Pulsed electric field treatment as a potential method for microbial inactivation in scaffold materials for tissue engineering: the inactivation of bacteria in collagen gel

Aims: To investigate the effectiveness of pulsed electric field (PEF) treatment as a new method for inactivation of micro-organisms in complex biomatrices and to assess this by quantifying the inactivation of Escherichia coli seeded in collagen gels. Methods and Results: PEF was applied to E. coli seeded collagen gels in static (nonflowing) chambers. The influence of electric field strength, pulse number and seeded cell densities were investigated. The highest level of inactivation was obtained at the maximum field strength of 45 kV cm⁻¹. For low levels of E. coli contamination (10³ CFU ml⁻¹), PEF treatment resulted in no viable E. coli being recovered from the gels. However, PEF treatment of gels containing higher cell densities (≥10⁴ CFU ml⁻¹) did not achieve complete inactivation of E. coli. Conclusions: PEF treatment successfully inactivated E. coli seeded in collagen gels by 3 log₁₀ CFU ml⁻¹. Complete inactivation was hindered at high cell densities by the tailing effect observed. Significance and Impact of the Study: PEF shows potential as a novel, nondestructive method for decontamination of collagen-based matrices. Further investigation is required to ensure its compatibility with other proteins and therapeutic drugs for tissue engineering and drug delivery applications.     

Characterization of Bacillus subtilis spore inactivation in low‐pressure,low‐temperature gas plasma sterilization processes

Aims: To identify structural components of Bacillus subtilis spores serving as targets for sterilization with microwave induced low‐pressure, low‐temperature nitrogen‐oxygen plasma. Methods and Results: The inactivation of spores followed a biphasic kinetics consisting of a log‐linear phase with rapid inactivation followed by a slow inactivation phase. In the course of plasma treatment, damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild‐type strain showed the highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid‐soluble proteins (ΔsspA ΔsspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV‐C radiation is the most effective inactivation agent in the plasma, whereby the splB and ΔcotE mutant spores were equally and slightly less sensitive, respectively, than the wild‐type spores. Finally, the extent of damages in the spore DNA determined by quantitative PCR correlated with the spore inactivation. Conclusions: Spore inactivation was efficiently mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. Significance and Impact of the Study: The results provide new evidence to the understanding of plasma sterilization processes. This knowledge supports the identification of useful parameters for novel plasma sterilization equipment to control process safety.     

Kinetic study of thermal inactivation for native and methoxypolyethylene glycol modified trypsin

Bovine pancreatic trypsin (Ti) has been modified with four kinds of methoxypolyethylene glycol (MPEG, molecular masses 350, 750, 2000 and 5000 Da) to enhance thermostability. The MPEG-modified Ti was more stable against temperature than the native form, the larger molecular mass moiety of MPEG showing higher thermostabillty. To investigate the mechanism of thermal inactivation, a new kinetic model, which has the ability of taking the thermal denaturation and autolysis effects of the proteases into account, has been used to analyze the thermal inactivation process of the native and modified Ti in detail. The kinetic analysis showed that the stabilization effect caused by MPEG modification was the result of a decrease in autolysis rate and a decrease in the rate of thermal denaturation. In addition, the possible mechanism of reduced autolysis and lower thermal denaturation rate were also discussed.     

Photo‐inactivation of Bacillus endospores: inter‐specific variability of inactivation efficiency

The aims of this work were to (a) evaluate the susceptibility of endospores of Bacillus cereus, B. licheniformis, B. sphaericus and B. subtilis to photodynamic inactivation using a tricationic porphyrin as photosensitizer, (b) assess the efficiency of adsorption of the photosensitizer in endospore material as a determinant of the susceptibility of endospores of different Bacillus species to photo‐inactivation, (c) determine the value of B. cereus as a model organism for studies of antimicrobial photodynamic inactivation of bacterial endospores. The results of irradiation experiments with endospores of four species of Bacillus showed that B. cereus was the only species for which efficient endospore photo‐inactivation (> 3 log reduction) could be achieved. Endospores of B. licheniformis, B. sphaericus and B. subtilis were virtually resistant to photo‐inactivation with tricationic porphyrin. The amount of porphyrin bound to endospore material was not significantly different between species, regardless of the presence of an exosporium or exosporium‐like outer layer. The sensitivity of endospores to photodynamic inactivation with a tricationic porphyrin is highly variable among different species of the genus Bacillus. The presence of an exosporium in endospores of B. cereus and B. sphaericus, or an exosporium‐like glycoprotein layer in endospores of B. subtilis, did not affect the amount of bound photosensitizer and did not explain the inter‐species variability in susceptibility to photodynamic inactivation. The results imply that the use of B. cereus as a more amenable surrogate of the exosporium‐producing B. anthracis must be carefully considered when testing new photosensitizers for their antimicrobial photo‐inactivation properties.     

A vitalistic model to describe the thermal inactivation ofListeria monocytogenes

Summary Thermal inactivation of microorganisms has traditionally been described as log-linear in nature, that is the reduction in log numbers of survivors decreases in a linear manner with time. This is despite a plethora of data that shows consistent deviations from such kinetics for a wide range of organisms and conditions and that cannot be accounted for by experimental artifacts. Existing thermal death models fail to take such deviations into account and also fail to quantify the effects of heating menstruum on heat sensitivity. The thermal inactivation ofListeria monocytogenes ATCC 19115 has been investigated using a factorially-designed experiment comparing 45 conditions of salt concentration, pH value and temperature. Heating was carried out using a Submerged Coil heating apparatus that minimized experimental artifacts. Low pH values increased, whilst high salt concentrations decreased heat sensitivity. Results showed a significant and consistent deviation from log-linear kinetics, particularly at low temperatures. A number of distributions were tested for suitability to describe the variability of heat sensitivity within the population of heated cells (vitalistic approach). The use of the logistic function and log dose (log time) allowed the development of an accurate unifying predictive model across the whole range of heating conditions. It is proposed that this approach should be tested as a generalized modeling technique for death kinetics of vegetative bacteria.     

Age‐dependent investment in death‐feigning behaviour in the sweetpotato weevil Cylas formicarius

Because life‐history theory predicts that risky behaviours such as mating should increase as life expectancy decreases, predatory avoidance is expected to decrease with age. However, this prediction has not been examined. In the present study, the effect of age on death‐feigning behaviour, a form of predatory avoidance behaviour in the sweetpotato weevil Cylas formicarius (Summers) (Coleoptera: Brentidae), is investigated by performing a longitudinal study. Because the effects of mating history and age usually cannot be distinguished, mating history is controlled. The results show that only female weevils decrease the investment in death‐feigning behaviour with age, whereas male weevils do not show any age‐related change. In addition, death‐feigning behaviour of mated females is longer than that of virgin females, possibly because additional mating partners would be not needed by mated females.     

Stabilization mechanism of MPEG modified trypsin based on thermal inactivation kinetic analysis and molecular modeling computation

Thermal inactivation kinetic analysis and molecular modeling computation were jointly utilized to illuminate the detailed stabilization mechanism of trypsin caused by methoxypolyethylene glycol (MPEG) modification. First, trypsin was modified by MPEG (molecular mass 350 Da) to enhance its thermal stability. As expected, the modified trypsin was more stable against temperature than the native form. Second, a new kinetic model, which has the ability of taking the thermal denaturation and autolysis effects of proteases into account, was established and used to analyze the thermal inactivation process of the native and modified trypsin. The kinetic analysis showed that the increased thermal stability of MPEG modified trypsin is the joint result of a reduction in autolysis and a decrease in thermal denaturation. Finally, the molecular modeling technique was also employed to calculate some structural information change, i.e. solvent accessible surface, intramolecular hydrogen bond and root mean square fluctuation, between the native and modified trypsin. The results of molecular modeling computation demonstrated that (i) the steric hindrance caused by MPEG chain would result in the decreased rate of autolysis, (ii) the decreased rate of thermal denaturation should be ascribed to the increased number of hydrogen bond, not the result of the increased molecular rigidity.     

Structure of 6‐diazo‐5‐oxo‐norleucine‐bound human gamma‐glutamyl transpeptidase 1, a novel mechanism of inactivation

Intense efforts are underway to identify inhibitors of the enzyme gamma‐glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma‐glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6‐diazo‐5‐oxo‐norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM ^?1 min^?1 and the K _i was 2.7 ± 0.7 mM . The crystal structure of DON‐inactivated hGGT1 contained a molecule of DON without the diazo‐nitrogen atoms in the active site. The overall structure of the hGGT1‐DON complex resembled the structure of the apo‐enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1‐DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α‐amine of Thr381. The structure of DON‐bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.     

Advanced Electrolytes for Fast‐Charging High‐Voltage Lithium‐Ion Batteries in Wide‐Temperature Range

LiNi_xMn_yCo_1?x?yO₂ (NMC) cathode materials with Ni ≥ 0.8 have attracted great interest for high energy‐density lithium‐ion batteries (LIBs) but their practical applications under high charge voltages (e.g., 4.4 V and above) still face significant challenges due to severe capacity fading by the unstable cathode/electrolyte interface. Here, an advanced electrolyte is developed that has a high oxidation potential over 4.9 V and enables NMC811‐based LIBs to achieve excellent cycling stability in 2.5–4.4 V at room temperature and 60 °C, good rate capabilities under fast charging and discharging up to 3C rate (1C = 2.8 mA cm^?2), and superior low‐temperature discharge performance down to ?30 °C with a capacity retention of 85.6% at C/5 rate. It is also demonstrated that the electrode/electrolyte interfaces, not the electrolyte conductivity and viscosity, govern the LIB performance. This work sheds light on a very promising strategy to develop new electrolytes for fast‐charging high‐energy LIBs in a wide‐temperature range.     

Mechanism‐based inactivation of cytochrome P450 2D6 by chelidonine

Chelidonine (CHE) is a major bioactive constituent of greater celandine, a plant used in traditional herbal medicines. CHE has widely been used as an analgesic in clinical settings. We evaluated the inhibitory effects of CHE on human cytochrome P450 enzymes. CHE produced time‐, concentration‐, and NADPH‐dependent inhibition of CYP2D6, with K _I and k _inact values of 20.49 μM and 11.05 min ^?1, respectively. Approximately 76% of CYP2D6 activity was suppressed after 9 minute incubation with CHE (50 μM). The loss of enzyme activity was not restored following dialysis. The estimated partition ratio of the inactivation was about 156. Quinidine, a competitive inhibitor of CYP2D6, attenuated the CHE‐mediated enzyme inactivation, while glutathione and catalase/superoxide dismutase did not markedly ameliorate the inhibitory effect. Upon oxidation using potassium ferricyanide, the 15.1% activity of CYP2D6 was restored. These findings indicate that CHE acted as a mechanism‐based inactivator of CYP2D6 and the observed effects may induce potential drug‐drug interactions.