Observed Inactivation Rates Research Articles

Salt Supersaturation as an Accelerator of Influenza A Virus Inactivation in 1 μL Droplets.

Influenza A virus (IAV) spreads through exhaled aerosol particles and larger droplets. Estimating the stability of IAV is challenging and depends on factors such as the respiratory matrix and drying kinetics. Here, we combine kinetic experiments on millimeter-sized saline droplets with a biophysical aerosol model to quantify the impact of NaCl on IAV stability. We show that IAV inactivation is determined by NaCl concentration, which increases during water evaporation and then decreases again when efflorescence occurs. When drying in air with relative humidity RH = 30%, inactivation follows an inverted sigmoidal curve, with inactivation occurring most rapidly when the NaCl concentration exceeds 20 mol/(kg H2O) immediately prior to efflorescence. Efflorescence reduces the NaCl molality to saturated conditions, resulting in a significantly reduced inactivation rate. We demonstrate that the inactivation rate k depends exponentially on NaCl molality, and after the solution reaches equilibrium, the inactivation proceeds at a first-order rate. Introducing sucrose, an organic cosolute, attenuates IAV inactivation via two mechanisms: first by decreasing the NaCl molality during the drying phase and second by a protective effect against the NaCl-induced inactivation. For both pure saline and sucrose-containing droplets, our biophysical model ResAM accurately simulates the inactivation when NaCl molality is used as the only inactivating factor. This study highlights the role of NaCl molality in IAV inactivation and provides a mechanistic basis for the observed inactivation rates.

Persistence of emerging viral fecal indicators in large-scale freshwater mesocosms

Fecal indicator bacteria (FIB) are typically used to monitor microbial water quality but are poor representatives of viruses due to different environmental fate. Viral fecal indicators have been proposed as alternatives to FIB; however, data evaluating the persistence of emerging viral fecal indicators under realistic environmental conditions is necessary to evaluate their potential application. In this study, we examined the persistence of five viral fecal indicators, including crAssphage and pepper mild mottle virus (PMMoV), and three bacterial fecal indicators (E. coli, enterococci and HF183/BacR287) in large-scale experimental ponds and freshwater mesocosms. Observed inactivation rate constants were highly variable and ranged from a minimum of −0.09 d−1 for PMMoV to a maximum of −3.5 d−1 for HF183/BacR287 in uncovered mesocosms. Overall, viral fecal indicators had slower inactivation than bacterial fecal indicators and PMMoV was inactivated more slowly than all other targets. These results demonstrate that bacterial fecal indicators inadequately represent viral fate following aging of sewage contaminated water due to differential persistence, and that currently used fecal indicator monitoring targets demonstrate highly variable persistence that should be considered during water quality monitoring and risk assessment.

Substrate inactivation of bacterial L-aspartate α-decarboxylase from Corynebacterium jeikeium K411 and improvement of molecular stability by saturation mutagenesis.

Bacterial L-aspartate α-decarboxylase (PanD) is a potential biocatalyst for the green production of β-alanine, an important block chemical for manufacturing nitrogen-containing chemicals in bio-refinery field. It was reported that the poor catalytic stability caused by substrate inactivation limited the large-scale application. Here, we investigated the characters of inactivation by L-aspartate of PanD from Corynebacterium jeikeium (PDCjei), and found that L-aspartate induced a time-, and concentration-dependent inactivation of PDCjei with the values of KI and kinact being 288.4mM and 0.235/min, respectively. To improve the catalytic stability of PDCjei, conserved amino acid residues essential to catalytic stability were analyzed by comparing the discrepancy in the observed inactivation rate of various sources. By an efficient colorimetric high-throughput screening method, four mutants with 3.18-24.69% higher activity were obtained from mutant libraries. Among them, the best mutation (R3K) also performed 66.38% higher catalytic stability than the wild type, showing great potential for industrial bio-production of β-alanine.

Sunlight Inactivation of Human Viruses and Bacteriophages in Coastal Waters Containing Natural Photosensitizers

Sunlight inactivation of poliovirus type 3 (PV3), adenovirus type 2 (HAdV2), and two bacteriophage (MS2 and PRD1) was investigated in an array of coastal waters to better understand solar inactivation mechanisms and the effect of natural water constituents on observed inactivation rates (k(obs)). Reactor scale inactivation experiments were conducted using a solar simulator, and k(obs) for each virus was measured in a sensitizer-free control and five unfiltered surface water samples collected from different sources. k(obs) values varied between viruses in the same water matrix, and for each virus in different matrices, with PV3 having the fastest and MS2 the slowest k(obs) in all waters. When exposed to full-spectrum sunlight, the presence of photosensitizers increased k(obs) of HAdV2, PRD1 and MS2, but not PV3, which provides evidence that the exogenous sunlight inactivation mechanism, involving damage by exogenously produced reactive intermediates, played a greater role for these viruses. While PV3 inactivation was observed to be dominated by endogenous mechanisms, this may be due to a masking of exogenous k(obs) by significantly faster endogenous k(obs). Results illustrate that differences in water composition can shift absolute and relative inactivation rates of viruses, which has important implications for natural wastewater treatment systems, solar disinfection (SODIS), and the use of indicator organisms for monitoring water quality.

Optimization and mechanism of copepod zooplankton inactivation using ozone oxidation in drinking water treatment

Bench-scale experiments were carried out to optimize ozone oxidation conditions for copepod inactivation by response surface methodology. Independent variables were ozone dosage (1, 3 and 5 mg/L), contact time (5, 15 and 25 min) and organic matter concentration (2, 5 and 8 mg/L). The results showed that all independent variables significantly influenced copepod inactivation rate. The observed inactivation rate was increased with the increasing ozone dosage and contact time. However, the trend of response gradually stabilized when the contact time was beyond 20 min. Copepod inactivation was more sensitive to high ozone dosage and short contact time under the same CT (dosage × contact time) value conditions. There was a negative effect of organic matter concentration on copepod inactivation. The polynomial response model for inactivation rate of copepod was established with R 2 = 0.9933 and adjusted- R 2 = 0.9865. The F -value of 165.93 implied the model significance and the p -value lower than 0.0001 indicated its good fit to use in the matrix. The ‘lack of fit (LOF) F -value’ of 0.0099 implied that the LOF was significant relative to pure error. The evaluation of the model showed it is able to make suitable predictions for the intended application.

Inactivation Models for Salmonella typhimurium, Staphylococcus aureus and Vibrio parahaemolyticus in Processed Vinegar.

Combined bactericidal effects of acetic acid, temperature, and sodium chloride on Salmonella typhimurium, Staphylococcus aureus and Vibrio parahaemolyticus were examined in processed vinegar. To express their effects, we established quadratic polynomial models distinguished into two types according to the presence or absence of sodium chloride. These models were also evaluated by the alternative variables of acetic acid concentration and pH value. Every model had high values for the correlation coefficients (r2=0.963-0.990) between predicted and observed inactivation rates. The fitness for the model prediction was observed independent of bacterial species, i.e., gram negative, positive, and halophilic bacteria.

Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site

Exchange-inert β,γ-bidentate Cr(H 2O) x(NH 3) yATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min −1 to 0.118 min −1 for Cr(NH 3) 4ATP and Cr(H 2O) 4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k obs by 3-PGA was proportional to the number of NH 3 ligands present in the coordination sphere of Cr 3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr 3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H 2O) 4ATP. Δ diastereomer of Cr(H 2O) 4ATP yielded an order of magnitude smaller K i value compared to the value observed with the Λ isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H 2O) 4ATP or Cr(NH 3) 4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr 3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme ·CrATP and enzyme·CrATP·3-PGA complexes.

How do MgATP analogues differentially modify high‐affinity and low‐affinity ATP binding sites of Na+/K+ ‐ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.

Acetylcholine receptor: evidence for a voltage-dependent regulatory site for acetylcholine. Chemical kinetic measurements in membrane vesicles using a voltage clamp

Acetylcholine receptor mediated ion translocation in membrane vesicles prepared from the Electrophorus electricus electroplax was investigated under voltage clamp conditions by using a quench-flow technique that allows the translocation to be measured in the millisecond to second time region. Two rate coefficients were measured over a 500-fold concentration range of acetylcholine, at a transmembrane voltage, Vm, of -45 mV, at pH 7.0, 1 degrees C. JA is the rate coefficient for ion translocation by the active state of the receptor in the absence of inactivation (desensitization), and alpha is the rate coefficient for the inactivation of the receptor by acetylcholine. (1) The values of JA and alpha increase with increasing acetylcholine concentration up to 300 microM. At higher concentrations, a concentration-dependent decrease in the ion flux rate was observed without a concomitant change in the inactivation rate. This inhibitory effect has not been reported previously and was not observed with acetylcholine or carbamoylcholine in the absence of a transmembrane voltage. (2) The value of the maximum influx rate coefficient, 26 s-1, is approximately twice that observed at 0 mV [JA(max) = 15 s-1]. This is consistent with previous interpretations that related JA(max) values to the channel-opening equilibrium constant, 1/phi, and with the relation of 1/phi to the mean lifetime of the open receptor channel in muscle cells, which is dependent on Vm. (3) The maximum observed inactivation rate coefficient [alpha(max) = 8.5 s-1] is somewhat larger than that observed at 0 mV [alpha(max) = 5 s-1].(ABSTRACT TRUNCATED AT 250 WORDS)

The Molecular Basis of Mutagenesis by Methyl and Ethyl Methanesulfonates

The molecular basis of the biological effect of alkylation, mutation induction and inactivation, was studied using transforming DNA.Comparative studies of mutation induction after methylation (by methyl methanesulfonate) and ethylation (by ethyl methanesulfonate) showed that methylation was five to ten times more effective than ethylation. This observation confirms chemical studies of the reactivities of the individual bases of DNA but conflicts sharply with observations that methyl methanesulfonate is much less mutagenic than ethyl methanesulfonate in bacteriophage and some higher organisms. In each case the number of mutational events caused in transforming DNA followed single‐hit kinetics. After termination of alkylation, the rates of decrease of mutants caused by methylation was twice that caused by ethylation, which fits chemical estimates of depurination rates.Reverse mutation studies with different mutagens and base analogs indicated that methyl methanesulfonate predominantly methylates guanine whereas ethyl methanesulfonate alkylates guanine and also adenine; in both cases the major product is an N7‐alkyl purine. Accordingly the cause of base‐pairing mistakes is unlikely to be related to steric effects of the alkyl group but rather due to ionization of the alkylated guanine.In addition, inactivation of transforming activity was observed to be linear with treatment time of DNA with methyl or ethyl methanesulfonate in a semilogarithmic plot. Since the observed inactivation rates cannot be solely explained as inactivating mutagenic events produced by base alkylation, it is likely that phosphate alkylation is inactivating.Lethal and mutagenic DNA alterations are shown to be dissociated processes by the following observations. (a) After termination of alkylation, the number of mutants caused by alkylation of the purine bases decreased linearly with time indicating that depurination is inactivating. (b) Under identical conditions, the number of total transformants decreased three times faster than the number of mutants, indicating that in addition to depurination triester breaks must be responsible for inactivation. Therefore, base alkylations are mutagenic and depurination and triester breaks are lethal DNA alterations.