Biolayer Interferometry for DNA-Protein Interactions v1

Biolayer interferometry (BLI) is a widely utilized technique for determining the interaction dynamics between macromolecules. Most BLI instruments, such as the Octet RED96e used throughout this protocol, are completely automated and detect changes in the interference pattern of white light reflected off a biosensor tip. Biosensors are initially loaded with a stationary macromolecule, then introduced into a solution containing macromolecules of interest. Binding to the stationary molecules creates a change in optical wavelength that is recorded by the instrument in real-time. The majority of published BLI experiments assess protein-protein (such as antibody-substrate kinetics) or protein-small molecule (such as drug discovery) interactions. However, a less-appreciated assay for BLI analysis is DNA-protein interactions. In our laboratory, we have shown the practicality of using biotinylated-DNA probes to determine the binding kinetics of transcription factors to specific DNA sequences. The following protocol describes these steps, including the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis through GraphPad Prism.

Modelling the efficacy of Neprilysin from various species in degrading different Amyloid-β peptides: Potential application in therapeutics of Alzheimer’s disease

Recombinant neprilysin due to its degradation potential against Amyloid-β (Aβ) peptides has been looked at as a potential therapeutic candidate for treating Alzheimer’s disease (AD). However the enzymatic activity of neprilysin against different Aβ peptides can variable which significantly limits the therapeutic optimization. Using the molecular interaction analysis and modelling it against the known enzyme-substrate kinetics, this study developed a novel approach to predicting biosimilar enzyme-substrate kinetics. The known enzyme-substrate kinetics of human recombinant neprilysin with Aβ1-40 peptide was used as the prototype to assess the affinity and efficacy of various inter and intra-species neprilysin- Aβ peptide enzyme kinetics based on the relative molecular interaction analysis. Significant inter and intra-species variations in neprilysin- Aβ peptide enzyme kinetics was observed which further validated the need for optimizing enzyme kinetics tailored to specific substrate degradation. The novel enzyme kinetics modelling approach described in this study can be helpful in the developing of recombinant enzymes/peptides for personalised therapeutic applications.

Application of monod two-substrate kinetics with an intermediate for anaerobic co-digestion of distillery wastewater and molasses/glycerol waste in batch experiments

Anaerobic digestion is a highly complex process, particularly in co-digestion between poorly-defined, complex co-substrates like distillery wastewater, molasses, and crude glycerine. Thus, in this article, the authors tackled the problems by using Monod two-substrate with an intermediate (M2SI) model to represent accumulated biomethane evolution (ABE) obtained from the co-substrates, including easily degradable, slowly degradable substrates and intermediate. The M2SI model predictions were compared with the traditional Monod model's simulation results to clarify an outstanding of the present model in the aspect of modeling and control. Different behaviors of ABE curves from batch experiments were used to calibrate the M2SI model prediction with sensitivity analysis of the model parameters. It was found that the M2SI model gives a correct trend to describe the co-digestion process with multiple substrates and complex microbial activities with satisfactory fitting accuracy. At the same time, simple Monod kinetics have a good fit for dilute pure distillery wastewater, but the estimated microbial growth kinetics were counterintuitive. Therefore, the M2SI Model has a broader range of applications for co-digestion dealing with the complexity of multiple microbial activities to consume inherently complex or artificial co-substrates.

MATHEMATICAL MODELLING AND STATIONARY CHARACTERISTICS OF A TWO-PHASE FLUIDISED-BED BIOREACTOR WITH EXTERNAL AERATION

A mathematical model for a two-phase fluidised bed bioreactor with liquid recirculation and an external aerator was proposed. A stationary nonlinear analysis of such a bioreactor for an aerobic process with double-substrate kinetics was carried out. The influences of a volumetric fraction of solid carriers in the liquid phase, the rate of active biomass transfer from the biofilm to the liquid, the concentration of carbonaceous substrate, the mean residence time of the liquid and the efficiency of the external aerator on the steady state characteristics of the bioreactor were described. A method for determination of the minimal recirculation ratio related to oxygen demand and fluidised bed conditions was presented. On the basis of the obtained results, it is possible to choose reasonable operating conditions of such plants and to determine constraints, while considering acceptable concentrations of a toxic substrate being degraded.