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A low-molecular-weight, solid CO surrogate that only requires a low-power LED for activation to release 2 equiv of CO is reported. The surrogate can be universally implemented in various palladium-catalyzed carbonylative transformations. It is also compatible with protocols that employ blue-light to activate conventionally inaccessible substrates such as nonactivated alkyl halides. Furthermore, we demonstrate that the photolabile CO-releasing scaffold can be installed into polymeric materials, thereby creating new materials with CO-releasing capabilities.

Extensive studies in the last few decades have led to the establishment of CO as an endogenous signaling molecule and subsequently to the exploration of CO's therapeutic roles. In the current state, there is a critical conundrum in CO-related research: the extensive knowledge of CO's biological effects and yet an insufficient understanding of the quantitative correlations between the CO concentration and biological responses of various natures. This conundrum is partially due to the difficulty in examining precise concentration-response relationships of a gaseous molecule. Another reason is the need for appropriate tools for the sensitive detection and concentration determination of CO in the biological system. We herein report a new chemical approach to the design of fluorescent CO probes through de novo construction of fluorophores by a CO insertion-initiated lactamization reaction, which allows for ultra-low background and exclusivity in CO detection. Two series of CO detection probes have been designed and synthesized using this strategy. Using these probes, we have extensively demonstrated their utility in quantifying CO in blood, tissue, and cell culture and in cellular imaging of CO from exogenous and endogenous sources. The probes described will enable many biology and chemistry labs to study CO's functions in a concentration-dependent fashion with very high sensitivity and selectivity. The chemical and design principles described will also be applicable in designing fluorescent probes for other small molecules.

Background Necrotizing soft tissue infections (NSTI) are characterized by fulminant tissue destruction and high mortality rates. Primary treatment is surgical debridement and antibiotics; hyperbaric oxygen therapy (HBOT) may be considered as adjuvant therapy. HBOT for NSTI remains controversial given the lack of quality evidence. HBOT is frequently used for NSTI at our institution; however, the early COVID-19 pandemic forced a significant decrease in its utilization. This practice change created an unusually high proportion of NSTI patients who did not receive HBOT at our institution and thus provided an opportunity to compare outcomes amongst NSTI patients who received HBOT against those who did not. Methods We performed a retrospective cohort study of patients aged 18 years and older admitted with the diagnosis of NSTI between January 2018 and December 2020, a period that included the first 6 months of the pandemic (Figure 1). We collected data on patient demographics, comorbidities, infection location, pathogen, severity of illness (APACHE II score), whether HBOT was provided and number of dives. Outcomes included 90-day mortality, amputations, inpatient antibiotic days, ventilator days, and hospital length of stay. Figure 1Proportion of NSTI patients admitted to R Adams Cowley Shock Trauma Center who received HBOT between January 2018 through December 2020. Proportions were averaged over six month blocks. Results 253 patients were included of whom 143 (56%) received HBOT and 110 (43.3%) did not. Baseline characteristics were similar between the groups except for Wound Surface Area (WSA) and APACHE II score (Table 1). Length of stay was significantly longer in HBOT patients (15.3 days vs 11.7 days p=0.005, Table 2). Fewer HBOT patients had amputations (7.5% vs 21% p=0.007) and fewer died within 90 days of admission (6.3% vs 14.5% p=0.029). When stratifying patients by APACHE II score, HBOT patients with APACHE II scores > 18 had significantly lower mortality than non-HBOT patients (9.7% vs 32.4% p=0.035). Conclusion In this population of NSTI patients who underwent surgical debridement, HBOT was associated with lower incidence of amputation, and lower mortality. Patients with APACHE II scores >18 who received HBOT had lower mortality than those who did not. A prospective study should further evaluate the impact of HBOT on mortality and amputations. Disclosures All Authors: No reported disclosures.

A systematic and simple approach to develop a 20 W audio frequency range switch mode amplifier is presented in this paper. A non-linear sliding mode (SM) technique-based low cost analog controller enables the realized amplifier to deliver highly linear and efficient operation throughout the audio frequency spectrum. The theoretical aspects and practical limitations in the design and realization of subsystems, such as the signal conditioning stage, power stage and sliding mode controller, are considered, while the viable solution is also stated and justified. The hardware realization scheme is also elaborated, based on which the laboratory prototype is fabricated. Hardware results with a 4 Ω resistive load are given on which the performance of the amplifier is evaluated. The total harmonic distortion (THD) below 1% and 73% efficiency at peak load make the amplifier well suited for high quality audio application.

Background Overdiagnosis of ventilator associated pneumonia (VAP) is common and may be due, in part, to excessive culturing of the respiratory tract in ventilated patients. We sought to evaluate the appropriateness of these cultures based on clinical characteristics leading to culture acquisition, and the relationship with antibiotic use. Methods We included mechanically ventilated adult patients in the Neuroscience and Cardiac Surgery Intensive Care Units at an academic medical center in Baltimore, MD, who had respiratory tract cultures obtained from March 1 to June 30, 2021. The appropriateness of respiratory cultures was evaluated by chart review in a standardized manner using a published algorithm (Figure 1). Clinical characteristics of patients with appropriate vs. inappropriate cultures were compared using Fisher’s exact test. Results 98 respiratory tract cultures (58 endotracheal aspirates, 25 mini bronchoalveolar lavages (BAL), 13 BALs, and 2 bronchial washings) were evaluated. Of these, 19 (19%) were deemed appropriate and 79 (81%) were inappropriate. Compared to patients with inappropriate cultures, those with appropriate cultures were more likely to demonstrate worsening oxygenation, new or increasing infiltrate on chest x-ray, or hemodynamic instability. The groups did not significantly differ with respect to fever or changes in WBC count (Figure 2). Of 79 inappropriate cultures, 31 (39%) received a clinical diagnosis of VAP from their provider. In 23 (29%) instances, antibiotics were started or changed based on culture results, and 9 (11%) were on empiric therapy for VAP that was continued unchanged. An additional 24 (30%) continued antibiotics not directed toward VAP. Excluding those on antibiotics not directed towards index respiratory culture results, patients received a median of 6 (IQR 2.5, 7) days of therapy. Conclusion A large majority of lower respiratory tract cultures in mechanically ventilated patients are potentially inappropriate and associated with overdiagnosis of VAP and unnecessary antibiotic exposure. Incorporating respiratory tract specific attributes into clinical decision making rather than “pan-culturing” for fever or leukocytosis may be an opportunity for diagnostic stewardship in this setting. Disclosures Kimberly C. Claeys, PharmD, BioFire Diagnostics: Honoraria.

Charge separation under solvation stress conditions is a fundamental process that comes in many forms in doped water clusters. Yet, the mechanism of intramolecular charge separation, where constraints due to the molecular structure might be intricately tied to restricted solvation structures, remains largely unexplored. Microhydrated amino acids are such paradigmatic molecules. Ab initio simulations are carried out at 300 K in the frameworks of metadynamics sampling and thermodynamic integration to map the thermal mechanisms of zwitterionization using Gly(H2O) n with n = 4 and 10. In both cases, a similar water-mediated proton transfer chain mechanism is observed; yet, detailed analyses of thermodynamics and kinetics demonstrate that the charge-separated zwitterion is the preferred species only for n = 10 mainly due to kinetic stabilization. Structural analyses disclose that bifurcated H-bonded water bridges, connecting the cationic and anionic sites in the fluctuating microhydration network at room temperature, are enhanced in the transition-state ensemble exclusively for n = 10 and become overwhelmingly abundant in the stable zwitterion. The findings offer potential insights into charge separation under solvation stress conditions beyond the present example.

Ruthenium-based CO releasing molecules (CO-RMs), CORM-2 and CORM-3, have been widely used as surrogates of CO for studying its biological effects in vitro and in vivo with much success. However, several previous solution-phase and in-vitro studies have revealed the ability for such CO-RMs to chemically modify proteins and reduce aromatic nitro groups due to their intrinsic chemical reactivity under certain conditions. In our own work of studying the cytoprotective effects of CO donors, we were in need of assessing chemical factors that could impact the interpretation of results from CO donors including CORM-2,3 in various in-vitro assays. For this, we examined the effects of CORM-2,3 toward representative reagents commonly used in various bioassays including resazurin, tetrazolium salts, nitrites and azide-based H(2)S probes. We have also examined the effect of CORM-2,3 on glutathione disulfide (GSSG), which is a very important redox regulator. Our studies show the ability for these CO-RMs to induce a number of chemical and/or spectroscopic changes for several commonly used biological reagents under near physiological conditions. These reactions/spectroscopic changes cannot be duplicated with CO-deleted CO-RMs (iCORMs), which are often used as negative controls. Further, both CORM-2 and -3 are capable of consuming and reducing GSSG in solution. We hope the results described will help future design of control experiments in using Ru-based CO-RMs in similar experiments.

We previously reported a series of p53-elevating anthraquinone compounds with considerable cytotoxicity for acute lymphatic leukemia (ALL) cells. To further develop this class of compounds, we examined the effect of a few key structural features on the anticancer structure-activity relationship in ALL cells. The active analogs showed comparable cytotoxicity and upregulation of p53 but did not induce significant downregulation of MDM2 as seen with the lead compound AQ-101, indicating the importance of the anthraquinone core scaffold for MDM2 regulation. The result from the current study not only contributes to the SAR framework of these anthraquinone derivatives but also opens up new chemical space for further optimization work.

Despite decades of research, the location and molecular identity of the proton release group together with the subsequent proton release pathway remain controversial even for the simplest light-driven proton pump, bacteriorhodopsin, according to the most recent experiments and simulations. Yet despite this nagging lack of knowledge for the generic case, even more complex pumps are currently under investigation. The proton release group disclosed by our large-scale simulations satisfies available experimental results, especially the broad Zundel continuum absorption subject to a striking anisotropy observed only recently. Moreover, our simulations delineate the seamless pathway by which the excess proton (being stored in an ultrastrong centered H-bond involving two glutamates) is finally translocated into the extracellular medium.

The quest to find novel strategies to tackle respiratory illnesses has led to the exploration of the potential therapeutic effects of carbon monoxide (CO) as an endogenous signaling molecule and a cytoprotective agent. Further, several studies have demonstrated the pharmacological efficacy of CO in animal models of respiratory disorders, such as acute lung injury and pulmonary hypertension. Because of the gaseous nature of CO and its affinity for multiple targets, its controlled delivery has been a challenge. Past studies have employed different delivery modalities, including CO gas, HO-1 inducers, and CO donors, sometimes leading to substantive variations in the resulting pharmacological effects for various reasons. Herein, this review summarizes and analyzes the differences among the profiles of various CO-delivery modalities in terms of their efficacy, dosing regimen, and pharmacokinetics in airways models. We believe that analysis of these issues will help in understanding the fundamental roles of CO in airways, and eventually, contribute to its development as a medicine for respiratory diseases.

GTPases play a crucial role in the regulation of many biological processes by catalyzing the hydrolysis of GTP into GDP. The focus of this work is on the dynamin-related large GTPase human guanine nucleotide binding protein-1 (hGBP1) which is able to hydrolyze GTP even to GMP. Here, we studied the largely unknown mechanisms of both GTP and GDP hydrolysis steps utilizing accelerated ab initio QM/MM metadynamics simulations to compute multi-dimensional free energy landscapes. We find an indirect substrate-assisted catalysis (SAC) mechanism for GTP hydrolysis involving transfer of a proton from the water nucleophile to a nonbridging phosphoryl oxygen via a proton relay pathway where the rate-determining first step is concerted-dissociative nature. A “composite base” consisting of Ser73, Glu99, a bridging water molecule, and GTP was found to activate the nucleophilic water, thus disclosing the complex nature of the general base in hGBP1. A nearly two-fold reduction in the free energy barrier was obtained for GTP hydrolysis in the enzyme in comparison to bulk solvent. The subsequent GDP hydrolysis in hGBP1 was also found to follow a water-mediated proton shuttle mechanism. It is expected that the proton shuttle mechanisms unravelled for hGBP1 apply to many classes of GTPases/ATPases that possess an optimally-arranged hydrogen bonding network, which connects the catalytic water to a proton acceptor.

The catalytic power of enzymes is usually ascribed to a few catalytically competent residues as revealed by site-directed mutagenesis studies in conjunction with biochemical, thermodynamic and structural analyses. Surprisingly, the mutations of such pivotal residues in a GTPase that can hydrolyse GTP even to GMP, namely hGBP1, have been reported to result only in marginal changes of the catalytic rate compared to the wild type. Our large-scale ab initio quantum-mechanical/molecular-mechanical (QM/MM) metadynamics simulations disclose that the replacement of catalytically competent residues by the inert amino acid alanine, S73A and E99A, opens a plethora of molecularly different reaction pathways featuring very similar energy barriers and thus rates. These hitherto unknown reaction channels are established by mechanistically involving far-distant residues using “floating” water molecules, which connect them via hydrogen-bonding bridges to the nucleophilic water molecule, thus allowing for efficient long-distance proton transfer via the Grotthuss mechanism. Given the generic nature of the disclosed detour mechanisms that provide the molecular underpinning of catalytic versatility and thus mutational robustness of hGBP1, it is expected that the same concept is operational for GTPases in a broad sense.

Understanding the molecular details of antibiotic resistance by the bacterial enzymes β-lactamases is vital for the development of novel antibiotics and inhibitors. In this spirit, the detailed mechanism of deacylation of the acyl-enzyme complex formed by cephalothin and class C β-lactamase is investigated here using hybrid quantum-mechanical/molecular-mechanical molecular dynamics methods. The roles of various active-site residues and substrate in the deacylation reaction are elucidated. We identify the base that activates the hydrolyzing water molecule and the residue that protonates the catalytic serine (Ser64). Conformational changes in the active sites and proton transfers that potentiate the efficiency of the deacylation reaction are presented. We have also characterized the oxyanion holes and other H-bonding interactions that stabilize the reaction intermediates. Together with the kinetic and mechanistic details of the acylation reaction, we analyze the complete mechanism and the overall kinetics of the drug hydrolysis. Finally, the apparent rate-determining step in the drug hydrolysis is scrutinized.

Enhanced sampling of large number of collective variables (CVs) is inevitable in molecular dynamics (MD) simulations of complex chemical processes such as enzymatic reactions. Because of the computational overhead of hybrid quantum mechanical/molecular mechanical (QM/MM)-based MD simulations, especially together with density functional theory, predictions of reaction mechanism, and estimation of free-energy barriers have to be carried out within few tens of picoseconds. We show here that the recently developed temperature-accelerated sliced sampling method allows one to sample large number of CVs, thereby enabling us to obtain rapid convergence in free-energy estimates in QM/MM MD simulation of enzymatic reactions. Moreover, the method is shown to be efficient in exploring flat and broad free-energy basins that commonly occur in enzymatic reactions. We demonstrate this by studying deacylation and reverse acylation reactions of aztreonam drug catalyzed by a class-C β lactamase (CBL) bacterial enzyme. Mechanistic details and nature of kinetics of aztreonam hydrolysis by CBL are elaborated here. The results of this study point to characteristics of the aztreonam drug that are responsible for its slow hydrolysis.\ud \ud

Endophthalmitis is still the major post operative complication of ocular surgery. Most commoncause for endophthalmitis to develop after ocular surgery is the inhabitant microbial flora ofconjunctiva and periocular tissue. This study was conducted with the aim to assess the effectof prophylactic intracameral moxifloacin on incidence of endophthalmitis in high volume surgerysetup. Patient were divided into two groups, group 1 which didn’t received intracameralmoxifloxacin at the end of surgery and group 2 which received prophylactic intracameralmoxifloxacin at the end of surgery. We have achieved zero incidences of endophthalmitis withintracameral moxifloxacin. Our findings consolidate the view that use of intracameral antibioticsdecreases the rate of enopthalmitis.

New Delhi metallo β-lactamase (NDM-1) is a recent addition to the metallo-β-lactamases family that is capable of hydrolyzing most of the available antibiotics, including the new generation carbapenems. Here, we report the mechanism of Meropenem hydrolysis catalyzed by NDM-1 based on hybrid quantum-mechanical/molecular-mechanical metadynamics simulations. Our work elicits the molecular details of the catalytic mechanism and free energy profiles along the reaction pathway. We identified the ring opening step involving the nucleophilic addition of the bridging hydroxyl group on the β-lactam ring of the drug as the rate-determining step. Subsequent protonation of β-lactam nitrogen occurs from a bulk water molecule that diffuses into the active site and is preferred over proton transfer from the bridging hydroxyl group or from the protonated Asp124. The roles of important active site residues of NDM-1 and change in the coordination environment of Zn ions during the hydrolysis are also scrutinized.

The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg

Critical care 21(1), 131 (2017). doi:10.1186/s13054-017-1690-5, Published by BioMed Central, London

GTP hydrolysis in hGBP1 occurs via indirect substrate-assisted catalysis involving a complex proton relay mechanism of concerted-dissociative nature., GTPases play a crucial role in the regulation of many biological processes by catalyzing the hydrolysis of GTP into GDP. The focus of this work is on the dynamin-related large GTPase human guanine nucleotide binding protein-1 (hGBP1) which is able to hydrolyze GTP even to GMP. Here, we studied the largely unknown mechanisms of both GTP and GDP hydrolysis steps utilizing accelerated ab initio QM/MM metadynamics simulations to compute multi-dimensional free energy landscapes. We find an indirect substrate-assisted catalysis (SAC) mechanism for GTP hydrolysis involving transfer of a proton from the water nucleophile to a nonbridging phosphoryl oxygen via a proton relay pathway where the rate-determining first step is concerted-dissociative nature. A “composite base” consisting of Ser73, Glu99, a bridging water molecule, and GTP was found to activate the nucleophilic water, thus disclosing the complex nature of the general base in hGBP1. A nearly two-fold reduction in the free energy barrier was obtained for GTP hydrolysis in the enzyme in comparison to bulk solvent. The subsequent GDP hydrolysis in hGBP1 was also found to follow a water-mediated proton shuttle mechanism. It is expected that the proton shuttle mechanisms unravelled for hGBP1 apply to many classes of GTPases/ATPases that possess an optimally-arranged hydrogen bonding network, which connects the catalytic water to a proton acceptor.

By employing computationally intensive molecular dynamics simulations using hybrid quantum-mechanical/molecular-mechanical approach, we analyze here the kinetic and thermodynamic stabilities of various active site protonation states of a fully solvated class C β-lactamase. We report the detailed mechanism of proton transfer between catalytically important active site residues and the associated free energy barriers. In the apoenzyme, significant structural changes are associated with the proton transfer, and the orientations of active site residues are distinctly different for various protonation states. Among several propositions on the protonation state of the apoprotein, we find that the one with Tyr150 deprotonated and both Lys67 and Lys315 residues being protonated is the most stable one, both thermodynamically and kinetically. However, the equilibrium structure at room temperature is a dynamic one, with Lys315Hζ delocalized between Tyr150Oη and Lys315Nζ. Of great importance, the kinetic and thermodynamic stability of protonation states are significantly affected on noncovalently complexing with cephalothin, an antibiotic molecule. The equilibrium structure of the enzyme-substrate (precovalent) complex has a dynamic protonation state where a proton shuttles frequently between the Tyr150Oη and Lys67Nζ. We examine here the genesis of the manifold change in stability at the molecular level. The importance of our observations toward understanding the reactivity of the enzyme is discussed and experimental observations are rationalized.

Summary The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg 2+ ions. While studying this reaction using X-ray crystallography, we captured snapshots of the pyrophosphate (product) as it exits along a pocket. Surprisingly, one of the active site Mg 2+ ions remains coordinated to the exiting pyrophosphate. This hints at the participation of Mg 2+ in the process of product release, besides its role in catalyzing nucleotidyl transfer. These observations are further supported by enhanced sampling molecular dynamics simulations. Free energy computations suggest that the product release is likely to be rate limiting in SNTs, and the origin of the high free energy barrier for product release could be traced back to the "slow" conformational change of an Arg residue at the exit end of the pocket. These results establish a dual role for Mg 2+ , and propose a general mechanism of product release during the nucleotidyl transfer by SNTs.

HCN is clearly associated with the prebiotic chemical evolution of life. It has been known for decades that the radiolysis of HCN solutions produces sugars, amino acids and nucleobases. Remarkably, recent experimental studies have shown that the photolytic reduction of aqueous HCN by a photoredox reagent [Cu(CN)3](2-) specifically yields sugars, which are the essential building blocks of RNA. Although a mechanistic understanding of such reductions with solvated electrons is poor, the general consensus is that they involve neutral free radicals. We show herein through the use of electronic structure studies and molecular simulations that the reduction of the nitrile bond of HCN is initiated through the formation of a molecular dipole-bound anion from the photoredox reagent. Our theoretical studies show how HCN binds to the photoexcited reagent and then extracts an electron from the reagent and is ultimately detached as a dipole-bound anion. The dipole-bound anionic form of [HCN](-) can easily convert into a solvated valence-bound form of [HCN](-). After the formation of solvated [HCN](-), an extraordinary chemical event ensues through a counter-intuitive coupling of two valence-bound anions to form a solvated molecular dianionic intermediate, [HCN]2(2-). Finally, a proton-coupled electron transfer occurs within the dianionic entity to complete the reduction. This mechanistic scenario is applicable to the reduction of other prebiotic nitrile species and avoids neutral radical-based pathways, thereby preventing the proliferation of reactive species and preserving chemical selectivity. Furthermore, we show how such similar nitrile reduction pathways operate to yield the sugar precursors.