutation, K117E, to compensate the effects of the K121E cancer variant. These predictions have been tested and confirmed experimentally. The single point mutation K117E was also experimentally observed to produce a stable A3H, and was then tested computationally. In this poster I will present and discuss our computational predictions and experimental results.

Characterization of APOBEC3H substrate binding orientation/selectivity and effect of a lung–cancer associated mutation and rescue mutation

Boron Neutron Capture Therapy (BNCT) is a binary radiotherapeutic cancer treatment that has shown great promise in clinical trials, in which delivery agents containing 10B atoms are selectively transported into tumor cells and then irradiated with low energy (0.025 eV) thermal neutrons. BNCT success rate is governed by selectivity of the drug towards tumor cells, and absolute cellular concentration of at least 10-30 μg of 10B atoms/g of tumor. Matrix Metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are involved in the remodeling and degradation of all components of the extracellular matrix (ECM). MMP enzymes play a key role in normal development, morphogenesis, bone remodeling, wound healing, and angiogenesis, yet inappropriately high MMP activity has been implicated in a number of disease states including tumor growth and metastasis and in the degradation of articular cartilage in arthritis. In particular, upregulation of gelatinases MMP-2 and MMP-9 have been reported to be crucial for tumor angiogenesis and metastasis in cancer. In order to halt disease progression resulting from exaggerated matrix remodeling mediated by MMPs, MMP inhibitors (MMPi's) have been extensively explored as promising antitumor targets due to their selectivity and tight binding to different MMPs. We selected to append a borane-rich carborane cluster onto an MMP-targeting pharmacophore to enable delivery of 10B atoms into a tumor. The piperidine α- sulfone hydroxamate pharmacophore of MMP inhibitors SC-76276, SC-78080, and SC-77964 was selected, as this series of compounds is potent for the desired target MMP-2 and MMP-9. The design and synthesis of novel BNCT agents is described. Furthermore, to understand the binding of synthesized BNCT analogs, the Molecular Operating Environment (MOE) software was utilized for docking studies with MMP2 (PDB 1HOV). To enable the docking experiments to be performed, the carborane cluster in each analog was replaced with a phenyl group. Docking suggests that the hydroxamate chelates the active site’s zinc atom, and the diaryl ether occupies the S1’ pocket.

Carborane-containing α-sulfone hydroxamate MMP inhibitors for boron neutron capture therapy (BNCT) in cancer treatment

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.

Presentations