Cytosine Research Articles

LncRNA FAM83H-AS1 inhibits ferroptosis of endometrial cancer by promoting DNMT1-mediated CDO1 promoter hypermethylation

Endometrial cancer (EC) is the most prevalent gynecological epithelial malignancy. DNA methylation is a promising cancer biomarker, but limited use for detecting EC. We previously found that the level of cysteine dioxygenase 1 (CDO1) promoter methylation was elevated in EC patients through methylomics, but the role and mechanism of CDO1 in EC remained unclear. Here, the methylation level of CDO1 promoter was detected by Bisulfite-sequencing PCR (BSP) and methylation-specific PCR (MSP) (bisulfite-conversion-based PCR methods, which remain the most commonly used techniques for methylation detection). Cells were incubated with erastin (the ferroptosis activator). Cell vitality was measured using the cell counting kit-8 (CCK-8) assay. FAM83H-AS1 cellular distribution was analyzed by the fluorescence in situ hybridization (FISH) assay. Lipid ROS level was examined by BODIPY-C11 staining. The interactions between FAM83H-AS1, CDO1, and DNA methyltransferase1 (DNMT1) were analyzed by RNA binding protein immunoprecipitation (RIP) or chromatin immunoprecipitation (ChIP) assay. The xenograft mouse model was utilized to test CDO1 and FAM83H-AS1's influence on tumor development in vivo. Results showed that CDO1 was hypermethylated and downregulated in EC. CDO1 knockdown reduced erastin-induced ferroptosis in EC cells. Mechanistically, DNMT1 is a DNA methyltransferase, which can transfer methyl groups to cytosine nucleotides in genomic DNA. Long non-coding RNA (lncRNA) FAM83H-AS1 increased CDO1 promoter methylation level and inhibited its expression in EC cells by recruiting DNMT1. CDO1 knockdown or FAM83H-AS1 overexpression promoted EC tumor growth in vivo. LncRNA FAM83H-AS1 inhibited ferroptosis in EC by recruiting DNMT1 to increase CDO1 promoter methylation level and inhibit its expression.

Identifying mutation region of herbicide-resistant Mutant Quinoa (Chenopodium quinoa Willd.) lines by molecular characterization

Quinoa can be considered as an alternative plant with its features being compatible with unfavorable climate and soil conditions. Moreover, the nutrient content of its seed is great. But there is not a selective herbicide for weeds in quinoa fields. This is a significant problem in the quinoa cultivation. Therefore, it is necessary to develop herbicide-resistant quinoa lines In the study, Tititcaca, a variety of quinoa was used. Sodium azide was used 1.5 mM for mutagenesis. Herbicide-resistant plants were determined by applying herbicide belonging to the IMI group in the M3 generation. These were named ET-6, ET-7, OT-11 and T-103. The 4 mutant lines obtained as a result of seed mutagenesis, and mutant line OT-11 plants occurred in the form of replacement of the cytosine nucleotide with the adenine nucleotide (CA) in the ALS gene. Line ET-6 plants, the thymine nucleotide was replaced with a guanine nucleotide (TG). The mutation occurring in the OT-11 and ET-6 genotypes was evaluated as a transversion-type mutation. A transition type mutation occurred in mutant line T-103 plants as well, and this mutation occurred in the form of substitution of thymine nucleotide with cytosine nucleotide (TC) at nucleotide 1114. According to the results, weed control in agriculture can be done effectively by developing varieties resistant to IMI group herbicides in quinoa plants. Herbicide resistance studies should be continued in the next generations with the ET-6, OT-11, and T-103 lines.

Salivary metabolomic biomarkers for esophageal and gastric cancers by liquid chromatography-mass spectrometry.

Early detection of esophageal and gastric cancers is essential for patients' prognosis; however, optimal noninvasive screening tests are currently not available. Saliva is a biofluid that is readily available, allowing for frequent screening tests. Thus, we explored salivary diagnostic biomarkers for esophageal and gastric cancers using metabolomic analyses. Saliva samples were collected from patients with esophageal (n = 50) and gastric cancer (n = 63), and patients without cancer as controls (n = 20). Salivary metabolites were analyzed by liquid chromatography-mass spectrometry to identify salivary biomarkers. We also examined the metabolic profiles of gastric cancer tissues and compared them with the salivary biomarkers. The sensitivity of the diagnostic models based on salivary biomarkers was assessed by comparing it with that of serum tumor markers. Additionally, using postoperative saliva samples collected from patients with gastric cancer, we analyzed the changes in the biomarkers' concentrations before and after surgery. Cytosine was detected as a salivary biomarker for gastric cancer, and cytosine, 2-oxoglutarate, and arginine were detected as salivary biomarkers for esophageal cancer. Cytidine, a cytosine nucleotide, showed decreased concentrations in gastric cancer tissues. The sensitivity of the diagnostic models for esophageal and gastric cancers was 66.0% and 47.6%, respectively, while that of serum tumor markers was 40%. Salivary cytosine concentration increased significantly postoperatively relative to the preoperative value. In summary, we identified salivary biomarkers for esophageal and gastric cancers, which showed diagnostic sensitivity at least comparable to that of serum tumor markers. Salivary metabolomic tests could be promising screening tests for these types of cancer.

Sequence-Dependent Surface Coverage of ssDNA Coatings on Single-Wall Carbon Nanotubes.

A combination of experimental measurements and molecular dynamics (MD) simulations was used to investigate how the surfaces of single-wall carbon nanotubes (SWCNTs) are covered by adsorbed ssDNA oligos with different base compositions and lengths. By analyzing the UV absorption spectra of ssDNA-coated SWCNTs before and after coating displacement by a transparent surfactant, the mass ratios of adsorbed ssDNA to SWCNTs were determined for poly-T, poly-C, GT-containing, and AT-containing ssDNA oligos. Based on the measured mass ratios, it is estimated that an average of 20, 22, 26, or 32 carbon atoms are covered by one adsorbed thymine, cytosine, adenine, or guanine nucleotide, respectively. In addition, the UV spectra revealed electronic interactions of varying strengths between the nucleobase aromatic rings and the nanotube π-systems. Short poly-T DNA oligos show stronger π-π stacking interactions with SWCNT surfaces than do short poly-C DNA oligos, whereas both long poly-C and poly-T DNA oligos show strong interactions. These experiments were complemented by MD computations on simulated systems that were constrained to match the measured ssDNA/SWCNT mass ratios. The surface coverages computed from the MD results varied with oligo composition in a pattern that correlates higher measured yields of nanotube fluorescence with greater surface coverage.

Silver induced chirality controlled spin filtration observed in ss-DNA functionalized with MoS2.

Chiral molecules can exhibit strong spin-orbit coupling, which can result in a large spin polarization. This is due to the fact that the energy levels of the electrons in a chiral molecule are strongly influenced by the chiral structure of the molecule, which can result in the separation of the energy levels for electrons with different spin orientations. We report a controlled spin-selective transmission of electrons through 20 base-paired poly-cytosine molecules functionalized with MoS2 flakes on ITO glass via the quantum mechanical tunneling effect. A reversion in spin polarization was observed after the silver ions interact with poly-cytosine due to the strong coordination of Ag(I) with cytosine-cytosine (C-C) mismatches, indicating the formation of duplex structural motifs, as confirmed by the circular dichroism spectroscopy at room temperature. Manipulating the spin of an electron through such a small molecule merely controlled by special cations could pave the way for major advances in spin-independent charge transport, advanced bioanalytical system design, and related applications.

Novel mutation in efflux pump Rv1258c (Tap) gene in drug resistant clinical isolates of Mycobacterium tuberculosis in Iran.

Tuberculosis (TB) remains a serious public health problem worldwide. Drug-resistant TB is considered a major and growing global threat. Despite the great variety of described mutations in Mycobacterium tuberculosis (MTB) resistance genes, the mechanisms of drug resistance are still controversial. Recently, a report on the role of efflux pump genes in drug resistance added to this complexity. Therefore, a thorough understanding of efflux pump genes in drug-resistant TB clinical isolates is needed. We performed molecular analysis of the efflux pump gene (Rv1258c) in 33 drug-resistant and 20 drug-sensitive clinical MTB isolates by sequencing the amplicons' targets in both the forward and reverse directions. A novel mutation of the Rv1258c gene was identified at G442A (Ala148Thr) in rifampicin mono-resistant clinical strain, as compared to the H37Rv reference strain. In addition, a cytosine nucleotide insertion was found between the positions 580 and 581 (denominated Tap580) in two drug-sensitive strains at identical gene positions. These results indicated the possibility of mutation in the efflux pump genes and the important role of Tap efflux pump genes in drug-resistant MTB isolates. However, further research is required to determine the direct association of these mutations with resistant MTB.

5-Chloro-2'-deoxycytidine Induces a Distinctive High-Resolution Mutational Spectrum of Transition Mutations In Vivo.

The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissues. Replication of a site-specific 5ClC in a viral DNA genome results in C → T mutations, which is consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune-cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, nonmutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool by exposing cells to the nucleoside 5-chloro-2'-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. High-resolution sequencing of DNA from MEFs derived from the gptΔ C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG → TA transitions in 5'-GC(Y)-3' contexts (Y = a pyrimidine) were dominant, while TA → CG transitions appeared at a much lower frequency. The high-resolution mutational spectrum of 5CldC revealed a notable similarity to the Catalogue of Somatic Mutations in Cancer mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively. SBS84 is associated with the expression of activation-induced cytidine deaminase (AID), a cytosine deaminase associated with inflammation, as well as immunoglobulin gene diversification during antibody maturation. The similarity between the spectra of AID activation and 5CldC could be coincidental; however, the administration of 5CldC did induce some AID expression in MEFs, which have no inherent expression of its gene. In summary, this work shows that 5CldC induces a distinct pattern of mutations in cells. Moreover, that pattern resembles human mutational signatures induced by inflammatory processes, such as those triggered in certain malignancies.

Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.

DNA Methylation Changes in Nucleated Red Blood Cells from Patients with Sickle Cell Disease: A Potential Marker of Engraftment after Stem Cell Transplantation

Hematopoietic cell transplant (HCT) is the only established cure for sickle cell disease (SCD). Engraftment (chimerism) of donor red blood cells (RBCs) determines HCT success but current methods measure only white blood cell chimerism and may be inaccurate or fail to indicate graft rejection in a timely manner. Measurement of differentially methylated regions (DMRs) in nucleated red blood cells (nRBCs) to distinguish individuals with sickle cell anemia (genotype HbSS) nRBCs from HbAA and HbAS nRBCs to quantify RBC chimerism could improve post-HCT monitoring by increasing sensitivity to detect rejection and allow for early intervention. Cell-free DNA derived from nRBCs represents a potential method to specifically measure RBC chimerism. Methylation of cytosine nucleotides that are followed by a guanine (methylated CpG sites) is a major means of cell-specific gene expression, and measuring regions of multiple differentially methylated cytosines (DMRs) enables the identification of cell-of-origin of DNA fragments. Cell-free DNA is released into the circulation from all body tissues and cells, including nRBCs, and retains their cell-specific methylation signatures (figure 1). We hypothesized that DMRs unique to healthy and/or SCD nRBC DNA could be identified and later quantified to assess RBC donor chimerism. We generated genome-wide methylomes of nRBC DNA from pediatric SCD (n=2) and non-SCD control (n=3) patients using enzymatic methyl-sequencing (figure 2). From these 5 novel nRBC methylomes and publicly available methylomes and hydroxymethylomes from 35 different tissues and hematopoietic cells we computationally identified 105 DMRs uniquely found in nRBCs compared to other tissues and cells. There were 34 DMRs that specifically distinguished control nRBCs from SCD nRBCs. The 105 DMRs unique to nRBCs were all relatively hypomethylated. These DMRs were intergenic and not associated with CpG islands or shores. DMRs that distinguished SCD nRBCs from control nRBCs were predominantly hypermethylated (76%) in SCD nRBCs and were commonly (21%-24%) located at promoter, exon or intron sequences while 47% were intergenic. 24% were in CpG islands and 5% were in CpG shores. The most significant gene ontology terms in SCD-specific nRBC DMRs were cardiac-related, including heart contraction and conduction. Other highly significant terms were related to cell signaling, transmembrane transporters, and ion channels. Enriched pathway analysis identified terms related to cell signaling, though these were associated with neuronal and immune signaling pathways. To validate our in silico findings, we amplified 8 DMRs from genomic DNA isolated from 23 different cells and tissues and compared methylation patterns. Four DMRs were relatively hypomethylated in control nRBCs compared to SCD nRBCs and other tissues and cells. Amplification of these DMRs from cell-free DNA in patient plasma before and after HCT will validate them as potential unique markers of successful RBC chimerism. Quantification in recipients with HbAA and HbAS donors will be performed. The successful identification of nRBC-specific DMRs in plasma cell-free DNA will enable novel methodology for the quantification of RBC chimerism after HCT for SCD that can be compared to conventional methods of chimerism measurement in future clinical trials.

Enhancing flame retardancy of poly(lactic acid) with a novel fully biobased flame retardant synthesized from phytic acid and cytosine

AbstractIn order to reduce environmental pollution and improve material safety, biobased flame‐retardant polymers are gradually becoming a new trend. We synthesized a fully biobased flame retardant called PACY by reacting phytic acid (PA) and cytosine (CY) in water, and then mixed the flame retardant with biodegradable poly(lactic acid) (PLA) to obtain composites, characterizing the performance of the composites by microscopic morphology, thermal properties, flame‐retardant performance and so on. The test results showed that the PLA composite with 2 wt% PACY (PLA/2PACY) achieved UL‐94V‐1 and the PLA composite with 4 wt% PACY (PLA/4PACY) had a limiting oxygen index of 23.6%. Compared to pure PLA, PLA/4PACY had good flame retardancy with 12% less peak heat release and 19% less total heat release. Analysis of the flame‐retardant mechanism showed that PACY could promote the formation of a graphitized carbon layer after burning PLA, therefore enhancing the flame‐retardant properties of PLA. Through a thermal stability study, it was found that the temperature required for PACY mass loss of 5% was 275 °C and the residual weight reached 38% at 750 °C, which indicated high thermal stability and residual carbon rate. The addition of PACY also improved the thermal conductivity of PLA, with that of PLA/4PACY being 3.84 times higher than that of pure PLA. In addition, the tensile strength, flexural modulus, maximum force during impact and maximum energy received during fracture of PLA/4PACY increased by 3.3%, 8.1%, 29.2% and 17.5% respectively over pure PLA, effectively improving the mechanical properties of PLA. Overall, a fully biobased flame retardant was synthesized for improving the fire resistance of PLA degradable polymers. © 2023 Society of Industrial Chemistry.

DNA methylation differences between stick insect ecotypes.

Epigenetic mechanisms, such as DNA methylation, can influence gene regulation and affect phenotypic variation, raising the possibility that they contribute to ecological adaptation. Beginning to address this issue requires high-resolution sequencing studies of natural populations to pinpoint epigenetic regions of potential ecological and evolutionary significance. However, such studies are still relatively uncommon, especially in insects, and are mainly restricted to a few model organisms. Here, we characterize patterns of DNA methylation for natural populations of Timema cristinae adapted to two host plant species (i.e. ecotypes). By integrating results from sequencing of whole transcriptomes, genomes and methylomes, we investigate whether environmental, host and genetic differences of these stick insects are associated with methylation levels of cytosine nucleotides in the CpG context. We report an overall genome-wide methylation level for T. cristinae of ~14%, with methylation being enriched in gene bodies and impoverished in repetitive elements. Genome-wide DNA methylation variation was strongly positively correlated with genetic distance (relatedness), but also exhibited significant host-plant effects. Using methylome-environment association analysis, we pinpointed specific genomic regions that are differentially methylated between ecotypes, with these regions being enriched for genes with functions in membrane processes. The observed association between methylation variation and genetic relatedness, and with the ecologically important variable of host plant, suggests a potential role for epigenetic modification in T. cristinae adaptation. To substantiate such adaptive significance, future studies could test whether methylation can be transmitted across generations and the extent to which it responds to experimental manipulation in field and laboratory studies.

Myricetin causes site-specific DNA damage via reactive oxygen species generation by redox interactions with copper ions.

Myricetin (MYR), found in tea and berries, may have preventive effects on diseases, including Alzheimer's disease and cancer. However, MYR is also a mutagen, inducing DNA damage in the presence of metal ions. We have studied the molecular mechanisms of DNA damage by MYR in the presence of Cu(II) (MYR+Cu). Using 32P-5'-end-labeled DNA fragments, we analyzed site-specific DNA damage caused by MYR+Cu. MYR+Cu caused concentration-dependent DNA strand breaks and base alterations, leading to cleavage of DNA at thymine, cytosine, and guanine nucleotides. Formation of the oxidative DNA damage indicator, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in calf thymus DNA was increased by MYR+Cu. The production of 8-oxodG in MYR-treated HL-60 cells was significantly higher than in HP100 cells, which are more resistant to H2O2 than are HL-60 cells. Reactive oxygen species (ROS) scavengers were used to elucidate the mechanism of DNA damage. DNA damage was not inhibited by typical free hydroxyl radical (•OH) scavengers such as ethanol, mannitol, or sodium formate. However, methional, catalase, and bathocuproine inhibited DNA damage induced by MYR+Cu. These results suggest that H2O2, Cu(I), and ROS other than •OH are involved in MYR+Cu-induced DNA damage. We conclude that the Cu(I)/Cu(II) redox cycle and concomitant H2O2 production via autoxidation of MYR generate a complex of H2O2 and Cu(I), probably Cu(I)-hydroperoxide, which induces oxidative DNA damage.

Evolution of High-Affinity ssDNA Sequence to Carbon Nanotube

Colloidal ssDNA-wrapped single walled carbon nanotube (ssDNA-SWCNT) has been used as the second near-infrared (NIR-II, 1000 -1700 nm) fluorescent nanoprobes with outstanding biocompatibility, and acts as various optical nano sensors for biological analytes such as a dopamine and serotonin. The stability and affinity of ssDNA on the SWCNT surface significantly depends on the sequence of ssDNA, however the large-scale quantitative analysis between ssDNA sequence library, which has high affinity to the SWCNT surface, was repeatedly selected from a random ssDNA(N30) library (~1018) and the resulting sequence data was analyzed. The binding affinity of ssDNA to SWCNT was quantitatively characterized. To quantitatively analyze the kinetics of the process which occurs solvatochromic shift by sodium cholate, assumed the dynamics follow a first-order kinetic model and fit the experimental data using exponential regression, from which decay time constants were determined. As a result, the highest binding affinity of ssDNA to SWCNT was quantitatively characterized by time constant (1.5 X106 sec) which increased by 100 times compared to the N30 random library. Through analysis of the ssDNA sequences with high affinity for the SWCNT surface, we found that the sequences contain high contents of cytosine and adenine nucleotides and complementary base pairs appeared at distance of 5 to 8 bases. This novel sequence library can be applied for the preparation of the ultra-stable ssDNA-SWCNT dispersion and used for anchoring surface ligands for SWCNT as biosensor. Figure 1

ASSOCIATION BETWEEN ATP1A1 GENE POLYMORPHISMS WITH SEMEN CHARACTERISTICS IN HOLSTEIN BULLS

This study was conducted to identify the polymorphism of ATP1A1 gene, distribution ratios, and its association with semen characteristics of Holstein bulls. Twenty-five Holstein bulls were used currently. Blood samples were collected via venipuncture to separate the DNA and identify the polymorphism of the ATP1A1 gene. Semen was collected using an artificial vagina for fresh evaluation. Results were showed the change of cytosine (C) nucleotide at site 15738 to adenine (A) at the exon 17 (SNP C15738A) of the ATP1A1 gene. Two polymorphisms were obtained according to this change, the first is wild (CC; allele frequency 0.84), and the second is heterogeneous (CA; allele frequency 0.16). The CC polymorphism of the ATP1A1 gene exhibited a highly significant (p<0.01) superiority for ejaculate volume, sperm concentration, and mass activity, and for sperm’s cell individual motility (p<0.05), compared with the CA polymorphism of Holstein bulls. The other semen characteristics did not differ between the two mentioned polymorphisms. This polymorphism exhibited obvious semen characteristics despite stress conditions of feed shortage and poor management practices accompanied with the covid-19 pandemic. This gene can be used to predict the fertility and stress resistance of bulls before they reach sexual maturity and its reflection on the economic return of breeding these bulls at the artificial insemination centers.

Hidden Compositional Heterogeneity of Fish Chromosomes in the Era of Polished Genome Assemblies

Fish chromosomes are considered homogeneous in their AT/GC nucleotide composition, and banding patterns enabling identification of homologs are largely missing. While cytogenomic approaches try to compensate for this issue by virtual karyotyping, they rely on the quality of genome assemblies available. Recently, soft-masked genome assemblies combining costly and arduous long- and short-read sequencing and new generation assemblers became available for two teleost fish species, climbing perch (Anabas testudineus) and channel bull blenny (Cottoperca gobio). Soft-masking turns repetitive sequences in a genome assembly into lower case letters, leaving unique sequences in upper case. This enables investigators to assess the proportion of guanine and cytosine nucleotides (GC%) of transposable elements as an indicator of AT/GC homogenisation in fish. We have developed a new version of our Python tool Evan, which utilises chromosome-level genome assemblies and combines the profiles of GC% and the proportion of repeats (rep%) along chromosomes. Our profiles of both of those fishes showed clear and abrupt but small-scale fluctuations in GC% along otherwise compositionally homogenised sequences. Our study also highlights the key role of the sliding window size in determining the resolution of GC% profiling. While the quality of the genome assemblies appeared to be sufficient for GC%/rep% profiling, more effective repeat masking is necessary to better distinguish to what extent repeats compositionally homogenize fish genomes.

DNA Hypomethylation Is Associated with Increased Inflammation in Peripheral Blood Neutrophils of Children with Autism Spectrum Disorder: Understanding the Role of Ubiquitous Pollutant Di(2-ethylhexyl) Phthalate.

Autism spectrum disorder (ASD) is a multidimensional disorder in which environmental, immune, and genetic factors act in concert to play a crucial role. ASD is characterized by social interaction/communication impairments and stereotypical behavioral patterns. Epigenetic modifications are known to regulate genetic expression through various mechanisms. One such mechanism is DNA methylation, which is regulated by DNA methyltransferases (DNMTs). DNMT transfers methyl groups onto the fifth carbon atom of the cytosine nucleotide, thus converting it into 5-methylcytosine (5mC) in the promoter region of the DNA. Disruptions in methylation patterns of DNA are usually associated with modulation of genetic expression. Environmental pollutants such as the plasticizer Di(2-ethylhexyl) phthalate (DEHP) have been reported to affect epigenetic mechanisms; however, whether DEHP modulates DNMT1 expression, DNA methylation, and inflammatory mediators in the neutrophils of ASD subjects has not previously been investigated. Hence, this investigation focused on the role of DNMT1 and overall DNA methylation in relation to inflammatory mediators (CCR2, MCP-1) in the neutrophils of children with ASD and typically developing healthy children (TDC). Further, the effect of DEHP on overall DNA methylation, DNMT1, CCR2, and MCP-1 in the neutrophils was explored. Our results show that the neutrophils of ASD subjects have diminished DNMT1 expression, which is associated with hypomethylation of DNA and increased inflammatory mediators such as CCR2 and MCP-1. DEHP further causes downregulation of DNMT1 expression in the neutrophils of ASD subjects, probably through oxidative inflammation, as antioxidant treatment led to reversal of a DEHP-induced reduction in DNMT1. These data highlight the importance of the environmental pollutant DEHP in the modification of epigenetic machinery such as DNA methylation in the neutrophils of ASD subjects.

Potential use of LIAD time-of-flight mass spectrometry for the detection of biomolecules: An example of detecting nucleobases in DNA

Deoxyribonucleic acid (DNA) carries the genetic information necessary for the synthesis of RNA and proteins; it is a biological macromolecule essential for the development and proper functioning of living organisms and is composed of nucleobases, deoxyribose, and phosphate. The four nucleobases in DNA are adenine (AD), guanine (GU), thymine (TY), and cytosine (CY). Abnormal concentrations of these four nucleobases in an organism have a significant impact on disease diagnosis. Therefore, the qualitative and quantitative detection of these DNA nucleobases in organisms is helpful to diagnose certain diseases. In this work, we report the simultaneous determination of purine (AD, GU) and pyrimidine (TY, CY) nucleobases in DNA using laser-induced acoustic desorption (LIAD) with electron ionization (EI)/time-of-flight mass spectrometry (TOFMS). The purine (MW 120 Da) samples were used as model compounds to assess the sensitivity and quantitative performance of the instrument. Its limits of detection assessed using the LIAD/EI/MS method were ∼0.5–1.2 pg under optimal conditions, and their calibration curves exhibited good linearity (R2 = 0.98). The LIAD/TOFMS was successfully applied in the simultaneous detection of AD, GU, TY, and CY in real DNA samples. The advantage of this technique is simple, fast, and without complex pre-treatment processes. In addition, a quartz-enhanced LIAD (QE-LIAD) source was used to improve the signal strength. The desorption for complex biomolecules shows that the QE-LIAD is still a “gentle” desorption source.

Synchronous electrochemical detection of nanomolar acetaminophen, cytosine and phenylephrine hydrochloride in drugs using Zn3V2O8/ZrO2@f-MWCNTs nanocomposite GC electrode

The fabrication of ternary nanocomposite is crucial since a multiphase interface provides an enormously sensitive and selective mechanism for the individual and synchronized electrochemical assessment of medicines. Here, we present an electrochemical sensor for concomitantly determining the drugs acetaminophen (AAP), cytosine (CTS), and phenylephrine hydrochloride (PPH) using Zn3V2O8/ZrO2@f-MWCNTs a ternary nanocomposite. At the electrochemical probe, we used CV and DPV to investigate the performance of the several modified electrodes and found that Zn3V2O8/ZrO2@f-MWCNTs nanocomposites were enormously selective and sensitive for detecting AAP, CTS, and PPH separately and simultaneously. The DPV technique yielded sensitive electrooxidation pinnacle potentials of 0.329, 1.276, and 0.658 V for AAP, CTS, and PPH, respectively. AAP, CTS, and PPH all have linear responses of 0.0055 to 435.6, 0.0094 to 365.5, and 0.0098 to 134.8 μM, respectively. The Zn3V2O8/ZrO2@f-MWCNTs composite exhibited stability, repeatability, and reproducibility. AAP, CTS, and PPH had lower detection limits of 0.00617, 0.00345, and 0.00654 M, respectively. Real sample analysis with Zn3V2O8/ZrO2@f-MWCNTs/GCE is useful for the concurrent detection of AAP, CTS, and PPH in pharmaceuticals, calf thymus DNA, and human serum samples.

Enzymatic properties of CARF-domain proteins in Synechocystis sp. PCC 6803.

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.

Adsorption of cytosine on prebiotic siliceous clay surface induced with metal dications: Relevance to origin of life

Adsorption of protobiomolecules on primigenial mineral surfaces transacts a very specific and selective role in prebiotic chemical evolution. Cytosine (CYN) is a core constituent of genetic macromolecules i.e., DNA and RNA. A prebiotic simulation experiment following adsorption behaviour of a nucleic acid base CYN, as a function of pH, concentration, time and temperature on the surface of Montmorillonite (MMT), a prominent siliceous clay of smectite group having specific surface area, was studied systematically and methodically using UV, FTIR, SEM and XRD. Biologically significant metal dication [M2+ = Mg2+, Ca2+, Fe2+ and Cu2+] embeded surfaces of MMT (MMT-M2+) were prepared separately by cation-exchange approach. Results observed through UV spectral data reveal a very significant role of metal ions on the quantity of CYN adsorbed on different solid surfaces. The adsorption behaviour fitted well with Langmuir isotherm model and the adsorption isotherm shows monolayer formation of CYN (as adsorbate) on the surface of MMT and MMT-M2+(as adsorbent) showing Langmuir type adsorption. The calculated Langmuir adsorption parameters (KL& Xm) delineate appreciable interaction of CYN on MMT-Fe2+ surface following MMT-Cu2+ as compared to MMT alone and to other metal induced MMTs.In terms of % binding and Xm data, the effectiveness of various adsorbents in adsorption of CYN on MMT/MMT-M2+ at pH 6.6 and temperature 298K was found in the order; MMT-Fe2+> MMT-Cu2+> MMT > MMT-Ca2+> MMT-Mg2+. The results were further confirmed by SEM, XRD and FTIR which also support the outcomes. The present work throws light on the affinity of MMT clay (having multifarious applications in biological systems) with and without biogenic metal diions towards the surface interaction of nucleic acid base mimicking primeval sea shore/sea bed or hydrothermal vent conditions deciphering the phenomena that how biomonomers could have been protected from extreme hazardous conditions of primitive Earth and stabilized during chemical evolution and availed them for consecutive molecular evolution. Results further embark better adsorption capability of transition metal ion incorporated MMTs than the clay without metal ion and exchanged with alkaline Earth dications.