Search Results (52)

Catalytic coacervates, or droplet reactors, represent a forefront research area in chemistry and materials science. Despite advancements in this field, challenges persist in achieving liquid–liquid phase separation (LLPS) droplet compartmentalization and site-specific reactant recruitment/localization for reaction catalysis, similar to those within biological systems. Herein, we describe the catalytic coacervates for aqueous photo-RAFT in dilution, focusing on the site-specific recruitment/localization of ionized monomer with the aid of macromolecular crowding and confinement. Cooperative hydrogen-bonded interpolymer complexation (IPC) of imidazolium-copolymers initiates the ion-cluster formation. Further hierarchical inter-cluster complexation (ICC) leads to the LLPS droplet compartmentalization into charged dense-phase and neutral dilute-phase compartments. Site-specific recruitment and localization of the oppositely charged monomer into dense-phase compartments are achieved by salt-bridging molecular recognition. “Substantial DMA-dilution” (that is, macromolecular crowding) results in sustainable dense-phase catalytic sites within dilute-phase crowding surroundings, enabling reaction catalysis in dilution (<2% w/w monomer) to 97% conversion in 12 min. These findings underscore the key roles of macromolecular crowding and confinement in the tailorable LLPS droplet compartmentalization and also the site-specific reactant recruitment/localization essential for enzyme reaction catalysis, and provide practical guidelines for creating catalytic coacervates towards lifelike reaction functions. Full article

A brief summary of the effect of nonspecific interactions upon chemical equilibria in solutions containing a high total concentration of macromolecular solutes comparable to that found in biological fluid media is presented. Analyses of experimental measurements permitting relatively direct quantitation of the free energy of nonspecific intermolecular interaction in solutions of one or two macrosolutes are described, and a table listing published experimental studies of both homo- and hetero-interactions is provided. Methods for calculating the free energy of nonspecific interaction via theory and computer simulation are described. Recommendations for further progress in both measurement and calculation of interaction free energies are presented. Full article

Recent advances in molecular dynamics (MD) simulations and the introduction of artificial intelligence (AI) have resulted in a significant increase in accuracy for structure prediction. However, the cell is a highly crowded environment consisting of various macromolecules, such as proteins and nucleic acids. The macromolecular crowding and solution conditions, such as temperature, ion concentration, and the presence of crowders, significantly influence the molecular interactions between and structural changes in proteins and nucleic acids. In this study, we investigate the presence of crowders and their effect on the melting of DNA molecules by analyzing melting profiles of short and long heterogeneous DNA duplexes. In particular, we examine how multiple inert crowders, randomly distributed along long DNA chains, influence DNA melting. We find that the presence of crowders stabilizes double-stranded DNA (dsDNA), with this effect being more pronounced in short DNA duplexes. These findings complement in vitro observations and improve our understanding of dsDNA in cell-like environments. Full article

This study aimed to investigate whether the water-soluble pharmaceutical form of phosphatidylcholine nanoparticles (wPC) stimulated the catalytic activity of CYP enzymes 2C9 and 2D6. We have shown that electroenzymatic CYP2C9 catalysis to nonsteroidal anti-inflammatory drug naproxen as a substrate was enhanced from 100% to 155% in the presence of wPC in media. Electroenzymatic CYP2D6 activity in the presence of the adrenoceptor-blocking agent bisoprolol as a substrate was elevated significantly from 100% to 144% when wPC was added to potassium phosphate buffer solution. These results indicate the ability of wPC in the form of the phospholipid ultra-small nanoparticles to work as a membrane additive and crowding agent to accelerate the electroenzymatic reactions of cytochrome P450. Full article

The 1-anilino-8-naphthalenesulfonate (ANS) fluorescent dye is widely used in protein folding studies due to the significant increase in its fluorescence quantum yield upon binding to protein hydrophobic regions that become accessible during protein unfolding. However, when modeling cellular macromolecular crowding conditions in protein folding experiments in vitro using crowding agents with guanidine hydrochloride (GdnHCl) as the denaturant, the observed changes in ANS spectral characteristics require careful consideration. This study demonstrates that crowding agents can form clusters that interact differently with ANS. Furthermore, GdnHCl can disrupt these clusters and directly affect the ANS spectral characteristics. A model for the interaction between GdnHCl, crowders, and ANS is proposed. Using bovine serum albumin (BSA) as a model protein, the limitations of using ANS for studying conformational transitions induced by GdnHCl in the presence of crowding agents are demonstrated. Full article

Despite the promising potential of cell-based therapies developed using tissue engineering techniques to treat a wide range of diseases, including limbal stem cell deficiency (LSCD), which leads to corneal blindness, their commercialization remains constrained. This is primarily attributable to the limited cell sources, the use of non-standardizable, unscalable, and unsustainable techniques, and the extended manufacturing processes required to produce transplantable tissue-like surrogates. Herein, we present the first demonstration of the potential of a novel approach combining collagen films (CF), hyaluronic acid (HA), human telomerase-immortalized limbal epithelial stem cells (T-LESCs), and macromolecular crowding (MMC) to develop innovative biomimetic substrates for limbal epithelial stem cells (LESCs). The initial step involved the fabrication and characterization of CF and CF enriched with HA (CF-HA). Subsequently, T-LESCs were seeded on CF, CF-HA, and tissue culture plastic (TCP). Thereafter, the effect of these matrices on basic cellular function and tissue-specific extracellular matrix (ECM) deposition with or without MMC was evaluated. The viability and metabolic activity of cells cultured on CF, CF-HA, and TCP were found to be similar, while CF-HA induced the highest (p < 0.05) cell proliferation. It is notable that CF and HA induced cell growth, whereas MMC increased (p < 0.05) the deposition of collagen IV, fibronectin, and laminin in the T-LESC culture. The data highlight the potential of, in particular, immortalized cells and MMC for the development of biomimetic cell culture substrates, which could be utilized in ocular surface reconstruction following further in vitro, in vivo, and clinical validation of the approach. Full article

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity—the condensates in oral epithelium disassembled within 1–2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4–7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic “macromolecular crowding”) confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm—the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid–liquid phase separation (LLPS) in cells of the oral mucosa. Full article

Even though tissue-engineered medicines are under intense academic, clinical, and commercial investigation, only a handful of products have been commercialised, primarily due to the costs associated with their prolonged manufacturing. While macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition in eukaryotic cell culture, possibly offering a solution in this procrastinating tissue-engineered medicine development, there is still no widely accepted macromolecular crowding agent. With these in mind, we herein assessed the potential of gum Arabic, gum gellan, gum karaya, and gum xanthan as macromolecular crowding agents in WS1 skin fibroblast cultures (no macromolecular crowding and carrageenan were used as a control). Dynamic light scattering analysis revealed that all macromolecules had negative charge and were polydispersed. None of the macromolecules affected basic cellular function. At day 7 (the longest time point assessed), gel electrophoresis analysis revealed that all macromolecules significantly increased collagen type I deposition in comparison to the non-macromolecular crowding group. Also at day 7, immunofluorescence analysis revealed that carrageenan; the 50 µg/mL, 75 µg/mL, and 100 µg/mL gum gellan; and the 500 µg/mL and 1000 µg/mL gum xanthan significantly increased both collagen type I and collagen type III deposition and only carrageenan significantly increased collagen type V deposition, all in comparison to the non-macromolecular crowding group at the respective time point. This preliminary study demonstrates the potential of gums as macromolecular crowding agents, but more detailed biological studies are needed to fully exploit their potential in the development of tissue-engineered medicines. Full article

Immunosensors based on field-effect transistors with nanowire channels (NWFETs) provide fast and real-time detection of a variety of biomarkers without the need for additional labels. The key feature of the developed immunosensor is the coating of silicon NWs with multilayers of polyelectrolytes (polyethylenimine (PEI) and polystyrene sulfonate (PSS)). By causing a macromolecular crowding effect, it ensures the “soft fixation” of the antibodies into the 3-D matrix of the oppositely charged layers. We investigated the interaction of prostate-specific antigen (PSA), a biomarker of prostate cancer, and antibodies adsorbed in the PEI and PSS matrix. In order to visualize the formation of immune complexes between polyelectrolyte layers using SEM and AFM techniques, we employed a second clone of antibodies labeled with gold nanoparticles. PSA was able to penetrate the matrix and concentrate close to the surface layer, which is crucial for its detection on the nanowires. Additionally, this provides the optimal orientation of the antibodies’ active centers for interacting with the antigen and improves their mobility. NWFETs were fabricated from SOI material using high-resolution e-beam lithography, thin film vacuum deposition, and reactive-ion etching processes. The immunosensor was characterized by a high sensitivity to pH (71 mV/pH) and an ultra-low limit of detection (LOD) of 0.04 fg/mL for PSA. The response of the immunosensor takes less than a minute, and the measurement is carried out in real time. This approach seems promising for further investigation of its applicability for early screening of prostate cancer and POC systems. Full article

The cellular environment is highly crowded, with up to 40% of the volume fraction of the cell occupied by various macromolecules. Most laboratory experiments take place in dilute buffer solutions; by adding various synthetic or organic macromolecules, researchers have begun to bridge the gap between in vitro and in vivo measurements. This is a review of the reported effects of macromolecular crowding on the compaction and extension of DNA, the effect of macromolecular crowding on DNA kinetics, and protein-DNA interactions. Theoretical models related to macromolecular crowding and DNA are briefly reviewed. Gaps in the literature, including the use of biologically relevant crowders, simultaneous use of multi-sized crowders, empirical connections between macromolecular crowding and liquid–liquid phase separation of nucleic materials are discussed. Full article

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division. Full article

Connective tissue models grown from cell monolayers can be instrumental in a variety of biomedical fields such as drug screening, wound healing, and regenerative engineering. However, while connective tissues contain abundant fibrillar collagen, achieving a sufficient assembly and retention of fibrillar collagen in vitro is challenging. Unlike the dilute cell culture environment, the body’s environment is characterized by a high density of soluble macromolecules (crowding) and macromolecular networks (confinement), which contribute to extracellular matrix (ECM) assembly in vivo. Consequently, macromolecular crowding (MMC) has been successfully used to enhance the processing of type I procollagen, leading to significant increases in fibrillar collagen assembly and accumulation during in vitro culture of a variety of cell types. In this study, we developed a combination approach using a carrageenan hydrogel, which released soluble macromolecules and served as a confinement barrier. We first evaluated the local carrageenan release and then confirmed the effectiveness of this combination approach on collagen accumulation by the human MG-63 bone cell line. Additionally, computational modeling of oxygen and glucose transport within the culture system showed no negative effects of the hydrogel and its releasates on cell viability. Full article

Pseudo isocyanine chloride (PIC) has been identified in a preceding work as a sensor suited to probe macromolecular crowding both in test tubes with solutions of synthetic crowding agents and in HeLa cells as a representative of living systems. The sensing is based on a delicate response of the self-assembly pattern of PIC towards a variation in macromolecular crowding. Based on a suitable selection of criteria established in the present study, four additional cyanine dyestuffs (TDBC, S071, S2275, and PCYN) were scrutinized for their ability to act as such a sensor, and the results were compared with the corresponding performance of PIC. UV-VIS and fluorescence spectroscopy were applied to investigate the photo-physical properties of the four candidates and, if possible, light scattering was used to characterize the self-assembly of the dyestuffs in solution. Finally, HeLa cells were exposed to solutions of the most promising candidates in order to analyze their ability to infiltrate the cells and to self-assemble therein. None of the dyestuff candidates turned out to be as similarly promising in probing crowding effects in cells as PIC turned out to be. S0271 and S2275 are at least stable enough and meet the photophysical requirements necessary to act as sensors responding to changes in macromolecular crowding. Full article

This article reviews the state of the art of the studies on charge regulation (CR) effects in flexible weak polyelectrolytes (FWPE). The characteristic of FWPE is the strong coupling of ionization and conformational degrees of freedom. After introducing the necessary fundamental concepts, some unconventional aspects of the the physical chemistry of FWPE are discussed. These aspects are: (i) the extension of statistical mechanics techniques to include ionization equilibria and, in particular, the use of the recently proposed Site Binding-Rotational Isomeric State (SBRIS) model, which allows the calculation of ionization and conformational properties on the same foot; (ii) the recent progresses in the inclusion of proton equilibria in computer simulations; (iii) the possibility of mechanically induced CR in the stretching of FWPE; (iv) the non-trivial adsorption of FWPE on ionized surfaces with the same charge sign as the PE (the so-called “wrong side” of the isoelectric point); (v) the influence of macromolecular crowding on CR. Full article

A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water–cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site. Full article