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Peptide modification is a popular strategy for developing an active targeting lipid nanoparticle (LNP). In modifying the surface of an LNP with a peptide, the sequence and structure of the peptide strongly affects the formation of the LNP. Specifically, a peptide with a high hydrophobicity can induce coarsening and aggregation of the LNP. In an attempt to prevent this from occurring, we incorporated monoacyl and diacyl group-conjugated poly(ethylene glycol) (PEG) into a LNP. We previously developed an original LNP, a multifunctional envelope type nanodevice (MEND) modified with an Epi-1 peptide, a ligand with a high affinity for the epithelial cell adhesion molecule (EpCAM). Using this peptide-modified MEND, the efficiency of delivery of a small interfering RNA (siRNA) encapsulated in the MEND was significantly improved. Although increasing the ratio of modification enhanced cellular uptake, the increase also induced aggregation of the LNP, particularly in the case of a large scale preparation. Our results indicate that a monoacyl PEG-lipid can prevent aggregation, even when the LNP is modified with higher molar ratios of peptide, but that this also results in a decrease in delivery efficiency. Moreover, the Epi-1-modified MEND exhibited a strong silencing effect in an ovarian cancer peritoneal dissemination model. Our results suggest that the simple incorporation of a monoacyl derivative into the PEG-lipid resulted in the formation of a peptide-modified LNP with improved characteristics.

The present study established systems to predict the chemo-sensitivity of muscle invasive bladder cancer (MIBC) for neoadjuvant chemotherapy (NAC) with methotrexate, vinblastine, doxorubicin plus cisplatin (M-VAC) and carboplatin plus gemcitabine (CaG) by analyzing microarray data. The primary aim of the study was to investigate whether the clinical response would increase by combining these prediction systems. Treatment of each MIBC case was allocated into M-VAC NAC, CaG NAC, surgery, or radiation therapy groups by their prediction score (PS), which was calculated using the designed chemo-sensitivity prediction system. The therapeutic effect of the present study was compared with the results of historical controls (n=76 patients) whose treatments were not allocated using the chemo-sensitivity prediction system. In addition, the overall survival between the predicted to be responder (positive PS) group and predicted to be non-responder (negative PS) group was investigated in the present study. Of the 33 patients with MIBC, 25 cases were positive PS and 8 were negative PS. Among the 25 positive PS cases, 7 were allocated to receive M-VAC NAC and 18 were allocated to receive CaG NAC according to the results of the prediction systems. Of the 8 negative PS cases, 3 received CaG NAC, 1 received surgery without NAC and 4 received radiation therapy. The total clinical response to NAC was 88.0% (22/25), which was significantly increased compared with the historical controls [56.6% (43/76) P=0.0041]. Overall survival of the positive PS group in the study was significantly increased compared with the negative PS group (P=0.027). In conclusion, the combination of the two prediction systems may increase the treatment efficacy for patients with MIBC by proposing the optimal NAC regimen. In addition, the positive PS group would have a better prognosis compared with the negative PS group. These results suggest that the two prediction systems may lead to the achievement of 'precision medicine'.

Objective The aim of the present study was to examine factors of nocturnal polyuria and blood pressure variability in male patients with lower urinary tract symptoms (LUTS) who were treated. Methods Two hundred and forty-two male patients with LUTS who were treated recorded frequency volume charts. We investigated their urinary condition and characteristics, medical history, and medications. Thirty-four of these patients underwent ambulatory blood pressure monitoring (ABPM) for 24 hours to evaluate blood pressure variability. Results In the present study, 194 patients (80.2%) had nocturia and 136 (56.2%) had nocturnal polyuria (NP). Among patients with nocturia (≥2 voids/night), 130 (67.0%) had nocturnal polyuria, and 26 of those with nocturia (13.4%) had reduced functional bladder capacity. The use of 2 or more antihypertensive medications was significantly higher in the NP than non-NP group (22.8% vs. 12.3%; P = .035). Significantly more patients in the NP group had non-dipping blood pressure (P = .037). Non-dipping blood pressure was considered a potential factor for NP. Conclusion We suggest that treatment of non-dipping blood pressure may improve NP.

The development of a specific, effective method for the delivery of therapeutics including small molecules and nucleic acids to tumor tissue remains to be solved. Numerous types of lipid nanoparticles (LNPs) have been developed in attempts to achieve this goal. However, LNPs are probably not taken up by target cells because cancer-targeting LNPs are typically modified with poly(ethylene glycol) (PEG), which inhibits the cellular uptake of LNPs, to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. It would clearly be important to develop a LNP with both a prolonged circulation and cancer-specific efficient uptake for use in an innovative nanodrug delivery system. Herein, we assessed the effect of nonstandard macrocyclic peptides against the epithelial cell adhesion molecule (EpCAM) Epi-1, which was discovered by a random nonstandard peptides integrated discovery (RaPID) system, on the cellular uptake and therapeutics delivery of LNPs. A liposomal siRNA delivery system (MEND) was modified with an Epi-1 lipid-derivative (EpCAM-targeting MEND; ET-MEND). The resulting ET-MEND showed a more than 27-fold increase in cellular uptake in EpCAM-positive cell lines. In the case of negative cells, cellular uptake and the efficiency of the ET-MEND for delivering therapeutics were comparable with those of nonmodified MEND. In addition, when systemically injected, the ET-MEND successfully inhibited gene expression in the tumor tissue at a dose of 0.5 mg siRNA/kg without any obvious toxicity. These results suggest that a combination of a specific peptide ligand can be used to identify a RaPID system and that the use of such a MEND for liposomal drug delivery has the potential for use in developing a system for the efficacious delivery of pharmaceuticals to various cancer cells.

We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla-Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis.

Purpose We investigated that preoperative membranous urethral length (MUL) would be associated with the recovery of urinary continence after robot-assisted laparoscopic prostatectomy (RALP). Patients and methods We studied 204 patients who underwent RALP between May 2013 and March 2016. All patients underwent pelvic magnetic resonance imaging (MRI) preoperatively to measure MUL. Urinary continence was defined as the use of one pad or less (safety pad). The 204 patients were divided into two groups: continence group, those who achieved recovery of continence at 3, 6, and 12 months after RALP, and incontinence group, those who did not. We retrospectively analyzed the patients in terms of preoperative clinical factors including age, body mass index (BMI), estimated prostate volume, neurovascular bundle salvage, history of preoperative hormonal therapy, and MUL. Results The safety pad use rate was 69.6%, 86.9%, and 91.1% at 3, 6, and 12 months, respectively. On univariate and multivariate analyses, MUL were significant factors in every term of recovery of urinary continence in both groups. According to the receiver operating characteristic (ROC) curve analysis, the preoperative MUL that could best predict early recovery of urinary continence at 3 months after RALP was 12 mm. Conclusions We suggest that preoperative MUL > 12 mm would be a predictor of early recovery of urinary continence after RALP.

This study was conducted to acquire novel insight into differences between bulk (16S rDNA) and metabolically active (16S rRNA) prokaryotic communities in the sediment of a hypereutrophic lake (Japan). In the bulk communities, the class Deltaproteobacteria and the order Methanomicrobiales were dominant among bacteria and methanogens. In the metabolically active communities, the class Alphaproteobacteria and the order Methanomicrobiales and the family Methanosaetaceae were frequently found among bacteria and methanogens. Unlike the bulk communities of prokaryotes, the composition of the metabolically active communities varied remarkably vertically, and their diversities greatly decreased in the lower 20 cm of sediment. The metabolically active prokaryotic community in the sediment core was divided into three sections based on their similarity: 0–6 cm (section 1), 9–18 cm (section 2), and 21–42 cm (section 3). This sectional distribution was consistent with the vertical pattern of the sedimentary stable carbon and nitrogen isotope ratios and oxidation–reduction potential in the porewater. These results suggest that vertical disturbance of the sediment may influence the communities and functions of metabolically active prokaryotes in freshwater lake sediments. Overall, our results indicate that rRNA analysis may be more effective than rDNA analysis for evaluation of relationships between actual microbial processes and material cycling in lake sediments.

BACKGROUND/AIM Secure dose escalation is required to compensate avoidance of concurrent chemotherapy in radiotherapy for increasing elderly bladder cancer. We aimed to evaluate the efficacy of lipiodol submucosally injected as a fiducial marker during image-guided radiotherapy (Lip-IGRT) for muscle invasive bladder cancer (BC). PATIENTS AND METHODS Twenty-three patients with T2a-4aN0-1M0 BC underwent whole-bladder irradiation of 46 Gy and Lip-IGRT of 20 Gy, conventionally. The bladder volume exposed to 19 Gy (bV19:%) on Lip-IGRT was referred as an index predicting cystitis. RESULTS Lipiodol consistently highlighted the boundaries of 20 tumors (88%) on planning and portal verification images. Three of 4 patients under oral anticoagulant agents usage were complicated with grade ≥2 hematuria for 3 days (a patient with a bV19 of >50%) or more than a year (2 patients with bV19 of

The contents and elution behavior of metals in consumer electronics parts were determined so as to understand their maximum environmental risk. Elements contained most in printed-circuit boards were Cu, Si, Br, Ca, Al, Sn, Pb, Sb, Ba, Fe, Ni, Ti, and Zn; in cathode-ray tube glass were Si, Pb, Ba, Sr, Zn, Zr, Ca, and Sb; in arsenic contained liquid-crystal displays were Si, Ca, Sr, Ba, As, and Fe; and in antimony contained liquid-crystal displays were Si, Ba, Ca, Sb, Sr, Fe, and Sn. The elements eluted most from printed-circuit boards were Zn, Pb, and Cu; from cathode-ray tube glass were Pb, Zn, B, Ba, and Si; and from liquid-crystal displays were B and Si, and the toxic As and Sb. The amount eluted was greatest at acidic pH. It was revealed that officially recommended 6-h-shaking with a pure water test was insufficient to understand the real environmental risk of waste electronics.

Recently, new and advanced ideas have been presented on the value of polymer-based syringes for improved safety, better strength, reduced aggregation, and the prevention of drug degradation. In this report, our findings on drug degradation from protein oxidation will be presented and discussed. Commonly, dissolved oxygen is one of the factors for causing protein degradation. Due to the nature of higher gas permeability in polymer-based syringes, it was thought to be difficult to control the oxygen level during storage. However, this report demonstrates the appropriateness of combining the use of an oxygen absorber within the secondary packaging as a deoxygenated packaging system. In addition, this report suggests that another factor to enhance protein oxidization is related to radicals on the syringe barrel from sterilization by irradiation. We demonstrate that steam sterilization can minimize protein oxidization, as the protein filled in steam sterilized syringe is much more stable. In conclusion, the main oxidation pathway of a protein has been identified as dissolved oxygen and radical generation within a polymer container. Possible solutions are herewith presented for controlling oxidation by means of applying a deoxygenated packaging system as well as utilizing steam sterilization as a method of sterilization for prefillable polymer syringes. LAY ABSTRACT: There have been many presentations and discussions about the risks associated with glass prefilled syringes. Advanced ideas are being presented on the value of polymer-based syringes for improved safety, better strength, reduced protein aggregation, and the prevention of drug degradation. Drug degradation based on protein oxidation is discussed in this report. Identification of the main factors causing this degradation and possible solutions available by using polymer-based syringes will be presented. The causes of protein oxidation have been identified as dissolved oxygen and radicals generated by the applied method of sterilization. The oxidation reaction created by dissolved oxygen within the drug product can be effectively inhibited by controlling the removal of the oxygen through the use of a deoxygenated packaging system. This packaging system can control the level or complete removal of oxygen from the primary container and the secondary packaging system. Protein oxidation induced by the formation of radicals from sterilization by irradiation is another critical aspect where it was thought that various sterilization methods were acceptable without loosing drug product quality. However, this report is first to demonstrate that gamma sterilized polymer-based syringes accelerated protein oxidation by radical generation; this effect can be prevented by means of steam sterilization.

The functionality of a newly developed silicone oil-free (SOF) syringe system, of which the plunger stopper is coated by a novel coating technology (i-coating™), was assessed. By scanning electron microscopy observations and other analysis, it was confirmed that the plunger stopper surface was uniformly covered with the designed chemical composition. A microflow imaging analysis showed that the SOF system drastically reduced both silicone oil (SO) doplets and oil-induced aggregations in a model protein formulation, whereas a large number of subvisible particles and protein aggregations were formed when a SO system was used. Satisfactory container closure integrity (CCI) was confirmed by means of dye and microorganism penetration studies. Furthermore, no significant difference between the break loose and gliding forces was observed in the former, and stability studies revealed that the SOF system could perfectly show the aging independence in break loose force observed in the SO system. The results suggest that the introduced novel SOF system has a great potential and represents an alternative that can achieve very low subvisible particles, secure CCI, and the absence of a break loose force. In particular, no risk of SO-induced aggregation can bring additional value in the highly sensitive biotech drug market. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1520–1528, 2014

Objective: Everolimus is positioned as second-line treatment for metastatic renal cell carcinoma resistant to vascular endothelial growth factor receptor-tyrosine kinase inhibitors. We investigated retrospectively the efficacy and safety of everolimus in Japanese patients with advanced renal cell carcinoma in the clinical setting. Methods: Nineteen patients who discontinued treatment with vascular endothelial growth factor receptor-tyrosine kinase inhibitors because of disease progression or adverse events were administered everolimus. We evaluated progression-free survival, overall survival and tumor response rate of everolimus treatment. We also compared laboratory abnormalities and adverse events of everolimus treatment with those of prior vascular endothelial growth factor receptor-tyrosine kinase inhibitors therapy. Results: In all patients, median progression-free survival was 8.4 months and median overall survival was not reached at 25 months. The best objective response was complete response in 1 patient and stable disease in 15 patients. Eleven patients (58%) were intolerant and 8 (42%) were refractory to prior vascular endothelial growth factor receptor-tyrosine kinase inhibitors treatment. Median overall survival was significantly longer (P , 0.01) in vascular endothelial growth factor receptor-tyrosine kinase inhibitor-intolerant (.25 months) than in vascular endothelial growth factor receptor-tyrosine kinase inhibitor-refractory subjects (4.3 months), and median progression-free survival tended to be better (P ¼ 0.06) in vascular endothelial growth factor receptor-tyrosine kinase inhibitor-intolerant (10.0 months) than in vascular endothelial growth factor receptor-tyrosine kinase inhibitor-refractory subjects (2.5 months). Two patients discontinued everolimus treatment because of adverse events. Conclusions: In this study, the overall survival and progression-free survival were better in vascular endothelial growth factor receptor-tyrosine kinase inhibitor-intolerant than in vascular endothelial growth factor receptor-tyrosine kinase inhibitor-refractory subjects. The adverse event profiles of everolimus and vascular endothelial growth factor receptor-tyrosine kinase inhibitors were different. Patients intolerant to vascular endothelial growth factor receptortyrosine kinase inhibitors may tolerate everolimus well and have greater survival benefit from switching to everolimus than those refractory to vascular endothelial growth factor receptortyrosine kinase inhibitors.

INTRODUCTION AND OBJECTIVES: To compare the rate of chemo-sensitivity in neoadjuvant chemotherapy for muscle invasive bladder cancer between historical control and this interventional prospective study by using our M-VAC and CaG prediction systems. METHODS: Muscle invasive bladder cancer patients (Stage II-III) were engaged in this study. They were assigned to M-VAC, CaG, operation or radiation therapy according to the result of the prediction system which we had already established. According to their responses to the neoadjuvant chemotherapy, we categorized the patients into two groups: 0responders0 who achieved significant tumor shrinking (>60%), and 0non-responders0 ( 60%). Patients were evaluated either 0accurate0 or 0 inaccurate0 according to a tumor shrinkage rate cut off line of a 60% decrease after chemotherapies. The primary endpoint of this study was comparison with the historical controls in terms of the rate of chemo-sensitivity and efficacy. The secondary endpoint was down staging ( pT1) and the overall survival. RESULTS: From March 2011 to July 2013, an intention-to-treat 35 cancer samples from patients were entered in this study. Seven patients M-VAC, 22 CaG, 2 operation and 4 radiation therapy were received respectively. The predicted responder rate for M-VAC was 42.9% (15/35) and for CaG was 57.1% (20/35). Moreover, 74.3% (26/35) of all patients could be expected to respond to both or either of these two regimens by applying our two prediction systems. While, in previous historical control studies (M-VAC 39, CaG 37, total 76 cases), the total predicted response rate of the 76 cases was 57.9% (44/76) (p1⁄40.09). And in the predicted 0responder0 cases, the response rate of M-VAC neoadjuvant chemotherapy was 85.7% (6/7) and CaG was 89.5% (17/19). Therefore, the combined response rate in this group was 88.5% (23/26). In the historical control cases, the observed response rate of M-VAC/CaG therapy was 59.0% (23/39)/54.1% (20/37), and combined observed response rate was 56.6% (43/76) (p1⁄40.004). Down staging and overall survival between this prospective study and the historical control were not significant statistical differences, however in overall survival this prospective study was better tendency than that of the historical control (p1⁄40.233). CONCLUSIONS: Combining the M-VAC and CaG prediction systems would improve the chemo-sensitivity for muscle invasive bladder cancers.

Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the molecular interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system.

Renal cell carcinoma is a typical hypervascular tumor in which neovascularization may have a large part in progression. We examined expression of the cancer regulating, p53 targeted angiogenesis inhibitor brain-specific angiogenesis inhibitor 1 in renal cell carcinoma tissue to elucidate the clinical significance of its expression.We examined brain-specific angiogenesis inhibitor 1 mRNA and protein expression in 47 renal cell carcinoma and 10 normal kidney tissues using real-time quantitative polymerase chain reaction and immunohistochemistry, respectively. Levels of VEGF and bFGF mRNA, and immunohistochemical expression of p53 protein were also investigated in the same renal cell carcinoma tissues.A significant decrease in BAI1 mRNA was noted in renal cell carcinoma tissue compared with that in normal kidney tissue (p0.001). Immunostaining for brain-specific angiogenesis inhibitor 1 was also decreased in carcinoma tissue compared with normal kidney tissue. BAI1 mRNA and protein expression were lower in advanced renal cell carcinoma (pT3-4) than in localized renal cell carcinoma (pT1-2) tissues (p0.03 and 0.003, respectively). A significant negative correlation was observed between microvessel density and brain-specific angiogenesis inhibitor 1 protein expression (r = -0.4056, p = 0.002). No significant correlation was noted between BAI1 and VEGF or bFGF mRNA levels. Brain-specific angiogenesis inhibitor 1 protein expression did not correlate with p53 protein expression.These observations suggest that down-regulation of brain-specific angiogenesis inhibitor 1 expression may be a critical factor in renal cell carcinoma development and BAI1 may be a promising candidate for gene therapy of renal cell carcinoma.

Myocardin (Mycd), which is essential for the differentiation of the smooth muscle cell lineage, is constitutively located in the nucleus, although its family members, myocardin-related transcription factors A and B (MRTF-A/B), mostly reside in the cytoplasm and translocate to the nucleus in response to Rho signaling. The mechanism for their nuclear import is unclear. Here we investigated the mechanism for the nuclear import of Mycd family members and demonstrated any correlation between such mechanism and the phenotype of vascular smooth muscle cells (VSMCs). In cultured VSMCs, the knockdown of importin β1 inhibited the nuclear import of Mycd and MRTF-A/B. Their NH(2)-terminal basic domain was identified as a binding site for importin α/β1 by in vitro analyses. However, Mycd had a higher affinity for importin α/β1 than did MRTF-A/B, even in the absence of G-actin, and Mycd affinity for importin α1/β1 was stronger than for any other importin α/β1 heterodimers. The binding of Mycd to importin α/β1 was insensitive to G-actin, whereas that of MRTF-A/B was differently inhibited by G-actin. In dedifferentiated VSMCs, the levels of importins α1 and β1 were reduced concomitant with down-regulation of Mycd, serum response factor, and smooth muscle cell markers. By contrast, in differentiated VSMCs, their expressions were up-regulated. Thus, the nuclear import of Mycd family members in VSMCs depends on importin α/β1, and their relative affinities for importin α/β1 heterodimers determine Mycd nuclear import. The expression of Mycd nuclear import machineries is related to the expression levels of VSMC phenotype-dependent smooth muscle cell markers.

RhoA is known to be involved in myogenic differentiation, but whether it acts as a positive or negative regulator is controversial. To resolve this issue, we investigated the differentiation stage-specific roles of RhoA and its effector, Rho-associated kinase, using C2C12 myoblasts. We found that proliferating myoblasts show high levels of RhoA and serum-response factor activities and strong expression of the downstream target of RhoA, myocardin-related transcription factor-A (MRTF-A or MAL); these activities and expression are markedly lower in differentiating myocytes. We further demonstrated that, in proliferating myoblasts, an increase in MRTF-A, which forms a complex with Smad1/4, strikingly activates the expression level of the Id3 gene; the Id3 gene product is a potent inhibitor of myogenic differentiation. Finally, we found that during differentiation, one of the forkhead transcription factors translocates into the nucleus and suppresses Id3 expression by preventing the association of the MRTF-A-Smad complex with the Id3 promoter, which leads to the enhancement of myogenic differentiation. We conclude that RhoA/Rho-associated kinase signaling plays positive and negative roles in myogenic differentiation, mediated by MRTF-A/Smad-dependent transcription of the Id3 gene in a differentiation stage-specific manner.

We investigated the effects of coexisting surfactants and high-molecular-weight organic compounds on the reductive dechlorination of trichloroethylene by zerovalent iron powder to determine whether these additives had utility as washing reagents for remediation of soil and groundwater pollution. During the dechlorination reaction, the amount of trichloroethylene decreased, and the formation of cis-1,2-dichloroethylene was observed. The decomposition of trichloroethylene was found to be first-order with respect to the trichloroethylene and zerovalent iron concentrations when the solution contained no additives. The rates of decomposition of trichloroethylene in the presence of the additives were lower than the rate in the absence of the additives: the rate constant was reduced by a factor of 0.7 for the cationic surfactant cetyltrimethylammonium bromide; by a factor of 0.5 for the anionic surfactants sodium n-dodecylbenzenesulfonate, sodium n-dodecylsulfate, and sodium n-dodecanesulfonate and for the high-molecular-weight organic compounds soluble starch, beta-cyclodextrin, and polyethyleneglycol 6000; and by a factor of 0.2 for sodium laurate and the nonionic surfactants Triton X-100, Tween 20, Tween 60, Brij 35, and Brij 58. Comparison of the concentrations of the nonionic surfactants with their critical micellar concentrations indicated that the rate-reducing effect of these additives was due to solubilization of trichloroethylene into the micellar phase. The adsorption of trichloroethylene onto the zerovalent iron surface was also affected by the presence of the additives. Thus, our results indicated that the changes in the decomposition rate of trichloroethylene were determined by several factors.

With the ultimate aim of developing bioremediation technology that use the optimum bacterial community for each pollutant, we performed polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis and identified communities of culturable bacteria in HgCl(2)- and trichloroethylene (TCE)-contaminated soil microcosms. PCR-DGGE band patterns were similar at 0 and 1 ppm HgCl(2), but changes in specific bands occurred at 10 ppm HgCl(2). Band patterns appearing at 10 and 100 ppm TCE were very different from those at 0 ppm. Phylogenetic analysis showed four bacterial groups in the HgCl(2)-contaminatied cultures: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Most high-density bands, decreased-density bands, and common bands were classified into the phyla Proteobacteria, Actinobacteria, and Firmicutes, respectively; the effects of HgCl(2) on culturable bacteria appeared to differ among phyla. Duganella violaceinigra [98.4% similarity to DNA Data Bank of Japan (DDBJ) strain], Lysobacter koreensis (98.2%), and Bacillus panaciterrae (98.6%) were identified as bacteria specific to HgCl(2)-contaminated soils. Bacteria specific to TCE-contaminated soils were distributed into three phyla (Firmicutes, Proteobacteria, and Actinobacteria), but there was no clear relationship between phylum and TCE effects on culturable bacteria. Paenibacillus kobensis (97.3%), Paenibacillus curdlanolyticus (96.3%), Paenibacillus wynnii (99.8%), and Sphingomonas herbicidovorans (99.4%) were identified as bacteria specific to TCE-contaminated soils. These bacteria may be involved in pollutant degradation.

The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with the unplanted system. Among the representative alkane hydroxylase genes alkB, CYP153, almA and ladA, the first two were detected in this study, and the genotypes of both genes were apparently different among the systems studied. Their diversity was also higher on the rhizoplanes of the grasses than in unplanted oil-contaminated soils. Actinobacteria-related genes in particular were among the most diverse alkane hydroxylase genes on the rhizoplane in this study, indicating that they are one of the main contributors to degrading alkanes in oil-contaminated soils during phytoremediation. Actinobacteria-related alkB genes and CYP153 genes close to the genera Parvibaculum and Aeromicrobium were found in significant numbers on the rhizoplanes of grasses. These results suggest that the increase in diversity and genotype differences of the alkB and CYP153 genes are important factors affecting petroleum hydrocarbon-degrading ability during phytoremediation. Electronic supplementary material The online version of this article (doi:10.1186/s40064-015-1312-0) contains supplementary material, which is available to authorized users.

Here, we report a fluorescent probe based on a macrocyclic peptide scaffold that specifically stains EpCAM-expressing MCF7 cells. The 14-mer macrocyclic peptide binding to the extracellular domain of EpCAM with a dissociation constant in the low nM range (1.7 nM) was discovered using the random non-standard peptide-integrated discovery system. Notably, this probe containing a fluorescence tag is less than 3000 Da in total and able to visualize nearly every live cell under high cell-density conditions, which was not achieved by the conventional mAb staining method. This suggests that the molecular probe based on the compact macrocyclic scaffold has great potentials as an imaging tool for the EpCAM biomarker as well as a delivery vehicle for drug conjugates.

A new most-probable-number (MPN) method using 96-well microtiter plates was developed to enumerate denitrifying bacteria. In this method, dilutions of samples are added to microtiter plates with a medium and incubated anaerobically using the AnaeroPouch culture system (Mitsubishi Gas Chemical, Inc., Tokyo, Japan). The microtiter plate MPN method gave precise estimates of the population densities of a culture of Paracoccus denitrificans, similar to the estimates obtained using a test tube MPN method and a colony count method. The population densities of denitrifying bacteria in soil samples estimated by the microtiter plate MPN method were higher than the population densities estimated by the test tube MPN method. Because the new method requires less equipment, labor, and time than the conventional test tube method. It will be valuable for estimating the biomass of denitrifying bacteria in natural samples.

We developed a new most-probable-number (MPN) method for enumeration of methanotrophs, using 48-well microtiter plates. With this method, a microtiter plate which has been inoculated with serial dilutions of a sample is placed in a plastic desiccator containing methane and shaken. The proposed method gave estimates of population densities of a pure culture of Methylocystis sp. strain M and of methanotrophs in a paddy soil sample similar to those obtained by a conventional MPN method using serum bottles. The new microtiter plate MPN method with an additional replica-plating technique made it possible to enumerate small populations of methanotrophs in cornfield and forest soils. It also requires less equipment, labor, and time than the conventional serum bottle MPN method.

We investigated spatial and temporal variations in bacterial community structures as well as the presence of three functional proteolytic enzyme genes in the sediments of a hypereutrophic freshwater lake in order to acquire an insight into dynamic links between bacterial community structures and proteolytic functions. Bacterial communities determined from 16S rRNA gene clone libraries markedly changed bimonthly, rather than vertically in the sediment cores. The phylum Firmicutes dominated in the 4–6 cm deep sediment layer sample after August in 2007, and this correlated with increases in interstitial ammonium concentrations (p < 0.01). The Firmicutes clones were mostly composed of the genus Bacillus. npr genes encoding neutral metalloprotease, an extracellular protease gene, were detected after the phylum Firmicutes became dominant. The deduced Npr protein sequences from the retrieved npr genes also showed that most of the Npr sequences used in this study were closely related to those of the genus Bacillus, with similarities ranging from 61% to 100%. Synchronous temporal occurrences of the 16S rRNA gene and Npr sequences, both from the genus Bacillus, were positively associated with increases in interstitial ammonium concentrations, which may imply that proteolysis by Npr from the genus Bacillus may contribute to the marked increases observed in ammonium concentrations in the sediments. Our results suggest that sedimentary bacteria may play an important role in the biogeochemical nitrogen cycle of freshwater lakes.

We developed a method for enumerating trichloroethylene (TCE) degrading Mycobacterium sp. TA27 by means of PCR. The PCR primers and probe were designed based on the 16S rRNA sequences of Mycobacterium sp. TA27, compared with corresponding regions of 8 phylogenetically related strains. The total DNAs of strain TA27 and the other 8 mycobacteria were extracted and used for PCR amplification by a 5'-nuclease real-time PCR assay. The highest specificity for strain TA27 was observed at an annealing temperature of 62°C and at forward and reverse primer concentrations of 0.2 μM each. The other 8 mycobacteria were not detected under these conditions. The detection limits for the DNA and cells of strain TA27 were 0.05 pg/tube and 25 cells/tube, respectively. The utility of the PCR assay for the quantification of strain TA27 cells was demonstrated in TCE-polluted groundwater.

Mycobacterium spp. strains TA5 and TA27 (ethane-utilizing bacteria), which can degrade trichloroethylene (TCE) and 1,1,1-trichloroethane (1,1,1-TCA), were isolated from soil. Both bacteria could cometabolically degrade dichloromethane, chloroform, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-TCA, 1,1,2-TCA, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, 1,1-dichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, and TCE with ethane as a carbon source. They could not degrade carbon tetrachloride, freon 113, or tetrachloroethylene. The TCE degradation characteristics of strain TA27 were determined. Under a head-space gas containing 3% ethane, strain TA27 degraded more than 95% of TCE at an initial concentration of 1 mg l–1 within 3 days. We observed good growth and TCE degradation between 25 and 35 °C. At an initial TCE concentration of 30 mg l–1, it degraded 30% of TCE within 7 days. Although growth was inhibited for more than 50 mg l–1 TCE at 3% ethane concentration, good growth and 50% degradation of TCE were observed at 12% ethane concentration within 14 days. High ethane concentration may mitigate the toxicity of TCE.

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l−1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.

Two strains of 1,1,1-trichloroethane (TCA)-degrading bacteria, TA5 and TA27, were isolated from soil and identified as Mycobacterium spp. Strains TA5 and TA27 could degrade 25 and 75 mg · liter of TCA −1 cometabolically in the presence of ethane as a carbon source, respectively. The compound 2,2,2-trichloroethanol was produced as a metabolite of the degradation process.

The chlorinated organic compounds, trichloroacetic acid (TCAA), 2, 2, 2-trichloroethanol (TCAol), dichloroacetic acid (DCAA) and chloral were produced from the biotransformation of trichloroethylene (TCE) by a methane utilizing bacterium Methylocystis sp. strain M. The degradation of TCE by strain M was influenced by pH, with an optimum between 6 and 7. The conversions to TCAA, TCAol and DCAA derived from added TCE were 7.8%, 0.04% and 1.8%, respectively, at pH 7, and varied with pH. The reactions that convert chloral to these metabolites were also influenced by pH. The conversions to TCAA, TCAol and DCAA from added chloral were 77%, 0.6% and 6.0%, respectively, at pH 7. Strain M did not degrade TCAA, TCAol and DCAA, which were shown to be end products of TCE degradation by strain M. The production of DCAA from chloral suggested a new pathway for producing DCAA via chloral, in addition to that via TCEoxide. Thrity-four percent and 66% of DCAA produced from TCE were derived via chloral and TCEoxide, respec...

Volatile aliphatic chlorinated compounds such as trichloroethylene (TCE), 1, 1, 1-trichloroethane (TCA), and tetrachloroethylene have been detected in groundwater throughout Japan. Various soil and groundwater cleanup technologies are now being developed, studied, and evaluated in Japan. For chlorinated compounds, soil vapor extraction, pumping up and treatment, and digging up and drying are very common approaches. Physical and chemical methods for remediation are expensive. Therefore, less expensive but more complete pollutant destruction technology is required. Bioremediation could be one of the most promising new technologies for cleaning up groundwater contamination, because of its low cost and the complete destruction of pollutants. In this paper, we reported TCE and TCA degrading bacteria and determined their fundamental characteristics for bioremediation.

Objective: Although cisplatin-based neoadjuvant chemotherapy followed by cystectomy was demonstrated to improve the survival among patients with locally advanced bladder cancer, its severe adverse events, including nephrotoxicity, are critical issues. We investigated the safety and activity of carboplatin, a mild nephrotoxic agent, combined with gemcitabine as a neoadjuvant chemotherapy compared with methotrexate, vinblastine, doxorubicin and cisplatin for patients with locally advanced bladder cancer. Methods: We retrospectively evaluated 68 patients with locally advanced bladder cancer who received neoadjuvant methotrexate, vinblastine, doxorubicin and cisplatin (n ¼ 34) or gemcitabine and carboplatin (n ¼ 34) followed by cystectomy at our institute. The adverse events, chemotherapy delivery profile, rate of down-stage and recurrence-free survival were assessed for methotrexate, vinblastine, doxorubicin and cisplatin compared with gemcitabine and carboplatin. Results: The mean cycles of methotrexate, vinblastine, doxorubicin and cisplatin, and gemcitabine and carboplatin, were 2.5 and 2.7, respectively. The hematologic adverse events of Grade 3 or 4 neutropenia, anemia and thrombocytopenia for methotrexate, vinblastine, doxorubicin and cisplatin were 15, 18 and 0%, respectively. The occurrences for gemcitabine and carboplatin were 53, 21 and 50%, respectively. Grade 3 or 4 non-hematologic toxicities for methotrexate, vinblastine, doxorubicin and cisplatin were nausea and vomiting in 24%, and were not observed for gemcitabine and carboplatin. The lowest median estimated glomerular filtration rate during methotrexate, vinblastine, doxorubicin, and cisplatin and gemcitabine and carboplatin was 55.8 and 70.6 ml/min/1.73 m 2 ,r espectively (P ¼ 0.002). The rate of downstage to pT1 or less was 59% for methotrexate, vinblastine, doxorubicin and cisplatin, and 53% for gemcitabine and carboplatin (P ¼ 0.624). The recurrence-free survival of methotrexate, vinblastine, doxorubicin and cisplatin, and gemcitabine and carboplatin, at 36 months from the diagnosis was 79 and 75%, respectively (P ¼ 0.85). Conclusions: Neoadjuvant gemcitabine and carboplatin showed less non-hematologic toxicity than methotrexate, vinblastine, doxorubicin and cisplatin, and especially less nephrotoxicity was demonstrated for gemcitabine and carboplatin. Although observed during the short term, the recurrence-free survival for gemcitabine and carboplatin was comparable to that for methotrexate, vinblastine, doxorubicin and cisplatin.

The mode of thioether macrocyclization of peptides containing an N-terminal 2-chloroacetyl group and two or three competing cysteine residues at downstream positions has been extensively studied, leading to a strategy for designated formation of overlapping-bicyclic peptides or dumbbell-type bicyclic peptides.

The optimum electrotransformation conditions were determined for Pseudomonas putida PpY101 with plasmid pSUP104 (9.5 kb) and pSR134 (18.6 kb). Field strength was a very important parameter for electrotransformation efficiency. Optimum efficiencies (1.1 x 10(5) transformants/micrograms DNA) with pSUP104 and pSR134 were obtained at a field strength of 12.5 kV/cm, a time constant of about 4.5 ms (resistance setting of 200 ohms), a supercoiled DNA concentration of 100 ng/ml, and a cell concentration of 10(9)/ml. Because the efficiency obtained is high enough, electrotransformation is useful for the direct cloning of P. putida PpY101. No significant relationship between plasmid size and electrotransformation efficiency was observed. These efficiencies were about 4.5 times higher than those using the MgCl2 method. Under these conditions, electrotransformation efficiencies of relaxed plasmid DNA treated with topoisomerase I and that linearized by EcoRI digestion were high.

This study investigated the expression of hypoxia-inducible protein 2 (HIG2), a novel renal cell carcinoma (RCC)-associated molecule and an essential growth factor for RCC, in kidneys to elucidate its clinical significance in RCC. An immunohistochemical study of HIG2 was conducted in 93 surgical samples of RCC tissues and 10 samples of normal kidney tissues obtained after nephrectomies for localized RCC. HIG2 expression was also correlated with clinicopathological characteristics and survival. Only faint or weak immunostaining for HIG2 was observed in normal kidney samples. HIG2 expression was found in 86% of RCC tissues (80/93). When analyzed by histological type, positive staining for HIG2 was detected in all papillary (7/7), chromophobe (1/1) and cyst-associated (3/3) RCC. In contrast, the HIG2 expression was observed in 85% of clear cell (68/80) and 50% of spindle cell (1/2) RCC. Labeling indices were 74.1, 45.4, 39, 24.8 and 12.1% in papillary, spindle, clear cell, cyst-associated and chromophobe RCC, respectively. A significant increase in HIG2 expression was noted in RCC tissues obtained from patients with high stage RCC, lymph node metastasis and high nuclear grade (p

Degradation rates of PCE and TCE were determined in lotus, rice and vegetable field soils. The lotus field soil had the highest ability to degrade tetrachloroethylene(PCE) and trichloroethylene(TCE). The values of T50(50% degradation time) in L-1 lotus soil were 8 and 15 days for PCE and TCE under the substrate concentration of 5 µg in 50ml of soil solution. PCE was biologically transformed to TCE in all soils. The material balance of PCE depletion and TCE production were determined. The degradation rates of PCE and TCE were significantly influenced by temperature and substrate concentration.

We report here the ribosomal synthesis of methyllanthionine-containing cyclic peptides involving a site-specific incorporation of vinylglycine under the reprogrammed genetic code, followed by the isomerization of the vinylglycine to dehydrobutyrine, and the subsequent intramolecular Michael addition of a cysteine residue placed at a downstream position of the vinylglycine.

Background: We reviewed our experience with orthotopic continent urinary reconstructionafter radical cystectomy to assess the feasibility of Studer ileal neobladder for patients whoare relatively advanced in age.Methods: Between June 1997 and January 2005, 31 consecutive male patients (mean age:64 years) underwent lower urinary tract reconstruction after radical cystoprostatectomy.Perioperative and late complications, functional outcome of the neobladder, urinary continence,upper urinary tract status and renal function with the metabolic balance were evaluated in allpatients.Results:There was no perioperative death, and perioperative and late complication rates were22.8% and 3.3%, respectively. All 31 patients were able to void urine. Although the meanmaximal functional capacity of the neobladder was 122 ml at 1 month after surgery, the meancapacities were increased to 247 ml at 6 months and 321 ml at 1 year after the operation.Urodynamic results at 3 years showed unchanged characteristics as to micturition pattern andvolume of residual urine and neobladder pressure remained low. Of 31 patients, 29 (93.5%)showed excellent or good continent status during the daytime and 9 (29%) were completely dryat night in 6 months after surgery. Even at 3 years after the operation, only 1 patient out of 21evaluated required single pad during nighttime. In a subgroup of five patients (24%) older than70 years, the status of continence was satisfactory at 3 years after the reconstruction, and onlyone patient required a pad during the night at that point. Renal function levels and metabolicstatus were comparable before surgery and 3 years after surgery. Moreover, pyelographyrevealed normal condition of the upper urinary tract 1 month postoperatively in almost allcases.Conclusions: These data provide evidence that Studer ileal neobladder is a satisfactorysurgical technique for selected patients at our institute. Even for patients older than 70 years,this urinary diversion procedure is safe in terms of morbidity and efficacious as indicated byfunctional outcome.Key words: Studer ileal neobladder – orthotopic continent urinary reconstruction – bladder cancer

The global gene expression of cultured Saccharomyces cerevisiae protoplasts was compared with that of cells using DNA microarray. Quantitative and qualitative analyses revealed that after 6 h of cultivation, 416 gene transcript levels (about 7.1% in all) in the cultured protoplasts were different from those in the cells. Various characteristics and functions of the protoplasts were predicted from the analysis of the gene functions. The cultured protoplasts were more sensitive to oxidative stress than the cultured cells. Their cell cycles were arrested at the G1 phase and cell wall synthesis was promoted. Carbohydrate metabolism was activated in cultured protoplasts, while amino acid biosynthesis was inhibited. Furthermore, some genes associated with the secretory pathway of metabolites were activated, leading to active secretion of these metabolites into the broth. As an example of the application of DNA microarray analysis, we developed two novel methods for the production of useful enzymes based on the characteristics of protoplasts. One was the production of invertase based on the activated secretory pathway, while the other was the production of alpha-glucosidase based on the activated carbohydrate metabolism. The secretion of invertase and alpha-glucosidase was promoted in cultured protoplasts. The invertase and alpha-glucosidase productivities in the cultured protoplasts were 657 U and 218 U, respectively. On the other hand, only 227 U of invertase was produced, while alpha-glucosidase was not detected, in the cultured cells. The fragile protoplasts were immobilized in agarose gel to protect them from hydrodynamic stress. Four repeated-batch cultures with the immobilized protoplasts were performed, leading to the production of 1574 U of invertase and 739 U of alpha-glucosidase. The same productivities were obtained when this system was scaled up by 10-fold (invertase: 13304 U; alpha-glucosidase: 7688 U).