Efficacy of combination therapy with pembrolizumab and axitinib for metastatic renal collecting duct cell carcinoma: A report on two cases.

Introduction: Immunotherapy-based combinations have become the standard first-line therapy for metastatic renal cell carcinoma. However, combined immunotherapy for renal collecting duct carcinoma had been reported, but its therapeutic efficacy had been unclear. Case presentation: The first case was a 62-year-old man treated with pembrolizumab and axitinib for renal collecting duct carcinoma with multiple bone metastases. After 7 months, the primary and metastatic lesions shrunk and were evaluated as a partial response. The second case was a 71-year-old man treated with pembrolizumab and axitinib for renal collecting duct carcinoma with lymph node and lung metastases. After 9 months, the primary and metastatic lesions shrunk and were evaluated as a partial response. In both cases, the tumor cell expression of programmed death ligand-1 was negative, and CD4+ and CD8+ cells were observed in the tumor. Conclusion: Combined immunotherapy with pembrolizumab and axitinib may be effective for metastatic renal collecting duct carcinoma.

Improved Stability of siRNA-Loaded Lipid Nanoparticles Prepared with a PEG-Monoacyl Fatty Acid Facilitates Ligand-Mediated siRNA Delivery.

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.

Vertical variation of bulk and metabolically active prokaryotic community in sediment of a hypereutrophic freshwater lake.

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.

Relationship between nocturnal polyuria and non-dipping blood pressure in male patients with lower urinary tract symptoms.

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.

Appropriate preoperative membranous urethral length predicts recovery of urinary continence after robot-assisted laparoscopic prostatectomy.

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.

A prospective study to examine the accuracies and efficacies of prediction systems for response to neoadjuvant chemotherapy for muscle invasive bladder cancer.

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'.

Partial Bladder Boost Using Lipiodol Marking During Image-guided Radiotherapy for Bladder Cancer.

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 <50%) after the injection. The 3-year overall survival and disease-free survival rates were 70.4% and 71.1%, respectively. CONCLUSION: Lipiodol marking is an effective way of demarcating BC. However, it is necessary to address the comorbidities of elderly patients.

Composition and Elution Behavior of Various Elements from Printed Circuit Boards, Cathode-ray Tube Glass, and Liquid-crystal Displays in Waste Consumer Electronics.

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.

Efficient siRNA Delivery by Lipid Nanoparticles Modified with a Nonstandard Macrocyclic Peptide for EpCAM-Targeting.

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.

Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake.

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.

A Fluorescent Imaging Probe Based on a Macrocyclic Scaffold That Binds to Cellular EpCAM.

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.

Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils.

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.

A strategy for the prevention of protein oxidation by drug product in polymer-based syringes.

UNLABELLED: 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.

Linking temporal changes in bacterial community structures with the detection and phylogenetic analysis of neutral metalloprotease genes in the sediments of a hypereutrophic lake.

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.

Efficacy of everolimus in patients with advanced renal cell carcinoma refractory or intolerant to VEGFR-TKIs and safety compared with prior VEGFR-TKI treatment.

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= 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 receptor-tyrosine kinase inhibitors may tolerate everolimus well and have greater survival benefit from switching to everolimus than those refractory to vascular endothelial growth factor receptor-tyrosine kinase inhibitors.

Functional evaluation and characterization of a newly developed silicone oil-free prefillable syringe system.

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.

Neoadjuvant gemcitabine plus carboplatin for locally advanced bladder cancer.

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 = 34) or gemcitabine and carboplatin (n = 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), respectively (P = 0.002). The rate of down-stage to pT1 or less was 59% for methotrexate, vinblastine, doxorubicin and cisplatin, and 53% for gemcitabine and carboplatin (P = 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 = 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.

Selective thioether macrocyclization of peptides having the N-terminal 2-chloroacetyl group and competing two or three cysteine residues in translation.

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.

Flexizymes: their evolutionary history and the origin of catalytic function.

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.