Emissions of VOCs, SVOCs, and mold during the construction process: Contribution to indoor air quality and future occupants' exposure.

Building materials and human activities are important sources of contamination indoors, but little information is available regarding contamination during construction process which could persist during the whole life of buildings. In this study, six construction stages on two construction sites were investigated regarding the emissions of 43 volatile organic compounds (VOCs), 46 semi-volatile organic compounds (SVOCs), and the presence of 4 genera of mold. Results show that the future indoor air quality does not only depend on the emissions of each building product but that it is also closely related to the whole implementation process. Mold spore measurements can reach 1400 CFU/m3 , which is particularly high compared with the concentrations usually measured in indoor environments. Relatively low concentrations of VOCs were observed, in relation to the use of low emissive materials. Among SVOCs analyzed, some phthalates, permethrin, and hydrocarbons were found in significant concentrations upon the delivery of building as well as triclosan, suspected to be endocrine disruptor, and yet prohibited in the treatment of materials and construction since 2014. As some regulations exist for VOC emissions, it is necessary to implement them for SVOCs due to their toxicity.

Analysis of co-assembly and co-localization of ameloblastin and amelogenin.

Epithelially-derived ameloblasts secrete extracellular matrix proteins including amelogenin, enamelin, and ameloblastin. Complex intermolecular interactions among these proteins are believed to be important in controlling enamel formation. Here we provide in vitro and in vivo evidence of co-assembly and co-localization of ameloblastin with amelogenin using both biophysical and immunohistochemical methods. We performed co-localization studies using immunofluorescence confocal microscopy with paraffin-embedded tissue sections from mandibular molars of mice at 1, 5, and 8 days of age. Commercially-available ameloblastin antibody (M300) against mouse ameloblastin residues 107-407 and an antibody against full-length recombinant mouse (rM179) amelogenin were used. Ameloblastin-M300 clearly reacted along the secretory face of ameloblasts from days 1-8. Quantitative co-localization was analyzed (QCA) in several configurations by choosing appropriate regions of interest (ROIs). Analysis of ROIs along the secretory face of ameloblasts revealed that at day 1, very high percentages of both the ameloblastin and amelogenin co-localized. At day 8 along the ameloblast cells the percentage of co-localization remained high for the ameloblastin whereas co-localization percentage was reduced for amelogenin. Analysis of the entire thickness on day 8 revealed no significant co-localization of amelogenin and ameloblastin. With the progress of amelogenesis and ameloblastin degradation, there was a segregation of ameloblastin and co-localization with the C-terminal region decreased. CD spectra indicated that structural changes in ameloblastin occurred upon addition of amelogenin. Our data suggest that amelogenin-ameloblastin complexes may be the functional entities at the early stage of enamel mineralization.

Localization and quantitative co-localization of enamelin with amelogenin.

Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 µm height, 33 µm width) and small (7 µm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.

Amelogenin processing by MMP-20 prevents protein occlusion inside calcite crystals.

Calcite crystals were grown in the presence of full-length amelogenin and during its proteolysis by recombinant human matrix metalloproteinase 20 (rhMMP-20). Recombinant porcine amelogenin (rP172) altered the shape of calcite crystals by inhibiting the growth of steps on the {104} faces and became occluded inside the crystals. Upon co-addition of rhMMP-20, the majority of the protein was digested resulting in a truncated amelogenin lacking the C-terminal segment. In rP172-rhMMP-20 samples, the occlusion of amelogenin into the calcite crystals was drastically decreased. Truncated amelogenin (rP147) and the 25-residue C-terminal domain produced crystals with regular shape and less occluded organic material. Removal of the C-terminal diminished the affinity of amelogenin to the crystals and therefore prevented occlusion. We hypothesize that HAP and calcite interact with amelogenin in a similar manner. In the case of each material, full-length amelogenin binds most strongly, truncated amelogenin binds weakly and the C-terminus alone has the weakest interaction. Regarding enamel crystal growth, the prevention of occlusion into maturing enamel crystals might be a major benefit resulting from the selective cleavage of amelogenin at the C-terminus by MMP-20. Our data have important implications for understanding the hypomineralized enamel phenotype in cases of amelogenesis imperfecta resulting from MMP-20 mutations and will contribute to the design of enamel inspired biomaterials.

Purification, characterisation and intracellular localisation of aryl hydrocarbon interacting protein-like 1 (AIPL1) and effects of mutations associated with inherited retinal dystrophies.

Mutations in AIPL1 are associated with Leber Congenital Amaurosis (LCA), a major cause of childhood blindness, yet the cellular function of the encoded protein has yet to be fully elucidated. In order to investigate the biochemistry of AIPL1, we have developed a system for the expression of the recombinant protein in bacteria and its subsequent purification. The secondary structure and thermostability of wild-type and mutant proteins have been examined by circular dichroism (CD) spectroscopy. Some of the variants, notably W278X and P376S, had markedly different secondary structure compositions, indicating that the proteins had not folded properly, whilst W278X and T114I were particularly thermolabile. When eukaryotic cells were transfected with the AIPL1 expression constructs, we show by immunofluorescence microscopy that wild-type protein is distributed throughout the nucleus and cytoplasm. Several of the mutants give similar results. With two of the disease-associated variants (W278X and A336Delta2), however, the protein remains in the cytoplasm in aggresome-like particles. These particles were shown to be ubiquitinated, indicating that the mutant protein had been tagged for proteosomal degradation. On this basis, we can conclude that wild-type protein is expressed in a soluble and folded manner, and that some of the disease-associated mutant proteins are nonfunctional because they are insoluble and are degraded by the cell. Other mutations appear to have a more localised effect on secondary structure, which does not result in insolubility or affect protein targeting, but reduces the stability of the protein at human body temperature.

Disease mechanism for retinitis pigmentosa (RP11) caused by mutations in the splicing factor gene PRPF31.

This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology.