Retraction Note: Autoantibodies detection in patients affected by autoimmune retinopathies.

The article "Autoantibodies detection in patients affected by autoimmune retinopathies", by M.R. Ceccarini, M.C. Medori, K. Dhuli, S. Tezzele, G. Bonetti, C. Micheletti, P.E. Maltese, S. Cecchin, K. Donato, L. Colombo, L. Rossetti, G. Staurenghi, A.P. Salvetti, M. Oldani, L. Ziccardi, D. Marangoni, G. Iarossi, B. Falsini, G. Placidi, F. D'Esposito, F. Viola, M. Nassisi, G. Leone, L. Cimino, L. De Simone, V. Mastrofilippo, T. Beccari, M. Bertelli, published in Eur Rev Med Pharmacol Sci 2023; 27 (6 Suppl): 57-63-DOI: 10.26355/eurrev_202312_34690-PMID: 38112948 has been retracted by the Editor in Chief for the following reasons. Following some concerns raised on PubPeer, the Editor in Chief has started an investigation to assess the validity of the results. The outcome of the investigation revealed that the manuscript presented major flaws in the following: -       Issues with ethical approval -       Undeclared conflict of interest In light of concerns regarding the potential manipulation of Supplementary Figure 2, the journal's inquiry has been unable to conclusively determine whether the alterations noted on PubPeer constitute figure manipulation. The investigation yielded divergent evaluations. However, given the aforementioned concerns, the Editor in Chief doubts the integrity of the findings presented and thus, has opted to retract the article. The authors disagree with this retraction. This article has been retracted. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/34690.

Autoantibodies detection in patients affected by autoimmune retinopathies.

OBJECTIVE: Autoimmune retinopathies (ARs) encompass a spectrum of immune diseases that are characterized by the presence of autoantibodies against retinal proteins in the bloodstream. These autoantibodies (AAbs) lead to a progressive and sometimes rapid loss of vision. ARs commonly affect subjects over 50 years of age, but also rare cases of kids under 3 years of age have been reported. PATIENTS AND METHODS: In this study, 47 unrelated Caucasian patients were enrolled. All subjects showed negative cancer diagnoses and negative results in their genetic screenings. We studied 8 confirmed retinal antigens using Western blotting analysis, with α-enolase followed by carbonic anhydrase II being the two most frequently found in the patients' sera. RESULTS: Nineteen patients were positive (40.4%), thirteen uncertain (27.7%), and fifteen were negative (31.9%). Their gender did not correlate with the presence of AAbs (p=0.409). CONCLUSIONS: AAbs are responsible for retinal degeneration in some cases, while in others, they contribute to exacerbating the progression of the disease; however, their detection is crucial to reaching a better diagnosis and developing more effective treatments for these conditions. Moreover, finding good biomarkers is important not only for AR monitoring and prognosis, but also for helping with early cancer diagnosis.

Albumin Proteins as Delivery Vehicles for PFAS Contaminants into Respiratory Membranes.

Poly- and perfluoroalkyl substances (PFAS) are a family of chemicals that have been used in a wide range of commercial products. While their use is declining, the prevalence of PFAS, combined with their chemical longevity, ensures that detectable levels will remain in the environment for years to come. As such, there is a pressing need to understand how PFAS contaminants interact with other elements of the human exposome and the consequences of these interactions for human health. Using serum albumin as a model system, we show that proteins can bind PFAS contaminants and facilitate their incorporation into model pulmonary surfactant systems and lipid bilayers. Protein-mediated PFAS delivery significantly altered the structure and function of both model membrane systems, potentially contributing to respiratory dysfunction and airway diseases in vivo. These results provide valuable insights into the synergistic interaction between PFAS contaminants and other elements of the human exposome and their potential consequences for human health.

The partitioning of primary alcohols into the aggregates of gemini amphiphiles determined from diffusion NMR experiments.

The partition constants (p-values) of primary alcohols in solutions containing aggregates of symmetric gemini surfactants of the family N,N'-dimethyl, N-dialkyl-α,ω-alkanediammonium dibromide (m-s-m = symmetric gemini surfactants) have been computed from the measured values of their diffusion coefficients obtained from NMR-diffusion experiments. From the p-values, both mole-fraction and concentration-based partition coefficients and Gibbs energies of transfer for the alcohols from the bulk D2O phase to the gemini aggregate phase have been calculated. As expected, the Gibbs energy of transfer decreased linearly with an increase in the alcohol carbon length for each of the primary alcohol/gemini amphiphile series studied. The Gibbs transfer energy increment per CH2 for the alcohols was consistent for all the alcohol/gemini amphiphile series and was in excellent agreement with the values measured for the same primary alcohol series in conventional single-headed, single-tailed surfactants. Surprisingly, the partition coefficients of the alcohols in the symmetric gemini aggregates exhibited little, if any, dependence on the spacer length of the gemini amphiphiles and were remarkably consistent as the length of the main surfactant chain increased at constant spacer length. When these results are compared to the partition coefficients of the same alcohols in corresponding monomeric surfactants, we observe little difference in the thermodynamic driving forces governing the transfer of alcohols from water to the aggregates of either monomeric or symmetric gemini surfactants.

Lactate shuttling as an allostatic means of thermoregulation in the brain.

Lactate, the redox-balanced end product of glycolysis, travels within and between cells to fulfill an array of physiologic functions. While evidence for the centrality of this lactate shuttling in mammalian metabolism continues to mount, its application to physical bioenergetics remains underexplored. Lactate represents a metabolic "cul-de-sac," as it can only re-enter metabolism by first being converted back to pyruvate by lactate dehydrogenase (LDH). Given the differential distribution of lactate producing/consuming tissues during metabolic stresses (e.g., exercise), we hypothesize that lactate shuttling vis-à-vis the exchange of extracellular lactate between tissues serves a thermoregulatory function, i.e., an allostatic strategy to mitigate the consequences of elevated metabolic heat. To explore this idea, the rates of heat and respiratory oxygen consumption in saponin-permeabilized rat cortical brain samples fed lactate or pyruvate were measured. Heat and respiratory oxygen consumption rates, and calorespirometric ratios were lower during lactate vs. pyruvate-linked respiration. These results support the hypothesis of allostatic thermoregulation in the brain with lactate.

Mixing behaviour of Pluronics with gemini surfactant/plasmid DNA condensates: effect of Pluronic composition.

Nanoparticles prepared from plasmid DNA (pDNA) and N,N-bis(dimethylhexadecyl)-1,3-propanediammonium bromide (16-3-16) have been mixed with various Pluronic block copolymers and investigated as binary surfactant systems in water using the previously demonstrated critical aggregation concentration of the surfactant-DNA complex. Surface tensiometry was used to determine critical micelle concentrations of mixed micelles formed within these Pluronic/16-3-16/pDNA mixtures. Use of mixed micelle theories reveals that mixed micelle composition and the interaction parameter, ß, are influenced by the structure, in particular hydrophobicity, of the Pluronic component. Ethidium bromide fluorescence studies demonstrate the ability of the Pluronics to de-condense the plasmid DNA from the cationic 16-3-16 gemini surfactant complex, and show some relationship to the interaction parameter and Pluronic composition.

m-s-m cationic gemini and zwitterionic surfactants - a thermodynamic analysis of their mixed micelle formation.

Micelle formation enthalpies (Δmic H values) have been calorimetrically determined at 298 K for three sets of mixed zwitterionic/cationic gemini systems consisting of N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12) and a series of structurally related gemini surfactants, the N,N'-bis(dimethyldodecyl)-α,ω-alkanediammonium dibromide (12-s-12) systems. From the experimental and the estimated ideal micelle formation enthalpies, the excess enthalpies were obtained. The degrees of nonideality of the interaction in the mixed micelle (ß m) from our previous work was used along with the excess enthalpy values to determine excess thermodynamic quantities of the surfactants in the mixed system according to Regular Solution Theory (RST) and Motomura's theory. The excess enthalpies for the ZW3-12/12-4-12 were positive in magnitude and rose sharply when small amounts of the zwittergent were distributed into the gemini micelles. The excess enthalpies for the ZW3-12/12-5-12 and the ZW3-12/12-6-12 systems were also >0 kJ mol-1, and as a function of zwittergent composition, were quite different to those of the ZW3-12/12-4-12 mixed micelles. These results indicate that the heat of mixed micelle formation is strongly dependent on electrostatic interactions and the structure of the surfactants involved, specifically, the length of the tether group for the 12-s-12 gemini surfactants. From the calorimetric data and the application of RST and Motomura's theory, we have obtained estimates of the excess Gibbs energy and entropy of mixing. An analysis of the three thermodynamic properties suggests that the relative contributions of enthalpic and entropic effects to nonideal behavior for mixed micelles involving gemini surfactants are strongly dependent on the gemini structure.

m-s-m Cationic Gemini and Zwitterionic Surfactants-Spacer-Dependent Synergistic Interactions.

Critical micelle concentration (cmc) values were determined for the mixed zwitterionic/cationic gemini systems of N-dodecyl- N, N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12)/ N, N'-bis(dimethyldodecyl)-α,ω-alkanediammonium dibromide (12-s-12) systems. The cmc values for the mixed systems were determined through conductivity measurements. The degree of nonideality of the interaction in the mixed micelle (ßm), for each system, was determined according to Rubingh's nonideal solution theory. In most cases, the systems exhibited negative deviations (-ßm values) at high surfactant mole fractions of zwittergent (αZW3-12). Specifically, the ZW3-12/12-4-12 system displayed -ßm values at αZW3-12 ≥ 0.5, while both the ZW3-12/12-5-12 and the ZW3-12/12-6-12 systems displayed -ßm values over the entire mole fraction range. Except for the low mole fraction range in the 12-4-12 system, these mixed surfactant systems demonstrated almost identical behavior to the n-dodecyltrimethylammonium bromide/12-2-12 system studied by Bakshi et al. providing further evidence that ZW3-12 tends to behave as a cationic surfactant in mixed surfactant systems. The manner in which the cosurfactants aggregate in the micelles was determined via two-dimensional (2D) NOESY spectroscopy. In the case of both the ZW3-12/12-5-12 and the ZW3-12/12-6-12 systems, the 2D NOESY spectra exhibited strong cross peaks between the gemini and zwitterionic surfactants over the entire micellar composition range. In the case of the ZW3-12/12-4-12 system, little cross peak intensity was observed between the gemini and the zwitterionic surfactant at low micellar compositions of the zwittergent. The results suggest some micelle demixing is occurring between the gemini and the zwittergent certain micellar composition ranges, a phenomenon rarely associated with hydrocarbon surfactants.

The micellar and surface properties of a unique type of two-headed surfactant--pentaerythritol based di-cationic surfactants.

The surface properties of some families of cationic two-headed surfactants based on a pentaerythritol backbone are described. The compounds have the following general structure (1), where R' are head groups and R are linear alkyl groups ranging from octyl to tetradecyl. The syntheses of these compounds has been published in detail previously. Critical micelle concentrations (cmc values) of these two-headed surfactants have been determined and compared to conventional ionic surfactants and gemini surfactants of similar structure. In addition, the surface activity of these two-headed surfactants, expressed as the C20 value and the surface tension at the cmc, have been determined. Transmission electron microscopy has been used to examine the morphology of the aggregates formed by these amphiphiles. In general, when compared to conventional ionic and two-headed surfactants, these new two-headed surfactants exhibit a remarkable efficiency in the tendency to self-assemble and are significantly more surface active than their conventional counterparts [structure: see text].

Nonaromatic hydrotropic cationic ammonium salts as a rheology modifier for an anionic/zwitterionic surfactant mixture.

In this article, we report additive-induced micellar growth and rheology modification for mixtures of anionic (sodium dodecyl sulfate, SDS) and zwitterionic (N-alkylated glycine derivative, Empigen BB or EBB) surfactants. Two nonaromatic hydrotropic salts (hexyltrimethylammonium bromide, C6TAB, and/or dibutylenebis(dimethylbutylammonium bromide), 4-4-4) are used as novel additives to induce micellar growth in these systems. Nuclear magnetic resonance (NMR), photon correlation spectroscopy (PCS), transmission electron microscopy (TEM), the Weissenberg effect, and rheology measurements were employed to assess mixed micelle formation, micellar growth, and rheology modifications. Finally, the manner in which the surfactants and hydrotropes self-assemble into aggregates has also been deduced from 2D NMR NOESY measurements. In this study, both hydrotropic ions have been found to contribute to similar structural modifications in the mixed micelles of the anionic and zwitterionic surfactants. However, the extent of the rheology modification in solution is found to be quite different when the gemini hydrotrope (4-4-4) versus the monomeric hydrotrope (C6TAB) is employed.

A longitudinal follow-up study of saffron supplementation in early age-related macular degeneration: sustained benefits to central retinal function.

Objectives. In a previous randomized clinical trial (Falsini et al. (2010)), it was shown that short-term Saffron supplementation improves retinal flicker sensitivity in early age-related macular degeneration (AMD). The aim of this study was to evaluate whether the observed functional benefits from Saffron supplementation may extend over a longer follow-up duration. Design. Longitudinal, interventional open-label study. Setting. Outpatient ophthalmology setting. Participants. Twenty-nine early AMD patients (age range: 55-85 years) with a baseline visual acuity >0.3. Intervention. Saffron oral supplementation (20 mg/day) over an average period of treatment of 14 (±2) months. Measurements. Clinical examination and focal-electroretinogram-(fERG-) derived macular (18°) flicker sensitivity estimate (Falsini et al. (2010)) every three months over a followup of 14 (±2) months. Retinal sensitivity, the reciprocal value of the estimated fERG amplitude threshold, was the main outcome measure. Results. After three months of supplementation, mean fERG sensitivity improved by 0.3 log units compared to baseline values (P < 0.01), and mean visual acuity improved by two Snellen lines compared to baseline values (0.75 to 0.9, P < 0.01). These changes remained stable over the follow-up period. Conclusion. These results indicate that in early AMD Saffron supplementation induces macular function improvements from baseline that are extended over a long-term followup.

Spectral and hydrodynamic studies on p-toluidine induced growth in cationic micelle.

The effect of p-toluidine (PTD) on the growth of cationic surfactant micelles in aqueous solutions was examined by viscosity, UV-visible spectroscopy, dynamic light scattering (DLS), (1)H NMR and nuclear Overhauser effect spectroscopy (NOESY). Viscosity and scattering results are used to follow the growth of the aggregates and examine the structural transitions that occur. The reduction of electrostatic repulsions between the surfactant headgroups in combination with the hydrophobicity of the additive caused micellar growth and viscoelasticity at high PTD concentration; the micellar growth was found to be more pronounced in the presence of salt. (1)H NMR chemical shift changes reflect the interaction of PTD molecules with surfactant micelles while 2D NOESY experiments reveal that PTD molecules insert themselves in micelles such that its -CH(3) group is in contact with the core region and the benzene ring resides near the palisade layer of micelle.

Mixed aggregate formation in gemini surfactant/1,2-dialkyl-sn-glycero-3-phosphoethanolamine systems.

An evaluation of the physical interactions between gemini surfactants, DNA, and 1,2-dialkyl-sn-glycero-3-phosphoethanolamine helper lipid is presented in this work. Complexation between gemini surfactants and DNA was first investigated using surface tensiometry where the surface tension profiles obtained were found to be consistent with those typically observed for mixed surfactant-polymer systems; that is, there is a synergistic lowering of the surface tension, followed by a first (CAC) and second (CMC) break point in the plot. The surfactant alkyl tail length was observed to exhibit a significant effect on the CAC, thus demonstrating the importance of hydrophobic interactions during complexation between gemini surfactants and DNA. The second study presented is an investigation of the mixing interactions between gemini surfactants and DOPE using Clint's, Rubingh's, and Motomura's theories for mixed micellar formation. The mixing interactions between the 16-3-16/16-7-16/16-12-16/16-7NH-16 gemini surfactants and DOPE were observed to be antagonistic, where the strength of antagonism was found to be dependent upon the gemini surfactant spacer group and the solution composition.

Simulations of a lattice model of two-headed linear amphiphiles: influence of amphiphile asymmetry.

Using a 2D lattice model, we conduct Monte Carlo simulations of micellar aggregation of linear-chain amphiphiles having two solvophilic head groups. In the context of this simple model, we quantify how the amphiphile architecture influences the critical micelle concentration (CMC), with a particular focus on the role of the asymmetry of the amphiphile structure. Accordingly, we study all possible arrangements of the head groups along amphiphile chains of fixed length N = 12 and 16 molecular units. This set of idealized amphiphile architectures approximates many cases of symmetric and asymmetric gemini surfactants, double-headed surfactants, and boloform surfactants. Consistent with earlier results, we find that the number of spacer units s separating the heads has a significant influence on the CMC, with the CMC increasing with s for s < N/2. In comparison, the influence of the asymmetry of the chain architecture on the CMC is much weaker, as is also found experimentally.

Micellization and solubilization of a model hydrophobic drug nimesulide in aqueous salt solutions of Tetronic T904.

The micellar and phase behavior of an ethylene oxide-propylene oxide branched octablock copolymer Tetronic T 904 (hereafter written as T904) in water and NaCl solutions was examined. The copolymer shows a cloud point (CP) ranging from 74-65°C in the concentration range of 1-10% and forms aggregates (micelles) with a hydrodynamic diameter around 10-12nm in the temperature range 30-40°C. Stable, bluish solutions containing aggregates of variable size (several hundred nm in some cases) were observed even at temperatures much less than the critical micellization temperature (CMT=30°C for a 2% solution in water). The CP and the CMT markedly decrease in the presence of NaCl due to the dehydration of the polyethylene oxide shell. The size of the micelles in water or salt solutions increases at temperatures close to the CP as inferred from viscosity measurments. A model drug compound (nimesulide, NIM) was solubilized in T904 micelles which showed a remarkable increase in solubilization at higher temperature; however, a decrease in solubilization was observed in salt solutions. The thermodynamic parameters for solubilization were obtained, and the location of NIM in the copolymer micelles was investigated by UV-Visible spectroscopy.

Induced micellization and micellar transitions in aqueous solutions of non-linear block copolymer Tetronic® T904.

Tetronics® (Poloxamines) are the least studied block copolymers with an X-shaped molecular geometry formed by four poly (propylene oxide, PPO) and poly (ethylene oxide, PEO) block chains, bonded to a central ethylenediamine group. Compared to their linear counterparts, the Pluronics, Tetronics® are novel, in that they possess superior physicochemical properties, and are relatively less studied. A complete understanding of their solution behavior under different solution conditions can make them competitive candidates for novel drug delivery systems. The micellization behavior and aqueous solution properties of Tetronic® T904 [(EO(15)PO(17)) (2)NCH(2)CH(2)N (PO(17)EO(15))(2)] have been determined by cloud point, viscosity, dynamic light scattering (DLS), small-angle neutron scattering (SANS), and nuclear magnetic resonance (NMR) measurements. The copolymer formed spherical micelles at 30°C with a core radius (R(c)) of about 2.5 nm, a hard sphere radius (R(hs)) of 5.2 nm and an aggregation number (N(agg)) of 10. The effect of copolymer concentration, temperature, pH and salt on the micellar and phase behavior is examined. The copolymer has been found to exist in aggregated form only at higher pH values of >8. An increase in micelle size and aggregation has been observed for an increase in temperature and salt concentration, mainly due to the dehydration of the hydrated PEO shell. The added salts (NaCl, Na(2)SO(4) and Na(3)PO(4)) induce micellization and favor the micellar transition at lower temperatures due to the "salting out effect"; the effect of anions follows the Hofmeister series (PO(4)(3-)>SO(4)(2-)>Cl(-)).

Synthesis of surfactants based on pentaerythritol. I. Cationic and zwitterionic gemini surfactants.

Simple strategies for the synthesis of five series of cationic gemini surfactants and one series of zwitterionic gemini surfactants from pentaerythritol have been developed. Two lipophilic groups were introduced onto pentaerythritol by alkylation of the known compound, O-benzylidenepentaerythritol, with 1-bromooctane, 1-bromodecane, 1-bromododecane, and 1-bromotetradecane. Hydrogenolysis of the benzylidene acetals gave diols which were converted into three different series of trimethylammonium derivatives. The diiodides derived from the diols could be displaced by dimethylamine, even though they are adjacent to a quaternary carbon atom. Alkylation with methyl iodide gave the first series. The iodides were easily displaced by cyanide ion and the resulting dinitriles were hydrolyzed, converted to N,N-dimethylamides, and reduced to give a second series. Oxidation of the diols to dialdehydes under Swern conditions followed by Horner-Wadsworth-Emmons reactions with diethyl N,N-dimethylcarbamoylmethylphosphonate followed by two-stage reduction gave a third series. The dialkoxides derived from the four di-O-alkylpentaerthritol diols were reacted with 2-dimethylaminoethyl chloride and 3-dimethylaminopropyl chloride in neighboring-group assisted double Williamson ether syntheses to give precursors to two more series. As expected because the neighboring group participation occurs through a four-membered-ring intermediate, considerably more vigorous conditions were required for the reactions with 3-dimethylaminopropyl chloride. 2-Diethylaminoethyl bromide was found to be less reactive than 2-dimethylaminoethyl chloride. The products were alkylated with methyl iodide and, in some cases, other alkyl halides to give cationic gemini surfactants. Alkylation of one series with ethyl bromoacetate followed by anion exchange resin catalyzed ester hydrolysis gave zwitterionic gemini surfactants. The members of all series have superior surfactant properties.

Spontaneous vesicle formation with an ionic liquid amphiphile.

A simple and effective method for the formation of stable multilamellar vesicles is reported as a potential application of ionic liquid materials (IL's) and as replacements for conventional surfactants used in such applications. The methodology is based on the various approaches for the formation of vesicles from oppositely charged surfactants. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) have been used to estimate the size of the aggregates; the TEM studies have also revealed morphological differences in the self-assembled systems with changing ionic liquid material. Size measurements from PCS indicate consistent growth of the ionic-liquid containing vesicles with increasing concentration of added anionic surfactant. 2D NOESY NMR spectroscopy have been used to examine the manner in which IL amphiphile self-assembles with the second surfactant in solution. A comparison has been made between the aggregates formed from hexylpyridinium tetrafluoroborate ([HexPy][BF4-])/sodium dodecylsulfate (SDS) and hexylpyridinium bromide ([HexPy][Br])/sodium dodecylsulfate (SDS).

Synergistic interactions in the mixed micelles of cationic gemini with zwitterionic surfactants: the pH and spacer effect.

Mixed micelle formation of binary cationic gemini (12-s-12, s=4, 6) and zwitterionic (N-dodecyl-N,N-dimethylglycine, EBB) surfactants has been investigated by measuring the surface tension of aqueous solution as a function of total concentration at various pH values from acidic to basic, under conditions of 298.15 K and atmospheric pressure. The results were analyzed by applying regular solution theory (RST), and Motomura's theory, which allows for the calculation of the excess Gibbs energy of micellization purely on the basis of thermodynamic equations. The synergistic interactions of all the investigated cationic gemini + zwitterionic surfactants mixtures were found to be dependent upon the pH of the solution and the length of hydrophobic spacer of gemini surfactant. The evaluated excess Gibbs free energy is negative for all the systems.

Interactions between zwitterionic and conventional anionic and cationic surfactants.

Critical micelle concentration (cmc) values have been determined for the mixed zwitterionic/anionic surfactant systems of N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12)/sodium dodecyl sulfate (SDS), N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB)/SDS, N-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-08)/sodium octyl sulfate (SOS), and the zwitterionic/cationic systems of ZW3-12/dodecyltrimethylammonium bromide (DTAB), DDMAB/DTAB. Conductivity studies and nuclear magnetic resonance (NMR) spectroscopy were the methods employed for cmc determinations. The degree of nonideality of the interaction in the micelle (beta(m)), for each system, was determined according to Rubingh's nonideal solution theory. Evidence was found for the existence of strong interactions between zwitterionic and anionic surfactants in each of the zwitterionic/anionic systems. The ZW3-08/SOS and DDMAB/SDS systems behaved synergistically at all mole fractions studied while the ZW3-12/SDS system exhibited synergistic behavior above mole fractions of 0.30. Greater negative deviations from ideal behavior were demonstrated in the DDMAB/SDS system than in the other two zwitterionic/anionic systems. The zwitterionic/cationic systems of ZW3-12/DTAB and ZW3-08/OTAB displayed only slight deviations from ideal behavior, therefore indicating near ideal mixing.