Crystal structure of heliorhodopsin.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

Specific zinc binding to heliorhodopsin.

Heliorhodopsins (HeRs), a recently discovered family of rhodopsins, have an inverted membrane topology compared to animal and microbial rhodopsins. The slow photocycle of HeRs suggests a light-sensor function, although the actual function remains unknown. Although HeRs exhibit no specific binding of monovalent cations or anions, recent ATR-FTIR spectroscopy studies have demonstrated the binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR) and 48C12. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding, suggesting a possible modification of the HeR function by Zn2+. The present study shows that Zn2+-binding lowers the thermal stability of TaHeR, and slows back proton transfer to the retinal Schiff base (M decay) during its photocycle. Zn2+-binding was similarly observed for a TaHeR opsin that lacks the retinal chromophore. We then studied the Zn2+-binding site by means of the ATR-FTIR spectroscopy of site-directed mutants. Among five and four mutants of His and Asp/Glu, respectively, only E150Q exhibited a completely different spectral feature of the α-helix (amide-I) in ATR-FTIR spectroscopy, suggesting that E150 is responsible for Zn2+-binding. Molecular dynamics (MD) simulations built a coordination structure of Zn2+-bound TaHeR, where E150 and protein bound water molecules participate in direct coordination. It was concluded that the specific binding site of Zn2+ is located at the cytoplasmic side of TaHeR, and that Zn2+-binding affects the structure and structural dynamics, possibly modifying the unknown function of TaHeR.

Inhibition of enamel demineralization by an ion-releasing tooth-coating material.

PURPOSE: To evaluate the inhibitory effect of a surface pre-reacted glass-ionomer (S-PRG) filler-containing tooth-coating material on enamel demineralization. The outer surface of the S-PRG filler is in a state in which ions are readily released. METHODS: Human enamel blocks were incubated in lactic acid solution (pH 4.0) with and without a disk (n=6) made of the cured tooth-coating material. Test solutions were changed every 24 hours and incubation was continued for 4 days. The pH and amount of fluoride released were measured with an electrode and ion meter, respectively. The concentrations of ions (aluminum, boron, calcium, phosphorus, silicon, sodium, and strontium) were measured by inductively coupled plasma atomic emission spectroscopy. The surface of the enamel block was observed with a scanning electron microscope. RESULTS: Enamel demineralization was not observed in an enamel block incubated with a disk of the tooth-coating material. Ions released from S-PRG filler had an acid buffering action in the low pH lactic acid solution. However, in the enamel block-only solution showing high levels of calcium ion release, the degree of demineralization was correlated with morphological changes of the enamel surface. CLINICAL SIGNIFICANCE: Due to the buffering effects of the pre-reacted glass-ionomer surface by ion release, the S-PRG filler-containing tooth-coating material inhibited enamel demineralization by neutralizing the acidic environment at an early time point.

Epigallocatechin Gallate-Modified Gelatin Sponges Treated by Vacuum Heating as a Novel Scaffold for Bone Tissue Engineering.

Chemical modification of gelatin using epigallocatechin gallate (EGCG) promotes bone formation in vivo. However, further improvements are required to increase the mechanical strength and bone-forming ability of fabricated EGCG-modified gelatin sponges (EGCG-GS) for practical applications in regenerative therapy. In the present study, we investigated whether vacuum heating-induced dehydrothermal cross-linking of EGCG-GS enhances bone formation in critical-sized rat calvarial defects. The bone-forming ability of vacuum-heated EGCG-GS (vhEGCG-GS) and other sponges was evaluated by micro-computed tomography and histological staining. The degradation of sponges was assessed using protein assays, and cell morphology and proliferation were verified by scanning electron microscopy and immunostaining using osteoblastic UMR106 cells in vitro. Four weeks after the implantation of sponges, greater bone formation was detected for vhEGCG-GS than for EGCG-GS or vacuum-heated gelatin sponges (dehydrothermal cross-linked sponges without EGCG). In vitro experiments revealed that the relatively low degradability of vhEGCG-GS supports cell attachment, proliferation, and cell-cell communication on the matrix. These findings suggest that vacuum heating enhanced the bone forming ability of EGCG-GS, possibly via the dehydrothermal cross-linking of EGCG-GS, which provides a scaffold for cells, and by maintaining the pharmacological effect of EGCG.

Inhibition of matrix metalloproteinases and toxicity of gold and platinum nanoparticles in L929 fibroblast cells.

This study evaluated the inhibition of matrix metalloproteases (MMPs) and cellular responses elicited by gold (Au) and platinum (Pt) nanoparticles (NPs). The interaction of MMP-1 and NPs was evaluated using an MMP assay kit. The cultured L929 cells were exposed to various concentrations of NPs. The cellular responses to NPs were examined using a cytotoxicity assay (that evaluated cell viability and lactic dehydrogenase production), real-time polymerase chain reaction (RT-qPCR), and transmission electron microscopy. Both types of NPs, when used at concentrations above 10 µg ml(-1), inhibited MMP-1 activity. No cytotoxic effects were found when the cells were exposed to AuNPs. In contrast, PtNPs, at both 100 and 400 µg ml(-1), induced cytotoxicity. No inflammatory responses (production of interleukin-6 and tumor necrosis factor-alpha) to NPs were identified by RT-qPCR. The negative surface charge of NPs (COOH(-)) binds to the Zn(2+) of the MMP active center by chelation, leading to MMP inhibition. Gold nanoparticles are plausible candidates for MMP inhibitors in resin-bonding materials because they effectively inhibit MMP-1 activity without cytotoxic or inflammatory effects.

Postoperative pneumatosis intestinalis (PI) and portal venous gas (PVG) may indicate bowel necrosis: a 52-case study.

BACKGROUND: The significance of pneumatosis intestinalis (PI) and portal venous gas (PVG) is controversial. This retrospective study evaluated the risk factors for bowel necrosis in patients with PI and/or PVG. METHODS: Between 2002 and 2015, 52 patients were diagnosed with PI and/or PVG and were included in this study. The patients were classified according to the presence or absence of bowel necrosis in surgical findings or at autopsy. Patient characteristics and clinical findings related to bowel necrosis were investigated. RESULTS: Bowel necrosis was diagnosed in 17 (32.7 %) patients. Amongst these 17, 10 patients received salvage surgical intervention, and seven of those diagnosed with bowel necrosis survived after the operation. The remaining 35 patients received conservative treatment with or without exploratory laparotomy. Between patients with and without bowel necrosis, laboratory data revealed significant differences in the levels of C-reactive protein (P = 0.0038), creatinine (P = 0.0054), and lactate (P = 0.045); clinical findings showed differences in abdominal pain (P = 0.019) and peritoneal irritation signs (P = 0.016); computed tomography detected ascites (P = 0.011) and changes of bowel wall enhancement (P = 0.03) that were significantly higher in patients with bowel necrosis. The rate of PI and/or PVG detected in patients postoperatively was significantly higher in patients with bowel necrosis (P < 0.0001). Multivariate analysis showed that bowel necrosis was significantly more likely when PI or PVG was detected in postoperative patients than in patients who had not had surgery (P = 0.003). CONCLUSIONS: PI and/or PVG, alone, are not automatically indicative of bowel necrosis. However, when these conditions occur postoperatively, they indicate bowel necrosis requiring reoperation.

Inhibition of enamel demineralization by buffering effect of S-PRG filler-containing dental sealant.

The buffering capacity and inhibitory effects on enamel demineralization of two commercially available dental sealants were evaluated in this study. The effects of filler particles were also examined. Disks of enamel and cured sealant materials of BeautiSealant (silica or S-PRG filler) or Teethmate F-1 were incubated in lactic acid solutions (pH 4.0) for 1-6 d. The pH changes and amounts of ions released in the solutions were assessed, and enamel surfaces were observed using a scanning electron microscope. The pH of the solution with BeautiSealant (S-PRG filler) was neutralized from pH 4.0 to pH 6.1 (after incubation for 1 d) and from pH 4.0 to pH 6.7 (after incubation for 6 d). In addition, no release of calcium ions was detected and the enamel surface was morphologically intact in scanning electron microscopy images. However, the pH of the solution with Teethmate F-1 remained below pH 4.0 during incubation from days 1 to 6. Calcium release was increased in solutions up to and after 6 d of incubation. Scanning electron microscopy images showed that the structures of hydroxyapatite rods were exposed at the specimen surfaces as a result of demineralization. Ions released from S-PRG filler-containing dental sealant rapidly buffered the lactic acid solution and inhibited enamel demineralization.

Internal Proton Transfer in the Activation of Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion of the protonated Schiff base, E108 in HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR). Upon light absorption, the M and O intermediates form in HeRs, as well as type-1 microbial rhodopsins, indicating that the proton transfer from the Schiff base leads to the activation of HeRs. The present flash photolysis study of TaHeR in the presence of a pH-sensitive dye showed that TaHeR contains a proton-accepting group (PAG) inside protein. Comprehensive mutation study of TaHeR found the E108D mutant abolishing the M formation, which is not only at pH 8, but also at pH 9 and 10. The lack of M observation does not originate from the short lifetime of the M intermediate in E108D, as FTIR spectroscopy revealed that a red-shifted K-like intermediate is long lived in E108D. It is likely that the K-like intermediate returns to the unphotolyzed state without internal proton transfer in E108D. E108 and D108 are the Schiff base counterions of the wild-type and E108D mutant TaHeR, respectively, whereas small difference in length of side chains determine internal proton transfer reaction from the Schiff base. Based on the present finding, we propose that the internal water cluster (four water molecules) constitutes PAG in the M intermediate of TaHeR. In the wild type TaHeR, a protonated water cluster is stabilized by forming a salt bridge with E108. In contrast, slightly shortened counterion (D108) cannot stabilize the protonated water cluster in E108D, and thus impairs internal proton transfer from the Schiff base.

[Three patients with local recurrence of ER-positive breast cancer for whom anastrozole was clinically effective].

We treated three postmenopausal female patients with unresectable local recurrence from breast cancer. All pathological diagnoses of the local recurrence lesions were ER-positive breast cancer. For treatment, we administered anastrozole to these three patients. One has been stable disease for 25 months after taking anastrozole. Another has also showed stable disease for 18 months, and the last patient has been a partial response. We performed a biopsy from a recurring lesion on these three patients, and made a diagnosis of ER-positive breast cancer. This strategy of unresectable local recurrence revealed that these three patients could have had a stable condition for a long duration by taking anastrozole.

Anterior graft migration in posterior lumbar interbody fusion: A case report and literature review.

Key Clinical Message: Spine surgeons should be aware of the possibility of anterior displacement of the grafted bone during PLIF and the potential for severe complications that may arise because of such displacement so that preparations can be made for a proper response. Abstract: We report two cases of anterior displacement of the grafted bone after posterior lumbar interbody fusion (Graphical Abstract A-D). The patients did not require additional surgery. The anterior migration of grafted bone or cage can cause damage to anterior organs and blood vessels. Therefore, a careful surgical procedure is necessary.

Bonding of self-etching adhesives on dehydrated dentin.

PURPOSE: This study examined the effect of dentin pretreatment with acetone or ethanol on bonding of one-bottle self-etching vs two-step self-etching adhesives to evaluate the effect of hydration on bond strength. MATERIALS AND METHODS: The bond strengths of resins were measured using two one-bottle and two two-step selfetching adhesives that bonded to wet dentin (control), or acetone- or ethanol-pretreated dentin. Wet dentin is the normal substrate for bond testing, and in the pretreatment groups, 95% acetone or ethanol was applied to the dentin surface for 30 s before bonding. Weight of resin adhesives before and after evaporation was measured to evaluate the extent of the solvent contents in the adhesives. Furthermore, phase separation of adhesives was morophologically investigated by in situ light-microscopic observation to evaluate the hydrophilicity of the adhesive resins. RESULTS: The bond strengths of the acetone and ethanol groups for one-bottle adhesives were significantly greater than for the control (p < 0.05). However, significantly lower bond strengths were found in the pretreatment groups for the two-step self-etching adhesives (p < 0.05). CONCLUSION: The bond strength depended on the wetness of the bonding substrate for both adhesive groups, with a positive effect in bond strength for one-bottle adhesives and a negative effect for two-bottle self-etching adhesives. The acetone- or ethanol-pretreated dentin surfaces may provide an improved substrate for resin adhesion of hydrophilic self-etching adhesives such as one-bottle self-etching adhesives due to dentin dehydration.

Chemical interaction of 4-META with enamel in resin-enamel bonds.

X-ray photoelectron spectroscopy (XPS) is used to analyze 4-META resin and enamel that are debonded at an adhesive interface. The XPS spectra showed two chemical states for Ca: one resulted from Ca of hydroxyapatite and the other, an unknown chemical state, suggested that Ca was chemically bonded with 4-META. We postulate that for a chemical reaction of 4-META and hydroxyapatite, the chemical structure of carboxyl groups will resemble that of calcium phthalate. Hence, calcium phthalate was used as a reference material. Additionally, the spectra obtained from the adhesive interface and the mixture of calcium phthalate with hydroxyapatite particles were compared using peak deconvolution analysis. XPS analysis revealed that the chemical bond of 4-META with enamel resembled the chemical state of Ca in calcium phthalate. Consequently, we suggest that Ca of the enamel and the carboxyl group of 4-META were chelate-bonded at the interface.

α-Methylacyl-CoA racemase: a useful immunohistochemical marker of breast carcinoma with apocrine differentiation.

Carcinoma with apocrine differentiation is an androgen receptor (AR)-positive subset of triple-negative breast carcinomas. In addition to carcinoma with apocrine differentiation, other AR-positive triple-negative breast carcinomas occur, albeit less frequently. We found that α-methylacyl-CoA racemase (AMACR), also known as P504S, is overexpressed in carcinoma with apocrine differentiation and non-neoplastic apocrine metaplasia. We aimed to evaluate AMACR as a possible marker of carcinoma with apocrine differentiation. We immunohistochemically examined the expression of AMACR in carcinoma with apocrine differentiation and nonapocrine carcinomas and compared it with that of gross cystic disease fluid protein-15 (GCDFP-15). In total, 212 breast carcinomas were evaluated: 39 carcinomas with apocrine differentiation, 28 ductal carcinomas in situ with apocrine morphology (ADCIS), and 145 nonapocrine breast carcinomas. AMACR was expressed in 38 of 39 (97.4%) carcinomas with apocrine differentiation and in 27 of 28 (96.4%) ADCIS, consistent with the expression of GCDFP-15. However, in nonapocrine carcinomas, AMACR expression was observed in 32 of 145 (22.0%) lesions, whereas GCDFP-15 expression was observed in 91 of 145 (62.7%) lesions. For carcinoma with apocrine differentiation, AMACR was as sensitive as GCDFP-15 (both 97.1%) but more specific (77.9% versus 37.2%). In selected cases, AMACR messenger RNA (mRNA) levels were quantitatively determined relative to that of TATA-binding protein mRNA, and they comprised 5.23, 1.33, and 0.60 for carcinomas with apocrine differentiation, nonapocrine carcinomas, and normal breast tissue, respectively. CONCLUSION: Our findings demonstrate that AMACR expression may be used for differentiating carcinoma with apocrine differentiation from nonapocrine carcinomas and indicate that AMACR is a more sensitive carcinoma with apocrine differentiation marker than GCDFP-15.

Crystal growth by restorative filling materials.

This study examined the activity and ability of materials to mineralize teeth in gaps, simulating microleakage between the materials and teeth. Three restorative materials (two glass ionomer cements and a compomer) were used in this study. Cured disks of restoratives were placed over flat human tooth surfaces (enamel and dentin), separated by a standardized 20-µm interfacial space, and stored in water for 24 h or 1 yr. After the water-storage period, the materials were detached from the teeth and the opposing surfaces were examined by scanning electron microscopy (SEM) and electron probe micro analysis (EPMA).Hemispherical-shaped precipitations, composed of minute semicircle plate-like crystals, were observed by SEM on the enamel surface after 1 yr of water storage for glass ionomer cement. The amount of crystal growth with the chemical-cured type of glass ionomer cement was greater than with the dual-cured type of glass ionomer cement. However, there was no crystal formation in the compomer. Moreover, no structural changes were observed on dentin surfaces for any material in water after 1 yr. The elements detected in the crystals by EPMA were calcium (Ca), phosphorus (P), and aluminum (Al). The two glass ionomer cements tested have the ability to induce crystals whose composition might be derived from cured glass ionomers.

Measurements of volatile compound contents in resins using a moisture analyzer.

The contents of volatile adhesive compounds, such as water, solvents, and residual unpolymerized monomers, affect the integrity and durability of adhesive bonding. However, there is no method available that can be used to rapidly assess the residual solvent or water contents of adhesive resins. This study examined the effectiveness of a digital moisture analyzer to measure the volatile compound contents of resins. Five self-etching adhesives and seven experimental light-cured resins prepared with different contents (0, 10, and 20% by weight) of water or solvents (acetone and ethanol) were examined in this study. The resins were prepared using different methods (with and without air blast or light-curing) to simulate the clinical conditions of adhesive application. Resin weight changes (% of weight loss) were determined as the residual volatile compound contents, using the moisture analyzer. After the measurements, the resin films were examined using a scanning electron microscope. The weight changes of the resins were found to depend on the amount of water or solvents evaporating from the resin. Water and solvents were evaporated by air blast or light-curing, but some of the water and solvents remained in the cured resin. The moisture analyzer is easy to operate and is a useful instrument for using to measure the residual volatile compound contents of adhesive resin.

Ten-years degradation of resin-dentin bonds.

The purpose of this study was to evaluate the durability of resin-dentin bonds in 10-yr water-storage testing. Resin-dentin bonded bulk specimens were prepared using six commercially available resin adhesives. The resin-dentin bonded specimens were stored in water for 24 h (control group) or for 10 yr (experimental groups). After each storage period, the specimens were sectioned to make specimen beams and then subjected to a microtensile bond test. After the bond test, fractured surfaces were examined by scanning electron microscopy (SEM). In addition, interfacial observation of silver nanoleakage was performed using the backscatter electron mode of SEM. The bond strengths of four of the six adhesive systems tested decreased significantly after 10 yr. However, no significant bond-strength reduction was recorded for the other two systems. The interfacial observations showed water tree propagation in the bonding resin layer as a typical morphological change after aging for five of the six adhesives tested. Water tree propagation may be a symptom of degradation in the resin bonding layer of resin-dentin bonds.

[A case of herpetic meningoencephalitis associated with massive intracerebral hemorrhage during acyclovir treatment: a rare complication].

We report a case of a 35-year-old female with herpetic meningoencephalitis confirmed by polymerase chain reaction and immunohistochemical study for herpes simplex virus-1 accompanied with a massive intracerebral hematoma as a complication. A hematoma localized at the medial temporal lobe and the medial frontal lobe occurred on the 11th day after initiation of acyclovir treatment. She subsequently required emergency surgery for temporal lobectomy, as well as hematoma and external decompression. Intracerebral hematoma with MR imaging showed gyral pattern along the cortex of the medial temporal lobe and the base of the medial frontal lobe. We speculate that the hemorrhage occurred by rupture of small vessels affected by vasculitis in addition to hypertension caused by increased intracranial pressure. We therefore emphasize the risk of intracerebral hemorrhage at an early stage or during acyclovir treatment, especially during one or two weeks after initiation of the treatment, and the necessity of careful observation during these periods.

Zinc Binding to Heliorhodopsin.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, has an inverted membrane topology compared to animal and microbial rhodopsins, and no ion-transport activity. The slow photocycle of HeRs suggests a light-sensor function, although the function remains unknown. HeRs exhibit no specific binding of monovalent cations or anions. Despite this, ATR-FTIR spectroscopy in the present study demonstrates binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR). The biding of Zn2+ to 0.2 mM Kd is accompanied by helical structural perturbations without altering its color. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding. Similar results were obtained for HeR 48C12. These findings suggest a possible modification of HeR function by Zn2+.

In vitro degradation of resin-dentin bonds with one-bottle self-etching adhesives.

The purpose of this study was to evaluate the durability of one-bottle self-etching adhesive during long-term water-storage testing. Resin-dentin bonded specimens were prepared using four commercially available one-bottle self-etching adhesives. The specimens were sectioned perpendicular to the adhesive interface to produce beam-shaped specimens that were stored in water for 24 h (control group) and 100, 200, and 300 d (experimental groups). After each storage period, the beams were subjected to a microtensile bond test. After the bond test, fractured surfaces were examined using a scanning electron microscope. In addition, interfacial observations of the silver tracer were performed using the secondary and back-scatter modes of the scanning electron microscope. The bond strength of all tested adhesives decreased significantly after 100 or more days in water. The interfacial observations showed an oxygen-inhibition zone as electron lucent in the adhesive-composite border in control specimens, displaying silver impregnation with breakage after aging. The deterioration of the oxygen-inhibition zone in the adhesive-resin composite junction resulted in a decrease in bond strength after 100 d in water for one-bottle self-etching adhesives.

Effect of dentinal water on bonding of self-etching adhesives.

This study examined the effect of dentinal water on bonding, comparing one-bottle and two-step self-etching adhesives using microtensile bond test and scanning electron microscope. The bond strength of resin to dentin was measured for wet dentin (control) and dry dentin substrates. Wet dentin is the normal substrate for bond testing, whereas dry dentin was dehydrated in a desiccator at different drying times (5 to 60 min) before bonding. After bond testing, the fractured surfaces were examined. Although no correlation was found for two-step self-etching adhesives, the bond strength of the dry-dentin was significantly increased with the increase in the drying-time for one-bottle adhesives. With increased drying-time, the amount of water-bubbles was decreased for one-bottle adhesives; however, no bubble formation was seen in two-step adhesives in any group. The hydrophilic resin adhesive may entrap the water from dentin by osmosis during and after bonding. This effect may depend on the "hydrophilicity" of adhesives.