Hydrophobic Fluorescent Probe Research Articles

A boron dipyrromethene-derivative fluorescent probes for β-cyclodextrin and maltooligotose hydrophobicity recognition

This study introduces a novel approach to oligosaccharide sensing utilizing hydrophobic interaction-driven fluorescent probes. The design involves three Boron dipyrromethene (BODIPY)-based Oligosaccharide sensing Fluorescent dyes (BOliFluor) with varying hydrophobic moieties synthesized to detect oligosaccharides precisely. The probes demonstrated sensitivity to linear maltooctaose and cyclic β-cyclodextrin, with a distinct response profile. The hydrophobic nature of the benzyl group in BOliFluor 2 enhanced interaction with β-cyclodextrin, resulting in a linear response with a low detection limit of 0.06 mM. In contrast, BOliFluor 1 and 3 exhibited a sigmoidal response according to Hill's equation, F = Fmax·cn/(Kd + cn), suggesting a complex interaction mechanism influenced by the conformational flexibility of the recognition groups. The study also explored the impact of chaotropic salts on the binding mode of BOliFluor to β-cyclodextrin, revealing a salt-dependent response. Overall, this research presents a promising strategy for selective oligosaccharide detection through hydrophobic interactions, paving the way for enhanced sensitivity and specificity in this field. The findings contribute to developing a comprehensive analytical method for oligosaccharides, addressing existing challenges in their detection and quantification.

Reconsideration of the ESIPT off mechanism for fluorescent probe MNC in aqueous solution

Fluorescent probes with excited state intramolecular proton transfer (ESIPT) properties play a significant role in the research of life science and material science. Guo et al. designed 3-hydroxy-2-(6-Methoxynaphthalen-2-yl)-4H-chromen-4-one (MNC) as a control to achieve the dual-color fluorescence imaging of lipid droplets and endoplasmic reticulum (ER). They deemed that the ESIPT process would be turned off in ER with high water content [J. Am. Chem. Soc. 2021, 143, 3169–3179]. However, contrary to the conventional ESIPT off case, the enol* state fluorescence intensity that should have been enhanced was severely quenched in water. Here, combined with ultrafast spectrum, steady-state fluorescence spectrum and potential energy surface, the mechanism of ESIPT process of MNC turned off in water is revised. Furthermore, the formation of aggregated states in water is responsible for the quenching of MNC fluorescence. This work is expected to provide broader ideas for the design of hydrophobic fluorescent probes.

The role of high molecular weight hyaluronic acid in mucoadhesion on an ocular surface model

Hyaluronic acid (HA) is frequently formulated in eye drops to improve the stability of the tear film by hydration and lubrication. Mucoadhesion is related to the ocular residence time and therefore to the effectiveness of the eye drops. The ocular residence time of the HA formulation is correlated with the ability of HA to create specific strong interactions in the ocular surface with the mucus layer, mainly composed of a mixture of secreted mucins (MUC; gel forming MUC5AC and MUC2) and shed membrane-bound soluble mucins (MUC1, MUC4, and MUC16). Dry eye disease (DED) is a multifactorial pathology of the preocular tear film with possible damage to the ocular surface classified in two types: (1) aqueous-deficient dry eye and (2) evaporative dry eye, caused by a decrease in goblet cell density that reduces MUC expression and/or by meibomian gland dysfunction, that results in a drop in the lipidic fraction of the tear film.In this work, the binding affinity between HA and MUC2 has been evaluated with three complementary approaches because the secreted MUCs play a pivotal role in the viscoelastic properties of the tear film: 1. Rheological analysis, measuring the mucoadhesive index and the complex viscosity in relation to MM (Molecular Mass) and concentration; 2. Fluorescence analysis, using a fluorescent hydrophobic probe, to investigate the conformational change of MUC2 during the interaction with the HA polymer; 3. Surface plasmon resonance analysis, used to measure the affinity between MUC2 (immobilized on the surface of a sensor chip) and the HA polymers that flowed on it at the molecular level.For all these tests, the mucoadhesive performance of the natural HA linearly increases with the MM, whereas cross-linked HA and other emollient and gelling agents (formulated in artificial tears) do not show the same mucoadhesive properties (with the exception of xanthan gum). The mucoadhesive performance of high MM HA has also been confirmed in conditions that simulate the pathological condition of the tear film during DED by decreasing the MUC2 or oleic acid concentration. Physico-chemical analysis of a series of marketed artificial tears confirms the linear correlation between the MM of the HA used in the products and the mucoadhesive index measured on the ocular surface model.

Disparate properties of Burkholderia multivorans and Pseudomonas aeruginosa regarding outer membrane chemical permeabilization to the hydrophobic substances novobiocin and triclosan.

Burkholderia multivorans causes opportunistic pulmonary infections and is intrinsically resistant to many antibacterial compounds including the hydrophobic biocide triclosan. Chemical permeabilization of the Pseudomonas aeruginosa outer membrane affects sensitization to hydrophobic substances. The purpose of the present study was to determine if B. multivorans is similarly susceptive suggesting that outer membrane impermeability properties underlie triclosan resistance. Antibiograms and conventional macrobroth dilution bioassays were employed to establish baseline susceptibility levels to hydrophobic antibacterial compounds. Outer membrane permeabilizers compound 48/80, polymyxin B, polymyxin B-nonapeptide, and ethylenediaminetetraacetic acid were used in attempts to sensitize disparate B. multivorans isolates to the hydrophobic agents novobiocin and triclosan, and to potentiate partitioning of the hydrophobic fluorescent probe 1-N-phenylnapthylamine (NPN). The lipophilic agent resistance profiles for all B. multivorans strains were essentially the same as that of P. aeruginosa except that they were resistant to polymyxin B. Moreover, they resisted sensitization to hydrophobic compounds and remained inaccessible to NPN when treated with outer membrane permeabilizers. These data support the notion that while both phylogenetically-related organisms exhibit general intrinsic resistance properties to hydrophobic substances, the outer membrane of B. multivorans either resists permeabilization by chemical modification or sensitization is mitigated by a supplemental mechanism not present in P. aeruginosa.

Influence of outer membrane permeabilization on intrinsic resistance to the hydrophobic biocide triclosan in opportunistic Serratia species

Triclosan is a hydrophobic antimicrobial agent commonly employed in health care settings. While it exhibits broad-spectrum antibacterial properties, the gram-negative nosocomial opportunists Pseudomonas aeruginosa and Serratia marcescens are atypically refractory. Intrinsic resistance to triclosan in P. aeruginosa is largely due to its outer membrane impermeability properties for hydrophobic and bulky substances. The present study was undertaken to determine the relationship between triclosan and the outer cell envelopes of thirteen strains of ten Serratia species reported to be opportunistic pathogens in humans. General intrinsic resistance to hydrophobic and other outer membrane impermeant compounds was assessed using cultural selection, disk agar diffusion, and macrobroth dilution bioassays. Uptake of the hydrophobic fluorescent probe 1-N-phenylnapthylamine was assessed in four disparate strains of S. marcescens. Batch culture kinetics in the presence of combinations of triclosan and outer membrane permeabilizer compound 48/80 allowed analysis of outer membrane involvement in intrinsic resistance. Aggregate results revealed that individual species ranged in response to hydrophobic and bulky molecules from generally refractory to extremely susceptible. Moreover, susceptivity to triclosan sensitization by chemical disruption of outer membrane exclusionary properties differed markedly among species which exhibited intrinsic resistance to triclosan. These data suggest that disparate opportunistic pathogens within the genus Serratia differ phenotypically regarding the degree to which outer membrane exclusion contributes to intrinsic resistance for impermeant molecules in general, and triclosan specifically. Ancillary resistance mechanisms appear to contribute in some species and may involve constitutive multi-drug efflux systems.ImportanceA paucity of knowledge exists regarding the cellular and molecular mechanisms by which opportunistically pathogenic members of the genus Serratia are able to infect immunocompromised and otherwise susceptible individuals, and then evade chemotherapy. This is especially true for species other than Serratia marcescens and Serratia liquefaciens, although much remains to be learned with regard to the nature of key virulence factors and infection mechanisms which allow for the typically nosocomial acquisition of even these species. The research described in the present study will provide a better understanding of the contribution of outer cell envelope permeability properties to the pathogenicity of these opportunistic species in an ever-increasing susceptible patient population. It is our hope that greater knowledge of the basic biology of these organisms will contribute to the mitigation of suffering they cause in patients with underlying diseases.

Fluorescent probe-encapsulated smart nanohydrogel to enhance sensitivity toward hydrogen peroxide in living cells

In this study, a fluorescent probe-encapsulated reduction-sensitive smart nanohydrogel as a new nanosensor was fabricated. The nanohydrogel was prepared via distillation-precipitation polymerization by using N, N′-bis(acryloyl) cystinamide both as the monomer and crosslinker. Besides, red fluorescent hydrophobic peroxyresorufin-1 (PR1) probe for H2O2 was employed and encapsulated into the nanohydrogel. We found that the nanohydrogel not only can encapsulate a large number of hydrophobic fluorescent probes with high loading capacity, but also can avoid the aggregation of hydrophobic fluorescent probes. Confocal laser fluorescence imaging of H2O2 in HeLa cells demonstrated that the PR1-encapsulated nanohydrogel allows efficient delivery of PR1 probe into living HeLa cells. In cytoplasm, the nanohydrogels reduced and degraded by glutathione, and the PR1 probes released from the nanohydrogel were enriched and concentrated in some local regions, resulting in enhanced imaging of endogenous H2O2. This work provides a facile nanohydrogel-assisted probe-encapsulation strategy, which is also applicable to multiple fluorescent probes, thus enabling imaging of diverse endogenous bioactive molecules.

5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells.

Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 μM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined.

Separated Micelles Formation of pH-Responsive Random and Block Copolymers Containing Phosphorylcholine Groups.

The self-assembly of pH-responsive random and block copolymers composed of 2-(N,N-diisopropylamino)ethyl methacrylate and 2-methacryloyloxyethyl phosphorylcholine was investigated in aqueous media. Their pH-responsive behaviors were investigated in aqueous media by dynamic light scattering (DLS) and fluorescence measurements using a pyrene hydrophobic fluorescence probe. In an acidic environment, these copolymers existed as single polymer chains that did not interact with each other. In contrast, upon increasing the pH of the solution above the critical value of ~8, separated micelles were formed in the mixture, which was indicated by bimodal distribution in DLS results with radius of 4.5 and 10.4 nm, corresponding to the random and block copolymer micelles, respectively. Fluorescence resonance energy transfer efficiencies were near to zero in the mixture of the donor labeled block and acceptor labeled random copolymers under both acidic and basic pH. These results demonstrated the coexistence of two distinct micelles.

Scrutinizing the stability of haemoglobin in 1,2,4-triazolium based ionic liquid

In this work, N-1 substituted 1,2,4-triazolium-based cation and trifluoromethanesulfonate anion based IL has been synthesized and used as a solvent system to stabilize haemoglobin (Hb). The conformational stability of Hb at different concentrations of developed IL was inspected through spectroscopic techniques like UV–Vis, steady-state fluorescence, fluorescence time-correlated single photon counting (TCSPC), temperature dependent UV–Vis, fluorescence, and circular dichroism (CD). Hb structure is stabilized in the IL concentration range 0.001 to 0.01 M; Hb structure starts opening up in the IL concentration range 0.05 to 0.1 M; at 0.2 M IL concentration, denaturation of Hb takes place. Further, the nature of the interaction between IL and Hb was verified through a competitive hydrophobic fluorescence probe, 1-Anilino-8-Naphthalene Sulfonate (ANS), and it was observed that ANS does not interact with IL till 0.01 M IL concentration. Finally, the experiments illustrate that the IL concentration plays a significant role in stabilizing/destabilizing a particular protein.

In-situ hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage

In-situ hydrophobic environment triggering reactive fluorescence probe to real-time monitor mitochondrial DNA damage

Real-time tracking of lipid droplets interactions with other organelles by a high signal/noise probe

Investigating the interactions between lipid droplets (LDs) and other organelles are greatly significant to the in-depth understand the physiology and pathology of LDs. Despite of commercial LDs probes available, the ER and other membrane structures are also stained due to their amphiphilicity, which limits their further applications. To address this issue, we herein report a highly hydrophobic fluorescent probe to specifically label intracellular LDs with high signal/noise ratio, avoiding undesirable staining of ER and thus achieving to real-time monitor the interactions between LDs and other organelles. The probe showed strong emission in lipid environments but no fluorescence in water and liposomes, enabling the probe with high signal/noise ratio. Using this probe, it was found that the contacts of LDs with ER and mitochondria clearly increased under the condition of starvation.

Revisiting the Rate-Limiting Step of the ANS-Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity.

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.

Life-Inspired Endogenous Dynamic Behavior of Lipid Droplet-like Microcompartments in Artificial Adipocyte-like Structures

Chemical systems that can replicate cellular behaviors are gaining increasing attention and are being used to study various biological processes. Here, a protein- or amylose-based assembly at an oi...

Disparate outer membrane exclusionary properties underlie intrinsic resistance to hydrophobic substances in Pseudomonas spp. isolated from surface waters under triclosan selection

Representative members of surface water microbiota were obtained from three unrelated municipal sites in Oklahoma by direct plating under selection by the hydrophobic biocide triclosan. Multiple methods were employed to determine if intrinsic triclosan resistance reflected resistance to hydrophobic molecules by virtue of outer membrane impermeability. While all but one organism isolated in the absence of triclosan were able to initiate growth on MacConkey agar, only one was able to initiate significant growth with triclosan present. In contrast, all bacteria selected with triclosan were identified as Pseudomonas spp. using 16S RNA gene sequencing and exhibited growth comparable to Pseudomonas aeruginosa controls in the presence of hydrophobic antibacterial agents to include triclosan. Two representative bacteria isolated in the absence of triclosan allowed for greater outer membrane association with the fluorescent hydrophobic probe 1-N-phenylnapthylamine than did two triclosan-resistant isolates. Compound 48/80 disruption of outer membrane impermeability properties for hydrophobic substances either partially or fully sensitized nine of twelve intrinsically resistant isolates to triclosan. These data suggest that outer membrane exclusion underlies intrinsic resistance to triclosan in some, but not all Pseudomonas spp. isolated by selection from municipal surface waters and implicates the involvement of concomitant triclosan resistance mechanisms.

Structure and properties of thermoresponsive gels formed by RAFT polymerization: effect of the RAFT agent content

We prepared the chemically crosslinked, cylindrical thermoresponsive poly(N-isopropylacrylamide) gels with changing the contents of reversible addition-fragmentation chain transfer (RAFT) agent. The environment in the gel was estimated by using a hydrophobic fluorescence probe. While the equilibrium swelling ratios were comparable, the deswelling response became fast as the content of RAFT agent increased with the formation of “bubble pattern”. This result indicates that the PNIPAAm gels prepared by RAFT still have spatial defects. In addition, the cooperative diffusion coefficients in swelling process of the gels prepared by RAFT polymerization were estimated.

Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition.

SummaryIn situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and β-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

Synthesis and Properties of Upper Critical Solution Temperature Responsive Nanogels.

A random copolymer ((U/A10)165) bearing pendent ureido groups and a small amount (10 mol %) of primary amino groups exhibits an upper critical solution temperature (UCST). We prepared a diblock copolymer (PMPC20P(U/A10)165) composed of water-soluble poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and (U/A10)165 blocks via reversible addition-fragmentation chain-transfer radical polymerization with postmodification reaction. The subnumbers are the degrees of polymerization of each block. Although in water PMPC20P(U/A10)165 dissolves as a unimer above the UCST phase transition temperature ( Tp), it forms polymer micelles composed of dehydrated (U/A10)165 cores and hydrophilic PMPC shells. A nanogel was prepared by cross-linking the pendent primary amines in the micelle core using (hydroxymethyl)phosphonium chloride below Tp. NMR and light-scattering data indicated that the nanogel core shrinks upon dehydration below Tp and swells upon hydration above Tp. The nanogel can encapsulate guest molecules such as hydrophobic fluorescence probes and bovine serum albumin (BSA) below Tp mainly owing to hydrophobic interactions in the core. Encapsulated BSA can be held in the nanogel core below Tp and subsequently released above Tp.

МОЛЕКУЛЯРНЫЕ ЭФФЕКТЫ ЭРИТРОПОЭТИНА НА ЛИПИДНЫЙ СОСТАВ МЕМБРАНЫ ЭРИТРОЦИТОВ КРОВИ ПУПОВИНЫ НОВОРОЖДЕННЫХ ПРИ ЦИТОМЕГАЛОВИРУСНОЙ ИНФЕКЦИИ В ПЕРИОД БЕРЕМЕННОСТИ

In order to identify the molecular effects of erythropoietin on the lipid composition and properties of the membrane of red blood cells of the umbilical cord, 35 newborn children born from cytomegalovirus seropositive women with exacerbation of infection in the third trimester of pregnancy (main group) were examined. The control group consisted of 30 newborns born from cytomegalovirus seronegative women. Plasma and red blood cells of the umbilical cord became the material for the study. In the membrane of red blood cells of the umbilical cord by spectrophotometry there were studied the products that react with thiobarbituric acid; membrane microviscosity was studied by lateral diffusion of hydrophobic fluorescent probe of pyrenes; lysophosphatidylcholine content by a thin-layer chromatography; the content of arachidonic, oleic and α-linolenic fatty acids by gas chromatography; ionized calcium content by analyzer of blood gases and electrolytes; erythropoietin in umbilical cord blood plasma by enzyme immunoassay. The number of erythrocytes was determined by an automatic hematological analyzer. The assessment of erythrocytes morphology and echinocytes counting was performed by light microscopy with further cytophotometrical data processing. In the umbilical cord blood of newborns born from cytomegalovirus seropositive women with exacerbation of infection in the third trimester of pregnancy, there was found out an increase in the level of erythropoietin by 20% (p<0.05), the number of erythrocytes by 21% (p<0.01), of products reacting with thiobarbituric acid by 60% (p<0.001), of lysophosphatidylcholine by 42% (p<0.001), of arachidonic acid by 31% (p<0.001)<0.001), of ionized calcium by 14% (p<0.05), of the amount of echinocytes by 91% (p<0.001); with a decrease in the content of oleic and α-linolenic fatty acids by 40% (p<0.001), there was an increase in the ratio of fluorescence of excimers and monomers (Fe/Fm) by 30% (p<0.001), the number of red blood cells by 21% (p<0.01). The results obtained suggested in the newborns from seropositive women to cytomegalovirus with exacerbation of the infection in the third trimester of pregnancy the development of ineffective erythropoiesis, enhancing oxidative damage of red blood cells, leading to echinocytosis and eryptosis

Dynamic pH-induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen.

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton-induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4 CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near-identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid-base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH-dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β-barrel domain in response to the fluctuating acidity of the thylakoid lumen.

Time-Resolved Structured Illumination Microscopy for Phase Separation Dynamics of Water and 2-Butoxyethanol Mixtures: Interpretation of "Early Stage" Involving Micelle-Like Structures.

Phase separation dynamics of a water/2-butoxyethanol (2BE) mixture was studied with newly developed time-resolved structured illumination microscopy (SIM). Interestingly, an employed hydrophobic fluorescent probe for SIM showed spectral shifts up to 500 ns after a laser-induced temperature jump, which suggests 2BE micellar-like aggregates become more hydrophobic at the initial stage of phase separation. This hydrophobic environment in 2BE aggregates, probably due to the ejection of water molecules, continued up to at least 10 μs. Time-resolved SIM and previously reported light scattering data clearly showed that the size of a periodic structure remained constant (ca. 300 nm) from 3 to 10 μs, and then the growth of periodic structures having the self-similarity started. We think that the former and the latter processes correspond to "early stage" (concentration growth) and "late stage" (size growth), respectively, in phase separation dynamics. Here we suggest that, in the early stage, the entity to bear 2BE phase be water-poor 2BE aggregates, and the number density of these aggregates would simply increase in time.