Diffusion Limitations in Root Uptake of Cadmium and Zinc, But Not Nickel, and Resulting Bias in the Michaelis Constant

It has long been recognized that diffusive boundary layers affect the determination of active transport parameters, but this has been largely overlooked in plant physiological research. We studied the short-term uptake of cadmium (Cd), zinc (Zn), and nickel (Ni) by spinach (Spinacia oleracea) and tomato (Lycopersicon esculentum) in solutions with or without metal complexes. At same free ion concentration, the presence of complexes, which enhance the diffusion flux, increased the uptake of Cd and Zn, whereas Ni uptake was unaffected. Competition effects of protons on Cd and Zn uptake were observed only at a very large degree of buffering, while competition of magnesium ions on Ni uptake was observed even in unbuffered solutions. These results strongly suggest that uptake of Cd and Zn is limited by diffusion of the free ion to the roots, except at very high degree of solution buffering, whereas Ni uptake is generally internalization limited. All results could be well described by a model that combined a diffusion equation with a competitive Michaelis-Menten equation. Direct uptake of the complex was estimated to be a major contribution only at millimolar concentrations of the complex or at very large ratios of complex to free ion concentration. The true K_m for uptake of Cd²⁺ and Zn²⁺ was estimated at <5 nm, three orders of magnitude smaller than the K_m measured in unbuffered solutions. Published Michaelis constants for plant uptake of Cd and Zn likely strongly overestimate physiological ones and should not be interpreted as an indicator of transporter affinity.Internalization of metals by biota is traditionally described by Michaelis-Menten kinetics (Wilkinson and Buffle, 2004). The K_m corresponds to the concentration in solution at which the uptake is one-half of the maximal uptake, F_max. The Michaelis-Menten equation relates the uptake flux, F, to the free ion concentration at the site of uptake, [M]_s:If diffusion of a metal across a diffusive boundary layer adjacent to the roots is the rate-limiting step for uptake, the concentration at the site of uptake will be lower than that in the bulk solution. As a result, diffusion limitations result in an overestimate of the K_m, if the concentration at the root surface is assumed to be the same as in the bulk solution, as is usually done. This bias in K_m has been discussed in detail by Winne (1973) and has, for instance, been demonstrated experimentally for uptake of Glc in rabbit jejunum (Thomson and Dietschy, 1980) and for uptake of several sugars, amino acids, and bile acids in rat ileum (Wilson and Dietschy, 1974).Models used to predict ion availability and toxicity of metals by plants usually rely on the assumption that uptake is controlled by the free metal ion activity and the activity of competing ions in the bulk solution. For instance, the biotic ligand model (BLM), originally developed to predict metal toxicity to aquatic organisms, assumes that toxicity of an ion is mitigated by the presence of competing ions that bind on the biotic ligand (Paquin et al., 2002). Hough et al. (2005) used a free ion activity model taking into account proton competition effects to predict cadmium (Cd) uptake by soil-grown ryegrass (Lolium perenne). The uptake was reasonably well predicted; however, as the authors pointed out, it was not clear whether the derived constants truly represented physiological affinity constants or were just fitting parameters in a rate-limited uptake process. In case of strong diffusion limitation, ion competition effects on the internalization are expected to have negligible effect on the uptake, as the uptake is controlled by diffusion and not by internalization (Campbell et al., 2002; Degryse and Smolders, 2012).In previous studies, we found strong evidence that uptake of Cd and zinc (Zn) is limited by the diffusive transport of the free metal ion to the root at low free ion concentration. At constant free ion concentration, the uptake of Cd and Zn increased in presence of metal complexes and the contribution of the complex increased with increasing dissociation rate of the complex (Degryse et al., 2006a, 2006c). In unbuffered solutions, i.e. solutions without metal complexes, stirring increased Cd uptake by plants (Degryse and Smolders, 2012). For nickel (Ni), however, contribution of complexes was small or undetectable, and stirring did not increase the uptake (Degryse and Smolders, 2012). Given this evidence that Cd and Zn uptake by plants is limited by diffusion, it is likely that published K_m values for uptake of Cd²⁺ and Zn²⁺ by plants overestimate true physiological values. This bias in the K_m when a diffusive boundary layer is present has been largely ignored in plant-physiological research. Indeed, in numerous studies the K_m value has been interpreted as a characteristic of the carrier-mediated transport process, while in many cases it may reflect mass transfer properties. In addition, these diffusion limitations may mask ion competition effects in the uptake.The aim of this article was (1) to present evidence that K_m values determined for Cd²⁺ and Zn²⁺ uptake by plants in general reflect transport limitations rather than transporter affinity; (2) to derive true K_m values by determining the K_m under conditions where the uptake is not transport limited; (3) to identify the consequence of diffusion limitations on competition effects; and (4) to describe uptake of Cd, Zn, and Ni by plants in a single comprehensive model that combines competitive Michaelis-Menten kinetics with a diffusion equation.

Theoretical Framework

In the following, we qualitatively discuss the bias in the K_m because of diffusion limitations, based on Figure 1. Equations are given in the “Materials and Methods” section. Figure 1A presents a case where the potential internalization flux by the plant at low concentrations is much larger than the maximal rate at which the free ion can be supplied through diffusive transport of the ion to the root surface. In this case, the actual uptake flux by the plant will approach the maximal diffusive flux, and the free ion concentration at the root surface is much smaller than that in the bulk solution. The apparent K_m (K_m*; determined as the concentration where the uptake flux is one-half of the maximal uptake) is much larger than the true K_m value. In Figure 1B, the potential internalization flux at low concentration is of the same order of magnitude as the maximal diffusion flux. In this case, the concentration at the root surface is slightly smaller than that in the bulk solution, and there is only a slight bias in the K_m value.Open in a separate windowFigure 1.Conceptual diagram of the internalization flux (Michaelis-Menten curve; full line), maximal diffusive supply from solution to root (dashed line), and actual uptake flux (dotted line) as a function of free ion concentration for two theoretical cases. Left and right sections show the same curves, on log (left) or linear (right) scale. In A, the potential internalization flux is much larger than the maximal diffusive supply at low concentrations, i.e. the uptake is strongly limited by the transport of the free ion to the root. The plant acts as a near-zero sink, and the actual plant uptake equals the maximal diffusive flux. The K_m* is much larger than the true K_m, and the experimental permeability P (slope of the actual uptake curve) is much smaller than the membrane permeability P_m (slope of the internalization curve). In B, the maximal diffusive flux is larger than the potential internalization flux. The uptake is not limited by diffusive transport, and the K_m* and true K_m are almost equal.Given the evidence that uptake of Cd and Zn by plants is diffusion limited, even in stirred nutrient solutions, we hypothesize that reported K_m values of Cd²⁺ and Zn²⁺ are biased, and that the physiological K_m values are much smaller. To test this hypothesis, we measured the uptake of Cd, Zn, and Ni in solution, in absence or in presence of labile hydrophilic metal complexes. If diffusion limitations prevail, the complexes dissociate within the diffusion layer and thus enhance the diffusion flux and therefore the metal uptake. By adding labile complexes in large amounts, it should be possible to abolish the diffusion barrier completely, in which case the physiological K_m can be determined.In addition, the effect of competitive ions on uptake was tested. The presence of competitive ions decreases the internalization flux. However, if uptake is rate limited by the diffusive transport to the uptake site and not by internalization, competition effects should theoretically not affect the uptake flux.     

Synthesis of biologically stable gold nanoparticles using imidazolium-based amino acid ionic liquids

A novel double-step reduction procedure for the synthesis of gold nanoparticles (AuNPs) using amino acid ionic liquids has been employed. 1-Dodecyl-3-methyl imidazolium tryptophan ([C(12)mim]Trp) and 1-ethyl-3-methyl imidazolium tryptophan ([C(2)mim]Trp) were used for this synthesis. The synthesized AuNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. The behavior of these AuNPs were also probed in a biological media. It was proven that AuNPs synthesized at [C(12)mim]Trp have more stability than AuNPs synthesized at [C(2)mim]Trp due to the longer alkyl chain of the imidazolium moiety. The solubility test shows that the resultant AuNPs have a hydrophilic nature. Finally, it was seen that due to the presence of a biomolecule, namely Trp, in the structure of AuNPs protecting shell, higher stability and biocompatibility was achieved in the biological media.     

Synthesis and characterization of cobalt oxide nanoparticles by thermal treatment process

The simple preparation of Co₃O₄ nanoparticles from a solid organometallic molecular precursor N-N′-bis(salicylaldehyde)-1,2-phenylenediimino cobalt(II); Co(salophen) has been achieved via two simple steps: firstly, the Co(salophen) precursor was precipitated from the reaction of cobalt(II) acetate and N-N′-bis(salicylaldehyde)-1,2-phenylenediimino; H₂salophen; in propanol under nitrogen condition; then, cubic phase Co₃O₄ nanoparticles with the size of mostly 30-50 nm could be produced by thermal treatment of the Co(salophen) in air at 773 K for 5 h. The as-synthesized products were characterized by powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and scanning electronic microscopy (SEM). These results confirm that the resulting oxide was pure single-crystalline Co₃O₄ nanoparticles. The optical property test indicates that the absorption peak of the nanoparticles shifts towards short wavelength, and the blue shift phenomenon might be ascribed to the quantum effect. The hysteresis loops of the obtained samples reveal the ferromagnetic behaviors the enhanced coercivity (H_c) and decreased saturation magnetization (M_s) in contrast to their respective bulk materials.     

Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran.Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.     

Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film

The composite film based on Nafion and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium chloride ([bmim]Cl) was used as an immobilization matrix to entrap myoglobin (Mb). The study of ionic liquid (IL)-Mb interaction by ultraviolet-visible (UV-vis) spectroscopy showed that Mb retains its native conformation in the presence of IL. The immobilized Mb displayed a pair of well-defined cyclic voltammetric peaks with a formal potential (E_o^’) of −0.35 V in a 0.1 M phosphate buffer solution (PBS) of pH 7.0. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which a mediator-free amperometric biosensor for hydrogen peroxide was designed. The linear range for the determination of hydrogen peroxide was from 1.0 to 180 μM with a detection limit of 0.14 μM at a signal/noise ratio of 3. The apparent Michaelis constant () for the electrocatalytic reaction was 22.6 μM. The stability, repeatability, and selectivity of the sensor were evaluated. The proposed biosensor has a lower detection limit than many other IL-heme protein-based biosensors and is free from common interference in hydrogen peroxide biosensors.     

An improved PCR-based amplification of unknown homologous DNA sequences

The PCR primers used for cloning of evolutionary conserved genes or homologous DNA sequences are usually guessmer oligonucleotides. We introduce a simple way using Pfu polymerase to overcome possible PCR amplification failure because of 3'-end mismatches of guessed primers with the target DNA.     

IL-4 down-regulates anaphylatoxin receptors in monocytes and dendritic cells and impairs anaphylatoxin-induced migration in vivo

Anaphylatoxins mobilize leukocytes to the sites of inflammation. In the present study we investigated the impact of GM-CSF, IL-4, and IFN-gamma on anaphylatoxin receptor expression in monocytes and dendritic cells (DC). IL-4 was identified as the strongest down-regulator of the receptors for C5a and C3a in monocytes and monocyte-derived DC (MoDC). To study the impact of IL-4 on anaphylatoxin-induced chemotaxis, an in vivo migration model was established. For this purpose, human monocytes and MoDC were injected i.v. into SCID mice that at the same time received anaphylatoxins into the peritoneal cavity. A peritoneal influx of human monocytes could be demonstrated by 4 h after injections of C5a and C3a. In line with receptor down-regulation, IL-4 treatment inhibited in vivo mobilization of human monocytes and MoDC in response to C5a and C3a. In addition to its effects on human cells, IL-4 reduced C5a receptors in murine bone marrow-derived DC and impaired recruitment of labeled bone marrow-derived DC in syngeneic BALB/c mice to i.p. injected C5a. Overall, these data suggest that inhibition of a rapid anaphylatoxin-induced mobilization of monocytes and DC to inflamed tissues represents an important anti-inflammatory activity of the Th2 cytokine IL-4.     

Enhanced Optical Transmission Through Metallic Holes Array: Role of TE Polarization in SPP Excitation

Optical transmission through double-layer metallic subwavelength holes array is studied under oblique incidence by split-field finite-difference time-domain method. Both TM and TE polarizations are investigated. It is proved that the transmission peaks can also be observed for TE polarization due to the excitation of surface plasmon polaritons (SPP) through diffraction orders. By changing the incident angle, these transmission peaks follow the SPP wavelength shift. The field profiles, even for the field components not present in the incident field, clearly show the SPP excitation. The mechanism of enhanced transmission will be fully discussed.     

Determination of the axial and circumferential mechanical properties of the skin tissue using experimental testing and constitutive modeling

The skin, being a multi-layered material, is responsible for protecting the human body from the mechanical, bacterial, and viral insults. The skin tissue may display different mechanical properties according to the anatomical locations of a body. However, these mechanical properties in different anatomical regions and at different loading directions (axial and circumferential) of the mice body to date have not been determined. In this study, the axial and circumferential loads were imposed on the mice skin samples. The elastic modulus and maximum stress of the skin tissues were measured before the failure occurred. The nonlinear mechanical behavior of the skin tissues was also computationally investigated through a suitable constitutive equation. Hyperelastic material model was calibrated using the experimental data. Regardless of the anatomic locations of the mice body, the results revealed significantly different mechanical properties in the axial and circumferential directions and, consequently, the mice skin tissue behaves like a pure anisotropic material. The highest elastic modulus was observed in the back skin under the circumferential direction (6.67 MPa), while the lowest one was seen in the abdomen skin under circumferential loading (0.80 MPa). The Ogden material model was narrowly captured the nonlinear mechanical response of the skin at different loading directions. The results help to understand the isotropic/anisotropic mechanical behavior of the skin tissue at different anatomical locations. They also have implications for a diversity of disciplines, i.e., dermatology, cosmetics industry, clinical decision making, and clinical intervention.     

Prolonged viral shedding and antibody persistence in patients with COVID-19

SARS-CoV-2 as a new global threat has affected global population for one year. Despite the great effort to eradicate this infection, there are still some challenges including different viral presentation, temporal immunity in infected individuals and variable data of viral shedding. We studied 255 COVID-19 suspected individuals to assess the viral shedding duration and also the antibody development against SARS-CoV-2 among the cases. Real Time RT-PCR assay was applied to determine the virus presence and SARS-CoV-2 antibodies were evaluated using SARS-CoV-2 IgM and IgG kits. 113 patients were confirmed for COVID-19 infection. The patients were followed until negative PCR achieved. The median viral shedding among studied population was obtained 34.16 (±17.65) days which was not significantly associated with age, sex and underlying diseases. Shiver and body pain were found in prolonged form of the infection and also patients who had gastrointestinal problems experienced longer viral shedding. Moreover, IgG was present in 84% of patients after 150 days. According to this data, the median viral shedding prolongation was 34.16 days which indicates that 14 days isolation might not be enough for population. In addition, IgG profiling indicated that it is persistent in a majority of patients for nearly 6 months which has brought some hopes in vaccine efficacy and application.