TFCP2 is a transcriptional regulator of heparan sulfate assembly and melanoma cell growth.

Heparan sulfate (HS) is a long, linear polysaccharide that is ubiquitously expressed in all animal cells and plays a key role in many cellular processes, including cell signaling and development. Dysregulation of HS assembly has been implicated in pathophysiological conditions, such as tumorigenesis and rare genetic disorders. HS biosynthesis occurs in a non-template-driven manner in the endoplasmic reticulum and Golgi through the activity of a large group of biosynthetic enzymes. While much is known about its biosynthesis, little is understood about the regulation of HS assembly across diverse tissue types and disease states. To address this gap in knowledge, we recently performed genome-wide CRISPR/Cas9 screens to identify novel regulatory factors of HS biosynthesis. From these screens, we identified the alpha globin transcription factor, TFCP2, as a top hit. To investigate the role of TFCP2 in HS assembly, we targeted TFCP2 expression in human melanoma cells using the CRISPR/Cas9 system. TFCP2 knockout cells exhibited decreased fibroblast growth factor binding to cell surface HS, alterations in HS composition, and slowed cell growth compared to wild-type cells. Additionally, RNA sequencing revealed that TFCP2 regulates the expression of multiple enzymes involved in HS assembly, including the secreted endosulfatase, SULF1. Pharmacological targeting of TFCP2 activity similarly reduced growth factor binding and increased SULF1 expression, and the knockdown of SULF1 expression in TFCP2 mutant cells restored melanoma cell growth. Overall, these studies identify TFCP2 as a novel transcriptional regulator of HS and highlight HS-protein interactions as a possible target to slow melanoma growth.

Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate.

Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.

ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis.

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.

Spatiotemporal diversity and regulation of glycosaminoglycans in cell homeostasis and human disease.

Glycosaminoglycans (GAGs) are long, linear polysaccharides that are ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These complex carbohydrates play important roles in many cellular processes and have been implicated in many disease states, including cancer, inflammation, and genetic disorders. GAGs are among the most complex molecules in biology with enormous information content and extensive structural and functional heterogeneity. GAG biosynthesis is a nontemplate-driven process facilitated by a large group of biosynthetic enzymes that have been extensively characterized over the past few decades. Interestingly, the expression of the enzymes and the consequent structure and function of the polysaccharide chains can vary temporally and spatially during development and under certain pathophysiological conditions, suggesting their assembly is tightly regulated in cells. Due to their many key roles in cell homeostasis and disease, there is much interest in targeting the assembly and function of GAGs as a therapeutic approach. Recent advances in genomics and GAG analytical techniques have pushed the field and generated new perspectives on the regulation of mammalian glycosylation. This review highlights the spatiotemporal diversity of GAGs and the mechanisms guiding their assembly and function in human biology and disease.

Sequence-Controlled Polymers Through Entropy-Driven Ring-Opening Metathesis Polymerization: Theory, Molecular Weight Control, and Monomer Design.

The bulk properties of a copolymer are directly affected by monomer sequence, yet efficient, scalable, and controllable syntheses of sequenced copolymers remain a defining challenge in polymer science. We have previously demonstrated, using polymers prepared by a step-growth synthesis, that hydrolytic degradation of poly(lactic- co-glycolic acid)s is dramatically affected by sequence. While much was learned, the step-growth mechanism gave no molecular weight control, unpredictable yields, and meager scalability. Herein, we describe the synthesis of closely related sequenced polyesters prepared by entropy-driven ring-opening metathesis polymerization (ED-ROMP) of strainless macromonomers with imbedded monomer sequences of lactic, glycolic, 6-hydroxy hexanoic, and syringic acids. The incorporation of ethylene glycol and metathesis linkers facilitated synthesis and provided the olefin functionality needed for ED-ROMP. Ring-closing to prepare the cyclic macromonomers was demonstrated using both ring-closing metathesis and macrolactonization reactions. Polymerization produced macromolecules with controlled molecular weights on a multigram scale. To further enhance molecular weight control, the macromonomers were prepared with cis-olefins in the metathesis-active segment. Under these selectivity-enhanced (SEED-ROMP) conditions, first-order kinetics and narrow dispersities were observed and the effect of catalyst initiation rate on the polymerization was investigated. Enhanced living character was further demonstrated through the preparation of block copolymers. Computational analysis suggested that the enhanced polymerization kinetics were due to the cis-macrocyclic olefin being less flexible and having a larger population of metathesis-reactive conformers. Although used for polyesters in this investigation, SEED-ROMP represents a general method for incorporation of sequenced segments into molecular weight-controlled polymers.

Targeting heparin and heparan sulfate protein interactions.

Heparin and heparan sulfate glycosaminoglycans are long, linear polysaccharides that are made up of alternating dissacharide sequences of sulfated uronic acid and amino sugars. Unlike heparin, which is only found in mast cells, heparan sulfate is ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These negatively-charged glycans play essential roles in important cellular functions such as cell growth, adhesion, angiogenesis, and blood coagulation. These biomolecules are also involved in pathophysiological conditions such as pathogen infection and human disease. This review discusses past and current methods for targeting these complex biomolecules as a novel therapeutic strategy to treating disorders such as cancer, neurodegenerative diseases, and infection.

Removing the "Silencer": Coverage and Protection of Physician Speech Under the First Amendment.

The physician-patient relationship rests on a bedrock of trust. Without trust, patients--and for that matter, physicians--are less willing to divulge information critical to providing accurate medical diagnoses and treatments. The state of Florida seemingly ignored this when its legislature, with support from the National Rifle Association and other pro-gun advocates, enacted the Firearm Owners Privacy Act (FOPA), a statute that restricts physicians from questioning their patients about firearm ownership. In Wollschlaeger v. Governor of Florida, the United States Court of Appeals for the Eleventh Circuit held that FOPA did not regulate physician speech but, instead, regulated physician conduct. As such, the law was exempted from First Amendment scrutiny. But almost one year to the day after publishing its first Wollschlaeger opinion, the Eleventh Circuit sua sponte vacated its original opinion and substituted in its place a brand new opinion--one holding that FOPA was subject to First Amendment scrutiny, but nonetheless passed constitutional muster. This Note uses the diverging Wollschlaeger opinions as a vehicle to analyze the First Amendment's coverage and protection of physician speech. Specifically, it argues that an uninhibited line of communication is required to protect the trust necessary for an effective physician-patient relationship. This logical underpinning leads to the conclusion that the First Amendment presumptively covers physician speech and, furthermore, that physician speech should be subject to intermediate scrutiny--a level of scrutiny that FOPA cannot meet.

IRF5 is a novel regulator of CXCL13 expression in breast cancer that regulates CXCR5(+) B- and T-cell trafficking to tumor-conditioned media.

Clinical studies using prognostic and predictive signatures have shown that an immune signal emanating from whole tumors reflects the level of immune cell infiltration--a high immune signal linked to improved outcome. Factors regulating immune cell trafficking to the tumor, however, are not known. Previous work has shown that expression of interferon regulatory factor 5 (IRF5), a critical immune regulator, is lost in ~80% of invasive ductal carcinomas examined. We postulated that IRF5-positive and -negative breast tumors would differentially regulate immune cell trafficking to the tumor. Using a focused tumor inflammatory array, differences in cytokine and chemokine expression were examined between IRF5-positive and -negative MDA-MB-231 cells grown in three-dimensional culture. A number of cytokines/chemokines were found to be dysregulated between cultures. CXCL13 was identified as a direct target of IRF5 resulting in the enhanced recruitment of B and T cells to IRF5-positive tumor-conditioned media. The ability of IRF5 to regulate mediators of cell migration was confirmed by enzyme-linked immunosorbent assay, chromatin immunoprecipitation assay, small interfering RNA knockdown and immunofluorescence staining of human breast tumor tissues. Analysis of primary immune cell subsets revealed that IRF5 specifically recruits CXCR5(+) B and T cells to the tumor; CXCR5 is the receptor for CXCL13. Analysis of primary breast tumor tissues revealed a significant correlation between IRF5 and CXCL13 expression providing clinical relevance to the study. Together, these data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may, in combination with CXCL13, serve as a novel prognostic marker for antitumor immunity.

Recurrent thrombosis and patency of the arteriovenous access in patients receiving hemodialysis.

BACKGROUND: Thrombosis of the vascular access in patients with end-stage renal disease requiring hemodialysis are common and require timely interventional procedures to restore patency. The aim of the current study was to identify factors having a significant effect on patency rates after access thrombosis. Our hypothesis was the length of time between the initial clotting of the access and the subsequent percutaneous declotting impacts the patency rates of the vascular access. METHODS: In this retrospective cohort study, patients with a clotted arteriovenous access between Jan 1, 2011, and Jan 1, 2016, were included. Demographics, access history, and associated details of the access procedure were reviewed from the electronic medical record. Statistical analysis was done using t-test and chi-square or fisher exact tests to compare arteriovenous fistulae (AVF) and arteriovenous grafts (AVG). Primary patency, defined as the time from index procedure to endpoint, was analyzed using the Kaplan-Meier method and log rank test. RESULTS: There were 883 percutaneous declotting procedures reviewed. About 351 procedures were performed in patients with an AVF and 532 with an AVG. The mean time from thrombosis to declotting was 1.71 ± 2.29 days. The overall median primary patency for both AVF and AVG was 43 days with no difference in patency between patients with AVF (39 days) versus AVG (42 days; p = 0.385). The time period from access thrombosis to declotting did not affect patency rates for either AVG or AVF (p = 0.385). On multivariable analysis, prior intervention (HR: 1.32, 95% CI: 1.14-1.53, p < 0.001) and cardiovascular disease (HR: 1.19, 95% CI: 1.03-1.37, p = 0.016) were independently associated with access patency. CONCLUSIONS: Time from thrombosis to declotting did not affect patency rates however once there was a thrombotic event, recurrent thrombosis requiring intervention was common with patency significantly decreased. Future prospective studies to validate our results and study pathogenic mechanisms of recurrent thrombosis are warranted.

Branched-Tail Lipid Nanoparticles for Intravenous mRNA Delivery to Lung Immune, Endothelial, and Alveolar Cells in Mice.

Lipid nanoparticles (LNPs) are proven safe and effective delivery systems on a global scale. However, their efficacy has been limited primarily to liver and immune cell targets. To extend the applicability of mRNA drugs, 580 ionizable lipidoids are synthesized and tested for delivery to extrahepatocellular targets. Of these, over 40 enabled protein expression in mice, with the majority transfecting the liver. Beyond the liver, several LNPs containing new, branched-tail ionizable lipidoids potently delivered mRNA to the lungs, with cell-level specificity depending on helper lipid chemistry. Incorporation of the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 16 mol% enabled highly specific delivery to natural killer and dendritic cells within the lung. Although inclusion of the cationic lipid 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) improved lung tropism, it decreased cell specificity, resulting in equal transfection of endothelial and lymphoid cells. DOTAP formulations are also less favorable than DOPE formulations because they elevated liver enzyme and cytokine levels. Together, these data identify a new branched-tailed LNP with a unique ability to selectively transfect lung immune cell populations without the use of toxicity-prone cationic helper lipids. This novel vehicle may unlock RNA therapies for lung diseases associated with immune cell dysregulation, including cancer, viral infections, and autoimmune disorders.

Heparan sulfate modified proteins affect cellular processes central to neurodegeneration and modulate presenilin function.

Mutations in presenilin-1 (PSEN1) are the most common cause of familial, early-onset Alzheimer's disease (AD), typically producing cognitive deficits in the fourth decade. A variant of APOE, APOE3 Christchurch (APOE3ch) , was found associated with protection from both cognitive decline and Tau accumulation in a 70-year-old bearing the disease-causing PSEN1-E280A mutation. The amino acid change in ApoE3ch is within the heparan sulfate (HS) binding domain of APOE, and purified APOEch showed dramatically reduced affinity for heparin, a highly sulfated form of HS. The physiological significance of ApoE3ch is supported by studies of a mouse bearing a knock-in of this human variant and its effects on microglia reactivity and Aß-induced Tau deposition. The studies reported here examine the function of heparan sulfate-modified proteoglycans (HSPGs) in cellular and molecular pathways affecting AD-related cell pathology in human cell lines and mouse astrocytes. The mechanisms of HSPG influences on presenilin- dependent cell loss and pathology were evaluated in Drosophila using knockdown of the presenilin homolog, Psn , together with partial loss of function of sulfateless (sfl) , a homolog of NDST1 , a gene specifically affecting HS sulfation. HSPG modulation of autophagy, mitochondrial function, and lipid metabolism were shown to be conserved in cultured human cell lines, Drosophila , and mouse astrocytes. RNAi of Ndst1 reduced intracellular lipid levels in wild-type mouse astrocytes or those expressing humanized variants of APOE, APOE3 , and APOE4 . RNA-sequence analysis of human cells deficient in HS synthesis demonstrated effects on the transcriptome governing lipid metabolism, autophagy, and mitochondrial biogenesis and showed significant enrichment in AD susceptibility genes identified by GWAS. Neuron-directed knockdown of Psn in Drosophila produced cell loss in the brain and behavioral phenotypes, both suppressed by simultaneous reductions in sfl mRNA levels. Abnormalities in mitochondria, liposome morphology, and autophagosome-derived structures in animals with Psn knockdown were also rescued by simultaneous reduction of sfl. sfl knockdown reversed Psn- dependent transcript changes in genes affecting lipid transport, metabolism, and monocarboxylate carriers. These findings support the direct involvement of HSPGs in AD pathogenesis.

Altering heparan sulfate suppresses cell abnormalities and neuron loss in Drosophila presenilin model of Alzheimer Disease.

We examined the function of heparan-sulfate-modified proteoglycans (HSPGs) in pathways affecting Alzheimer disease (AD)-related cell pathology in human cell lines and mouse astrocytes. Mechanisms of HSPG influences on presenilin-dependent cell loss were evaluated in Drosophila using knockdown of the presenilin homolog, Psn, together with partial loss-of-function of sulfateless (sfl), a gene specifically affecting HS sulfation. HSPG modulation of autophagy, mitochondrial function, and lipid metabolism were shown to be conserved in human cell lines, Drosophila, and mouse astrocytes. RNA interference (RNAi) of Ndst1 reduced intracellular lipid levels in wild-type mouse astrocytes or those expressing humanized variants of APOE, APOE3, and APOE4. Neuron-directed knockdown of Psn in Drosophila produced apoptosis and cell loss in the brain, phenotypes suppressed by reductions in sfl expression. Abnormalities in mitochondria, liposomes, and autophagosome-derived structures in animals with Psn knockdown were also rescued by reduction of sfl. These findings support the direct involvement of HSPGs in AD pathogenesis.

Glycosaminoglycan-mediated lipoprotein uptake protects cancer cells from ferroptosis.

Lipids are essential for tumours because of their structural, energetic, and signaling roles. While many cancer cells upregulate lipid synthesis, growing evidence suggests that tumours simultaneously intensify the uptake of circulating lipids carried by lipoproteins. Which mechanisms promote the uptake of extracellular lipids, and how this pool of lipids contributes to cancer progression, are poorly understood. Here, using functional genetic screens, we find that lipoprotein uptake confers resistance to lipid peroxidation and ferroptotic cell death. Lipoprotein supplementation robustly inhibits ferroptosis across numerous cancer types. Mechanistically, cancer cells take up lipoproteins through a pathway dependent on sulfated glycosaminoglycans (GAGs) linked to cell-surface proteoglycans. Tumour GAGs are a major determinant of the uptake of both low and high density lipoproteins. Impairment of glycosaminoglycan synthesis or acute degradation of surface GAGs decreases the uptake of lipoproteins, sensitizes cells to ferroptosis and reduces tumour growth in mice. We also find that human clear cell renal cell carcinomas, a distinctively lipid-rich tumour type, display elevated levels of lipoprotein-derived antioxidants and the GAG chondroitin sulfate than non-malignant human kidney. Altogether, our work identifies lipoprotein uptake as an essential anti-ferroptotic mechanism for cancer cells to overcome lipid oxidative stress in vivo, and reveals GAG biosynthesis as an unexpected mediator of this process.

Glycosaminoglycan Analysis: Purification, Structural Profiling, and GAG-Protein Interactions.

Glycosaminoglycans (GAGs) are long, linear polysaccharides that are ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These complex carbohydrates are composed of alternating glucosamine and uronic acids that can be heterogeneously N- and O-sulfated. The arrangement and orientation of the sulfated sugar residues specify the location of distinct ligand binding sites on the cell surface, and their capacity to bind ligands impacts cell growth and development, the ability to form tissues and organs, and normal physiology. The heterogeneous nature of GAGs and their inherent structural diversity across different tissues, cell types, and disease states creates challenges to characterizing their structure and function. Here, we describe detailed methods to investigate GAG-protein interactions in vitro and evaluate the structural composition of two classes of sulfated GAGs, heparan sulfate and chondroitin/dermatan sulfate, using liquid chromatography, mass spectrometry, and radiolabeling techniques. Overall, these methods facilitate the evaluation of GAG structure and function to uncover the unique roles these molecules play in cell biology and human disease.

The mixing method used to formulate lipid nanoparticles affects mRNA delivery efficacy and organ tropism.

mRNA is a versatile drug molecule with therapeutic applications ranging from protein replacement therapies to in vivo gene engineering. mRNA delivery is often accomplished using lipid nanoparticles, which are formulated via mixing of aqueous and organic solutions. Although this has historically been accomplished by manual mixing for bench scale science, microfluidic mixing is required for scalable continuous manufacturing and batch to batch control. Currently, there is limited understanding on how the mixing process affects mRNA delivery efficacy, particularly in regard to tropism. To address this knowledge gap, we examined the influence of the type of mixing and microfluidic mixing parameters on the performance of lipid nanoparticles in mice. This was accomplished with a Design of Experiment approach using four nanoparticle formulations with varied ionizable lipid chemistry. We found that each formulation required unique optimization of mixing parameters, with the total delivery efficacy of each lipid nanoparticle generated with microfluidics ranging from 100-fold less to 4-fold more than manually mixed LNPs. Further, mixing parameters influenced organ tropism, with the most efficacious formulations disproportionately increasing liver delivery compared to other organs. These data suggest that mixing parameters for lipid nanoparticle production may require optimization for each unique chemical formulation, complicating translational efforts. Further, microfluidic parameters must be chosen carefully to balance overall mRNA delivery efficacy with application-specific tropism requirements.

Colopathy Associated with Pentosan Polysulfate Use.

Introduction: We describe a novel colopathy associated with pentosan polysulfate (PPS) use and measure the strength of the drug-disease association. Methods: Two-part investigation. In the cohort study of individuals with a history of prior long-term PPS use, case histories were obtained and gastrointestinal disease course was followed with review of endoscopy records and histopathology specimens. Findings were summarized with descriptive statistics. In the cross-sectional study of individuals with interstitial cystitis, drug exposure and medical histories were obtained for patients seen at a single clinical center. Strength of association between PPS use and diagnoses of inflammatory bowel disease (IBD) and/or irritable bowel syndrome (IBS) was measured with multivariate logistic regression. Results: In the cohort study of 13 participants, median PPS exposure was 2.04 kg (0.99-2.54). Eleven (84.6%) developed symptoms suggestive of IBD and/or IBS after initiation of PPS therapy. Of the 10 participants whose endoscopic and histopathologic findings we reviewed, six had abnormal-appearing colonic mucosa on endoscopy and all 10 had abnormal mucosal changes on histology. Clinical and histologic improvement was observed after PPS cessation. In the cross-sectional study of 219 subjects with interstitial cystitis, PPS use was a statistically significant predictor of both the IBD [adjusted odds ratio=3.3 (95% confidence interval, 1.2-8.8, p=0.02)] and the composite IBD+IBS [adjusted odds ratio=3.3 (95% confidence interval, 1.5-7.3, p=0.002)] outcomes. Discussion: We describe a strong association between PPS use and a clinical diagnosis of IBD and/or IBS. Histopathologic findings suggest a novel drug-associated colopathy, with some subjects requiring colectomy for dysplasia.

Periodic incorporation of pendant hydroxyl groups in repeating sequence PLGA copolymers.

A series of repeating sequence poly(lactic-co-glycolic acid) copolymers (RSC PLGAs) has been prepared with the precise incorporation of a pendant benzyl-ether substituted monomer derived from serine. Copolymers were synthesized from the assembly of sequence-specific, stereopure dimeric, and trimeric segmers of lactic, glycolic, and (S)-3-benzyloxy-2-hydroxypropionic acids with controlled and varied tacticities. Deprotection of the hydroxyl groups was accomplished by catalytic hydrogenolysis to yield highly functionialized, hydrophilic polyesters. The (1)H and (13)C NMR spectra for all of the copolymers were consistent with sequence and stereochemical retention and lacked the signal broadening that is inherent with more random copolymers.

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.