Evaluation of a cell-based osteogenic formulation compliant with good manufacturing practice for use in tissue engineering

Molecular Biology Reports - Proper bony tissue regeneration requires mechanical stabilization, an osteogenic biological activity and appropriate scaffolds. The latter two elements can be combined...     

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic,calorimetric and computational molecular methods

Abstract

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.

Communicated by Ramaswamy H. Sarma     

Protonation state of the selectivity filter of bacterial voltage-gated sodium channels is modulated by ions

The selectivity filter (SF) of bacterial voltage-gated sodium channels consists of four glutamate residues arranged in a C₄ symmetry. The protonation state population of this tetrad is unclear. To address this question, we simulate the pore domain of bacterial voltage-gated sodium channel of Magnetococcus sp. (Na_vMs) through constant pH methodology in explicit solvent and free energy perturbation calculations. We find that at physiological pH the fully deprotonated as well as singly and doubly protonated states of the SF appear feasible, and that the calculated pKa decreases with each additional bound ion, suggesting that a decrease in the number of ions in the pore can lead to protonation of the SF. Previous molecular dynamics simulations have suggested that protonation can lead to a decrease in the conductance, but no pKa calculations were performed. We confirm a decreased ionic population of the pore with protonation, and also observe structural symmetry breaking triggered by protonation; the SF of the deprotonated channel is closest to the C₄ symmetry observed in crystal structures of the open state, while the SF of protonated states display greater levels of asymmetry which could lead to transition to the inactivated state which possesses a C₂ symmetry in the crystal structure. We speculate that the decrease in the number of ions near the mouth of the channel, due to either random fluctuations or ion depletion due to conduction, could be a self-regulatory mechanism resulting in a nonconducting state that functionally resembles inactivated states.     

Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome

Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔV_Tot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔV_Tot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔV_Tot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔV_Tot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.     

Learning a functional grammar of protein domains using natural language word embedding techniques

In this paper, using Word2vec, a widely-used natural language processing method, we demonstrate that protein domains may have a learnable implicit semantic “meaning” in the context of their functional contributions to the multi-domain proteins in which they are found. Word2vec is a group of models which can be used to produce semantically meaningful embeddings of words or tokens in a fixed-dimension vector space. In this work, we treat multi-domain proteins as “sentences” where domain identifiers are tokens which may be considered as “words.” Using all InterPro (Finn et al. 2017) pfam domain assignments we observe that the embedding could be used to suggest putative GO assignments for Pfam (Finn et al. 2016) domains of unknown function.     

A Bayesian nonparametric testing procedure for paired samples

We propose a Bayesian hypothesis testing procedure for comparing the distributions of paired samples. The procedure is based on a flexible model for the joint distribution of both samples. The flexibility is given by a mixture of Dirichlet processes. Our proposal uses a spike-slab prior specification for the base measure of the Dirichlet process and a particular parametrization for the kernel of the mixture in order to facilitate comparisons and posterior inference. The joint model allows us to derive the marginal distributions and test whether they differ or not. The procedure exploits the correlation between samples, relaxes the parametric assumptions, and detects possible differences throughout the entire distributions. A Monte Carlo simulation study comparing the performance of this strategy to other traditional alternatives is provided. Finally, we apply the proposed approach to spirometry data collected in the United States to investigate changes in pulmonary function in children and adolescents in response to air polluting factors.     

Combined Force-Torque Spectroscopy of Proteins by Means of Multiscale Molecular Simulation

Assessing the structural properties of large proteins is important to gain an understanding of their function in, e.g., biological systems or biomedical applications. We propose a method to examine the mechanical properties of proteins subject to applied forces by means of multiscale simulation. Both stretching and torsional forces are considered, and these may be applied independently of each other. As a proof of principle, we apply torsional forces to a coarse-grained continuum model of the antibody protein immunoglobulin G using fluctuating finite element analysis and use it to identify the area of strongest deformation. This region is essential to the torsional properties of the molecule as a whole because it represents the softest, most deformable domain. Zooming in, this part of the molecule is subjected to torques and stretching forces using molecular dynamics simulations on an atomistically resolved level to investigate its torsional properties. We calculate the torsional resistance as a function of the rotation of the domain while subjecting it to various stretching forces. From this, we assess how the measured twist-torque profiles develop with increasing stretching force and show that they exhibit torsion stiffening, in qualitative agreement with experimental findings. We argue that combining the twist-torque profiles for various stretching forces effectively results in a combined force-torque spectroscopy analysis, which may serve as a mechanical signature for a biological macromolecule.     

Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.     

Microhabitat contributes to microgeographic divergence in threespine stickleback

Since the New Synthesis, most migration-selection balance theory has predicted that there should be negligible differentiation over small spatial scales (relative to dispersal), because gene flow should erode any effect of divergent selection. Nevertheless, there are classic examples of microgeographic divergence, which theory suggests can arise under specific conditions: exceptionally strong selection, phenotypic plasticity in philopatric individuals, or nonrandom dispersal. Here, we present evidence of microgeographic morphological variation within lake and stream populations of threespine stickleback (Gasterosteus aculeatus). It seems reasonable to assume that a given lake or stream population of fish is well-mixed. However, we found this assumption to be untenable. We examined trap-to-trap variation in 34 morphological traits measured on stickleback from 16 lakes and 16 streams. Most traits varied appreciably among traps within populations. Both between-trap distance and microhabitat characteristics such as depth and substrate explained some of the within-population morphological variance. Microhabitat was also associated with genotype at particular loci but there was no genetic isolation by distance, implying that heritable habitat preferences may contribute to microgeographic variation. Our study adds to growing evidence that microgeographic divergence can occur across small spatial scales within individuals’ daily dispersal neighborhood where gene flow is expected to be strong.