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Superhydrophobic surfaces garner tremendous research attention due to their potential applications in the fields of oil–water separation membrane, self‐cleaning, and anti‐icing systems. However, the reported superhydrophobic materials that achieve low‐surface‐tension (<60 mN m−1) liquid repellency are typically prepared using perfluorinated compounds (PFCs), which pose significant risks to the natural environment and human health. Here a concept based on the excluded volume effect in polymer dilute solutions is explored to achieve a hydrophobic flexible polymer‐chain wrapping of multidimensional particles, which forms a nonadherent soft‐shell‐hard‐core structure and maintains the original structural integrity of particles. The prepared fluoro‐free super‐repellent multidimensional particles can be spray‐coated and dip‐coated on different substrates to prepare large‐scale superhydrophobic coatings, which exhibit excellent low‐surface‐tension (42.6−50 mN m−1) liquid repellency superior to the reported fluoro‐free superhydrophobic materials. Benefiting from the strong interaction of super‐repellent particles with flexible polymer chains, the superhydrophobic coatings show unexpected wear resistance and durability. This first demonstration of hydrophobic polymer‐wrapped particles for repelling low‐surface‐tension liquids provides insights into next‐generation fluoro‐free superhydrophobic materials. [ABSTRACT FROM AUTHOR]

Crowding environment has a significant impact on the folding and stability of protein in biological systems. In this work, we have used four different sizes of a molecular crowder, polyethylene glycol (PEG), to analyze the unfolding and refolding kinetics of an iLBP protein, CRABP I, using urea as chemical denaturant. In general, the stability of the native state of the protein is boosted by the presence of crowding agents in the solution. However, our findings show that not only the type of crowder but also the crowder size played a key role in the effects of excluded volume. In case of lower molecular weight of PEG (M.W. 400), even at 200 g/L concentration, only the viscosity effect is observed, whereas for higher molecular weight of PEG (M.W. 1000), both the viscosity effect and excluded volume effect are noticed, and even at a higher concentration (200 g/L) of PEG 1000, the excluded volume predominates over the viscosity effect. Using the transition state theory, we were also able to determine the free energies of activation for the unfolding and refolding studies from their respective rate constants. Additionally, MD simulation studies provide strong support for our experimental observation. Analysis of secondary structure propensity (SSP) reveals a marked decline in the presence of structural elements (β‐sheet, β‐bridge, turn, and α‐helix) from 81% to 43% over the 1 μs time scale unfolding MD simulation under 8 M urea conditions. Conversely, in a 200 ns refolding simulation, the rate of refolding notably increases at a concentration of 200 g/L PEG 1000. [ABSTRACT FROM AUTHOR]

In queuing systems, the dynamics of the excluded volume effect, which describes the movement of objects with a physical volume, are generally not considered. Recent studies have presented a theoretical formulation that incorporates the excluded volume effect to describe the delayed dynamics in M/M/1 queuing systems. We attempted to extend these results to the GI/G/1 queuing systems. Although the GI/G/1 queuing model is a stochastic process that cannot be completely described by a Markov chain, recent studies have indicated that it can be solved numerically using matrix geometry methods in discrete time under certain conditions. We propose a theoretical and analytical methodology in addition to approximation formulas for a discrete-time GI/G/1 queuing model that incorporates the excluded volume effect by utilizing the matrix-geometric method. The approximation methods were validated under single-server conditions for practical applications. Furthermore, using a theoretical perspective based on the aggregation method, we conducted numerical experiments on multiple servers to evaluate the performance of the discrete-time GI/PH/c queuing model. This approach adapted the excluded volume effect to the geometric matrix method. After testing it with a hyper-gamma distribution, we observed practical agreement, albeit to a limited extent.

Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition in eukaryotic cell culture. Single hyaluronic acid (HA) molecules have not induced a notable increase in the amount and rate of deposited ECM. Thus, herein we assessed the physicochemical properties and biological consequences in equine bone marrow mesenchymal stromal cell cultures of single and mixed HA molecules and correlated them to the most widely used MMC agents, the FicollⓇ cocktail (FC) and carrageenan (CR). Dynamic light scattering analysis revealed that all HA cocktails had significantly higher hydrodynamic radius than the FC and CR; the FC and the 0.5 mg/ml 100 kDa and 500 kDa single HA molecules had the highest charge; and, in general, all molecules had high polydispersity index. Biological analyses revealed that none of the MMC agents affected cell morphology and basic cell functions; in general, CR outperformed all other macromolecules in collagen type I and V deposition; FC, the individual HA molecules and the HA cocktails outperformed CR in collagen type III deposition; FC outperformed CR and the individual HA molecules and the HA cocktails outperformed their constituent HA molecules in collagen type IV deposition; FC and certain HA cocktails outperformed CR and constituent HA molecules in collagen type VI deposition; and all individual HA molecules outperformed FC and CR and the HA cocktails outperformed their constituent HA molecules in laminin deposition. With respect to tri-lineage analysis, CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. This work opens new avenues in mixed MMC in eukaryotic cell culture. Mixed macromolecular crowding (MMC) in eukaryotic cell culture is still under-investigated. Herein, single and double hyaluronic acid (HA) macromolecules, along with the traditional MMC agents FicollⓇ cocktail (FC) and carrageenan (CR), were used as MMC agents in equine mesenchymal stromal cell cultures. Biological analysis showed that none of the MMC agents affected cell morphology and basic cell functions. Protein deposition analysis made apparent that CR outperformed all other macromolecules in collagen type I and collagen type V deposition, whilst FC, the individual HA macromolecules and the HA cocktails outperformed CR in collagen type III deposition. Tri-lineage analysis revealed that CR and HA enhanced chondrogenesis and osteogenesis, whilst FC enhanced adipogenesis. These data illustrate that MMC agents are not inert macromolecules. [Display omitted] [ABSTRACT FROM AUTHOR]

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Crowding environment has a significant impact on the folding and stability of protein in biological systems. In this work, we have used four different sizes of a molecular crowder, polyethylene glycol (PEG), to analyze the unfolding and refolding kinetics of an iLBP protein, CRABP I, using urea as chemical denaturant. In general, the stability of the native state of the protein is boosted by the presence of crowding agents in the solution. However, our findings show that not only the type of crowder but also the crowder size played a key role in the effects of excluded volume. In case of lower molecular weight of PEG (M.W. 400), even at 200 g/L concentration, only the viscosity effect is observed, whereas for higher molecular weight of PEG (M.W. 1000), both the viscosity effect and excluded volume effect are noticed, and even at a higher concentration (200 g/L) of PEG 1000, the excluded volume predominates over the viscosity effect. Using the transition state theory, we were also able to determine the free energies of activation for the unfolding and refolding studies from their respective rate constants. Additionally, MD simulation studies provide strong support for our experimental observation. Analysis of secondary structure propensity (SSP) reveals a marked decline in the presence of structural elements (β-sheet, β-bridge, turn, and α-helix) from 81% to 43% over the 1 μs time scale unfolding MD simulation under 8 M urea conditions. Conversely, in a 200 ns refolding simulation, the rate of refolding notably increases at a concentration of 200 g/L PEG 1000., (© 2024 John Wiley & Sons Ltd.)

Observable properties of neutron stars are studied within a hadronic equation of state derived from the quark level. The effect of short-range repulsion is incorporated within the excluded volume framework. It is found that one can sustain neutron stars with masses as large as 2.2 M ⊙ even including hyperons in β equilibrium, while producing radii and tidal deformabilities consistent with current constraints. [ABSTRACT FROM AUTHOR]

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macromolecular crowding (traditional equine tenocyte culture), the highest number of significantly decreased readouts were observed for cells in foetal bovine serum, at passage 3, at day 5 and day 7 and without macromolecular crowding. Again, in comparison to traditional equine tenocyte culture, the highest number of significantly increased readouts were observed for cells in equine serum, at passage 3 and passage 6, at day 7 and with macromolecular crowding. Our data advocate the use of an allogeneic serum and tissue-specific extracellular matrix for effective expansion of equine tenocytes. [ABSTRACT FROM AUTHOR]

The paper reviews the scientific achievements reported by the school of polymers founded by Acad. Cristofor I. Simionescu in the field of pleistomers (ultrahigh molecular weight polymers). During the 80’s and 90’s, many efforts were devoted to their synthesis and characterization, and here Simionescu and coworkers have made a substantial contribution. Plasma-induced polymerization was successfully used for the first time to obtain very long macromolecular chains with unusual properties. By using different techniques, systematic studies were carried out in dilute solution and solid state, for determining the conformational characteristics of pleistomers. New thermodynamic approaches were tested and validated experimentally for the very high molecular weight range of polymers. Semidilute solutions of pleistomers were used as model fluids for testing the new rheological concepts for polymers in entangled state.

Successful in vitro culture of small-diameter tissue-engineered vascular grafts (TEVGs) requires rapid deposition of biomacromolecules secreted by vascular smooth muscle cells in a polyglycolic acid mesh scaffold's three-dimensional (3D) porous environment. However, common media have lower crowding conditions than in vivo tissue fluids. In addition, during the early stages of construction, most of the biomolecules secreted by the cells into the medium are lost, which negatively affects the TEVG culture process. In this study, we propose the use of macromolecular crowding (MMC) to enhance medium crowding to improve the deposition and self-assembly efficiency of major biomolecules in the early stages of TEVG culture. The addition of carrageenan significantly increased the degree of MMC in the culture medium without affecting cell viability, proliferation, and metabolic activity. Protein analysis demonstrated that the deposition of collagen types I and III and fibronectin increased significantly in the cell layers of two-dimensional and 3D smooth muscle cell cultures after the addition of a MMC agent. Collagen type I in the culture medium decreased significantly compared with that in the medium without a MMC agent. Scanning electron microscopy demonstrated that MMC agents considerably enhanced the formation of matrix protein structures during the early stages of 3D culture. Hence, MMC modifies the crowding degree of the culture medium, resulting in the rapid formation of numerous matrix proteins and fiber structures. Impact Statement Small-diameter tissue-engineered vascular grafts (TEVGs) are one of the most promising means of treating cardiovascular diseases; however, the in vitro construction of TEVGs has some limitations, such as slow deposition of extracellular matrix (ECM), long culture period, and poor mechanical properties. We hypothesized that macromolecular crowding can increase the crowding of the culture medium to construct a more bionic microenvironment, which enhances ECM deposition in the medium to the cell layer and reduces collagen loss, accelerating and enhancing TEVG culture and construction in vitro .

In multicellular organisms, the intracellular and extracellular spaces are considerably packed with a diverse range of macromolecular species. Yet, standard eukaryotic cell culture is performed in dilute, and deprived of macromolecules culture media, that barely imitate the density and complex macromolecular composition of tissues. Essentially, we drown cells in a sea of media and then expect them to perform physiologically. Herein, we argue the use of macromolecular crowding (MMC) in eukaryotic cell culture for regenerative medicine and drug discovery purposes. In eukaryotic cell culture, macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition. Negative charge and polydispersity are key modulators of enhanced and accelerated ECM deposition. MMC augments dynamic cell-ECM reciprocity in cell culture. We argue that MMC, by allowing the development of functional and truly 3D cell-assembled tissue surrogates, may be the missing link between developing and translating cell-based regenerative medicine and drug discovery concepts. [ABSTRACT FROM AUTHOR]

In spray dried emulsions, frequently milk proteins are used as interfacial active components and starch conversion products are added as matrix material at high concentrations. To characterize interfacial properties at the oil/water interface by commonly applied methods, low protein, and carbohydrate concentrations from 1 to 2% are usually analyzed. The impact of a higher concentration of starch conversion products was not investigated so far. Therefore, the formation and rheological properties of β-lactoglobulin (β-LG) stabilized films at the oil/water interface were investigated via short and long-time adsorption behavior using pendant drop tensiometry as well as dilatational and interfacial shear rheology. Suitability of the applied methods to the chosen samples with higher concentrations >1–2% was verified by calculation of selected key numbers like capillary number and by detailed reviewing of the results which is summarized further on as key indicators. It is hypothesized, that the increase in concentration via presence of starch conversion products will delay interfacial stabilization as a result of increased bulk viscosity with decreasing degree of degradation (dextrose equivalent) of the starch. Furthermore, this increase in concentration leads to more stable interfacial films due to thermodynamic incompatibility effects between protein and starch conversion products which results in increases of local protein concentration. Key indicators proved a general suitability of applied methods for the evaluation of the investigated samples. Moreover, results showed an increase in interfacial film stability and elastic properties alongside a decreased interfacial tension if starch conversion products were present in a high concentration. [ABSTRACT FROM AUTHOR]

The development of therapeutic regenerative medicine and accurate drug discovery cell-based products requires effective, with respect to obtaining sufficient numbers of viable, proliferative, and functional cell populations, cell expansion ex vivo. Unfortunately, traditional cell culture systems fail to recapitulate the multifaceted tissue milieu in vitro, resulting in cell phenotypic drift, loss of functionality, senescence, and apoptosis. Substrate-, environment-, and media-induced approaches are under intense investigation as a means to maintain cell phenotype and function while in culture. In this context, herein, the potential of macromolecular crowding, a biophysical phenomenon with considerable biological consequences, is discussed. [ABSTRACT FROM AUTHOR]

Cell-assembled tissue engineering strategies hold great potential in regenerative medicine, as three-dimensional tissue-like modules can be produced, even from a patient's own cells. However, the development of such implantable devices requires prolonged in vitro culture time, which is associated with cell phenotypic drift. Considering that the cells in vivo are subjected to numerous stimuli, multifactorial approaches are continuously gaining pace towards controlling cell fate during in vitro expansion. Herein, we assessed the synergistic effect of simultaneous and serial growth factor supplementation (insulin growth factor-1, platelet-derived growth factor ββ, growth differentiation factor 5 and transforming growth factor β3) to macromolecular crowding (carrageenan) in human tenocyte function; collagen synthesis and deposition; and gene expression. TGFβ3 supplementation (without/with carrageenan) induced the highest (among all groups) DNA content. In all cases, tenocyte proliferation was significantly increased as a function of time in culture, whilst metabolic activity was not affected. Carrageenan supplementation induced significantly higher collagen deposition than groups without carrageenan (without/with any growth factor). Of all the growth factors used, TGFβ3 induced the highest collagen deposition when used together with carrageenan in both simultaneous and serial fashion. At day 13, gene expression analysis revealed that TGFβ3 in serial supplementation to carrageenan upregulated the most and downregulated the least collagen- and tendon- related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis- and adipose- related trans-differentiation genes. Collectively, these data clearly advocate the beneficial effects of multifactorial approaches (in this case, growth factor and macromolecular crowding supplementation) in the development of functional cell-assembled tissue surrogates.

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macromolecular crowding (traditional equine tenocyte culture), the highest number of significantly decreased readouts were observed for cells in foetal bovine serum, at passage 3, at day 5 and day 7 and without macromolecular crowding. Again, in comparison to traditional equine tenocyte culture, the highest number of significantly increased readouts were observed for cells in equine serum, at passage 3 and passage 6, at day 7 and with macromolecular crowding. Our data advocate the use of an allogeneic serum and tissue-specific extracellular matrix for effective expansion of equine tenocytes.

Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer. [ABSTRACT FROM AUTHOR]

Within the crowded and complex environment of the cell, a protein experiences stabilizing excluded-volume effects and destabilizing quinary interactions with other proteins. Which of these prevail, needs to be determined on a case-by-case basis. PAPS synthases are dimeric and bifunctional enzymes, providing activated sulfate in the form of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) for sulfation reactions. The human PAPS synthases PAPSS1 and PAPSS2 differ significantly in their protein stability as PAPSS2 is a naturally fragile protein. PAPS synthases bind a series of nucleotide ligands and some of them markedly stabilize these proteins. PAPS synthases are of biomedical relevance as destabilizing point mutations give rise to several pathologies. Genetic defects in PAPSS2 have been linked to bone and cartilage malformations as well as a steroid sulfation defect. All this makes PAPS synthases ideal to study protein unfolding, ligand binding, and the stabilizing and destabilizing factors in their cellular environment. This review provides an overview on current concepts of protein folding and stability and links this with our current understanding of the different disease mechanisms of PAPSS2-related pathologies with perspectives for future research and application.

The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240–300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein.

Hadrons formed in heavy-ion collisions are not point-like objects, they cannot occupy too close space-time points. When the two bosons are too close to each other, their constituents start to mix and they cannot be considered as bosons subjected to Bose–Einstein statistics, this effect is called the excluded volume effect. We study the excluded volume effect on Hanbury Brown–Twiss (HBT) for the sources with various sizes. The effect on HBT was shown in out, side and long directions, and it is more obvious for the source with a narrow space-time distribution. The correlation functions for high transverse momenta are more suppressed by the excluded volume effect. [ABSTRACT FROM AUTHOR]

Graphical abstract Polydispersed and negatively charged, as opposed to mono-domain and neutral, crowders are more effective in enhancing and accelerating extracellular matrix deposition in human dermal fibroblast culture. Abstract Macromolecular crowding is a biophysical phenomenon that stems from the volume excluded by macromolecules, as they undergo steric repulsion and electrostatic interactions. The excluded volume depends on the shape, size, charge and polydispersity of the molecules. Although theoretical/computational models have been used to assess the influence of macromolecular crowding in biological media, real-time experiments are scarce. Herein, we evaluated the influence of hydrodynamic radius, charge and polydispersity of (a) various concentrations of different crowders (carrageenan, Ficoll™ and dextran sulphate); (b) various molecular weights of different crowders (70, 400 and 100 kDa of Ficoll™ and 10, 100 and 500 kDa of dextran sulphate) and (c) various cocktails of the same crowders (cocktails of various concentrations of different molecular weights Ficoll™ and dextran sulphate) on extracellular matrix deposition in human dermal fibroblast culture. The use of crowding cocktails with different molecular weight/concentrations of Ficoll™ or dextran sulphate molecules led to increased polydispersity and enhanced collagen type I deposition in comparison to their mono-domain counterparts. Carrageenan, however, induced the highest deposition of collagen type I due to its negative charge and inherent polydispersity. Our data contribute to a better understanding of the influence of the biophysical properties of the crowders on extracellular matrix deposition in vitro. Statement of Significance Macromolecular crowding is a biophysical phenomenon that accelerates and enhances extracellular matrix deposition in cell culture systems. Herein, we demonstrate that negatively charged and polydispersed macromolecules or cocktails of macromolecules, as opposed to neutral and monodomain macromolecules, induce highest extracellular matrix deposition in human dermal fibroblast cultures. [ABSTRACT FROM AUTHOR]

The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240-300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein. [ABSTRACT FROM AUTHOR]

The use of macromolecular crowding in the development of extracellular matrix-rich cell-assembled tissue equivalents is continuously gaining pace in regenerative engineering. Despite the significant advancements in the field, the optimal macromolecular crowder still remains elusive. Herein, the physicochemical properties of different concentrations of different molecular weights hyaluronic acid (HA) and their influence on equine adipose-derived stem cell cultures were assessed. Within the different concentrations and molecular weight HAs, the 10 mg/mL 100 kDa and 500 kDa HAs exhibited the highest negative charge and hydrodynamic radius, and the 10 mg/mL 100 kDa HA exhibited the lowest polydispersity index and the highest % fraction volume occupancy. Although HA had the potential to act as a macromolecular crowding agent, it did not outperform carrageenan and Ficoll®, the most widely used macromolecular crowding molecules, in enhanced and accelerated collagen I, collagen III and collagen IV deposition.

Introduction: As the available space inside the tooth becomes smaller because of the continuous formation of secondary dentin, the pulp may suffer from a physiologic static compression. The hypothesis: The dental pulp is lifelong under a static compression because of the continuous formation of secondary dentin, so that both cellular reduction and pulp fibrosis can also represent adaptive changes caused by the compression. Evaluation of the Hypothesis: The physiologic compression of the dental pulp can lead not only to the development of a hypoxia followed by cell death but also to the development of excluded volume effect, which helps convert the procollagen into collagen and form a collagen fiber network.

Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an "optimum" [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions. [ABSTRACT FROM AUTHOR]

We derived a model to predict the concentration profile, or the center-of-mass distribution, of rod-like particles near a wall under shear flows. Various excluded volume mechanisms of a rod near a wall for each Peclet number (a ratio of shear rate and diffusivity) regime were incorporated into the model through a steric factor concept. At low and moderate Peclet numbers, the steric factor is mainly determined by the ratio of the restricted/unrestricted rod orientation distributions. However, at high Peclet number, the ratio between the rod penetration time in a depletion layer and the Jeffery orbit frequency mainly affects the steric factor. The predicted concentration profiles showed a good agreement with the results from previous works. [ABSTRACT FROM AUTHOR]

The role of crowding agents on structure and activities of heme proteins has been established. Analysis of kinetic and thermodynamic parameters measured for CO-dissociation reaction of natively-folded carbonmonoxycytochrome c (NCO) and carbonmonoxymyoglobin (MbCO) at different [GdnHCl] or [Urea] in the presence of crowding agents (dextran 40, dextran 70 and ficoll 70) demonstrate that (i) at low denaturant concentrations, crowder presence enhances the denaturant-mediated restricted dynamics of NCO and MbCO, and (ii) at higher denaturant concentrations, large scale unfolding-fluctuations dominate the dynamics and inclusion of crowder counteracts the structural-fluctuations causing the unfolding of proteins. Thermodynamic analysis of thermal and urea-unfolding curves of cytochrome c (Cyt c ) and myoglobin (Mb) measured at different [GdnHCl] in presence of crowding agents reveals that crowder presence counterbalances and strengthens the destabilizing action of GdnHCl on stability of Cyt c and Mb, respectively. This study further demonstrates that the size, shape and concentration of crowding agent modulate the effect of crowder on denaturant-mediated dynamics and thermodynamic stability of heme proteins. [ABSTRACT FROM AUTHOR]

The thermal stability of apo α–lactalbumin (α–LA) and lysozyme was measured in the presence of mixtures of glucose, fructose, and galactose. Mixtures of these monosaccharides in the appropriate stoichiometric ratio were found to have a greater stabilizing effect on each of the two proteins than equal weight/volume concentrations of di- tri- and tetrasaccharides with identical subunit composition (sucrose, trehalose, raffinose, and stachyose). The excluded volume model for the effect of a single saccharide on the stability of a protein previously proposed by Beg et al. [Biochemistry 54 (2015) 3594] was extended to treat the case of saccharide mixtures. The extended model predicts quantitatively the stabilizing effect of all monosaccharide mixtures on α–LA and lysozyme reported here, as well as previously published results obtained for ribonuclease A [Biophys. Chem. 138 (2008) 120] to within experimental uncertainty. [ABSTRACT FROM AUTHOR]

Macromolecular crowding is known to affect protein folding, binding of small molecules, interaction with nucleic acids, enzymatic activity, protein-protein interactions, and protein aggregation. Although for a long time it was believed that the major mechanism of the action of crowded environments on structure, folding, thermodynamics, and function of a protein can be described in terms of the excluded volume effects, it is getting clear now that other factors originating from the presence of high concentrations of “inert” macromolecules in crowded solution should definitely be taken into account to draw a more complete picture of a protein in a crowded milieu. This review shows that in addition to the excluded volume effects important players of the crowded environments are viscosity, perturbed diffusion, direct physical interactions between the crowding agents and proteins, soft interactions, and, most importantly, the effects of crowders on solvent properties.

In this paper, we demonstrate that 20 μm microbeads can be preferentially assembled into substrate trenches of similar width by employing a polymer (depletant) that induces the depletion volume effect (depletion attraction). In previous works, we proved that this strategy is useful to assemble mesoscale parts in a site-specific manner. Here, we show that it is also applicable to assemble functional parts, such as fluorescent particles, into trenches engraved on the surface of two- and three-dimensional template components. A convenient advantage of this strategy is that it is independent of material properties for assembling mesoscale functional components into desired patterns.

Introduction: As the available space inside the tooth becomes smaller because of the continuous formation of secondary dentin, the pulp may suffer from a physiologic static compression. The hypothesis: The dental pulp is lifelong under a static compression because of the continuous formation of secondary dentin, so that both cellular reduction and pulp fibrosis can also represent adaptive changes caused by the compression. Evaluation of the Hypothesis: The physiologic compression of the dental pulp can lead not only to the development of a hypoxia followed by cell death but also to the development of excluded volume effect, which helps convert the procollagen into collagen and form a collagen fiber network. [ABSTRACT FROM AUTHOR]

The change of conformation of pepsin and trypsin in absence and presence of a high molecular crowding agent has been characterized using dynamic light scattering (DLS). Structural properties were investigated as a function of chemical denaturant concentrations, the guanidine hydrochloride (GdmCl). The results showed that Ficoll 400, macromolecular crowding, has a strong effect on the chemical denaturation process of these two proteins. The changes of measured hydrodynamic radius are attributed to the enhancement effect of the crowder agent due to the excluded volume effects. The data proved that the large size of a macromolecular crowder plays a crucial role on the conformation of a protein in its unfolded states. The values of interactions Parameter k d of complex particles and a number of proteins n pr attached on the Ficoll 400 measured in different GdmCl concentrations. The effect of aging on the structure of complex are studied by small angle light scattering (SALS). [ABSTRACT FROM AUTHOR]

Background The environment inside cells in which proteins fold and function are quite different from that of the dilute buffer solutions often used during in vitro experiments. The presence of large amounts of macromolecules of varying shapes, sizes and compositions makes the intracellular milieu extremely crowded. Scope of review The overall concentration of macromolecules ranges from 50 to 400 g l − 1 , and they occupy 10–40% of the total cellular volume. These differences in solvent conditions and the level of crowdedness resulting in excluded volume effects can have significant consequences on proteins' biophysical properties. A question that arises is: how important is it to examine the roles of shape, size and composition of macromolecular crowders in altering the biological properties of proteins? This review article aims at focusing, gathering and summarizing all of the research investigations done by means of in vitro and in silico approaches taking into account the size-dependent influence of the crowders on proteins' properties. Major conclusions Altogether, the internal architecture of macromolecular crowding environment including size, shape and concentration of crowders, appears to be playing an extremely important role in causing changes in the biological processes. Most often the small sized crowders have been found more effective crowding agents. However, thermodynamic stability, structure and functional activity of proteins have been governed by volume exclusion as well as soft (chemical) interactions. General significance The article provides an understanding of importance of internal architecture of the cellular environment in altering the biophysical properties of proteins. [ABSTRACT FROM AUTHOR]

The excluded volume effect, or depletion attraction, is the phenomenon describing induced aggregation of colloidal particles suspended in a densely crowded macromolecule solution. In this work, we attempted to utilize this effect for the self-assembly of artificially manufactured microcomponents on a 10–100 μm scale. The bonding energy does not originate from the nature of the surfaces, but is generated by an increase of the translational entropy of macromolecules in solution. Thus, simple immersion of the microcomponents in the macromolecule solution allowed us to observe their assembly, based on shape complementarity, to minimize the free energy of the system. However, it became apparent that elaborate design is required for specific bonding between complementary shapes, in addition to merely increasing the contact surface area. [ABSTRACT FROM AUTHOR]

We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5–5.5). In terms of drug targeting, the question is whether c-MYC promoter sequence i-motifs will exist in the nucleus at neutral pH. In this work, we have investigated the stability of a mutant c-MYC i-motif in solutions containing a molecular crowding agent. The crowded nuclear environment was modeled by the addition of up to 40% w/w polyethylene glycols having molecular weights up to 12,000 g/mol. CD and DSC were used to establish the presence and stability of c-MYC i-motifs in buffer solutions over the pH range 4 to 7. We have shown that the c-MYC i-motif can exist as a stable structure at pH values as high as 6.7 in crowded solutions. Generic dielectric constant effects, e.g., a shift in the pKa of cytosine by more than 2 units (e.g., 4.8 to 7.0), or the formation of non-specific PEG/DNA complexes appear to contribute insignificantly to i-motif stabilization. Molecular crowding, largely an excluded volume effect of added PEG, having a molecular weight in excess of 1,000 g/mol, appears to be responsible for stabilizing the more compact i-motif over the random coil at higher pH values.

In vitro studies on macromolecules, like proteins and nucleic acids, are mostly carried out in highly diluted systems where the molecules are studied under artificial conditions. These experimental conditions are optimized for both the system under investigation and the technique used. However, these conditions often do not reflect the in vivo situation and are therefore inappropriate for a reliable prediction of the native behavior of the molecules and their interactions under in vivo conditions. The intracellular environment is packed with cosolutes (macromolecules, metabolites, etc.) that create 'macromolecular crowding'. The addition of natural or synthetic macromolecules to the sample solution enables crowding to be mimicked. In this surrounding most of the studied biomolecules show a more compact structure, an increased activity, and a decrease of salt requirement for structure formation and function. Herein, we refer to a collection of examples for proteins and nucleic acids and their interactions in crowding environments and present in detail the effect of cosolutes on RNA folding and activity using a group II intron ribozyme as an example.

The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly mimicked by traditional physiological conditions, where low concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution containing high concentrations of various polymers that serve as model crowding agents. An expected outcome of the presence of such macromolecular crowding agents is their ability to increase conformational stability of a globular protein due to the excluded volume effects. In line with this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas the chemical nature of a macromolecular crowder should not play a role in its ability to modulate conformational properties. In this study, the effects of different crowding agents (polyethylene glycols (PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70) on the spectral properties and unfolding-refolding processes of the super-folder green fluorescent protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that, in addition to the expected excluded volume effects, there are some changes in the solvent properties. [ABSTRACT FROM AUTHOR]

A theoretical model for the translocation process of biomacromolecule is developed based on the self-consistent field theory (SCFT), where the biomacromolecule is regarded as a self-avoiding polymer chain actuated by the external potential. In this theoretical model, the external potential, the Coulomb electrostatic potential of the charged ions (the electrolyte effect), and the attractive interaction between the polymer and the nanopore (the excluded volume effect) are all considered, which have effects on the free energy landscape and conformation entropy during the translocation stage. The result shows that the entropy barrier of the polymer in the solution with high valence electrolyte is much larger than that with low valence electrolyte under the same condition, leading to that the translocation time of the DNA molecules in the solution increases when the valence electrolyte increases. In addition, the attractive interaction between the polymer and the nanopore increases the free energy of the polymer, which means that the probability of the translocation through the nanopore increases. The average translocation time decreases when the excluded volume effect parameter increases. The electrolyte effect can prolong the average translocation time. The simulation results agree well with the available experimental results. [ABSTRACT FROM AUTHOR]

Myoglobin (Mb) undergoes pronounced heme loss under denaturing conditions wherein the proximal histidine gets protonated. Our data show that macromolecular crowding agents (both synthetic and protein based) can appreciably influence the extent of heme retention in Mb. Interestingly, glucose and sucrose, the monomeric constituents of dextran and ficoll-based crowders were much more effective in preventing heme dissociation of Mb, albeit, at much higher concentrations. The protein crowders BSA and lysozyme show very interesting results with BSA bringing about the maximum heme retention amongst all the crowding agents used while lysozyme induced heme dissociation even in the native state of Mb. The stark difference that these protein crowders exhibit when interacting with the heme protein is a testament to the varied interaction potentials that a test protein might be exposed to in the physiological (crowded) milieu. [ABSTRACT FROM AUTHOR]

Carbonmonoxycytochrome c refolds to a native-like compact state (NCO-state), where the non-native Fe 2 + -CO interaction persists. Structural and molecular properties extracted from CD, fluorescence and NMR experiments reveal that the NCO-state shows the generic properties of molten globules. Slow thermal-dissociation of CO transforms the NCO-state to native-state (N-state), where the native Fe 2 + –M80 bond recovers. To determine the role of crowding agents and salt ions on the structural-fluctuation of NCO, the kinetic and thermodynamic parameters for CO-dissociation from NCO (NCO → N + CO) were measured at varying concentrations of crowding agents (dextran 70, dextran 40, ficoll 70) and salt ions (anion: ClO 4 − , I − , Br − , NO 3 − , Cl − ; cation: NH 4 + , K + , Na + ). As [crowding agent] or [ion] is increased, the rate coefficient of CO-dissociation ( k diss ) decreases exponentially. Furthermore, the extent of decrease in k diss is found to be dependent on (i) size, charge density and charge dispersion of the ion, and (ii) size, shape, and viscosity of the crowding agent. [ABSTRACT FROM AUTHOR]

A ternary self-condensing vinyl polymerization (SCVP) system consisting of monomers, inimers and core initiators is studied by the principle of statistical mechanics. Starting from partition functions for polymerization that constructed from two different viewpoints, explicit expressions of the size distribution function (SDF), equilibrium free energy, and the law of mass action are obtained. For two types of hyperbranched polymers (HBPs) formed in the system, the first, the second, and the third mean-square radii of gyration (MSRG) are presented under various conditions. Furthermore, the solvent effect and excluded volume effect are taken into account in the calculation of the MSRG. With these results, the effects of monomers, inimers and core initiators on the spatial dimension of HBPs are discussed. It is shown that the competition between polymers with and without a core in the growing process results in a significant influence on the average properties of the system. [ABSTRACT FROM AUTHOR]

The cellular environment is crowded with different kinds of molecules with varying sizes, shapes and compositions. Most of the experiments studying the nature and behaviour of a protein have been done on the isolated protein in dilute buffer solutions which actually do not imitate the in vivo situation. To understand the consequences of such crowded environment, we investigated the effect of macromolecular crowding on the stability and activity of hen egg white lysozyme. Two crowding agents, dextran 70 and ficoll 70 which have different shapes and composition, have been employed in this study. To mimic the cellular condition from physiological point of view, the effect of mixtures of both the crowding agents has been also studied. The results indicate that owing to volume exclusion, lysozyme is stabilized while its activity decays with the increasing concentration of both the crowders elucidating the hypothesis of stability-activity trade-off. Mixed macromolecular crowding exerts greater effect than the sum of constituent crowding agents (dextran 70 and ficoll 70). [ABSTRACT FROM AUTHOR]

Background Cellular interior is known to be densely crowded due to the presence of soluble and insoluble macromolecules, which altogether occupy ~ 40% of the total cellular volume. This results in altered biological properties of macromolecules. Scope of Review Macromolecular crowding is observed to have both positive and negative effects on protein folding, structure, stability and function. Significant data has been accumulated so far on both the aspects. However, most of the review articles so far have focused on the positive aspect of macromolecular crowding and not much attention has been paid on the deleterious aspect of crowding on macromolecules. In order to have a complete knowledge of the effect of macromolecular crowding on proteins and enzymes, it is important to look into both the aspects of crowding to determine its precise role under physiological conditions. To fill the gap in the understanding of the effect of macromolecular crowding on proteins and enzymes, this review article focuses on the deleterious influence of crowding on macromolecules. Major Conclusions Macromolecular crowding is not always good but also has several deleterious effects on various macromolecular properties. Taken together, the properties of biological macromolecules in vivo appears to be finely regulated by the nature and level of the intracellular crowdedness in order to perform their biological functions appropriately. General Significance The information provided here gives an understanding of the role played by the nature and level of cellular crowdedness in intensifying and/or alleviating the burden of various proteopathies. [ABSTRACT FROM AUTHOR]

Scaffold-free in vitro organogenesis exploits the innate ability of cells to synthesise and deposit their own extracellular matrix to fabricate tissue-like assemblies. Unfortunately, cell-assembled tissue engineered concepts require prolonged ex vivo culture periods of very high cell numbers for the development of a borderline three-dimensional implantable device, which are associated with phenotypic drift and high manufacturing costs, thus, hindering their clinical translation and commercialisation. Herein, we report the accelerated (10 days) development of a truly three-dimensional (338.1 ± 42.9 μm) scaffold-free tissue equivalent that promotes fast wound healing and induces formation of neotissue composed of mature collagen fibres, using human adipose derived stem cells seeded at only 50,000 cells/cm2 on an poly (N -isopropylacrylamide-co- N - tert -butylacrylamide (PNIPAM86-NTBA14) temperature-responsive electrospun scaffold and grown under macromolecular crowding conditions (50 μg/ml carrageenan). Our data pave the path for a new era in scaffold-free regenerative medicine. Human adipose derived stem cells seeded on temperature-responsive electrospun scaffolds under macromolecular crowding conditions allowed the accelerated development of truly three-dimensional cell assembled micro tissues, the functionality of which was demonstrated in a wound healing model. [Display omitted] [ABSTRACT FROM AUTHOR]

Although cell-derived matrices are at the forefront of scientific research and technological innovation for the development of in vitro tumour models, their two-dimensional structure and low extracellular matrix composition restrict their capacity to accurately predict toxicity of candidate molecules. Herein, we assessed the potential of macromolecular crowding (a biophysical phenomenon that significantly enhances and accelerates extracellular matrix deposition, resulting in three-dimensional tissue surrogates) in improving cell-derived matrices in vitro tumour models. Among the various decellularisation protocols assessed (NH 4 OH, DOC, SDS/EDTA, NP40), the NP40 appeared to be the most effective in removing cellular matter and the least destructive to the deposited matrix. Among the various cell types (mammary, skin, lung fibroblasts) used to produce the cell-derived matrices, the mammary fibroblast derived matrices produced under macromolecular crowding conditions and decellularised with NP40 resulted in significant increase in focal adhesion molecules, matrix metalloproteinases and proinflammatory cytokines, when seeded with MDA-MB-231 cells. Further, macromolecular crowding derived matrices significantly increased doxorubicin resistance and reduced the impact of intracellular reactive oxygen species mediated cell death. Collectively our data clearly illustrate the potential of macromolecular crowding in the development of cell-derived matrices-based in vitro tumour models that more accurately resemble the tumour microenvironment. NP40 decellularised mammary fibroblast derived matrices produced under macromolecular crowding conditions more accurately resemble the tumour microenvironment, thus enabling the development of more predictive drug screening models. [Display omitted] [ABSTRACT FROM AUTHOR]