The author acknowledges Dr. Christian Derntl from the Department for Biotechnology and Microbiology at the TU Wien and the Department of Medicine III, Division of Nephrology and Dialysis at the Medical University of Vienna, especially Dr. Markus Wahrmann for technical and scientific support. Protein expression in mammalian cells was supported by the Department of Biotechnology at the University of Natural Resources and Life Sciences (BOKU) Vienna. The author wants to express her deep acknowledgement to Dr. Nico Dankbar from XanTec bioanalytics in Duesseldorf, Germany, for helpful scientific discussions on performing the SPR binding assays.

For the present study, a CVN-small ubiquitin-like modifier (SUMO) fusion protein has been used in enzyme-linked immunosorbent assays instead of CV-N and is suitable for cell-based assays. Recombinant full-length influenza A virus HA H3 protein is obtained commercially (see Table of Materials) or expressed in mammalian HEK293 cell lines and baculovirus-infected insect cells according to standard protocols12. Wuhan-1 spike protein is expressed in mammalian HEK293 cells. The synthesis of monomannosylated peptide (MM) and dimannosylated peptide (DM) allows the detection of homogeneous ligands to CVN2 and monomannosylated small molecule10.
1. Creating CV-N constructs
<ol>
	<li>For each of the CVN2 variants and CVN2L0 protein (PDB ID 3S3Y), obtain the gene construct with an N-terminal pelB leader sequence and His-tag in pET27b(+) vector from commercial sources (see Table of Materials, Supplementary File 1).</li>
	<li>Obtain CVN2L0 and its variants (V2, V3, V4, and V5; Figure 2A,C) in the background of a CVN2L0 template gene consisting of two distinct DNA sequences for each CV-N repeat.</li>
	<li>Dissolve the lyophilized plasmid DNA in sterile deionized distilled water (ddH2O) to a final concentration of 100 ng/&#181;L.</li>
</ol>
2. Preparation of LB-agar plates with plasmid DNA transformed cells
<ol>
	<li>Prepare culture medium LB-Lennox by dissolving 10 g/L of peptone, 5 g/L of yeast extract, and 5 g/L of NaCl in ddH2O (see Table of Materials), and adjust the pH to 7.4. Perform the transformation into competent E. coli BL21 (DE3) for each variant (V2-V5) by chemical method following a previously published report10.</li>
	<li>Split the solution (900 &#181;L and 100 &#181;L), transfer 100 &#181;L on LB-agar plates (50 &#181;g/mL kanamycin), and gently use a sterile cell spreader. Incubate the agar plates overnight at 37 &#176;C.</li>
</ol>
3. Cloning
<ol>
	<li>Subclone the gene for CV-N into the NdeI and BamHI sites of pET11a (see Table of Materials) for transformation (electroporation) into electrocompetent cells following reference27.</li>
</ol>
4. Site-directed mutagenesis
<ol>
	<li>To generate CVN2L029 and mutant CVN-E41A in the background of a CVN2L0 template gene containing two distinguished DNA sequences for each CV-N repeat27.</li>
	<li>Make mutations using a site-directed mutagenesis kit (see Table of Materials) and specific mutagenic primers 5&#39;-gagaaccgtcaacgtttgcgataacagagttcagg-3&#39; and 5&#39;-cctgaactctgttatcgcaaacgttgacggttctc-3&#39; for running the PCR31.
	<ol>
		<li>Start a series of sample reactions using multiple concentrations of double-stranded DNA (dsDNA) template ranging from 5-50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA template). Keep the primer concentration constant. 
		NOTE: The PCR mix and thermal cycling protocol are generally used as described in the instruction manual for the site-directed mutagenesis kit32.</li>
	</ol>
	</li>
	<li>Add the Dpn I restriction enzyme (1 &#181;L, 10 U/&#181;L, see Table of Materials) below the mineral oil overlay. Thoroughly and gently mix reactions, spin down in a table-top microcentrifuge for 1 min, and incubate immediately at 37 &#176;C for 1 h for digesting the parental supercoiled dsDNA.</li>
</ol>
5. Transformation of bacterial cells
<ol>
	<li>Thaw the XL1-Blue supercompetent cells (see Table of Materials) gently on ice. To transform each control and sample reaction, aliquot the supercompetent cells (50 &#181;L) to a prechilled polypropylene round-bottom tube (14 mL).
	<ol>
		<li>Transfer 1 &#181;L of the Dpn I-treated single-stranded DNA (ssDNA) from each control and sample reaction (mutated ssDNA) to separate aliquots of the supercompetent cells, which synthesize the complementary strand. Swirl the transformation reactions carefully to mix and incubate the reactions on ice for 30 min. 
		NOTE: Before transferring the Dpn I-treated DNA to the transformation reaction, it is recommended to remove any remaining mineral oil carefully from the pipette tip. As an optional control, the transformation efficiency of the XL1-Blue supercompetent cells needs to be checked by mixing 0.1 ng/&#181;L of the pUC18 control plasmid (1 &#181;L) with a 50 &#181;L aliquot of the supercompetent cells.</li>
	</ol>
	</li>
	<li>Apply heat pulse to the transformation reactions at 42 &#176;C for 45 s, and then place the reactions on ice for 2 min. 
	NOTE: The applied heat pulse has already been optimized for the mentioned conditions in polypropylene round-bottom tubes (14 mL).</li>
	<li>Add 0.5 mL of NZY+ broth (containing per liter: 10 g of NZ amine (casein hydrolysate), 5 g of yeast extract, 5 g of NaCl, 12.5 mL of 1 M MgCl2, 12.5 mL of 1 M MgSO4, 10 mL of 2 M glucose, pH 7.5, and preheated to 42 &#176;C) and incubate the transformation reactions at 37 &#176;C with shaking at 225-250 rpm for 1 h. Plate the correct volume of each transformation reaction (5 &#181;L from control plasmid transformation; 250 &#181;L from sample transformation) on LB-ampicillin agar plates. 
	NOTE: For the transformation controls and mutagenesis, spread cells on LB-ampicillin agar plates having 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal, 80 &#181;g/mL) and isopropyl-1-thio-&#946;-D-galactopyranoside (IPTG, 20 mM) (see Table of Materials). Inoculate 50 mL of the cell cultures with a single colony of transformed E. coli cells to purify mutated plasmid DNA for analyses. Mutagenesis is confirmed by DNA sequencing at an external facility.</li>
</ol>
6. Expression and protein purification
<ol>
	<li>For a large-scale culture, inoculate a small amount of LB (containing ampicillin) with a single colony from the transformed plate.</li>
	<li>Using the overnight culture, inoculate the expression culture with additives, such as 10 mM of MgCl2, 10 mM of MgSO4, and 20 mM of glucose, diluting the seed culture to 1/100.
	<ol>
		<li>Grow cells with vigorous shaking at 37 &#176;C. Grow cells to an Abs 600 nm between 0.4-0.6 (mid-log phase) before cooling the cells to 20 &#176;C. Induce with 1 mM IPTG and grow overnight.</li>
		<li>Then, harvest the cells by centrifuging at 4,000 x g for 15 min at 4 &#176;C, and discard the supernatant with a pipette.</li>
	</ol>
	</li>
	<li>Resuspend the cell pellet in phosphate-buffered saline (PBS) buffer and re-centrifuge at 4,000 x g for 15 min at 4 &#176;C. Then, discard the supernatant with a pipette. Resuspend the remaining pellet in 10 mL of lysis buffer and incubate the suspension for 1 h at 37 &#176;C. 
	NOTE: Composition of lysis buffer: (50 mM of NaH2PO4, 300 mM of NaCl, 2% Triton-X100, 500 ng/mL of lysozyme, 1 mM of phenylmethylsulfonylfluoride (PMSF), 1 mM of dithiothreitol, 1 mM of MgCl2, pH 8, see Table of Materials).
	<ol>
		<li>Subject the mixture to two freeze-thaw cycles (-80 &#176;C). Separate soluble and insoluble fractions by centrifugation at 4,000 x g for 15 min at 4 &#176;C and analyze them using polyacrylamide gel electrophoresis (PAGE), in particular, sodium dodecyl sulfate (SDS)-PAGE33(Figure 2B,C). 
		NOTE: Cells can be lysed in several ways, such as freeze-thaw, sonication, homogenization, enzymatic lysis, or a combination of these methods. Purification from inclusion bodies is recommended for collecting high protein yields26.</li>
	</ol>
	</li>
	<li>Purify proteins using Ni-NTA column chromatography (see Table of Materials). Load the soluble fraction onto a regenerated Ni-NTA bead column (1 mL/min). Wash the system with TBS buffer (50 mM of Tris, 150 mM of sodium chloride, pH 7.5) before starting a gradient (0-100% 500 mM imidazole in TBS over 60 min) and collecting fractions (1 mL/min). Dialyze the purified proteins for biochemical characterization against 100 mM PBS (Figure 2C,D).</li>
	<li>Alternatively, use His-Select Ni2+ affinity gel (see Table of Materials) in 14 mL tubes to bind and re-suspend his-tagged recombinantly expressed CV-N in buffer solutions with 20 mM imidazole and 250 mM imidazole, respectively. Incubate in batch for at least 30 min. 
	NOTE: Apply these semi-purified proteins to a single-use prepacked column for buffer exchange and cleanup of biological samples, for example, carbohydrates and proteins, which can load 1-1.5 mL eluate from immobilized metal affinity chromatography.</li>
	<li>Transfer the protein solutions to centrifugation tubes with a 10 kDa cut-off filter (see Table of Materials) and concentrate them by centrifuging for 10 min at 4,500 x g and 4 &#176;C. For SPR measurements, exchange the analyte solutions to 10 mM HEPES, 150 mM sodium chloride, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% Tween20, pH 7.4 (HBS-EP(+), see Table of Materials).
	<ol>
		<li>Add this SPR running buffer to a dilution factor of 1:10 and centrifuge four times to the initial volume for 10 min at 4,500 x g and 4 &#176;C.</li>
	</ol>
	</li>
	<li>Determine the protein concentration at 280 nm using a NanoDrop UV-Vis spectrophotometer (see Table of Materials) based on the calculated extinction coefficient (20,440 M-1 cm-1) for the main protein CVN2L0 showing a size of 23,474 Da34. Use PBS (100 mM, pH 7.0) or SPR buffer as a blank, and measure the protein concentration at three dilution steps (1:1, 1:10, and 1:100).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63541/63541fig02.jpg" /> 
Figure 2: CV-N sequences and expression. (A) CVN2 without a linker between each CV-N repeat (101 amino acids each) and four disulfide bridges is expressed in pET11a vector in E. coli. (B) Expressions of two independent colonies for CV-N (monomer) and CVN2 (dimer). (C) Disulfide bond variants are purified and analyzed on SDS-PAGE. A low molecular weight marker (6 &#181;L) is used as a reference. WT = CVN2L0 bearing four disulfide bridges as marked in (A). V2 is a variant with a disulfide bond replacement by polar residues at positions 58 and 73. V3-V5 are variants with two remaining S-S bonds and either polar (C58E-C73R) or non-polar (C58W-C73M) substituting amino acids or a combination of these residue pair substitutions. (D) HPLC chromatograms of purified CVN2L0 are elutated at a flow rate of 1 mL/min with a linear gradient from 5%-65% buffer B in buffer A over 30 min. Buffer A is: 0.1% (v/v) trifluoroacetic acid in ddH2O, buffer B is: 0.08% (v/v) trifluoroacetic acid in acetonitrile. Protein is analyzed on a high-performance silica gel 300-5-C4 (150 x 4.6 mm) column at 214 nm and 280 nm. <a href="https://www.jove.com/files/ftp_upload/63541/63541fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. SPR spectroscopy
<ol>
	<li>Use the Dual Channel SPR system (see Table of Materials) with running buffer HBS-EP(+) and 10 mM glycine HCl pH 1.5-1.6 as the regeneration buffer. Turn on the instrument, degasser, auto-sampler, and pump and wash the entire system with ddH20 for 1 h. Place ready-to-use running buffer in a separate bottle.</li>
	<li>Drop emersion oil onto the detector and mount a glass sensor chip (see Table of Materials) coated with a thin gold film and on the upper side functionalized with carboxymethyldextran hydrogel directly onto the detector below the three-port flow cell. Fix the setting by pulling down the handling. 
	NOTE: C19RBDHC30M 200 nm streptavidin derivatized carboxymethyldextran hydrogel with a medium density of biotinylated severe acute respiratory syndrome coronavirus-2 RBD protein, is a ready-to-use sensorchip with the pre-immobilized ligand.</li>
</ol>
8. SPR binding assay for CV-N binding to HA, S protein, and RBD
<ol>
	<li>Immobilize the proteinaceous ligands to sensor chips following the steps below.
	<ol>
		<li>Open a run table by clicking on Form in the menu bar and Run Table Editor in the integrated SPRAutoLink software (see Table of Materials). Choose and click on BASIC_Immobilization from the list of available run tables and follow the steps of the experimental procedure on the computer screen. The respective Sample Editor used is shown in the right upper corner.</li>
		<li>Click on Sample Set Editor in the Form section to fill out the reagents list for two racks placed in the autosampler for further analyses. Click on Autosampler Direct Control as a &#34;Tool&#34; in the menu bar to bring the racks forward or back home. Choose 4 &#176;C as the operating temperature. 
		NOTE: The SPR software allows for &#34;SPR Instrument Direct Control&#34; and &#34;Pump Direct Control&#34; via selecting the corresponding tools, as well as autosampler-handling, and by clicking on Form; also Run Table Editor, Data-Plot, or Post-Processing can be chosen to perform data analysis. Files are directly saved in the default directory and exported as scrubber.files from the Post-Processing window.</li>
	</ol>
	</li>
	<li>Start the pump to infuse ddH20 by clicking on Tools and Pump Direct Control and record data by clicking on SPR Instrument Direct Control, and each time Start&#160;in the newly appearing windows. Put the coupling reagents (step 8.3) in 300 &#181;L vials, put them into the autosampler racks and start the run table by clicking on Run. 
	NOTE: Chip surfaces are either conditioned with 10 mM glycine buffer pH 9.0 or may have been flushed with 1 M sodium chloride, 0.1 M sodium borate buffer pH 9.0 to condition carboxyl derivatized chip surface for EDC/NHS activation mix30.</li>
	<li>For this simple protein-protein interaction, use the CMD500D chip (see Table of Materials) to generate a micro-refractive index units (&#181;RIU) = 2500 - 3000 flow cell with immobilized HA and &#181;RIU = 400 flow cell with spike protein. At an infuse flow rate of 15 &#181;L/min, inject an aqueous and equal mixture of 0.4 M N-ethyl-N&#39;-(dimethylaminopropyl) carbodiimide hydrochloride (EDC*HCl) and 0.1 M N-hydroxysuccinimide (NHS) by applying the following sequential steps.
	<ol>
		<li>Refill pump refill at 25,000 &#181;L/min, perform baseline adjustment for 30 s, inject 90 &#181;L of sample activation solution (EDC/NHS) over 6 min contact time, and then hold for another 5 min.</li>
		<li>Repeat this cycle after baseline running for 1 min at 10 &#181;L/min on only the left flow cell (blue) to inject and immobilize chemically synthesized peptides10, HA, and spike protein at 20 &#181;g/mL, and allow for the subsequent baseline adjustment with ddH2O for 1.5 min before quenching the activated chip surface with 1 M ethanolamine HCl pH 8.5.</li>
	</ol>
	</li>
	<li>Switch the tubes from the liquid sampler to the degasser from ddH20 into the bottle with HBS-EP(+) (Supplementary Figure 1).</li>
	<li>Analyze the SPR sensorgrams.
	<ol>
		<li>To perform kinetic studies, use various analyte concentrations (10-5-10-8 M), with a regeneration step after each injection and blank measurements after different analytes. Change the flow rate to 10 &#181;L/min and start injections for 4 min contact time, then 5 min baseline generation, and two regeneration steps of 2 min each with an interval of 30 s.</li>
		<li>Inject the buffer solution for blank measurements whose sensorgrams are subtracted from sample runs to normalize different protein concentrations.</li>
	</ol>
	</li>
	<li>Click on Form, scroll down, and change to &#34;Post-Processing&#34; by clicking this operation mode. Click on Add to select binding curves generated over time in the Data Plot Form for each flow cell, and export the overlay as a scrubber file (.ovr). Click on File to open the file saving options. Obtain response curves by aligning left and right curves and subtracting signals of the second reference channel from those of the ligand channel. 
	NOTE: Data is operated in &#34;Post-Processing&#34; by defining sensorgrams computationally. It is put into an overlay of sensorgrams from left and right flow cells, or represented as sensorgrams from the difference of both channels.</li>
	<li>Clean the entire fluidics with 50-100 mM glycine buffer pH 9.5, water, and 20% ethanol before and after binding measurements to remove traces of salt or any protein contamination, or more stringent, with 0.5% SDS and glycine. 
	NOTE: To prevent instrument damage, it is recommended to check the mechanical stability of the glass chip before re-inserting the chip cartridge into the instrument if chips have been stored under buffer or at 100% humidity.</li>
</ol>

The author has nothing to disclose.

CV-N&#39;s binding affinity is correlated with the number of functional binding sites [2H on domains B, and 2L on domain(s) A when engineered as domain-swapped dimer]. A variant with an altered binding affinity (CVN2L0-V2, a homodimeric stable fold of CV-N comprising a disulfide bridge knock-out) is expressed in E. coli, purified, and positively tested for binding to HA-protein (H3N2) using SPR10, and shows a conformational change upon binding HA with either H or L carbohydrate-binding sites and KD1 = 49 nM and KD2 = 8 &#181;M38. As the number of disulfide bridges near the glycan-targeting pocket in CVN2 decreased from 4 to 2, binding affinity to HA protein decreased (Figures 3B-D and Supplementary Table 1). Disulfide bond variants are created by substituting Cys and inserting polar residue pairs Glu - Arg with slightly decreasing stability. The variants otherwise represent the same molecule, and the four binding sites are functional, although multisite binding on the chip is affected for all variants based on two disulfide substitutions. A non-polar substitution of both disulfide bonds in the two high-affinity binding pockets makes variant CVN2L0-V3, which actively binds the HA-peptide in both its monomannosylated and dimannosylated form. The interactions with CVN2L0-V3 are confirmed for micromolar concentrations and binding with the synthetic peptides (MM and DM) in solution via STD-NMR. Notably, CVN2 SPR binding experiments to MM are not analyzable due to insufficient calculation of Rmax, a general drawback of the SPR technique, and weak binding affinities to the immobilized MM10. Increased ligand density of this peptide and other peptides reduces binding selectivity. Using SPR, dimeric CVN2L0 (2H+2L) with high stability shows multivalent binding to HA, which is expressed in insect cells, and specificity for dimannose on a single chemically engineered biomimicry for high-mannose oligosaccharides, assuming a binding model 1:1. Binding to oligo-mannosides is usually measured by isothermal titration calorimetry, which requires a higher concentration of ligand to be tittered against the lectin17.
Competitive binding to monomannose or any glycan smaller than Man(7) has not been found before either15, indicating the involvement of the chemical linkage with the peptide backbone in recognition of these glycan moieties. The number of mannose-mannose linkages in the target was varied, deciphering tryptophan interactions in the high-affinity glycan pocket. The type of modification of peptides with triazole-linked monomannose or triazole-linked dimannose may determine KD values, in particular to H. Concerning binding affinities of CVN2L0 (2H+2L) and CVN2L0-V2 (1H+2L) to DM, koff is to the maximum 10-fold higher concerning CVN2L0-V2 dissociation from DM versus HA10. Besides measuring kinetic rate constants, SPR allows to estimate the equilibrium constants for very slowly equilibrating systems without actually reaching equilibrium (Table 1). By contrast, kon expresses good relative response values for all binding curves of domain-swapped CVN2L0 and its disulfide bond variants (V2, V4, and V5) to HA (Supplementary Table 1), or DM, while the dissociation rate constant koff shows faster off-rates for V4 and V5, two variants bearing two functional L, and V4 with non-analyzable KD concerning DM (Figures 4B). Assigning domain B to H and domain A the respective L is a previous finding that basis for the disulfide bond variants to reveal different binding affinities in SPR, specified, to HA18,28.
SPR is one of the leading tools for biomolecular interaction analysis in biomedical research35. If a specific, timed control of the bulk analyte concentration is feasible in the SPR instrument, the quantitative evaluation of the kinetics of binding progress between macromolecules is possible, with increasing interest in analyzing multi-protein complexes39. The challenge in its use is the controlled positioning of one of the interaction components on the widely used carboxymethyl dextran, or HC polycarboxylate hydrogel surface, which captures small and large biomolecules. To optimize immobilization and binding capacity of the chip surface, streptavidin-biotin sandwich immobilization and immobilization via protein His-tag to an anti-his antibody are available40. The biophysical characteristics of proteins or functional groups of small molecules might interfere with immobilization results, but any difficulties are experienced with the herein described glycoproteins and glycopeptides that are covalently bound via EDC/NHS chemistry. The immobilized glycans, either glycoprotein or the synthetic homogenously mono- or dimannosylated glycopeptides, are used on re-usable chips to screen various engineered CV-N variants, and binding assays are applied to fully purified and recombinantly expressed lectins. Impurities in injected protein solution are damaging the SPR channel system. Although experimental procedures are described on an in vitro system in this study, the glycosylation pattern on viral spikes may be recognized selectively by each analyte concentration of this small model protein as injected over time with optimized mass transfer limitation36. The size of CV-N is ~11 kDa and supports reproducible SPR data for its binding to viral spike proteins.
CV-N&#39;s binding specificity to high-mannose glycans is confirmed by the bivalent binding interaction with the dimannosylated peptide mimetic rather than binding to structurally functional glycans and the mono-mannosylated peptide using isothermal titration calorimetry (data not shown). A variant with the point mutation Glu41Ala expresses the monomeric 101-residue protein with two intertwined carbohydrate-binding sites on each protomer. Therefore, this mutation site abolishes a contacting residue between the high- and low-affinity carbohydrate sites and reduces molecular binding strength to N-acetyl-D-glucosamine. Binding of CVN-E41A to SARS-CoV-2 spike protein, bearing complex-type N-linked glycosylation and O-glycosylation, is achieved in the SPR, but CV-N binding to this spike is for both the spike protein and RBD without physiological relevance, as measured at micromolar concentrations (Figure 4,5).
Mannosylated peptides developed and generated in this study are used as protein scaffolds to screen the antiviral agents&#39; binding characteristics by SPR and NMR, as they represent invariable ligands for carbohydrate-binding assays attached to a defined amino acid sequence10. Moreover, STD-NMR spectroscopy is a label-free technique, such as SPR, which allows the characterization of carbohydrate-binding by conformational selection10,41. In such settings, STD-NMR allows for the development of rapid screening methods for a large library of compounds to identify bioactive ligands via binding, epitope mapping, and direct determination of KD under different experimental conditions. Accurate KD values can be obtained by analyzing the protein-ligand association curve utilizing STD values at the limit of zero saturation time41,42. Therefore, the interrogation of protein-ligand interactions by the STD-NMR method was accomplished, but KDs are calculated on the SPR system.
Human 2019-nCoV (Wuhan-Hu-1-2019 novel coronavirus) is estimated to differ among 11 (out of 18) predicted N-linked glycosylation sites to SARS-CoV43. Epitope/paratope mapping reveals a few interplays with host-derived N-glycans and negligible contributions of antibody somatic hypermutations to epitope contacts37. The ability of antiviral lectins and broadly neutralizing antibodies to bind highly conserved epitopes on the influenza HA glycoprotein is most important to the rational design of vaccines for preventive and therapeutic use. Further investigation, however, is needed to evaluate the effect of immune-suppressive N-linked glycosylation around the host receptor binding site. The data may provide insight into the potential of virus spike proteins to escape from antibodies elicited during infection or to bind non-immunogenic CV-N and thus guide structure-based computational protein design for new optimized antiviral CVN2 variants. By contrast, the affinity of glycosylated Fc amino acid mutant to its receptor to elicit effector functions in vivo, uses a composition of glycosylation sites, and the number of glycans involved, may therefore be important to binding affinity but may not be determined for neutralization ability.
Taken together, fewer rotamers are predicted to bind carbohydrate ligands to proteins by computational protein design than protein-protein interactions alone. Domain-swapped CVN2, however, bearing various numbers of carbohydrate-binding sites, provides the necessary stability and flexibility to reveal different binding-affinities attributed to H and to form the multivalent interactions between oligomannose clusters on viral envelope spikes and neutralizing antibodies (NAb), thus allowing for inhibition assays to be experimentally validated.

Tetherin-associated antiviral activity is induced by interferon-&#945;, and it comprises protein-based tethers, that leads to the retention of fully formed virions on infected cell surfaces1. The necessity for tetherin glycosylation in the inhibition of virus release remains uncertain, implying the importance of glycosylation patterns on recombinantly expressed glycans for in vitro studies1,2, which depends on the conformation of (in the case of influenza virus) surface-expressed influenza hemagglutinin HA3,4. It has been noted that modification of oligosaccharide tethered to N-linked glycosylation is enough for tetherin-mediated restriction of HIV type-1 release2, while dimerization plays an essential role in preventing virus release, thereby involving the transmembrane domain or glycosyl-phosphatidyl-inositol (GPI)-anchor for tethering the budding virions5. Unique features are described for human and murine tetherin to block multiple enveloped viruses, retroviruses, and filoviruses. BST-2/tetherin is an interferon-inducible antiviral protein of the innate immunity1,6, acting with broad-spectrum antiviral activity and is antagonized by envelope glycoproteins5 to either translocate tetherin or disrupt the structure of tetherin6. For example, surface-expressed envelope glycoprotein HA and neuraminidase on influenza A virus are well known for tetherin antagonism in a strain-specific manner7, facilitating the recognition of host receptor binding sites8. Glycan-targeting antibodies are studied in the stoichiometry of their interactions with the rapidly customizing glycan shields on HA, resulting in binding affinity to influenza A H3N2 and H1N1 subtypes4.
To elucidate the binding mechanisms between antiviral agents and virus envelope spikes, i.e., carbohydrate ligands, and complementary immunological and spectroscopic methods, mono-, di- and tri-mannose moieties are chemically synthesized. The mannosylated peptides are created via azido glycosylation of glycosyl {beta}-peracetates to 1,2-trans glycosyl azides transformation9, mimicking the typically found N-acetyl glucosamine and high-mannose oligosaccharides on the surface of life-threatening viruses. Triazole bioisosteres are utilized to mimic linkages that form the mannosylated residue of HA peptide10 and facilitate site-specific interactions with antiviral CV-N derivatives around the second N-linked glycosylation spot on the HA head domain (HA top with 4 N-linked glycans N54, N97, N181, N301)8,11,12. Interactions between glutamic acid (Glu) and arginine (Arg) and the resulting helix dipole manifestated good stability of both model peptides and proteins but are visualized using SPR. If compared with recognizing a single chemically synthesized glycosylation site on HA10 by directly inhibiting receptor binding on the glycan moieties, a higher affinity of a four-site mutated Fc structure to its receptor is shown to elicit effector functions in vivo, revealing the unrelated composition of N-linked glycans attached to Fc mutant to be mechanistically determined13.
CV-N displays antiviral activity against HIV14,15, influenza16, and Ebola virus, which is mediated by nanomolar binding to high-mannose oligosaccharide modifications on envelope spike proteins12,17,18,19. Influenza HA binding to one high-affinity carbohydrate-binding site (H) in CV-N or two Hs in covalently linked dimeric CVN2 is determined to have equilibrium dissociation constants (KD) = 5.7 nM (Figure 1A) and KD = 2.7 nM, respectively. Both CV-N and CVN2 harbor another one or two low-affinity carbohydrate-binding sites (L)s12,17,20,21. Ebola GP1,2 binds to 2H of CVN2 with affinities in the lower nanomolar range (KD = 26 nM). CV-N WT binding to Ebola GP1,2 and HA exhibits affinities from KD = 34 nM to KD = 5.7 nM (A/New York/55/04)12. Lectins, such as CV-N, which specifically target high-mannose glycans on the viral envelopes, further inhibit replication of hepatitis C virus, SARS-CoV, herpesvirus, Marburg virus, and measles virus22.
The small CV-N molecule has been studied thoroughly for more than 20 years as it functionalizes to bind a wide range of viruses to inhibit viral entry16,18. Structural analyses and binding affinity assays indicate cross-linking of two Ls in a domain-swapped CVN2 dimer by bivalent binding in the micromolar range to enhance avidity to viral envelope glycoproteins10,19. Selective binding of Man&#945;1-2Man&#945; on Man(8) D1D3 arms and Man(9) comprises two binding sites of differing affinities located on opposite protein protomers20, thereby reaching nanomolar binding affinities (Figure 1B). Thus, CVN2 is considered a pseudo-antibody concerning its application to bind epitopes on HIV gp120, similar to virus-neutralizing antibodies17. Herein, the author is interested in investigating the potential binding of CVN2 to the SARS-CoV-2 spike via its receptor-binding domain (RBD). Binding curves of immobilized human angiotensin-converting enzyme (ACE)-2 with the SARS-CoV-2 RBD result in KD = 4.7 nM for this biologically relevant binding interaction23.
By contrast, selected immunoglobulin classes recognize specific and consistent structural protein patterns, which impart a substrate for affinity maturation in the membrane-anchored HA regions24. CV-N shows highly potent activity in almost all influenza A and B viruses16, and it is a broadly neutralizing antiviral agent. Our knowledge is incomplete on the location of targeted epitopes on the stem of HA1 and HA2 that possibly involve epitopic structures for glycan-targeting by highly neutralizing antibodies and as compared with lectin binding25.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63541/63541fig1v5.jpg" /> 
Figure 1: Schematic representation of the SPR binding assay for CV-N to virus envelope spikes. (A) SPR Assay for CV-N binding to ligand: HA full-length protein (90 kDa). Kinetic data set (5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 0 nM) showing real-time double-referenced binding to influenza HA A/New-York/55/04 (H3N2). (B) CVN2L0 variant V2 binding to immobilized ligand DM within a concentration range of 500 nM to 16 &#181;M. Sequence: L residues are highlighted in yellow. H residues are highlighted in gray. E58 and R73 are a replacement for cysteines in the wildtype protein and make V2 a stable protein fold with three instead of four disulfide bonds <a href="https://www.jove.com/files/ftp_upload/63541/63541fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Whereas the glycan shield on the membrane-distal HA top part induces high-affinity binding to CV-N12, CVN2 binding to HA adjacent to a disulfide bridge of the HA top part has further been observed at its low-affinity sites10,12. Various polar interactions and interaction sites are identified in carbohydrate-binding by CV-N. These interactions are verified by generating knock-out variants in the binding site to correlate binding affinities to in silico predicted glycosylation12. Thus, the project aims to compare previously tested chemically mannosylated HA peptides in binding affinity and specificity with short peptide sequences from SARS-related 2019-nCoV spikes and SARS-CoV-2, naturally occurring modified by a small number of different N-linked glycosylation sites and O-linked glycosylation. Using cryo-electron microscopy and binding assays, Pinto and coworkers report a monoclonal antibody, S309, that potentially recognizes an epitope on SARS-CoV-2 spike protein containing a conserved glycan within the Sarbecovirus subgenus, without competing with receptor attachment26. The protocol of this study describes how designing, expressing, and characterizing CV-N variants are important to study how CV-N and CVN2 bind to glycosylated proteins and synthetic mannosylated peptides using the SPR technology10,12.
Tandem-linked dimer CVN2L027 and binding-site variants (V2-V5) are recombinantly expressed and variants are with disulfide bond replacements (C58E and C73R) (Figure 2A). Also, a mutant with a single-point mutation E41A is prepared because this position has been seen as an intermolecular cross-contacting residue. This mutant is another interesting molecule for SPR binding measurements between the lectin and high-mannose oligosaccharides deciphering binding domains and allows comparison with the dimeric form. The domain-swapped crystal structure of CVN2 shows a flexible linker, that extends between 49 and 54 residues. The two domains can continue moving around the hinge as rigid bodies, developing either a monomer through intramolecular domain interactions (domain A -residues 1-39;90-101- with domain B -residues 40-89) or a dimer by intermolecular domain swapping [domain A (of the first monomer) with domain B (of the second), and domain B (of the first monomer) with domain A (of the second copy)]. There are no close interactions between the two protomers&#39; A and B domains, except for Glu4128. The gene for CV-N can be developed using a repetitive PCR method with 40-mer synthesized oligos29 and is then subcloned into the NdeI and BamHI sites of pET11a for transformation (electroporation) into electrocompetent cells as described by Keeffe, J.R.27. The protein, which is used for achieving the respective crystal structure (PDB ID 3S3Y), includes an N-terminal 6-histidine purification tag followed by a Factor Xa protease cleavage site. Site-directed mutagenesis is utilized to make point mutations, switch codons, and insert or delete single or multiple bases or codons for amino acid exchange. These transformations provide invaluable insight into protein function and structure. Recombinantly expressed and purified CV-N, CVN2, and CVN3 have been biophysically well studied20,21,27, are cheap to produce, and therefore used to characterize binding assays to glycans immobilized onto SPR sensor chips. Conventional enzyme-linked immunosorbent assay (ELISA) provides less reproducibility concerning the immobilization technique of glycan ligands and transforms real-time binding of various binding-site variants, which is shown for SPR, into endpoint assays.
Binding-affinity variant CVN2L0-V2 (an intact fold of homodimeric CV-N with a disulfide bridge substitution10) is expressed with a His-tag in Escherichia coli (E. coli), purified over Ni-NTA column applying affinity chromatography and tested for binding to HA (H3N2), monomannosylated HA-peptide and dimannosylated HA-peptide using SPR. Chemically mannosylated peptides, or HA and S protein, all are ligands and amine coupled to the hydrophilic chip surface via reactive esters or biotin-streptavidin protein engineering. The same procedure of sequential runs is applied to those ligands, injecting various dilutions of CV-N and variants of CV-N (and CVN2) to obtain kinetic information for the molecular interaction analyses as described below30. RBD-immobilized SPR sensor chip is used for binding studies on CV-N to S peptides, and affinities are compared to SARS-CoV-2 binding with the human ACE2.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#196;kta primeplus</td>
			<td>Cytiva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon tubes</td>
			<td>Merck</td>
			<td>C7715</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampillicin</td>
			<td>Sigma-Aldrich</td>
			<td>A5354</td>
			<td></td>
		</tr>
		<tr>
			<td>Beckmann Coulter Cooler Allegra X-30R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B06320</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell spreader</td>
			<td>Sigma-Aldrich</td>
			<td>HS86655</td>
			<td>silver stainless steel, bar L 33&#160;mm</td>
		</tr>
		<tr>
			<td>Custom DNA Oligos</td>
			<td>Sigma-Aldrich</td>
			<td>OLIGO</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Gensynthesis</td>
			<td>GenScript</td>
			<td>#1390661&#160;</td>
			<td>cloning vector: pET27b(+)&#160;</td>
		</tr>
		<tr>
			<td>Cytiva&#160;HBS-EP+ Buffer 10, 4x50mL</td>
			<td>Thermo Scientific</td>
			<td>50-105-5354</td>
			<td></td>
		</tr>
		<tr>
			<td>Dionex UlitMate 3000</td>
			<td>Thermo Scientific</td>
			<td>IQLAAAGABHFAPBMBFB</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpn I restriction enzyme (10 U/&#956;L)&#160;</td>
			<td>Fisher Scientific</td>
			<td>ER1701</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Merck</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC</td>
			<td>Merck</td>
			<td>39391</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Merck</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Minispin&#160;and MiniSpin Plus personal microcentrifuge</td>
			<td>Sigma-Aldrich</td>
			<td>Z606235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>51976</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine HCl</td>
			<td>Merck</td>
			<td>E6133</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap, Sterile, 25/Pack</td>
			<td>Corning</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine HCl</td>
			<td>Merck</td>
			<td>55097</td>
			<td></td>
		</tr>
		<tr>
			<td>HA H3 protein</td>
			<td>Abcam</td>
			<td>ab69751</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Merck</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>His-select Ni2+</td>
			<td>Merck</td>
			<td>H0537</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Merck</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Merck</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin A</td>
			<td>Sigma-Aldrich</td>
			<td>K1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Kromasil 300-5-C4</td>
			<td>Nouryon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Merck</td>
			<td>52062</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Merck</td>
			<td>19344</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Lennox</td>
			<td>Merck</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Merck</td>
			<td>10837059001</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Merck</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Merck</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2P04</td>
			<td>Merck</td>
			<td>S0751</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop UV-Vis2000c spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND2000CLAPTOP</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS</td>
			<td>Merck</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>NZ amine (casein hydrolysate)</td>
			<td>Merck</td>
			<td>C0626</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Merck</td>
			<td>806552</td>
			<td></td>
		</tr>
		<tr>
			<td>PD MidiTrap G-10</td>
			<td>Sigma-Aldrich</td>
			<td>GE28-9180-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck</td>
			<td>70171</td>
			<td></td>
		</tr>
		<tr>
			<td>pET11a</td>
			<td>Merck Millipore (Novagen)</td>
			<td>69436&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Merck</td>
			<td>PMSF-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (1000)</td>
			<td>Qiagen</td>
			<td>27106X4</td>
			<td></td>
		</tr>
		<tr>
			<td>Reichert Software Package Autolink1-1-9</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reichert SPR SR7500DC Dual Channel System</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scrubber2-2012-09-04 for data analysis</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Merck</td>
			<td>11667289001</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-directed mutagenesis kit incl pUC18 control plasmid</td>
			<td>Stratagene</td>
			<td>#200518</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodim chloride</td>
			<td>Merck</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate.Trihydrate</td>
			<td>Merck</td>
			<td>236500</td>
			<td></td>
		</tr>
		<tr>
			<td>SPR sensor chip C19RBDHC30M</td>
			<td>XanTec bioanalytics</td>
			<td>SCR C19RBDHC30M</td>
			<td></td>
		</tr>
		<tr>
			<td>SPR sensor chip CMD500D</td>
			<td>XanTec bioanalytics</td>
			<td>SCR CMD500D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilin Standard 90mm Petri Dishes</td>
			<td>Thermo Scientific</td>
			<td>101R20</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS</td>
			<td>Merck</td>
			<td>T5912</td>
			<td>10x, solution</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Merck</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Merck</td>
			<td>93657</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Merck</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2 Mixer</td>
			<td>Merck</td>
			<td>Z258423</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Merck</td>
			<td>XGAL-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>XL1-Blue Supercompetent Cells</td>
			<td>Stratagene</td>
			<td>#200236</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Merck</td>
			<td>Y1625</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering antiviral agents via surface plasmon resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions between mannosylated peptides and CV-N's high-affinity binding site. Virus envelope spikes gp120, HA, and Ebola glycoprotein (GP) 1,2 have been reported to bind both high- and low-affinity binding sites on dimeric CVN2. Dimannosylated HA peptide is also bound at the two low-affinity binding sites to an engineered molecule of CVN2, which is bearing a high-affinity site for the respective ligand and mutated to replace a stabilizing disulfide bond in the carbohydrate-binding pocket, thus confirming multivalent binding. HA binding is shown to one high-affinity binding site of pseudo-antibody CVN2 at a dissociation constant (KD) of 275 nM that further neutralizes human immunodeficiency virus type 1 (HIV-1) through oligomerization. Correlating the number of disulfide bridges in domain-swapped CVN2, which are decreased from 4 to 2 by substituting cystines into polar residue pairs of glutamic acid and arginine, results in reduced binding affinity to HA. Among the strongest interactions, Ebola GP1,2 is bound by CVN2 with two high-affinity binding sites in the lower nanomolar range using the envelope glycan without a transmembrane domain. In the present study, binding of the multispecific monomeric CV-N to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is measured at KD = 18.6 &#181;M as compared with nanomolar KD to those other virus spikes, and via its receptor-binding domain in the mid-&#181;-molar range.

A dimeric domain-swapped CVN2L0 molecule is tested for binding to the HA top region in three separate SPR experiments and binding affinity is presented in KD values. Domain B is assumed to comprise H-binding sites, which are impacted by replacing a disulfide bond into ionic residues, and domain A forms L10,18. Single injections of CVN2L0 and variants V2 (three disulfide bridges) and V5 (two disulfide bridges) are first tested for binding to the HA-coupled sensor chip to estimate binding capacities before setting up the kinetics measurement using the autosampler and running table editor (Figure 3A and Supplementary Figure 2). Repeated injections are automated for a series of CVN2L0, V2, and V5 at various concentrations in the nanomolar and &#181;-molar range in the system over time (Figure 3B-D). Correlating the number of intact Cys-Cys bonds, CVN2L0 is binding immobilized HA at KD = 255 nM when reaching good saturation. In this case, both KD values, calculated either from kinetic data or by fitting the equilibrium data to the Langmuir binding isotherm, are in moderate agreement (Table 1 and Supplementary Figure 3, Supplementary Figure 4).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63541/63541fig03.jpg" /> 
Figure 3: SPR binding assay for ligand binding via multivalent interactions. CVN2 (WT) and binding site-variants V2 and V5 with disulfide replacements in the high-affinity carbohydrate-binding pocket are tested for binding covalently immobilized HA. (A) Left and right flow cell binding curves of CVN2, V2, and V5 can be extracted from the SPR run protocol, and sensorgrams are summarized in an overlay. (B) Sensorgram from conventional SPR experiment shown as kinetic analyses for binding of CVN2L0 to HA, injecting analyte concentrations from 10-4 to 10-8 M.&#160;CVN2L0 is measured up to the highest concentration at 1.5 &#181;M (top line) and up to 500 nM (bottom line), for which no saturation on the chip surface nor equilibrium binding is achieved. RU = Response units. A CMD500D sensor chip is used. (C) SPR sensorgram for V2 binding to HA. (D) V5 binding to HA. The figure (B-D) is reproduced with permission from reference10. <a href="https://www.jove.com/files/ftp_upload/63541/63541fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>ka (M-1s-1)</td>
			<td>kd (s-1)</td>
			<td>KD [HA] 
			Kinetic</td>
			<td>KD [HA] 
			Equilibrium</td>
		</tr>
		<tr>
			<td>CVN2</td>
			<td>5.1 e3</td>
			<td>1.3 x10-3</td>
			<td>255 nM</td>
			<td>246 nM</td>
		</tr>
		<tr>
			<td>V2</td>
			<td>4.0 e3</td>
			<td>1.1 x10-3</td>
			<td>275 nM</td>
			<td>255 nM</td>
		</tr>
		<tr>
			<td>V5</td>
			<td>1.2 e3</td>
			<td>5.8 x10-2</td>
			<td>5 &#181;M</td>
			<td>4.9 &#181;M</td>
		</tr>
		<tr>
			<td rowspan="2">CVN2L0</td>
			<td>2.07 e4</td>
			<td>3.0 x10-3</td>
			<td>147 nM</td>
			<td>600 nM</td>
		</tr>
		<tr>
			<td>2.27 e4</td>
			<td>3.0 x10-3</td>
			<td>133 nM</td>
			<td>490 nM</td>
		</tr>
	</tbody>
</table>
Table 1: Binding affinities of CVN2 and variants to HA measured via SPR. Scrubber 2.0 (a BioLogic software) is utilized to fit real-time SPR association and dissociation curves and to calculate the equilibrium dissociation constants (KD) from kinetic and equilibrium data. Ka = association rate constant. Kd = dissociation rate constant.
Whereas KD values for binding of CVN2L0 and V2 to HA are both in the nanomolar range, V5 is diminished in binding (Table 1). In V5, two disulfide bonds are replaced by ion-pairing residues that retrain potential ionic interactions between mutated Glu and Arg residues (Figure 2A,3D). Primary data is analyzed following the integrated rate of association and rate of dissociation equations, which describe the binding kinetics of the interaction of soluble CV-N with immobilized ligand and the dissociation of the formed complex from the chip surface. Two independent measurements are performed, and sensorgrams, equivalent to those obtained from replicates are analyzed. KD is calculated as the quotient of koff/kon (Supplementary Table 1)10,35. However, KD is related to the equilibrium data that fit the Langmuir binding isotherm36, where the equilibrium binding response is plotted as a function of the analyte concentration (Supplementary Figure 3A,4A,3B,4B). In a concentration-dependent manner, dissociation rate constant kd is determined post analyses and incorporated into association phase fitting (kon) to estimate the association rate constant ka. (Table 1, Supplementary Figure 3C, Supplementary Figure 4C).
Next, the binding of CV-N to HA is compared to its interaction with RBD. CV-N WT binding to SARS-CoV-2 RBD is measured at weaker affinity (KD = 260 &#181;M, Figure 4A) as compared with binding to HA (KD = 5.7 nM)8,10,12 and binding to S protein (KD = 18.6 &#181;M, Figure 5). Concentration versus response is plotted for CV-N WT binding to RBD, assuming specific targeting of possibly conserved carbohydrates on the RBD S1 subunit (Figure 4A). The same affinity plot is shown for CVN2L0-V4 binding to DM that is no longer analyzable due to the replacement of disulfide bonds which interfere with high-affinity binding to small glycosylated peptides (Figure 4B).
CVN2L0-V4 (2 disulfide bonds opposite to unsymmetric replacement of cysteine residues by either Glu-Arg residues or amphipathic amino acids Trp and Met) can form non-polar hydrogen bond networks around the pseudo-domain B carbohydrate-binding site10. Higher density of glycans on HA protein reaches binding with polar Glu-Arg residues instead of cystines (Supplementary Table 1), and an association with mannosylated peptides (KD = 10 &#181;M for DM) between CVN2L0 and modifications into Glu-Arg, but shows discontinuous dissociation of variants from this monoglycosylated peptide, in particular when H is modified on both monomers. For the interaction between CVN2L0-V4 with DM, the response of 56 &#181;M CVN2L0-V4 injection yields a higher response than Rmax. (Figure 4B). V5 (2x Glu-Arg) fails in dissociation from surface-bound DM (data not shown). In sum, response curves of lower concentrations decline before ending the injection for all sensorgrams on CVN2 binding to peptide without native H and monomer binding to RBD.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63541/63541fig04.jpg" /> 
Figure 4: SPR analyses of (A) CV-N WT monomer binding to RBD. KD is calculated by fitting kinetic data (left side, KD = 260 &#181;M) and compared with affinity data (right side) using a simple biomolecular model for 1:1 binding between ligand and analyte (KD equil = 274 &#181;M). Equilibrium binding response or binding capacity is plotted as a function of concentration in comparison with the SPR binding curves (0-605 &#181;M CVN). H = High-affinity binding site. L = Low-affinity binding site. Both are found on CV-N and CVN2L0. (B) Dimeric CVN2L0-V4 binding to DM, assuming 2L&#160;without analyzable KD. CVN2L0-V4 concentrations are 56 &#181;M, 28 &#181;M, 14 &#181;M, 7 &#181;M, 3.5 &#181;M. <a href="https://www.jove.com/files/ftp_upload/63541/63541fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63541/63541fig05.jpg" /> 
Figure 5: Mutant CVN-E41A and CV-N WT binding to (A) S glycoprotein. Arrows indicate the beginning of the association and dissociation phase.&#160;(B) CVN-E41A binding to RBD on SARS-CoV-2. The ligands are pre-immobilized onto HC polycarboxylate and CMD500D sensor chips. Covid-19 S1 subunit [Pinto, D. et al.26; and Barnes, C. et al.37] is used for RBD immobilization. Flow rate: 30 &#181;L/min, 25 &#176;C; plotted raw data. In comparison, the binding of human blood serum antibodies to RBD with a dilution factor of 1:200 is depicted. (C) SPR assay of CV-N WT binding to S glycoprotein that is immobilized to a response level of 400 &#181;RIU to capture CV-N. <a href="https://www.jove.com/files/ftp_upload/63541/63541fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It has been shown before that monomeric CV-N with a single L cannot sufficiently bind gp120, but that CV-N&#39;s neutralization ability requires cross-linking of two carbohydrate-binding sites and is predominantly restored by dimerization to functionalize with 2H12,19. Hence, a monomeric mutant CVN-E41A is expressed, which is suspected to destabilize pseudo-domain B or can interrupt connectivity with the second domain A, such as found in CV-N WT. CVN-E41A monomer reveals stability upon binding to S protein similar to CV-N monomer. Although SPR response for 605-680 &#181;M analyte concentrations results in high response units and typical SPR sensorgrams for CV-N WT and E41A binding, respectively, the mutant is unstable when diluted (Figure 5).
Supplementary Figure 1: SPR sensorgram for capturing DM mimicry as part of influenza HA top.&#160;Screenshot: SPR data plot showing the SPR run protocol for the immobilization of DM onto SPR sensor chip CMD500D, which is coated with 500 nm carboxymethyl dextran hydrogel and suitable for kinetics analyses of low molecular weight analytes on high ligand density. Sensorchips are directly mounted onto the detector with emersion oil and fixed below a three-port flow cell. After quenching the activated chip surface with 1 M ethanolamine HCl pH 8.5, CVN2 is injected twice (concentration =1 &#181;M and 2 &#181;M, respectively). Purple: Difference between flow cell 1 and flow cell 2; response is recorded at an interval of 0.2 s. Blue: Flow cell 1: 3000-4000 micro-Refractive Index Units (&#181;RIU) coated from a 400 nM solution of the ligand DM to an activated carboxylated surface. Red: Reference flow cell 2. <a href="https://www.jove.com/files/ftp_upload/63541/Supplemental Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Manual run on HA-functionalized CMD500D sensor chip showing binding of dimeric CVN2L0-V5, V2, and CVN2L0. At infuse flow rates from 30-50 &#181;L/min, CVN2L0 and disulfide bond variants expressed with a His-tag and purified over Ni-NTA are injected in the mid-&#181;M-range and tested for binding to HA with an association phase of 3 min and dissociation phase of 2 min. Injections are V5 (15 &#181;M), V2 (2.4 &#181;M), 0 &#181;M, CVN2L0 subcloned from a SUMO-fusion protein (1 &#181;M), and CVN2L0 (2 &#181;M). Running buffer at physiological pH is used for all experiments at 25 &#176;C. All solutions are degassed and filtered (0.2 &#181;m) before injection into the system. Purple: Difference between flow cell 1 and flow cell 2; response is recorded at an interval of 0.2 sec. Blue: Flow cell 1: 2540 &#181;RIU HA. Red: Reference flow cell 2. <a href="https://www.jove.com/files/ftp_upload/63541/Supplemental Figure 2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: CVN2 binding to HA. Analyses of sensorgrams when curves are auto-aligned. (A) Sensorgrams zeroed and aligned using Scrubber software (left) and binding isotherm showing concentration-dependent response curve to bound analytes (right). (B) Binding isotherm expressed in percent capacity. (C) Computationally fitted theoretical association and dissociation curves. (D) Kinetics data on SPR sensorgrams without fitted curves. <a href="https://www.jove.com/files/ftp_upload/63541/Supplemental Figure 3.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: CVN2 binding to HA. Analyses of sensorgrams when curves are manually aligned. (A) Sensorgrams zeroed and aligned using Scrubber software (left) and binding isotherm showing concentration-dependent response curve to bound analytes (right). (B) Binding isotherm expressed in percent capacity. (C) Computationally fitted theoretical association and dissociation curves. (D) Kinetics data on SPR sensorgrams without fitted curves. <a href="https://www.jove.com/files/ftp_upload/63541/Supplemental Figure 4.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Kinetic data obtained from SPR sensorgrams for the binding of CVN2L0 and Cys-Cys bond variants V2, V4, and V5 to HA using a Langmuir 1:1 binding model. KD [M] = koff/kon&#160;or&#160;kd/ka. All data is generated at 25 &#176;C in HBS-EP(+) buffer.: <a href="https://www.jove.com/files/ftp_upload/63541/Supplementary Table 1.zip" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Engineering Antiviral Agents via Surface Plasmon Resonance at JoVE.com

Engineering Antiviral Agents via Surface Plasmon Resonance

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Engineering Antiviral Agents via Surface Plasmon Resonance

Surface plasmon resonance (SPR) is used to measure hemagglutinin (HA) binding to domain-swapped Cyanovirin-N (CV-N) dimer and to monitor interactions ...

engineering-antiviral-agents-via-surface-plasmon-resonance

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

2.2K Views.  University of California, Los Angeles. The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans.

Video: Engineering Antiviral Agents via Surface Plasmon Resonance

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...