Enhancing Cordycepin in Functional Porridge through Rice Varietal Fermentation with Cordyceps militaris (Ascomycetes) and Utilizing Non-Targeted Metabolomics for Process Enhancement.

Cordyceps militaris, a medicinal fungus rich in cordycepin, shows promise in treating diseases such as cancer, respiratory issues, and COVID-19. This study examines the impact of different Taiwanese rice varieties on its solid-state fermentation, focusing on optimizing cordycepin production. The results indicated that the cordycepin yield was indeed affected by the type of rice used. In terms of the fruiting bodies, germ rice resulted in the highest yield (13.1 ± 0.36 mg/g), followed by brown rice (11.9 ± 0.26 mg/g). In the rice culture medium (RCM), brown rice led to the highest yield (4.77 ± 0.06 mg/g). Using gas chromatography-mass spectrometry and untargeted metabolomics, the study identifies four key volatile components linked to cordycepin, providing insights into developing functional rice porridge products. These findings are significant for advancing cordycepin mass production and offering dietary options for older individuals.

Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution.

The discovery of N6-methyldeoxyadenine (6mA) across eukaryotes led to a search for additional epigenetic mechanisms. However, some studies have highlighted confounding factors that challenge the prevalence of 6mA in eukaryotes. We developed a metagenomic method to quantitatively deconvolve 6mA events from a genomic DNA sample into species of interest, genomic regions, and sources of contamination. Applying this method, we observed high-resolution 6mA deposition in two protozoa. We found that commensal or soil bacteria explained the vast majority of 6mA in insect and plant samples. We found no evidence of high abundance of 6mA in Drosophila, Arabidopsis, or humans. Plasmids used for genetic manipulation, even those from Dam methyltransferase mutant Escherichia coli, could carry abundant 6mA, confounding the evaluation of candidate 6mA methyltransferases and demethylases. On the basis of this work, we advocate for a reassessment of 6mA in eukaryotes.

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine.

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

Identification of an N'-Nitrosonornicotine-Specific Deoxyadenosine Adduct in Rat Liver and Lung DNA.

The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered to be two of the most important carcinogens in unburned tobacco and its smoke. They readily cause tumors in laboratory animals and are classified as "carcinogenic to humans" by the International Agency for Research on Cancer. DNA adduct formation by these two carcinogens is believed to play a critical role in tobacco carcinogenesis. Among all the DNA adducts formed by NNN and NNK, 2'-deoxyadenosine (dAdo)-derived adducts have not been fully characterized. In the study reported here, we characterized the formation of N6-[4-(3-pyridyl)-4-oxo-1-butyl]-2'-deoxyadenosine (N6-POB-dAdo) and its reduced form N6-PHB-dAdo formed by NNN 2'-hydroxylation in rat liver and lung DNA. More importantly, we characterized a new dAdo adduct N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N6-HPB-dAdo) formed after NaBH3CN or NaBH4 reduction both in vitro in calf thymus DNA reacted with 5'-acetoxy-N'-nitrosonornicotine and in vivo in rat liver and lung upon treatment with NNN. This adduct was specifically formed by NNN 5'-hydroxylation. Chemical standards of N6-HPB-dAdo and the corresponding isotopically labeled internal standard [pyridine-d4]N6-HPB-dAdo were synthesized using a four-step method. Both NMR and high-resolution mass spectrometry data agreed well with the proposed structure of N6-HPB-dAdo. The new adduct coeluted with the synthesized internal standard under various LC conditions. Its product ion patterns of MS2 and MS3 transitions were also consistent with the proposed fragmentation patterns. Chromatographic resolution of the two diastereomers of N6-HPB-dAdo was successfully achieved. Quantitation suggested a dose-dependent response of the levels of this new adduct in the liver and lung of rats treated with NNN. However, its level was lower than that of 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine, a previously reported dGuo adduct that is also formed from NNN 5'-hydroxylation. The identification of N6-HPB-dAdo in this study leads to new insights pertinent to the mechanism of carcinogenesis by NNN and to the development of biomarkers of NNN metabolic activation.

Effects of Substrates on the Production of Fruiting Bodies and the Bioactive Components by Different Cordyceps militaris Strains (Ascomycetes).

Cordyceps militaris is a type of food and medicinal species and is widely cultured in Asia. Substrate and strain are important factors for the production of fruiting bodies and bioactive components contents in fruiting bodies of C. militaris. This study aimed to select the excellent strains and suitable substrates by six strains of C. militaris cultivated on rice, wheat, and tussah (Antheraea pernyi) pupae. The results showed that the rice and wheat were suitable for fruiting body formation of strain CM3, with yields of 23.19 and 19.07 g per bottle, and biological efficiency of strain CM3 were 62.26% and 54.48%, respectively, which were significantly higher than other strains. Tussah pupae is suitable for fruiting body formation of strain CM9, with fruiting body length, yield, and biological efficiency of 5.57 cm, 6.80 g per each, and 291.70%, respectively, which were significantly higher than other strains. The content of adenosine in fruiting bodies of strain CM9 cultivated on tussah pupae was 2.62 mg g-1, followed by that of strain CM3 on rice of 2.51 mg g-1. The content of cordycepin in fruiting bodies of strain CM4 cultivated on wheat was 5.68 mg g-1, followed by that of strain CM9 on wheat of 5.41 mg g-1. To improve the product quality and the contents of bioactive components, C. militaris strains and substrates should both be considered, that is, different strains should be appropriate for different substrates.

Development of a solid-phase microextraction LC-MS/MS method for determination of oxidative stress biomarkers in biofluids.

Recently the connection between oxidative stress and various diseases, including cancer and Alzheimer's, attracts notice as a pathway suitable for diagnostic purposes. 8-Oxo-deoxyguanosine and 8-oxo-deoxyadenosine produced from the interaction of reactive oxygen species with DNA become prominent as biomarkers. Several methods have been developed for their determination in biofluids, including solid-phase extraction and enzyme-linked immunosorbent assays. However, still, there is a need for reliable and fast analytical methods. In this context, solid-phase microextraction offers many advantages such as flexibility in geometry and applicable sample volume, as well as high adaptability to high-throughput sampling. In this study, a solid-phase microextraction method was developed for the determination of 8-oxo-deoxyguanosine and 8-oxo-deoxyadenosine in biofluids. The extractive phase of solid-phase microextraction consisted of hydrophilic-lipophilic balanced polymeric particles. In order to develop a solid-phase microextraction method suitable for the determination of the analytes in saliva and urine, several parameters, including desorption solvent, desorption time, sample pH, and ionic strength, were scrutinized. Analytical figures of merit indicated that the developed method provides reasonable interday and intraday precisions (<15% in both biofluids) with acceptable accuracy. The method provides a limit of quantification for both biomarkers at 5.0 and 10.0 ng/mL levels in saliva and urine matrices, respectively.

A rapid assay to screen adenosine deaminase inhibitors from Ligustri Lucidi Fructus against metabolism of cordycepin utilizing ultra-high-performance liquid chromatography-tandem mass spectrometry.

Cordycepin has recently received increased attention owing to its extensive pharmacological activity. Adenosine deaminase (ADA) is widely distributed in mammalian blood and tissues; as a result, cordycepin is quickly metabolized upon entering into the body and converted into the inactive metabolite 3'-deoxyinosine, thus limiting its activity when administered alone. We herein present a novel ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for screening ADA inhibitors against the metabolism of cordycepin. Cordycepin and 3'-deoxyinosine were chosen as substrate and product, respectively. A proper separation was achieved for all analytes within 3 min. 3'-Deoxyinosine was quantified in the presence or absence of potential ADA inhibitors to evaluate ADA activity. The assay can simultaneously determine substrate and product, with the endogenous substance and ADA inhibitors added not interfering in its activity. After optimizing the enzymatic incubation and UHPLC-MS/MS conditions, Km and Vmax values for ADA deamination of cordycepin were 95.18 ± 7.85 µm and 363.90 ± 12.16 µmol/min/unit, respectively. Oleanolic acid and ursolic acid from Ligustri Lucidi Fructus were chosen as ADA inhibitors with half maximal inhibitory concentration values of 21.82 ± 0.39 and 18.41 ± 0.14 µm, respectively. A non-competitive inhibition model was constructed and this assay can be used to screen other potential ADA inhibitors quickly and accurately.

An Efficient Strategy for Enhancement of Bioactive Compounds in the Fruit Body of Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), by Spraying Biotic Elicitors.

Cordyceps militaris is a mushroom species with high nutritive and medicinal values based on diverse bioactive metabolites. The contents of bioactive ingredients are indicative of the quality of commercially available fruit body of this fungus. Although the application of biotic elicitors has been an efficient strategy to induce the accumulation of valuable bioactive compounds in vivo, related research in C. militaris is rarely reported. In this study, five biotic elicitors in different concentrations (0.05, 0.5, 1, and 2 mg/mL), including chitosan (CHT), 2,4-dichlorophenoxyacetic acid (2,4-D), methyl jasmonate (MeJA), gibberellic acid (GA), and triacontanol (TRIA), were first introduced to enhance the production of 10 kinds of major bioactive components in the fruit body of C. militaris. Results showed that the effect of biotic elicitors on bioactive compounds in the fruit body of C. militaris was elicitor-specific and concentration-dependent. Overall, 1 mg/L CHT was considered the most favorable for the production of 10 bioactive ingredients in C. militaris fruit body, which could increase the content of protein, polysaccharides, polyphenol, triterpenoids, flavonoids, cordyceps acid, cordycepin, and anthocyanins by 20.38-, 1.41-, 0.7-, 0.47-, 11.90-, 1.09-, 0.34-, and 2.64-fold, respectively, compared with the control. The results of this study would provide an efficient strategy for the production of a superior quality fruit body of and contribute to further elucidation of the effects of biotic elicitors on metabolite accumulation in C. militaris.

High levels of oxidatively generated DNA damage 8,5'-cyclo-2'-deoxyadenosine accumulate in the brain tissues of xeroderma pigmentosum group A gene-knockout mice.

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, a pathway that eliminates a wide variety of helix-distorting DNA lesions, including ultraviolet-induced pyrimidine dimers. In addition to skin diseases in sun-exposed areas, approximately 25% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of an oxidatively generated type of DNA damage called purine 8,5'-cyclo-2'-deoxynucleoside (cyclopurine). However, that hypothesis has not been verified. In this study, we tested that hypothesis by using the XP group A gene-knockout (Xpa-/-) mouse model. To quantify cyclopurine lesions in this model, we previously established an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (CdA-1) that specifically recognizes 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA). By optimizing conditions, we increased the ELISA sensitivity to a detection limit of ˜one cyclo-dA lesion/106 nucleosides. The improved ELISA revealed that cyclo-dA lesions accumulate with age in the brain tissues of Xpa-/- and of wild-type (wt) mice, but there were significantly more cyclo-dA lesions in Xpa-/- mice than in wt mice at 6, 24 and 29 months of age. These findings are consistent with the long-standing hypothesis that the age-dependent accumulation of endogenous cyclopurine lesions in the brain may be critical for XP neurological abnormalities.

1H-NMR-Based Metabolic Profiling of Cordyceps militaris to Correlate the Development Process and Anti-Cancer Effect.

The study of metabolomics in natural products using the diverse analytical instruments including GC-MS, LC-MS, and NMR is useful for the exploration of physiological and biological effects and the investigation of drug discovery and health functional foods. Cordyceps militaris has been very attractive to natural medicine as a traditional Chinese medicine, due to its various bioactive properties including anti-cancer and anti-oxidant effects. In this study, we analyzed the metabolite profile in 50% ethanol extracts of C. militaris fruit bodies from three development periods (growth period, matured period, and aging period) using 1H-NMR, and identified 44 metabolites, which are classified as 16 amino acids, 10 organic acids, 5 carbohydrates, 3 nucleotide derivatives, and 10 other compounds. Among the three development periods of the C. militaris fruit body, the aging period showed significantly higher levels of metabolites including cordycepin, mannitol (cordycepic acid), and ß-glucan. Interestingly, these bioactive metabolites are positively correlated with antitumor growth effect; the extract of the aging period showed significant inhibition of HepG2 hepatic cancer cell proliferation. These results showed that the aging period during the development of C. militaris fruit bodies was more highly enriched with bioactive metabolites that are associated with cancer cell growth inhibition.

Metabolic profiling of natural and cultured Cordyceps by NMR spectroscopy.

Cordyceps, a type of Chinese herbal medicine that exhibits anti-angiogenesis and tumor growth suppression effects, has recently gained increasing popularity. However, high-quality, natural Cordyceps, such as Ophiocordyceps sinensis, is very rare and difficult to obtain in large amounts. Cordyceps is cultured instead of harvested from natural sources, but the quality with respect to the ingredients has not been fully studied. In this study, we performed an NMR metabolic profiling of aqueous extracts of Cordyceps without any sample treatment to evaluate the proper species and medium and influence of two different disinfection methods. It was discovered that Cordyceps militaris fungus and silkworm chrysalis medium were suitable for cultivation of Cordyceps. Furthermore, cordycepin, a Cordyceps-specific functional compound, was produced at different growth stages during different cultivation processes, even at the mycelial stage, and was found at three times higher concentrations in cultured C. militaris compared to that in naturally occurring C. militaris.

Effect of Culture Time on the Bioactive Components in the Fruit Bodies of Caterpillar Mushroom, Cordyceps militaris CM-H0810 (Ascomycetes).

Cordyceps militaris are widely cultivated in China for an important raw material for health foods. CM-H0810 is a C. militaris strain used in the production of C. militaris in Shanghai, the surrounding areas of Shanghai, and Guangdong province in China. We evaluated the effect of culture time on the bioactive components in the fruit bodies of C. militaris CM-H0810 to provide scientific references for production of C. militaris fruit bodies with good quality. The results showed that the polysaccharide contents increased gradually during 35-45 d, but it declined with the prolongation of culture time. The highest polysaccharide content was 3.46% at 45 d. With the prolongation of culture time the cordycepin content gradually increased; the highest cordycepin content was 3.57 µg/mg at 60 d, which increased 321% compared to that at 35 d. Contrary to cordycepin, the adenosine content declined gradually, with the highest content of 1.86 µg/mg at 35 d and the lowest content of 1.48 µg/mg at 60 d. Our study indicates that it is necessary to select suitable harvest times in view of different compounds that are desirable to obtain in high quantities.

Pediococcus pentosaceus-Fermented Cordyceps militaris Inhibits Inflammatory Reactions and Alleviates Contact Dermatitis.

Cordyceps militaris is a medicinal mushroom used to treat immune-related diseases in East Asia. We investigated the anti-inflammatory effect of the extract of C. militaris grown on germinated Rhynchosia nulubilis (GRC) fermented with Pediococcus pentosaceus ON89A isolated from onion (GRC-ON89A) in vivo as well as in vitro. The anti-inflammatory effect of GRC-ON89A was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The total polyphenol content (TPC) and total flavonoid content (TFC) in the GRC-ON89A ethanol extract were significantly increased compared to that in GRC. GRC-ON89A hexane fraction (GRC-ON89A-Hex) inhibited the release of nitric oxide (NO) compared to that of the LPS-treated control without cytotoxicity in LPS-stimulated RAW 264.7 macrophages. GRC-ON89A-Hex decreased the inducible NO synthase (iNOS), cyclooxygenase 2 (COX2), and tumor necrosis factor (TNF)-α mRNA expression in LPS-stimulated RAW 264.7 macrophages. In addition, pre-treatment with GRC-ON89A-Hex significantly inhibited LPS-stimulated phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB. To induce allergic contact dermatitis (ACD), 1-fluoro-2, 4-dinitrofluorobenzene (DNFB) was applied to the surface of the right ears of C57BL/6N mice. GRC-ON89A reduced the ear swelling and thickness in DNFB-induced ACD mice. This study demonstrates the potential usefulness of GRC-ON89A as an anti-inflammatory dietary supplement or drug.

Analysis and Identification of 2'-Deoxyadenosine-Derived Adducts in Lung and Liver DNA of F-344 Rats Treated with the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are carcinogenic in animal models and are believed to play an important role in human lung carcinogenesis for cigarette smokers. Cytochrome P450-mediated metabolism of these tobacco-specific nitrosamines produces reactive species that alkylate DNA in the form of pyridyloxobutyl (POB)- or pyridylhydroxybutyl (PHB)-DNA adducts. Understanding the formation mechanism and overall levels of these adducts can potentially enhance cancer prevention methods through the identification of particularly susceptible smokers. Previous studies have identified and measured a panel of POB- and PHB-DNA base adducts of dGuo, dCyd, and Thd; however, dAdo adducts have yet to be determined. In this study, we complete this DNA adduct panel by identifying and quantifying levels of NNK- and NNAL-derived dAdo adducts in vitro and in vivo. To accomplish this, we synthesized standards for expected dAdo-derived DNA adducts and used isotope-dilution LC-ESI+-MS/MS to identify POB adducts formed in vitro from the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) with calf thymus DNA. Adduct levels were then quantified in lung and liver DNA of rats chronically treated with NNK or NNAL for 50 weeks using similar LC-MS detection methods. The in vitro studies identified N6-POB-dAdo and N1-POB-dIno as products of the reaction of NNKOAc with DNA, which supports our proposed mechanism of formation. Though both N6-dAdo and N1-dIno adducts were found in vitro, only N6-dAdo adducts were found in vivo, implying possible intervention by DNA repair mechanisms. Analogous to previous studies, levels of N6-POB-dAdo and N6-PHB-dAdo varied both with tissue and treatment type. Despite the adduct levels being relatively modest compared to most other POB- and PHB-DNA adducts, they may play a biological role and could be used in future studies as NNK- and NNAL-specific DNA damage biomarkers.

A new cordycepin-producing caterpillar fungus Ophiocordyceps xuefengensis with artificial infection to the host, cultivation of mycelia and stromata.

Caterpillar fungi have numerous pharmacological and therapeutic applications in traditional medicine, due to a variety of active chemical constituents, such as cordycepin and adenosine. It is imperative to discover new resource for artificial cultivation and biometabolite production since the traditional natural species are endangered. In this study, a new strain HACM 001 was isolated and identified as Ophiocordyceps xuefengensis by rDNA-ITS sequencing. This strain showed the potential of artificial infection to caterpillar larvae leading to mummification, as well as fermentation mycelia in liquid culture and cultivation stromata in solid medium. Eight nucleosides and nucleobases, especially cordycepin and adenosine, were determined and analyzed with HPLC-DAD-Q-TOF-MS/MS technology. Cordycepin was detected in all forms of present O. xuefengensis strain at different contents, among which the highest content (37.1 µg/g) appeared in the stromata cultivated on solid medium. The content of adenosine in mycelia and stromata, respectively, reached 1155 µg/g and 1470 µg/g. Therefore, O. xuefengensis might be an alternative source for obtaining artificial fungus-caterpillar-larvae complex and producing cordycepin and adenosine.

Comparison of Major Bioactive Compounds of the Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), Fruiting Bodies Cultured on Wheat Substrate and Pupae.

In this study, the main bioactive compounds of the fruit bodies of Cordyceps militaris-such as adenosine, cordycepin, polysaccharides, mannitol, superoxide dismutase (SOD), and carotenoids-were cultivated on wheat and pupae, as well as sclerotium (the pupae portion) and sclerotium with fruiting bodies. The amounts of adenosine and polysaccharide in all the tested samples (except for the polysaccharides of sclerotium) are higher than the quality standards (adenosine ≥0.055% and polysaccharide ≥2.5%) determined by the Ministry of Health of the People's Republic of China. As the most important bioactive compound in C. militaris, cordycepin is the highest in the fruiting bodies on pupae than in other samples, whereas it is the lowest in the sclerotium. The amounts of cordycepin, carotenoids, and SOD were higher in the fruiting bodies on pupae than that in the fruiting bodies on wheat, whereas the amounts of adenosine, polysaccharides, and mannitol were higher in the fruiting bodies on wheat than in the fruiting bodies on pupae. There was no significant difference in the amounts of cordycepin, carotenoids, and SOD in the sclerotium with fruiting bodies and the fruiting bodies on wheat. The adenosine, polysaccharide, and mannitol contents in the sclerotium with fruiting bodies were significantly lower than those of the fruiting bodies on wheat. Overall, the results of this evaluation could not distinguish which is better: the fruiting bodies on pupae or those on wheat; each has its own merits. The fruiting bodies of C. militaris cultivated on both wheat and pupae are important candidates for medicinal and tonic use for the welfare of humankind.

Identification of a Fused-Ring 2'-Deoxyadenosine Adduct Formed in Human Cells Incubated with 1-Chloro-3-buten-2-one, a Potential Reactive Metabolite of 1,3-Butadiene.

1-Chloro-3-buten-2-one (CBO) is an in vitro metabolite of 1,3-butadiene (BD), a carcinogenic air pollutant. CBO exhibited potent cytotoxicity and genotoxicity that have been attributed in part to its reactivity toward DNA. Previously, we have characterized the CBO adducts with 2'-deoxycytidine and 2'-deoxyguanosine. In the present study, we report on the reaction of CBO with 2'-deoxyadenosine (dA) under in vitro physiological conditions (pH 7.4, 37 °C). We used the synthesized standards and their decomposition and acid-hydrolysis products to characterize the CBO-DNA adducts formed in human cells. The fused-ring dA adducts (dA-1 and dA-2) were readily synthesized and were structurally characterized as 1,N(6)-(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine and 1,N(6)-(1-hydroxy-1-chloromethylpropan-1,3-diyl)-2'-deoxyadenosine, respectively. dA-1 exhibited a half-life of 16.0 ± 0.7 h and decomposed to dA at pH 7.4 and 37 °C. At similar conditions, dA-2 decomposed to dA-1 and dA, and had a half-life of 0.9 ± 0.1 h. These results provide strong evidence for dA-1 being a degradation product of dA-2. dA-1 is formed by replacement of the chlorine atom of dA-2 by a hydroxyl group. The slow decomposition of dA-1 to dA, along with the detection of hydroxymethyl vinyl ketone (HMVK) as another degradation product, suggested equilibrium between dA-1 and a ring-opened carbonyl-containing intermediate that undergoes a retro-Michael reaction to yield dA and HMVK. Acid hydrolysis of dA-1 and dA-2 yielded the corresponding deribosylated products A-1D and A-2D, respectively. In the acid-hydrolyzed reaction mixture of CBO with calf thymus DNA, both A-1D and A-2D could be detected; however, the amount of A-2D was significantly larger than that of A-1D. Interestingly, only A-2D could be detected by LC-MS analysis of acid-hydrolyzed DNA from cells incubated with CBO, suggesting that dA-2 was stable in DNA and thus may play an important role in the genotoxicity and carcinogenicity of BD. In addition, A-2D could be developed as a biomarker of CBO formation in human cells.

Boronate Affinity Fluorescent Nanoparticles for Förster Resonance Energy Transfer Inhibition Assay of cis-Diol Biomolecules.

Förster resonance energy transfer (FRET) has been essential for many applications, in which an appropriate donor-acceptor pair is the key. Traditional dye-to-dye combinations remain the working horses but are rather nonspecifically susceptive to environmental factors (such as ionic strength, pH, oxygen, etc.). Besides, to obtain desired selectivity, functionalization of the donor or acceptor is essential but usually tedious. Herein, we present fluorescent poly(m-aminophenylboronic acid) nanoparticles (poly(mAPBA) NPs) synthesized via a simple procedure and demonstrate a FRET scheme with suppressed environmental effects for the selective sensing of cis-diol biomolecules. The NPs exhibited stable fluorescence properties, resistance to environmental factors, and a Förster distance comparable size, making them ideal donor for FRET applications. By using poly(mAPBA) NPs and adenosine 5'-monophosphate modified graphene oxide (AMP-GO) as a donor and an acceptor, respectively, an environmental effects-suppressed boronate affinity-mediated FRET system was established. The fluorescence of poly(mAPBA) NPs was quenched by AMP-GO while it was restored when a competing cis-diol compounds was present. The FRET system exhibited excellent selectivity and improved sensitivity toward cis-diol compounds. Quantitative inhibition assay of glucose in human serum was demonstrated. As many cis-diol compounds such as sugars and glycoproteins are biologically and clinically significant, the FRET scheme presented herein could find more promising applications.

Aristolochic acid exposure in Romania and implications for renal cell carcinoma.

BACKGROUND: Aristolochic acid (AA) is a nephrotoxicant associated with AA nephropathy (AAN) and upper urothelial tract cancer (UUTC). Whole-genome sequences of 14 Romanian cases of renal cell carcinoma (RCC) recently exhibited mutational signatures consistent with AA exposure, although RCC had not been previously linked with AAN and AA exposure was previously reported only in localised rural areas. METHODS: We performed mass spectrometric measurements of the aristolactam (AL) DNA adduct 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) in nontumour renal tissues of the 14 Romanian RCC cases and 15 cases from 3 other countries. RESULTS: We detected dA-AL-I in the 14 Romanian cases at levels ranging from 0.7 to 27 adducts per 10(8) DNA bases, in line with levels reported in Asian and Balkan populations exposed through herbal remedies or food contamination. The 15 cases from other countries were negative. INTERPRETATION: Although the source of exposure is uncertain and likely different in AAN regions than elsewhere, our results demonstrate that AA exposure in Romania exists outside localised AAN regions and provide further evidence implicating AA in RCC.

Detection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5'-Cyclo-2'-deoxyadenosine by ³²P-Postlabeling Assay.

8,5'-Cyclopurine-2'-deoxynucleotides represent a class of oxidative DNA lesions that are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. The 32P-postlabeling assay is an ultrasensitive method that has been extensively used for the detection of carcinogen-DNA adducts in laboratory animal and epidemiological studies. This assay under modified chromatographic conditions is also a suitable and sensitive method for the detection of 8,5'-cyclo-2'-deoxyadenosine (cA). After enzymatic digestion of DNA, and enrichment of the oxidative products from the DNA digest, four dinucleotides containing cA, i.e., Ap-cAp, Cp-cAp, Gp-cAp, and Tp-cAp, are 5'-labeled with [32P]orthophosphate from [γ-32P]ATP, mediated by polynucleotide kinase (PNK). The 32P-labeled cA products are separated by two-dimensional thin-layer chromatography (TLC) and quantified by Instant Imager or by a scintillation counter. The assay only requires 1 to 10 µg of DNA sample and is capable of detecting cA lesions at frequencies as low as 1 in 1010 normal nucleotides. © 2015 by John Wiley & Sons, Inc.