Infantile hypophosphatasia due to a new compound heterozygous TNSALP mutation - functional evidence for a hydrophobic side-chain?

BACKGROUND: Infantile hypophosphatasia (IH) is an inherited disorder characterized by defective bone mineralization and a deficiency of alkaline phosphatase activity. OBJECTIVE/DESIGN: The aim of the study was to evaluate a new compound heterozygous TNSALP mutation for its residual enzyme activity and localization of the comprised amino acid residues in a 3D-modeling. PATIENT: We report on a 4-week old girl with craniotabes, severe defects of ossification, and failure to thrive. Typical clinical features as low serum alkaline phosphatase, high serum calcium concentration, increased urinary calcium excretion, and nephrocalcinosis were observed. Vitamin D was withdrawn and the patient was started on calcitonin and hydrochlorothiazide. Nonetheless, the girl died at the age of 5 months from respiratory failure. RESULTS: Sequence analysis of the patient's TNSALP gene revealed two heterozygous mutations [c.653T>C (I201T), c.1171C>T (R374C)]. Transfection studies of the unique I201T variant in COS-7 cells yielded a mutant TNSALP protein with only a residual enzyme activity (3.7%) compared with wild-type, whereas the R374C variant was previously shown to reduce normal activity to 10.3%. 3D-modeling of the mutated enzyme showed that I201T resides in a region that does not belong to any known functional site. CONCLUSION: We note that I201, which has been conserved during evolution, is buried in a hydrophobic pocket and, therefore, the I>T-change should affect its functional properties. Residue R374C is located in the interface between monomers and it has been previously suggested that this mutation affects dimerization. These findings explain the patient's clinical picture and severe course.

Development and characterization of fluorescent pH sensors based on porous silica and hydrogel support matrices

The hydrogen ion activity (pH) is a very important parameter in environment monitoring, biomedical research and other applications. Optical pH sensors have several advantages over traditional potentiometric pH measurement, such as high sensitivity, no need of constant calibration, easy for miniaturization and possibility for remote sensing. Several pH indicators has been successfully immobilized in three different solid porous materials to use as pH sensing probes. The fluorescent pH indicator fluorescein-5-isothiocyanate (FITC) was covalently bound onto the internal surface of porous silica (pore size ~10 nm) and retained its pH sensitivity. The excited state pK* a of FITC in porous silica (5.58) was slightly smaller than in solution (5.68) due to the free silanol groups (Si-OH) on the silica surface. The pH sensitive range for this probe is pH 4.5 - 7.0 with an error less than 0.1 pH units. The probe response was reproducible and stable for at least four month, stored in DI water, but exhibit a long equilibrium of up to 100 minutes. Sol-gel based pH sensors were developed with immobilization of two fluorescent pH indicators fluorescein-5-(and-6)-sulfonic acid, trisodium salt (FS) and 8-hydroxypyrene- 1,3,6-trisulfonic acid (HPTS) through physical entrapment. Prior to immobilization, the indicators were ion-paired with a common surfactant hexadecyltrimethylammonium bromide (CTAB) in order to prevent leaching. The sol-gel films were synthesized through the hydrolysis of two different precursors, ethyltriethoxysilane (ETEOS) and 3- glycidoxypropyltrimethoxysilane (GPTMS) and deposited on a quartz slide through spin coating. The pK a of the indicators immobilized in sol-gel films was much smaller than in solutions due to silanol groups on the inner surface of the sol-gel films and ammonium groups from the surrounding surfactants. Unlike in solution, the apparent pK a of the indicators in sol-gel films increased with increasing ionic strength. The equilibrium time for these sensors was within 5 minutes (with film thickness of ~470 nm). Polyethylene glycol (PEG) hydrogel was of interest for optical pH sensor development because it is highly proton permeable, transparent and easy to synthesize. pH indicators can be immobilized in hydrogel through physical entrapment and copolymerization. FS and HPTS ion-pairs were physically entrapped in hydrogel matrix synthesized via free radical initiation. For covalent immobilization, three indicators, 6,8-dihydroxypyrene-1,3- disulfonic acid (DHPDS), 2,7-dihydroxynaphthalene-3,6-disulfonic acid (DHNDS) and cresol red were first reacted with methacrylic anhydride (MA) to form methacryloylanalogs for copolymerization. These hydrogels were synthesized in aqueous solution with a redox initiation system. The thickness of the hydrogel film is controlled as ~ 0.5 cm and the porosity can be adjusted with the percentage of polyethylene glycol in the precursor solutions. The pK a of the indicators immobilized in the hydrogel both physically and covalently were higher than in solution due to the medium effect. The sensors are stable and reproducible with a short equilibrium time (less than 4 minutes). In addition, the color change of cresol red immobilized hydrogel is vivid from yellow (acidic condition) to purple (basic condition). Due to covalently binding, cresol red was not leaching out from the hydrogel, making it a good candidate of reusable "pH paper".

Development of Vapor Deposited Silica Sol-Gel Particles for a Bioactive Materials System to Direct Osteoblast Behavior

Tissue engineering and regenerative medicine have emerged in an effort to generate replacement tissues capable of restoring native tissue structure and function, but because of the complexity of biologic system, this has proven to be much harder than originally anticipated. Silica based bioactive glasses are popular as biomaterials because of their ability to enhance osteogenesis and angiogenesis. Sol-gel processing methods are popular in generating these materials because it offers: 1) mild processing conditions; 2) easily controlled structure and composition; 3) the ability to incorporate biological molecules; and 4) inherent biocompatibility. The goal of this work was to develop a bioactive vaporization system for the deposition of silica sol-gel particles as a means to modify the material properties of a substrate at the nano- and micro- level to better mimic the instructive conditions of native bone tissue, promoting appropriate osteoblast attachment, proliferation, and differentiation as a means for supporting bone tissue regeneration. The size distribution, morphology and degradation behavior of the vapor deposited sol-gel particles developed here were found to be dependent upon formulation (H2O:TMOS, pH, Ca/P incorporation) and manufacturing (substrate surface character, deposition time). Additionally, deposition of these particles onto substrates can be used to modify overall substrate properties including hydrophobicity, roughness, and topography. Deposition of Ca/P sol particles induced apatite-like mineral formation on both two- and three-dimensional materials when exposed to body fluids. Gene expression analysis suggests that Ca/P sol particles induce upregulation osteoblast gene expression (Runx2, OPN, OCN) in preosteoblasts during early culture time points. Upon further modification-specifically increasing particle stability-these Ca/P sol particles possess the potential to serve as a simple and unique means to modify biomaterial surface properties as a means to direct osteoblast differentiation.

Aza-Michael addition of amines to activated alkenes catalyzed by Silica supported perchloric acid under a solvent-free condition

An efficient aza-Michael addition of amines to a series of ,-unsaturated ketones, carboxylic esters, nitriles and chalcones has been carried out using perchloric acid supported over silica gel (HClO4-SiO2) at room temperature in high yields under solvent-free reaction conditions.

Fourier transform infrared spectroscopy, a new method for rapid determination of total organic and inorganic carbon and biogenic silica concentration in lake sediments

Abstract We demonstrate the use of Fourier transform infrared spectroscopy (FTIRS) to make quantitative measures of total organic carbon (TOC), total inorganic carbon (TIC) and biogenic silica (BSi) concentrations in sediment. FTIRS is a fast and costeffective technique and only small sediment samples are needed (0.01 g). Statistically significant models were developed using sediment samples from northern Sweden and were applied to sediment records from Sweden, northeast Siberia and Macedonia. The correlation between FTIRS-inferred values and amounts of biogeochemical constituents assessed conventionally varied between r = 0.84–0.99 for TOC, r = 0.85– 0.99 for TIC, and r = 0.68–0.94 for BSi. Because FTIR spectra contain information on a large number of both inorganic and organic components, there is great potential for FTIRS to become an important tool in paleolimnology.

Modeling Responses of Diatom Productivity and Biogenic Silica Export to Iron Enrichment in the Equatorial Pacific Ocean

Using a three-dimensional physical-biogeochemical model, we have investigated the modeled responses of diatom productivity and biogenic silica export to iron enrichment in the equatorial Pacific, and compared the model simulation with in situ (IronEx II) iron fertilization results. In the eastern equatorial Pacific, an area of 540,000 km(2) was enhanced with iron by changing the photosynthetic efficiency and silicate and nitrogen uptake kinetics of phytoplankton in the model for a period of 20 days. The vertically integrated Chl a and primary production increased by about threefold 5 days after the start of the experiment, similar to that observed in the IronEx II experiment. Diatoms contribute to the initial increase of the total phytoplankton biomass, but decrease sharply after 10 days because of mesozooplankton grazing. The modeled surface nutrients (silicate and nitrate) and TCO(2) anomaly fields, obtained from the difference between the "iron addition'' and "ambient'' (without iron) concentrations, also agreed well with the IronEx II observations. The enriched patch is tracked with an inert tracer similar to the SF6 used in the IronEx II. The modeled depth-time distribution of sinking biogenic silica (BSi) indicates that it would take more than 30 days after iron injection to detect any significant BSi export out of the euphotic zone. Sensitivity studies were performed to establish the importance of fertilized patch size, duration of fertilization, and the role of mesozooplankton grazing. A larger size of the iron patch tends to produce a broader extent and longer-lasting phytoplankton blooms. Longer duration prolongs phytoplankton growth, but higher zooplankton grazing pressure prevents significant phytoplankton biomass accumulation. With the same treatment of iron fertilization in the model, lowering mesozooplankton grazing rate generates much stronger diatom bloom, but it is terminated by Si(OH)(4) limitation after the initial rapid increase. Increasing mesozooplankton grazing rate, the diatom increase due to iron addition stays at minimum level, but small phytoplankton tend to increase. The numerical model experiments demonstrate the value of ecosystem modeling for evaluating the detailed interaction between biogeochemical cycle and iron fertilization in the equatorial Pacific.

An in vitro toxicity evaluation of gold-, PLLA- and PCL-coated silica nanoparticles in neuronal cells for nanoparticle-assisted laser-tissue soldering.

The uptake of silica (Si) and gold (Au) nanoparticles (NPs) engineered for laser-tissue soldering in the brain was investigated using microglial cells and undifferentiated and differentiated SH-SY5Y cells. It is not known what effects NPs elicit once entering the brain. Cellular uptake, cytotoxicity, apoptosis, and the potential induction of oxidative stress by means of depletion of glutathione levels were determined after NP exposure at concentrations of 10(3) and 10(9)NPs/ml. Au-, silica poly (ε-caprolactone) (Si-PCL-) and silica poly-L-lactide (Si-PLLA)-NPs were taken up by all cells investigated. Aggregates and single NPs were found in membrane-surrounded vacuoles and the cytoplasm, but not in the nucleus. Both NP concentrations investigated did not result in cytotoxicity or apoptosis, but reduced glutathione (GSH) levels predominantly at 6 and 24h, but not after 12 h of NP exposure in the microglial cells. NP exposure-induced GSH depletion was concentration-dependent in both cell lines. Si-PCL-NPs induced the strongest effect of GSH depletion followed by Si-PLLA-NPs and Au-NPs. NP size seems to be an important characteristic for this effect. Overall, Au-NPs are most promising for laser-assisted vascular soldering in the brain. Further studies are necessary to further evaluate possible effects of these NPs in neuronal cells.

Independent measurement of biogenic silica in sediments by FTIR spectroscopy and PLS regression

We present an independent calibration model for the determination of biogenic silica (BSi) in sediments, developed from analysis of synthetic sediment mixtures and application of Fourier transform infrared spectroscopy (FTIRS) and partial least squares regression (PLSR) modeling. In contrast to current FTIRS applications for quantifying BSi, this new calibration is independent from conventional wet-chemical techniques and their associated measurement uncertainties. This approach also removes the need for developing internal calibrations between the two methods for individual sediments records. For the independent calibration, we produced six series of different synthetic sediment mixtures using two purified diatom extracts, with one extract mixed with quartz sand, calcite, 60/40 quartz/calcite and two different natural sediments, and a second extract mixed with one of the natural sediments. A total of 306 samples—51 samples per series—yielded BSi contents ranging from 0 to 100 %. The resulting PLSR calibration model between the FTIR spectral information and the defined BSi concentration of the synthetic sediment mixtures exhibits a strong cross-validated correlation ( R2cv = 0.97) and a low root-mean square error of cross-validation (RMSECV = 4.7 %). Application of the independent calibration to natural lacustrine and marine sediments yields robust BSi reconstructions. At present, the synthetic mixtures do not include the variation in organic matter that occurs in natural samples, which may explain the somewhat lower prediction accuracy of the calibration model for organic-rich samples.

Maxillary sinus grafting with a synthetic, nanocrystalline hydroxyapatite-silica gel in humans: histologic and histomorphometric results.

The aim of this study was to evaluate in humans the amount of new bone after sinus floor elevation with a synthetic bone substitute material consisting of nanocrystalline hydroxyapatite embedded in a highly porous silica gel matrix. The lateral approach was applied in eight patients requiring sinus floor elevation to place dental implants. After elevation of the sinus membrane, the cavities were filled with 0.6-mm granules of nanocrystalline hydroxyapatite mixed with the patient's blood. A collagen membrane (group 1) or a platelet-rich fibrin (PRF) membrane (group 2) was placed over the bony window. After healing periods between 7 and 11 months (in one case after 24 months), 16 biopsy specimens were harvested with a trephine bur during implant bed preparation. The percentage of new bone, residual filler material, and soft tissue was determined histomorphometrically. Four specimens were excluded from the analysis because of incomplete biopsy removal. In all other specimens, new bone was observed in the augmented region. For group 1, the amount of new bone, residual graft material, and soft tissue was 28.7% ± 5.4%, 25.5% ± 7.6%, and 45.8% ± 3.2%, respectively. For group 2, the values were 28.6% ± 6.90%, 25.7% ± 8.8%, and 45.7% ± 9.3%, respectively. All differences between groups 1 and 2 were not statistically significant. The lowest and highest values of new bone were 21.2% and 34.1% for group 1 and 17.4% and 37.8% for group 2, respectively. The amount of new bone after the use of nanocrystalline hydroxyapatite for sinus floor elevation in humans is comparable to values found in the literature for other synthetic or xenogeneic bone substitute materials. There was no additional beneficial effect of the PRF membrane over the non-cross-linked collagen membrane.

Early bone apposition to hydrophilic and hydrophobic titanium implant surfaces: a histologic and histomorphometric study in minipigs.

OBJECTIVE The first objective of this pilot study was to evaluate the impact of the hydrophilicity on the early phases of osseointegration. The second objective was to compare two hydrophilic implant surfaces with different geometries, surface roughness, and technologies achieving hydrophilicity. MATERIAL AND METHODS Twelve weeks after extraction, all four quadrants of nine minipigs received three dental implants, alternating between hydrophilic microrough surfaces (INICELL and SLActive) and a conventional hydrophobic microrough surface. After 5, 10, and 15 days of submerged healing, ground sections were prepared and subjected to histologic and histomorphometric analysis. RESULTS The histologic analysis revealed a similar healing pattern among the hydrophilic and hydrophobic implant surfaces, with extensive bone formation occurring between day 5 and day 10. With BIC values of greater than 50% after 10 days, all examined surfaces indicated favorable osseointegration at this very early point in healing. At day 15, the mean new bone-to-implant contact (newBIC) of one hydrophilic surface (INICELL; 55.8 ± 14.4%) was slightly greater than that of the hydrophobic microrough surface (40.6 ± 20.2%). At day 10 and day 15, an overall of 21% of the implants had to be excluded from analysis due to inflammations primarily caused by surgical complications. CONCLUSION Substantial bone apposition occurs between day 5 and day 10. The data suggest that the hydrophilic surface can provoke a slight tendency toward increased bone apposition in minipigs after 15 days. A direct comparison of two hydrophilic surfaces with varying geometries is of limited relevance.