Inactivation Step Research Articles

Rapid one-step extraction method for the identification of molds using MALDI-TOF MS

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized fungal identification. Previously, we developed a MALDI-TOF MS mold extraction procedure and comprehensive database. While MALDI-TOF MS has become routine in a few laboratories, it has not yet become widespread. A major obstacle is the lack of a simple, reproducible and uniform protein extraction procedure. In this study, we developed and validated a rapid one-step protein extraction protocol for filamentous fungi. Excised molds were placed into tubes containing zirconia-silica beads and extraction solution without washing or ethanol inactivation steps. Extraction solutions containing different ratios of acetonitrile and formic acid were evaluated. Samples were then processed using a PowerLyzer high power bead based homogenizer and supernatants spotted for MALDI-TOF MS. The rapid method was evaluated prospectively and in parallel to our current mold extraction protocol for 3 months. Analysis of 106 clinical mold isolates resulted in an improved performance and a decrease in extraction time by 30 minutes to a total of 5 minutes of hands-on time. Acceptable identification scores (≥ 2.00) were achieved for up to 63.0% of mold isolates by the rapid method compared with 52.8% of isolates by the current routine protocol. Score comparisons between duplicate spots showed higher reproducibility of the rapid method as compared to the routine method. The rapid extraction method allows efficient analysis of clinical mold isolates both in scheduled batch runs and on an in-demand basis while providing a simple starting platform for laboratories adopting MALDI-TOF MS for mold identification.

Cationic Antimicrobial Peptides Inactivate Shiga Toxin-Encoding Bacteriophages.

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

Low hepatitis E virus RNA prevalence in a large-scale survey of United States source plasma donors.

Hepatitis E virus (HEV) is a small, nonenveloped, single-stranded, RNA virus of emerging concern in industrialized countries. HEV transmission through transfusion of blood components has been reported, but not via plasma-derived medicinal products (PDMPs) manufactured with virus inactivation and/or removal steps. This study aimed to determine the prevalence of HEV among US source plasma donors. Samples were collected from US source plasma donors at centers across the United States and were initially screened for HEV RNA in 96-sample minipools using the Roche cobas HEV test on the cobas 8800 system. Assuming a sensitivity of 18.6IU/mL, the minipool screening strategy allowed for reliable detection of individual donations with HEV RNA titers of more than 2×103 IU/mL. Reactive minipools were resolved to individual donations, which were further analyzed to quantify viral RNA concentration, determine HEV genotype, and immunoglobulin (Ig)G and IgM HEV antibody status. A total of 128,020 samples were collected from 96 CSL Plasma centers in the United States, representing 27 states. The prevalence of HEV RNA-positive samples was 0.002% with three unique HEV-positive donors identified, all HEV Subgenotype 3a. Virus titers of HEV-positive samples were relatively low (103 -104 IU HEV RNA/mL). One positive donation was HEV IgG seropositive. Routine screening of US source plasma donations for HEV would not substantially improve the safety of most PDMPs. The low prevalence and potential viral load of HEV, together with effective virus reduction steps in manufacturing processes, results in a low residual risk and acceptable safety margins for PDMPs derived from US plasma donors.

Design and operation of a continuous integrated monoclonal antibody production process.

The realization of an end-to-end integrated continuous lab-scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter-based cell-retention, a continuous two column capture process, a virus inactivation step, a semi-continuous polishing step (twin-column MCSGP), and a batch-wise flow-through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity-yield trade-off of classical batch-wise bind-elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight-through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end-to-end integration. The steady-state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303-1313, 2017.

Rational Optimization of Mechanism-Based Inhibitors through Determination of the Microscopic Rate Constants of Inactivation.

Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine because of their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated because of time-dependent inhibition resulting from multistep inactivation mechanisms. The global kinetic parameters kinact and KI have been used to characterize MBIs, but they provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone 1. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min-1).

Evaluation of plant-produced Clostridium perfringens type D epsilon toxoid in a vaccine against enterotoxaemia in sheep.

Enterotoxaemia (pulpy kidney) is a common bacterial disease of sheep caused by Clostridium perfringens type D epsilon toxin. It has mortality rates of up to 30% in non-vaccinated animals. Current vaccines from whole cell cultures are expensive to manufacture and can induce local inflammatory responses in sheep. They usually have reduced immunogenicity because of the difficulty of standardising the inactivation step in vaccine manufacturing. In the current study, we evaluated the safety and potency of a recombinant plant-made epsilon toxoid protein (r-Etox) as an affordable and safer alternative vaccine for developing countries. Results of injection site reactions, rectal temperature and toxin neutralisation test in single and prime–boost inoculations of mice, guinea pigs and sheep suggest that the product is not toxic to animals and could protect sheep against enterotoxaemia.

Direct high-pressure NMR observation of dipicolinic acid leaking from bacterial spore: A crucial step for thermal inactivation

A bacterial spore protects itself with an unusually high concentration (~10% in dry weight of spore) of dipicolinic acid (DPA), the release of which is considered the crucial step for inactivating it under mild pressure and temperature conditions. However, the process of how the spore releases DPA in response to pressure remains obscure. Here we apply 1H high-resolution high-pressure NMR spectroscopy, for the first time, to the spore suspension of Bacillus subtilis natto and monitor directly and in real-time the leaking process of DPA in response to pressure of 200MPa at 20°C. We find that about one third of the total DPA leaks immediately upon applying pressure, but that the rest leaks slowly in hrs upon decreasing the pressure. Once DPA is fully released from the spore, the proteins of the spore become easily denatured at a mild temperature, e.g., 80°C, much below the temperature commonly used to inactivate spores (121°C). The success of the present experiment opens a new avenue for studying bacterial spores and cells at the molecular level in response to pressure, temperature and other perturbations.

Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae.

The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories.

Use of Humanized RS-ATL8 Reporter System for Detection of Allergen-Specific IgE Sensitization in Human Food Allergy.

Allergen-specific Immunoglobulin E (IgE) determination lies at the heart of diagnosis of sensitization to food and other allergens. In the past few years, reporter systems capable of detecting the presence of allergen-specific IgE have been developed by several labs. These rely on humanized rat basophil leukemia cell lines stably transfected with reporter genes such as firefly luciferase. In this chapter, we describe protocols for the use of the RS-ATL8 cell line (IgE cross-linking-induced luciferase expression; EXiLE) in 96-well and 384-well formats. We also describe optional treatment steps for enveloped virus and complement inactivation.

Inactivation of Zika virus by solvent/detergent treatment of human plasma and other plasma-derived products and pasteurization of human serum albumin.

In 2016 the World Health Organization declared the mosquito-borne Zika virus (ZIKV) a "public health emergency of international concern." ZIKV is a blood-borne pathogen, which therefore causes concerns regarding the safety of human plasma-derived products due to potential contamination of the blood supply. This study investigated the effectiveness of viral inactivation steps used during the routine manufacturing of various plasma-derived products to reduce ZIKV infectivity. Human plasma and intermediates from the production of various plasma-derived products were spiked with ZIKV and subjected to virus inactivation using the identical techniques (either solvent/detergent [S/D] treatment or pasteurization) and conditions used for the actual production of the respective products. Samples were taken and the viral loads measured before and after inactivation. After S/D treatment of spiked intermediates of the plasma-derived products Octaplas(LG), Octagam, and Octanate, the viral loads were below the limit of detection in all cases. The mean log reduction factor (LRF) was at least 6.78 log for Octaplas(LG), at least 7.00 log for Octagam, and at least 6.18 log for Octanate after 60, 240, and 480 minutes of S/D treatment, respectively. For 25% human serum albumin (HSA), the mean LRF for ZIKV was at least 7.48 log after pasteurization at 60°C for 120 minutes. These results demonstrate that the commonly used virus inactivation processes utilized during the production of human plasma and plasma-derived products, namely, S/D treatment or pasteurization, are effective for inactivation of ZIKV.

Pharmacokinetic Profile of Nonacog Gamma (recombinant factor IX) in Previously-Treated Patients with Severe or Moderately Severe Hemophilia B

Pharmacokinetic (PK) parameters are important surrogate indicators for hemostatic efficacy replacement therapy in the treatment of haemophilia patients. Nonacog gamma (Rixubis) is a recombinant factor IX (rFIX) concentrate that is manufactured using two viral inactivation steps (solvent/detergent treatment and nanofiltration) and without any materials of animal origin. Clinical trials of nonacog gamma included a comprehensive evaluation of PK in hemophilia B patients across multiple age groups and a direct comparison to determine equivalence with another licensed, commercially-available factor IX product. The objective of this evaluation was to assess the factor IX activity across clinical development with nonacog gamma.PK parameters for nonacog gamma were evaluated in a non-bleeding state in previously-treated patients with severe (FIX level < 1%) or moderately severe (FIX level 1-2%) hemophilia B in three prospective clinical trials (a pivotal trial in adults,1 a pediatric trial in children aged 2 to 12 years,2 and a third trial in patients undergoing surgical procedures3).Factor IX activity was determined using a one-stage clotting assay. In the pivotal trial, PK equivalence with a comparator recombinant factor IX product (nonacog alfa) was determined by the ratio of AUC0-72 h (area under the plasma concentration versus time curve from 0 to 72 hours post-infusion), per dose with a type I error of 5% for the two-sided 90% confidence interval (equivalence margin: 80% to 125%).Windyga et al. (2014) established bioequivalence of nonacog gamma with a comparator recombinant factor IX in the pivotal trial (N=27), as the 90% CI for the ratio of the AUC0-72 h per dose ranged from 103% to 109%.1 A plot of mean factor IX activity levels versus time after start of infusion on a linear scale and log scale (figure 1a and 1b) depicts the equivalence throughout the 72-hour follow-up period. A similar pattern can be observed in the linear and log regression for mean factor IX activity in pediatric patients (N=23). These results are further supported by consistent PK parameters determined prior to surgical intervention in the surgery trial (N=12).3 Two important PK parameters determined in each study were as follows: mean [STD] T ½ 26.70h (9.55), incremental recovery (IR) 0.87 (0.22) for nonacog gamma and 27.87h (9.22), IR 0.76 (0.20) for the comparator rFIX in the pivotal trial, 25.31h (3.130), IR 0.67 (0.16) in the pediatric trial, and 23.60h (3.60), IR 1.06 (0.35) in the surgery trial.ConclusionFactor IX activity of nonacog gamma throughout its clinical development support previous finding of its equivalence with another licensed recombinant factor IX concentrate in patients with severe to moderately severe hemophilia B.

Inactivation of Zika Virus By Solvent/Detergent Treatment or Pasteurization

▪ BACKGROUNDSince 2015 Zika virus (ZIKV) has spread within the Americas and even before in other regions. In 2016, the World Health Organisation (WHO) declared the ZIKV as a "Public Health Emergency of International Concern" (PHEIC). Therefore, it is very important to confirm the pathogen safety of blood plasma products. These products generally demand an effective donor selection as well as robust pathogen elimination/reduction steps within the manufacturing processes. Here, we provide experimental evidence that both solvent/detergent (S/D) treatment and pasteurization effectively inactivate ZIKV. STUDY DESIGN AND METHODSThe ZIKV inactivation capacity of the pasteurization step and the S/D treatment for different products and process conditions were investigated. Therefore, in-process material from commercial batches was collected and spiked with ZIKV in a ratio of approximately 1:10. Viral loads were determined prior to the safety steps and at pre-defined intervals during the individual processes to investigate the inactivation kinetics. Each process was investigated in duplicate runs. RESULTSSixty minutes after S/D treatment the viral titer was below detection limit during manufacturing of human plasma resulting in mean ZIKV reduction factor (LRF) of ≥ 6.78 log10. For immunoglobulin (IgG) after 240 min of S/D treatment the LRF was determined to be ≥ 7.00 log10. ZIKV was inactivated ≥ 6.18 log10 after 480 min by S/D treatment during the manufacture of factor VIII.During pasteurization of human albumin ZIKV infectivity was already below the detection limit after the heat-up phase. This finding was confirmed by subsequent test samples up to 2 hours. A LRF of ≥ 7.48 log10 was demonstrated. CONCLUSIONWe showed that different S/D treatment procedures inactivate the ZIKV to below the detection limit. This effect was seen independently of various product matrices or the choice of S/D reagent at various concentrations and temperatures, while taking product-specific treatment times into consideration. We also showed that pasteurization is a very efficient inactivation step for the ZIKV. These results demonstrate the effectiveness of S/D treatment against even newly emerging lipid-enveloped viruses like ZIKV in the manufacturing of human plasma and derivatives thereof, such as IgG or factor VIII. DisclosuresSchmidt:Octapharma: Employment. Kuehnel:Octapharma: Employment. Mueller:Octapharma: Employment. Pichotta:Octapharma: Employment. Radomski:Octapharma: Employment. Volk:Octapharma: Employment. Knaub:Octapharma: Employment.

Current methods to manufacture human platelet lysates for cell therapy and tissue engineering: possible trends in product safety and standardization

Human platelet lysates (HPLs), obtained from therapeutic‐grade platelet concentrates (PC), are rich in growth factors and other bioactive molecules. HPL is increasingly evaluated as a substitute for foetal bovine serum (FBS), to likely become the next ‘gold standard’ supplement of growth media for clinical‐grade ex vivo propagation of human cells. HPL avoids the risks of immunological reactions against xenoproteins. HPL quality and safety benefit from the pathogen safety measures in place for producing PC for transfusion. Numerous studies support that HPL‐expanded mesenchymal stromal cells show improved proliferation and decreased doubling time than when using FBS‐supplemented media and maintain characteristic immunophenotype, differentiation capacity and T‐cell immunosuppressive effect. Several methods are used to prepare HPL; they differ in the selection and formulation of the starting PC, the consideration or not for blood groups, the pool size, the membrane lysis procedure, the depletion of fibrinogen, and the implementation or not of a pathogen inactivation step on PC materials or during manufacture. Allogeneic ‘off‐the‐shelf’ pooled HPLs, made from approximately fifty, or more, PC, are currently produced by blood establishments and/or available through commercial suppliers. Improved HPL standardization and safety profile, through larger pools and dedicated viral inactivation and removal treatments, is expected in the next few years, broadening applications in cell therapy and regenerative medicine. Systematic studies of the impact of the mode of production of HPL on the isolation and expansion of various human cell types should be encouraged.

Development and evaluation of an in-house single step loop-mediated isothermal amplification (SS-LAMP) assay for the detection of Mycobacterium tuberculosis complex in sputum samples from Moroccan patients.

BackgroundTuberculosis (TB) is a major global health problem and remains the leading cause of morbidity and mortality in developing countries. Routinely used TB diagnostic methods, in most endemic areas, are time-consuming, often less-sensitive, expensive and inaccessible to most patients. Therefore, there is an urgent need for the development of early, easy to use and effective diagnosis tools of TB, which can be effectively integrated into resource limited settings, to anticipate the early treatment and limit further spread of the disease.Over the last decade, Loop-mediated isothermal amplification (LAMP) assays have become a powerful tool for rapid diagnosis of infectious diseases because of the simplicity of device requirements. Indeed, LAMP is a simple, quick and cost effective Isothermal Nucleic Acid Amplification diagnostic test (INAAT) that has the potential to be used in TB endemic settings of resource-poor countries.MethodsIn the present study, we have developed a simple and rapid TB molecular diagnostic test using a Single-Step Loop-mediated isothermal DNA amplification (SS-LAMP) method for the detection of Mycobacterium tuberculosis complex (MTBC) strains, with a simplified sample preparation procedure, eliminating DNA extraction prior to LAMP amplification, DNA initial denaturation and enzymatic inactivation steps during the amplification process.To perform our in-house SS-LAMP assay, a set of six specific primers was specifically designed to recognize eight distinct regions on the MTBC species-specific repetitive insertion sequence 6110 (IS6110). The amplification of the targeted DNA was carried out under isothermal conditions at 65 °C within 1 h. Our protocol was firstly optimized using 60 of confirmed MTBC isolates and a recombinant pGEMeasy-IS6110 vector for sensitivity testing. Thereafter, the assay was evaluated on liquefied sputum specimens collected from 157 Moroccan patients suspected of having TB.ResultsOur SS-LAMP developed assay was able to detect MTBC DNA directly from liquefied sputum samples without any prior DNA extraction, denaturation nor the final enzymatic inactivation step. When compared to routinely used Löwenstein Jensen (LJ) Culture method, our SS-LAMP assay is rapid and showed specificity and sensitivity of 99.14 % and 82.93 % respectively which are within the international standards. In addition, the limit of detection of our assay was found to be as little as 10 copies of bacterial DNA.ConclusionTo our knowledge, this is the first study using a single step LAMP (SS-LAMP) procedure as a rapid, easy to perform and cost effective testing for TB early detection. This innovative assay could be suitable for low-income countries with restricted health equipment facilities.

Celecoxib non-competitively inhibits acetylcholinesterase

Isocitrate lyase from the mycelium of Phycomyces blakesleeanus was inactivated with thiol-reactive reagents, 5,5′-dithiobis-(2-nitrobenzoic)acid, p-hydroxymercuribenzoic acid, N-ethylmaleimide or iodoacetate, at pH 6.8 and 25°C. In all cases the inactivation is characterized by a biphasic kinetic profile. The rapid initial phase of inactivation does not increase linearly with increasing reagent concentration, but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. Re-activation of the enzyme was observed under thiol excess treatment. The pH dependence of the initial phase of inactivation suggests that a group on the enzyme with pKa = 6.8 is being modified. The effect of ligands was tested on the inactivation reaction. Mg2+-Ds-isocitrate and Ds-isocitrate provided total protection, whereas Mg2+ ions, succinate and oxalate provided only partial protection of the enzyme against inactivation. On the basis of these results, we would suggest that the thiol-reactive reagents modify at least one thiol group crucial for the enzymatic activity and probably located in the interface between succinate and glyoxylate subsite.

Dynamical characterization of inactivation path in voltage-gated Na+ ion channel by non-equilibrium response spectroscopy

ABSTRACTInactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena.

Modeling and Validation of the Ecological Behavior of Wild-Type Listeria monocytogenes and Stress-Resistant Variants.

Listeria monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress-resistant variants. This study aimed to evaluate the impact of the presence of stress-resistant variants of Listeria monocytogenes and their corresponding trade-offs on population composition under different environmental conditions. A set of stress robustness and growth parameters of the wild type (WT) and an rpsU deletion variant was obtained and used to model their growth behavior under combined mild stress conditions and to model their kinetics under single- and mixed-strain conditions in a simulated food chain. Growth predictions for the WT and the rpsU deletion variant matched the experimental data generally well, although some deviations from the predictions were observed. The data highlighted the influence of the environmental conditions on the ratio between the WT and variant. Prediction of performance in the simulated food chain proved to be challenging. The trend of faster growth and lower stress robustness for the WT than for the rpsU variant in the different steps of the chain was confirmed, but especially for the inactivation steps and the time needed to resume growth after an inactivation step, the experimental data deviated from the model predictions. This report provides insights into the conditions which can select for stress-resistant variants in industrial settings and discusses their potential persistence in food processing environments. Listeria monocytogenes exhibits a heterogeneous stress response which can partially be attributed to the presence of genetic variants. These stress-resistant variants survive better under severe conditions but have, on the other hand, a reduced growth rate. To date, the ecological behavior and potential impact of the presence of stress-resistant variants is not fully understood. In this study, we quantitatively assessed growth and inactivation behavior of wild-type L. monocytogenes and its stress-resistant variants. Predictions were validated under different conditions, as well as along a model food chain. This work illustrates the effects of environmental factors on population dynamics of L. monocytogenes and is a first step in evaluating the impact of population diversity on food safety.

Bioelectrochemical analysis of thermodynamics of the catalytic cycle and kinetics of the oxidative inactivation of oxygen-tolerant [NiFe]-hydrogenase

Membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus (HmMBH) is an O2-tolerant enzyme and allows direct electron transfer (DET)-type bioelectrocatalysis for the H2 oxidation. Very fast interfacial electron transfer occurs between the [NiFe]-active site of HmMBH and the electrode, and the potential dependence of the steady-state DET-type catalytic current has been analyzed on a thermodynamic model of a two-step one-electron transfer to get a Pourbaix diagram of the catalytic center. A reversible and oxidative inactivation that occurs when the [NiFe]-hydrogenases are suffering from the oxidative stress at high electrode potentials or high solution potentials has been kinetically analyzed for the time-dependence of the steady-state catalytic current as a measure. The kinetic analysis has shown that the rate-determining step of the oxidative inactivation is not electrochemical but chemical process and that the rate of the reductive reactivation is determined by the electrochemical process. The observed catalytic waves, especially the dependence of the waves on the scan rate and the hydrogen concentration, have been well reproduced by simulation with the thermodynamic and kinetic parameters evaluated here.

Continuous viral inactivation at low pH value in antibody manufacturing

Due to the evolving biopharmaceutical market, small scale and flexible production processes for continuous antibody manufacturing become more important. Consequently, various continuously operated processing steps like perfusion fermentation or continuous chromatography have been developed. However, no continuously operated viral inactivation at low pH value is available so far. In this article, the coiled flow inverter (CFI) is identified as a suitable reactor for this application and two different design approaches are presented. The logarithmic reduction value (LRV) approach targets at the same LRV in the continuous viral inactivation when compared to current batch operation. The minimum residence time (MRT) approach guarantees a residence time for all molecules entering the continuous viral inactivation of at least the inactivation time of the batch inactivation step. The viral reduction value and the monomer loss during inactivation are characterized for each design approach. Moreover, a linear regression model is developed, which predicts the Bodenstein number as a function of two CFI design parameters under defined boundary conditions. This model allows designing the CFI according to the inactivation time and the chosen design approach.

Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols.

We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine-ferrocenyltriazole-proline-tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol-disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection.