Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Porphyromonas gingivalis Fimbriation via Random Mutant Library Construction.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli-Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure-function relationships of proteins of interest.

The AtoC family response regulator upregulates an operon encoding putative outer membrane proteins sorted by type IX secretion system in Porphyromonas gingivalis.

OBJECTIVES: Porphyromonas gingivalis, a keystone periodontopathogen, has multiple two-component systems that are thought to modulate virulence. In this study, we focused on PGN_0775 response regulator (RR), an AtoC homolog, and attempted to identify the target gene that it regulates in P. gingivalis. METHODS: Comparative proteomic analyses comprising two-dimensional electrophoresis and peptide mass fingerprinting were applied to total protein samples from parent (WT) and atoC gene knockout (KO) strains to screen for affected protein spots. Fluctuations in the expression of corresponding genes were further confirmed using relative quantitative real-time polymerase chain reaction (RQPCR). RESULTS: Five protein spots with fluctuating expression levels were identified in pgn_0775 KO strains along with their masses and physiological features, which contained two hypothetical proteins with higher expression levels in the WT than in the KO strains. RQPCR analysis confirmed that mRNA levels were consistently decreased in KO and recovered in pgn_0775-complemented KO strains. The two hypothetical proteins appeared to be the products of an operon that comprises four genes encoding three hypothetical but putative type IX secretion system sorting domain-containing proteins and an N-terminal region of the C25 cysteine peptidase. CONCLUSIONS: The AtoC RR homolog in P. gingivalis upregulates the expression of the operon encoding potentially antigenic proteins retained on the cell surface; thus, it could be a promising target for P. gingivalis-specific antivirulence therapy.

Environmental Risk Assessment of Active Human Pharmaceutical Ingredients in Urban Rivers in Japan.

Active pharmaceutical ingredients (APIs) have become a public concern owing to their possible adverse effects on aquatic organisms. Ministry of Health, Labor and Welfare in Japan (MHLW) issued "Guidance on the Environmental Risk Assessment (ERA) in new pharmaceutical development" in 2016. To evaluate the validity of phase 1 in the MHLW's ERA guidance, we monitored the measured environmental concentrations (MECs) of approved APIs in urban rivers and sewage treatment plants (STPs) in Japan and compared these MECs with the predicted environmental concentration (PEC). We collected water samples from urban seven rivers and three STPs during each season. Fifty-one APIs for human and veterinary use and the artificial sweetener sucralose were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Forty-four APIs were observed in the rivers and 42 were found in the influent and effluent of STPs, with levels ranging from nanograms to micrograms per liter. The action limit in phase I of the MHLW's guidance was set to 10 ng/L, and there was no API except for ketoprofen, for which PEC of the MHLW's guidance (PECjapan) was lower than 10 ng/L and the maximum MEC (MECmax) was 10 ng/L or greater. Almost all APIs also had median MECs that were lower than those of the respective PECjapan. These results indicate that the PECjapan values in phase I of the MHLW's guidance were appropriate. However, some APIs had MECmax values that were greater than those of the respective PECjapan due to overestimation of the dilution factor of river water and/or underestimation of API production.

Expressive Arts Therapy with Hospitalized Children: A Pilot Study of Co-Creating Healing Sock Creatures©.

A novel form of expressive arts therapy was developed in a pediatric unit and received enthusiastic support from hospital staff and family members because of their impressions that the children were calmer following therapy, as well as throughout the remainder of the hospital stay. A pilot study was conducted to assess the feasibility of quantifying such impressions by measuring changes in the children's mood by self-report. Twenty-five children (mean age 8.34 years, SD 3.77) were recruited for the study, coming from diverse social-economic backgrounds, ethnicities, and an array of medical diagnoses. The results document improvements in mood for children following therapy sessions, compared to children in a wait-list control group. Additionally, a meta-analysis examining external influences and changes in salivary cortisol levels measured before and after therapy sessions illustrates the importance of considering aspects of the clinical setting when assessing the effectiveness of this and other expressive arts therapies for reducing stress during hospitalization.

Stepwise regulation of hole transport in DNA by control of triplex formation.

A functionality for regulating hole transport efficiency is a prerequisite for the utilization of DNA duplexes as nanodevices. Herein, we report the regulation of hole transport in anthraquinone-tethered DNA with dual triplex forming sites. Long-range photooxidation experiments showed that hole transport was effectively suppressed by the formation of triplex at low temperature, while it was recovered by dissociation to the duplex at higher temperature. Variation of temperature induced the formation and dissociation of the third strand at each triplex region individually, leading to the stepwise regulation of hole transport in DNA.

Selenium content in seafood in Japan.

Selenium is an essential micronutrient for humans, and seafood is one of the major selenium sources, as well as red meat, grains, eggs, chicken, liver and garlic. A substantial proportion of the total amount of selenium is present as selenium containing imidazole compound, selenoneine, in the muscles of ocean fish. In order to characterize the selenium content in seafood, the total selenium levels were measured in the edible portions of commercially important fish and shellfish species. Among the tested edible portions, alfonsino muscle had the highest selenium levels (concentration of 1.27 mg/kg tissue). High levels of selenium (1.20-1.07 mg/kg) were also found in the salted ovary products of mullet and Pacific herring. In other fish muscles, the selenium levels ranged between 0.12 and 0.77 mg/kg tissue. The selenium levels were closely correlated with the mercury levels in the white and red muscles in alfonsino. The selenium content in spleen, blood, hepatopancreas, heart, red muscle, white muscle, brain, ovary and testis ranged between 1.10 and 24.8 mg/kg tissue in alfonsino.

Co-expression of somatostatin receptor subtypes and estrogen receptor-α mRNAs by non-functioning pituitary adenomas in young patients.

The expression by non-functioning adenomas (NFoma) of somatostatin receptor (SSTR) subtypes and estrogen receptor (ERα) is poorly understood. Consequently, the mRNAs of SSTR subtypes (SSTR) 1, 2, 3, and 5, dopamine receptor (D2R), and ERα were measured by real-time quantitative RT-PCR in 59 NFomas and 50 functioning adenomas; the latter included 30 GH-secreting adenomas (GHomas) and 20 prolactinomas (PRLomas). NFomas expressed higher levels of SSTR3 than functioning adenomas but had lower levels of SSTR2, SSTR5 and D2R mRNAs than GHomas. Their ERα levels were higher than those of GHomas. The SSTR subtype mRNA levels in NFomas correlated significantly with each other; there was also a good correlation between the SSTR subtypes and ERα in NFomas. These correlations were largely only observed in younger patients (<50 years). The present study describes the differential expression of SSTR subtypes in the largest number of NFoma patients studied thus far, and further proposes possible involvements of SSTR3 and estrogen in the pathophysiology of NFomas.

Photoelectrochemical evaluation of pH effect on hole transport through triplex-forming DNA immobilized on a gold electrode.

We characterized pH effect on hole transport through DNA duplexes possessing a partial triplex-forming region. Direct electrochemical measurement of the current response of photosensitizer-tethered DNA immobilized on a gold electrode revealed that the partial triplex formation under acidic conditions suppressed photocurrent due to hole transport, while dissociation of the triplex into the duplex as occurred upon increasing pH values recovered the photocurrent efficiency. Reversible conversion between duplex and triplex induced upon cyclic alternation of pH values resulted in a rise and fall of photocurrent responses, indicating that pH change may feature in the switching function of hole transport in DNA. These electrochemical behaviors could be correlated to the results obtained in long-range photo-oxidative DNA cleavage experiments, in which DNA cleavage at the hole trapping site beyond the triplex region was significantly suppressed under triplex-forming acidic conditions.

Electrochemical evaluation of alternating duplex-triplex conversion effect on the anthraquinone-photoinjected hole transport through DNA duplex immobilized on a gold electrode.

Amperometry was employed to characterize the anthraquinone (AQ)-photoinjected hole transport through a 20-mer oligodeoxynucleotide (ODN) duplex, as immobilized on the surface of a gold electrode, and its triplex forms converted by association with several third oligopyrimidine (OPD) short strands. While the cathodic photocurrent was observed upon irradiation at 365 nm of the AQ photosensitizer linked to the end of DNA duplex, a marked lowering of the current density was identified to occur by the triplex formation of a duplex with a given third OPD short strand. The photocurrent through the DNA duplex showed a reversible fall-rise response concomitant with alternating association-dissociation cycle of the OPD short-strand, as regulated by temperature change around the corresponding melting temperature of the DNA triplex. Both the switched photoirradiation and the thermally alternating duplex-triplex conversion could provide tools of regulating the DNA hole transport.

Switching of charge transport in DNA by regulated triplex formations at the dual sites.

A functionality of on/off switching to regulate conductivity should be among the prerequisites for the utilization of DNA duplex as a conducting material. We have reported that variation of temperatures can regulate the triplex formation and dissociation, thereby regulating charge transport in DNA. Based on this result, the on/off switching of conductivity at dual triplex forming sites in DNA was attempted, using two types of oligonucleotide (ODNs) with different strand lengths. Variation of temperatures produced DNA duplexes without any triplex site and with single and double triplex sites, by which the intramolecular charge transport in DNA duplex could be regulated at the respective triplex-forming sites. This may promote the utility of DNA duplex as a potential constituent of electronic nanodevices.

The effect of triple helix forming on charge transport in DNA.

The charge transport efficiency in DNA duplex could be regulated by thermal conversion between the formation and dissociation of partial triplex structure with a short strand oligonucleotide (ODN). Photoinduced long-distance one-electron oxidation in duplex ODNs with partial triplex structural region was examined at various temperatures to evaluate the charge transport efficiency. Under these conditions, the apparent charge transport was suppressed dramatically at 0 degrees C relative to 25 degrees C. These results strongly indicate that variation of temperatures could regulate the triplex formation and dissociation, resulting in the regulation of charge transport in DNA. Thus, triplex formation is expected to be a constituent of new well regulated biomaterial that will applicable to nano-scale electronic devices.