Ethylene glycol metabolism in the poly(ethylene terephthalate)-degrading bacterium Ideonella sakaiensis.

Poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis produces hydrolytic enzymes that convert PET, via mono(2-hydroxyethyl) terephthalate (MHET), into the monomeric compounds, terephthalic acid (TPA), and ethylene glycol (EG). Understanding PET metabolism is critical if this bacterium is to be engineered for bioremediation and biorecycling. TPA uptake and catabolism in I. sakaiensis have previously been studied, but EG metabolism remains largely unexplored despite its importance. First, we identified two alcohol dehydrogenases (IsPedE and IsPedH) and one aldehyde dehydrogenase (IsPedI) in I. sakaiensis as the homologs of EG metabolic enzymes in Pseudomonas putida KT2440. IsPedE and IsPedH exhibited EG dehydrogenase activities with Ca2+ and a rare earth element (REE) Pr3+, respectively. We further found an upregulated dehydrogenase gene when the bacterium was grown on EG, whose gene product (IsXoxF) displays a minor EG dehydrogenase activity with Pr3+. IsPedE displayed a similar level of activity toward various alcohols. In contrast, IsPedH was more active toward small alcohols, whereas IsXoxF was the opposite. Structural analysis with homology models revealed that IsXoxF had a larger catalytic pocket than IsPedE and IsPedH, which could accommodate relatively bulkier substrates. Pr3+ regulated the protein expression of IsPedE negatively; IsPedH and IsXoxF were positively regulated. Taken together, these results indicated that the combination of IsPedH and IsXoxF complements the function of IsPedE in the presence of REEs. IsPedI exhibited dehydrogenase activity toward various aldehydes with the highest activity toward glycolaldehyde. This study demonstrated a unique alcohol oxidation pathway of I. sakaiensis, which could be efficient in EG utilization. KEY POINTS: • IsPedH and IsXoxF complement IsPedE function in the presence of REEs. • IsPedI displayed the highest dehydrogenase activity toward glycolaldehyde. • Unique alcohol oxidation pathway of I. sakaiensis identified for EG utilization.

A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea.IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.

A case of synchronous advanced gastric cancer and locally advanced prostate cancer with combined laparoscopic and robotic surgery: A case report.

INTRODUCTION: The optimal management strategy for synchronous gastric cancer (GC) and prostate cancer (PCa) remains unclear, particularly in cases in which two cancers are progressive. PRESENTATION OF CASE: A 68-year-old man diagnosed with synchronous advanced GC and locally advanced PCa was referred to our institution. Laparoscopic total gastrectomy (LTG) and robotic-assisted radical prostatectomy were simultaneously performed. The postoperative course was similar to the standard postoperative course of LTG alone. Pathological diagnoses were T3N3aM0 gastric adenocarcinoma and T3N0M0 prostatic adenocarcinoma. Adjuvant chemotherapy and adjuvant androgen deprivation therapy (ADT) for GC and PCa were initiated on postoperative days 15 and 27, respectively. Six months subsequent to surgery, the patient received adjuvant chemotherapy and ADT, and no evidence of cancer recurrence was observed. DISCUSSION: In terms of survival, curative resection with adjuvant therapy is advantageous for patients with advanced GC or locally advanced PCa. At present, treatment for synchronous cancer should be combined with optimal management for individual cancers. Minimally invasive surgery may play an important role in the multidisciplinary treatment of synchronous advanced cancer. CONCLUSION: Combined laparoscopic and robotic surgery for synchronous GC and PCa allows for minimally invasive radical resection and appropriate adjuvant therapy.

A rare case of for synchronous advanced cancer of ascending colon and urinary bladder with simultaneous laparoscopic resection: A case report.

INTRODUCTION: Advances in diagnostic techniques have resulted in an increase in the diagnosis of numerous patients with multiple primary cancers. However, the diagnosis of synchronous primary colorectal cancer and bladder cancer remains rare. PRESENTATION OF CASE: A 69-year-old man diagnosed with synchronous advanced cancer of the ascending colon and urinary bladder underwent simultaneous laparoscopic resection. His postoperative course was similar to that of routine colorectal cancer. The patient refused adjuvant therapy. The patient was diagnosed as having recurrence of bladder cancer 3 months after surgery; he died 9 months after surgery. DISCUSSION: For multiple primary malignant tumors, simultaneous tumor resection is preferred. Simultaneous laparoscopic resection may be proposed for postoperative multidisciplinary treatment. If an established regimen is determined in the future, neoadjuvant chemotherapy may be an option for the treatment of synchronous advanced cancer. CONCLUSION: Simultaneous laparoscopic surgery is a greatly beneficial approach for synchronous cancer requiring multidisciplinary treatment. Additionally, an appropriate support system for patients is indispensable for completing multidisciplinary treatment.

Combined laparoscopic and robotic surgery for synchronous colorectal and genitourinary cancer: A case series.

INTRODUCTION: Advances in diagnostic techniques and treatment have resulted in an increase in patients with synchronous cancer. Surgical reports of combined laparoscopic and robotic resection for synchronous colorectal and genitourinary cancer are rare. MATERIALS AND METHODS: Between August 2015 and November 2017, three patients underwent combined laparoscopic and robotic surgery for synchronous colorectal and genitourinary cancer in our hospital. RESULTS: Case 1 was a 59-year-old man with synchronous rectal and prostate cancer treated by combined laparoscopic anterior resection and robotic-assisted prostatectomy. Case 2 was a 77-year-old man with synchronous cancer of transverse colon and left kidney treated by combined laparoscopic transverse colectomy and robotic-assisted partial nephrectomy. Case 3 was a 74-year-old man with synchronous adenocarcinoma of descending colon and prostate treated by combined laparoscopic left hemicolectomy and robotic-assisted prostatectomy. DISCUSSION: In simultaneous endoscopic surgery, it is necessary to consider sequence of resection, intraoperative position of patient and port arrangement. Simultaneous surgery allows promptly for postoperative adjuvant chemotherapy. CONCLUSION: Combined laparoscopic and robotic surgery for synchronous colorectal and genitourinary cancer is suitable for advanced cancer cases requiring multidisciplinary treatment.

Evaluation of a rapid serological test for leprosy classification using human serum albumin as the antigen carrier.

The presence of anti-BSA antibodies may interfere in serological tests, as ELISA or immunochromatographic assays. BSA is frequently used as a blocking agent or as "inert" carrier of antigens, such as the NT-P-BSA, the semi-synthetic trisaccharide analogue of the PGL-I (phenolic glycolipid-I) antigen from the cell wall of the Mycobacterium leprae. PGL-I was prepared and linked to human serum albumin based in the hypothesis that replacing BSA by a human protein carrier would enhance the performance of leprosy serological tests. A total of 1162 serum samples were tested by ELISA and by the ML Flow rapid test using NT-P-BSA or NT-P-HSA antigens. When grouping leprosy patients as paucibacillary (PB) or multibacillary (MB) according to the Ridley & Jopling classification, ML Flow BSA and ML Flow HSA tests correctly allocated 70.9% and 68.6% of patients in the PB group, and 87% and 81% of patients in the MB group, respectively. Concordant results were found in 82.0% (953/1162) (kappa value=0.637; sd=0.023) of samples between ML Flow tests and 85.7% (996/1162) (kappa value=0.703; sd=0.021) between ELISA tests. ML Flow results were statistically similar and the same was true for ELISA tests using HSA or BSA. However, we noticed a tendency to decreased capacity to detect MB patients and an increased positivity among PB patients, HHC, TB patients and healthy controls by the HSA carrier in both ML Flow and ELISA. The PGL-I serology performed by the ML Flow test with BSA or HSA as antigen carriers can be a useful, friendly auxiliary tool to identify patients with higher bacterial load.

Serological diagnosis of leprosy in patients in vietnam by enzyme-linked immunosorbent assay with Mycobacterium leprae-derived major membrane protein II.

A serological diagnostic test using phenolic glycolipid-I (PGL-I) developed in the 1980s is commercially available, but the method is still inefficient in detecting all forms of leprosy. Therefore, more-specific and -reliable serological methods have been sought. We have characterized major membrane protein II (MMP-II) as a candidate protein for a new serological antigen. In this study, we evaluated the effectiveness of the enzyme-linked immunosorbent assay (ELISA) using the MMP-II antigen (MMP-II ELISA) for detecting antibodies in leprosy patients and patients' contacts in the mid-region of Vietnam and compared to the results to those for the PGL-I method (PGL-I ELISA). The results showed that 85% of multibacillary patients and 48% of paucibacillary patients were positive by MMP-II ELISA. Comparison between the serological tests showed that positivity rates for leprosy patients were higher with MMP-II ELISA than with PGL-I ELISA. Household contacts (HHCs) showed low positivity rates, but medical staff members showed comparatively high positivity rates, with MMP-II ELISA. Furthermore, monitoring of results for leprosy patients and HHCs showed that MMP-II is a better index marker than PGL-I. Overall, the epidemiological study conducted in Vietnam suggests that serological testing with MMP-II would be beneficial in detecting leprosy.

Distamycin A enhances the cytotoxicity of duocarmycin A and suppresses duocarmycin A-induced apoptosis in human lung carcinoma cells.

Duocarmycin A (Duo), which is one of well-known antitumor antibiotics, efficiently alkylates adenine N3 at the 3' end of AT-rich sequences in the DNA. The addition of a minor groove binder, distamycin A (Dist), not only accerelates the reactivity of Duo with oligonucleotide duplex but also switches the DNA-alkylation site to guanine in GC-rich sequences. Here we examined cytotoxic effect of Duo in the coexistence of Dist using human lung carcinoma (HLC-2) cells. The cytotoxicity of Duo to HLC-2 cells was enhanced 10 times by the addition of 0.5microg/ml Dist, which was much lower than the IC(50) value of 16microg/ml. Addition of Duo alone to HLC-2 cells resulted in typically apoptotic changes, including chromatin condensation, sub-G1 accumulation in DNA histogram pattern, and decrease in procaspase-3 and 9 levels. Interestingly, these apoptotic characteristics in Duo-treated cells were suppressed by the addition of 0.5microg/ml Dist, and the G2/M population in the cell cycle progression of HLC-2 cells was largely unchanged in the coexistence of Dist along with the extremely low accumulation of p53 and higher induction of p21. In contrast, the treatment of HLC-2 cells with Dist (16microg/ml) alone was observed to induce the accumulation of p53 and cell cycle arrest at the G1 phase. These results indicate that Dist suppresses apoptosis induced by Duo as well as enhances Duo-induced cytotoxicity in living cells, and may contribute to chemotherapy for tumors resistant to inducing apoptotic cell death.

An unnatural base pair system for in vitro replication and transcription.

The development of unnatural base pairs that function in replication, transcription, and translation could expand the genetic alphabet and enable the site-specific incorporation of functional components into nucleic acids and proteins. We present an unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (denoted by Ds) and pyrrole-2-carbaldehyde (denoted by Pa). In replication, the Ds-Pa pair exhibits high selectivity in combination with the usual and modified triphosphate substrates and exonuclease-proficient DNA polymerases. In transcription, the Ds-Pa pair mediates the site-specific incorporation of the substrates of both Ds and Pa into RNA by T7 RNA polymerase. This unnatural base pair system could facilitate the specific incorporation of functional components into RNA molecules at desired positions using DNA templates containing the unnatural base pair, which can be amplified by PCR.

An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA.

Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions. In replication, this unnatural base pair exhibits high selectivity in combination with the usual triphosphates and modified triphosphates, gamma-amidotriphosphates, as substrates of 3' to 5' exonuclease-proficient DNA polymerases, allowing PCR amplification. In transcription, the unnatural base pair complementarity mediates the incorporation of these base substrates and their analogs, such as a biotinylated substrate, into RNA by T7 RNA polymerase (RNAP). With this system, functional components can be site-specifically incorporated into a large RNA molecule.

A two-unnatural-base-pair system toward the expansion of the genetic code.

Toward the site-specific incorporation of amino acid analogues into proteins, a two-unnatural-base-pair system was developed for coupled transcription-translation systems with the expanded genetic code. A previously designed unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was used for the site-specific incorporation of yTP into RNA opposite s in templates by T7 RNA polymerase. For the site-specific incorporation of sTP into RNA, a newly developed unnatural base, imidazolin-2-one (denoted by z), is superior to y as a template base for pairing with s in T7 transcription. The combination of the s-y and s-z pairs provides a powerful tool to prepare both y-containing mRNA and s-containing tRNA for efficient coupled transcription-translation systems, in which the genetic code is expanded by the codon-anticodon interactions mediated by the s-y pair. In addition, the nucleoside of s is strongly fluorescent, and thus the s-z pair enables the site-specific fluorescent labeling of RNA molecules. These unnatural-base-pair studies provide valuable information for understanding the mechanisms of replication and transcription.

Unnatural base pairs between 2- and 6-substituted purines and 2-oxo(1H)pyridine for expansion of the genetic alphabet.

An unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y) shows high selectivity in transcription and translation. Toward the further development of unnatural base pairs that also have exclusive selectivity in replication, we examined the roles of the 2-amino and 6-thienyl groups of s using base pairs between y and purine-analogs, 6-thienylpurine and 2-amino-6-furanylpurine, as well as s. The results obtained from the thermal stability and DNA polymerase single-nucleotide insertion experiments suggest that the 2-amino group of s contributes toward the shape complementarity of the pairing with y, rather than the hydrogen bonding with the 2-keto group of y. In addition, the bulkiness of positions 2 and 6 of the unnatural purines cooperatively determines the selectivity of the noncanonical pairing with y or the natural pyrimidines in replication. This information is useful not only for the development of unnatural, orthogonal base pairs, but also for understanding the mechanisms of base pair formation in replication.

Observation of electrostatically released DNA from gold electrodes with controlled threshold voltages.

DNA oligo-nucleotides, localized at Au metal electrodes in aqueous solution, are found to be released when applying a negative bias voltage to the electrode. The release was confirmed by monitoring the intensity of the fluorescence of cyanine dyes (Cy3) linked to the 5' end of the DNA. The threshold voltage of the release changes depending on the kind of linker added to the DNA 3'-terminal. The amount of released DNA depends on the duration of the voltage pulse. Using this technique, we can retain DNA at Au electrodes or Au needles, and release the desired amount of DNA at a precise location in a target. The results suggest that DNA injection into living cells is possible with this method.

Excessive counterion condensation on immobilized ssDNA in solutions of high ionic strength.

We present experiments on the bias-induced release of immobilized, single-stranded (ss) 24-mer oligonucleotides from Au-surfaces into electrolyte solutions of varying ionic strength. Desorption is evidenced by fluorescence measurements of dye-labeled ssDNA. Electrostatic interactions between adsorbed ssDNA and the Au-surface are investigated with respect to 1), a variation of the bias potential applied to the Au-electrode; and 2), the screening effect of the electrolyte solution. For the latter, the concentration of monovalent salt in solution is varied from 3 to 1600 mM. We find that the strength of electric interaction is predominantly determined by the effective charge of the ssDNA itself and that the release of DNA mainly occurs before the electrochemical double layer has been established at the electrolyte/Au interface. In agreement with Manning's condensation theory, the measured desorption efficiency (etarel) stays constant over a wide range of salt concentrations; however, as the Debye length is reduced below a value comparable to the axial charge spacing of the DNA, etarel decreases substantially. We assign this effect to excessive counterion condensation on the DNA in solutions of high ionic strength. In addition, the relative translational diffusion coefficient of ssDNA in solution is evaluated for different salt concentrations.

An unnatural base pair for incorporating amino acid analogs into proteins.

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

An unnatural base pair between imidazolin-2-one and 2-amino-6-(2-thienyl)purine in replication and transcription.

Nucleosides of imidazolin-2-one (designated by z) were designed and synthesized as pairing partners of 2-amino-6-(2-thienyl)purine (designated by s). Previously, we developed an unnatural base pair between s and pyridine-2-one (designated by y), and polymerases specifically incorporated the substrate of y into DNA and RNA opposite s in templates. Although s was efficiently incorporated opposite y, A was also incorporated opposite y with high efficiency. The replacement of y by z effectively improved the incorporation selectivity of s. The incorporation efficiency of A opposite z decreased, but the efficient incorporation of s opposite z was maintained as compared to that of the s-y pairing.

Sequence-Specific DNA Alkylation by Hybrid Molecules between Segment A of Duocarmycin A and Pyrrole/Imidazole Diamide.

In the absence of distamycin A (Dist), hybrids 1 (X=N, CH) selectively alkylate the 3' end of adenine in AT-rich DNA sequences. However, these hybrids can form a heterodimer with Dist to alkylate G residues of predetermined DNA sequences efficiently and with high selectivity.