Detection of trifluridine in tumors of patients with metastatic colorectal cancer treated with trifluridine/tipiracil.

PURPOSE: Trifluridine (FTD) is the active component of the nucleoside chemotherapeutic drug trifluridine/tipiracil (FTD/TPI), which is approved worldwide for the treatment of patients with metastatic gastrointestinal cancer. FTD exerts cytotoxic effects via its incorporation into DNA, but FTD has not been detected in the tumor specimens of patients. The purpose of this study was to detect FTD in tumors resected from metastatic colorectal cancer (mCRC) patients who were administered FTD/TPI. Another purpose was to investigate the turnover rate of FTD in tumors and bone marrow in a mouse model. METHODS: Tumors and normal tissue specimens were obtained from mCRC patients who were administered FTD/TPI or placebo at Kyushu University Hospital. Tumors and bone marrow were resected from mice with peritoneal dissemination treated with FTD/TPI. To detect and quantitate FTD incorporated into DNA, immunohistochemical staining of paraffin-embedded specimens (IHC-p staining) and slot-blot analysis of DNA purified from these tissues were performed using an anti-BrdU antibody. IHC-p staining of proliferation and apoptosis markers was also performed. RESULTS: FTD was detected in metastatic tumors obtained from mCRC patients who were administered FTD/TPI, but who had discontinued the treatment several weeks before surgery. In a peritoneal dissemination mouse model, FTD was still detected in tumors 13 days after the cessation of FTD/TPI treatment, but had disappeared from bone marrow within 6 days. CONCLUSION: These results indicate that FTD persists longer in tumors than in bone marrow, which may cause a sustained antitumor effect with tolerable hematotoxicity.

A novel mass spectrometry method based on competitive non-covalent interaction for the detection of biomarkers.

We report a novel biosensor platform based on competitive non-covalent interaction between ssDNA and a mass tag towards AuNPs, which detects PSA biomarkers sensitively, observed using MALDI MS. A detection limit of 57 pg mL-1 has been achieved, showing an improvement of two orders of magnitude compared to the traditional spectroscopic method.

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine as well as 5-(hydroxymethyl)-2'-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt's lymphoma lymphoblasts (Raji), EBV-negative Burkitt's lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of TET1, TET2, TET3, SMUG1, and TDG genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2'-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.

Zn(2+)-cyclen-based complex enable a selective detection of single-stranded thymine-rich DNA in aqueous buffer.

It is a big challenge to develop fluorescent probes for selective detection of DNA with specific sequences in aqueous buffers. We report a new tetraphenylethene-based Zn(2+)-cyclen complex (TPECyZn), and a chemo-sensing ensemble of the Zn complex with phenol red. TPECyZn showed significant fluorescence enhancement upon binding to thymine-rich DNA in HEPES buffers. But its selectivity was not high enough to eliminate the interference from some random DNA. By constructing the chemo-sensing ensemble of TPECyZn with phenol red, the background fluorescence was eliminated due to the energy transfer from TPECyZn to phenol red. Moreover, this chemo-sensing ensemble revealed high selectivity in detecting thymine-rich single-stranded DNA over other DNA in aqueous buffer. It can detect poly deoxythymidylic acid sequence as short as 2 nt. This detection in aqueous media makes this probe feasible in real application.

Synthesis and photophysical properties of pyrene-labeled 3-deaza-2'-deoxyadenosines comprising a non-π-conjugated linker: fluorescence quenching-based oligodeoxynucleotide probes for thymine identification.

Pyrene-labeled 3-deaza-2'-deoxyadenosine comprising a non-π-conjugated linker (py3z)A (1) was synthesized and its photophysical properties were investigated. Oligodeoxynucleotide (ODN) probes containing (py3z)A (1) exhibited remarkable fluorescence quenching only when the opposite base of the complementary strand was the perfectly matched thymine. Such fluorescence quenching-based ODN probes exhibited excellent on-off switching properties, making them useful tools for single nucleotide polymorphism (SNP) genotyping and for the identification of target genes and structural studies of nucleic acids.

CuFe2O4 magnetic nanocrystal clusters as a matrix for the analysis of small molecules by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

CuFe2O4 magnetic nanocrystal clusters (CuFe2O4 MNCs) were proposed as a new matrix for small molecule analysis by negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the first time. We demonstrated its advantages over conventional organic matrices in the detection of small molecules such as amino acids, peptides, nucleobases, fatty acids, and steroid hormones. A systematic comparison of CuFe2O4 MNCs with different ionization modes revealed that MS spectra obtained for the CuFe2O4 MNC matrix in the negative ion mode was only featured by deprotonated ion peaks with a free matrix background, which was different from the complicated alkali metal adducts produced in the positive ion mode. The developed method was found relatively tolerant to salt contamination and exhibited good reproducibility. A detection limit down to the subpicomolar level was achieved when testosterone was analyzed. In addition, by comparison of the MS spectra obtained from bare Fe3O4 and MFe2O4 MNC (M = Co, Ni, Cu, Zn) matrices, two main factors of MFe2O4 MNC matrices were revealed to play a vital role in assisting the negative ion desorption/ionization (D/I) process: doping transition metals into ferrite nanocrystals favoring laser absorption and energy transfer and a good match between the UV absorption of MFe2O4 MNCs and the excitation of nitrogen laser source facilitating LDI efficiency. This work creates a new branch of application for MFe2O4 MNCs and provides an alternative solution for small molecule analysis.

Molecular design of an environmentally sensitive fluorescent nucleoside, 3-deaza-2'-deoxyadenosine derivative: distinguishing thymine by probing the DNA minor groove.

An environmentally sensitive fluorescent nucleoside containing a 3-deazaadenine skeleton has been developed, and its photophysical properties were investigated. Newly developed C3-naphthylethynylated 3-deaza-2'-deoxyadenosine ((3nz) A, 1) exhibited dual fluorescence emission from an intramolecular charge-transfer state and a locally excited state, depending upon molecular coplanarity. DNA probes containing 1 clearly discriminated a perfectly matched thymine base on the complementary strand by a distinct change in emission wavelength.

Design of an environmentally sensitive fluorescent 8-aza-7-deaza-2'-deoxyadenosine derivative with dual fluorescence for the specific detection of thymine.

A novel environmentally sensitive fluorescent (ESF) purine nucleoside, (cna)A, was synthesized and its photophysical properties were investigated. The base-modified fluorescent nucleoside (cna)A exhibited remarkable solvatochromicity and environmentally sensitive dual fluorescence. By using (cna)A, we have developed highly thymine (T) selective fluorescent DNA probes that can sense the corresponding T in a target DNA/RNA sequence, as measured by a distinct change in emission wavelength.

Analysis of RANKL gene polymorphism (rs9533156 and rs2277438) in Iranian patients with chronic periodontitis and periimplantitis.

OBJECTIVE: RANK/OPG/RANKL pathway plays a significant role in osteoclastogenesis, osteoclast activation, and regulation of bone resorption. The aim of this study was to investigate the association of RANKL gene polymorphisms (rs9533156 and rs2277438) with chronic periodontitis and peri-implantitis in an Iranian population. DESIGN: 77 patients with chronic periodontitis, 40 patients with peri-implantitis and 89 periodontally healthy patients were enrolled in this study. 5cc of blood was obtained from the cephalic vein of subjects arms and transferred into tubes containing EDTA. Genomic DNA was extracted using Miller's Salting Out technique. The DNA was transferred into 96 division plates, transported to Kbioscience Institute in United Kingdom and analyzed using the Kbioscience Competitive Allele Specific PCR (KASP) technique. Differences in the frequencies of genotypes and alleles in the disease and control groups were analyzed using Chi-square and Fisher's exact statistical tests. RESULTS: Comparison of frequency of alleles in SNP rs9533156 of RANKL gene between the chronic periodontitis group with the control and peri-implantitis groups revealed statistically significant differences (P=0.024 and P=0.027, respectively). Comparison of genotype expression of SNP rs9533156 on RANKL gene between the peri-implantitis group with chronic periodontitis and control groups revealed statistically significant differences (P=0.001); the prevalence of CT genotype was significantly higher amongst the chronic periodontitis group. Regarding SNP rs2277438 of RANKL gene, comparison of prevalence of genotypes and frequency of alleles did not reveal any significant differences (P=0.641/P=0.537, respectively). CONCLUSIONS: The results of this study indicate that CT genotype of rs9533156 RANKL gene polymorphism was significantly associated with peri-implantitis, and may be considered as a genetic determinant for peri-implantitis.

Real-time sensing and discrimination of single chemicals using the channel of phi29 DNA packaging nanomotor.

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This article reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of 12 protein subunits encircled into a 3.6 nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Da), thymine (167 Da), and benzene (105 Da) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete stepwise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices.

[Determination of nucleosides in Rhizoma Pinelliae by high performance liquid chromatography].

A high performance liquid chromatographic (HPLC) method was established to determine nucleosides in Rhizoma Pinelliae, which is a dried stem tuber of Pinellia pedatisecta Schott in Pinellia plant belonging to Araceae family and has multiple efficiencies about down-bear counterflow and check vomiting, eliminating dampness and phlegm, etc. The separation of adenine, hypoxanthine, xanthine, uridine, thymine, adenosine and guanosine was achieved on a Lichrospher C18 column (150 mm x 4.6 mm, 5 microm) with the detection at 254 nm and gradient elution by acetonitrile-water containing 0.1% formic acid as the mobile phase. The linear ranges were from 1.6 mg/L to 50 mg/L for adenine, hypoxanthine, xanthine, uridine and guanosine, while from 1.2 mg/L to 40 mg/L for thymine and adenosine with correlation coefficients above 0.999 5. The average recoveries were between 98.9% and 101.2% with the relative standard deviations below 3%. The results of methodological study demonstrated that the method met the requirements of the determination. The nucleosides in Rhizoma Pinelliae from different districts were determined. The method is convenient and accurate with good reproducibility and can be used to evaluate the quality of Rhizoma Pinelliae.

Genomic bipyrimidine nucleotide frequency and microbial reactions to germicidal UV radiation.

The role of the genomic bipyrimidine nucleotide frequency in pyrimidine dimer formation caused by germicidal UV radiation was studied in three microbial reference organisms (Escherichia coli K12, Deinococcus radiodurans R1, spores and cells of Bacillus subtilis 168). The sensitive HPLC tandem mass spectrometry assay was used to identify and quantify the different bipyrimidine photoproducts induced in the DNA of microorganisms by germicidal UV radiation. The yields of photoproducts per applied fluence were very similar among vegetative cells but twofold reduced in spores. This similarity in DNA photoreactivity greatly contrasted with the 11-fold range determined in the fluence causing a decimal reduction of survival. It was also found that the spectrum of UV-induced bipyrimidine lesions was species-specific and the formation rates of bi-thymine and bi-cytosine photoproducts correlated with the genomic frequencies of thymine and cytosine dinucleotides in the bacterial model systems.

Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding.

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE binding, whereas the DNA architectural properties of HMGB are dispensable. Specific PRE DNA inhibited HMGB binding to the CTE, indicating that DNA and HMGB-CTE interactions are mutually exclusive. Exogenous CTE peptide increased PR-binding affinity for PRE as did deletion of the CTE. In a PR-binding site selection assay, A/T sequences flanking the PRE were enriched by HMGB, indicating that PR DNA-binding specificity is also altered by HMGB. We conclude that a transient HMGB-CTE interaction alters a repressive conformation of the flexible CTE enabling it to bind to preferred sequences flanking the PRE.

Sequence analysis of p53 response-elements suggests multiple binding modes of the p53 tetramer to DNA targets.

The p53 tetramer recognizes specifically a 20-bp DNA element. Here, we examined symmetries encoded in p53 response elements (p53REs). We analyzed base inversion correlations within the half-site, as well as in the full-site palindrome. We found that p53REs are not only direct repeats of half-sites; rather, two p53 half-sites couple to form a higher order 20 bp palindrome. The palindrome couplings between the half-sites are stronger for the human than for the mouse genome. The full-site palindrome and half-site palindrome are controlled by insertions between the two half-sites. The most notable feature is that the full-site palindrome with coupling between quarter-sites one and four (H14 coupling) dominates the p53REs without insertions. The most frequently observed insertion in human p53REs of 3 bp enhances the half-site palindrome. The statistical frequencies of the coupling between the half-sites in the human genome correlate with grouped experimental p53 affinities with p53REs. Examination of known p53REs indicates the H14 couplings are stronger for positive regulation than for negatively regulated p53REs, with repressors having the lowest H14 couplings. We propose that the palindromic sequence couplings may encode such potential preferred multiple binding modes of the p53 tetramer to DNA.

Qualitative and quantitative analyses of nucleosides and nucleobases in Ganoderma spp. by HPLC-DAD-MS.

A high-performance liquid chromatography-diode array detector-mass spectrometry (HPLC-DAD-MS) analytical method was developed for detection of the nucleosides and nucleobases in two species of Lingzhi, the dried sporophore of Ganoderma lucidum and G. sinense. The method, combining advantages of both DAD and MS, was successfully used to qualitatively identify for six nucleosides namely, adenosine, cytidine, guanosine, inosine, thymidine, uridine and five nucleobases namely, adenine, guanine, hypoxanthine, thymine and uracil in Lingzhi samples. Quantitative analyses showed that uridine was the most abundant nucleoside in these Lingzhi samples and the contents of nine target analytes were found to be different in pileus and stipes of the fruiting bodies and among the different species of G. spp. The established method might apply as an alternative approach for the quality assessment of Lingzhi.

Alterations of the USP26 gene in Caucasian men.

The Ubiquitin Specific Protease 26 gene is a testis-specific gene that is located on the X chromosome. Sequence variants of this gene were previously reported in men with azoospermia caused by defects at the level of spermatogenesis. Especially a cluster of three changes (c.370_371insACA, c.494T>C and c.1423C>T) was frequently observed. To further define the role of this cluster of sequence variants in the USP26 gene, we have now analysed 202 control samples and 146 patients of Caucasian origin with cryptozoospermia or oligozoospermia. The detection method was based on a restriction reaction, by which the change c.494T>C can be detected. In none of the patients, the change c.494T>C was observed. Only in one man with normal spermatogenesis this sequence variant was detected. Sequencing can confirm the presence of the three changes of the USP26 gene. These data indicate that the cluster of changes is not restricted to men with severe testicular dysfunction.

Polymerase chain reaction inhibition assay documenting the amotosalen-based photochemical pathogen inactivation process of platelet concentrates.

BACKGROUND: The INTERCEPT Blood System (Baxter Healthcare Corp.) for platelets (PLTs) uses amotosalen-HCl (S-59) in conjunction with ultraviolet A (UVA) light to inactivate contaminating pathogens by modifying the nucleic acids of pathogens. The success of this photochemical treatment (PCT) process can be documented indirectly with a high-performance liquid chromatography assay measuring the photodegradation of amotosalen and measurement of the UVA light dose delivered by the illumination system. STUDY DESIGN AND METHODS: To develop an assay that documents the success of PCT directly on the effector molecule DNA, the effect of PCT on PLT-derived mitochondrial DNA (mtDNA) was examined. mtDNA-specific polymerase chain reaction (PCR) assays were tested with regard to their susceptibility for PCT, their reliability in terms of PCR performance, and the absence of polymorphic sites in primer hybridization loci. RESULTS: Suitable PCR amplification targets were found in the regions of 16S rDNA, cytochrome c oxidase I, and cytochrome c oxidase III of mitochondria. Amplicon sizes between 868 and 1248 bp gave consistent signals before PCT and complete inhibition of the PCR signal after PCT. Amplicons of less than 300 bp were found to be transparent to PCT. CONCLUSION: Based on PCT-mediated mtDNA modifications in PLTs, a PCR inhibition assay was established with a large amplicon documenting the success of PCT and a small amplicon serving as an internal control.

DNA binding and helicase actions of mouse MCM4/6/7 helicase.

Helicases play central roles in initiation and elongation of DNA replication. We previously reported that helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine-rich single-stranded DNA. Here, we examined its substrate preference and helicase actions using various synthetic DNAs. On a bubble substrate, Mcm4/6/7 makes symmetric dual contacts with the 5'-proximal 25 nt single-stranded segments adjacent to the branch points, presumably generating double hexamers. Loss of thymine residues from one single-strand results in significant decrease of unwinding efficacy, suggesting that concurrent bidirectional unwinding by a single double hexameric Mcm4/6/7 may play a role in efficient unwinding of the bubble. Mcm4/6/7 binds and unwinds various fork and extension structures carrying a single-stranded 3'-tail DNA. The extent of helicase activation depends on the sequence context of the 3'-tail, and the maximum level is achieved by DNA with 50% or more thymine content. Strand displacement by Mcm4/6/7 is inhibited, as the GC content of the duplex region increases. Replacement of cytosine-guanine pairs with cytosine-inosine pairs in the duplex restored unwinding, suggesting that mammalian Mcm4/6/7 helicase has difficulties in unwinding stably base-paired duplex. Taken together, these findings reveal important features on activation and substrate preference of the eukaryotic replicative helicase.

Studies on the synthesis and characterization of four trans-planaramineplatinum(II) complexes of the form trans-PtL(NH3)CL2 where L = 2-hydroxypyridine, 3-hydroxypyridine, imidazole, and imidazo(1,2-alpha)pyridine.

Four trans-planaramineplatinum(II) complexes code named YH9, YH10, YH11 and YH12, each of the form trans-PtL(NH(3))Cl(2) where L = 2-hydroxypyridine and 3-hydroxypyridine, imidazole, and imidazo(1,2-alpha)pyridine for YH9, YH10, YH11 and YH12, respectively. All of the compounds have significant anticancer activity against human cancer cell lines. YH12 is found to be significantly more active than cisplatin against cisplatin-resistant ovary cell line A2780(cisR).

Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells.

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.