Structure-dependent detection of polyphenols using crown ether-immobilized gold nanoparticles.

Gold nanoparticles functionalized with 18-crown 6-ether (18C6-AuNPs) can be used for detection of tannic acid, epigallocatechin gallate, and epicatechin gallate by color change in the µg mL-1 range. 18C6-AuNPs were insensitive to l-ascorbic acid and l-tyrosine unlike conventional detection methods, such as Folin & Ciocalteu assay, whose principle is based on the redox reaction of polyphenols. Although 18C6-AuNPs did not respond to some polyphenols, such as gallic acid and epicatechin, if the polyphenols of interest are responsive to this approach, these are expected to be effective nanomaterial for simple sensing of polyphenols.

Relapsed urachal carcinoma responding to first-line chemotherapy with capecitabine-oxaliplatin plus bevacizumab.

Introduction: Advanced urachal carcinoma has a poor prognosis; however, a standard systemic treatment has not been established. We treated a patient with relapsed urachal carcinoma with capecitabine-oxaliplatin plus bevacizumab, a standard regimen for colon cancer, and obtained favorable responses. Case presentation: A 47-year-old woman presented with hematuria. Under the diagnosis of non-metastatic urachal carcinoma, an extended partial cystectomy was performed. Histopathological examination revealed adenocarcinoma with negative surgical margins and lymph nodes. Thirty-two months postoperatively, lung metastases and local recurrence were confirmed, along with elevated carcinoembryonic antigen levels, and nine chemotherapy cycles were administered. Subsequently, the recurrent lesion regressed, and tumor marker levels normalized. Fourteen months after treatment discontinuation, the disease remained stable without progression. Conclusion: This is the first report of advanced urachal carcinoma treated with capecitabine-oxaliplatin plus bevacizumab, demonstrating the potential of this treatment as first-line chemotherapy for this disease.

Introduction of pseudo-base benzimidazole derivatives into nucleosides via base exchange by a nucleoside metabolic enzyme.

In alternate organic synthesis, biocatalysis using enzymes provides a more stereoselective and cost-effective approach. Synthesis of unnatural nucleosides by nucleoside base exchange reactions using nucleoside-metabolizing enzymes has previously shown that the 5-position recognition of pyrimidine bases on nucleoside substrates is loose and can be used to introduce functional molecules into pyrimidine nucleosides. Here we explored the incorporation of purine pseudo bases into nucleosides by the base exchange reaction of pyrimidine nucleoside phosphorylase (PyNP), demonstrating that an imidazole five-membered ring is an essential structure for the reaction. In the case of benzimidazole, the base exchange proceeded to give the deoxyribose form in 96 % yield, and the ribose form in 23 % yield. The reaction also proceeded with 1H-imidazo[4,5-b]phenazine, a benzimidazole analogue with an additional ring, although the yield of nucleoside was only 31 %. Docking simulations between 1H and imidazo[4,5-b]phenazine nucleoside and the active site of PyNP (PDB 1BRW) supported our observation that 1H-imidazo[4,5-b]phenazine can be used as a substrate by PyNP. Thus, the enzymatic substitution reaction using PyNP can be used to incorporate many purine pseudo bases and benzimidazole derivatives with various functional groups into nucleoside structures, which have potential utility as diagnostic or therapeutic agents.

Reagentless Sensing of Vancomycin Using an Indium Tin Oxide Electrode Grafted with Molecularly Imprinted Polymer including Ferrocenyl Group.

Vancomycin (VCM) is a first-line antimicrobial agent against methicillin-resistant Staphylococcus aureus, a cause of nosocomial infections. Therapeutic drug monitoring is strongly recommended for VCM-based chemotherapy. The authors attempted to develop a simple VCM sensor based on molecularly imprinted polymer (MIP), which can be used with simple operations. Methacrylic acid (MAA), acrylamide, methylenebisacrylamide, and allylamine carboxypropionate-3-ferrocene (ACPF) were copolymerized in the presence of VCM and grafted from the surface of indium-tin oxide (ITO) to obtain MIP-coated electrodes. The MIP-grafted ITO electrode was used for differential pulse voltammetry (DPV) measurements in a buffer solution containing VCM or whole bovine blood. The obtained current depends on the VCM concentration with high linearity. The dynamic range covered the therapeutic range (20-40 µg/mL) of the VCM but was almost insensitive to teicoplanin, which has a similar structure to VCM. The ITO electrodes grafted by the same procedure except for omitting either VCM or APCF were not sensitive to VCM. The sensitivity of the MIP electrodes to VCM in whole blood and buffered saline, but the background current in blood was higher than that in saline. This high background current was also seen in the deproteinized plasma. Thus, the current is probably originated from the oxidation of low molecular weight reducing agents in the blood. The MIP-grafted ITO electrode using ACPF as a functional monomer would be a promising highly selective sensor for real-time monitoring of VCM with proper correction of the background current.

Significance of PSA Screening in Niigata, Japan: Survey of Actual Status of New Cases of Prostate Cancer.

PURPOSE: This study aims to investigate the utility of prostate-specific antigen (PSA) screening by conducting an all-case survey of newly diagnosed prostate cancer patients at Niigata Prefecture, Japan. PATIENTS AND METHODS: Depending on whether patients were subjected to screening, information was prospectively collected on all prostate cancer patients newly diagnosed between October 1, 2019, and September 30, 2020, at all institutions in Niigata Prefecture where urologists performing prostate biopsy routinely work and differences in clinical parameters were investigated. RESULTS: PSA was measured in 478 out of 1332 patients (35.8%) as part of a community health screening. The rate of metastatic carcinoma (M1) in all patients was 14.9%. When patients were divided into three categories of population-based screening (community health screening and workplace health screening), opportunistic screening (PSA measurements at complete medical check-ups or on patient request), and testing triggered by clinical symptoms or findings, the proportion of metastatic cancer was 4.5%, 3.7%, and 30.6%, respectively, demonstrating that the number of distant metastases was significantly lesser in all patients who underwent screening. CONCLUSION: The one-year all-case survey of newly diagnosed prostate cancer patients demonstrated that PSA screening significantly contributed to the early diagnosis of current prostate cancer in Japan.

A "Single-Use" Ceramic-Based Electrochemical Sensor Chip Using Molecularly Imprinted Carbon Paste Electrode.

An inexpensive disposable electrochemical drug sensor for the detection of drugs (vancomycin, meropenem, theophylline, and phenobarbital) is described. Molecularly imprinted polymer (MIP) templated with the target drugs was immobilized on the surface of graphite particles using a simple radical polymerization method and packed into the working electrode of a three-electrode ceramic-based chip sensor. Differential pulse voltammetry (DPV) was used to determine the relationship between the response current and the concentration of the targeted drug while using one sensor chip for one single operation. The time required for each DPV measurement was less than 2 min. Concentrations corresponding to the therapeutic range of these drugs in plasma were taken into account while performing DPV. In all the cases, the single-used MIP sensor showed higher sensitivity and linearity than non-imprinted polymer. The selectivity test in drugs with a structure similar to that of the target drugs was performed, and it was found that MIP-based sensors were more selective than the untreated ones. Additionally, the test in whole blood showed that the presence of interfering species had an insignificant effect on the diagnostic responses of the sensor. These results demonstrate that the disposable MIP-sensor is promising for quick and straightforward therapeutic drug monitoring to prevent the toxic side effects and the insufficient therapeutic effect due to the overdose and underdose, respectively.

Parallel motif triplex formation via a new, bi-directional hydrogen bonding pattern incorporating a synthetic cyanuryl nucleoside into the sense chain.

This research presents the first example of the formation of a triplex via hydrogen bonding of a synthetic cyanuryl nucleoside (Cn) composed of a 6-membered ring compound (triazine-2,4,6-trione) which functions as a pyrimidine base. The Cn was able to form the triad via hydrogen bonding in two directions with two adenines, one in the antisense and one in the parallel chain. The thermal stability of the duplex between the antisense and sense chains was evaluated via its UV melting temperature. The melting curves of triplexes possessing the cyanuryl nucleoside (sense chain) and two adenines (antisense and parallel chains) were biphasic. To prove the formation of hydrogen bonding between the cyanuryl nucleoside and the adenine base toward the major groove, structural analysis via NMR was undertaken. A cyanuryl nucleoside containing three 15N in triazine-2,4,6-trione was synthesized first, and then the 15N cyanuryl nucleoside was incorporated into target sequences. The triplex containing the A·Cn[double bond, length as m-dash]A triad was analyzed via 1H NMR, coupled and decoupled with 15N. This triad has two imino proton signals derived from the cyanuryl nucleoside, split according to the 15N coupling condition, at low field. These results are evidence of the formation of hydrogen bonds between the Cn and adenosine. The solution structure of the triplex was analyzed via NOE information from the imino proton. The cyanuryl nucleoside-containing triplex forms a right-handed helical structure similar to natural triplex DNA, albeit DNA having an enhanced pyrimidine analog in the sense chain capable of bidirectional hydrogen bonding with high sequence selectivity.

Use of a deoxynojirimycin-fluorophore conjugate as a cell-specific imaging probe targeting α-glucosidase on cell membranes.

Molecules designed for cell-specific imaging were studied, taking advantage of an enzyme-inhibitor interaction. 1-Deoxynojirimycin (DNJ) can be actively captured by cells which express the surface membrane protein α-glucosidase. New probes composed of DNJ for recognition linked to a fluorophore signal portion were prepared (DNJ-CF31, DNJ-Dans 2 and DNJ-DEAC 3). Docking simulations revealed that the inhibitors acarbose and miglitol and the inhibitor portion of the probes bind at the same position in the pocket of α-glucosidase (human-derived PDB: 3TON). The ability of probes 1-3 to detect the difference between HeLa cells (from human cervical cancer tissue), Neuro-2a cells (from a mouse neuroblastoma C1300 tumor), N1E-115 cells (from a mouse brain neuroblastoma C1300 tumor), A1 cells (from the astrocyte of a newborn mouse brain), and Caco-2 cells (from a human colon carcinoma) was evaluated, and cell-specific fluorescence imaging was possible for conjugate probes 1 and 2. Caco-2 cells treated with probes 1 and 2 showed blue and green fluorescence, respectively, from the cell membrane, and did not stain the Caco-2 cells inside. These results show that DNJ-CF31 and DNJ-Dans 2 recognize an α-glucosidase protein on the surface of Caco-2 cells. Probes 1 and 2 did not stain any part of the other cells. This cell-specific imaging strategy is applicable for a variety of therapeutic agents for many diseases.

Synthesis and characterization of novel, conjugated, fluorescent DNJ derivatives for α-glucosidase recognition.

A series of five new fluorescent deoxynojirimycin (DNJ) conjugates were synthesized and evaluated for their inhibitory effect (IC50) on several α- and ß-glucosidases. Three of the conjugates showed enhanced activity. The two synthetic conjugates, DNJ-CF31 and DNJ-Me 2, exhibited improved α-glucosidase inhibitory effects compared to DNJ and miglitol. Interestingly, conjugates 1 and 2 showed strong inhibition of almond-derived ß-glucosidase, in contrast to the inhibition tendencies of other inhibitors. Conjugate 5 strongly inhibited rat intestinal maltase, even at 0.10µM. A docking study indicated that all five conjugates bind to the active site of α-glucosidase (PDB: 3L4V, derived from Homo sapiens). The DNJ portion of the conjugate fits into the cavity of the enzyme, and the fluorescent part locates randomly on the outside surface. Thus, it is likely that these conjugates can specifically recognize intestinal cells, specifically the α-glucosidase on cell membranes.

Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety.

Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells.

One-pot approach to functional nucleosides possessing a fluorescent group using nucleobase-exchange reaction by thymidine phosphorylase.

Herein, we describe ß-selective coupling between a modified uracil and a deoxyribose to produce functionalized nucleosides catalyzed by thymidine phosphorylase derived from Escherichia coli. This enzyme mediates nucleobase-exchange reactions to convert unnatural nucleosides possessing a large functional group such as a fluorescent molecule, coumarin or pyrene, linked via an alkyl chain at the C5 position of uracil. 5-(Coumarin-7-oxyhex-5-yn)uracil (C4U) displayed 57.2% conversion at 40% DMSO concentration in 1.0 mM phosphate buffer pH 6.8 to transfer thymidine to an unnatural nucleoside with C4U as the base. In the case of using 5-(pyren-1-methyloxyhex-5-yn)uracil (P4U) as the substrate, TP also could catalyse the reaction to generate a product with a very large functional group at 50% DMSO concentration (21.6% conversion). We carried out docking simulations using MF myPrest for the modified uracil bound to the active site of TP. The uracil moiety of the substrate binds to the active site of TP, with the fluorescent moiety linked to the C5 position of the nucleobase located outside the surface of the enzyme. As a consequence, the bulky fluorescent moiety binding to uracil has little influence on the coupling reaction.

Solution structure of S-DNA formed by covalent base pairing involving a disulfide bond.

Here, we present the solution structure of a DNA duplex containing a disulfide base pair (S-DNA). The unnatural nucleoside "S" possessing a thiophenyl group as base was incorporated into a self-complementary singled-stranded oligonucleotide. Crosslinking of the disulfide base pair was analyzed by non-denaturing polyacrylamide gel electrophoresis. Under oxidizing conditions a high molecular weight band as 18 mer, corresponding to the double-stranded molecule (5'-GCGASTCGC: 3'-CGCTSAGCG), was found, whereas single-stranded self-complementary 9 mer oligonucleotide GCGASTCGC was detected in the presence of a reducing agent. These results suggest that the oligonucleotide is covalently linked by disulfide bonding under oxidizing conditions, which can be reversibly reduced to two thiol groups under reducing conditions. CD spectrum of S-DNA (CGASTCG) under oxidizing conditions suggested that the duplex had a right-handed double-stranded structure similar to that of natural DNA (B-form, CGATCG). NMR studies confirmed that this CGASTCG resembled natural B-DNA and that the two phenyl rings derived from the disulfide base pairing intercalated into the duplex. However, these two phenyl rings were not positioned in the same plane as the other base pairs. Specifically, NOEs suggest that although CGASTCG adopts a structure similar to B-type DNA, the S-DNA duplex is bent at the point of disulfide base pairing to face the major groove.

Inhibitory effects on microbial growth using the derivatives of benzyl phenyl sulfide.

We investigated the antimicrobial activities of twelve derivatives of benzyl phenyl sulfide by using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values against 10 microbial strains. These derivatives of benzyl phenyl sulfides were synthesized by means of the nucleophilic coupling reaction at our laboratory. MIC testing revealed that all synthetic derivatives of benzyl and 4-methoxybenzyl phenyl sulfides had no effect against the tested microbial strains. However, the compounds of 4-nitrobenzyl phenyl sulfide showed antimicrobial activity against many of the tested strains. Above all, 4-nitrobenzyl 4-chlorophenyl sulfide 11 exhibited the strongest and widest ranging inhibitory effects among the twelve synthetic compounds. We researched the antimicrobial activities of the coupling materials of sulfide. As a result, it was considered important for the expression of antimicrobial activities that the sulfide had a 4-nitrobenzyl group and 4-chlorophenyl group in the same molecule as in the case of benzyl phenyl sulfide.

[A randomized controlled study comparing clinical effects of naftopidil and tamsulosin on benign prostatic hyperplasia].

A prospective randomized controlled study was performed to compare the clinical effects of naftopidil and tamsulosin. Men complaining of lower urinary tract symptoms due to benign prostatic hyperplasia were randomized into two groups : one receiving 50mg naftopidil once daily (Naf group, n=36 patients), and the other receiving 0.2 mg tamsulosin once daily (Tam group, n=32 patients). In the Naf group at 12 weeks, 7 items of the International Prostate Symptom Score (IPSS), storage and voiding symptoms, total IPSS, quality of life (QOL) index (QOLI) and Qmax were improved significantly. In the Tam group at 12 weeks, 6 items of IPSS except urgency, storage and voiding symptoms, total IPSS, QOLI and Qmax were improved significantly. Improvement of residual urine volume (PVR) was insignificant in both groups. In intergroup comparison between the Naf and the Tam groups, variations of 7 items of IPSS, storage and voiding symptoms, total IPSS, QOLI, Qmax and PVR at 4 and 12 weeks after treatment were not statistically significant. There was almost no difference in clinical efficacy between Naf and Tam.

Impact of unnatural nucleosides on the control of microbial growth.

This research investigated the antimicrobial activities of unnatural nucleosides. We tested the MIC and MBC of 17 synthetic nucleoside analogues against 10 microbial strains. These nucleoside analogues were classified into four groups according to their structural characteristics. Inhibition was observed with compounds 1-1, 3-1, and 4-3. In particular, 5'-deoxythymidine (3-1) was most effective at 50 micro g/mL against Bacillus subtilis and Staphylococcus aureus. This analogue has had the hydroxyl group at the 5' position replaced with a hydrogen atom. All compounds had weak effects against various species of mold. The MBC of 5'-deoxythymidine was 50 g/mL in 0.5 h against S. aureus. These results showed that 5'-deoxythymidine had the most effective antimicrobial activity of the 17 different unnatural nucleosides. The inhibitory effect of 3-1 suggests that it may be useful as an antibacterial agent in medical situations.

Kinetic parameters and recognition of thymidine analogues with varying functional groups by thymidine phosphorylase.

Thymidine phosphorylase (TP, EC 2.4.2.4) recognized the structure of the substrate with high specificity, via both the base and the ribosyl moieties. The replacement of 3'-OH of thymidine markedly influenced its catalytic activity with TP. The conversion of pyrimidine nucleosides with modified base moieties to the corresponding 1-phosphate form was poor. The leaving group activity decreased with an increase in aromaticity of the pyrimidine base moiety, because of increased difficulty in polarizing the base by the amino acids local to the active site. The replacement of 3' and 5' functional groups tended to decrease the reaction rate and the percentage conversion with TP. In particular the ribosyl 3' hydroxyl group was structurally important for the binding of the substrate by the enzyme. The kinetic assay clearly showed high K(m) and low V(max) values on replacing the 3' hydroxyl group with hydrogen.

Aerobic oxidation of thiols to disulfides catalyzed by trichlorooxyvanadium.

Thiols were converted into disulfide by the aerobic oxidation catalyzed by trichlorooxyvanadium in the presence of molecular sieves 3A.

Synthesis and characterization of a DNA analogue stabilized by mercapto C-nucleoside induced disulfide bonding.

A redox-active nucleobase analogue of a nucleotide was synthesized and incorporated into DNA using phosphoramidite chemistry. An analogue-containing oligonucleotide in the absence of a reducing reagent formed a stable duplex with a substantially higher melting temperature compared to that of a standard DNA duplex of the same length.

[Persistent müllerian duct Syndrome in brothers].

Persistent Müllerian duct syndrome is associated with cryptorchidism and transverse testicular ectopia. Such gonads are at an increased risk of malignant transformation. Furthermore, most patients have azoospermia. Here in we report about two brothers with persistent Müllerian duct syndrome. The diagnosis was made during surgical operation for testicular cancer in younger brother. In the other one, persistent Müllerian duct syndrome was diagnosed during examination for infertility.