Concentration Of Binding Sites Research Articles

The role of cell surface sulfhydryl and amine binding sites in the removal of Cr(VI) from solution by Bacillus subtilis bacterial cells

A range of bacterial species are known to be capable of reducing soluble Cr(VI) to Cr(III), causing the precipitation of Cr(III) phases from solution, but the mechanism responsible for the initial interaction between anionic aqueous Cr(VI) and the negatively charged cell surface has not been identified. Our study examines Cr(VI) removal from solution by the Gram-positive soil bacterial species Bacillus subtilis. We measured the kinetics of Cr(VI) removal as a function of pH in B. subtilis suspensions with different Cr(VI):cell ratios, and we measured the effect of pH on the extent of Cr(VI) removal at a fixed time in experiments with B. subtilis biomass (cells plus exudates) and in experiments involving B. subtilis exudates alone. The roles of sulfhydryl and amine binding sites in Cr(VI) removal were constrained using site–specific blocking molecules, and the impact of the site blocking on Cr(VI) removal was studied as a function of pH. Our results indicate that Cr(VI) removal by B. subtilis cells under the experimental conditions is at least partially non-reversible and is dependent on binding site concentration, pH, and metal loading. Our results are consistent with a two step removal process: Cr(VI) first adsorbs reversibly onto a cell wall binding site, followed by Cr(VI) reduction to Cr(III) likely via electron transfer from cell wall electron transport chain molecules. B. subtilis exudates are capable of removing a relatively small fraction of Cr(VI) from solution, and hence our results indicate that the dominant mechanism of Cr(VI) removal requires interaction with bacterial biomass. The presence of either the sulfhydryl-specific blocking molecule or the amine-specific blocking molecule or both in the experimental systems dramatically reduces the extent of Cr(VI) reduction, especially under circumneutral pH conditions, strongly suggesting that both sulfhydryl and amine binding site types participate in the initial attachment of Cr(VI) onto the cell surface. The experiments with either sulfhydryl or amine sites blocked both exhibited a similar reduction in Cr(VI) removal to the experiments with both types of sites blocked, strongly suggesting that both types of sites are involved simultaneously in binding Cr(VI) species to the cell surface. For example, these sites could both be involved in forming a bidentate bond with Cr(VI) species, or the positive charge of an amine site in close proximity to a sulfhydryl site could be necessary in order to enable the Cr(VI) species to approach the cell wall and to bind to a sulfhydryl site. The results of this study are the first to propose a viable mechanism that can explain the binding of anionic Cr(VI) onto an overall negatively charged cell surface as a first step in the reduction to Cr(III) and subsequent removal of Cr from solution.

Porous Triple-Layered Supramolecular Heptazine-Based MOF with High Concentration of Binding Sites for Selective Separation of Methane

Porous Triple-Layered Supramolecular Heptazine-Based MOF with High Concentration of Binding Sites for Selective Separation of Methane

Microfluidic Diffusional Sizing (MDS) Measurements of Secretory Neutralizing Antibody Affinity Against SARS-CoV-2

SARS-CoV-2 has rampantly spread around the globe and continues to cause unprecedented loss through ongoing waves of (re)infection. Increasing our understanding of the protection against infection with SARS-CoV-2 is critical to ending the pandemic. Serological assays have been widely used to assess immune responses, but secretory antibodies, the essential first line of defense, have been studied to only a limited extent. Of particular interest and importance are neutralizing antibodies, which block the binding of the spike protein of SARS-CoV-2 to the human receptor angiotensin-converting enzyme-2 (ACE2) and thus are essential for immune defense. Here, we employed Microfluidic Diffusional Sizing (MDS), an immobilization-free technology, to characterize neutralizing antibody affinity to SARS-CoV-2 spike receptor-binding domain (RBD) and spike trimer in saliva. Affinity measurement was obtained through a contrived sample and buffer using recombinant SARS-CoV-2 RBD and monoclonal antibody. Limited saliva samples demonstrated that MDS applies to saliva neutralizing antibody measurement. The ability to disrupt a complex of ACE2-Fc and spike trimer is shown. Using a quantitative assay on the patient sample, we determined the affinity and binding site concentration of the neutralizing antibodies.

Analysis of total thiol concentrations in environmental samples using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)

In this study, we developed an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) approach to measure the concentration of total reactive thiol binding sites in a range of types of environmental samples by monitoring the decrease of monobromo-(trimethylammonio)-bimane (qBBr) caused by thiol-qBBr binding reactions. We first examined the stability of qBBr and potential side reactions that could reduce the accuracy of the UHPLC-MS approach. We found that significant loss of qBBr occurs in the absence of thiols in aqueous solutions after 2–4 h due to qBBr degradation and other side reactions, such as a chloride-for-bromide substitution reaction. To address these issues, in our approach we conduct the thiol-qBBr binding reaction in the presence of excessive qBBr for only 1 h, and we compare the MS signal of unbound qBBr in a thiol-free qBBr standard with that in a thiol-bearing qBBr standard in order to calculate the decrease in the concentration of free qBBr in solution that is caused by thiol-qBBr binding. Using 100–600 nM of N-Acetyl-L-cysteine (ACYS) as a model thiol in a 2 μM qBBr standard, we found that the measured decrease in the MS signal of unbound qBBr linearly correlates to the concentration of added ACYS, with a R2 of 0.988, demonstrating that monitoring the decrease in unbound qBBr can be used to calculate the concentration of total thiol sites in a sample. We demonstrate that the new UHPLC-MS approach yields accurate results when measuring a series of single and mixed thiol standards in the 200–500 nM range, with measured values ranging from 86% to 109% of the known concentration of thiols. In addition to the above control tests, we also used the UHPLC-MS approach to measure dissolved thiol concentrations in several lake water samples. These measurements revealed that each lake contained low μM levels of total thiols, not only demonstrating the ability of the analytical approach to determine thiol site concentrations in natural waters, but also suggesting the widespread importance of these sites in controlling chalcophile element behavior in these systems. The UHPLC-MS approach not only can be used to determine the concentration of dissolved thiol sites at nM levels, but can also be adapted to measure the concentration of thiol binding sites on other environmental samples such as soil, sediment, and solid-phase organic matter surfaces.

Dissolved organic matter thiol concentrations determine methylmercury bioavailability across the terrestrial-marine aquatic continuum

The most critical step for methylmercury (MeHg) bioaccumulation in aquatic food webs is phytoplankton uptake of dissolved MeHg. Dissolved organic matter (DOM) has been known to influence MeHg uptake, but the mechanisms have remained unclear. Here we show that the concentration of DOM-associated thiol functional groups (DOM-RSH) varies substantially across contrasting aquatic systems and dictates MeHg speciation and bioavailability to phytoplankton. Across our 20 study sites, DOM-RSH concentrations decrease 40-fold from terrestrial to marine environments whereas dissolved organic carbon (DOC), the typical proxy for MeHg binding sites in DOM, only has a 5-fold decrease. MeHg accumulation into phytoplankton is shown to be directly linked to the concentration of specific MeHg binding sites (DOM-RSH), rather than DOC. Therefore, MeHg bioavailability increases systematically across the terrestrial-marine aquatic continuum as the DOM-RSH concentration decreases. Our results strongly suggest that measuring DOM-RSH concentrations will improve empirical models in phytoplankton uptake studies and will form a refined basis for modeling MeHg incorporation in aquatic food webs under various environmental conditions.

Tunable PEG Hydrogels for Discerning Differential Tumor Cell Response to Biomechanical Cues.

Increased extracellular matrix (ECM) density in the tumor microenvironment has been shown to influence aspects of tumor progression such as proliferation and invasion. Increased matrix density means cells experience not only increased mechanical properties, but also a higher density of bioactive sites. Traditional in vitro ECM models like Matrigel and collagen do not allow these properties to be investigated independently. In this work, a poly(ethylene glycol)-based scaffold is used which modifies with integrin-binding sites for cell attachment and matrix metalloproteinase 2 and 9 sensitive sites for enzyme-mediated degradation. The polymer backbone density and binding site concentration are independently tuned and the effect each of these properties and their interaction have on the proliferation, invasion, and focal complex formation of two different tumor cell lines is evaluated. It is seen that the cell line of epithelial origin (Hs 578T, triple negative breast cancer) proliferates more, invades less, and forms more mature focal complexes in response to an increase in matrix adhesion sites. Conversely, the cell line of mesenchymal origin (HT1080, fibrosarcoma) proliferates more in 2D culture but less in 3Dculture, invades less, and forms more mature focal complexes in response to an increase in matrix stiffness.

A Rapid Method for Direct Quantification of Antibody Binding-Site Concentration in Serum.

The quantitation of the available antibody binding-site concentration of polyclonal antibodies in serum is critical in defining the efficacy of vaccines against substances of abuse. We have conceptualized an equilibrium dialysis (ED)-based approach coupled with fluorimetry (ED-fluorimetry) to measure the antibody binding-site concentration to the ligand in an aqueous environment. The measured binding-site concentrations in monoclonal antibody (mAb) and sera samples from TT-6-AmHap-immunized rats by ED-fluorimetry are in agreement with those determined by a more established equilibrium dialysis coupled with ultraperformance liquid chromatography tandem mass spectrometry (ED-UPLC-MS/MS). Importantly, we have shown that the measured antibody binding-site concentrations to the ligand by ED-fluorimetry were not influenced by the sample serum matrix; thus, this method is valid for determining the binding-site concentration of polyclonal antibodies in sera samples. Further, we have demonstrated that under appropriate analytical conditions, this method resolved the total binding-site concentrations on a nanomolar scale with good accuracy and repeatability within the microliter sample volumes. This simple, rapid, and sample preparation-free approach has the potential to reliably perform quantitative antibody binding-site screening in serum and other more complex biological fluids.

Surface tension and super-stoichiometric surface enrichment in two-component biomolecular condensates

SummaryCells can achieve internal organization by exploiting liquid-liquid phase separation to form biomolecular condensates. Here we focus on the surface properties of condensates composed of two multivalent associative polymers held together by one-to-one “sticker” bonds. Using coarse-grained molecular-dynamics simulations, we study the influence of component stoichiometry on condensate surface properties. We find that unequal stoichiometry results in enrichment of the majority species at the interface and a sharp reduction of surface tension. To relate these two effects, we show that the reduction in surface tension scales linearly with the excess concentration of free binding sites at the interface. Our results imply that each excess free site contributes an approximately fixed additional energy and entropy to the interface, with the latter dominating such that enrichment of free majority sites lowers the surface tension. Our work provides insight into novel physical mechanisms by which cells can regulate condensate surface properties.

Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site.

Although myriad protein-protein interactions in nature use polyvalent binding, in which multiple ligands on one entity bind to multiple receptors on another, to date an affinity advantage of polyvalent binding has been demonstrated experimentally only in cases where the target receptor molecules are clustered prior to complex formation. Here, we demonstrate cooperativity in binding affinity (i.e., avidity) for a protein complex in which an engineered dimer of the amyloid precursor protein inhibitor (APPI), possessing two fully functional inhibitory loops, interacts with mesotrypsin, a soluble monomeric protein that does not self-associate or cluster spontaneously. We found that each inhibitory loop of the purified APPI homodimer was over three-fold more potent than the corresponding loop in the monovalent APPI inhibitor. This observation is consistent with a suggested mechanism whereby the two APPI loops in the homodimer simultaneously and reversibly bind two corresponding mesotrypsin monomers to mediate mesotrypsin dimerization. We propose a simple model for such dimerization that quantitatively explains the observed cooperativity in binding affinity. Binding cooperativity in this system reveals that the valency of ligands may affect avidity in protein-protein interactions including those of targets that are not surface-anchored and do not self-associate spontaneously. In this scenario, avidity may be explained by the enhanced concentration of ligand binding sites in proximity to the monomeric target, which may favor rebinding of the multiple ligand binding sites with the receptor molecules upon dissociation of the protein complex.

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.

Equilibrium calculations of iron speciation and apparent iron solubility in the Celtic Sea at ambient seawater pH using the NICA-Donnan model

We used a combined ion pairing - organic matter speciation model (NICA-Donnan) to predict the organic complexation of iron (Fe) at ambient pH and temperature in the Celtic Sea. We optimized our model by direct comparison with Fe speciation determined by Adsorptive Cathodic Stripping Voltammetry using the added Fe-binding ligand 1-nitroso-2-naphthol (HNN) in the presence and absence of natural organic matter. We compared determined Fe speciation with simulated titrations obtained via application of the NICA-Donnan model with four different NICA parameter sets representing a range of binding site strengths and heterogeneities. We tested the assumption that binding sites scale to dissolved organic carbon (DOC) concentrations in marine waters. We found that a constant low DOC concentration resulted in an improved fit of our titration data to the simulated titrations, suggesting that inputs of autochthonous marine DOM may not increase the heterogeneity or concentrations of Fe binding sites. Using the optimal parameter set, we calculated pFe(III)´ (−log(∑Fe(OH)i3−i)) and apparent Fe(III) solubility (SFe(III)app) at ambient pH and temperature in the water column of the Celtic Sea. SFe(III)app was defined as the sum of aqueous inorganic Fe(III) species and Fe(III) bound to DOM formed at a free Fe (Fe3+) concentration equal to the limiting solubility of Fe hydroxide (Fe(OH)3(s)). SFe(III)app was within range of the determined dissolved Fe concentrations observed after winter mixing on the shelf and in waters >1500 m depth at our most offshore stations. Our study supports the hypothesis that the ocean dissolved Fe inventory is controlled by the interplay between Fe solubility and Fe binding by organic matter, although the overall number of metal binding sites in the marine environment may not be directly scalable to DOC concentrations.

Miscalculating Equilibrium Constants for Multivalent Protein Complexes: Site-Binding Concentration or Protein Molecular Concentration?

Because the authors continue to note instances in the scientific literature of failure to use the correct receptor binding site concentration for determining binding constants, herein we discuss the fundamental concepts that need to be considered to determine correct binding constants or conversely calculate accurate reactant concentrations with known equilibrium constants. We also show the derivation and analytical solutions of the cubic and quartic equations that give the exact free ligand concentration in bivalent and trivalent receptor systems at equilibrium as a function of the macroscopic equilibrium dissociation constants and the total concentrations of ligand and multivalent protein. These equations and solutions strongly reemphasize the critical dependency of deriving the correct concentrations of bound or free ligand and multivalent protein on the choice of the correct concentration basis for the multivalent protein, which is in turn dependent upon the type of equilibrium constant used. These results demonstrate the importance of choosing the proper multivalent protein concentration for the determination of either valid microscopic or valid macroscopic equilibrium dissociation constants from binding isotherms of ligand-multivalent protein complexes.

A machine learning-based chemoproteomic approach to identify drug targets and binding sites in complex proteomes

Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.

Overexpression of Sedoheptulose-1,7-Bisphosphatase Enhances Photosynthesis in Chlamydomonas reinhardtii and Has No Effect on the Abundance of Other Calvin-Benson Cycle Enzymes.

The productivity of plants and microalgae needs to be increased to feed the growing world population and to promote the development of a low-carbon economy. This goal can be achieved by improving photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7-bisphosphatase (SBP1) up to threefold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the nuclear transgene represented ∼0.3% of total cell protein. Photosynthetic rate and growth were significantly increased in SBP1-overexpressing lines under high-light and elevated CO2 conditions. Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This analysis also revealed that the 11 CBC enzymes represent 11.9% of total cell protein in Chlamydomonas. Here, the range of concentrations of CBC enzymes turned out to be much larger than estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates are often but not always higher than the binding site concentrations of the enzymes for which they act as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates for overexpression in subsequent engineering steps.

Role of bacterial cell surface sulfhydryl sites in cadmium detoxification by Pseudomonas putida

Understanding bacterial metal detoxification systems is crucial for determining the environmental impacts of metal pollution and for developing advanced bioremediation and water disinfection strategies. Here, we explore the role of cell surface sulfhydryl sites in bacterial detoxification of Cd, using Pseudomonas putida with surface sulfhydryl sites mostly on its EPS molecules as a model organism. Our results show that 5 and 20 ppm Cd in LB growth medium affects the lag phase of P. putida, but not the overall extent of cell growth at stationary phase, indicating that P. putida can detoxify Cd at these concentrations. EXAFS analysis of Cd bound to biomass from the different growth stages indicates that Cd binds to both sulfhydryl and non-sulfhydryl sites, but that the importance of Cd-sulfhydryl binding increases from early exponential to stationary phase. Cell growth is positively correlated to the measured sulfhydryl concentration on different biomass samples, but is independent of the measured non-sulfhydryl binding site concentration on the cell surfaces. Taken together, our results demonstrate that the sulfhydryl binding sites on EPS molecules can play an important role in binding and detoxifying toxic metals, significantly decreasing the bioavailability of the metal by sequestering it away from the bacterial cells.

Independent control of matrix adhesiveness and stiffness within a 3D self-assembling peptide hydrogel

A cell’s insoluble microenvironment has increasingly been shown to exert influence on its function. In particular, matrix stiffness and adhesiveness strongly impact behaviors such as cell spreading and differentiation, but materials that allow for independent control of these parameters within a fibrous, stromal-like microenvironment are very limited. In the current work, we devise a self-assembling peptide (SAP) system that facilitates user-friendly control of matrix stiffness and RGD (Arg-Gly-Asp) concentration within a hydrogel possessing a microarchitecture similar to stromal extracellular matrix. In this system, the RGD-modified SAP sequence KFE-RGD and the scrambled sequence KFE-RDG can be directly swapped for one another to change RGD concentration at a given matrix stiffness and total peptide concentration. Stiffness is controlled by altering total peptide concentration, and the unmodified base peptide KFE-8 can be included to further increase this stiffness range due to its higher modulus. With this tunable system, we demonstrate that human mesenchymal stem cell morphology and differentiation are influenced by both gel stiffness and the presence of functional cell binding sites in 3D culture. Specifically, cells 24 hours after encapsulation were only able to spread out in stiffer matrices containing KFE-RGD. Upon addition of soluble adipogenic factors, soft gels facilitated the greatest adipogenesis as determined by the presence of lipid vacuoles and PPARγ-2 expression, while increasing KFE-RGD concentration at a given stiffness had a negative effect on adipogenesis. This three-component hydrogel system thus allows for systematic investigation of matrix stiffness and RGD concentration on cell behavior within a fibrous, three-dimensional matrix. Statement of SignificancePhysical cues from a cell’s surrounding environment—such as the density of cell binding sites and the stiffness of the surrounding material—are increasingly being recognized as key regulators of cell function. Currently, most synthetic biomaterials used to independently tune these parameters lack the fibrous structure characteristic of stromal extracellular matrix, which can be important to cells naturally residing within stromal tissues. In this manuscript, we describe a 3D hydrogel encapsulation system that provides user-friendly control over matrix stiffness and binding site concentration within the context of a stromal-like microarchitecture. Binding site concentration and gel stiffness both influenced cell spreading and differentiation, highlighting the utility of this system to study the independent effects of these material properties on cell function.

Turmeric Powder as a Natural Heavy Metal Chelating Agent: Surface Characterisation

The present study was conducted to investigate surface properties of turmeric in order to evaluateits detoxification potential and ability to sequester toxic metals ions. Scanning Electron Microscopy (SEM),Energy Dispersion Spectroscopy (EDS), Infra-Red (IR) spectroscopy and potentiometric titrations wereemployed for characterisation of the surface of turmeric powder. Spectroscopic studies revealed that thesurface of turmeric powder was porous mainly composed of polymeric -OH , -NH , -CH2 , -COO and-OH groups of polysaccharides. From potentiometric titrations and modelling of batch titration data, it wasfound that surface of the turmeric contains at least four binding sites with pKa values 3.56 (pK1), 4.83(pK2), 7.68 (pK3) and 10.4 (pK4). Turmeric powder contains highest concentration of amino and hydroxylgroups for the pK4 values i.e., 0.55 mmol/ g. The total binding sites concentration for turmeric powderwas 1.2 mmol/ g.

The productive cellulase binding capacity of cellulosic substrates.

Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc.

Surface reactivity of the anaerobic phototrophic Fe(II)-oxidizing bacterium Rhodovulum iodosum: Implications for trace metal budgets in ancient oceans and banded iron formations

It is widely accepted that anoxygenic photosynthetic bacteria (photoferrotrophs) oxidized dissolved Fe(II) in anoxic Archean seawater, leading to the sedimentation of cellular biomass and the Fe(III) minerals that comprised banded iron formations (BIF). However, it remains unknown to which extent the sedimented bacteria may have transported trace metals to BIF through their ability to sorb ions from seawater. We therefore chose the marine anoxygenic photoferrotroph Rhodovulum iodosum to quantify Cd2+, Co2+, Cu2+, Mn2+, Ni2+ and Zn2+ adsorption to bacteria/Fe(III)-oxyhydroxide composites at neutral pH and an ionic strength of 0.1M. Acid base titration data were modeled using a linear programming optimization method to yield pKa values of 4.83±0.13, 6.21±0.18, 7.74±0.24 and 9.28±0.27 and corresponding site densities of 5.7, 12.0, 3.3 and 6.5×10−4mol/g, respectively. FTIR spectroscopy confirmed the presence of carboxyl groups as the most acidic sites corresponding to the lowest pKa values. The analysis of Cd2+ sorption data showed two metal complexing sites with pKS values of 3.44±0.14 and 4.80±0.21 and corresponding binding site concentrations of 1.1±0.3×10−4 and 0.9±0.2×10−4mol/g on the ferrihydrite/R. iodosum composite. The pKS values were used to calculate metal-ligand binding constants, Km. This confirmed Cd2+ binding to the most acidic carboxyl groups on the ferrihydrite/R. iodosum composite surface, as the calculated Km was consistent with reported Cd2+ binding constants for simple organic acids (e.g., lactic, acetic, pyruvic and citric acid). This was used to further calculate the concentration of Co2+, Cu2+, Mn2+, Ni2+ and Zn2+ sorbed to ferrihydrite/R. iodosum composites. The resulting concentrations were ultimately compared to the concentrations of trace elements in BIF to demonstrate that is plausible that the trace metal content in BIF was derived from plankton growing in the ancient ocean's photic zone.

Direct influence of culture dimensionality on human mesenchymal stem cell differentiation at various matrix stiffnesses using a fibrous self-assembling peptide hydrogel.

Much is unknown about the effects of culture dimensionality on cell behavior due to the lack of biomimetic substrates that are suitable for directly comparing cells grown on two-dimensional (2D) and encapsulated within three-dimensional (3D) matrices of the same stiffness and biochemistry. To overcome this limitation, we used a self-assembling peptide hydrogel system that has tunable stiffness and cell-binding site density as well as a fibrous microarchitecture resembling the structure of collagen. We investigated the effect of culture dimensionality on human mesenchymal stem cell differentiation at different values of matrix stiffness (G'=0.25, 1.25, 5, and 10 kPa) and a constant RGD (Arg-Gly-Asp) binding site concentration. In the presence of the same soluble induction factors, culture on top of stiff gels facilitated the most efficient osteogenesis, while encapsulation within the same stiff gels resulted in a switch to predominantly terminal chondrogenesis. Adipogenesis dominated at soft conditions, and 3D culture induced better adipogenic differentiation than 2D culture at a given stiffness. Interestingly, initial matrix-induced cell morphology was predictive of these end phenotypes. Furthermore, optimal culture conditions corresponded to each cell type's natural niche within the body, highlighting the importance of incorporating native matrix dimensionality and stiffness into tissue engineering strategies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2356-2368, 2016.