Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA.

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000 bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.

Observation of the Mott insulator to superfluid crossover of a driven-dissipative Bose-Hubbard system.

Dissipation is ubiquitous in nature and plays a crucial role in quantum systems such as causing decoherence of quantum states. Recently, much attention has been paid to an intriguing possibility of dissipation as an efficient tool for the preparation and manipulation of quantum states. We report the realization of successful demonstration of a novel role of dissipation in a quantum phase transition using cold atoms. We realize an engineered dissipative Bose-Hubbard system by introducing a controllable strength of two-body inelastic collision via photoassociation for ultracold bosons in a three-dimensional optical lattice. In the dynamics subjected to a slow ramp-down of the optical lattice, we find that strong on-site dissipation favors the Mott insulating state: The melting of the Mott insulator is delayed, and the growth of the phase coherence is suppressed. The controllability of the dissipation is highlighted by quenching the dissipation, providing a novel method for investigating a quantum many-body state and its nonequilibrium dynamics.

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA.

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.

Percutaneous endoscopic gastrostomy with Funada-style gastropexy greatly reduces the risk of peristomal infection.

BACKGROUND AND AIMS: Peristomal wound infections are common complications of percutaneous endoscopic gastrostomy (PEG). The Funada-style gastropexy device has two parallel needles with a wire loop and suture thread, and was developed about 20 years ago in Japan. This kit has allowed us to perform dual gastropexy very easily; PEG with gastropexy has become a very popular technique in Japan. The present study aimed to compare the advantages and disadvantages of PEG with the gastropexy technique with the standard 'pull' method. METHODS: We retrospectively reviewed 182 consecutive, non-randomized patients undergoing PEG in our hospital, and a comparative analysis was made between the gastropexy (87 patients) and non-gastropexy (95 patients) groups. RESULTS: The rates of patients having erythema (11.6% vs. 47.9%; P < 0.001), exudates (2.3% vs. 14.9%; P < 0.01) and infection (0% vs. 6.4%; P = 0.01) in the peristomal area were lower in the gastropexy than in the non-gastropexy group. The rate of minor bleeding from the peristomal area was higher in the gastropexy than in the non-gastropexy group (12.8% vs. 2.1%; P < 0.01), but no patient required a blood transfusion. Mean procedure time was longer in the gastropexy group than in the non-gastropexy group (31 vs. 24 min; P < 0.001). The 30-day mortality rates were 4.7% and 5.3% respectively, and these deaths were not related to the gastrostomy procedure. CONCLUSION: PEG with gastropexy markedly reduces peristomal inflammation. Although minor bleeding and a longer procedure time were disadvantages, there were no severe complications. The findings suggested that PEG with Funada-style gastropexy was a safe and feasible method for reducing early complications of PEG.

Primary leiomyosarcoma of the colon.

Primary leiomyosarcomas of the gastrointestinal (GI) tract are extremely rare and highly aggressive neoplasms, and only a small number of true cases have been reported since the concept of GI stromal tumors was established. Here, we report a case of a primary leiomyosarcoma of the transverse colon. A 46-year-old Japanese male with a large mass in the right upper abdomen was admitted to our hospital. Computed tomography and magnetic resonance imaging revealed long segments of wall thickening of the transverse colon with large consecutive tumors measuring 12 cm in diameter. A projecting irregular mass with marked mucosal necrosis was found on colonoscopy. Pathological examination revealed a spindle cell tumor growing circumferentially and transmurally to replace the muscularis propria in the transverse colon. The spindle cells were positive for smooth muscle actin, and negative for KIT, CD34, DOG-1, and S-100 protein. The patient has shown repeat recurrence in spite of sufficient surgical excision being promptly performed.

Gene manipulation using artificial restriction DNA cutter.

Artificial restriction DNA cutter (ARCUT), which we developed recently, was used for manipulation of plasmid DNA. PCR product was inserted into pBR322 plasmid vector using ARCUT, and E. coli cells were transfected with this recombinant plasmid DNA. Successful growth of cells shows that the recombination proceeds without any unexpected mutations. Furthermore, this artificial system was applied to PCR-free construction of chimera protein.

Manipulation of double-stranded DNA by artificial restriction enzyme composed of Ce(IV)/EDTA and PNA.

Through the invasion of pseudo-complementary PNA (pePNA) to double-stranded DNA, gap-like structures were formed at predetermined sites in both strands of PBR322 plasmid DNA. These gap-like sites were selectively hydrolyzed by Ce(IV)/EDTA complex, and two designed fragments were obtained. Furthermore, the scission fragment by this artificial restriction enzyme was successfully ligated with foreign DNA.

PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection.

By the combination of peptide nucleic acid (PNA) with single-stranded DNA specific nucleases, alteration of a single base to another in DNA has been detected with high accuracy. Only the DNAs in DNA/PNA duplexes involving a mismatch are efficiently hydrolyzed by these enzymes, whereas fully matching sequences are kept intact. This difference is visually scored by adding 3,3'-diethylthiadicarbocyanine, which changes its color from blue to purple upon binding to DNA/PNA duplexes. These findings are applied to the convenient and straightforward detection of single nucleotide polymorphisms (SNPs). When the target site in the sample DNA is completely complementary with the PNA, a notable amount of DNA/PNA duplex remains and thus the solution exhibits purple color. In the presence of even one mismatch between PNA and DNA, however, the DNA is completely digested by the enzyme and therefore the dye shows its intrinsic blue color. The SNPs in the apolipoprotein E gene of human DNA have been successfully genotyped by this method.

Simultaneous detection of multiple single nucleotide polymorphism by single-strand-specific nuclease and PNA probe.

The combination of PNA (peptide nucleic acid) and single-strand-specific nuclease have been used for detection of single nucleotide polymorphisms (SNPs). When DNA is perfectly complementary to PNA, it is protected from digestion by the nuclease. If there exists a single-base mismatch between them, however, the DNA is completely digested. These differences are visualized by using 3,3'-diethylthiadicarbocyanine (DiSc2(5)), which changes its color from blue to purple upon binding to PNA/DNA hybrids. In terms of this methodology, homozygous and heterozygous SNPs in apoE gene have been successfully analyzed. Furthermore, the multiplex SNPs are simultaneously genotyped. This technique provides a simple, straightforward, facile, and visual genetic screening, with no need for expensive and complicated equipment.