Shielding of Viruses such as SARS-CoV-2 from Ultraviolet Radiation in Particles Generated by Sneezing or Coughing: Numerical Simulations of Survival Fractions

Shielding of Viruses such as SARS-CoV-2 from Ultraviolet Radiation in Particles Generated by Sneezing or Coughing: Numerical Simulations of Survival Fractions Steven C. Hill, David C. Doughty, and Daniel W. Mackowski DEVCOM Army Research Laboratory, 2800 Powder Mill Rd., Adelphi, Maryland, USA (Hill and Doughty); Auburn University, Auburn, Alabama, USA (Mackowski) #Address correspondence to Steve Hill, [email protected] ABSTRACT SARS-CoV-2 and other microbes within aerosol particles can be partially shielded from UV radiation. The particles refract and absorb light, and thereby reduce the UV intensity at various locations within the particle. Shielding has been demonstrated in calculations of UV intensities within spherical approximations of SARS-CoV-2 virions that are within spherical particles approximating dried-to-equilibrium respiratory fluids. The purpose of this paper is to calculate the survival fractions of virions (i.e., the fractions of virions that can infect cells) within spherical particles approximating dried respiratory fluids, and to investigate the implications of these calculations for using UV light for disinfection. The particles may be on a surface or in air. In this paper the survival fraction (S) of a set of virions illuminated with a UV fluence (F, in J/m2) is approximated as S=exp(-kF), where k is the UV inactivation rate constant (m2/J). The average survival fractions (Sp) of all the simulated virions in a particle are calculated using the calculated decreases in fluence. The results show that virions in particles of dried respiratory fluids can have significantly larger Sp than do individual virions. For individual virions, and virions in 1, 5, and 9 µm particles illuminated (normal incidence) on a surface with 260-nm UV light, the Sp = 0.00005, 0.0155, 0.22 and 0.28, respectively, when kF=10. The Sp decrease to <10-7, <10-7, 0.077 and 0.15, respectively, for kF=100. Calculated results also show that illuminating particles with UV beams from widely separated directions can strongly reduce the Sp. These results suggest that the size distributions and optical properties of the dried particles of virion-containing respiratory fluids are likely important in effectively designing and using UV germicidal irradiation systems for microbes in particles. The results suggest the use of reflective surfaces to increase the angles of illumination and decrease the Sp. The results suggest the need for measurements of the Sp of SARS-CoV-2 in particles having compositions and sizes relevant to the modes of disease transmission.

Quasi-Irreversible Inhibition of CYP2D6 by Berberine

In our previous study, Hwang-Ryun-Hae-Dok-Tang, which contains berberine (BBR) as a main active ingredient, inhibited cytochrome P450 (CYP) 2D6 in a quasi-irreversible manner. However, no information is available on the detailed mechanism of BBR-induced CYP2D6 inhibition. Thus, the present study aimed to characterize the inhibition mode and kinetics of BBR and its analogues against CYP2D6 using pooled human liver microsomes (HLM). BBR exhibited selective quasi-irreversible inhibition of CYP2D6 with inactivation rate constant (kinact) of 0.025 min−1, inhibition constant (KI) of 4.29 µM, and kinact/KI of 5.83 mL/min/µmol. In pooled HLM, BBR was metabolized to thalifendine (TFD), demethyleneberberine (DMB), M1 (proposed as demethylene-TFD), and to a lesser extent berberrubine (BRB), showing moderate metabolic stability with a half-life of 35.4 min and a microsomal intrinsic clearance of 7.82 µL/min/mg protein. However, unlike BBR, those metabolites (i.e., TFD, DMB, and BRB) were neither selective nor potent inhibitors of CYP2D6, based on comparison of half-maximal inhibitory concentration (IC50). Notably, TFD, but not DMB, exhibited metabolism-dependent CYP2D6 inhibition as in the case of BBR, which suggests that methylenedioxybenzene moiety of BBR may play a critical role in the quasi-irreversible inhibition. Moreover, the metabolic clearance of nebivolol (β-blocker; CYP2D6 substrate) was reduced in the presence of BBR. The present results warrant further evaluation of BBR–drug interactions in clinical situations.

A simulation model of microbe overlapping for the correct estimation of UV-C device log-reduction

Background Candida auris is an emerging pathogen responsible for several outbreaks within healthcare facilities. It can be found on hospital surfaces and patient care devices. UV- C sanitisation may constitute an effective adjunct to routine room cleaning to prevent the spreading of this yeast. Previous findings with this technology suggest to investigate different sources of variability in the study of the biocidal effect of UV devices on C. auris. In this study we develop a computer simulation of surface distribution of microorganisms on a stainless steel carrier, to optimize UV-device experiments. Methods Based on the literature about C. auris studies and its estimated average size (about 5 μm diameter), several Matlab simulations have been performed to include as many microorganisms as possible to be ideally placed on a 20 cm2 stainless steel support, avoiding cell overlapping. This was done in order to maximize the effectiveness of UV exposure. Results Two initial simulations were performed to evaluate a random arrangement of a very large number of microorganisms (8x107 CFU) on the steel support, widely overlapped. In this case, due to the poor UV-C penetrability, we would not exceed two log10 reduction. By randomly distributing 1x106 CFU, the probability of overlapping was about 1.3%, but even a partial overlap limits the log10 reduction. By randomly distributing 1x105 CFU, the overlaps were at least 10 times less likely. Conclusions The simulation results allowed us to evaluate the most appropriate microorganism distribution model able to optimize the biocidal effects of UV-C devices. The overlapping of microorganisms reduces UV-C penetrability. Our simulated study is consistent with literature results where we observed a lower log-reduction by increasing the concentration of microorganisms and therefore the probability of overlapping. The resulting model would simulate any log-reduction scenario, at any distance and any concentration, with and without overlapping. Key messages The complete or partial microorganism overlapping plays a relevant role in the outcome of UV-C biocidal experiments. To identify the exponential CFU reduction curve and estimate accurately the inactivation rate constant, simulated experiments should be performed to assess the real effectiveness of UV devices.

A two-point IC50 method for evaluating the biochemical potency of irreversible enzyme inhibitors

Irreversible (covalent) enzyme inhibitors cannot be unambiguously ranked for biochemical potency by using IC50 values determined at a single point in time, because the same IC50 value could originate either from relatively low initial binding affinity accompanied by high chemical reactivity, or the other way around. To disambiguate the potency ranking of covalent inhibitors, here we describe a data-analytic procedure relying on two separate IC50 values, determined at two different reaction times. In the case of covalent inhibitors following the two-step kinetic mechanism E + I ⇌ E I → EI, the two IC50 values alone can be used to estimate both the inhibition constant (Ki) as a measure of binding affinity and the inactivation rate constant (kinact) as a measure of chemical reactivity. In the case of covalent inhibitors following the one-step kinetic mechanism E + I → EI, a simple algebraic formula can be used to estimate the covalent efficiency constant (kinact/Ki) from a single experimental value of IC50. The two simplifying assumptions underlying the method are (1) zero inhibitor depletion, which implies that the inhibitor concentrations are always significantly higher than the enzyme concentration; and (2) constant reaction rate in the uninhibited control assay. The newly proposed method is validated by using a simulation study involving 64 irreversible inhibitors with covalent efficiency constants spanning seven orders of magnitude.

Deciding between one-step and two-step irreversible inhibition mechanisms on the basis of “kobs” data: A statistical approach

This paper describes an objective statistical approach that can be used to decide between two alternate kinetic mechanisms of covalent enzyme inhibition from kinetic experiments based on the standard “kobs” method. The two alternatives are either a two-step kinetic mechanism, which involves a reversibly formed noncovalent intermediate, or a one-step kinetic mechanism, proceeding in a single bimolecular step. Recently published experimental data [Hopper et al. (2020) J. Pharm. Exp. Therap.372, 331–338] on the irreversible inhibition of Bruton tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) by ibrutinib (PCI-32765) and acalabrutinib are used as an illustrative example. The results show that the kinetic mechanism of inhibition was misdiagnosed in the original publication for at least one of the four enzyme/inhibitor combinations. In particular, based on the available kobs data, ibrutinib behaves effectively as a one-step inhibitor of the TEC enzyme, which means that it is not possible to reliably determine either the inhibition constant Ki or the inactivation rate constant kinact, but only the covalent efficiency constant keff = kinact/Ki. Thus, the published values of Ki and kinact for this system are not statistically valid.

Inactivation kinetics and thermodynamic properties of polygalacturonase produced by Aspergillus awamori CICC 2040 on pretreated orange and plantain peels

This study investigated the effect of pretreatment of orange and plantain peels on the inactivation kinetics and thermodynamic properties of polygalacturonase (PG) produced by Aspergillus awamori CICC 2040. Orange and plantain peel powders were subjected to microwave-assisted NaOH pretreatment and used as substrates for PG production. Un-treated peels served as controls. The PG was purified using acetone precipitation and column chromatography, and the inactivation kinetics, temperature dependency, and thermodynamic properties of the crude and purified PGs were determined. Higher inactivation rate constant was obtained for crude PG produced using pretreated orange peel (CPOF) and plantain peel (CPPF) compared to PG produced using untreated orange peel (Uo) and plantain peel (Up). At all the temperatures considered, higher half-life and decimal reduction time were recorded for CPOF and CPPF compared to Uo and Up. The highest half-life (45.60 min) and decimal reduction time (151.49 min) were recorded for CPOF at 60 ?C. Lower half-life and decimal reduction time were obtained for purified PGs compared to the crude PG. Polygalacturonase produced from pretreated peels had lower activation energy than those produced from untreated ones. The higher activation energy was recorded for the PG produced using orange peel compared to the one from plantain peels. The enthalpy of CPOF and CPPF was slightly lower than Uo and Up. The pretreatment of the peels resulted in a reduction of Gibbs free energy (?G ) and entropy (?S) of crude and purified PG. Higher ?G and ?S were recorded for the purified PG compared to the crude PG. Negative entropy and enthalpy were recorded for all the PGs. The findings from this study showed that the kinetic and thermodynamic properties of PG, produced by Aspergillus awamori CICC 2040, were enhanced by the pretreatment of orange and plantain peels.

SPECIFIC ASPECTS OF AVIBACTERIUM PARAGALLINARUM INACTIVATION WITH FORMALDEHYDE AND THIOMERSAL

The paper demonstrates results of testing different modes of Avibacterium paragallinarum inactivation with formaldehyde and thiomersal. The bacterium destruction by 0.20% and 0.10% formaldehyde proceeds at the constant rate thus indicating exponential dependence of the microorganism inactivation processes. This fact allows for calculation of the inactivation rate constant that amounts to 2.94 ± 0.37 h-1 for 0.10% formaldehyde and 5.86 ± 0.72 h-1 for 0.20% formaldehyde. Inactivation using formaldehyde at final concentration of 0.10% at 37 °С and continuous stirring (60 rpm) produces 7.0 dm3 of bacterin at concentration of 9.5 ± 0.2 lg microbial cells (mc)/cm3 in 4.3 ± 0.1 h. Thiomersal demonstrated bactericidal action against Avibacterium paragallinarum at concentration of 0.04% (1:2500) or higher. Herewith, inactivation process is specified by linearity and the inactivation rate constant amounts to 7.92±1.12 h-1. Under thiomersal sublethal concentration of 0.2% (1:5000) the survival curve is of irregular shape. However, the process of the microorganism death is not exponential, and under continuous decrease, the inactivation rate is going to zero thus making impossible the calculation of the inactivation rate constant. Inactivation mode involving use of 0.04% thiomersal at 37 °С allows production of 7.0 dm3 of bacterin at 9.5 ± 0.2 lg mc/cm3 concentration in 5.8 ± 0.1 h. Right after production, the hemagglutination activity of the thiomersal inactivated antigen was higher as compared to formaldehyde inactivated antigen (Р 0.05). The antigen produced using formaldehyde maintains high hemagglutination activity during storage that is critical for high quality vaccine production.

Inactivation of particle-associated Escherichia coli with chlorine dioxide

In this paper, the inactivation of both free Escherichia coli (FE) and particle-associated E. coli (PAE) with chlorine dioxide (ClO2) were investigated using granular activated carbon effluent water samples. The inactivation rate of FE was higher than that of PAE and the reactivation ratio of PAE was higher than that of FE, indicating the threat of particle-associated bacteria. Response surface methodology (RSM) was applied to determine the factors influencing the disinfection efficiency of ClO2 in inactivating PAE. The experimental results indicated that particle concentration was a principal factor influencing the PAE inactivation efficiency, presenting a negative correlation, while exposure time and ClO2 dosage revealed a positive correlation. The inactivation kinetics of PAE using ClO2 was also investigated and the results demonstrated that PAE inactivation with ClO2 fitted the Chick–Watson kinetic model. The inactivation rate constants of PAE were found to follow the Arrhenius expression with an activation energy of 107.5 kJ/mol, indicating a relatively strong temperature dependence. However, there are minor effects of pH and initial ClO2 dosage on PAE inactivation rate constant.

Kinetic Analysis of Guanidine Hydrochloride Inactivation of β-Galactosidase in the Presence of Galactose

Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant Km and Vmax values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([P]∞−[P]t) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[P]∞ versus 1/[S] yielded k+0 and k'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k+0 and k'+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed.

Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

ABSTRACTClass A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated aKmvalue of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed aKmvalue of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (kinact) was 0.034 ± 0.003 s−1. When covalently bound to S70, PSR-3-226 forms atrans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.