Compressive Strength Evaluation of Ordinary Portland Cement Mortar Blended with Hydrogen Nano-Bubble Water and Graphene.

Abnormal climate changes have occurred all over the world due to greenhouse gases (GHGs). Various countries are targeting emission reductions of GHGs in 2020 and trying to GHGs those in multiple fields. Cement-based structures account for a large part in the construction industry. One ton of carbon dioxide is produced during the manufacturing process for a ton of cement. Therefore, decreasing cement usage is essential for carbon dioxide reduction. However, strength characteristics of cement are necessary conditions to meet the required strength of a structure. Therefore, it is necessary to develop cement substitutes and economic additives. In this study, we proposed an eco-friendly blend ratio by comparing the compressive strength of Ordinary Portland Cement (OPC) mortar and two variations. One was a mortar with a small amount of APTMS-sGO added. The other was a mortar mixed with HNBW (hydrogen nano-bubble water) as an enhanced material instead of ordinary water. The mortar added with 0.1% of APTMS-sGO showed improved early strength compared with OPC mortar. Its strength was enhanced 31.1% by using HNBW as functional water. Strength was improved 20.4% for cement mortar added with Graphene Oxide after reacting with SAM containing APTMS. When the texture of both mortars became denser, early compressive strength at 7 days each was 20.4-31.1% higher than that of OPC mortar. Finally, the strength was increased by 10.2% at 28 days.

Risk assessment of chlortetracycline, oxytetracycline, sulfamethazine, sulfathiazole, and erythromycin in aquatic environment: are the current environmental concentrations safe?

To understand potential risks of major pharmaceutical residues in waters, we evaluated ecotoxicities of five major veterinary pharmaceuticals, i.e., chlortetracycline, oxytetracycline, sulfamethazine, sulfathiazole, and erythromycin, which have been frequently detected in freshwater environment worldwide. We conducted acute and chronic toxicity tests using two freshwater invertebrates (Daphnia magna and Moina macrocopa) and a fish (Oryzias latipes). In general, D. magna exhibited greater sensitivity than M. macrocopa, and chronic reproduction was the most sensitive endpoints for both organisms. The population growth rate was adversely influenced by exposure to chlortetracycline, sulfamethazine, or sulfathiazole in water fleas, but reduction in population size was not expected. In O. latipes, the tested pharmaceuticals affected several reproduction related endpoints including time to hatch and growth. Based on the toxicity values from the present study and literature, algae appeared to be the most sensitive organism, followed by Daphnia and fish. Hazard quotients derived from measured environmental concentrations (MECs) and predicted no effect concentrations (PNECs) for erythromycin and oxytetracycline exceeded unity, suggesting that potential ecological effects at highly contaminated sites cannot be ruled out. Long-term consequences of veterinary pharmaceutical contamination in the environment deserve further investigation.

Influence of a five-day vegetarian diet on urinary levels of antibiotics and phthalate metabolites: a pilot study with "Temple Stay" participants.

Diet is purported to be means of exposure to many environmental contaminants. The purpose of this study is to understand the influence of dietary change on the levels of exposure to several environmental chemicals - in particular, antibiotics and phthalates. For this purpose, we examined the extent to which short-term changes in diet influenced the inadvertent exposure levels to these chemicals in an adult population. We recruited participants (n=25) of a five-day 'Temple Stay' program in Korea and collected urine samples before and after the program. We also conducted a questionnaire survey on participants' dietary patterns prior to their participation. During the program, participants followed the daily routines of Buddhist monks and maintained a vegetarian diet. Urinary levels of three antibiotics and their major metabolites, metabolites of four major phthalates, and malondialdehyde (MDA) as an oxidative stress biomarker were analyzed. The frequency and levels of detection for antibiotics and phthalates noticeably decreased during the program. Urinary MDA levels were significantly lower than before program participation (0.16 versus 0.27mg/g creatinine). Although the exposure to target compounds might be influenced by other behavioral patterns, these results suggest that even short-term changes in dietary behavior may significantly decrease inadvertent exposure to antibiotics and phthalates and hence may reduce oxidative stress levels.

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2.

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.

Quantitative proteomics of intracellular Porphyromonas gingivalis.

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.

Transcriptional organization of the Porphyromonas gingivalis fimA locus.

Two different transcriptional start sites, as well as promoter regions and translational starts, have been proposed for the fimA gene encoding a long fimbriae subunit protein of Porphyromonas gingivalis. In this study, the fimA promoter regions and organization of the fimA operon were characterized. The two putative promoter regions for fimA were fused with a lacZ reporter gene, cloned into the shuttle plasmid vector pT-COW, and the recombinant plasmids were introduced to P. gingivalis 33277. Reverse transcriptase-polymerase chain reaction demonstrated mRNA production from the promoter proximal to the translational start. LacZ activities of P. gingivalis containing the recombinant plasmids showed that maximal expression of fimA was promoted by the proximal promoter in combination with distal regulatory sequences. A polycistronic message spanning PG2130, PG2131 and fimA (PG2132) was observed, thus fimA transcripts may also be generated by processing of the polycistronic message.

Intrinsic apoptotic pathways of gingival epithelial cells modulated by Porphyromonas gingivalis.

Porphyromonas gingivalis can inhibit chemically induced apoptosis in primary cultures of gingival epithelial cells through blocking activation of the effector caspase-3. The anti-apoptotic phenotype of P. gingivalis is conserved across strains and does not depend on the presence of fimbriae, as fimbriae-deficient mutants and a naturally occurring non-fimbriated strain were able to impede apoptosis. To dissect the survival pathways modulated by P. gingivalis, protein and gene expression of a number of components of apoptotic death pathways were investigated. P. gingivalis infection of epithelial cells resulted in the phosphorylation of JAK1 and Stat3. Quantitative real-time reverse transcription polymerase chain reaction showed that expression of Survivin and Stat3 itself, targets of activated Stat3, were elevated in P. gingivalis-infected cells. siRNA knockdown of JAK1, in combination with knockdown of Akt, abrogated the ability of P. gingivalis to block apoptosis. In contrast, cIAP-1 and cIAP-2 were not differentially regulated at either the protein or mRNA levels by P. gingivalis. One mechanism by which P. gingivalis can block apoptotic pathways in gingival epithelial cells therefore is through manipulation of the JAK/Stat pathway that controls the intrinsic mitochondrial cell death pathways. Induction of a pro-survival phenotype may prevent programmed host cell death and aid survival of P. gingivalis within gingival epithelial cells.

Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: non-label methods comparison, q-values and LOWESS curve fitting.

Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

Expression of the short fimbriae of Porphyromonas gingivalis is regulated in oral bacterial consortia.

The Mfa1 protein of Porphyromonas gingivalis is the structural subunit of the short fimbriae and mediates coadhesion between P. gingivalis and Streptococcus gordonii. We utilized a promoter-lacZ reporter construct to examine the regulation of mfa1 expression in consortia with common oral plaque bacteria. Promoter activity of mfa1 was inhibited by S. gordonii, Streptococcus sanguinis and Streptococcus mitis. In contrast, Streptococcus mutans, Streptococcus cristatus, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum did not affect mfa1 expression. Expression of SspA/B, the streptococcal receptor for Mfa1, was not required for regulation of mfa1 promoter activity. Proteinaceous molecule(s) in oral streptococci may be responsible for regulation of Mfa1 expression. Porphyromonas gingivalis is capable of detecting heterologous organisms, and responds to selected organisms by specific gene regulation.

LuxS involvement in the regulation of genes coding for hemin and iron acquisition systems in Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe(2+) ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.

Role of the Porphyromonas gingivalis InlJ protein in homotypic and heterotypic biofilm development.

The oral pathogen Porphyromonas gingivalis expresses a homolog of the internalin family protein InlJ. Inactivation of inlJ reduced monospecies biofilm formation by P. gingivalis. In contrast, heterotypic P. gingivalis-Streptococcus gordonii biofilm formation was enhanced in the InlJ-deficient mutant. The results indicate a nuanced role for InlJ in regulating biofilm accumulations of P. gingivalis.

Short fimbriae of Porphyromonas gingivalis and their role in coadhesion with Streptococcus gordonii.

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, attaches and forms biofilms on substrata of Streptococcus gordonii. Coadhesion and biofilm development between these organisms requires the interaction of the short fimbriae of P. gingivalis with the SspB streptococcal surface polypeptide. In this study we investigated the structure and binding activities of the short fimbriae of P. gingivalis. Electron microscopy showed that isolated short fimbriae have an average length of 103 nm and exhibit a helical structure with a pitch of ca. 27 nm. Mfa1, the major protein subunit of the short fimbriae, bound to SspB protein, and this reaction was inhibited by purified recombinant Mfa1 and monospecifc anti-Mfa1 serum in a dose-dependent manner. Complementation of a polar Mfa1 mutant with the mfa1 gene restored the coadhesion phenotype of P. gingivalis. Hence, the Mfa1 structural fimbrial subunit does not require accessory proteins for binding to SspB. Furthermore, the interaction of Mfa1 with SspB is necessary for optimal coadhesion between P. gingivalis and S. gordonii.

Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components.

The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were identified in four independent analyses; 479 of these proteins showed evidence of differential expression after exposure of P. gingivalis to either conditioned epithelial cell growth medium or control conditions: i.e., they were only detected under one set of conditions. Moreover, 276 genes annotated as hypothetical were found to encode expressed proteins. Among the proteins up-regulated in the presence of epithelial cell components were a homolog of the internalin proteins of Listeria monocytogenes and subunits of the ATP-dependent Clp protease complex. Insertional inactivation of clpP, encoding the Clp proteolytic subunit, resulted in approximately a 50% reduction in invasion of P. gingivalis. These results suggest that adaptation to an epithelial cell environment induces a major shift in the expressed proteome of the organism. Furthermore, ClpP, that is up-regulated in this environment, is required for optimal invasive activity of P. gingivalis.

Identification of Porphyromonas gingivalis genes specifically expressed in human gingival epithelial cells by using differential display reverse transcription-PCR.

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, can invade and survive within host epithelial cells. The molecular mechanisms by which P. gingivalis induces uptake and adapts to an intracellular environment are not fully understood. In this study, we have investigated the genetic responses of P. gingivalis internalized within human gingival epithelial cells (GECs) in order to identify factors involved in invasion and survival. We compared the differential display of arbitrarily PCR-amplified gene transcripts in P. gingivalis recovered from GECs with the display of transcripts in P. gingivalis control cultures. Over 20 potential differentially expressed transcripts were identified. Among these, pepO, encoding an endopeptidase, and genes encoding an ATP-binding cassette (ABC) transporter and a cation-transporting ATPase were upregulated in GECs. To investigate the functionality of these gene products, mutants were generated by insertional inactivation. Compared to the parental strain, mutants of each gene showed a significant reduction in their invasion capabilities. In addition, GEC cytoskeletal responses to the mutants were distinct from those induced by the parent. In contrast, adhesion of the mutant strains to GECs was not affected by lack of expression of the gene products. These results suggest that PepO, a cation-transporting ATPase, and an ABC transporter are required for the intracellular lifestyle of P. gingivalis.

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression.

We compare typical qualitative protein identification data from two-dimensional (2D) polyacrylamide gel electrophoresis and reconstructed protein arrays, in the context of measuring protein expression by the Gram-negative periodontal pathogen Porphyromonas gingivalis. The arrays were assembled computationally from genome annotations and tandem mass spectrometry data from an off-line HPLC fractionation combined with 2D capillary HPLC analysis of whole proteome enzymatic digests. The 2D separation was carried out with a standard binary gradient HPLC system, modified only slightly with readily available components. Compared to 2D gels, the number of annotated open reading frames identified using the 3D HPLC approach was typically larger by at least a factor of 30. However, the newer technology is currently limited in its ability to reflect the many protein variants derived from posttranscriptional and posttranslational processing.

Role of the Streptococcus gordonii SspB protein in the development of Porphyromonas gingivalis biofilms on streptococcal substrates.

Porphyromonas gingivalis is an aggressive periodontal pathogen that persists in the mixed-species plaque biofilm on tooth surfaces. P. gingivalis cells attach to the plaque commensal Streptococcus gordonii and this coadhesion event leads to the development of P. gingivalis biofilms. Binding of these organisms is multimodal, involving both the P. gingivalis major fimbrial FimA protein and the species-specific interaction of the minor fimbrial Mfa1 protein with the streptococcal SspB protein. This study examined the contribution of the Mfa1-SspB interaction to P. gingivalis biofilm formation. P. gingivalis biofilms readily formed on substrata of S. gordonii DL1 but not on Streptococcus mutans cells which lack a coadhesion-mediating homologue of SspB. An insertional inactivation of the mfa1 gene in P. gingivalis resulted in a phenotype deficient in S. gordonii binding and unable to form biofilms. Furthermore, analysis using recombinant streptococci and enterococci showed that P. gingivalis biofilms formed on Enterococcus faecalis strains expressing SspB or translational fusions of SspB with SpaP (the non-adherent SspB homologue in S. mutans) containing the P. gingivalis adherence domain (SspB adherence region, BAR) of SspB. In contrast, an isogenic Ssp null mutant of S. gordonii DL1 was unable to support biofilm growth, even though this strain bound to P. gingivalis FimA at levels similar to wild-type S. gordonii DL1. Finally, site-specific mutation of two functional amino acid residues in BAR resulted in SspB polypeptides that did not promote the development of P. gingivalis biofilms. These results suggest that the induction of P. gingivalis biofilms on a streptococcal substrate requires functional SspB-minor fimbriae interactions.