Crowding Environment Research Articles

Mimicking a Cellular Crowding Environment for Enzyme-Free Paper-Based Nucleic Acid Tests at the Point of Care.

Point of care (PoC) nucleic acid amplification tests (NAATs) are a cornerstone of public health, providing the earliest and most accurate diagnostic method for many communicable diseases in the same location where the patient receives treatment. Communicable diseases, such as human immunodeficiency virus (HIV), disproportionately impact low-resource communities where NAATs are often unobtainable due to the resource-intensive enzymes that drive the tests. Enzyme-free nucleic acid detection methods, such as hybridization chain reaction (HCR), use DNA secondary structures for self-driven amplification schemes, producing large DNA nanostructures, capable of single-molecule detection in cellulo. These thermodynamically driven DNA-based tests have struggled to penetrate the PoC diagnostic field due to their inadequate limits of detection or complex workflows. Here, we present a proof-of-concept NAAT that combines HCR-based amplification of a target nucleic acid sequence with paper-based nucleic acid filtration and enrichment capable of detecting sub-pM levels of synthetic DNA. We reconstruct the favorable hybridization conditions of an in cellulo reaction in vitro by incubating HCR in an evaporating, microvolume environment containing poly(ethylene glycol) as a crowding agent. We demonstrate that the kinetics and thermodynamics of DNA-DNA and DNA-RNA hybridization is enhanced by the dynamic evaporating environment and inclusion of crowding agents, bringing HCR closer to meeting PoC NAAT needs.

Induced Circular Dichroism Analysis of Thermally Induced Conformational Changes on Protein Binding Sites Under a Crowding Environment.

Protein-ligand interactions in crowded cellular environments play a crucial role in biological functions. The crowded environment can perturb the overall protein structure and local conformation, thereby influencing the binding pathway of protein-ligand reactions within the cellular milieu. Therefore, a detailed understanding of the local conformation is crucial for elucidating the intricacies of protein-ligand interactions in crowded cellular environments. In this study, we investigated the feasibility of induced circular dichroism (ICD) using 8-anilinonaphthalene-1-sulfonic acid (ANS) for local conformational analysis at the binding site in a crowding environment. Bovine serum albumin (BSA) concentration-dependent measurements were performed to assess the feasibility of ANS-ICD for analyzing protein interior binding sites. The results showed distinct changes in the ANS-ICD spectra of BSA solutions, indicating their potential for analyzing the internal conformation of proteins. Moreover, temperature-dependent measurements were performed in dilute and crowding environments, revealing distinct denaturation pathways of BSA binding sites. Principal component analysis of ANS-ICD spectral changes revealed lower temperature pre-denaturation in the crowded solution than that in the diluted solution, suggesting destabilization of binding sites owing to self-crowding repulsive interactions. The established ANS-ICD method can provide valuable conformational insights into protein-ligand interactions in crowded cellular environments.

Fibrillization Process of Human Amyloid-Beta Protein (1-40) under a Molecular Crowding Environment Mimicking the Interior of Living Cells Using Cell Debris.

Molecular crowding environments play a crucial role in understanding the mechanisms of biological reactions. Inside living cells, a diverse array of molecules coexists within a volume fraction ranging from 10% to 30% v/v. However, conventional spectroscopic methods often face difficulties in selectively observing the structures of particular proteins or membranes within such molecularly crowded environments due to the presence of high background signals. Therefore, it is crucial to establish in vitro measurement conditions that closely resemble the intracellular environment. Meanwhile, the neutron scattering method offers a significant advantage in selectively observing target biological components, even within crowded environments. Recently, we have demonstrated a novel scattering method capable of selectively detecting the structures of targeted proteins or membranes in a closely mimicking intracellular milieu achieved utilizing whole-cell contents (deuterated-cell debris). This method relies on the inverse contrast matching technique in neutron scattering. By employing this method, we successfully observed the fibrillization process of human amyloid beta-protein (Aβ 1-40) under a molecular crowding environment (13.1% w/v cell debris, Aβ/cell debris = ~1/25 w/w) that closely mimics the interior of living cells. Aβ protein is well known as a major pathogenic component of Alzheimer's disease. The present results combining model simulation analyses clearly show that the intracellular environment facilitates the potential formation of even more intricate higher-order aggregates of Aβ proteins than those previously reported.

Social- or task-oriented: how does social crowding shape consumers' preferences for chatbot conversational styles?

PurposeDriven by artificial intelligence technology, chatbots have begun to play an important customer service role in the online retail environment. This study aims to explore how conversational styles improve the interaction experience between consumers and chatbots in different social crowding environments, and the moderating role of product categories is considered.Design/methodology/approachThree studies are conducted to understand the influences of conversational styles, social crowding and product categories on consumer acceptance, assessed using situational experiments and questions.FindingsIn a low social crowding environment, consumers prefer chatbots with a social-oriented (vs. task-oriented) conversational style, while in a high social crowding environment, consumers prefer a task-oriented (vs. social-oriented) conversational style, and warmth and competence mediate these effects. The moderating effect of product categories is supported.Originality/valueThis study expands the application of the stereotype content model to improve the interaction experience level between consumers and chatbots in online retail. The findings can provide managerial suggestions for retailers to select a chatbot's conversational style and promote a more continuous interaction between consumers and chatbots.

New insights into the effect of molecular crowding environment induced by dimethyl sulfoxide on the conformation and stability of G-quadruplex

Various structures of G-quadruplex in biosystems play an important role in different diseases and are often regulated by a variety of molecular crowding environments induced by internal and even external factors (e.g., a solvent). Dimethyl sulfoxide (DMSO), a universal solvent, has been widely used in biological studies and for drug therapy, but little is known regarding its effect on G-quadruplex structure and stability. Here, we report the influence of molecular crowding environment induced by DMSO on the conformation and stability of G-quadruplex structure. We show that the G-quadruplex-forming sequences such as human telomeric sequence, which may have diverse conformations in different environments, tend to convert their topologies to parallel structures under the molecular crowding stimulated by DMSO. Moreover, DMSO can increase the stability of the parallel and antiparallel topologies, especially the parallel G-quadruplex sequence c-kit, but not the hybrid topologies. Further analysis of c-kit using the CD and NMR technique, combined with the unique structural characteristics of c-kit, reveals that the crowding, dehydration and interaction of DMSO are conductive to the formation and stability of the parallel G-quadruplex. The present study suggests that, DMSO, a common solvent used in DNA experiments, may have a nonnegligible influence on the structure and stability of G-quadruplex.

Alignment of Colloidal Rods in Crowded Environments.

Understanding the hydrodynamic alignment of colloidal rods in polymer solutions is pivotal for manufacturing structurally ordered materials. How polymer crowding influences the flow-induced alignment of suspended colloidal rods remains unclear when rods and polymers share similar length scales. We tackle this problem by analyzing the alignment of colloidal rods suspended in crowded polymer solutions and comparing that to the case where crowding is provided by additional colloidal rods in a pure solvent. We find that the polymer dynamics govern the onset of shear-induced alignment of colloidal rods suspended in polymer solutions, and the control parameter for the alignment of rods is the Weissenberg number, quantifying the elastic response of the polymer to an imposed flow. Moreover, we show that the increasing colloidal alignment with the shear rate follows a universal trend that is independent of the surrounding crowding environment. Our results indicate that colloidal rod alignment in polymer solutions can be predicted on the basis of the critical shear rate at which polymer coils are deformed by the flow, aiding the synthesis and design of anisotropic materials.

Investment in adult reproductive tissues is affected by larval growth conditions but not by evolution under poor larval growth conditions in Drosophila melanogaster.

In many insects, the larval environment is confined to the egg-laying site, which often leads to crowded larval conditions, exposing the developing larvae to poor resource availability and toxic metabolic wastes. Larval crowding imposes two opposing selection pressures. On one hand, due to poor nutritional resources during developmental stages, adults from the crowded larval environment have reduced investment in reproductive tissues. On the other hand, a crowded larval environment acts as a cue for future reproductive competition inducing increased investment in reproductive tissues. Both these selection pressures are likely affected by the level of crowding. The evolutionary consequence of adaptation to larval crowding environment on adult reproductive investment is bound to be a result of the interaction of these two opposing forces. In this study, we used experimentally evolved populations of Drosophila melanogaster adapted to larval crowding to investigate the effect of adaptation to larval crowding on investment in reproductive organs (testes and accessory glands) of males. Our results show that there is a strong effect of larval developmental environment on absolute sizes of testes and accessory glands. However, there was no effect of the developmental environment when testis size was scaled by body size. We also found that flies from crowded cultures had smaller accessory gland sizes relative to body size. Moreover, the sizes of the reproductive organs were not affected by the selection histories of the populations. This study highlights that adaptation to two extremely different developmental environments does not affect the patterns of reproductive investment. We discuss the possibility that differential investment in reproductive tissues could be influenced by the mating dynamics and/or investment in larval survival traits, rather than just the developmental environment of the populations.

Evolution of pathogen-specific improved survivorship post-infection in populations of Drosophila melanogaster adapted to larval crowding.

The environment experienced by individuals during their juvenile stages has an impact on their adult stages. In holometabolous insects like Drosophila melanogaster, most of the resource acquisition for adult stages happens during the larval stages. Larval-crowding is a stressful environment, which exposes the larvae to scarcity of food and accumulation of toxic waste. Since adult traits are contingent upon larval stages, in larval-crowding like conditions, adult traits are prone to get affected. While the effect of resource limited, poor-developmental environment on adult immune response has been widely studied, the effect of adaptation to resource-limited developmental environment has not been studied, therefore in this study we assayed the evolution of ability to survive infection in adult stages as a correlated response to adaptation to larval crowding environments. Using four populations of Drosophila melanogaster adapted to larval crowding for 240 generations and their respective control populations, we show that populations adapted to larval crowding show an improved and evolved post-infection survivorship against a gram-negative bacteria Pseudomonas entomophila. Whereas, against a gram-positive bacteria Enterococcus faecalis, no difference in post-infection survivorship was observed across control and selected populations. In this study, we report the co-related evolution of pathogen-specific increased survivorship post-infection in populations of Drosophila melanogaster as a result of adaptation to larval crowding environment.

An equation for biomimicking macromolecular crowding using Escherichia coli MG1655 strain

Macromolecules present in the intracellular environment of a cell are densely packed, resulting in a highly crowded cytosolic environment. This crowded milieu influences several biochemical equilibria such as diffusibility and association constant of biomolecules which impose a serious impact on cellular functions as well as its processes. A number of in silico and in vitro studies have been reported till date about using synthetic crowding agents for resembling such a crowding environment within the cell. Lately, it has been realized that synthetic crowders are not suitable for mimicking the intrinsic environment of the cell. In this study, proteins were assumed to be the major biological molecule which contributes to the crowding environment. We have semi-theoretically determined the total protein concentration within an individual E. coli MG1655 cell which changes notably as the growth curve proceeds from 0.2 to 1.0 OD600. The average range of total cellular protein concentration throughout the batch culture was found to be in the range of 15.2 to 178 fg/fL of cytoplasmic volume. The fundamental knowledge gained through the study was translated to applied research in the form of an equation. We propose an equation that could help to mimic the OD600 dependent crowding environment present within a single cell of E. coli in the desired volume of reaction solution. In a nutshell, the equation provides quantitative estimation of the volume of culture required to prepare the cell lysate for biomimicking the intracellular crowding environment in vitro. This finding provides a new insight into the cellular cytosolic environment that could be used as a platform to frame more cells like environment in cell-free protein synthesis (CFPS) system for synthetic biology applications.

Saccharides Create a Crowding Environment for Gene Expression in Cell-Free Systems.

Cellular physical microenvironment such as crowding shows great influence on enzymatic reactions. Herein, we report a new finding that saccharides with low molecular weight create an effective crowding microenvironment for gene expression in cell-free protein synthesis, which provides valuable implications for living systems. Four saccharides including sorbose, galactose, sucrose, and cellobiose are screened out as effective crowders. At a low concentration range of saccharides, both the mRNA and protein amounts present an upward trend with the concentration increment of saccharides; when the concentrations exceed a critical value, the mRNA and protein amounts decrease. A mechanism is proposed that at low concentrations of saccharides, the effective concentrations of reactants increase due to the coexistence of crowders and reactants in a finite volume; when the concentrations exceed a critical value, the molecular diffusion of reactants is dominantly restricted due to the increased viscosity. Our finding opens a new view that saccharides with low molecular weight could be crowders and provides a new insight that substances with low molecular weight in cells would produce a crowding effect on biochemical reactions in living systems.

Preferential Intercalation of Human Amyloid-β Peptide into Interbilayer Region of Lipid-Raft Membrane in Macromolecular Crowding Environment.

This study focuses on the interaction of human amyloid β-peptide (Aβ) with a lipid-raft model membrane under macromolecular crowding conditions that mimic the intracellular environment. Aβ is central to the development of Alzheimer's disease (AD) and has been studied extensively to determine the molecular mechanisms of Aβ-induced cellular dysfunctions underlying the pathogenesis of AD. According to evidence from spectroscopic studies, ganglioside clusters are key to the fibrillization process of Aβ. Gangliosides are a major component of glycosphingolipids and are acidic lipids of the central nervous system known to form so-called lipid rafts. In this study, the small unilamellar vesicle (SUV) membrane, composed of monosialogangliosides, cholesterol, and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine, did not show any structural changes after the addition of Aβ under noncrowding conditions. However, the addition of Aβ under crowding conditions induced shape deformation and aggregation to SUV resulting in multilamellar stacking. The time evolution of the lamellar peak suggested the preferential cohesion or intercalation of the Aβ peptide into the interbilayer region. This phenomenon was only observed at the gel (Lβ) phase. These results suggest that an intracellular crowding environment promotes Aβ-membrane interaction and a selective accumulation of Aβ peptides into the interbilayer regions.

The power of information on customers’ social withdrawal and citizenship behavior in a crowded service environment

PurposeCrowding in service environments is a constant concern for many firms due to the negative consequences it has on consumers and companies alike. Yet, scant empirical research exists on firm-generated initiatives aimed at improving customer service experiences in crowded situations. The purpose of this paper is to investigate how information, a managerially actionable variable, influences social interactions (in the form of customer social withdrawal and citizenship behavior) and service experience.Design/methodology/approachTwo experimental studies were conducted using an extended service context.FindingsThis research demonstrates that receiving information about crowds in advance results in heightened social withdrawal, which improves customer service experience. However, providing consumers with a platform to share crowding information increases customers’ citizenship behavior toward service employees and other customers, which, in turn, improves customer service experience.Practical implicationsFor extended service encounters (e.g. air travel) where social interactions are inevitable, companies should encourage customers to share their real-time experiences with other customers in hopes of creating more positive social interactions (e.g. citizenship behavior) within the crowded environment.Originality/valueExisting investigations of crowding stem from an overemphasis on the physical and atmospheric aspects of the environment by treating crowds as a “fixture” in the servicescape, rather than as “active participants” involved in the crowding environment. While the mere presence of crowds alone has negative effects, this research takes it a step further by examining interactions among and between customers and service employees within the crowded service environment.

Hsa‐miR‐1587 G‐quadruplex formation and dimerization induced by NH4+, molecular crowding environment and jatrorrhizine derivatives

A guanine-rich human mature microRNA, miR-1587, was discovered to form stable intramolecular G-quadruplexes in the presence of K+, Na+ and low concentration of NH4+ (25mM) by electrospray ionization mass spectrometry (ESI-MS) combined with circular dichroism (CD) spectroscopy. Furthermore, under high concentration of NH4+ (100mM) or molecular crowding environments, miR-1587 formed a dimeric G-quadruplex through 3′-to-3′ stacking of two monomeric G-quadruplex subunits with one ammonium ion sandwiched between the interfaces. Specifically, two synthesized jatrorrhizine derivatives with terminal amine groups could also induce the dimerization of miR‐1587 G‐quadruplex and formed 1:1 and 2:1 complexes with the dimeric G-quadruplex. In contrast, jatrorrhizine could bind with the dimeric miR‐1587 G‐quadruplex, but could not induce dimerization of miR‐1587 G‐quadruplex. These results provide a new strategy to regulate the functions of miR-1587 through induction of G-quadruplex formation and dimerization.

The Influence of Social Crowding on Brand Attachment

Abstract Feeling crowded in a shopping environment can decrease consumers’ evaluations of a product or service and lower customer satisfaction. However, the present research suggests that a crowded environment can sometimes have a positive impact on consumer behavior. Although feeling crowded motivates consumers to avoid interacting with others, it leads them to become more attached to brands as an alternative way of maintaining their basic need for belongingness. The effect does not occur (a) when the crowding environment is composed of familiar people (and, therefore, is not considered aversive); (b) when individuals have an interdependent self-construal (and consequently, high tolerance for crowdedness); (c) when people are accompanied by friends in the crowded environment; (d) when the social function of the brands is made salient; (e) when people have never used the brand before; or (f) when the brand is referred to as a general product rather than a specific brand.

Asymmetric Catalytic 1,2-Hydroperoxidation of Isatin-Derived Ketimine with Hydrogen Peroxide in the Crowding Environment of PEGs.

The first enantioselective catalytic 1,2-hydroperoxidation has been achieved in the presence of PEG-600 using an acid-base bifunctional chiral squaramide as the organocatalyst, affording a range of enantioenriched α-N-substituted hydroperoxides bearing an oxindole moiety with excellent stereoselectivities (up to 99% ee).

Energy transfer at heterogeneous protein-protein interfaces to investigate the molecular behaviour in the crowding environment

Investigation of the behaviour of proteins in crowded environments is crucial for understanding the role of proteins in biological environments. In this study, the behaviour of bovine serum albumin (BSA) in crowded (highly concentrated) environments was investigated using time-resolved fluorescence spectroscopy as a model system. By using energy transfer as a molecular ruler, the crowding effect was clearly observed in the time resolved spectra. In addition, by using both time resolved anisotropy measurement and Raman spectroscopy, more detail insights from conformational and dynamic points of view were described. Consequently, it was revealed that in the highly concentrated solution, most of the BSA molecules are in the fast-reversible oligomeric state and the association at the “hard” and “soft” interfaces between protein surfaces occurred in a highly crowded environment with the aid of a charge-charge and short-range attractive interface. From both the conformational and dynamic aspects, the detail spectroscopic understanding of the behaviour of BSA in the crowding environment was obtained.

Water Dynamics in Cytoplasm-Like Crowded Environment Correlates with the Conformational Transition of the Macromolecular Crowder.

Polyethylene glycol (PEG) is a unique polymer material with enormous applicability in many industrial and scientific fields. Here, its use as macromolecular crowder to mimic the cellular environment in vitro is the focus of the present study. We show that femtosecond mid-IR pump-probe spectroscopy using three different IR probes, HDO, HN3, and azido-derivatized crowder, provides complete and stereoscopic information on water structure and dynamics in the cytoplasm-like macromolecular crowding environment. Our experimental results suggest two distinct subpopulations of water molecules: those that interact with other water molecules and those that are part of a hydration shell of crowder on its surface. Interestingly, water dynamics even in highly crowded environment remains bulk-like in spite of significant perturbation to the tetrahedral H-bonding network of water molecules. That is possible because of the formation of water aggregates (pools) even in water-deficient PEGDME-water solutions. In such a crowded environment, the conformationally accessible phase space of the macromolecular crowder is reduced, similar to biopolymers in highly crowded cytoplasm. Nonetheless, the hydration water on the surface of crowders slows down considerably with increased crowding. Most importantly, we do not observe any coalescing of surface hydration water (of the crowder) with bulk-like water to generate collective hydration dynamics at any crowder concentration, contrary to recent reports. We anticipate that the present triple-IR-probe approach is of exceptional use in studying how conformational states of crowders correlate with structural and dynamical changes of water, which is critical in understanding their key roles in biological and industrial applications.

Nonlinear response and emerging nonequilibrium microstructures for biased diffusion in confined crowded environments.

We study analytically the dynamics and the microstructural changes of a host medium caused by a driven tracer particle moving in a confined, quiescent molecular crowding environment. Imitating typical settings of active microrheology experiments, we consider here a minimal model comprising a geometrically confined lattice system (a two-dimensional striplike or a three-dimensional capillary-like system) populated by two types of hard-core particles with stochastic dynamics (a tracer particle driven by a constant external force and bath particles moving completely at random). Resorting to a decoupling scheme, which permits us to go beyond the linear-response approximation (Stokes regime) for arbitrary densities of the lattice gas particles, we determine the force-velocity relation for the tracer particle and the stationary density profiles of the host medium particles around it. These results are validated a posteriori by extensive numerical simulations for a wide range of parameters. Our theoretical analysis reveals two striking features: (a) We show that, under certain conditions, the terminal velocity of the driven tracer particle is a nonmonotonic function of the force, so in some parameter range the differential mobility becomes negative, and (b) the biased particle drives the whole system into a nonequilibrium steady state with a stationary particle density profile past the tracer, which decays exponentially, in sharp contrast with the behavior observed for unbounded lattices, where an algebraic decay is known to take place.

Unexpected Swelling of Stiff DNA in a Polydisperse Crowded Environment.

We investigate the conformations of DNA-like stiff chains, characterized by contour length (L) and persistence length (lp), in a variety of crowded environments containing monodisperse soft spherical (SS) and spherocylindrical (SC) particles, a mixture of SS and SC, and a milieu mimicking the composition of proteins in the Escherichia coli cytoplasm. The stiff chain, whose size modestly increases in SS crowders up to ϕ ≈ 0.1, is considerably more compact at low volume fractions (ϕ ≤ 0.2) in monodisperse SC particles than in a medium containing SS particles. A 1:1 mixture of SS and SC crowders induces greater chain compaction than the pure SS or SC crowders at the same ϕ, with the effect being highly nonadditive. We also discover a counterintuitive result that the polydisperse crowding environment, mimicking the composition of a cell lysate, swells the DNA-like polymer, which is in stark contrast to the size reduction of flexible polymers in the same milieu. Trapping of the stiff chain in a fluctuating tube-like environment created by large-sized crowders explains the dramatic increase in size and persistence length of the stiff chain. In the polydisperse medium, mimicking the cellular environment, the size of the DNA (or related RNA) is determined by L/lp. At low L/lp, the size of the polymer is unaffected, whereas there is a dramatic swelling at an intermediate value of L/lp. We use these results to provide insights into recent experiments on crowding effects on RNA and also make testable predictions.

2P003 Crowding環境下でのタンパク質構造の熱安定性(01A. 蛋白質:構造,ポスター,第52回日本生物物理学会年会(2014年度))

2P003 Crowding環境下でのタンパク質構造の熱安定性(01A. 蛋白質:構造,ポスター,第52回日本生物物理学会年会(2014年度))