Competing Protonation and Halide Elimination as a Probe of the Character of Thiamin-Derived Reactive Intermediates.

Decarboxylation reactions from comparable thiamin diphosphate- and thiamin-derived adducts of p-(halomethyl)benzoylformic acids in enzymic and non-enzymic reactions, respectively, reveal critical distinctions in otherwise similar Breslow intermediates. The ratio of protonation to chloride elimination from the Breslow intermediate is 102-fold greater in the enzymic process. This is consistent with a lower intrinsic barrier to proton transfer on the enzyme, implicating formation of a localized tetrahedral (sp3) carbanion that is formed as CO2 is produced. In contrast, slower protonation in solution of the decarboxylated intermediate is consistent with formation of a delocalized planar carbanionic enol/enamine. The proposed structural and reactive character of the enzymic Breslow intermediate is consistent with Warshel's general theory of enzymic catalysis, structural characterization of related intermediates, and the lower kinetic barrier in reactions that occur without changes in hybridization.

Charge Dispersion and Its Effects on the Reactivity of Thiamin-Derived Breslow Intermediates.

The enzymic decarboxylation of 2-ketoacids proceeds via their C2-thiazolium adducts of thiamin diphosphate (ThDP). Loss of CO2 from these adducts leads to reactive species that are known as Breslow intermediates. The protein-bound adducts of the 2-ketoacids and ThDP are several orders of magnitude more reactive than the synthetic analogues in solution. Studies of enzymes are consistent with formulation of protein-bound Breslow intermediates with localized carbanionic character at the reactive C2α position, reflecting the charge-stabilized transition state that leads to this form. Our study reveals that nonenzymic decarboxylation of the related thiamin adducts proceeds to the alternative charge-dispersed enol form of the Breslow intermediate. These differences suggest that the greatly enhanced rate of decarboxylation of the precursors to Breslow intermediates in enzymes arises from maintenance of the carbanionic character at the position from which the carboxyl group departs, avoiding charge dispersion by stabilizing electrostatic interactions with the protein as formulated by Warshel. Applying Guthrie's "no-barrier" addition to Marcus theory also leads to the conclusion that maintaining the tetrahedral carbanion at C2α of the resulting adduct minimizes associated kinetic barriers by avoiding rehybridization as part of steps to and from the intermediate. Finally, maintenance of the reactive energetic carbanion agrees with the concepts of Albery and Knowles as the outcome of evolved enzymic processes.

The Need for an Alternative to Radicals as the Cause of Fragmentation of a Thiamin-Derived Breslow Intermediate.

Mandelylthiamin (1) is a conjugate of benzoylformate and thiamin that loses CO2 to form the classic Breslow intermediate (2), whose expected fate is formation of the thiamin conjugate of benzaldehyde (3). Surprisingly, it was observed that 2 decomposes to 4 and 5 and rearranges to 6 in competition with the expected protonation to give 3. Recent reports propose that the alternatives to protonation arise from homolysis followed by radical-centered processes. It is now found, instead, that the spectroscopic observations cited in support of the proposed radical pathways are likely to be the result of other events. An alternative explanation is that ionization of the enolic hydroxy group of 2 and resultant electronic reorganization leads to C-C bond cleavage and non-radical intermediates that readily form 4, 5, and 6.

Lithium-stabilized nucleophilic addition of thiamin to a ketone provides an efficient route to mandelylthiamin, a critical pre-decarboxylation intermediate.

Mandelylthiamin (MTh) is an accurate model of the covalent intermediate derived from the condensation of thiamin diphosphate and benzoylformate in benzoylformate decarboxylase. The properties and catalytic susceptibilities of mandelylthiamin are the subjects of considerable interest. However, the existing synthesis gives only trace amounts of the precursor to MTh as it is conducted under reversible conditions. An improved approach derives from the unique ability of lithium ions to drive to completion the otherwise unfavorable condensation of the conjugate base of thiamin and methyl benzoylformate. The unique efficiency of the condensation reaction in the presence of lithium ions is established in contrast to the effects of other Lewis acids. Interpretation of the pattern of the results indicates that the condensation of the ketone and thiamin is thermodynamically controlled. It is proposed that the addition of lithium ions displaces the equilibrium toward the product through formation of a stable lithium-alkoxide.

Multimodal Comparisons of Results Achieved by Different Side Branch Ballooning Techniques for Bifurcation Provisional Stenting.

BACKGROUND: Stepwise provisional stenting is the gold standard for percutaneous coronary intervention (PCI) on bifurcation lesions, but the optimal ballooning technique for eventual side branch treatment is not established. The objective of the present study was to compare the stent configuration obtained by 2 different side branch optimization techniques performed after main vessel (MV) stent implantation: proximal optimization technique+kissing balloon inflation+final proximal optimization technique (POT/KBI/POT [PKP]) versus proximal optimization technique+isolated side branch dilation+final proximal optimization technique (POT-side-POT [PSP]). METHODS: We realized a 1:1 prospective randomized trial comparing bifurcation PCI conducted (under angiographic and angioscopic visualization) with either PKP or PSP in reanimated swine hearts using commercially available drug-eluting stents. After PCI, the obtained stent configuration (expansion, eccentricity, apposition) was assessed by optical coherence tomography and micro-computed tomography dividing the stent in 4 segments. Primary study end point was minimum stent expansion at the distal MV segment. RESULTS: A total of 30 PCIs were successfully performed according to randomization obtaining overall good results (average minimum stent expansion >90% at optical coherence tomography and micro-computed tomography) with PSP or PKP. Minimum stent expansion at the distal MV segment was significantly higher with PKP as compared with PSP at optical coherence tomography (97.9±4.2% versus 91.0±7.7%; P=0.002) and micro-computed tomography (98.1±4.1% versus 91.3±7.9%; P=0.006). Other significant findings included higher stent eccentricity index at proximal MV with PSP, higher side branch scaffolding length and lower malapposition (at bifurcation core and distal MV) with PKP. CONCLUSIONS: This first prospective randomized trial in a unique non-atherosclerotic preclinical environment showed that bifurcation PCI conducted with PSP and PKP achieves different stent configurations. These findings might be useful in bifurcation PCI practice and call for further evaluations in clinical ground.

A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants.

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

Base-catalyzed decarboxylation of mandelylthiamin: direct formation of bicarbonate as an alternative to formation of CO2.

The decarboxylation of mandelylthiamin is subject to general base catalysis (ß = 0.26), an outcome that is inconsistent with the expected dissociative transition state in which CO(2) forms along with a residual carbanion. The results implicate a previously unrecognized associative route in which addition of water to a carboxylate followed by base-catalyzed proton transfer and C-C cleavage produces bicarbonate directly. Various reports of the presence or absence of base catalysis in decarboxylation reactions are consistent with the associative route's occurrence in cases where nucleophiles would be generated along with CO(2) in the usual dissociative route.

Multimodal Imaging of a Chimney-Stenting Procedure Performed Simultaneously with a Transcatheter Aortic Valve Replacement (TAVR) in a Reanimated Human Heart including Post-Implant Analyses.

Transcatheter aortic valve replacement (TAVR) has become a popular treatment option for severe aortic stenosis for patients with a high risk for mortality with surgical aortic valve replacement (SAVR). Coronary artery occlusion (CAO) following the implantation of the device is a potential and sometimes devastating complication of this procedure, that provokes a sudden deterioration of hemodynamic status followed by cardiogenic shock and electrical instability. With patients that present a high risk for coronary obstruction, coronary protection with a chimney stenting technique is an effective strategy that can ensure coronary perfusion during TAVR in case of acute CAO. Utilizing Visible Heart® methodologies, a human heart was reanimated. A chimney stenting technique was implemented simultaneously with the deployment of a Medtronic Evolut™ Pro+ valve (Medtronic PLC; Minneapolis, MN, USA). The entire procedure was recorded utilizing endoscopic cameras, fluoroscopy, optical coherence tomography, and echocardiography. In addition to these procedural visualizations, post-procedural micro-computed tomography (micro-CT) was conducted to provide post-implantation imaging with approximately 60-micron resolution. Utilizing these imaging modalities in a reanimated human heart allows for the unique opportunity to collect data for TAVR procedures in real human anatomies for the subsequent educational uses by the physicians treating aortic valvular disease and/or the designers of future TAVR technologies and procedures.

Axonopathy precedes cell death in ocular damage mediated by blast exposure.

Traumatic brain injuries (TBI) of varied types are common across all populations and can cause visual problems. For military personnel in combat settings, injuries from blast exposures (bTBI) are prevalent and arise from a myriad of different situations. To model these diverse conditions, we are one of several groups modeling bTBI using mice in varying ways. Here, we report a refined analysis of retinal ganglion cell (RGC) damage in male C57BL/6J mice exposed to a blast-wave in an enclosed chamber. Ganglion cell layer thickness, RGC density (BRN3A and RBPMS immunoreactivity), cellular density of ganglion cell layer (hematoxylin and eosin staining), and axon numbers (paraphenylenediamine staining) were quantified at timepoints ranging from 1 to 17-weeks. RNA sequencing was performed at 1-week and 5-weeks post-injury. Earliest indices of damage, evident by 1-week post-injury, are a loss of RGC marker expression, damage to RGC axons, and increase in glial markers expression. Blast exposure caused a loss of RGC somas and axons-with greatest loss occurring by 5-weeks post-injury. While indices of glial involvement are prominent early, they quickly subside as RGCs are lost. The finding that axonopathy precedes soma loss resembles pathology observed in mouse models of glaucoma, suggesting similar mechanisms.