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Ultrathin bronchoscopy aids in the diagnosis of peripheral pulmonary lesions. However, both the working channel and the specimens are small. A 1.1-mm ultrathin cryoprobe that can enter the working channel of the ultrathin bronchoscope is now available, which may overcome the limitations of small specimen size. The aim of this study was to evaluate the feasibility, efficacy and safety of ultrathin bronchoscopic cryobiopsy using an ultrathin cryoprobe for diagnosing peripheral pulmonary lesions.Patients with peripheral pulmonary lesions ≤30 mm in diameter were prospectively enrolled in the study. All patients underwent forceps biopsy followed by cryobiopsy using a 3.0-mm ultrathin bronchoscope under radial probe endobronchial ultrasound guidance, virtual bronchoscopic navigation and fluoroscopic guidance. The primary endpoint was the feasibility of cryobiopsy.In total, 50 patients with peripheral pulmonary lesions were enrolled in the study; the median longest diameter on computed tomography was 17.9 mm. Cryobiopsy was performed successfully in 49 patients (98%). Forceps biopsy, cryobiopsy and the combination of these two methods provided a specific diagnosis in 54% (27/50), 62% (31/50) and 74% (37/50) of patients, respectively. The median size of specimens obtained via cryobiopsy was significantly larger than the median size obtained via forceps biopsy (7.0 vs. 1.3 mmUltrathin bronchoscopic cryobiopsy is feasible, effective and sufficiently safe for the diagnosis of peripheral pulmonary lesions.

OBJECTIVE To investigate the activities of gelatinases (matrix metalloproteinase [MMP]-2 and MMP-9) and serine proteases in the colorectal mucosa of Miniature Dachshunds (MDs) with inflammatory colorectal polyps (ICRPs). ANIMALS 15 MDs with ICRPs and 5 dogs with non–ICRP-related large bowel diarrhea (controls). PROCEDURES Zymographic methods were used to evaluate the activities of MMP-2, MMP-9, latent forms of MMP-2 and MMP-9 (pro–MMP-2 and pro–MMP-9), and serine proteases in inflamed and noninflamed tissue samples from MDs with ICRPs and in noninflamed tissue samples from control dogs. The associations of serine protease activities with MMP-2 or MMP-9 activity were also analyzed. RESULTS Activities of pro–MMP-2 and pro–MMP-9 were detected in most tissue samples, regardless of the tissue type, whereas activities of MMP-2 and MMP-9 were not detected in control tissue samples. In the inflamed tissue samples from MDs with ICRPs, the activities of MMP-2, pro–MMP-9, and MMP-9 were significantly higher than those in the noninflamed tissue samples from those dogs. Serine protease activities were significantly higher in the inflamed and noninflamed tissue samples from MDs with ICRP, compared with findings for control tissue samples. A weak correlation was detected between serine protease activities and MMP-9 activity. CONCLUSIONS AND CLINICAL RELEVANCE Study results suggested that gelatinase and serine protease activities are upregulated in the colorectal mucosa of MDs with ICRPs, possibly contributing to the pathogenesis of this disease through the functions of these enzymes in degradation of extracellular matrix and promotion of inflammatory cell migration and inflammatory responses.

Background: Airway stenting is a useful form of palliation for patients with airway stenosis/fistulas; the stent can be removed after addressing the cause of the airway disorder. Patients with airway stents often complain of coughing and difficulty with expectoration, so the use of such stents can negatively affect pulmonary function and worsen symptoms. Objectives: The aim of this study was to compare pulmonary function and respiratory symptoms before and after stent removal. Methods: Patients who would later undergo simple airway stent removal were prospectively recruited in two institutions. All stents were removed using both rigid and flexible bronchoscopes with patients under general anesthesia. Pulmonary function tests were performed before stent removal and at 1 and 4 weeks after stent removal. All patients self-reported their respiratory symptoms using a 100-mm visual analog scale (VAS). Results: Of the 31 patients enrolled, 28 (23 with malignant stenoses, 3 with benign stenoses, and 2 with fistulas [21 silicone and 7 metallic stents]) were included in analyses. Pulmonary function measurements before stent removal and at 1 and 4 weeks after stent removal were as follows: vital capacity, 3.00, 3.04, and 3.08 L (p = 0.387); forced expiratory volume in 1 s, 1.96, 1.96, and 2.12 L (p = 0.034); and peak expiratory flow, 3.60, 4.28, and 5.06 L/s, respectively (p < 0.001). Symptoms (cough, sputum production, difficulty with expectoration, and dyspnea) evaluated using the VAS improved significantly after stent removal. No complications were encountered during removal. Conclusion: Removal of unnecessary airway stents improves pulmonary function and respiratory symptoms. Any stent that is no longer functioning should be removed.

Bronchial carcinoid is a rare malignant tumor that is categorized as a typical carcinoid or atypical carcinoid. Many institutions use flexible bronchoscopy for diagnosis. However, due to the hemorrhagic nature of the tumor, the amount of specimen obtained is often small, making it difficult to obtain an accurate diagnosis. The use of rigid bronchoscopy may not only contribute to obtaining a diagnosis but also be beneficial in the treatment plan. The aim of this study was to evaluate the efficacy of rigid bronchoscopic interventions for the diagnosis and treatment of bronchial carcinoids. All patients with bronchial carcinoids who underwent rigid bronchoscopic intervention under general anesthesia at our institution between June 2006 and August 2018 were analyzed retrospectively. Eight patients [3 men and 5 women; median age, 71 years (range 45-82 years)] were eligible for the analysis. None of the cases had accurate subtyping preoperatively before intervention. In contrast, all cases were diagnosed as carcinoid with subtypes (5 patients had typical carcinoid and 3 had atypical carcinoid) following rigid bronchoscopic intervention. All respiratory symptoms improved immediately after the procedure. One instance of bleeding occurred, and was easily controlled by argon plasma coagulation and intraluminal administration of epinephrine under flexible and rigid bronchoscopy. Four patients (3 with typical carcinoid and 1 with atypical carcinoid) underwent radical surgery sequentially, and no recurrences were observed. We conclude that rigid bronchoscopic intervention is safe and effective for accurate diagnosis and improvement of respiratory symptoms in patients with bronchial carcinoids.

Accurate staging and evaluation of therapeutic effects are important in managing plasma-cell neoplasms. Diffusion-weighted imaging with body signal suppression magnetic resonance imaging (DWIBS-MRI) allows for acquisition of whole-body volumetric data without radiation exposure. This study aimed to investigate the usefulness of DWIBS-MRI in plasma-cell neoplasms. We retrospectively analyzed 29 and 8 Japanese patients with multiple myeloma and monoclonal gammopathy of undetermined significance, respectively, who underwent DWIBS-MRI. We conducted a histogram analysis of apparent diffusion coefficient values. The correlations between each histogram parameter and staging, cell maturation, prognosis, and treatment response were evaluated. We found that the apparent diffusion coefficient values in patients with monoclonal gammopathy of undetermined significance were lower than those in patients with multiple myeloma. Pretreatment apparent diffusion coefficient values of immature myeloma were lower than those of mature myeloma. Moreover, these values decreased in proportion to stage progression in Durie-Salmon classification system but showed no significant correlation with other staging systems or prognosis. Patients were stratified as responder, stable, and non-responder based on the International Myeloma Working Group criteria. The magnitude of changes in apparent diffusion coefficients differed significantly between responders and non-responders (0.154 ± 0.386 ×10–3 mm2/s vs. -0.307 ± 0.424 ×10–3 mm2/s, p = 0.003). Although its usefulness has yet to be established, DWIBS-MRI combined with apparent diffusion coefficient measurement allowed for excellent response evaluation in patients with multiple myeloma. Furthermore, apparent diffusion coefficient analysis using DWIBS-MRI may be useful in predicting cell maturation and total tumor volume.

Non-tuberculous mycobacterial (NTM) disease is a rare cause of neutropenic fever in patients with hematological malignancies. There are few studies on the optimal management for such patients with NTM. We report a case of myelodysplastic syndrome (MDS) treated by umbilical cord blood transplantation (CBT) after Mycobacterium kansasii (M. kansasii) pneumonia. A 38-year-old man diagnosed with MDS developed severe pneumonia during induction chemotherapy. Repeated sputum culture uncovered mycobacterium infection. Then, by the polymerase chain reaction of the bronchial lavage fluid, M. kansasii infection was proven. After 140 days of anti-NTM therapy, CBT was successfully carried out and the patient recovered without recurrence of NTM infection. This case provides valuable evidence that hematopoietic stem cell transplantation is feasible after a reliable diagnosis and continuous anti-NTM therapy.

OBJECTIVES Stent migration is a common complication of airway stent placement for upper tracheal stenosis and tracheoesophageal fistula. Although several researchers have reported that external fixation is effective in preventing stent migration, the usefulness and safety of external fixation have not been proved because their cohorts were small. We therefore investigated the efficacy and safety of external fixation during upper tracheal stenting. METHODS Records of patients who underwent airway stent placement from May 2007 to August 2018 in a single centre were retrospectively reviewed. We included only patients whose stent had been placed in the upper trachea with external fixation to the tracheal wall. The primary endpoint of this study was the rate of stent migration. RESULTS Altogether, 51 procedures were performed in 45 patients (32 males, 13 females; median age 60 years, range 14–91 years). The median follow-up period was 9 months (range 0.3–90 months). Among the procedures, 15 were performed for benign disease and 36 for malignancy. Stents were composed of either silicone (n = 42) or metal (n = 9). Stent migration occurred in 3 (6%) patients. The stents with migration were all composed of silicone. Other sequelae were granulation tissue formation in 10 (20%) patients, sputum obstruction in 6 (12%), cellulitis in 3 (6%) and pneumonia in 1 (2%). CONCLUSIONS External fixation was an effective method for preventing migration of airway stents placed for upper tracheal stenosis and tracheoesophageal fistula. The complications were acceptable in terms of safety.

Human herpesvirus-6 (HHV-6) reactivation is an important complication in patients receiving umbilical cord blood transplantation (CBT). Chromosomally integrated human herpesvirus-6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germline genome and is transmitted in a Mendelian manner. The influence of ciHHV-6 in recipients or donors in cases of CBT is unknown. We report the first case with ciHHV-6 that received CBT twice for acute lymphoblastic T-cell leukemia. HHV-6 DNA in peripheral blood leukocytes (PBLs) was examined over time through two CBTs. After the first CBT, the HHV-6 viral load was significantly reduced by conversion to PBLs derived from the first donor. During the second CBT, an increase in HHV-6 DNA in PBLs and plasma were observed. However, HHV-6 mRNA was not detected in either the sample before 2nd CBT or at the time of HHV-6 DNA elevation. It is considered that the HHV-6 DNA detected in PBLs and plasma samples might be the HHV-6 genome released due to tissue damage. This case suggests that physicians should be aware of HHV-6 DNA variability during allogeneic hematopoietic stem cell transplantation in ciHHV-6 patients.

Treatment options for patients with relapsed/refractory small cell lung cancer (R/R SCLC) are limited, and the efficacy of salvage therapies for heavily treated patients should be assessed. Here, we evaluated the efficacy of paclitaxel (PTX) in R/R SCLC patients. A single-institute retrospective chart review was conducted. The primary endpoint was overall survival (OS), whereas the secondary endpoints were progression-free survival (PFS), overall response rate, disease control rate (DCR), and safety. Thirty-one patients (median age, 69 [range, 56–80] years) were analyzed. The median follow-up period was 122 (range, 28–1121) days. The median OS and PFS were 4.4 and 2.2 months, respectively. Adverse events of grade 3 or higher, other than hematological toxicity, were febrile neutropenia and neuropathy. Multivariate analyses identified the following independent predictors of poor OS: performance status and lactate dehydrogenase at the upper limit of normal. PTX monotherapy showed moderate efficacy with acceptable toxicity in heavily treated patients with R/R SCLC patients.

We report the development of environmentally responsive fluorescent polymers. The reversible temperature-induced phase transition of copolymers composed of N-isopropylacrylamide and a fluorescent monomer based on the fluorescein (FL), coumarin (CO), rhodamine (RH), or dansyl (DA) skeleton was used as a molecular switch to control the fluorescence intensity. The poly(N-isopropylacrylamide) (PNIPAAm) chain showed an expanded coil conformation below the lower critical solution temperature (LCST) due to hydration, but it changed to a globular form above the LCST due to dehydration. Through the combination of a polarity-sensitive fluorophore with PNIPAAm, the synthetic fluorescent polymer displayed a response to external temperature, with the fluorescence strength dramatically changing close to the LCST. Additionally, the P(NIPAAm-co-FL) and P(NIPAAm-co-CO) polymers, containing fluorescein and coumarin groups, respectively, exhibited pH responsiveness. The environmental responsiveness of the reported polymers is derived directly from the PNIPAAm and fluorophore structures, thus allowing for the cellular uptake of the fluorescence copolymer by RAW264.7 cells to be temperature-controlled. Cellular uptake was suppressed below the LCST but enhanced above the LCST. Furthermore, the cellular uptake of both P(NIPAAm-co-CO) and P(NIPAAm-co-RH) conjugated with a fusogenic lipid, namely, l-α-phosphatidylethanolamine, dioleoyl (DOPE), was enhanced. Such lipid-conjugated fluorescence probes are expected to be useful as physiological indicators for intracellular imaging.

Mast cells are derived from hematopoietic stem cells and play important roles in allergic responses. Mast cells are long-lived compared with other granular cell types. Since the response of the individual mast cell after FcεRI-induced degranulation is unclear, the aim of this study was to analyze morphological changes in individual mast cells after restimulation. To observe plasma and granule membrane dynamics, AcGFP-actb (β-actin) and DsRed-monomer (DRM)- CD63 fusion constructs were introduced into bone marrow-derived mast cells (BMMCs). Furthermore, AcGFP-CD63 and DRM-Cma1 (mMCP-5) were introduced into BMMCs. Re-stimulation resulted in increased β-hexosaminidase release and cytokine mRNA expression similar to those observed during initial stimulation. Moreover, expression of FcεRI on BMMCs 24 h after initial stimulation was similar to that measured before initial stimulation. Changes in morphology of the plasma membrane and colocalization of granules and plasma membrane were observed after initial stimulation. BMMCs returned to normal 120 min after the initial stimulation. These phenomena were also observed in BMMCs after re-stimulation. BMMC chymase content decreased 20 min after stimulation but returned to near normal 24 h after stimulation. These findings suggest that mast cell functions can be maintained and that these cells can be repeatedly degranulated after FcεRI-mediated stimulation.

Streptococcus criceti is a cariogenic organism that belongs to the mutans streptococci. Of the four S. criceti strains, strain OMZ 61 has been identified as being resistant to erythromycin. Antimicrobial susceptibility testing showed that strain OMZ 61 is also resistant to azithromycin, josamycin and clindamycin but susceptible to tetracycline and tiamulin. DNA hybridization analysis of the 23S rRNA genes revealed that the hybridization patterns in strain OMZ 61 differed from those in the other three strains. We further analyzed the nucleotide sequences of a ribosomal RNA operon, the rrnD operon, and the rpsJ-rpsQ region including rplC and rplD genes for ribosomal proteins L3 and L4, respectively, in the four strains studied. Nucleotide sequence analysis indicated that strain OMZ 61 contains an A-to-G substitution at nucleotide position 2059, equivalent to Escherichia coli numbering 2058, in a 23S rRNA gene (rrlD) and a G-to-A substitution at nucleotide position 439 in the rplC gene, suggesting an amino acid residue change at position 147 from valine to isoleucine, whereas no mutation in the rplD gene was found. DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism analysis showed that most or all of the 23S rRNA genes in strain OMZ 61 contain the A2059G mutation. These findings suggest that the resistance to erythromycin, azithromycin, josamycin and clindamycin in strain OMZ 61 is conferred by alterations in 23S rRNA and/or ribosomal protein L3. This is the first description of mutations in the 23S rRNA and rplC genes in mutans streptococci.

Background Recent studies have reported that immune checkpoint inhibitors (ICI) are ineffective in non-small cell lung cancer (NSCLC) patients harboring the epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) receptor. Methods This study was designed as a single-institute, retrospective chart review. We reviewed 103 NSCLC patients treated with an ICI regimen (Nivolumab, Pembrolizumab, and Atezolizumab) between January 2009 and November 2018 at Nagoya Medical Center. The inclusion criteria were: metastatic or recurrent NSCLC with or without a driver oncogene, normal organ function, and ECOG Performance Status (PS) 0-2. The primary endpoint was progression-free survival (PFS) and secondary endpoints were overall survival (OS, calculated from the first day of ICI administration) and overall response rate (ORR) Results Eighty-two pts (median age, 69 years; range, 33-88) met the inclusion criteria. Among them, 13 pts had driver oncogenes (mt+) (EGFR 12, ALK 1), and 69 pts did not have driver oncogenes (mt-). ECOG-PS was 0, 1, and 2 in 27, 40, and 15 pts, respectively. Median number of regimen prior to ICI treatment was 2 (range 1-8, including EGFR-tyrosine kinase inhibitors (EGFR-TKI) and ALK inhibitors) in mt+ pts and 1 (range 0-11) in mt- pts. Programmed cell death ligand 1 (PD-L1) expression Conclusions This study showed that PFS following ICI therapy is longer in mt- than mt+ pts, suggesting that ICI is less efficacious in mt+ than mt- NSCLC pts.

Streptococcus mutans, a cariogenic agent, has a glucan-binding protein gene, gbpC, and S. criceti possesses four gbpC homologs, including dblA and dblB, as does S. sobrinus. The S. criceti dblB gene encodes a 1,717-amino-acid protein having two repetitive alanine-rich and proline-rich regions and an LPXTG motif, which is recognized by the sortase SrtA, near the C terminus. Reverse transcription-PCR analysis indicated no cotranscription of the dblA and dblB genes of S. criceti. As we could not obtain a dblB mutant of S. criceti, the dblB gene was characterized in S. mutans strain GS-5, which has genetic mutations in both gbpC and spaP genes and shows an inability to agglutinate triggered by dextran. A dextran-induced agglutination assay showed that S. mutans cells carrying dblB agglutinated in the presence of dextran. A hydrophobicity assay showed that the cells containing dblB were hydrophobic. A biofilm formation assay showed that the dblB gene was associated with biofilm formation by cells cultivated in brain heart infusion broth supplemented with glucose and maltose, but not sucrose. Nucleotide sequence analysis of the S. criceti strains studied revealed a frameshift mutation in the srtA gene encoding sortase, but intact dblA and dblB genes were found in dextran-induced agglutination-negative strains, whereas intact dblA, dblB and srtA genes were found in dextran-induced agglutination-positive strains. These results suggest the cell-surface localization of dblA and dblB gene products by SrtA and the responsibility of dblB for dextran-induced agglutination, cell-surface hydrophobicity and biofilm formation in S. criceti.

A novel insertion sequence element of the IS982 family, ISScr1, was previously identified in Streptococcus criceti strain E49 as a disrupted paaB gene encoding an antigen I/II homologous protein. In this study, we identified two divergent inserted regions of ISScr1 in S. criceti E49 by inverse polymerase chain reaction, the gene-walking method, and screening of the partial plasmid library. Nucleotide sequence analysis indicated the possible explanation that transposition generated 8- and 9-bp direct repeat sequences. DNA hybridization analysis revealed that an identical hybridization pattern to ISScr1 was observed in the four S. criceti strains studied and that at least three copies of ISScr1 were preserved in S. criceti strains. In addition, we found different susceptibility to erythromycin and diverse agglutination properties induced by dextran in S. criceti. Furthermore, DNA hybridization analysis showed that no ISScr1-like copy was detected in the other 14 strains of oral streptococci tested.

We developed a new chemically amplified molecular resist that showed high resolution by introducing an efficient acid-leaving group to 1,3,5-Tris(p-(p- hydroxyphenyl) phenyl) benzene (THTPPB). The lithographic properties such as sensitivity, developing rate, and adhesion are considered to be controlled using a suitable acid-leaving group. A molecular resist of THTPPB to which is attached with an alicyclic acid-leaving group, hyperlactyl group (HPVE) showed a high resolution for electron beam (EB) lithography and good etch resistance. Half-pitch (hp) 36 nm L&S (1:1) positive pattern was fabricated using 100 keV EB with a new molecular resist based on HPVETPPB.

AtlA is a major cell-lytic enzyme called autolysin in Streptococcus mutans. In this study, we identified the atlg gene-encoding autolysin (Atlg), consisting of 863 residues from Streptococcus sobrinus 6715DP, and confirmed lytic activity of recombinant Atlg by zymography of S. sobrinus cells. An atlA-inactivated mutant was constructed in S. mutans Xc, and the atlg gene product was characterized by plasmid complementation. Microscopic analysis, saliva-induced aggregation assay and autolysis assay of static cultures in air revealed that the atlg gene product partially complemented the role of AtlA. Furthermore, the capability of biofilm formation of the atlA-deficient mutant cultivated in air was restored by plasmid comprising the atlg gene. These findings suggest that Atlg may be involved in cell separation and biofilm formation in S. sobrinus.

A novel molecular resist based on a new amorphous molecule, a truxene derivative, was designed and synthesized. Truxene is characterized as an amorphous solid with a high glass transition temperature (Tg). H alf-pitch (hp) 50 nm line-and-space (1 : 1) positive pattern was fabricated by the exposure of an electron beam (30 keV) using the novel molecular resist. Design of novel molecular resists based on the truxene derivative is promising for development of more efficient molecular resists.

Geographic variations in two social behavioural characters, waving display and barricade building, and genetic population structure were examined in the tidal‐flat‐inhabiting dotillid crab Ilyoplax pusilla (de Haan, 1835), in six localities between the northern and southern limits of its distributional range (four localities from the Japanese mainland and two southern localities from the Ryukyu Islands). Although waving motion followed the same lateral–circular pattern in all of the local populations, cheliped‐extension at the wave peak was most prominent in the Yakugachi River population (near the distribution's southern limit in the Rykukyu Islands), followed by Haneji Inlet (the southern limit in the Ryukyu Islands) and four localities on the Japanese mainland in that order. The frequency of barricade building showed a similar trend, namely lowest in Yakugachi River, followed by Haneji Inlet and the four mainland localities. Genetic relationships among the six local populations revealed that the two so...

An autolysin gene, atlh, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. Atlh protein encoded by atlh is composed of 879 amino acids, with a molecular weight of 95,902.26. Atlh possesses four 15-amino-acid residue repeats in the putative cell-wall-binding domain and has a catalytic domain in the C-terminus. The deduced amino acid sequence of atlh showed homology to S. mutans autolysin AtlA (68.4% similarity). Inactivation of atlh resulted in elongated chain formation compared to the parent strain. Recombinant proteins Atlh and its derivatives were constructed and analyzed by zymography. Zymographic analysis revealed that the Asp-771 residue of Atlh was essential for lytic activity and that lytic activity was not diminished by the deletion of repetitive regions in the putative cell-wall-binding domain of Atlh. Biofilm assay showed that the wild-type strain formed glucose- and sucrose-dependent biofilms, the atlh mutant diminished this ability. These results suggest that Atlh is associated with cell separation and biofilm formation.

The small dotillid crab Ilyoplax pusilla builds an earthen structure, termed barricade, close to its neighbour's burrow. Barricades function for territorial defence by deterring invasion by neighbours. Comparison of the frequency of barricade building in the breeding season among 5 localities across the geographical range of I. pusilla, showed higher values at more northern localities. In two localities differing in the frequency of barricade building, the response of crabs to artificial barricades placed near their burrows was examined. The artificial barricade was less often broken and more often avoided in the locality with the higher frequency of barricades. These results suggest that the local difference in frequency of barricade building is correlated with a difference in how crabs respond to this earthen barrier.

Streptococcus criceti is a cariogenic organism that belongs to the mutans streptococci. Of the four S. criceti strains, strain OMZ 61 has been identified as being resistant to erythromycin. Antimicrobial susceptibility testing showed that strain OMZ 61 is also resistant to azithromycin, josamycin and clindamycin but susceptible to tetracycline and tiamulin. DNA hybridization analysis of the 23S rRNA genes revealed that the hybridization patterns in strain OMZ 61 differed from those in the other three strains. We further analyzed the nucleotide sequences of a ribosomal RNA operon, the rrnD operon, and the rpsJ-rpsQ region including rplC and rplD genes for ribosomal proteins L3 and L4, respectively, in the four strains studied. Nucleotide sequence analysis indicated that strain OMZ 61 contains an A-to-G substitution at nucleotide position 2059, equivalent to Escherichia coli numbering 2058, in a 23S rRNA gene (rrlD) and a G-to-A substitution at nucleotide position 439 in the rplC gene, suggesting an amino acid residue change at position 147 from valine to isoleucine, whereas no mutation in the rplD gene was found. DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism analysis showed that most or all of the 23S rRNA genes in strain OMZ 61 contain the A2059G mutation. These findings suggest that the resistance to erythromycin, azithromycin, josamycin and clindamycin in strain OMZ 61 is conferred by alterations in 23S rRNA and/or ribosomal protein L3. This is the first description of mutations in the 23S rRNA and rplC genes in mutans streptococci.

Many cases of hepatopathy including deaths have frequently occurred after ingestion of Chinese dietary supplements for weight loss containing N-nitrosofenfluramine (N-fen), a nitroso derivative of fenfluramine (Fen), which was used for the treatment of obesity in the United States. Since Fen decreases appetite by decreasing the serotonin level and exhibits an antibiotic effect, N-fen may have been added, expecting a similar effect. Thus, we synthesized N-fen and orally administered it to mice, and investigated its effect on the liver as well as on the cerebral serotonin nervous system to investigate whether N-fen exhibits an anorectic effect. Three doses of N-fen were orally administered once daily to mice for 1 week. No significant changes in body weight, food intake, and general condition were noted. The liver and kidney weights were significantly increased. On blood chemistry, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased, and total bilirubin and albumin were slightly decreased. On histopathological examination, acidophilic changes and mild cellular swelling were noted in the liver. The liver drug-metabolizing enzyme (P-450) level was significantly higher. The effect of N-fen on the serotonin (5HT) nervous system was examined by quantitative autoradiography of the mouse brain, and it was found that N-fen did not decrease the 5HT nerve activity. Effects of reuptake and release of monoamine neurotransmitters [dopamine (DA), 5HT, and norepinephrine (NE)] were investigated. N-fen inhibited a little 5HT reuptake, and did not inhibit reuptakes of DA and NE. Moreover, N-fen did not affect release of the three monoamines. The above findings suggested that N-fen did not exhibit a serotonin nerve fiber-mediated anorectic effect in mice, but induced hepatopathy.

Anchored osmotic pressure inducer was examined. Salt-containing filter paper attached to an osmotic membrane increased the water flux to more than 10% from the value of non-treated filter paper at 1 MPa. The salt-containing filter paper was fabricated via graft polymerization of acrylic acid onto a filter paper followed by alkaline treatment. The filter paper was characterized by thermo gravimetric analysis (TGA), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), scanning electronic microscopy-energy dispersive X-ray spectrometry (SEM-EDS).

Introduction: To measure levels of phosphatidylcholine (PtdCh) in various mouse tissues, we developed a rapid and precise method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. To generate an example data set, the effect of methoxamine (an α 1 -adrenergic agonist) on the PtdCh levels was examined by this method in the artery and the submandibular gland of the mouse in vivo. Methods: Under our modifications of the method of Zapata et al. [J. Neurosci. 18 (1998) 3597], the mixture of lipophilic choline metabolites (PtdCh, lyso-PtdCh, and sphingomyelin) extracted by chloroform from the tissue homogenate was dried without prior separation and hydrolyzed with free choline by a 1-N perchloric acid solution containing ethylhomocholine (an internal standard for choline assay) at 90 °C for 1 h. Subsequently, the hydrolyzed mixture was injected directly into the HPLC system for PtdCh assay. Results: The present method permitted PtdCh assay within 5 min in one chromatographic run. Recovery of an authentic PtdCh sample was 99% ( n =10). The within-run coefficients of variation for choline derived from PtdCh in the same tissue samples were 0.6% ( n =10) and 1.3% ( n =30). Under the present method, the lowest and highest PtdCh values in tissue samples were about 2 μmol/g (eye ball) and 29 μmol/g (spinal cord), respectively. Methoxamine significantly decreased PtdCh levels and increased free choline levels in mouse artery and submandibular gland. Discussion: Under the present sample processing procedure, the choline values originating from lyso-PtdCh and sphingomyelin were much less than those originating from PtdCh hydrolysis. Thus, it was possible to inject the hydrolyzed mixture directly into the HPLC system for PtdCh assay. Since the present method provides simple, rapid, and highly reliable PtdCh determination, it is suitable for routine assay of PtdCh in a large number of samples.

Antigen I/II protein is a cell surface protein antigen of Streptococcus mutans and is implicated in the adhesion mechanism to tooth surfaces. We have identified the antigen I/II homologous gene (paaA) from Streptococcus cricetus, a member of mutans streptococci. To clarify the mechanism of paaA expression, we sequenced the flanking regions of the paaA gene utilizing the gene-walking method in this study. In the upstream region of paaA, three homologous genes found in the corresponding region of Streptococcus sobrinus were identified. One of the corresponding genes in S. sobrinus was par, which has been characterized as a transcription represser of the antigen I/II (spaA) gene. Therefore, the homologous gene in S. cricetus could have a role in transcription of paaA. In the downstream region of paaA, the paaA homologous gene was found and designated as paaB. The paaB gene was interrupted by a novel insertion sequence element (ISScr1), a member of the IS982 family. Southern hybridization analysis of S. cricetus par indicated that no gene homologous to S. cricetus par was found in the oral streptococci used in this study. Furthermore, the downstream region of antigen I/II (spaA) in S. sobrinus was sequenced using the gene-walking method. No similar gene to antigen I/II gene was found in the region. It was suggested that the paaA might be regulated by the par in S. cricetus.

e20620 Background: Re-biopsy is important to decide the treatment after EGFR-tyrosine kinase inhibitor (TKI) failure in non-small cell lung cancer (NSCLC) patients. We hypothesized that the T790M mutation in EGFR might show heterogeneity depending on the re-biopsy site. Methods: NSCLC patients who had received initial EGFR-TKI since January 2009 to December 2016, at any stage and recurrence after surgery and at any line of treatment, were included. Results: In total, 128 patients were included. Median age at EGFR-TKI therapy initiation was 73 (range, 38–97) years; 67% patients were female, all were Asian, 56% had never smoked, and 99% had adenocarcinoma. Of total 128 patients, 109 showed progressive disease. Median progression-free survival (PFS) was 10 (0.56–57) months. Median period since EGFR-TKI failure until the first re-biopsy was 197 (0–1322) days. Re-biopsy was performed 50 times in 42 patients; the number of T790M positive, negative, and pathologically negative patients was 20, 17, and 5, respectively, and the number of re-biopsies in these patients was 20, 22, and 8, respectively. Median PFS was longer in T790M positive patients than in negative patients significantly (17 [11–24] vs. 7.6 [4.3–11] months, P = 0.007). Characteristics such as gender, smoking status, proportion of stage IV, time between EGFR-TKI failure and first re-biopsy, and number of biopsies did not affect the T790M status in the biopsies. T790M positive group had more exon 19 deletions than negative group significantly (75% vs. 23%, P = 0.012). Biopsies at primary lesion, distant, and pleural effusion (PE) were 25% vs. 50%, 60% vs. 36%, and 15% vs. 14%, respectively, in the T790M positive vs. negative groups. Compared with the biopsy-site at diagnosis, the site was same as before in 35% vs. 50% cases (primary lesion [20% vs. 45%], distant [10% vs. 4.5%], and PE [5% vs. 0%]) and was new in 55% vs. 41% cases (distant lesions [45% vs. 27%] and PE [10% vs. 14%]) in the T790M positive vs. negative groups, respectively. Conclusions: In NSCLC patients treated with EGFR-TKI, re-biopsy was performed in distant lesions more frequently in the T790M positive cases than in negative cases. However, the T790M status was not correlated with the re-biopsy site.

Streptococcus mutans, a cariogenic agent, has a glucan-binding protein gene, gbpC, and S. criceti possesses four gbpC homologs, including dblA and dblB, as does S. sobrinus. The S. criceti dblB gene encodes a 1,717-amino-acid protein having two repetitive alanine-rich and proline-rich regions and an LPXTG motif, which is recognized by the sortase SrtA, near the C terminus. Reverse transcription-PCR analysis indicated no cotranscription of the dblA and dblB genes of S. criceti. As we could not obtain a dblB mutant of S. criceti, the dblB gene was characterized in S. mutans strain GS-5, which has genetic mutations in both gbpC and spaP genes and shows an inability to agglutinate triggered by dextran. A dextran-induced agglutination assay showed that S. mutans cells carrying dblB agglutinated in the presence of dextran. A hydrophobicity assay showed that the cells containing dblB were hydrophobic. A biofilm formation assay showed that the dblB gene was associated with biofilm formation by cells cultivated in brain heart infusion broth supplemented with glucose and maltose, but not sucrose. Nucleotide sequence analysis of the S. criceti strains studied revealed a frameshift mutation in the srtA gene encoding sortase, but intact dblA and dblB genes were found in dextran-induced agglutination-negative strains, whereas intact dblA, dblB and srtA genes were found in dextran-induced agglutination-positive strains. These results suggest the cell-surface localization of dblA and dblB gene products by SrtA and the responsibility of dblB for dextran-induced agglutination, cell-surface hydrophobicity and biofilm formation in S. criceti.

Removal of phenol from saline water was carried out with chelating resin. A polyamine chelating resin, Diaion CR-20, removed phenol compounds selectively from industrial wastewater containing 2% salt. From saline water containing 20 mg/L phenol, 70% of the phenol was removed. After treatment, phenol was eluted from the resin by aqueous NaOH, and the resin could also be regenerated by heating in air. Diaion CR-20 adsorbed phenol even in the presence of FeCl3, indicating that treatment with this resin of wastewater containing metal can remove phenol and metal cations in a single step.

A novel insertion sequence element of the IS982 family, ISScr1, was previously identified in Streptococcus criceti strain E49 as a disrupted paaB gene encoding an antigen I/II homologous protein. In this study, we identified two divergent inserted regions of ISScr1 in S. criceti E49 by inverse polymerase chain reaction, the gene-walking method, and screening of the partial plasmid library. Nucleotide sequence analysis indicated the possible explanation that transposition generated 8- and 9-bp direct repeat sequences. DNA hybridization analysis revealed that an identical hybridization pattern to ISScr1 was observed in the four S. criceti strains studied and that at least three copies of ISScr1 were preserved in S. criceti strains. In addition, we found different susceptibility to erythromycin and diverse agglutination properties induced by dextran in S. criceti. Furthermore, DNA hybridization analysis showed that no ISScr1-like copy was detected in the other 14 strains of oral streptococci tested.

AtlA of Streptococcus mutans is a major autolysin and belongs to glycoside hydrolase family 25 with cellosyl of Streptomyces coelicolor. The autolysin gene (atlA) encoding AtlA was identified from S. criceti. AtlA of S. criceti comprises the signal sequence in the N-terminus, the putative cell-wall-binding domain in the middle, and the catalytic domain in the C-terminus. Homology modeling analysis of the catalytic domain of AtlA showed the resemblance of the spatial arrangement of five amino acids around the predicted catalytic cavity to that of cellosyl. Recombinant AtlA and its four point mutants, D655A, D747A, W831A, and D849A, were evaluated on zymogram of S. criceti cells. Lytic activity was destroyed in the mutants D655A and D747A and diminished in the mutants W831A and D849A. These results suggest that Asp655 and Asp747 residues are critical for lytic activity and Trp831 and Asp849 residues are also associated with enzymatic activity.

Molecular resists are expected to offer the advantages of high resolution and low line width roughness (LWR) for the next-generation lithography. We developed a new molecular resist that showed high resolution by introducing an efficient acid-leaving group to an amorphous molecule, 1,3,5-Tris(p-(p-hydroxy- phenyl) phenyl) benzene (THTPPB). The lithographic properties such as sensitivity, developing rate, and adhesion are considered to be controlled using a suitable acid-leaving group. A molecular resist of THTPPB to which is attached with an alicyclic acid-leaving group, hyperlactyl vinyl ether group (HPVE) showed a high resolution for electron beam (EB) lithography and good etch resistance. Half-pitch 36 nm line-and-space (1:1) positive pattern was fabricated using 100 keV EB with chemically amplified molecular resist based on HPVETPPB.

The functional role of CD14 and Toll-like receptor (TLR) in the Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-induced activation of B cells was assessed, using CD14-, TLR2- and TLR4-overexpressed murine B cells (CH12.LX). After stimulation with Pg-LPS, CD14- and TLR4-transfected, but not TLR2-transfected, CH12.LX showed higher induction of nuclear factor (NF)-κB activation. Although Pg-LPS induced control CH12.LX to proliferate accompanying by up-regulations of TGF-β and IL-6 mRNA, CD14-transfected cells showed the up-regulation of TGF-β mRNA, but not IL-6 mRNA nor proliferative responses to Pg-LPS. Thus, Pg-LPS could induce B cell activation both in CD14-dependent and independent pathways: In a CD14-dependent pathway, TGF-β production could be induced through TLR4 and NF-κB activation. In contrast, Pg-LPS induced proliferation and IL-6 production in the CD14-independent pathway.

Our previous study indicated that oleic acid prevented apoptotic cell death induced by trans10, cis12 (t10, c12)-conjugated linoleic acid in rat hepatoma dRLh-84 cells. The intracellular mechanism of action oleic acid is still unknown. Here, we showed that p38 mitogen-activated protein kinase (MAPK) inhibition using its specific inhibitor SB203580 cancelled the ameliorative effect of oleic acid on the cytotoxicity of t10, c12-conjugated linoleic acid. In addition, SubG1 cell population analysis showed that p38 MAPK played an essential role in the prevention of apoptotic cell death by oleic acid. In fact, p38 phosphorylation level was upregulated in cells treated with oleic acid irrespective of t10, c12-conjugated linoleic acid stimulation. Interestingly, t10, c12-conjugated linoleic acid increased intracellular triglyceride accumulation. However, oleic acid completely inhibited this effect. These observations indicated the involvement of blockade of a p38 MAPK pathway in the ameliorative effect of oleic acid on apoptosis induced by t10, c12-conjugated linoleic acid.

Antigen I/II of Streptococcus mutans is a cell surface protein involved in the adherence of cells to tooth surfaces. In this study, an antigen I/II homologous gene, pah, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. The pah gene encodes a cell-wall-anchoring protein, PAh, containing 1565 amino acids. At the deduced amino acid sequence level, PAh shows a strong similarity to PAg of S. sobrinus (97.6% identity). Southern hybridization analysis indicated that a single copy of the pah gene was preserved in the chromosomal DNA of S. downei. Two pah mutants, SES-1 and SES-2, were constructed and analyzed by Western blotting. Two bands corresponding to 200- and 160-kDa proteins were observed in the parent strain, whereas no band was detected in pah mutant strains. In an adhesion assay of cells, pah mutants failed to adhere to tube surfaces in contrast to the parent strain. Furthermore, saliva-induced aggregation was decreased in pah mutants compared to the parent strain. Together, PAh is associated with the adhesion of cells to abiotic surfaces and whole saliva.

The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene-walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200-amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell-surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C-terminus was partially purified and showed dextranase activity on blue-dextran sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDS-PAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD-SDS-PAGE, whereas no dextranase activity of the cells was observed on blue-dextran brain heart infusion (BD-BHI) agar plates. Furthermore, PCR-based mutations of dextranase indicated that a deletion mutant of the C-terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C-terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp-453 and Trp-793 residues of S. criceti dextranase are critical to the enzyme's activity.

The flanking region of the antigen I/II gene, paaA, in Streptococcus cricetus was examined using the gene-walking technique. In the region downstream of the paaA gene, another antigen I/II gene designated as paaB was found. The paaB gene was disrupted at the alanine-rich region (A region) by a novel insertion sequence element, ISScr1. ISScr1 is a member of the IS982 family and is composed of a 962-bp sequence and duplicated target DNA (the sequence 5'-TAGCTAAAT-3') resulting from its insertion. To clarify the structural divergence of the two antigen I/II proteins (PAaA and PAaB), computational analysis of the paaB gene was performed and the two structures were compared. The amino acid sequence homology indicated that PAaB resembled PAaA, but the middle region showed little similarity to that of PAaA. Phylogenetic analysis showed that PAaB was better classified in a major group with S. mutans PAc and S. gordonii SspA and SspB than with PAaA. The transcriptional expression of paaA and paaB was demonstrated by reverse transcription (RT)-PCR. In the region upstream of the paaA gene, three genes homologous to the genes located in the region upstream of the S. sobrinus antigen I/II gene (pag) were found. Of the three genes, ORF3 showed homology to the par gene encoding a transcriptional repressor for the pag gene in S. sobrinus. Therefore, ORF3 was designated the par gene of S. cricetus. Southern hybridization revealed that the par gene of S. cricetus was not found in other oral streptococci examined in this study.