The Dynamics of SARS-CoV-2 Infectivity with Changes in Aerosol Microenvironment

Understanding the factors that influence the airborne survival of viruses such as SARSCoV2 in aerosols is important for identifying routes of transmission and the value of various mitigation strategies for preventing transmission. We present measurements of the stability of SARSCoV2 in aerosol droplets (5 to 10 micrometres equilibrated radius) over timescales spanning from 5 seconds to 20 minutes using a novel instrument to probe survival in a small population of droplets (typically 5-10) containing ~1 virus/droplet. Measurements of airborne infectivity change are coupled with a detailed physicochemical analysis of the airborne droplets containing the virus. A decrease in infectivity to 10 % of the starting value was observable for SARS-CoV-2 over 20 minutes, with a large proportion of the loss occurring within the first 5 minutes after aerosolisation. The initial rate of infectivity loss was found to correlate with physical transformation of the equilibrating droplet; salts within the droplets crystallise at RHs below 50% leading to a near instant loss of infectivity in 50 to 60% of the virus. However, at 90% RH the droplet remains homogenous and aqueous, and the viral stability is sustained for the first 2 minutes, beyond which it decays to only 10% remaining infectious after 10 minutes. The loss of infectivity at high RH is consistent with an elevation in the pH of the droplets, caused by volatilisation of CO2 from bicarbonate buffer within the droplet. Three different variants of SARS-CoV-2 were compared and found to have a similar degree of airborne stability at both high and low RH.

Theoretical and Experimental Considerations for a Rapid and High Throughput Measurement of Catalase In Vitro

A rapid and high throughput protocol to measure the catalase activity in vitro has been designed. Catalase is an enzyme with unusual kinetic properties because it does not follow the standard Michaelis–Menten model and is inactivated by H2O2. This makes the analysis of the two rate equations of the second-ordered reactions of the kinetic model rather complex. A two-degree polynomial fitting of the experimental data is proposed after transforming the exponential form of the integrated rate equation of the [H2O2] into a polynomial using the Taylor series. The fitting is validated by establishing an experimental linear relationship between the initial rate of the H2O2 decomposition and the protein concentration, regardless of the suicide inactivation that catalase might undergo beyond t > 0. In addition, experimental considerations are taken into account to avoid statistical bias in the analysis of the catalase activity. ANOVA analyses show that the proposed protocol can be utilized to measure the initial rate of the H2O2 decomposition by catalase in 32 samples in triplicates if kept below 8 mM min−1 in the microplate wells. These kinetic and statistical analyses can pave the way for other antioxidant enzyme activity assays in microplate readers at small scale and low cost.

Relative Rates of Gluten Digestion by Nine Commercial Dietary Digestive Supplements

Endopeptidases containing supplements may digest gluten and reduce the impact on celiac and gluten-sensitive subjects who inadvertently consume gluten. We investigated the relative rate of disappearance of coeliac relevant epitopes in extracts of nine commercial supplements, using two competitive enzyme-linked immunosorbent assays (ELISAs)—Ridascreen (detects QQPFP, QQQFP, LQPFP, and QLPFP) and Gluten-Tec (detects Glia-α20 and PFRPQQPYPQ). All epitopes are destroyed by cleavage after P and Q amino acids. Rates at pH 3.5 and pH 7.0 were measured. These experiments were designed to measure relative rates of epitope digestion not to mimic in vivo digestion. The supplements were: 1 GluteGuard, 2 GlutenBlock, 3 GliadinX, 4 GlutnGo, 5 GlutenRescue, 6 Eat E-Z Gluten+, 7 Glutenease, 8 Glutezyme, and 9 Gluten Digest. The mean initial rate and half-lives of epitope digestion were deduced and extrapolated to rates at the recommended dose of one supplement in a fasting stomach volume. At pH 7, supplement 1 was the fastest acting of the supplements, with Ridascreen ELISA, more than twice as fast as the next fastest supplements, 5, 6, 7, and 8. Supplements 2, 3, and 4 showed little activity at pH 7.0. Supplement 1 was also the fastest acting at pH 7 with Gluten-Tec ELISA, more than three times the rate for supplements 2 and 3, with supplements 4–9 showing minimal activity. At pH 3.5, supplement 1 acted more than five times as fast as the next fastest supplements, 2 and 3, when measured by Ridascreen, but supplements 2 and 3 were over two times faster than supplement 1 when measured by Gluten-Tec. Supplements 4–9 demonstrated minimal activity at pH 3.5 with either ELISA. Supplement 1 most rapidly digested the key immuno-reactive gluten epitopes identified by the R5 antibody in the Codex-approved competitive Ridascreen ELISA method and associated with the pathology of celiac disease.

Emerging Roles on Immunological Effect of Indoleamine 2,3-Dioxygenase in Liver Injuries

Indoleamine 2,3-dioxygenase (IDO) is one of the initial rate-limiting enzymes of the kynurenine pathway (KP), which causes immune suppression and induction of T cell anergy. It is associated with the imbalance of immune homeostasis in numerous diseases including cancer, chronic viral infection, allergy, and autoimmune diseases. Recently, IDO has extended its role to liver field. In this review, we summarize the dysregulation and potentials of IDO in the emerging field of liver injuries, as well as current challenges for IDO targets. In particular, we discuss unexpected conclusions against previous work published. IDO is induced by pro-inflammatory cytokines in liver dysfunction and exerts an immunosuppressive effect, whereas the improvement of liver injury may require consideration of multiple factors besides IDO.

Modification of Cu2+ in polyphenol oxidase extract from purple eggplant for phenol degradation in coal wastewater treatment

Cracking coal-forming organic compounds during the gasification process produces liquid waste containing phenolic compounds that require special handling based on their toxicity. As one of the components, there is liquid waste resulting from the coal gasification process. The purpose of this research was to study the activity of polyphenol oxidase (PPO) on phenol after the addition of Cu2+ to purple eggplant (Solanum melongena L.) extract and its potential to work more effectively in phenol biodegradation for coal wastewater containing phenol. Enzyme activity and phenol determination were carried out spectrophotometrically. The results showed PPO activity of 25.90-38.10 U/mL; 4.0 mM phenol and the activity of PPO-Cu2+ was 21.58-46.32 U/mL; 2-10 mM CuSO4; 2.0-4.0 mM phenol. Based on Michaelis Menten’s graph, the initial rate of PPO-Cu2+ was 0.015 mM/min and the initial rate of PPO was 0.15 mM/min using 2 mM phenol as a substrate. Lineweaver-Burk’s graph shows the KM of PPO-Cu2+ = 6.92 mM, which is lower than KM of PPO = 13.05 mM. Its means that the phenol response has a higher affinity for PPO-Cu2+ than PPO. The application of PPO-Cu2+ in purple eggplant extract works effectively as much as 46.7% for artificial coal liquid waste containing phenol.

128. Mutations that Inactivate the Tricarboxylic Acid Cycle in Staphylococcus aureus Arise During Persistent MRSA Bacteremia

Background Persistent MRSA bacteremia is common with high morbidity and mortality despite appropriate antibiotics. Persistent infections are associated with antibiotic tolerance and can arise from perturbations in cellular pathways. We performed whole genome sequencing of clinical isolates to identify the genetic bases of antibiotic tolerance. Methods Whole genomes of MRSA from patients with persistent bacteremia were sequenced, which identified 8 isolates harboring different citZ mutations. To assess the effect of the mutations directly on citrate synthase activity, purified recombinant enzymes were assayed using a commercially available kit. The same kit was used to measure activity in whole cell lysates of MRSA isolates harboring wild-type and mutant citZ alleles. Enzyme kinetic parameters were determined by fitting initial rate data to the Michaelis-Menten equation using nonlinear regression. Figure 1. Whole Genome Sequencing of Persistent MRSA Bacteremia Whole genome sequencing of 206 blood cultures from 20 patients with persistent MRSA bacteremia (defined as >7 days) under the hypothesis that continuous antibiotic exposure will give rise to - and enrich for - mutations that convey antibiotic tolerance through in-host evolution. Results Analysis of whole genomes from 206 blood cultures from 20 patients with persistent MRSA bacteremia identified citZ, which encodes for citrate synthase, the first step of the tricarboxylic acid cycle, as the most repeatedly mutated gene. The data revealed parallelism in its evolution, as citZ was mutated 8 independent times, a rate far greater than from chance alone. To characterize the impact of the mutations on enzyme activity, recombinant proteins were expressed in E. coli and purified for enzymatic assays. As compared to wildtype enzyme, two mutants had reduced activity, and four mutants had near-absent activity; two mutants were unable to be expressed due to destabilizing mutations. Michaelis-Menten analysis of the two mutants with residual activity show that, as compared to wildtype, both had lower affinity for its substrates, and lower maximum activity. Furthermore, we found the cell was unable to compensate for the loss of citrate synthase activity, as lysates harboring these mutations also displayed analogous reductions in activity. Figure 2. Purified citrate synthase mutant proteins have reduced enzyme activity Citrate synthase catalyzes the reaction between acetyl-CoA and oxaloacetic acid to form citric acid with CoA-SH as a byproduct. CoA-SH can interact with DTNB to form TNB, which can be measured spectrophotometrically at A412nm to indirectly measure the activity of citrate synthase. Wildtype citrate synthase and the D141N mutant have comparable activity whereas the remaining study mutants have either reduced (mutants A313P and A313V) or near-absent (mutants G7D, G7D + D141N, S201P, P354S) activity. The three active site mutants (H248G, D309G, H219G) also have near-absent activity and serve as negative controls. Positive control is citrate synthase provided in the Sigma Citrate Synthase Assay Kit. No enzyme control replaces enzyme with assay buffer. All error bars are ± 1 SD, calculated from at least three replicate experiments. Figure 3. A313P and A313V mutants have decreased substrate affinity and decreased maximal activity Panels A and B show plots of the initial rate of citrate synthase activity (Y-axis, in units of µmole/mL/sec) versus OAA/AcCoA substrate concentration (X-axis, in millimolar units), for wildtype citrate synthase and the A313P and A313V mutants. Panel (A) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with variable OAA (0-0.625 mM) and fixed AcCoA (0.3 mM). Panel (B) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with fixed OAA (0.5 mM) and variable AcCoA (0-2.5 mM). Three replicates were performed for each condition, with error bars showing ± 1 SD. Figure 4. Citrate synthase mutants disrupt functional dimerization and destabilize alpha-helix packing Identified citrate synthase mutants mapped onto the crystal structure of T. thermophilus citrate synthase (PDB 1IOM) show that the mutations are located either at dimerization interfaces or within the interior of hydrophobic packing interfaces. Conclusion Cellular metabolism and virulence regulation are interconnected in S. aureus, as alterations in TCA cycle activity lead to increased persister formation and host macrophage inactivation. Our findings that inactivating citZ mutations are enriched can provide a potential explanation for the mechanism of persistent bacteremia. Disclosures All Authors: No reported disclosures

Recycling Crushed Waste Beer Bottle Glass in Fired Clay Bricks

Waste glass is a readily available domestic material. Each year, around 257,000 tonnes of glass waste are produced in Victoria, and the majority is glass packings. Typically, mixed waste glass cullet is deposited in landfills due to the limited recycling techniques. As a result, landfills are facing a growing issue. Therefore, this study investigates the addition of waste beer bottle glass (BG) in fired clay bricks and examines the effects of varying firing temperatures on the physical and mechanical properties of the manufactured samples. Clay bricks containing 10% BG at a firing temperature of 950 °C depicted similar compressive strength results (41 MPa) to the control samples (42 MPa). The results of all tested bricks were found to be below the water absorption limit of 17%. The thermal conductivity of the bricks incorporating BG was investigated, and it was found that the thermal performance improved with the decreasing firing temperature. Moreover, an initial rate of absorption (IRA), XRD, and XRF analysis was conducted. The experimental results have been discussed and compared with the recommended acceptable properties for standard bricks.

SPECIAL FEATURES OF COPPER AND NICKEL SORPTION ON LOW-BASE ANIONITES

In the work, the regularities of the process of sorption of copper and nickel ions from model solutions by low-base anionites containing coordination secondary groups are investigated. The main kinetic and concentration coordination characteristics of the process of complexation of copper and nickel ions with ligand groups of a low-base sorbent are determined.Potentiometric titration shows that anionite contains low-base functional groups, the pK of which is equal to 7.2.It is established that the process of sorption of copper ions by low-base macroporous anionite is characterized by a high initial rate and rapid achievement of equilibrium, which ensures absorption of copper ions about 70% of the equilibrium values in the first 10- 15 minutes.The main parameters of the sorption process are determined. Analytical description of sorption isotherm in coordinates of Langmuir equation is carried out. It was shown that due to high rates, the degree of extraction from solutions with a concentration of 10- 15 mmol/l under equilibrium conditions reaches 99.6-98.3%.Studies of the sorption process in a dynamic mode showed the possibility of separating heavy metal ions from aqueous solutions on new low-base anionites.

Sequential Injection Analysis for Automation and Evaluation of Drug Liberation Profiles: Clotrimazole Liberation Monitoring

A fully automated sequential injection system was tested in terms of its application in liberation testing, and capabilities and limitations were discussed for clotrimazole liberation from three semisolid formulations. An evaluation based on kinetic profiles obtained in short and longer sampling intervals and steady-state flux values were applied as traditional methods. The obtained clotrimazole liberation profile was faster in the case of Delcore and slower for Clotrimazol AL and Canesten cream commercial formulations. The steady-state flux values for the tested formulations were 52 µg cm−2 h−1 for Canesten, 35 µg cm−2 h−1 for Clotrimazol AL, and 7.2 µg cm−2 h−1 for Delcore measured in 4 min sampling intervals. A simplified approach for the evaluation of the initial rate based on the gradient between the second and third sampling points was used for the first time and was found to correspond well with the results of the conventional methods. A comparison based on the ratio of the steady-state flux and the initial rate values for Canesten and Clotrimazol AL proved the similarity of the obtained results. The proposed alternative was successfully implemented for the comparison of short-term kinetic profiles. Consequently, a faster and simpler approach for dissolution/liberation testing can be used.

Anionic Copolymerization of Acrylonitrile with Ethyl Acrylate under the Action of the Initiating System of 1,4-Diazabicyclo[2.2.2]Octane – Ethylene Oxide

It was found, studying acrylonitrile copolymerization with ethyl acrylate in dimethyl sulfoxide under the action of anionic initiating system of 1,4-diazabicyclo [2.2.2] octane – ethylene oxide, that the obtained copolymers have a branched structure. An increase in the molar fraction of ethyl acrylate in the reaction medium leads to a decrease in the initial rate of acrylonitrile polymerization. Thermal behavior of copolymer samples was investigated; it was found that ethyl acrylate, being introduced into the polyacrylonitrile structure, both reduces thermal effects related to the reactions taking place during heat treatment of copolymers, and increases the half-width of the heat release peak.