Pseudo-equilibrium equations for calcium phosphate precipitation with multi-unit particles.

The chemistry underlying bone mineral formation in vertebrates is the reaction of calcium phosphate precipitation. In a near-neutral solution, an amorphous phase and hydroxyapatite nanoparticles appear successively, and the reaction system containing either of the two kinds of precipitates is in a non-equilibrium state. Here, we propose a pseudo-equilibrium approach to the solution chemistry of the precipitation reactions. We employed two series of reaction systems, collected samples at various stages, and analyzed the solution chemistry data on the basis of a simplified model of reaction. We derived two types of pseudo-equilibrium equations from the two series, respectively. These equations reveal the existence of multiple structural units in a precipitate particle and correlate the ionic product with the surface proportion per structural unit (m). The surface proportion, in turn, is related to the whole particle through a particle-surface equation. Notably, the two types of pseudo-equilibrium constants have the common expression of "Kd = ionic product" if the number of the structural units (u) is large enough. Together, these findings have revealed some aspects of the non-equilibrium thermodynamics of precipitation reactions, indicating the solution chemistry route to the equilibrium state. The concept of the multi-unit particle may shed new light on the study of precipitation reactions of other slightly soluble electrolytes. And the relationship between the ionic product and the surface proportion of a structural unit is not only fundamental in chemistry, but may also apply to non-equilibrium systems in nature and biology, such as marine sedimentation, human vascular calcification, and bone mineral metabolism.

Hyperprogression under treatment with immune-checkpoint inhibitors in patients with gastrointestinal cancer: A natural process of advanced tumor progression?

Immunotherapy has shown great promise in treating various types of malignant tumors. However, some patients with gastrointestinal cancer have been known to experience rapid disease progression after treatment, a situation referred to as hyperprogressive disease (HPD). This minireview focuses on the definitions and potential mechanisms of HPD, natural disease progression in gastrointestinal malignancies, and tumor immunological microenvironment.

Research on Mechanism of miR-214 Packaged with Lipidosome Nanoparticles on Prompting the Apoptosis of Intestinal Cancer Through Regulating p53 Pathway.

Our study aimed at studying mechanism of miR-214 packaged with lipidosome nanoparticles on prompting apoptosis of intestinal cancer through regulating p53 pathway. SW480 cells were divided into blank group, empty carrier group, agonist group and group with carrier and antagonist. The negative control group was set, and groups related to p53 pathway were set as agonist group, inhibitor group and group with antagonist and inhibitor. The effect of miR-214 packaged with lipidosome nanoparticles on proliferation and apoptosis of intestinal cancer cells and p53 pathway in intestinal cancer cells was observed. Expression level of miR-214 in group with carrier and antagonist was lower than in other groups. The proportion of active cells in the group with carrier and antagonist started to be reduced notably from the second day. There was no notable declining tendency active cells' proportion from other groups. The quantity of cell apoptosis in group with carrier and antagonist was higher than in the other groups. The expression level of cleaved Caspase-3 in the group with carrier and antagonist was notably higher than in the other groups. Moreover, expression of Bcl-2/Bax protein was reversed, while expression of p53 protein in the carrier and antagonist groups was notably higher than in the other groups. The antagonist of miR-214 packaged with lipidosome nanoparticles could target on p53 pathway. The activity of p53 pathway was reduced by miR-214, and expression of Bcl-2 was increased. The expressions levels of Bax and cleaved Caspase-3 were also reversed, and molecular mechanism was mainly related with restraining of p53 signal pathway.

Correction to: Protein-bound calcium phosphate in uremic rat serum: a quantitative study.

In the original article, few equations and units were published incorrectly.

Protein-bound calcium phosphate in uremic rat serum: a quantitative study.

Protein-bound calcium (prCa) constitutes about 40% of serum total calcium, in which albumin is the most dominant protein. Given the chemical interaction between calcium and phosphate (Pi), the increased serum Pi in chronic kidney disease may cause changes in the composition and structure of the prCa fraction. Here, we report the phosphate binding on the protein-bound calcium in uremic rat serum. Using adenine-fed rats as a uremic model, we separated the calcium and phosphate fractions in rat serum by ultrafiltration, and found that the level of protein-bound phosphate (prPi) in the uremic serum was markedly higher than in control. The elevated prPi level was comparable to the prCa level, consistent with the presence of protein-bound calcium phosphate pr(Ca)j-m(CaPi)m. We then confirmed its presence by ex vivo X-ray absorption near-edge structure spectroscopy, revealing the discrete state of the calcium phosphate clusters associated with protein. Finally, in a quantitative investigation using Ca- and Pi-boosted serum, we discovered the threshold concentration for the Pi binding on prCa, and determined the binding constant. The threshold, while preventing Pi from binding to prCa in normal condition, allows the reaction to take place in hyperphosphatemia conditions. The protein-bound calcium phosphate could act as a link between the metabolism of serum proteins and the homeostasis of phosphate and calcium, and it deserves further investigation whether the molar ratio of (prPi/prCa)⋅100% may serve as a serum index of the vascular calcification status in chronic kidney disease.

Pseudo-equilibrium equation of calcium phosphate precipitation from aqueous solution.

An X-ray amorphous phase is frequently present at the early stage of calcium phosphate crystallization, and the relevant solution chemistry is essential for understanding the mechanism of reaction. Here, we report a quantitative study of a series of reaction systems at pseudo-equilibrium states. We determined the composition of solutions and the quantities of the precipitate samples, and characterized the long- and short-range order of the precipitate using X-ray diffraction and synchrotron X-ray absorption near-edge structure spectroscopy, respectively. We found that, in a particle with multiple structural units, only a fraction of the units was able to reach pseudo-equilibrium with the solution composition, which represents the average number of surficial clusters per unit. These findings enabled us to propose a general form of the equilibrium constant equation. The equation fits the pseudo-equilibrium data well, and it converts to the "solubility product (Ksp)" and the conventional "reaction quotient" in two limit cases, respectively. Further, using a cube model, we derived a "particle equation" that reveals the connection between the particle structure and the form of equilibrium constant equation. The dependency of the form of pseudo-equilibrium equation on the structure and size of the precipitate reveals a fundamental relation in chemistry, and its applicability remains to be examined in other reaction systems, such as those involving nanocrystals and porous materials.

Two-dimensional liquid chromatography with active solvent modulation for studying monomer incorporation in copolymer dispersants.

A pseudo-comprehensive two-dimensional liquid chromatography approach with size exclusion chromatography in the first dimension and gradient reversed-phase liquid chromatography in the second dimension was successfully developed for the characterization of vinyl acetate/acrylic acid copolymers and vinyl acetate/itaconic acid/acrylic acid terpolymers. Active solvent modulation was exploited to prevent the polymer breakthrough in the second dimension separation caused by the strong solvent used in the first dimension. The conditions of the active solvent modulation valve were optimized to achieve sufficient on-line dilution and to completely prevent polymer breakthrough without adding excessive time to the modulation cycle. Using this approach, copolymers made with different monomer ratios and processes were studied. Heterogeneous composition distribution due to insufficient monomer incorporation was detected in some of the copolymer samples. We demonstrated that with active solvent modulation, the two-dimensional liquid chromatography approach is no longer limited to water-soluble polymers and can be used for a broader range of polymers and copolymers.

Ex vivo detection of calcium phosphate and calcium carbonate in rat blood serum.

The total calcium (tCa) in blood serum comprises free Ca2+ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η2-HPO4)(H2O)4 and Ca(η1-HCO3)(H2O)5+, in which HPO42- and HCO3- serve as bidentate and unidentate ligands, respectively. The remained H2O molecules on the coordination sphere of Ca2+ enable these complexes to behave partially like aquated Ca2+ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.

Selenoprotein P as the major transporter for mercury in serum from methylmercury-poisoned rats.

Selenium (Se) has been found to promote weight gain, decrease hepatic damage, but redistribute mercury (Hg) in brains and livers in methylmercury (MeHg)-poisoned rats. The aims of the present work were to examine the effects of Se on the levels of Hg in serum and the role of serum selenoproteins in binding with Hg in MeHg-poisoned rats. The concentration of Se, Hg and MeHg were studied using ICP-MS and CVAFS. The Hg- and Se-binding selenoproteins were separated and quantified using affinity chromatography with post-column isotope dilution analysis using both enriched 78Se and 199Hg. It was found that Se treatment reduced Hg levels in serum in MeHg-poisoned rats. Among the three separated selenoproteins, the amounts of SelP-bound Hg and Se increased to 73% and 93.6%, from 64.4% and 89.3% of the total Hg and Se, respectively after Se treatment, suggesting that SelP acts as a major transporter for Hg and pool for Se in serum. Over 90% of the total Hg was MeHg in serum, and the molar ratios of MeHg to Se as 1:4 and 1:9 in the formed MeHg-Se-SelP complex in the control and the Se treatment group, respectively. The elevated Se level binding with SelP facilitated the Hg extraction from tissues and organs, as well as its redistribution in brains and livers through blood circulation in the MeHg-poisoned rats. Together, our findings provide direct evidence that serum SelP is the major Hg transporter in MeHg-poisoned rats.

Influences of LaCl3 on the mineral phase transformation during osteoblast mineralization in vitro.

Rat calvarial osteoblasts were treated with lanthanum chloride (LaCl3) to explore its effect on the mineral crystalline phase during the process of osteoblast calcification in vitro. The results confirmed that La was readily deposited in the mineral component of the matrix. Employing high-resolution transmission electron microscopy and Fourier transform infrared microspectroscopy techniques, we demonstrated that features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate, and hydroxyapatite (HAP) were detected in the mineral phases in vitro. Particularly, LaCl3 treatment retarded conversion from DCPD-like phase into HAP during mineralization. In addition, La was introduced in DCPD powder during wet chemical synthesis. When compared with that of La-free DCPD, the dissolution rate of La-incorporated DCPD was lower, thereby leading to a delayed DCPD-to-HAP phase transformation. Thus, it can be concluded that LaCl3 treatment influences the kinetics of inorganic phase transition by decreasing the dissolution rate of DCPD.

Calcium phosphate nanoparticles are associated with inorganic phosphate-induced osteogenic differentiation of rat bone marrow stromal cells.

In the present study, we demonstrated that calcium phosphate (CaP) nanoparticles formed in cell culture media were implicated in the process of high inorganic phosphate (Pi) mediated osteogenic differentiation of rat bone marrow stromal cells (BMSCs). Exposure of BMSCs in vitro to high Pi-containing media reduced alkaline phosphatase (ALP) activity and the expressions of osteoblast-specific genes. The sediments of CaP nanoparticles were observed at the cell surface and some of them were concomitantly found inside cells at high Pi concentration. In addition, treatment the cells with pyrophosphate (PPi), an inhibitor of calcium crystal formation, abrogated the ALP activity induced by high Pi, suggesting the contribution of CaP nanoparticles. Moreover, for isolated CaP nanoparticles, there was a trend of conversion from amorphous calcium phosphate to hydroxyapatite with elevated Pi. The particle size of CaP increased and the surface morphology changed from spherical to irregular due to increased concentrations of serum proteins incorporated into CaP nanoparticles. The study demonstrated that those physicochemical properties of CaP nanoparticles played an important role in modulating BMSCs differentiation. Furthermore, the addition of Pi in the osteogenic media resulted in a dose-dependent increase in matrix mineralization, while treatment of the cells with PPi suppressed Pi-induced calcium deposition. The findings indicated that calcium deposition in the matrix partly came from the spontaneous precipitation of CaP nanoparticles.

[Molecular structure and fractal analysis of oligosaccharide].

OBJECTIVE: To propose a calculation method of oligosaccharides' fractal dimension, and to provide a new approach to studying the drug molecular design and activity. METHODS: By using the principle of energy optimization and computer simulation technology, the steady structures of oligosaccharides were found, and an effective way of oligosaccharides fractal dimension's calculation was further established by applying the theory of box dimension to the chemical compounds. RESULTS: By using the proposed method, 22 oligosaccharides' fractal dimensions were calculated, with the mean 1.518 8 ± 0.107 2; in addition, the fractal dimensions of the two activity multivalent oligosaccharides which were confirmed by experiments, An-2 and Gu-4, were about 1.478 8 and 1.516 0 respectively, while C-type lectin-like receptor Dectin-1's fractal dimension was about 1.541 2. The experimental and computational results were expected to help to find a class of glycoside drugs whose target receptor was Dectin-1. CONCLUSION: Fractal dimension, differing from other known macro parameters, is a useful tool to characterize the compound molecules' microscopic structure and function, which may play an important role in the molecular design and biological activity study. In the process of oligosaccharides drug screening, the fractal dimension of receptor and designed oligosaccharides or glycoclusters can be calculated respectively. The oligosaccharides with fractal dimension close to that of target receptor should then take priority compared with others, to get the drug molecules with latent activity.

Differentiation-stimulating potency of differentiated HL60 cells after drug treatment.

Differentiation therapy in the treatment of leukemia is often hampered by limitations on using certain pharmaceutical regents or on the required doses due to various reasons, such as drug-resistance and retinoic acid syndrome. To circumvent these problems, a strategy might be developed on the basis of the ability of drug-differentiated cells to stimulate differentiation in leukemia cells. Using the promyelocytic leukemia cell line HL60 as a cell model, we assessed the differentiation-stimulating potency of differentiated granulocytes and monocytes/macrophages after treatments with all-trans retinoic acid (ATRA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. ATRA- and TPA-differentiated cells were able to stimulate differentiation in fresh HL60 cells, accompanied by inhibition on cell growth to various extents. The differentiated cells of the second generation, especially those originated from TPA treatment, were as potent as the drugs themselves in stimulating differentiation in fresh HL60 cells. On the basis of "differentiation induced by differentiated cells", we explored the feasibility of ex vivo therapy.

Identification of dicalcium phosphate dihydrate deposited during osteoblast mineralization in vitro.

The hydroxyapatite (HAP) with variable chemical substitutions has been considered as the major component in the mineralized part of bones. Various metastable crystalline phases have been suggested as transitory precursors of HAP in bone, but there are no consensuses as to the nature of these phases and their temporal evolution. In the present study, we cultured rat calvarial osteoblasts with ascorbate and ß-glycerophosphate to explore which calcium phosphate precursor phases comprise the initial mineral in the process of osteoblast mineralization in vitro. At the indicated time points, the deposited calcium phosphate was analyzed after removing organic substances from the extracellular matrix with hydrazine. The features comparable to dicalcium phosphate dihydrate (DCPD) and octacalcium phosphate (OCP), in addition to HAP, were detected in the mineral phases by high resolution transmission electron microscopy. And there was a trend of conversion from DCPD- and OCP-like phases to HAP in the course of mineralization, as indicated by Fourier-transform infrared microspectroscopy, energy-dispersive X-ray spectroscopy and synchrotron X-ray powder diffraction analyses. Besides, biochemical assay showed a progressive decrease in the ratio of mineral-associated proteins to calcium with time. These findings suggest that DCPD- and OCP-like phases are likely to occur on the course of osteoblast mineralization, and the mineral-associated proteins might be involved in modulating the mineral phase transformation.

In situ solution study of calcium phosphate crystallization kinetics.

Calcium phosphate minerals are the main inorganic component of the bones and teeth. It is of fundamental importance to discover the effect of biomolecules on kinetics of nucleation and crystal growth of calcium phosphate crystals, which shed light on the understanding of the mechanism of biomineralization. Here, we introduce some general solution-based in situ detection methods, including analyses of chemical composition and pH and stopped-flow spectrophotometry, to study the crystallization kinetics of calcium phosphate in solution. We present the details of experimental components and considerations, such as stock solution preparation and preservation, working solution preparation, and the protocols for mixing and solution crystallization. The factors that might influence the crystallization process such as temperature control, stirring and flow rate, ionic strength, ionic species ratio, and foreign particles are also discussed here. Finally, we describe the protocols for each method and the processing of experimental data to extract the kinetics of nucleation and crystal growth. The advantages and disadvantages for each method are summarized in this chapter.

Stability of flavin semiquinones in the gas phase: the electron affinity, proton affinity, and hydrogen atom affinity of lumiflavin.

Examination of electron transfer and proton transfer reactions of lumiflavin and proton transfer reactions of the lumiflavin radical anion by Fourier transform ion cyclotron resonance mass spectrometry is described. From the equilibrium constant determined for electron transfer between 1,4-naphthoquinone and lumiflavin the electron affinity of lumiflavin is deduced to be 1.86 ± 0.1 eV. Measurements of the rate constants and efficiencies for proton transfer reactions indicate that the proton affinity of the lumiflavin radical anion is between that of difluoroacetate (331.0 kcal/mol) and p-formyl-phenoxide (333.0 kcal/mol). Combining the electron affinity of lumiflavin with the proton affinity of the lumiflavin radical anion gives a lumiflavin hydrogen atom affinity of 59.7 ± 2.2 kcal/mol. The ΔG298 deduced from these results for adding an H atom to gas phase lumiflavin, 52.1 ± 2.2 kcal/mol, is in good agreement with ΔG298 for adding an H atom to aqueous lumiflavin from electrochemical measurements in the literature, 51.0 kcal/mol, and that from M06-L density functional calculations in the literature, 51.2 kcal/mol, suggesting little, if any, solvent effect on the H atom addition. The proton affinity of lumiflavin deduced from the equilibrium constant for the proton transfer reaction between lumiflavin and 2-picoline is 227.3 ± 2.0 kcal mol(-1). Density functional theory calculations on isomers of protonated lumiflavin provide a basis for assigning the most probable site of protonation as position 1 on the isoalloxazine ring and for estimating the ionization potentials of lumiflavin neutral radicals.

The intracellular controlled release from bioresponsive mesoporous silica with folate as both targeting and capping agent.

A smart mesoporous silica nanocarrier with intracellular controlled release is fabricated, with folic acid as dual-functional targeting and capping agent. The folate not only improves the efficiency of the nanocarrier internalized by the cancer cells, but also blocks the pores of the mesoporous silica to eliminate premature leakage of the drug. With disulfide bonds as linkers to attach the dual-functional folate within the surface of mesoporous silica, the controlled release can be triggered in the presence of reductant dithiothreitol (DTT) or glutathione (GSH). The cellular internalization via folate-receptor-mediated endocytosis and the intracellular controlled release of highly toxic anticancer drug DOX were demonstrated with an in vitro HeLa cell culture, indicating an efficient cancer-targeted drug delivery.

Modeling of angiotensin II-angiotensin-(1-7) counterbalance in disease progression in spontaneously hypertensive rats treated with/without perindopril.

Angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) are biologically active effectors in the renin-angiotensin system (RAS) and have been demonstrated to have potential function in predicting cardiovascular diseases. We developed mechanism-based mathematical models to characterize the up/down-regulation of Ang II/Ang-(1-7), and the effects of perindopril on hypertension progression in spontaneously hypertensive rats (SHR). SHR were randomly assigned to the control group (n=6) and treatment group (n=6). Rats in the treatment group received oral perindopril (5mgkg(-1)day(-1)). Systolic blood pressure (SBP) was measured by the tail-cuff method. Serum Ang II and Ang-(1-7) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Three linked turnover models were developed to describe Ang II, Ang-(1-7) and SBP profiles. All parameters were estimated using nonlinear mixed-effects modeling. The results showed that Ang II, Ang-(1-7) and SBP gradually increased in the control group. These counterbalance mechanisms were reflected in the models with two feedback cycles. It was assumed that the Ang-(1-7) production rate constant (K(in_Ang17)) was stimulated by Ang II, and the Ang II output rate constant (K(out_Ang2)) reflecting Ang II degradation was stimulated by Ang-(1-7). The decrease in Ang II and increase in Ang-(1-7) were observed in rats treated with perindopril. The models described the counterbalance relationship of Ang II and Ang-(1-7) well, and provided insights into ACE inhibition using perindopril. The models could be extended to incorporate other biomarkers and the effects of various ACE inhibitors (ACEIs).

Lanthanum chloride bidirectionally influences calcification in bovine vascular smooth muscle cells.

Vascular calcification (VC) is frequent prevalence in patients with chronic kidney disease (CKD) and atherosclerosis. Lanthanum carbonate is used as an orally administered phosphate-binding agent to reduce the gastrointestinal absorption of phosphate and ameliorate VC in advanced CKD. In this study, we used bovine vascular smooth muscle cells as a model VC in vitro and studied the effects of lanthanum chloride on calcium deposition. Exposure of cells to LaCl(3) at the concentration of 0.1 µM suppressed the ß-glycerophosphate-induced alkaline phosphatase activity and calcium deposition. Furthermore, LaCl(3) upregulated the ß-glycerophosphate-suppressed expression of calcium-sensing receptor. In contrast to the inhibitory effect of LaCl(3) on calcium deposition, higher level lanthanum (50 µM) was found to promote immediately precipitation of calcium phosphate in cell culture medium. At this concentration, LaCl(3) was found to induce cell apoptosis which involves caspases-9 and -3. These data indicate that the promotory effect of LaCl(3) on calcium deposition is likely mediated by induction of apoptosis. Our in vitro findings do suggest that, in the context of raised lanthanum, greater attention should be paid to potential toxic effects associated to the use of lanthanide-based drugs.

Gadolinium inhibits prostate cancer PC3 cell migration and suppresses osteoclast differentiation in vitro.

This study examined whether Gd (gadolinium) could suppress prostate cancer cell migration and prostate cancer cell-induced osteoclast differentiation. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and colony forming assay showed that GdCl3 treatment inhibited both cell viability and colony forming ability in PC3 cells more significantly than that in DU145 cells. Annexin/PI (propidium iodide) staining showed an increase in apoptotic death of PC3 cells in the presence of GdCl3. Wound healing and adhesion assay indicated that GdCl3 suppressed PC3 cell migration. Western-blot analysis demonstrated that GdCl3 treatment inhibited phosphorylation of ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase). Pretreatment with PTx (pertussis toxin), a Gi protein inhibitor, conferred resistance to GdCl3-induced colony formation, ERK and p38 phosphorylation in PC3 cells. Moreover, GdCl3 inhibited PC3 cell-induced osteoclast differentiation. RT-PCR (reverse transcription-PCR) indicated that GdCl3 decreased the expression of RANKL (receptor activator of nuclear factor-κB ligand) in PC3 cells, whereas it increased the expression of OPG (osteoprotegerin) in PC3 and DU145 cells. In conclusion, the present study indicated that GdCl3 inhibited PC3 cell migration mediated by the inactivation of both ERK and p38 MAPK pathways via PTx-sensitive G proteins, and also suppressed PC3 cell-induced osteoclast differentiation via regulating the mRNA expression of OPG and RANKL.