Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes

Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.     

Degradation of PEG and non-PEG alginate-chitosan microcapsules in different pH environments

Bioencapsulation allows the protection of biologically active substances or cells from the biological environment. As such, bioencapsulation is often used for the delivery of drugs, growth factors and therapeutically useful cells. Depending on the site of implantation, the biocapsules are subjected to different pH environments, which will affect the degradation properties, mechanical properties and swelling behaviour of the biocapsules. As such, the encapsulation material plays an important role in the long term stability and performance of the biocapsules in vivo. In this study, five types of encapsulation materials were investigated: (i) alginate (A), (ii) alginate-chitosan (AC), (iii) alginate-chitosan-alginate (ACA), (iv) alginate-chitosan-polyethylene glycol (PEG) (ACP) and (v) alginate-chitosan-polyethylene glycol (PEG)-alginate (ACPA). Degradation studies were carried out by immersing the microcapsules in solutions of different pH values to investigate the role of the material as well as the number of encapsulation layers in maintaining the stability of the microcapsules in the different pH environments. Compression testing indicated that even with the presence of PEG on the surface membrane, there was not much difference in mechanical strength between ACA and ACPA microcapsules. However, the use of PEG did affect the weight change of the ACPA microcapsules when immersed in water and three different pH solutions. For the swelling test, the ACPA microcapsules showed a lower water uptake than ACA microcapsules. For degradation, the presence of PEG led to a lower increase in weight change compared to non-PEG chitosan microcapsules. Hence, the study revealed that PEG influenced the integrity of the surface membrane and not the mechanical strength of the microcapsules. With the inclusion of PEG, the interpenetrating network on the surface membrane would be further reinforced. As such, the addition of PEG to the alginate-chitosan microcapsules led to protection against an acidic environment, whilst the number of coating layers only influences the swelling properties and not the degradation and Young’s modulus of the microcapsules.     

An experimental approach probing the conformational transitions and energy landscape of antibodies: a glimmer of hope for reviving lost therapeutic candidates using ionic liquid

Understanding protein folding in different environmental conditions is fundamentally important for predicting protein structures and developing innovative antibody formulations. While the thermodynamics and kinetics of folding and unfolding have been extensively studied by computational methods, experimental methods for determining antibody conformational transition pathways are lacking. Motivated to fill this gap, we prepared a series of unique formulations containing a high concentration of a chimeric immunoglobin G4 (IgG4) antibody with different excipients in the presence and absence of the ionic liquid (IL) choline dihydrogen phosphate. We determined the effects of different excipients and IL on protein thermal and structural stability by performing variable temperature circular dichroism and bio-layer interferometry analyses. To further rationalise the observations of conformational changes with temperature, we carried out molecular dynamics simulations on a single antibody binding fragment from IgG4 in the different formulations, at low and high temperatures. We developed a methodology to study the conformational transitions and associated thermodynamics of biomolecules, and we showed IL-induced conformational transitions. We showed that the increased propensity for conformational change was driven by preferential binding of the dihydrogen phosphate anion to the antibody fragment. Finally, we found that a formulation containing IL with sugar, amino acids and surfactant is a promising candidate for stabilising proteins against conformational destabilisation and aggregation. We hope that ultimately, we can help in the quest to understand the molecular basis of the stability of antibodies and protein misfolding phenomena and offer new candidate formulations with the potential to revive lost therapeutic candidates.

Probing the energy landscape and thermodynamics of biomolecules for drug design.     

Combinatorial effect of different alginate compositions, polycations, and gelling ions on microcapsule properties

Microencapsulation technology is commonly used to deliver cells and drugs for therapeutic applications. The encapsulation material has a direct influence over the properties of microcapsules and will eventually dictate the efficacy of this delivery system. In this study, the combinatory effect of different alginate compositions, polycations and gelling ions was investigated to determine their roles in affecting the properties of the microcapsules. A multifactorial relationship was found between the three factors, in which certain factors took priority over others in influencing the overall property of the microcapsules. As the size of the microcapsules was kept constant throughout the investigation, further insights into the role of fabrication parameters on microcapsules size were also obtained. From the results, poly-l-lysine-coated microcapsules fabricated from 40/60 sodium alginate and cross-linked with barium chloride were the most ideal for applications that require both good mechanical as well as diffusion properties.     

Improving Learner-Computer Interaction through Intelligent Learning Material Delivery Using Instructional Design Modeling

This paper describes an innovative and sophisticated approach for improving learner-computer interaction in the tutoring of Java programming through the delivery of adequate learning material to learners. To achieve this, an instructional theory and intelligent techniques are combined, namely the Component Display Theory along with content-based filtering and multiple-criteria decision analysis, with the intention of providing personalized learning material and thus, improving student interaction. Until now, the majority of the research efforts mainly focus on adapting the presentation of learning material based on students’ characteristics. As such, there is free space for researching issues like delivering the appropriate type of learning material, in order to maintain the pedagogical affordance of the educational software. The blending of instructional design theories and sophisticated techniques can offer a more personalized and adaptive learning experience to learners of computer programming. The paper presents a fully operating intelligent educational software. It merges pedagogical and technological approaches for sophisticated learning material delivery to students. Moreover, it was used by undergraduate university students to learn Java programming for a semester during the COVID-19 lockdown. The findings of the evaluation showed that the presented way for delivering the Java learning material surpassed other approaches incorporating merely instructional models or intelligent tools, in terms of satisfaction and knowledge acquisition.