[A case in which endoscopic ultrasound-guided transrectal drainage was effective for pelvic abscess].

The patient, a 33-year-old female, presented to her local doctor with lower abdominal pain. She was referred to our hospital for a plain CT scan, which revealed signs of ileitis. Because acute appendicitis could not be ruled out, the patient was hospitalized. On the third day of hospitalization, she underwent laparoscopic appendicectomy for perforated appendicitis. However, the inflammatory response persisted despite continued antibacterial treatment; we diagnosed this to be due to the formation of a postoperative residual abscess. Since drainage of the abscess was deemed necessary, the patient underwent EUS-guided transrectal drainage on the 26th day of hospitalization. The patient had a favorable postoperative course and was discharged on the 31st day of hospitalization. Along with some literature review, this report details a case in which transrectal drainage under ultrasound endoscopy was effective in treating a pelvic abscess. We report a case of a pelvic abscess that was drained through the rectum under EUS guidance and an internal and external fistula tube was placed. The abscess resolved without complications.

[Laparoscopic Resection of a Liver and Ovarian Metastasis of Transverse Colon Cancer-A Case Report].

A 60s woman was diagnosed to transverse colon cancer and she underwent laparoscopic right hemicolectomy. Localized peritoneal dissemination surrounding tumor was detected during surgery. She was administrated to chemotherapy due to a hepatic metastasis in S2/3 postoperatively. Subsequently, PET-CT revealed a left ovarian metastasis in addition to a liver metastasis during chemotherapy. Laparoscopic hepatic left lateral segmentectomy and bilateral adnexectomy was performed at 1 year and 9 months after the first surgery and histopathological examination showed a metastasis of transverse colon cancer. The growth of liver and lung metastases and peritoneal disseminations was detected at 6 months later after the second surgery and the patient is currently receiving palliative treatment. Previous literatures described that ovarian metastasis of colon cancer showed bilateral metastasis and resistance to chemotherapy frequently and ruptured in some cases. We should consider to resect bilateral ovary even if unilateral metastasis alone was detected by imaging examination.

Influence of endoscopic submucosal dissection on additional gastric resections.

BACKGROUND: Widespread application of endoscopic submucosal dissection (ESD) for early gastric cancer (EGC) results in noncurative resection in some patients. The influence of preceding ESD on additional gastric resections has not been completely evaluated. METHODS: Endoscopic, surgical, and pathological records of 255 patients who underwent additional gastrectomy after noncurative ESD at a single prefectural cancer center from September 2002 to December 2010 were reviewed. The estimated gastric resection based on endoscopic images before ESD was compared with the actual gastric resection performed after ESD. RESULTS: Altered gastric resection was performed in 4 (1.6%) of the 255 patients. In 3 patients, total gastrectomy was performed instead of distal gastrectomy; in 1 patient, distal gastrectomy was performed instead of pylorus-preserving gastrectomy because of an insufficient distance from the cardia or pylorus caused by contraction of the ESD scar. Standard gastrectomy including total or distal gastrectomy with D2 lymph node dissection was performed in 33 patients because of deep submucosal invasion with positive/indefinite vertical margins. The final pathology revealed pT2 or deeper in 10 patients. CONCLUSIONS: In conclusion, 98.4% patients underwent the scheduled gastric resection before ESD, and the preceding gastric ESD had almost no influence on changing the gastric resection of the additional surgery. Although rare, the preceding ESD may necessitate alterations in gastric resection to widen the surgical area because of contraction of ESD scar for lesions near the cardia or pylorus. MINI ABSTRACT: A retrospective study of additional gastrectomy after noncurative ESD showed that the preceding ESD had almost no influence on changing the gastric resection of the additional surgery.

Polypoid leiomyosarcoma of the esophagus treated by endoscopic submucosal dissection.

We report a rare case of polypoid leiomyosarcoma of the esophagus that was treated by endoscopic submucosal dissection (ESD). A 63-year-old man with complaints of progressive dysphagia was referred to Hyogo Cancer Center for treatment of esophageal tumor. Esophagoscopy revealed a polypoid tumor 25 mm in diameter on the left side of the upper esophagus. Despite several biopsy specimens, the diagnosis could not be confirmed. Computed tomography showed a protruded, homogeneously enhancing mass in the upper esophagus, but no lymph node enlargement or metastasis. After 1.5 months, the esophagogram showed a filling defect 47 mm in diameter in the upper esophagus. Given this rapid tumor growth, en bloc resection was done by ESD for therapeutic diagnosis. After this treatment, the tumor seemed to grow larger, showing a short stalk and occupying the esophageal lumen. Histopathologically, the tumor comprised pleomorphic spindle cells with mitosis. Tumor invasion involved the lumina propria mucosae and contact with the muscularis mucosae, but not involving the submucosa. Immunohistochemical examination showed positive staining for smooth muscle actin and HHF35, but negative for desmin, caldesmon, CD34, c-kit, DOG1, ALK, S-100 protein and cytokeratin. These histopathological findings were compatible with a diagnosis of esophageal leiomyosarcoma derived from the muscularis mucosae.

Percutaneous endoscopic gastrostomy for decompression of malignant bowel obstruction.

BACKGROUND: Previous reports on percutaneous endoscopic gastrostomy (PEG) for bowel decompression have included a relatively small number of patients and the details of post-procedural outcomes and complications are lacking. The aim of the present study was to evaluate the outcomes and safety of PEG for bowel decompression in a relatively large number of patients with malignant bowel obstruction. PATIENTS AND METHODS: Over a 10-year period, 76 patients with malignant bowel obstruction were referred to the main referral cancer center in Shizuoka prefecture for PEG to obtain decompression. The method for gastrostomy was carried out by the pull-method, the modified introducer method and the percutaneous endoscopic gastrojejunostomy method. Patient demographics, procedural success, complications, elimination of nasal intubation, and survival were reviewed. RESULTS: Successful placement was achieved in 93% of patients (71/76). Procedure-related complications occurred in 21% ofpatients (15/71), of which the majority involved stomal leakage (eight patients), and wound infection (six patients). There were no procedure-related deaths. Among the 55 patients who required nasal intubation before PEG, a trans-gastrostomy intestinal tube was inserted in 16 patients. The need for further nasal intubation was eliminated in 96% of the patients (53/55). The median survival time was 63 days (range, 8-444 days) after PEG placement. CONCLUSIONS: PEG for bowel decompression in patients with malignant obstruction can be carried out with an acceptable risk of minor complications. In combination with a trans-gastrostomy intestinal tube insertion, the elimination of nasal intubation can be achieved in most patients.

A case of esophageal squamous cell carcinoma accompanied by pancreatic and bile duct metastases: Successful treatment starts with an accurate diagnosis.

ABSTRACT: Pancreatic and bile duct metastases from esophageal cancer are extremely rare. We report a case of advanced esophageal cancer successfully treated with chemotherapy, selected on the basis of an accurate pathologic diagnosis. A 69-year-old man with chronic renal dysfunction presented with persistent abdominal pain and anorexia. Upper gastrointestinal endoscopy revealed an irregular-shaped tumor in the lower esophagus. Computed tomography and ultrasonography revealed swollen para-aortic lymph nodes, a pancreatic mass, and distal bile duct stenosis. Histopathological examination showed that all of the lesions were squamous cell carcinoma with unique immunohistochemical characteristics of p40+ and cytokeratin 7+. The final diagnosis was esophageal squamous cell carcinoma accompanied by lymph node, pancreas, and bile duct metastases. Taking his renal dysfunction into consideration, modified FOLFOX was administered as the first-line chemotherapy. The patient survived for 15 months since his first presentation. The favorable outcome was attributed to the accurate diagnosis based on comprehensive tissue sampling.

Synthesis of modified siRNA bearing C-5 polyamine-substituted pyrimidine nucleoside in their 3'-overhang regions and its RNAi activity.

Short interfering RNA (siRNA) induces specific gene silencing by the RNA interference (RNAi) pathway. Nucleosides in the 3'-overhang regions of siRNAs were replaced with 5-bis(aminoethyl)aminoethylcarbamoylmethyl-2'-deoxyuridine or thymidine. siRNA bearing modified nucleoside was more active in silencing the gene expression of hepatocyte nuclear factor 4α (HNF4α) compared with siRNA bearing thymidine.

Effect of 3'-end capping of aptamer with various 2',4'-bridged nucleotides: Enzymatic post-modification toward a practical use of polyclonal aptamers.

The capping of the 3'-ends of thrombin binding aptamers (TBAs) with bridged nucleotides increased the nuclease resistances and the stabilities in human serum. The binding abilities of the aptamers were not affected by the capping. The capping could be simply executed via a one step enzymatic process using 2',4'-bridged nucleoside 5'-triphosphate and terminal deoxynucleotidyl transferase.

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.

In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2',4'-bridged nucleotides and three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2'-O,4'-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2',4'-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2',4'-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2',4'-bridged nucleotides into extending strands using 2',4'-bridged nucleoside-5'-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.

Study on suitability of KOD DNA polymerase for enzymatic production of artificial nucleic acids using base/sugar modified nucleoside triphosphates.

Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA) which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2'-ONHCH2CH2-4' linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.

Smart conferring of nuclease resistance to DNA by 3'-end protection using 2',4'-bridged nucleoside-5'-triphosphates.

Incorporation of 2',4'-bridged nucleotides into the 3'-end of oligodeoxyribonucleotide (ODN) was examined using terminal deoxynucleotidyl transferase (TdT). The three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures used were incorporated efficiently into the 3'-end of DNA by TdT, although only single nucleotide incorporation was observed. Nuclease resistance was conferred on DNA, depending on the types of bridging nucleotides added.

Transcription and reverse transcription of artificial nucleic acids involving backbone modification by template-directed DNA polymerase reactions.

Oligodeoxyribonucleotides (ODN) where the phosphodiester linkage had been replaced with an amide-type linker [-CH(2)C=ONH-] or an amine-type linker [-CH(2)CH(2)NH-] were synthesized to investigate the effect of these backbone modifications on polymerase reactions. In addition, a triphosphate analogue of thymidine dinucleotide with the amide-type linker was synthesized and enzymatic insertion of the amide linkage into ODN was attempted using this analogue for the polymerase reaction. Primer extension reactions using three types of thermostable DNA polymerases, KOD(exo-), Vent(exo-) and Taq were performed for the assays. Analysis of these data indicate that (i) the polymerase reaction tends to be affected much more by insertion of the cationic flexible amine-type linker than by insertion of the neutral rigid amide-type linker; (ii) the backbone modification has a greater effect on the polymerase reaction when it is adjacent to the 3'-end of a primer as the elongation terminus than when it is on the template, as well as in base or sugar modification; (iii) although the modified linker in the modified DNA template is passed beyond by the polymerase, it still affects the extension reaction several bases downstream from its location; (iv) the modified linker in the template, in some cases, also affects the extension reaction upstream from its location; (v) further improvement of the chemical structure is required for dinucleotide-mimic incorporation.

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides.

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by approximately 20 degrees C) than that of the hairpin (by approximately 10 degrees C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.

Fluorescence-enhanced europium-diethylenetriaminepentaacetic (DTPA)-monoamide complexes for the assessment of renal function.

Real-time, noninvasive assessment of glomerular filtration rate (GFR) is essential not only for monitoring critically ill patients at the bedside, but also for staging and monitoring patients with chronic kidney disease. In our pursuit to develop exogenous luminescent probes for dynamic optical monitoring of GFR, we have prepared and evaluated Eu(3+) complexes of several diethylenetriamine pentaacetate (DTPA)-monoamide ligands bearing molecular "antennae" to enhance metal fluorescence via intramolecular ligand-metal fluorescence resonance energy transfer process. The results show that Eu-DTPA-monoamide complex 18b, which contains a quinoxanlinyl antenna, exhibits large (ca. 2700-fold) Eu(3+) fluorescence enhancement. Indeed, complex 18b exhibits the highest fluorescent enhancement observed thus far in the DTPA-type metal complexes. The renal clearance property was assessed using the corresponding radioactive (111)In complex 18a, and the data suggest that this complex clears via a complex mechanism that includes glomerular filtration.

Acridone-tagged DNA as a new probe for DNA detection by fluorescence resonance energy transfer and for mismatch DNA recognition.

Acridone is highly fluorescent and stable against photodegradation, oxidation, and heat. It is also a small molecule with no charge, making it a promising fluorescent agent for use in a DNA probe. Thus, we have prepared 5'-terminal acridone-labeled DNAs by post-modification, and have examined their photophysical properties and their use as donors for a fluorescence resonance energy transfer (FRET) system in combination with a 3'-terminal dabcyl-tagged DNA as an acceptor, which can detect the target DNA by emission-quenching caused by FRET. The FRET with an acridone and dabcyl pair has been found to complement that with fluorescence and dabcyl and other fluorescence-quencher pairs. Significant amounts of quenching of the acridone emissions by guanine in the DNA were observed when guanine was close to acridone, which can be applied as a quencher-free probe for the detection of special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could discriminate the opposite T-T base mismatch, although enhancement of discrimination ability is needed for the practical use of SNP typing.

Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

Arginine-modified DNA aptamers that show enantioselective recognition of the dicarboxylic acid moiety of glutamic acid.

We have screened glutamic acid-binding aptamers from a modified DNA pool containing arginine residues using the method of systematic evolution of ligands by exponential enrichment (SELEX). Thirty-one modified DNA molecules were obtained from the enriched pool after the 17th round of selection, and their binding affinities for the target were evaluated by binding assays using affinity gels. Three modified DNA molecules having higher affinity were sequenced and we determined their affinity and specificity for the target by surface plasmon resonance (SPR) measurements. The SPR studies indicated that two of these three aptamers distinguished the dicarboxylic acid moiety of the D-isomer from that of the L-isomer; however, the third aptamer did not show enantioselectivity.

Differences in substrate specificity of C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides by DNA polymerases from thermophilic bacteria, archaea, and phages.

The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of T is substituted with a Me group. Miller et al. hypothesized that various C(5)-substituted uracil derivatives were formed during chemical evolution, and that C(5)-substituted U derivatives may have played important roles in the transition from an 'RNA world' to a 'DNA-RNA-protein world'. Hyperthermophilic bacteria and archaea are considered to be primitive organisms that are evolutionarily close to the universal ancestor of all life on earth. Thus, we examined the substrate specificity of several C(5)-substituted or C(5)-unsubstituted dUTP and dCTP analogs for several DNA polymerases from hyperthermophilic bacteria, hyperthermophilic archaea, and viruses during PCR or primer extension reaction. The substrate specificity of the C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides varied greatly depending on the type of DNA polymerase. The significance of this difference in substrate specificity in terms of the origin and evolution of the DNA replication system is discussed briefly.

Clinical Factors of Delayed Perforation after Endoscopic Submucosal Dissection for Gastric Neoplasms.

BACKGROUND: Delayed perforation is a rare but severe complication of endoscopic submucosal dissection (ESD) for early gastric neoplasm (EGN). The aim of this study was to clarify clinical factors related to delayed perforation after ESD. METHODS: A total of 1158 consecutive patients with 1199 EGNs underwent ESD at our hospital between January 2000 and December 2015. Univariate analysis was used to identify clinicopathological factors related to delayed perforation. Moreover, duration of cautery needed for hemostasis was measured by comparison between perforated and nonperforated points in patients with delayed perforation. RESULTS: Delayed perforation occurred in 5 of 1158 consecutive patients with 1199 EGNs who underwent ESD (0.42%). All cases were diagnosed within 24 h after ESD and recovered with conservative management. On univariate analysis, location in the upper stomach was the factor most significantly associated with delayed perforation (P < 0.01). Duration of cautery needed for hemostasis was significantly longer at perforated points (9 s) than at nonperforated points (3.5 s) in five patients. CONCLUSIONS: Location in the upper stomach was the risk factor most prominently associated with delayed perforation after ESD for EGNs. In addition, delayed perforation appears associated with excessive electrocautery for hemostasis.

Molecular therapy of human neuroblastoma cells using Auger electrons of 111In-labeled N-myc antisense oligonucleotides.

UNLABELLED: Auger electrons can create breaks in nucleic acids, giving them possible therapeutic utility. We investigated the therapeutic effect of Auger electrons emitted by 111In-labeled phosphorothioate antisense oligonucleotides on human neuroblastoma cells in which N-myc was overexpressed. METHODS: Human SK-N-DZ neuroblastoma cells (5 x 10(6) cells) were treated with cationic reverse-phase evaporation vesicles (REVs) encapsulating 111In-labeled antisense (40 MBq/2 nmol of oligonucleotides/mumol of total phospholipids) that had an average diameter of 250 nm. Hybridization of the radiolabeled oligonucleotides with N-myc messenger RNA (mRNA), N-myc expression, and cell proliferation were investigated. The tumorigenicity of treated cells was analyzed in nude mice. Nonradiolabeled antisense, 111In-labeled sense, or empty cationic REVs were used as controls. RESULTS: 111In-Labeled antisense, which hybridized with N-myc mRNA, was detected in cells at 12 and 24 h after the initiation of treatment. Reduced N-myc expression and inhibited cell proliferation were shown in the same cells at 48 h after the completion of treatment. N-myc expression-suppressed cells produced intraperitoneal tumors in nude mice, but the average weight of the tumors was lower than that of tumors in control mice. CONCLUSION: Auger electrons emitted from 111In in close proximity to their target N-myc mRNA may prolong the time to cell proliferation in human neuroblastoma cells due to inhibition of the translation of N-myc. Auger electron therapy therefore has potential as an internally delivered molecular radiotherapy targeting the mRNA of a tumor cell.