Role of Polyprotic Acids as Inhibitors of Calcium Oxalate Crystallization

Crystallization is a ubiquitous phenomenon in many synthetic, natural, and biological systems that is often mediated by the action of foreign molecules (or modifiers) to tune the physicochemical properties of crystalline materials. Pathological biomineralization is an example of an undesirable crystallization process where modifiers act as inhibitors to reduce the rate of crystal growth. Here, we focus on the effects of modifiers and parametric studies of the growth medium on the crystallization of calcium oxalate monohydrate (COM), which is the most prevalent constituent of human kidney stones. We performed a systematic study to observe the specificity and efficacy of polyprotic organic acids, including the current drug citrate and its structural analogues, on COM crystallization. Interestingly, we identified modifiers that exhibit different modes of inhibitory action. A notable example is that of hydroxycitrate, which induces dissolution of the crystal surface in highly supersaturated growth solutions. Combined results from in situ atomic force microscopy studies and density functional theory calculations support our hypothesis that modifier-crystal interactions induce localized strain on the crystal lattice, which, in turn, leads to surface dissolution. We also studied the effects of solute and modifier speciation on COM crystallization under varying solution alkalinity that encompasses physiological conditions. We observe a pronounced disparity in the efficacy of modifiers with solution alkalinity owing to changes in local supersaturation near the crystal surface. Collectively, the improved fundamental understanding of modifier-crystal interactions coupled with high-resolution characterization techniques serve as a platform to developing new therapeutics for kidney stone disease as well as elucidating the role of natural biomolecules in the pathological biomineralization of calcium oxalate stones.Chemical and Biomolecular Engineering, Department o

Inhibition of Calcium Oxalate Monohydrate Crystallization Using Organic Growth Modifiers

Inhibition of calcium oxalate monohydrate (COM), the most common component in human kidney stone diseases, was investigated using various structural derivatives of citrate, a known inhibitor of COM crystallization. Bulk crystallization studies revealed the specific binding of modifiers to COM crystal surfaces. Kinetic studies were performed to quantify both the efficacy and potency of growth inhibitors. These studies demonstrated that increased number of hydroxyl and carboxyl groups play a crucial role in interacting with specific COM crystal surface. Designing effective inhibitors require a fundamental understanding of modifier-crystal interactions at the molecular level. Consequently, we have used in situ AFM to observe the COM surface growth in the absence and in the presence of a growth inhibitor. Results obtained in this study may serve as a general platform to investigate the molecular recognition between modifiers and COM crystal surfaces as a step towards designing preventative drugs for kidney stone disease.Chemical and Biomolecular Engineering, Department o

Elucidating the Effects of Polyprotic Acid Speciation in Calcium Oxalate Crystallization

Polyprotic acids tend to be very effective modifiers of crystals in synthetic, natural, and biological systems. Examples include calcium biomineralization where proteins and organic acids decorated with carboxylic acids act as inhibitors of crystal growth. For crystals implicated in pathological diseases, large variations in the pH of the growth medium can alter the speciation of polyprotic acids. This is particularly true for calcium minerals comprised of polyprotic counterions wherein changes in solute speciation affect supersaturation, and thus the kinetics of crystal growth. Here, we explore the combined effects of solute and modifier speciation, selecting calcium oxalate monohydrate (COM) as a representative system for calcification. COM is a major constituent of human kidney stones where crystallization <i>in vivo</i> occurs over a broad range of pH spanning 5–8. Common modifiers of COM and its solute (oxalate) are polyprotic acids. Few studies report the effects of oxalate speciation on COM growth. Moreover, it remains to be determined how pH influences the efficacy of polyprotic molecules used to inhibit COM growth, such as citrate (CA) and its molecular analogue hydroxycitrate (HCA). Here, we show that there is a dramatic reduction in the rate of COM growth in the lower limit of physiological pH, commensurate with the loss of oxalate net negative charge. Our findings reveal that CA and HCA exhibit dual modes of action as promoters and inhibitors of COM crystallization at low and high pH, respectively. We also observe distinct differences in the efficacy of each modifier and discuss how local changes in pH near charged crystal interfaces can have a marked impact on local supersaturation and the speciation of adsorbed modifiers. On the basis of our observations of COM crystallization, it is reasonable to expect that changes in pH, or more specifically the speciation of solute and modifier(s), could similarly impact the growth (and growth inhibition) of other crystals employing polyprotic acids

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction

Background: Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Results: Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. Conclusions: We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology

Agrobacterium strains and strain improvement : present and outlook

Almost 40 years ago the first transgenic plant was generated through Agrobacterium tumefaciens-mediated transformation, which, until now, remains the method of choice for gene delivery into plants. Ever since, optimized Agrobacterium strains have been developed with additional (genetic) modifications that were mostly aimed at enhancing the transformation efficiency, although an optimized strain also exists that reduces unwanted plasmid recombination. As a result, a collection of very useful strains has been created to transform a wide variety of plant species, but has also led to a confusing Agrobacterium strain nomenclature. The latter is often misleading for choosing the best-suited strain for one's transformation purposes. To overcome this issue, we provide a complete overview of the strain classification. We also indicate different strain modifications and their purposes, as well as the obtained results with regard to the transformation process sensu largo. Furthermore, we propose additional improvements of the Agrobacterium-mediated transformation process and consider several worthwhile modifications, for instance, by circumventing a defense response in planta. In this regard, we will discuss pattern-triggered immunity, pathogen-associated molecular pattern detection, hormone homeostasis and signaling, and reactive oxygen species in relationship to Agrobacterium transformation. We will also explore alterations that increase agrobacterial transformation efficiency, reduce plasmid recombination, and improve biocontainment. Finally, we recommend the use of a modular system to best utilize the available knowledge for successful plant transformation

Specificity of Growth Inhibitors and their Cooperative Effects in Calcium Oxalate Monohydrate Crystallization

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitorschondroitin sulfate, serum albumin, and transferrinusing analytical techniques capable of resolving inhibitor–crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was confirmed by bulk crystallization, fluorescent confocal microscopy, and atomic force microscopy. Kinetic studies of COM growth at varying inhibitor concentrations revealed marked differences in their efficacy and potency. Systematic analysis of inhibitor combinations, quantified via the combination index, identified various binary pairings capable of producing synergistic, additive, and antagonistic effects. Collectively, our investigation of physiologically relevant biomolecules suggests potential roles of COM inhibitors in pathological crystallization and provides guiding principles for biomimetic design of molecular modifiers for applications in crystal engineering

Specificity of Growth Inhibitors and their Cooperative Effects in Calcium Oxalate Monohydrate Crystallization

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitorschondroitin sulfate, serum albumin, and transferrinusing analytical techniques capable of resolving inhibitor–crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was confirmed by bulk crystallization, fluorescent confocal microscopy, and atomic force microscopy. Kinetic studies of COM growth at varying inhibitor concentrations revealed marked differences in their efficacy and potency. Systematic analysis of inhibitor combinations, quantified via the combination index, identified various binary pairings capable of producing synergistic, additive, and antagonistic effects. Collectively, our investigation of physiologically relevant biomolecules suggests potential roles of COM inhibitors in pathological crystallization and provides guiding principles for biomimetic design of molecular modifiers for applications in crystal engineering

Natural Promoters of Calcium Oxalate Monohydrate Crystallization

Crystallization is often facilitated by modifiers that interact with specific crystal surfaces and mediate the anisotropic rate of growth. Natural and synthetic modifiers tend to function as growth inhibitors that hinder solute attachment and impede the advancement of layers on crystal surfaces. There are fewer examples of modifiers that operate as growth promoters, whereby modifier–crystal interactions accelerate the kinetic rate of crystallization. Here, we examine two proteins, lysozyme and lactoferrin, which are observed in the organic matrix of three types of pathological stones: renal, prostatic, and pancreatic stones. This work focuses on the role of these proteins in the crystallization of calcium oxalate monohydrate (COM), the most prominent constituent of human kidney stones. Using a combination of experimental techniques, we show that these proteins, which are rich in l-arginine and l-lysine amino acids, promote COM growth. The synthesis and testing of peptides derived from contiguous segments of lysozyme’s primary amino acid sequence revealed subdomains within the protein that operate either as an inhibitor or promoter of COM growth, with the latter exhibiting efficacies that nearly match that of the protein. We observed that cationic proteins promote COM growth over a wide range of modifier concentration, which differs from calcification promoters in the literature that exhibit dual roles as promoters and inhibitors at low and high concentration, respectively. This seems to suggest a unique mechanism of action for lysozyme and lactoferrin. Possible explanations for their effects on COM growth and crystal habit are proposed on the basis of classical colloidal theories and the physicochemical properties of peptide subdomains, including the number and spatial location of charged or hydrogen-bonding moieties