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Biomedical Technologies: Imaging, Diagnostics, & Other Integrative Approaches to Study & Model Diseases: Unique Metamaterials for Imaging and Diagnostics Applications
02:00pm - 04:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Yonghyun Kim, Organizer, The University of Alabama; Maryam Raeeszadeh Sarmazdeh, Organizer, University of Nevada, Reno; Adam Melvin, Presider, Louisiana State University; Divya Chandra, Presider, ‍ ; Christopher Canova, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

A comprehensive understanding of diseases coupled with their effective diagnosis and treatment is inherently linked to the technologies available to doctors, clinicians, and researchers. With the advent of personalized medicine, many times bioanalytical and biochemical approaches to study and treat diseases are limited by the available technology. This has led to new approaches to perform (i) high-throughput single cell analysis, (ii) point-of-care detection of biomolecules, viruses, and cells, (iii) large scale analysis and processing of massive data sets, and (iv) low volume detection of disease biomarkers. Researchers have harnessed a range of tools and approaches including nanoparticles, microfluidics, tissue engineering, peptides, proteins, DNA, RNA, 3D printing, microscopy, and spectroscopy to name a few. This has led to selective and sensitive biosensors with low limits of detection and high signal-to-noise ratios that can be incorporated into high-throughput or field ready approaches to rapidly analyze biological samples. Recent years have also produced novel in vitro models that better recapitulate the in vivo environment to increase the fundamental understanding of disease progression. This has led to new approaches in cell culture co-culture and 3D culture that incorporate more realistic biomaterials to better represent tissue.

Sunday
NIRDye 812: An optimized molecular platform in immuconjugate form for the NIR-I fluorescence and photoacoustic-guided imaging of glioblastoma tumor issue
02:00pm - 02:20pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The primary treatment for malignant tumors is surgical removal of the diseased tissue. The presence or absence or residual diseased tissue at the tumor margin is the strongest predictor of postoperative prognosis and recurrence. Accordingly, reliance on the ability of surgeons to visually distinguish diseased from healthy tissue is crucial. The use of luorescence-guided surgery (FGS) has aided surgeons in visualizing such tissue by color-coding the surgical field in real time with contrasting pseudo colors deriving from NIR-I fluorescence emission of a fluorophore that’s concurrently employed. Antibody-fluorophore constructs, namely cetuximab-IRDye 800 (CTB-IRDye 800), are FDA-approved for clinical trial usage for the fluorescence-guided resection of diseased tissue. Unfortunately, CTB-IRDye 800 affords limited identification of diseased tissue and tumor margin delineation because the immunoconjugate generates suboptimal tumor-to-background ratios (TBRs) due to the spectral/photophysical profiles of IRDye 800 not aligning optimally with fixed optical windows of pre-/clinical setups. As such, CTB-IRDye 800 affords incomplete resection compared to if TBRs were higher due to otherwise. To aid in accurately identifying deep-seated diseased tissue, photoacoustic (PA) tomography has been implemented alongside CTB-IRDye 800 to achieve PA signals that could result in higher TBRs. However, in clinical practice, PA imaging also results in subpar TBRs due to IRDye 800 affording low PA signals. Here, we developed NIRDye 812, a structurally-modified topological equivalent of IRDye 800, so as to confer it the capability to provide both higher TBRs and superior PA signal than that of the equivalent CTB conjugate and the fluorophore itself. Indeed, CTB-NIRDye 812 demonstrated a red-shifted maximum absorption wavelength at 796 nm and peak NIR-I fluorescence emission wavelength at 820 nm, which is well-positioned within the fixed windows of preinstalled emission filters in standard pre-/clinical NIR-I fluorescence imaging instrumentation. CTB-NIRDye 812 provided a 2-fold increase in TBRs compared to those of CTB-IRDye 800 in vivo. Also, NIRDye 812 displayed an ~60% higher PA signal than IRDye 800. Collectively, we improved upon the spectral/photophysical and PA properties of IRDye 800 with subtle modifications to it in hopes of obtaining fast track or breakthrough designation of the immunoconjugate by the FDA for the FGS of tumor tissue.
Sunday
Dielectric metasurfaces as refractive index sensors: Towards high-sensitivity label-free optical sensing
02:20pm - 02:40pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Metamaterials are arrays of meticulously engineered sub-wavelength structures that exhibit properties that cannot be found in bulk materials. We engineer 2D metamaterial surfaces – metasurfaces – as optical sensors with high sensitivity and Quality factor (Q-factor) for bio and gas sensing applications. These are made up of arrays of dielectric nanostructures by state-of-the-art nanofabrication techniques. Dielectrics — contrary to the commonly used plasmonic materials — are CMOS compatible and have no associated heat loss, making them advantageous for integration into portable, digital point-of-care devices for biosensing applications in the future. Biosensing relies on the enhancement of the electromagnetic field when light interacts with the sub-wavelength particles composing the metasurfaces. These fields are highly sensitive to the surface refractive index, allowing them to be used as label-free refractive index sensors. Generally, in response to the presence of molecules on the metasurface, a shift in resonant wavelength is observed. However, these measurements are sensitive to the intensity fluctuations of the illumination source and its wavelength calibration. To bypass this limitation, we explored a novel sensing concept based on diffraction-based metagrating, that combines the resonant behavior with the tuned diffraction from the metagrating. To assess our metagrating sensors' performance, we explored its behavior in a concentration range of glycerol with the refractive index ranging from 1.331 to 1.401. Additionally, the sensors were benchmarked using selective capturing of anti-Mouse IgG by surface-immobilized Mouse IgG (antibody) bioreceptors (a model antibody system) on the surface that demonstrated a promising limit of detection of ~770 fM. Additionally, these dielectric metasurfaces, unlike plasmonic metasurfaces, can be reused by removal of the covalently attached molecular layers quite easily. These metasurface sensors are explored to develop point-of-care diagnostic devices for the detection of diabetes or multiple sclerosis.
Sunday
Development of polymer dot @ silicon quantum dots nanocomposite for fluorescence imaging
02:40pm - 03:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Mr. Di Sun, Presenter, University of North Dakota; Xu Wu; Diane Darland; Julia Zhao
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Luminescent semiconductor quantum dots (QDs) would have a wide range of biological applications due to their strong photostability and multiple emission wavelengths under a single excitation. However, the majority of traditional QDs are heavy metal-based (CdSe, CdTe). The toxicity of these heavy metals limits QDs’ applications in biological fields. To solve this problem, silicon as a nontoxic element has been used to generate QDs (SiQDs). However, the SiQDs are very unstable and are susceptible to aggregation, which would significantly reduce the quantum yield of SiQDs. In order to improve the stability and fluorescence quantum yield of SiQDs, using highly florescent pi-conjugated polymer dots (Pdots) we constructed a Pdot@SiQDs nanocomposite. In the nanocomposite, the Förster resonance energy transfer (FRET) occurred where the Pdots was used as an energy donor to excite SiQDs to emit strong fluorescence. Two polymers were used, poly(9-vinylcarbazole) (PVK) as a light-emitting polymer, and poly (styrene-co-maleic anhydride) (PSMA) was used as an amphiphilic polymer linked to the amine functional group terminated SiQDs. Through the coprecipitation method, the Pdot@SiQDs nanocomposite was generated. As a result, the SiQDs doped onto Pdots were protected from the aggregation. The stable and strong fluorescent hybrid was developed for bioimaging and biosensing.
Sunday
Reversible near-infrared fluorescent nanosensor reports oxytocin release in the brain
03:00pm - 03:20pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Nicole Navarro, Presenter
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Neuropeptide oxytocin is central to maternal and reproductive behaviors, anxiolysis, and social interaction, and is thus critical to our social well-being. Oxytocin has also been implicated in the pathogenesis of various social impairment disorders, including Autism Spectrum Disorder (ASD). Our understanding of oxytocin’s function in the brain is incomplete, however, as both normal and aberrant oxytocin signaling pathways remain uncharacterized. There is a demonstrable need for an imaging probe with the specificity and spatiotemporal resolution requisite for endogenous oxytocin signaling (seconds, microns). Leveraging the optical properties of single-walled carbon nanotubes (SWNT) and the molecular recognition capacity of biopolymers, our lab has previously developed sensors for imaging dopamine in the brain. Herein, we present a peptide fragment of the endogenous oxytocin receptor protein (OXTp) as the molecular recognition element when conjugated to near-infrared fluorescent SWNT, to produce a peptide-SWNT oxytocin imaging probe. To synthesize the OXTp-SWNT probe, SWNT are first functionalized with triazine handles and carboxylated. The receptor peptide is then conjugated to the carboxyl groups through EDC/NHS chemistry, and the OXTp-SWNT is subsequently wrapped with C12 DNA to impart colloidal stability. In solution, the OXTp(C12)-SWNT oxytocin probe binds to oxytocin reversibly and demonstrates a dF/F0 of >150% in response to 100 µM oxytocin. In the paraventricular nucleus (PVN) of mouse brain slices, where oxytocin is centrally released, the OXTp(C12)-SWNT probe enables imaging of oxytocin upon 0.5 mA electrical stimulation and maintains this response after incubation with 1 µM quinporole, a dopamine receptor agonist. Furthermore, OXTp(C12)-SWNT both respond to and can be used to image oxytocin in acute brain slice in the presence of atisoban, an oxytocin receptor antagonist. Our results herein demonstrate that the OXTp(C12)-SWNT probe can be employed to image neuropeptide oxytocin both in solution and in acute slices, towards a more complete understanding of oxytocin signaling in the brain and its role in neurological disorders such as ASD.
Sunday
Self-immobilizing near-infrared probe for in vivo imaging of senescence
03:20pm - 03:40pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Cellular senescence is a mechanism by which a cell enters stable cell cycle arrest triggered by various types of stress such as telomere shortening, oxidative stress and DNA damage. It is implicated in a number of pathophysiological conditions including age-related diseases, degenerative disorders and cancer. Despite its importance, the current toolbox for studying or monitoring senescence in vivo is limited. Here we present a new near-infrared (NIR) fluorescent probe that can be activated by senescence-associated β-galactosidase (SA-β-Gal), a widely used marker for senescence, and can be immobilized in targeted cells via quinone methide chemistry to provide longer retention. Using in vitro labeling experiments and in vivo animal imaging, we demonstrated that this new self-immobilizing near-IR probe for senescence allows for deeper tissue penetration, higher imaging contrast and longer imaging time window.

Sunday
Background-free modulated photoacoustic imaging
03:40pm - 04:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Detection of cancer at its earliest stages requires development of low-background, clinically relevant imaging modalities. We have utilized optically modulatable dyes inside silica nanoparticles as novel modulatable photoacoustic contrast agents to provide truly background-free photoacoustic images in near real-time. Because photoacoustic (PA) imaging uses photogenerated and minimally scattered acoustic waves for image reconstruction, PA imaging enables optical contrast deep within tissue. However, PA imaging is typically plagued by large background signals from endogenous chromophores, thus we utilize a two-pulse PA excitation scheme to directly and selectively recover only our contrast agent signals without any background signals. Contrast agents in several spectral regions have been utilized and are being used in vivo to target sentinel lymph nodes. Contrast agents with high photogenerated dark state yields produce the necessary nonlinearity in signal to produce excellent modulated PA contrast. Initially, correlating fluorescence and PA modulation studies, we have demonstrated excellent sensitivity using Xanthene dyes (Rose Bengal, Eosin Y) embedded in silica nanoparticles. The different dark state lifetimes provide the opportunity for PA lifetime imaging to separate contrast agents that are excited by the same excitation pulse. This additional dimension in PA imaging enables simultaneous discrimination of multiple targeted pathologies, while monitoring biological markers in vivo, in real-time. Compared with spectroscopic PA – the standard for background signal elimination, SAPhIRe (Synchronously Amplified Photoacoustic Image Recovery) offers improved background suppression with far fewer acquisitions, thereby reducing motion artifacts and increasing frame rate.
Enzyme Engineering & Biocatalysis: Enzyme Engineering & Biocatalysis
02:00pm - 04:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Nik Nair, Organizer; Zhe Rui, Organizer, UC Berkeley; Jerome Fox, Presider, University of Colorado, Boulder; Jovana Nazor, Presider
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Enzymes enable the synthesis of structurally complex molecules under ambient—or otherwise mild—conditions; they offer a sustainable means of building a striking variety of chemicals. Recent years have witnessed the expansion of biocatalytic processes across the pharmaceutical industry and fine chemical sector. Advances in enzyme engineering have enabled the assembly of sophisticated biocatalytic cascades and accelerated the design and optimization of enzymes with novel functions and stabilities (e.g., activity at high temperatures or in mixed solvents). This session invites abstracts focused on enzyme engineering, broadly defined. Topics include advances in process development; the design, discovery, and evolution of enzymes and biocatalytic systems; and high throughput screens, structure-function analyses, and modeling. Papers relevant to biocatalytic cascades, green chemistry, environmental and waste management, biocatalysis under non-natural conditions (e.g., organic solvents), or that highlight applications in the pharmaceutical, food, textile, detergent, and paper industries are particularly encouraged. Unlisted topics relevant to enzyme engineering and biocatalysis are also welcome.

Sunday
Introductory Remarks
02:00pm - 02:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Sunday
Enhancing biocatalysis with solvent-free liquid enzymes and ionic liquids
02:00pm - 02:20pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Dr Alex Brogan, Presenter, King's College London
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Enzymes can perform many industrially relevant reactions, such as esterification, hydrolysis, oxidation, reduction, and C-C bond formation, with high specificity and under significantly milder conditions than their chemical counterparts. They can perform these reactions on a wide range of substrates, making them highly attractive for many applications. As a result, research into the use of enzymes as industrial biocatalysts has been gaining ground, particularly in conjunction with emerging solvent systems such as ionic liquids. However, enzymes often have very low solubilities in nonaqueous environments and are frequently unstable, limiting the window of usability. Consequently, there is a need to develop new biotechnologies that improve solubility and stability of biocatalysts in nonaqueous media.

Surface modification of enzymes, to yield solvent-free liquid enzymes, has been demonstrated as a robust method for stabilizing enzymes against temperature, aggregation, and non-aqueous environments. Recent work showed that these new biomaterials are soluble in both hydrophilic and hydrophobic ionic liquids, with enzyme structure preserved in the non-aqueous environment. Using the enzyme glucosidase we were able to demonstrate that in ionic liquids, the optimal temperature for enzyme activity shifted to 110 °C accompanied by activity towards water insoluble cellulose. Solvent-induced promiscuity showed that it was possible to reduce the number of enzymes require for cellulose degradation from 3 to 1. As a result, this nascent technology in conjunction with emerging solvent systems such as ionic liquids could provide a versatile platform for industrial biocatalysis. Ongoing work seeks to demonstrate how this technology can be used to broaden the substrate scope for a range of enzymes and using the broad electrochemical window of ionic liquids to circumvent the requirement for expensive co-factors.

Sunday
Improved chemistry by combining enzyme engineering, enzyme immobilization, and flow chemistry
02:20pm - 02:40pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Biocatalysis, protein engineering, and flow chemistry are key enabling technologies that can yield drastically shorter and greener chemical processes. We sought to leverage these technologies to realize an improved synthesis. A cheap and green chemical commodity was identified as a starting material that allowed the generation of a key intermediate in the synthesis in a stepwise reaction. As an initial proof of concept, an enzyme-catalyzed reaction was demonstrated with modest yields and selectivity. However, the initial enzyme and process would not be sufficient to support large scale synthesis. To improve the synthesis, we engaged in enzyme evolution, enzyme immobilization, and flow chemistry. Through several rounds of evolution the enzyme was evolved to reach high conversion, selectivity and stability in the presence of organic solvents.
Sunday
Toward engineering an efficient and thermostable α-amino ester hydrolase (AEH): Minimizing substrate inhibition and deactivation for continuous production of cephalexin
02:40pm - 03:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
β-lactam antibiotics are continually the most prescribed antibiotics in the world. Semi-synthetic β-lactam antibiotics can be produced enzymatically as an environmentally conscious and cost-effective alternative to traditional chemical synthesis. The enzymatic reaction couples an activated acyl side chain with a β-lactam nucleus to produce the desired β-lactam antibiotics.
α-Amino ester hydrolases (AEHs) comprise a small class of enzymes capable of enantioselective production of semi-synthetic β-lactam antibiotics and are a potential alternative to the industrially used penicillin G acylases (PGA). Despite their rapid kinetics, AEHs have had limited use primarily due to their low stability, aggregation, and rapid deactivation. Recently, two quadruple variants (termed QVG & QVH) of wildtype AEH from Xanthomonas campestris pv. campestris were developed to improve AEH stability; however, AEH half-life is still on the order of one hour, which is far from ideal.
In addition, although AEH substrate specificity has been thoroughly studied, the full kinetic mechanism of AEH catalyzed synthesis of β-lactam antibiotics has yet to be fully realized. While the general mechanism for β-lactam antibiotic synthesis by Youshko and Svedas is applicable under low reactant concentrations, we demonstrate experimentally that AEH suffers from substrate inhibition and present a new kinetic model herein to fully describe the AEH catalyzed synthesis of cephalexin.
Based on results from the newly derived kinetic model, AEH has significant potential with cephalexin productivity >600 g/L/hr at 5 µM AEH compared to 200 g/L/hr at 5 µM of PGA. However, substrate inhibition and deactivation severely limit reasonable use of AEH. Reactant concentrations must remain low to maximize cephalexin productivity, but 7-ADCA conversion is limited under most configurations. In addition, AEH would need to be replaced each residence time to offset deactivation.
To address both substrate inhibition and AEH stability, computational protein engineering has been explored. Through use of Autodock Vina and CaverDock, substrate and product shuttling were found to be potentially limited by AEH tunnels which could lead to substrate inhibition. HotSpot Wizard and Solubis were used to predict mutations that minimize aggregation and increase overall AEH thermostability. Finally, the combination of mutations was assessed using FireProt to eliminate mutations that could be detrimental in combination.

Sunday
Rational engineering of polysaccharide-degrading enzymes: A case study on cellulases for improved biomass conversion to biofuels
03:00pm - 03:20pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Polysaccharides are an important class of biomolecules and the most abundant biopolymers on the planet. They are found in diverse natural milieus ranging from cellulose in plant biomass to hyaluronan in extracellular matrices of human tissues. Nature has evolved enzyme machineries which can deconstruct these polysaccharides to form useful bioproducts. These enzyme machineries can be tapped for biotechnological applications such as production of bioethanol from pretreated cellulosic biomass. However, non-productive binding of cellulases and associated carbohydrate-binding modules (CBMs) to cellulose and lignin in pretreated biomass has been a key bottleneck for cost-effective biomass conversion. To firstly understand the molecular mechanism of non-productive binding to cellulose, we monitored the single-molecule processive motility of full-length exocellulases and developed a single-molecule CBM-cellulose rupture assay employing optical tweezers to characterize the binding of a well-studied Type-A CBM and its mutant to cellulose allomorphs. Next, we examined the subtle interplay of CBM binding and cellulose hydrolysis activity for three model Type-A CBMs (families 1, 3a, and 64) tethered to a cellulase catalytic domain (CD) on two distinct cellulose allomorphs (i.e., cellulose I and III). We finally generated a small-library of mutant CBMs with varying cellulose affinity followed by monitoring cellulose hydrolysis activity of multiple CD-CBM fusion constructs to identify novel mutants with enhanced activity towards cellulose I. Kinetic binding assays using quartz crystal microbalance with dissipation (QCM-D) were then employed to measure mutant CBM adsorption and desorption rate constants and , respectively, towards nanocrystalline cellulose. However, development of efficient enzymes for biomass conversion needs to account for non-productive binding to lignin as well. To this end, we deployed a novel protein engineering strategy called supercharging and generated a library of endocellulase mutants with varying net charges. Overall, this presentation highlights our efforts to understand non-productive binding using a suite of analytical techniques and showcases enzymes with up to 80% improved activity on cellulose.
Sunday
Biocatalytic reactor with improved catalytic efficiency and longevity for entrapped diffusion-limited enzyme
03:20pm - 03:40pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Dr. Yue Yuan, Presenter, North Carolina State University; Sonja Salmon
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Enzyme immobilization has been used an alternative approach to enzyme engineering to produce robust biocatalytic products, as well as to improve the enzyme longevity in industrial applications. Among the various immobilization methods, enzyme entrapment exhibits advantages in versatility and high enzyme loading. However, entrapped enzymes can experience limitations in mass transfer efficiency, especially when diffusion-limited enzymes are entrapped. In this study, a robust biocatalytic matrix with controlled liquid transport properties was fabricated to address this problem. Catalase, an enzyme that catalyzes peroxide decomposition (H2O2 H2O + O2) at room temperature with ultrafast kinetics (107 s-1 turnover rate), was entrapped in a thin chitosan layer at a mild condition and then applied to a cellulosic fibrous support to examine the impact of controlled liquid transport on catalytic efficiency. Mild immobilization conditions and good affinity between the two polysaccharides with opposite charges minimized enzyme inactivation during the preparation steps and prevented enzyme from leaching during peroxide decomposition testing and washing, providing a novel, durable and versatile enzyme immobilization strategy. The catalytic efficiency of entrapped diffusion-limited enzymes in a reaction containing solid, liquid and gas phases was facilitated when the fibrous matrix was used as a flow-through configuration, where dissolved enzyme substrate was transported by liquid flowing through the coated fibrous structure. The flow-through configuration decomposed at least two times more peroxide in a twenty-times smaller reaction zone volume compared to a stirred tank configuration, and the mechanism of the improved catalytic efficiency was revealed by a neutron imaging technique. This new class of sustainable and flexible biocatalytic matrices has beneficial multifunctional properties that are applicable for numerous small- and large-scale applications including controlled flow reactors and reactive filtration.
Biocatalytic yarn with controlled liquid transport for improved biocatalytic efficiency and enzyme longevity in peroxide decomposition.

Biocatalytic yarn with controlled liquid transport for improved biocatalytic efficiency and enzyme longevity in peroxide decomposition.


Sunday
Structural mining of metagenomic data define the diversity of gut microbial enzymes
03:40pm - 04:00pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The gut microbiota play key roles in human health and disease but the mechanisms the microbiota use to drive host outcomes remain unclear. Enzymes produced by the gut microbiota have been implicated in such outcomes as they directly alter the chemical structure of small molecules traversing the intestinal tract, thereby modifying their bioavailability and potential biological effects. Gut microbial enzymes have been shown by us and others to exhibit dramatic structural diversity that annotate conserved core features and make the resultant proteins highly distinct between different phylogenetic groups. Despite their prevalence and diversity, only a tiny fraction of metabolically active enzymes produced by the gut microbiota have been identified and characterized. However, recent advances in metagenomic sequencing platforms have made it possible to obtain the amino acid sequences of gut microbial enzymes residing in the GI. Here, we employ an interdisciplinary approach combining microbiology, chemistry, bioinformatics, biochemistry, and structural biology, to unravel the mechanistic underpinnings of microbiome-dependent metabolism influencing health outcomes. Specifically, we identify key residues in protein structures that provide amino acid sequence information enabling us to pinpoint proteins with similar structure and function across a variety of gut metagenomes. By doing so, we facilitate the functional assignment and proper annotation of orphan gene products across these datasets. We have adapted this approach to assemble families of gut bacterial enzymes across a vast metabolic landscape. Strikingly, we observe drastic structural and functional diversity within each enzyme family, implicating subsets of these enzymes in targeted metabolism. Our pipeline can be applied broadly to advance our mechanistic understanding of functional enzymology and the human gut microbiota.
Systems Biology & Omics-Tools & Applications: Systems Biology & Omics-Tools & Applications
04:30pm - 06:10pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Melisa Carpio, Organizer; Danielle Ercek, Organizer, Northwestern University; Nitya Jacobs, Organizer; Kevin Solomon, Presider, University of Delaware; Davinia Salvachua, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Living systems are complex and dynamic requiring understanding of both individual components and their interactions. Systems biology addresses this challenge with large scale data and/or mechanism-driven modeling to generate hypotheses that are tested and validated. Systems of interest range from molecules and single cells to multicellular organisms or microbial consortia. This session will cover recent progress in the development and use of integrated methodologies (both experimental and computational) to elucidate or exploit the internal mechanisms of biological systems. Areas of interest include the development and application of -omic analyses, biological network models, metabolic flux analysis, metabolic pathway simulations, and protein and/or genome engineering based on systems-level understanding. Overall, this session will highlight recent discoveries and opportunities provided by these tools to drive biological systems to new levels of performance.

Sunday
Knowledge graph-based approaches to drug repurposing for COVID-19
04:30pm - 04:50pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The large-scale disruption caused by the COVID-19 pandemic has motivated researchers in academic, commercial and government sectors around the world to develop anti-SARS-CoV-2 therapeutics and vaccines. Drug repurposing has become an important approach to accelerate these efforts. By combining CAS REGISTRY, the world’s most authoritative collection of chemical information curated by hundreds of CAS information scientists, with data available from external databases, we developed a knowledge graph to support global research of drug repurposing for COVID-19. We identified 20 biological processes important for COVID-19 pathogenesis and utilized published data on host gene expression changes in response to SARS-CoV-2 infection to identify 16 more processes upregulated post-infection. These processes were then used as the starting point of a knowledge graph query to identify small molecules that target relevant genes. Our approach identified 1,350 potentially repurposeable drugs. We next developed a small molecule ranking method focused on rarity of the genes and biological processes within the graph to emphasize novel drug candidates. Among the top 50 small molecules identified by our algorithm, the two most common drug classes were kinase inhibitors and histone deacetylase inhibitors. Of the top 50 small molecules, 11 have been tested in COVID-19 clinical trials listed on clinicaltrial.gov. The high number of clinically tested drugs in our results suggests that the other 39 drug candidates should be investigated as potential therapeutics as well. We believe that our knowledge graph provides researchers an opportunity to accelerate innovation and streamline the investigative process not just for the COVID-19 pandemic but also in other biomedical and scientific research.
Sunday
Network-based analysis of CD4(+) and CD8(+) cells’ transcriptional data to reveal potential human targets for the decreasing of HIV progression
04:50pm - 05:10pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Acquired immunodeficiency syndrome (AIDS) is a dangerous outcome of the human immunodeficiency virus (HIV) infection developed because of the absence or ineffective antiretroviral therapy. A decline in the numbers of CD4(+) and CD8(+) lymphocytes is the most important pathogenic mechanism of HIV infection that mediates AIDS development. The decline can be caused by direct infection of CD4(+) lymphocytes and by indirect cytokine-based continuous activation of CD4(+) and CD8(+) cells, which leads to their dysfunction and apoptosis. Our study aimed at the identification of human protein targets whose modulation can decelerate HIV progression. We developed a pipeline to identify potential anti-HIV human targets in signaling networks based on the analysis of transcription changes in CD4(+) and CD8(+) lymphocytes from HIV-infected people. The pipeline includes two steps. First, we identified master regulators (MRs), the key proteins in signaling networks of CD4(+) and CD8(+) lymphocytes, which may be responsible for the observed transcription changes in HIV-infected people compared to healthy controls. The modulation of MRs can change gene transcription to those in uninfected individuals and decelerate HIV progression. Second, we applied the dichotomic dynamic modeling of cellular signaling in CD4(+) and CD8(+) lymphocytes to verify the obtained potential targets. As a result, particular cellular processes affected by the modulation of identified targets, which is essential for lymphocyte dysfunction and death, were identified. Based on an analysis of structure-activity relationships, we predicted the interactions of the approved drugs and biologically active compounds with the revealed human targets. These pharmaceutical agents may be potentially applied to decelerate HIV progression and increase the efficacy of antiretroviral therapy. The developed pipeline can be used to discover new opportunities for the treatment of any other viral infection.
Sunday
Using a multi-omic approach to understand and model eukaryotic signal transduction
05:10pm - 05:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Eukaryotic signaling networks are composed of interconnected systems of regulatory proteins and are involved in controlling nearly all biological processes. To study these networks, our collaborative team is working toward combining phosphoproteomic and transcriptomic data with genetic-algorithm programming to build dynamic mathematical models.

As a model system, we are studying the fungal Cell Wall Integrity Signaling (CWIS) pathway, which is activated upon wall stress, leading to expression of several wall-repair genes. To assess system-wide dynamic response to stress, we perturbed A. nidulans cultures with CWIS activating agent micafungin, collected 13 phosphoproteomic and transcriptomic samples over 10 and 120 min respectively and found over 1800 genes and 430 phosphorylation sites showed significant dynamic change. Omics-based hypotheses were confirmed with experiments, and our collective data indicate previously unknown connections with (i) actin regulation, (ii) endocytosis, and (iii) septum formation as critical cellular processes responding to activation of CWIS.

While the CWIS pathway induces expression of some wall repair genes, it is unclear how numerous others are regulated. To address this question we used a strain lacking the last kinase in the CWIS pathway, MpkA, rendering the pathway inactive. To begin, we compared MpkA+/- strains, at a single time point, in the absence of wall stress. Over 2500 genes and 200 phosphorylation sites showed differential expression and occupancy respectively. Omics-based hypotheses were confirmed with experiments, and our collective data indicate previously unknown connections with wall maintenance, branching regulation and iron metabolism. Seeking alternate regulatory pathways, we carried out dynamic experiments with the MpkA- strain taking phosphoproteomic and transcriptomic samples over 10 and 120 min respectively. We will report on our findings from these experiments and how we are using these data, along with a genetic algorithm programming approach, to develop models to describe signal transduction and resultant changes in gene expression.

Sunday
Engineering the unfolded protein response and downstream crosstalk for improving therapeutic protein production in CHO cells
05:30pm - 05:50pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Secretion levels needed for industrial Chinese hamster ovary (CHO) cell lines can lead to an imbalance in ER homeostasis, and accumulation of improperly folded proteins, or ER stress, is a particularly challenging bottleneck in cell line engineering. The unfolded protein response (UPR) is initiated to overcome ER stress and reestablish homeostasis. This research aims to demonstrate higher specific productivity results in unavoidable ER stress by measuring the UPR in cell lines engineered for high-levels of recombinant protein production. In fed-batch culture, we achieve high specific productivity with two distinct CHO cell lines, one producing immunoglobulin G (IgG) and one producing erythropoietin (EPO-Fc), respectively. The IgG line grew to a lesser viable cell density than the EPO-Fc producer and a host cell line, but all cell lines maintained viability in culture. Both producers exhibited high titer, productivity, and product mRNA expression. Western blot and quantitative polymerase chain reaction (qPCR) were used to investigate the UPR time course, correlated to product titer. In comparison to the host cell line control, both protein producers exhibited early activation of the IRE1 and PERK pathways. By Day 3 of fed-batch culture, spliced XBP1 mRNA expression increased up to 26 fold, and both ATF4 and CHOP mRNA expression increased up to 23 fold. High throughput sequencing was used to conduct a differential gene expression analysis: either each protein producer compared to the host cell line at a respective timepoint or each cell line compared to its respective Day 0 timepoint. These analyses revealed other protein processing genes were activated in addition to the UPR in both protein producers. These identified genes exhibited associations with the UPR, calcium-binding, and oxidative stress. Furthermore, genes associated with other signaling transduction pathways, such as glutathione synthesis, were found to be differentially expressed in both protein-producing cell lines. To determine if highly productive lines have been selected for the ability to cope with ER stress, the UPR was monitored in fed-batch cultures of CHO cell pools with reduced IgG and EPO production via shRNA interference. Lastly, we explore the impacts of engineering the protein-producing cell lines using protein processing targets identified in our transcriptomic analysis. It is anticipated this work will lead to advancements in CHO cell engineering and protein production.
Sunday
A semi-synthetic regulon in Saccharomyces cerevisiae identifies factors needed for rapid growth on pentoses
05:50pm - 06:10pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 04
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Our lab previously demonstrated galactose (GAL) regulon of Saccharomyces cerevisiae can serve as an excellent global regulatory infrastructure to aid the growth of this yeast on a non-native substrate, xylose. By engineering the sensor protein, Gal3p, to respond xylose, we created a semi-synthetic regulon where this pentose could activate its own metabolic genes and thousands of endogenous yeast genes required for rapid growth. Using minimal engineering approach, the semi-synthetic regulon exhibited higher growth rate compared to traditionally metabolically engineered S. cerevisiae. In this talk we will discuss our latest efforts to extend semi-synthetic regulon for utilization of another abundant pentose, arabinose. We found that the xylose-responsive Gal3p variant (called Syn4.1) exhibited strong interaction with arabinose as well. We incorporated the arabinose isomerase pathway genes - araA, araB, and araD from Lactobacillus plantarum under the control of GAL promoters (ARA-REG) or under constitutive promoter (ARA-CONS) as control. ARA-REG strain exhibited approximately 2.5-fold higher growth rate and 3.5-fold higher endpoint OD600compared to the ARA-CONS strain when grown in YP-arabinose suggesting that a regulon-based strategy may be a general strategy to yield superior for engineering non-native sugar utilization when compared to the conventional strategy that employs only constitutive heterologous gene expression. We improved the growth rate further by 2-fold using mutagenic gene-promoter shuffling and directed evolution strategy on the three arabinose catabolic genes. Gene expression analysis indicated requirement of high expression of AraA for optimum growth. Using transcriptomic analysis, we compared genes that are differentially expressed between pentose (xylose and arabinose) and hexose (galactose) to identify factors that specifically promote growth on pentoses and elucidate how information from metabolic processes percolates and permeates through the yeast regulatory network.
Downstream Processing: Advances in Chromatographic Separations for Novel Antibody Structures & Drug Conjugates: Advances in Chromatographic Separations beyond mAbs 2
04:30pm - 06:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Daniel Bracewell, Organizer, UCL Dept Biochemical Engr; Wai Chung, Organizer, Biogen Inc; Elizabeth Goodrich, Organizer, MilliporeSigma; Minni Aswath, Presider, Boehringer Ingelheim; Scott Husson, Presider, Clemson University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

This session calls for papers focused on the downstream processing of novel antibody structures which may include, but are not limited to, bispecific antibodies, antibody drug conjugates (ADC), single-chain variable fragments (scFv), antigen binding fragments (Fab), novel antibody structures, or other protein conjugates. The scope may range from theory/modeling, early stage screening, early/late stage development, process scale-up, and/or large-scale manufacturing. The following topics are particularly encouraged and may include HTPD, process optimization, troubleshooting, and/or case studies focused on antibody derivatives or other protein conjugates: 1. Investigations for new drug modalities and novel chromatographic ligands (e.g., affinity, HIC, multimodal), 2. Optimization of conjugation chemistry/unit operations, 3. Purification of conjugation products addressing challenges in removing undesired conjugation byproducts and/or difficult-to-remove impurities, and 4. Creative approaches to handling unstable products.

Sunday
The preparation and application of a dry-compressed packed-bed of hydroxyapatite nanoparticles for fast, high-resolution separation of proteins
04:30pm - 04:50pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Prof. Raja Ghosh, Presenter, McMaster University; Yves Durocher; Paul Gatt
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
A method for preparing a shallow cuboid packed-bed of crystalline hydroxyapatite nanoparticles using a simple dry-compression technique, and its integration within a chromatography device is described and discussed. The application of this device for fast, high-resolution protein separation is then discussed. In the flow rate range examined in this study, the pressure drop across the packed-bed increased linearly with flow rate, indicating negligible media compaction during use. Binary protein mixtures could be separated using this device, in the bind and elute mode, in less than a minute. Contrary to that observed in most packed-bed chromatographic separations, the efficiency in protein separation using this device increased with increase in flow rate. The suitability of using this device for preparative protein purification is demonstrated by purification of a monoclonal antibody (Trastuzumab) from mammalian cell culture supernatant. This study opens up the possibility for developing dry-compressed cuboid packed-bed chromatography devices for efficient protein separations, at both analytical and preparative scales.
Sunday
Scalable high productivity membrane adsorbers for process chromatography
04:50pm - 05:10pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Alternative stationary phases are being developed to resolve short-comings of bead chromatography processes, namely high diffusional resistance leading to long processing cycles (4-6 h). Targeted life-times of 75 -150 bind and elute cycles, taking many months or even years to be achieved, requiring their re-use, cleaning and respective validation, storage, sometimes even column un- and repacking.

Membranes, due to intrinsically high mass transfer rates, overcome these limitations, enabling residence times of seconds, where full life-time utilization (75-150 cycles) can be achieved in 1-2 shifts. Purely convective membranes offer high mass transfer rates and acceptable binding capacity, however, due to relatively small convective pore sizes they exhibit elevated back pressures and create challenges regarding scalability due to limitations with respect to applicable bed height.

Alternative chromatographic membranes are presented, combining material and performance aspects of beads and purely convective materials. These membrane adsorbers provide a high binding gel phase with short diffusional path lengths (2-3 µm). Large convective pores provide for transport to the gel phase, low fouling propensity and easy cleanability. More over, these large convective pores ensure sufficient permeability to allow bed heights of up to 4-8 mm that can still be used at residence times of 12 sec while maintaining pressure drops of less than 0.2 MPa.

Supported by modelling approaches we will briefly introduce to the mass transfer concept of this novel membrane material and show performance data exemplifying its capabilities. Focus will be on data generated for a protein-A affinity membrane, including yield, purity, productivity for a rapid cycling process using clarified mAb feedstream. Furthermore, we will show the scalability of this novel membrane technology over a range of device formats enabling large scale production with dedicated rapid cycling chromatography systems.

Sunday
Methodology for optimized development and implementation of multi-column capture chromatography
05:10pm - 05:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Mohamed Agoub, Presenter, Bristol-Myers Squibb; James Angelo, Presenter; Xuankuo Xu; Sanchayita Ghose
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Biologics manufacturing still remains dominated by batch operations. As upstream titers continue to increase due to developments in higher producing cell lines, the downstream purification process must adapt to meet the demands of productivity and processing cadence as to avoid becoming the bottleneck in manufacturing operations. In this work, we propose a methodology for developing a new process utilizing multi-column capture (MCC) operation using model assisted design practices. Through in-depth knowledge of column breakthrough behavior gained though simulation and column modeling, an optimal loading strategy for 2-column MCC can be derived based on product titer and column parameters (i.e. flowrate, dimensions, etc.). The outputs of this approach may be seamlessly integrated into GMP distributed control systems in order to alleviate operator intervention and decrease the barrier for entry for implementing continuous operations in the manufacturing environment.
Sunday
Impact of antibody disulfide bond reduction during harvest on downstream processing and product stability
05:30pm - 05:50pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Therapeutic monoclonal antibodies (mAbs) are primarily produced in mammalian cell culture systems, which can be susceptible to disulfide bond reduction during harvest. This reduction, which is dependent on cell line and cell culture process, has been correlated with the release of reducing components from the cells and depletion of dissolved oxygen before, during and after harvest. Consequently, these factors can lead to disulfide reduction during storage at room temperature prior to initial purification via Protein A chromatography.

This work seeks to understand the impact of disulfide reduction on product quality and on the downstream unit operations. mAb disulfide bond reduction appears to be reversible as the redox potential changes during the purification, however, we observed that the levels of fragment and aggregate increase during the low pH treatment proportional to the amount of free thiol present. The increase of such product related impurities impact the process yield and in process intermediate stability. Furthermore, the drug substance produced from clarified culture with disulfide reduction clearly showed a higher susceptibility to temperature induced changes on stability, potentially indicating incomplete assembly. In addition, for several mAb cases where disulfide bond reduction has been observed, we compared it with DTT induced mAb disulfide reduction. This comparison showed a different mAb sensitivity order to reduction by DTT than the observed at harvest indicating a potential different mechanism for disulfide reduction.

Sunday
Microfluidics and liquid-handling stations: Comprehensive comparison for chromatographic high-throughput process development
05:50pm - 06:10pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Tiago Silva, Presenter, Technical University Delft; Michel Eppink; Marcel Ottens
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
High-Throughput Screening is the state-of-the-art for process development and optimization in the biopharmaceutical industry, enabling process understanding and accelerating development and implementation. Chromatographic processes still represent the majority of operating costs for the production of several products, such as monoclonal antibodies. Optimal processes rely on several parameters and a good characterization of protein adsorption behavior is paramount, which can be derived from proper characterization of protein adsorption isotherms. The cost of the materials and sample demand technologies that can provide valuable and accurate data at a minimal both time and costs.
While liquid-handling stations have been the go-to technology for industry, microfluidics poses as an attractive alternative to further miniaturize assays. While both technologies offer miniaturization options, microfluidics reduces materials’ consumption manifold, even when compared to liquid-handling stations. Liquid-handling stations offer a great automation, reducing human-related errors to the maximum. Together, they simultaneously represent modern tools that allow for low sample consumption and fast data generation, essential for the screening and evaluation of different adsorbents and operating conditions.
In this work we present a novel microfluidic chip for the determination of protein adsorption isotherms in batch uptake mode. Protein isotherms determined using microfluidics are compared to those obtained via liquid-handling stations and a cost assessment for both techniques is presented. An assessment of the key features of each technology is finally provided, to further evaluate the implementability of both methodologies.

Sunday
Push-button high throughput chromatography
06:10pm - 06:30pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 05
Arjun Bhadouria, Presenter, Sanofi; Brian Murray
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
High throughput (HT) chromatography is an essential tool for clinical and commercial purification development, enabling more efficient workflows and more robust biopharmaceutical processes. However, as usage of HT chromatography increases, additional challenges remain. Within purification development, such challenges can be categorized into three areas: (1) bottlenecks due to pre- and post-run manual activities, (2) steep learning curves for end-users that limit adoption and use of HT equipment, and (3) handling and analysis of larger data sets. Here, we have developed an automation package that is designed to address all three challenges.
Leveraging a Tecan automated liquid handler, a simple graphical user interface, and a Visual Basic back end, we have extended the standard automation capabilities beyond HT chromatography execution to include all pre-run and post-run activities. Available modules include automated buffer preparation, load pH and conductivity adjustments, chromatography execution, and fraction pooling. The user defines key experimental parameters for chromatography operation and the run is then executed from a single Tecan script, which handles the variable conditions for the selected modules. Finally, a record of the run is saved in a standardized file format to enable streamlined analysis of larger data sets. This application allows for any chromatography experiment within our platform space to be executed start-to-finish with hands-free automation. This work eliminates significant bottlenecks within our chromatography experiments, increases adoption and usage of HT chromatography, and simplifies and accelerates data analysis. Ultimately, this fully automated HT chromatography approach enables more effective and efficient development of chromatography operations, reducing the timelines and costs to bring medicines to patients.

Protein Engineering Application in Therapeutics, Diagnostics & Sensors: Protein Engineering Application in Therapeutics, Diagnostics & Sensors
04:30pm - 06:35pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
Nik Nair, Organizer; Zhe Rui, Organizer, UC Berkeley; Carl Denard, Presider, ‍ ; Harun Rashid, Presider, ‍
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Division/Committee: [BIOT] Division of Biochemical Technology

Our ability to leverage and engineer desirable properties in proteins has ushered novel and improved ways to treat and diagnose diseases and to design sensitive and selective biosensors. This section focuses on newly-developed protein-based therapeutics, diagnostics, and sensors. Protein therapeutics may include studies about molecular engineering of antibodies for improved targeting and immunomodulation. Moreover, studies about engineered therapeutic proteins based on non-antibody scaffolds, as well as enzyme therapeutics, are encouraged. Protein-based diagnostics and sensors may include but are not limited to: genetically-encoded fluorescent and bioluminescent sensors, split and activatable proteins, small molecule-and light-responsive transcription factors, engineered CRISPR-Cas proteins for improved molecular targeting and engineered specificity.

Sunday
Introductory Remarks
04:30pm - 04:35pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual

Sunday
Exploring the use of a β-hairpin sequence as an alternative degron and CPP for a peptide-based PROTAC
04:35pm - 04:55pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
Hannah Hymel, Presenter; Jeffery Anderson; Ted Gauthier; Adam Melvin, Louisiana State University
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Proteolysis targeting chimeras (PROTACs) have emerged as a new class of therapeutics that utilize the ubiquitin-proteasome system (UPS) to facilitate proteasomal degradation of “undruggable” targets. Peptide-based PROTACs contain three essential components: a binding motif for the target protein, a short amino acid sequence recognized by an E3 ligase called a degron, and a cell penetrating peptide to facilitate uptake into intact cells. While peptide-based PROTACs have been shown to successfully degrade numerous targets, they have often been found to exhibit low cell permeability and high protease susceptibility. Prior work from Houston et al and Safa et al identified two peptides containing a β-hairpin sequence motif that function not only as protecting elements, but also as CPPs and degrons. These two sequences, RWRWR (RWRVpGRWIRQ) and OWRWR (OWRVpGRWIRQ), were rapidly internalized into cells and demonstrated enhanced intracellular stability compared to unstructured peptides. The goal of this study was to investigate if a single β-hairpin sequence could replace commonly used unstructured peptides sequences as the degron and CPP needed for PROTAC uptake and function. The degradation of Tau protein was selected as a model system as several published works have identified a Tau binding element that could easily be conjugated to the β-hairpin sequence. The experimental PROTACs were compared to a previously published PROTAC using a degron from HIF-1α and a poly-D-arginine CPP. A library of novel PROTACs was synthesized combining either RWRWR or OWRWR with the binding sequence for Tau. A series of time- and concentration-dependent studies confirm that the β-hairpin sequence was an adequate alternative CPP and degron to facilitate the proteasome-mediated degradation of Tau. A degradation assay confirmed that the β-hairpin conjugated PROTAC had a greater lifetime in the cells coupled with enhanced uptake kinetics as determined by fluorometry. Interestingly, Tau was found to be degraded to a greater extent using the β-hairpin conjugated PROTAC when compared to the control PROTAC which can be attributed to its enhanced uptake kinetics and intracellular lifetime. These findings highlight the of the β-hairpin sequence as an alternative degron and CPP to be incorporated into future PROTACs with the potential for enhanced performance and lifetime in cells leading to a greater degree of knockdown of target proteins.
Sunday
Deep mutational scanning guided directed evolution reveals multiple mechanisms to improve enzyme function
04:55pm - 05:15pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Phenylalanine ammonia lyase (EC 4.3.1.24; PAL) is a non-hydrolytic enzyme which catalyzes the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid and ammonia. This enzyme has widespread application in industry, agriculture and medicine. PAL has gained interest due to its use in treatment for phenylketonuria (PKU), tyrosinemia, and as amino acid deprivation approach for cancer. Considerable research has been focused on improving the catalytic activity of PAL, but the efforts have been far from comprehensive. Though extensive body of research exists on function, structure, and mechanism of PAL, a systematic study exploring the sequence-function space has not been attempted. Previously, we developed a directed evolution strategy to engineer PAL in E. coli. Here, we report detailed mutational landscape of PAL by performing deep DNA sequencing of the libraries obtained after growth enrichment. Based on the deep mutational scanning (DMS) analysis of PAL, we identified 79 mutational hotspots which exhibited positive change in fitness. Using structure-function approach we picked seven sites for comprehensive single or multi-site saturation mutagenesis in an attempt to further improve the catalytic activity of PAL. We observed that while most mutations were deleterious in combinations, few mutants showed improved fitness when present in combination. Using this new DMS-guided library we were able to identify new combination of mutants with 2.5-fold improvement in the catalytic activity. Further, to understand the mechanistic role of these mutants, we performed QM/MM and observed that different mutants contributed in a unique way to improve the catalytic activity. This included i) decreased root mean square fluctuation (RMSF) of substrate in the active site, ii) greater proximity of the substrate to catalytic residues, iii) stabilization of the substrate in the near attack conformation, iv) stabilization of the transition state, and v) improved tunnelling of the substrate to the active site. In summary, this study significantly advances basic and applied enzymology of PALs.
Graphical Abstract

Graphical Abstract


Sunday
Directed evolution of single chain antibodies targeting ADAM-17
05:15pm - 05:35pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
Mari Rita Toumaian, University of Nevada, Reno; Maryam Raeeszadeh Sarmazdeh, Presenter, University of Nevada, Reno
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The metalloproteinase (MP) family of proteases, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs), plays a crucial role in the extracellular matrix (ECM) remodeling and degradation activities. ADAMs, in particular ADAM-17, play an integral role in cell regulation, through the activation or inhibition of cytokines, growth factors, and their receptors, as well as adhesion molecules via cleavage of ectodomains. ADAM-17’s integral role in cell regulation also makes it a top contender for various human diseases, such as heart disease, cancer, Alzheimer’s disease, and diabetes. The use of small molecule therapeutics targeting ADAM-17 have failed in clinical trials due to non-specific targeting of MPs, which led to severe side effects in patients. Protein-based therapeutics such as antibodies or tissue inhibitors of metalloproteinases offer higher binding selectivity.
We used directed evolution and yeast surface display to screen a synthetic single chain antibody (anti-fluorescein scFv) library, previously engineered to reduce non-specific binding1, for improved binding to the ADAM-17 catalytic domain using fluorescent-activated cell sorting (FACS). DNA sequence analysis from each round of FACS, conducted via DNA sequence alignment bioinformatic tools, indicated that frequent antibody mutations within non-complementarity-determining regions (CDRs), light chain CDRs, and heavy chain CDR-2 are responsible for improving ADAM-17 binding. The individual scFv clones with improved binding affinity for ADAM-17 were further tested for MP binding affinities. This study set the stage for further engineering and design of protein scaffolds targeting MPs with high affinity and selectivity using directed evolution and rational design.

Sunday
Engineering of substrate specificity of direct electron transfer type glucose dehydrogenase complex which has a unique substrate binding cavity
05:35pm - 05:55pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
joseph kerrigan Jr., Presenter, UNC Chapel Hill/ NC State; Junko Okuda-Shimazaki; Koji Sode
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
The bacterial flavin adenine dinucleotide (FAD) glucose dehydrogenase (GDH) complex is composed of a catalytic subunit which contains an FAD cofactor and Fe/S cluster in its redox center, showing catalytic activity, a hitchhiker protein subunit functioning as the bacterial TAT secretion system, and a membrane-bound subunit with three heme c moieties that is responsible for the transfer of electrons between the active-site cofactor and external electron acceptors.

The FADGDH complex has the potential to directly transfer electrons (DET) to an electrode because of the presence of the heme c subunit, and is recognized as DET-type FADGDH. The catalytic subunit belongs to the family of glucose-methanol-choline (GMC) oxidoreductase family. The issue with this enzyme is that the enzyme does not have enough substrate specificity towards glucose as compared to other glucose sensing enzymes such as glucose oxidase. The recent elucidation of its X-ray structure revealed the unique substrate binding cavity of this enzyme. Unlike other GMC-oxidoreductase family glucose oxidoreductases, DET-type FADGDH catalytic subunit is characterized with a large cavity in the active site, and fewer number of residues, which are responsible for the recognition of glucose. The mutations have been designed through the crystal structure of DET-type GDH ( PDB: 6A2U) and then engineered into the wild type enzyme. The strategy of mutation and the characterization of mutant enzymes, which show drastic decrease of enzyme activity toward sugars other than glucose, will be presented.

Sunday
Engineered antibody-oligonucleotide conjugate for use in insulin detection
05:55pm - 06:15pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
We propose an innovated semisynthetic biosensing molecule, an antibody-oligonucleotide one-to-one complex. An important consideration in the design of biosensors is the molecular recognition element (MRE) from which the signal to be transduced is generated. Among MREs utilized for affinity principle-based biosensors, antibodies are commonly employed due to their high sensitivity and selectivity towards specific targets. However, antibodies’ structural rigidity proves limiting in biosensing applications. DNA and RNA oligonucleotides, however, are among other biomolecules used in sensor development as they are inherently flexible which allows for pronounced conformational changes upon binding.

To this end, we propose a new biosensing molecule for biosensor applications based on antibody-oligonucleotide conjugates, which includes advantages of both antibodies and oligonucleotides; e.g. high sensitivity, selectivity with molecular dynamics upon target binding, facilitating signals for sensing. We propose to use insulin as a model target for this novel MRE based sensor. A sensor which can detect insulin levels in persons with diabetes would provide another useful metric in diabetes technology, as insulin measurements are typically done via ELISA.

A fusion molecule consisting of an insulin single chain antibody (scFv) and monomeric streptavidin was designed and recombinantly produced using Escherichia coli as a host microorganism. The fusion molecule which was inserted downstream of a signal sequence for secretion was successively produced as a soluble protein, extracellularly. The fusion protein showed recognition of insulin, comparable with that of the original scFv. This fusion molecule bound with high affinity to biotin, which facilitates the preparation of a one-to-one antibody-oligonucleotide conjugate. The characterization of thus created antibody-oligonucleotide conjugate for insulin sensing will be presented.

Sunday
Directed evolution of superinhibitors of quorum sensing signaling in Staphylococcus aureus
06:15pm - 06:35pm USA / Canada - Eastern - August 22, 2021 | Room: Zoom Room 06
Division: [BIOT] Division of Biochemical Technology
Session Type: Oral - Virtual
Methicillin-resistant Staphylococcus aureus infections are very common and resistant to most antibiotics. Like many pathogens, S. aureus uses cell-cell communication aka quorum sensing (QS) to coordinate attack and regulate expression of toxins. Using the model Gram-positive organism Bacillus megaterium, we have developed a high-throughput fluorescence-based assay for quorum sensing inhibition and activation of S. aureus signaling. Using a designed library of ~5,000 variants of the QS autoinducer peptide, we found and characterized over 150 potential inhibitors of quorum sensing and several superactivators. Because they are ribosomally templated, these peptides can be secreted by live biotherapeutics as a potential non-antibiotic treatment for S. aureus infection.