Macromolecular Crowding Research Articles

Switch of cell migration modes orchestrated by changes of three-dimensional lamellipodium structure and intracellular diffusion

Cell migration plays important roles in many biological processes, but how migrating cells orchestrate intracellular molecules and subcellular structures to regulate their speed and direction is still not clear. Here, by characterizing the intracellular diffusion and the three-dimensional lamellipodium structures of fish keratocyte cells, we observe a strong positive correlation between the intracellular diffusion and cell migration speed and, more importantly, discover a switching of cell migration modes with reversible intracellular diffusion variation and lamellipodium structure deformation. Distinct from the normal fast mode, cells migrating in the newly-found slow mode have a deformed lamellipodium with swollen-up front and thinned-down rear, reduced intracellular diffusion and compartmentalized macromolecule distribution in the lamellipodium. Furthermore, in turning cells, both lamellipodium structure and intracellular diffusion dynamics are also changed, with left-right symmetry breaking. We propose a mechanism involving the front-localized actin polymerization and increased molecular crowding in the lamellipodium to explain how cells spatiotemporally coordinate the intracellular diffusion dynamics and the lamellipodium structure in regulating their migrations.

Dilution induced spectral and structural changes in allophycocyanin

Allophycocyanin (APC) is a key molecule of the light harvesting complex - phycobilisome of Cyanobacteria and Red Algae. Its bright color and fluorescent nature make it a suitable molecular for biomedical applications. Here, we report the dilution induced changes in the spectral and structural properties of APC, isolated from marine cyanobacterium, Phormidium sp. A09DM. We observe measurable blue shift in the light absorption and fluorescence emission properties of APC upon dilution. The blue shift in spectral properties is originated from the disruption of hydrophobic interactions and systematic dissociation of the APC into its building blocks. APC, a trimer of αβ-heterodimer, dissociates into monomer and further into individual subunits upon dilution. The observed dilution induced spectral and structural changes can be moderated in the presence of physiological osmolytes with the moderation strength in the order, Sucrose > Trehalose > Betaine = Potassium Phosphate buffer > Glycine. Our results conclude that the spectral properties of APC is a function of osmolytes concentrations, which makes it a potential candidate to be explored as the molecular probe for measuring the molecular crowding.

Minimal Coarse-Grained Model for Immunoglobulin G: Diffusion and Binding under Crowding.

Immunoglobulin G (IgG) is the most common type of antibody found in blood and extracellular fluids and plays an essential role in our immune response. However, studies of the dynamics and reaction kinetics of IgG-antigen binding under physiological crowding conditions are scarce. Herein, we develop a coarse-grained model of IgG consisting of only six beads that we find minimal for a coarse representation of IgG's shape and a decent reproduction of its flexibility and diffusion properties measured experimentally. Using this model in Brownian dynamics simulations, we find that macromolecular crowding affects only slightly the IgG's flexibility, as described by the distribution of angles between the IgG's arms and stem. Our simulations indicate that, contrary to expectations, crowders slow down the translational diffusion of an IgG less strongly than they do for a smaller Ficoll 70, which we relate to the IgG's conformational size changes induced by crowding. We also find that crowders affect the binding kinetics by decreasing the rate of the first binding step and enhancing the second binding step.

Micro- and nanochamber array system for single enzyme assays

Arrays of small reaction containers, ranging from 624 femtoliters (10–15 L) to 270 attoliters (10–18 L), for capturing a single enzyme molecule and measuring the activity were developed along with a new reversible sealing system based on a pneumatic valve actuator made of polydimethylsiloxane (PDMS). The valve was actuated by PBS solution, effectively preventing evaporation of the solution from the micro- and nanochambers and allowing the assay to be performed over a long period of time. The hydrolysis rates of β-D-galactosidase (β-gal), kcat, were decreased according to the decrease of the chamber size, and the overall tendency seems to be symmetrically related to the specific surface area of the chambers even under the prevented condition of non-specific adsorption. The spatial localization of the protons in the chambers, which might could affect the dissociation state of the proteins, was also investigated to explain the decrease in the hydrolysis rate. The developed chamber system developed here may be useful for artificially reproducing the confined intracellular environment and molecular crowding conditions.

Carrageenan as a macromolecular crowding agent in human umbilical cord derived mesenchymal stromal cell culture

Cell sheet tissue engineering requires prolonged in vitro culture for the development of implantable devices. Unfortunately, lengthy in vitro culture is associated with cell phenotype loss and substantially higher cost of goods, which collectively hinder clinical translation and commercialisation of tissue engineered medicines. Although macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition, whilst maintaining cellular phenotype, the optimal macromolecular crowding agent still remains elusive. Herein, we evaluated the biophysical properties of seven different carrageenan molecules at five different concentrations and their effect on human umbilical cord-derived mesenchymal stromal cell morphology, viability, metabolic activity, proliferation, extracellular matrix deposition and surface marker expression. All types of carrageenan (CR) assessed demonstrated a hydrodynamic radius increase as a function of increasing concentration; high polydispersity; and negative charge. Two iota CRs were excluded from further analysis due to poor solubility in cell culture. Among the remaining five carrageenans, the lambda medium viscosity type at concentrations of 10 and 50 μg/ml did not affect cell morphology, viability, metabolic activity, proliferation and expression of surface markers and significantly increased the deposition of collagen types I, III and IV, fibronectin and laminin. Our data highlight the potential of lambda medium viscosity carrageenan as a macromolecular crowding agent for the accelerated development of functional tissue engineered medicines.

Staging a comeback? The influencing mechanism of tourist crowding perception on adaptive behavior

An increasing number of studies have recently elucidated the serious negative effects of tourist crowding. However, how tourists cope with crowding and adopt adaptive responses remains unclear. Thus, this study qualitatively identified the mechanism of tourist crowding perception of adaptive behavior from interviewees and then quantitatively validated the mechanism through the mediation of tourist fatigue and negative emotions, as well as the moderation of peer emotion contagion. Tourists were more likely to return to a destination and engage in temporal replacement behavior through the mediating effect of tourist fatigue, but it was also found that peer emotional contagion interferes with two paths that explain how tourist crowding perception influences tourist emotion change or state variation, impacting adaptive behavior responses. Consequently, we recommend diverting tourists, focusing on alleviating fatigue and negative emotions and channeling the peers’ negative emotions to mitigate the negative impact of tourist crowding.

Molecular Crowding Facilitates Ribozyme-Catalyzed RNA Assembly.

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg2+, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity at low concentrations of Mg2+. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lower the Mg2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. Additionally, we show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally in the emergence of RNA-based cellular life.

Cortical Reorganization After Optical Alignment in Strabismic Patients Outside of Critical Period.

To measure visual crowding, an essential bottleneck on object recognition and reliable psychophysical index of cortex organization, in older children and adults with horizontal concomitant strabismus before and after strabismus surgery. Using real-time eye tracking to ensure gaze-contingent display, we examined the peripheral visual crowding effects in older children and adults with horizontal concomitant strabismus but without amblyopia before and after strabismus surgery. Patients were asked to discriminate the orientation of the central tumbling E target letter with flankers arranged along the radial or tangential axis in the nasal or temporal hemifield at different eccentricities (5° or 10°). The critical spacing value, which is the minimum space between the target and the flankers required for correct discrimination, was obtained for comparisons before and after strabismus surgery. Twelve individuals with exotropia (6 males, 21.75 ± 7.29 years, mean ± SD) and 15 individuals with esotropia (6 males, 24.13 ± 5.96 years) participated in this study. We found that strabismic individuals showed significantly larger critical spacing with nasotemporal asymmetry along the radial axis that related to the strabismus pattern, with exotropes exhibiting stronger temporal field crowding and esotropes exhibiting stronger nasal field crowding before surgical alignment. After surgery, the critical spacing was reduced and rebalanced between the nasal and temporal hemifields. Furthermore, the postoperative recovery of stereopsis was associated with the extent of nasotemporal balance of critical spacing. We find that optical realignment (i.e., strabismus surgery) can normalize the enlarged visual crowding effects, a reliable psychophysical index of cortical organization, in the peripheral visual field of older children and adults with strabismus and rebalance the nasotemporal asymmetry of crowding, promoting the recovery of postoperative stereopsis. Our results indicated a potential of experience-dependent cortical organization after axial alignment even for individuals who are out of the critical period of visual development, illuminating the capacity and limitations of optics on sensory plasticity and emphasizing the importance of ocular correction for clinical practice.

The Effects of Hypoxia and Macromolecular Crowders on Extracellular Matrix Production by Human Endometrial Mesenchymal Stromal Cells

The Effects of Hypoxia and Macromolecular Crowders on Extracellular Matrix Production by Human Endometrial Mesenchymal Stromal Cells

Mechanosensitive ion channels MSL8, MSL9, and MSL10 have environmentally sensitive intrinsically disordered regions with distinct biophysical characteristics in vitro.

Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations over time. In the past several decades, IDRs have emerged as a major component of many proteomes, comprising ~30% of all eukaryotic protein sequences. Proteins with IDRs function in a wide range of biological pathways and are notably enriched in signaling cascades that respond to environmental stresses. Here, we identify and characterize intrinsic disorder in the soluble cytoplasmic N-terminal domains of MSL8, MSL9, and MSL10, three members of the MscS-like (MSL) family of mechanosensitive ion channels. In plants, MSL channels are proposed to mediate cell and organelle osmotic homeostasis. Bioinformatic tools unanimously predicted that the cytosolic N-termini of MSL channels are intrinsically disordered. We examined the N-terminus of MSL10 (MSL10N) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10N adopted a predominately helical structure when exposed to the helix-inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8N, MSL9N, and MSL10N have sharply differing propensities for self-assembly into condensates, both inherently and in response to salt, temperature, and molecular crowding. Taken together, these data establish the N-termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond uniquely to changes in their physiochemical environment.

The role of water in reactions catalysed by hydrolases under conditions of molecular crowding.

Under macromolecular crowding (MC) conditions such as cellular, extracellular, food and other environments of biotechnological interest, the thermodynamic activity of the different macromolecules present in the system is several orders of magnitude higher than in dilute solutions. In this state, the diffusion rates are affected by the volume exclusion induced by the crowders. Immiscible liquid phases, which may arise in MC by liquid-liquid phase separation, may induce a dynamic confinement of reactants, products and/or enzymes, tuning reaction rates. In cellular environments and other crowding conditions, membranes and macromolecules provide, on the whole, large surfaces that can perturb the solvent, causing its immobilisation by adsorption in the short range and also affecting the solvent viscosity in the long range. The latter phenomenon can affect the conformation of a protein and/or the degree of association of its protomers and, consequently, its activity. Changes in the water structure can also alter the enzyme-substrate interaction, and, in the case of hydrolytic enzymes, where water is one of the substrates, it also affects the reaction mechanism. Here, we review the evidence for how macromolecular crowding affects the catalysis induced by hydrolytic enzymes, focusing on the structure and dynamics of water.

Ice recrystallization inhibition and acceleration by cellulose nanocrystals in the presence of anionic and neutral polymers

Understanding mixed stabilizers' ice recrystallization inhibition (IRI) effect is important for ice cream formulation. The IRI effect of cellulose nanocrystals (CNCs) was investigated in the presence of sodium alginate (SA), carboxymethyl cellulose (CMC), polyethylene glycol (PEG), and inulin. Adding low concentrations of SA (≤0.13%) and CMC (≤0.05%) enhanced the IRI effect of CNCs. The enhancement became less at higher concentrations of SA and CMC. Recrystallization was even accelerated with a further increase in concentration of SA (≥0.5%) or CMC (≥0.2%). PEG and inulin had little influence on the IRI effect of CNCs. Rheological analysis of polymer overlap concentration, particle size analysis of CNCs dispersion, and microscopical observation of ice crystals accretion suggested that the dual effects of excluded volume might explain the IRI effect of CNCs in the presence of polymers: (1) adding polymers resulted in the macromolecular crowding, reducing the available volume for CNCs and increasing their effective concentration to interact with ice crystals, and led to an enhanced IRI effect; (2) depletion interaction promoted the aggregation of CNCs and merging of ice crystals, resulting in less enhancement and recrystallization acceleration. Our findings advance our understanding of the IRI effect for mixed stabilizers and are critical to ice cream formulation.

Macromolecular Crowding Promotes Re-entrant Liquid-Liquid Phase Separation of Human Serum Transferrin and Prevents Surface-Induced Fibrillation.

Protein aggregation and inactivation upon surface immobilization are major limiting factors for analytical applications in biotechnology-related fields. Protein immobilization on solid surfaces often requires multi-step surface passivation, which is time-consuming and inefficient. Herein, we have discovered that biomolecular condensates of biologically active human serum transferrin (Tf) can effectively prevent surface-induced fibrillation and preserve the native-like conformation of phase-separated Tf over a period of 30 days. It has been observed that macromolecular crowding promotes homotypic liquid-liquid phase separation (LLPS) of Tf through enthalpically driven multivalent hydrophobic interactions possibly via the involvement of its low-complexity domain (residues 3-20) containing hydrophobic amino acids. The present LLPS of Tf is a rare example of salt-mediated re-entrant phase separation in a broad range of salt concentrations (0-3 M) solely via the involvement of hydrophobic interactions. Notably, no liquid-to-solid-like phase transition has been observed over a period of 30 days, suggesting the intact conformational integrity of phase-separated Tf, as revealed from single droplet Raman, circular dichroism, and Fourier transform infrared spectroscopy measurements. More importantly, we discovered that the phase-separated condensates of Tf completely inhibit the surface-induced fibrillation of Tf, illustrating the protective role of these liquid-like condensates against denaturation and aggregation of biomolecules. The cell mimicking compact aqueous compartments of biomolecular condensates with a substantial amount of interfacial water preserve the structure and functionality of Tf. Our present study highlights an important functional aspect of biologically active protein condensates and may have wide-ranging implications in cell physiology and biotechnological applications.

Evaluation of the relationship between incisor torque and profile aesthetics in patients having orthodontic extractions compared to non-extractions

ObjectiveThe aim of the present study was to evaluate the relationship between soft tissues aesthetics and incisor torque, as well as the effect of crowding, anchorage, and extraction pattern, in adult patients treated without extraction or with two or four extractions.Materials and methodsSeventy-seven subjects with permanent dentition were selected retrospectively. Among these, 24 patients were treated with four extractions, 24 with two extractions and 29 without extractions. Lateral cephalograms and photographies taken before (T0) and after (T1) treatment were retrieved. The amount of crowding and the type of anchorage were recorded, and a cephalometric analysis was performed. A one-way ANOVA was used to compare the variables within and between groups. Linear regressions were performed to evaluate the effect of different predictors on soft tissues variables at T1.ResultsThe statistical analysis showed no differences within and between groups for soft tissue aesthetics. A significant reduction of the angle obtained from the intersection of Frankfurt plane and mandibular plane was observed in the four-extractions group, and a significant proclination of the lower incisors was observed in the two-extraction group. Linear regressions revealed that the change in soft tissue profile aesthetics was affected by the type of anchorage and the two-extractions pattern.ConclusionsSimilar soft tissue aesthetics were observed after treatment in the three groups, despite the presence of some skeletal and dental differences.Clinical relevanceA well-controlled incisor torque helps to preserve soft tissues aesthetics. The type of anchorage could influence soft tissues.

Concentration, Life Stage, Feeding, Density, Flow, and Strain Effects on Formalin Sensitivity in Rainbow Trout (Oncorhynchus mykiss).

Formalin is one of the most widely used and effective chemotherapeutic compounds for treatment of fungal infections and external parasites of fish eggs and fish. However, exposure to formalin can cause mortality in eggs and fingerlings, dependent upon the concentration used and the rearing conditions in which fish are treated. Additionally, strains within a species can exhibit differential susceptibility to formalin. Four experiments were conducted to evaluate the differential sensitivity to formalin of four rainbow trout (Oncorhynchus mykiss) strains in both the egg and fingerling life stages. Eggs were exposed to concentrations of 1667, 2000, and 5000 ppm formalin, and sensitivity differed among the strains when formalin concentration exceeded 2000 ppm. Exposure to higher formalin concentrations (i.e., 5000 ppm) as eggs did not increase mortality when fish were re-exposed to concentrations of 0, 167, 250, or 500 ppm formalin at 77 mm total length (TL). Fish size affected formalin sensitivity, with larger fish (128 mm TL) exhibiting higher rates of mortality than fish ≤ 77 mm TL when exposed to 250 ppm formalin. The effects of crowding, feeding, flow, and density on the formalin sensitivity of 77 mm TL fish were also investigated. Mortality increased in fish crowded away from the inflow to prevent contact with formalin as it entered the tank, potentially the result of an increase in density index within the crowded tanks. Feeding fish on the day they were treated caused mortality to increase by 5.4 to 8.8% in fish exposed to 167 and 250 ppm formalin, respectively, and mortality differed by strain. Reducing flows by half resulted in doubled to quadrupled mortality, and increased densities resulted in increased mortality in some strains but not others. Hatchery managers should consider what effect rearing conditions and formalin concentrations might have on the strain of fish being treated, prior to large-scale treatment.

The effect of macromolecular crowding degree on the self-assembly of fatty acid and lipid hydrolysis

Investigation on the physiochemical nature involved in the production of fatty acid catalyzed by the vesicles is of importance to understand the digestion of lipid. In this paper, the effects of crowding degree, which was constructed by polyethylene glycol (PEG), on the autocatalytic production of fatty acid with different chain lengths was studied. The results showed that the higher crowding degree led to the slower production rate of decanoic acid but the faster rate of oleic acid. The reason lies in that the presence of macromolecules resulted in the increased sizes of decanoic acid vesicles, but decreased sizes of oleic acid vesicles. Meanwhile, decanoic acid vesicles in more crowded medium exhibited viscous behavior, whereas oleic acid displayed elastic behavior. This research provides useful information for understanding the unusual autocatalyzed production of fatty acid in macromolecular crowding and may also draw an attention to the physiologically relevant lipid digestion.

Observation of Rich Defect Dynamics in Monolayer MoS2.

Defects play a pivotal role in limiting the performance and reliability of nanoscale devices. Field-effect transistors (FETs) based on atomically thin two-dimensional (2D) semiconductors such as monolayer MoS2 are no exception. Probing defect dynamics in 2D FETs is therefore of significant interest. Here, we present a comprehensive insight into various defect dynamics observed in monolayer MoS2 FETs at varying gate biases and temperatures. The measured source-to-drain currents exhibit random telegraph signals (RTS) owing to the transfer of charges between the semiconducting channel and individual defects. Based on the modeled temperature and gate bias dependence, oxygen vacancies or aluminum interstitials are probable defect candidates. Several types of RTSs are observed including anomalous RTS and giant RTS indicating local current crowding effects and rich defect dynamics in monolayer MoS2 FETs. This study explores defect dynamics in large area-grown monolayer MoS2 with ALD-grown Al2O3 as the gate dielectric.

Effect of visual crowding in packaging on consumers' buying intention

PurposeBased on the construal level theory, the purpose of this study is to prove the effect of interaction between construal level and visual crowding on consumers' buying intention. The study tries to explain the reasons behind the different buying intention toward visual crowding among consumers with different construal level.Design/methodology/approachThis study was conducted through two situational simulation experiments. The main data analysis methods are ANOVA and bootstrap analysis.Findings(1) the matching of construal level and visual crowding has a significant effect on consumers’ buying intention. (2) Perceptual fluency mediates the interaction between the construal level and visual crowding on buying intention.Research limitations/implicationsThis study measures consumers' buying intention through situational experiments but does not measure consumers' buying behavior through real scenarios.Practical implicationsAccording to the study conclusions, consumers prefer visually crowded packaging that matches their construal level. Enterprises should consider the impact of the construal level on the effect of packaging stimulation.Social implicationsThis study enriches the theory related to construal level and highlights the mediating role of perceptual fluency. The addition of perceptual fluency explains the mechanism by which visual crowding affects consumers' buying intention. This extends the research on the antecedents and effects of perceptual fluency.Originality/valueThis study innovatively introduces visual crowding into packaging and matches visual crowding to construal level, explaining why different consumers buy different visually crowded packaging.

Volume exclusion effects in perovskite charge transport modeling

Due to their flexible material properties, perovskite materials are a promising candidate for many semiconductor devices such as lasers, memristors, LEDs and solar cells. For example, perovskite-based solar cells have recently become one of the fastest growing photovoltaic technologies. Unfortunately, perovskite devices are far from commercialization due to challenges such as fast degradation. Mathematical models can be used as tools to explain the behavior of such devices, for example drift-diffusion equations portray the ionic and electric motion in perovskites. In this work, we take volume exclusion effects on ion migration within a perovskite crystal lattice into account. This results in the formulation of two different ionic current densities for such a drift-diffusion model – treating either the mobility or the diffusivity as density-dependent while the other quantity remains constant. The influence of incorporating each current density description into a model for a typical perovskite solar cell configuration is investigated numerically, through simulations performed using two different open source tools.

Metastability mediated grain size control in PH 17-4 stainless steel fabricated using Laser-Powder Bed Fusion (LPBF)

Varying the chemical composition and cooling rates during additive manufacturing (AM) can enable the formation and, in some cases, the retention of metastable phases affecting the solidification pathways. Altering the solidification pathways directly affects the microstructure and in turn the mechanical properties of the parts. In this study, we show that modifying the solidification pathway through the deliberate retention of metastable austenite in PH 17–4 stainless steel (SS) leads to significant grain refinement (one order of magnitude smaller grains) and improvement in tensile strength (30% higher ultimate tensile strength) of parts printed using Laser-Powder Bed Fusion (LPBF). Nitrogen (N2)-atomized feedstock powder containing higher concentrations of austenite-stabilizing elemental nitrogen was used to print parts with retained austenite. Parts absent of retained austenite printed using argon-atomized feedstock powder were used for comparison of microstructure and tensile properties. The grain refinement has been attributed to the “crowding effect” observed due to the simultaneous growth and coexistence of metastable austenitic phase with the stable ferrite. We also show that employing a three-step heat treatment procedure can eliminate the unwanted yield point behavior associated with the softer austenite while preserving the superior tensile properties in N2-atomized samples.