Rose Bengal Binding to Collagen and Tissue Photobonding

We investigated two critical aspects of rose Bengal (RB) photosensitized protein cross-linking that may underlie recently developed medical applications. Our studies focused on the binding of RB to collagen by physical interaction and the effect of this binding and certain amino acids on RB photochemistry. Molecular dynamics simulations and free-energy calculation techniques, complemented with isothermal titration calorimetry, provided insight into the binding between RB and a collagen-like peptide (CLP) at the atomic level. Electrostatic interactions dominated, which is consistent with the finding that RB bound equally well to triple helical and single chain collagen. The binding free energy ranged from −5.7 to −3 kcal/mol and was strongest near the positively charged amino groups at the N-terminus and on lysine side chains. At high RB concentration, a maximum of 16 ± 3 bound dye molecules per peptide was found, which is consistent with spectroscopic evidence for aggregated RB bound to collagen or the CLP. Within a tissue-mimetic collagen matrix, RB photobleached rapidly, probably due to electron transfer to certain protein amino acids, as was demonstrated in solutions of free RB and arginine. In the presence of arginine and low oxygen concentrations, a product absorbing at 510 nm formed, presumably due to dehalogenation after electron transfer to RB. In the collagen matrix without arginine, the dye generated singlet oxygen as well as the 510 nm product. These results provide the first evidence of the effects of a tissue-like environment on the photochemical mechanisms of rose Bengal

Evaluation of plant-expressed RBD protein functionality, folding and binding kinetics.

A comprehensive assessment of protein function of the RBD produced in N. benthamiana as it pertains to protein folding and binding to ACE2 receptor, recognition and neutralization by antibodies in sera from SARS-CoV-2 exposed individuals. (A) Indirect ELISA demonstrating binding kinetics of soluble human ACE2 to immobilized mammalian RBD (red circle) and plant RBD (blue triangle). (B) Binding and recognition of immobilized mammalian and plant produced RBD by IgG, IgM and IgA polyclonal antibodies in sera of pooled naïve (unvaccinated, uninfected individuals; n>100), pooled convalescent (pooled Conv.) (n>100), convalescent and vaccinated with one dose (Conv. + 1 dose), convalescent and vaccinated with two doses (Conv. + 2 dose), vaccinated with one dose (1 dose), vaccinated with 2 doses (2 doses) of Pfizer (BNT162b2) and naïve (PCR negative confirmed). Pooled sera are samples pooled from a surveillance study of 100 individuals with (pooled convalescent) or without (pooled naïve) prior SARS-CoV-2 infection. While the rest of the samples were collected from single individuals from each category described above. (C) Binding and recognition of immobilized mammalian and plant produced RBD by conformation dependant monoclonal IgG, IgM, and IgA CR3022 antibodies and (D) their half-maximal inhibitory dilution (ID50) values. (E) Relative inhibition percentage of anti-SARS-CoV-2 neutralizing antibodies in blocking immobilized mammalian and plant produced RBD from binding to soluble ACE2 by snELISA. This assay is representative of technical triplicates or quadruplicates and presented as mean ± standard deviation. (F) Reciprocal ID50 values from (E) for mammalian- and plant-expressed RBD.</p

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Schematic representations of the different types of ELISA used to characterize plant-expressed RBD.

(A) Indirect ELISA (Kd) set up for the evaluation of binding kinetics of soluble human ACE2 to immobilized RBD. (B) Indirect ELISA (serology) set up for the evaluation of binding and recognition of commercial monoclonal and serum IgG, IgM and IgA antibodies to immobilized RBD. (C) Surrogate neutralization ELISA (snELISA) set up to evaluate relative inhibition of anti-SARS-CoV-2 neutralizing antibodies in blocking immobilized RBD from binding soluble ACE2.</p

Biochemical evaluation of plant-expressed RBD by circular dichroism (CD).

A) Spectral profiles of mammalian standard (black) and plant (blue) expressed RBD in PBS buffer pH 7.4 at 37°C (200–250 nm). Data expressed in molar circular dichroism (Δε) calculated from averaging 5 independent spectra of each sample. B) Secondary structure content in percentage calculated for mammalian and plant-derived RBD in PBS buffer pH 7.4 at 37°C (200–250 nm). Content was calculated using BeStSel analysis server by direct analysis of the raw data (mDeg) from the CD system. Molecular weights for both proteins were considered as 35 kDa.</p

Expression and purification of SARS-CoV-2 RBD in <i>Nicotiana benthamiana</i>.

(A) Agro-infiltrated Nicotiana benthamiana growing in greenhouse. (B) Schematic representation of genetic construct used to express SARS-CoV-2 RBD in planta. The SARS-CoV-2 sequence was expressed as a recombinant protein with a dual 8xHis and Twin-Strep II tag, interspersed with Gly-Ser linkers (gold boxes). An ER-retention KDEL sequence was positioned at the C-terminus, and a Thrombin cleavage site (LVPRGS) was included for tag removal. (C) Left panel: Anti-His IB of samples obtained from N. benthamiana 2 to 5 days post-infiltration (dpi) with RBD construct in B. Loading control at 5 dpi reproducibly demonstrates reduced abundance of protein due to initiation of tissue necrosis at this time. Right panel: NT control (lane 1) compared to RBD expressed in N. benthamiana with calreticulin (lane 2). Anti-his IB. (D) Co-infiltration of human calreticulin (CRT) increases expression levels of RBD in N. benthamiana. Samples collected 4dpi. Anti-his IB. (E) Anti-S1 IB of purified RBD expressed in N. benthamiana (lane 1) and control RBD expressed in mammalian 293F cells (lane 2). Arrow indicates expected migration of a protein corresponding to 31.3 kDa. (F) CBB-stained SDS-PAGE of purified RBD expressed in N. benthamiana (lane 1) and control RBD expressed in mammalian 293F cells (lane 2). (G) CBB-stained SDS-PAGE of plant-derived RBD treated with (+) and without (-) the amidase Peptide-N-Glycosidase F (PNGaseF), an enzyme that cleaves N-linked glycan chains. SP, signal peptide. IB, immunoblot. CBB, Coomassie Brilliant Blue. NT, non-transformed.</p

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Reaction Kinetics of Phenolic Antioxidants toward Photoinduced Pyranine Free Radicals in Biological Models

8-Hydroxy-1,3,6-pyrenetrisulfonic acid (pyranine, PyOH) free radicals were induced by laser excitation at visible wavelengths (470 nm). The photochemical process involves photoelectron ejection from PyO– to produce PyO• and PyO•– with maxima absorption at 450 and 510 nm, respectively. The kinetic rate constants for phenolic antioxidants with PyO•, determined by nanosecond time-resolved spectroscopy, were largely reliant on the ionic strength depending on the antioxidant phenol/phenolate dissociation constant. Further, the apparent rate constant measured in the presence of Triton X100 micelles was influenced by the antioxidant partition between the micelle and the dispersant aqueous media but limited by its exit rates from the micelle. Similarly, the rate reaction between ascorbic acid and PyO• was markedly affected by the presence of human serum albumin responding to the dynamic of the ascorbic acid binding to the protein

Deterministic Encapsulation of Human Cardiac Stem Cells in Variable Composition Nanoporous Gel Cocoons To Enhance Therapeutic Repair of Injured Myocardium

Although cocooning explant-derived cardiac stem cells (EDCs) in protective nanoporous gels (NPGs) prior to intramyocardial injection boosts long-term cell retention, the number of EDCs that finally engraft is trivial and unlikely to account for salutary effects on myocardial function and scar size. As such, we investigated the effect of varying the NPG content within capsules to alter the physical properties of cocoons without influencing cocoon dimensions. Increasing NPG concentration enhanced cell migration and viability while improving cell-mediated repair of injured myocardium. Given that the latter occurred with NPG content having no detectable effect on the long-term engraftment of transplanted cells, we found that changing the physical properties of cocoons prompted explant-derived cardiac stem cells to produce greater amounts of cytokines, nanovesicles, and microRNAs that boosted the generation of new blood vessels and new cardiomyocytes. Thus, by altering the physical properties of cocoons by varying NPG content, the paracrine signature of encapsulated cells can be enhanced to promote greater endogenous repair of injured myocardium