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We have investigated the impact of microsolvation on shape-type resonance states of nucleobases, taking cytosine as a case study. To characterize the resonance position and decay width of the metastable states, we employed the newly developed DLPNO-based EA-EOM-CCSD method in conjunction with the resonance via Padé (RVP) method. Our calculations show that the presence of water molecules causes a redshift in the resonance position and an increase in the lifetime for the three lowest-lying resonance states of cytosine. Furthermore, there are some indications that the lowest resonance state in isolated cytosine may get converted to a bound state in the presence of an aqueous environment. The obtained results are extremely sensitive to the basis set used for the calculations. [ABSTRACT FROM AUTHOR]

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Biologically active compounds are highly sought‐after materials for developing novel structures applicable to industry. Cytosine and pyridine‐2,3‐dicarboxylic acid (quinolinic acid) are notably significant environmentally. Cytosine, a pyrimidine derivative, features a six‐membered ring with a ketone and an amino group, constituting a fundamental nitrogenous base found in deoxyribonucleic acid (DNA). The present synthesis yielded a salt of dipyridine‐2,3‐dicarboxylic acid with cytosine, wherein a proton was transferred from a carboxyl group of quinolinic acid to a ring N atom in the cytosine molecule giving the salt 6‐amino‐2‐oxo‐2,3‐dihydropyrimidin‐1‐ium 3‐carboxypyridine‐2‐carboxylate, C4H6N3O+·C7H4NO4−. A Hirshfeld surface analysis was conducted to examine the contribution of contacts within the salt. The structure of the salt was compared to other structures containing quinolinic acid in the Cambridge Structural Database (CSD). [ABSTRACT FROM AUTHOR]

This study delves into the nature of individual hydrogen bonds and the relationship between metal cations and hydrogen bonding in the Watson–Crick guanine–cytosine (GC) base pair and its alkali and alkaline earth cation‐containing complexes (Mn+–GC). The findings reveal how metal cations affect the nature and strength of individual hydrogen bonds. The study employs interacting quantum atoms (IQA) analysis to comprehensively understand three individual hydrogen bonds within the GC base pair and its cationic derivatives. These analyses unveil the nature and strength of hydrogen bonds and serve as a valuable reference for exploring the impact of cations (and other factors) on each hydrogen bond. All the H⋯D interactions (H is hydrogen and D is oxygen or nitrogen) in the GC base pair are primarily electrostatic in nature, with the charge transfer component playing a substantial role. Introducing a metal cation perturbs all H⋯D interatomic interactions in the system, weakening the nearest hydrogen bond to the cation (indicated by a) and reinforcing the other (b and c) interactions. Notably, the interaction a, the strongest H⋯D interaction in the GC base pair, becomes the weakest in the Mn+–GC complexes. A broader perspective on the stability of GC and Mn+–GC complexes is provided through interacting quantum fragments (IQF) analysis. This approach considers all pairwise interactions between fragments and intra‐fragment components, offering a complete view of the factors that stabilize and destabilize GC and Mn+–GC complexes. The IQF analysis underscores the importance of electron sharing, with the dominant contribution arising from the inter‐fragment exchange‐correlation term, in shaping and sustaining GC and Mn+–GC complexes. From this point of view, alkaline and alkaline earth cations have distinct effects, with alkaline cations generally weakening inter‐fragment interactions and alkaline earth cations strengthening them. In addition, IQA and IQF calculations demonstrate that the hydration of cations led to small changes in the hydrogen bonding network. Finally, the IQA interatomic energies associated with the hydrogen bonds and also inter‐fragment interaction energies provide robust indicators for characterizing hydrogen bonds and complex stability, showing a strong correlation with total interaction energies. [ABSTRACT FROM AUTHOR]

Base editors enable the direct conversion of target bases without inducing double-strand breaks, showing great potential for disease modeling and gene therapy. Yet, their applicability has been constrained by the necessity for specific protospacer adjacent motif (PAM). We generate four versions of near-PAMless base editors and systematically evaluate their editing patterns and efficiencies using an sgRNA-target library of 45,747 sequences. Near-PAMless base editors significantly expanded the targeting scope, with both PAM and target flanking sequences as determinants for editing outcomes. We develop BEguider, a deep learning model, to accurately predict editing results for near-PAMless base editors. We also provide experimentally measured editing outcomes of 20,541 ClinVar sites, demonstrating that variants previously inaccessible by NGG PAM base editors can now be precisely generated or corrected. We make our predictive tool and data available online to facilitate development and application of near-PAMless base editors in both research and clinical settings. Systematic evaluation of near-PAMless base editors enables a robust deep learning model for predicting editing outcomes, facilitating broader and more precise targeting of disease-associated variants for research and therapy. [ABSTRACT FROM AUTHOR]

The engineered TadA variants used in cytosine base editors (CBEs) present distinctive advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employ TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieves robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enriches the base editing toolkits for gene-editing therapy and other potential applications. Engineered TadA variants in cytosine base editors (CBEs) offer advantages like smaller size and reduced off-target effects. Here, authors develop an advanced CBE (aTdCBE) through ortholog screening and protein engineering, successfully enabling exon skipping in Duchenne muscular dystrophy model. [ABSTRACT FROM AUTHOR]

Due to different solvent conditions, double helix DNA exists in various conformations, such as B-DNA, A-DNA, C-DNA, and Z-DNA. Studies have found that A-DNA is present in complexes with proteins and has an important biological role in the context of cellular defense mechanisms under harsh conditions. In this study, the well-tempered meta-dynamics (WTM-eABF) were used to explore the free energy barriers for base flipping of the four natural bases, adenine, guanine, cytosine, and thymine, in both A-form and B-form DNA duplex. The results show that the free energy barriers for base flipping were lower in A-DNA than that in B-DNA for all of the four natural bases. We analyzed the factors that may affect base flipping, such as π-π stacking, SASA, H-bonding, and conformational changes, and concluded that conformational changes and π-π stacking are the most important factors affecting base flipping. [ABSTRACT FROM AUTHOR]

Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, we report the development of a Continuous Directed Evolution system in Mammalian cells (CDEM). CDEM enables the BE evolution in a full-length manner with Cas9 nickase. We harness CDEM to evolve the deaminases of cytosine base editor BE3 and adenine base editors, ABEmax and ABE8e. The evolved cytidine deaminase variants on BE4 architecture show not only narrowed editing windows, but also higher editing purity and low off-target activity without a trade-off in on-targeting activity. The evolved ABEmax and ABE8e variants exhibit narrowed or shifted editing windows to different extents, and lower off-target effects. The results illustrate that CDEM is a simple but powerful approach to continuously evolve BEs without size restriction in the mammalian environment, which is advantageous over continuous directed evolution system in bacteria cells. Continuous directed evolution of base editors (BEs) has been successful in bacteria cells, but not yet in mammalian cells. Here, the authors report a Continuous Directed Evolution system in Mammalian cells (CDEM) enabling the BE evolution in a full-length manner with Cas9 nickase. [ABSTRACT FROM AUTHOR]

RNA modifications play an important role in actively controlling recently created formation in cellular regulation mechanisms, which link them to gene expression and protein. The RNA modifications have numerous alterations, presenting broad glimpses of RNA's operations and character. The modification process by the TET enzyme oxidation is the crucial change associated with cytosine hydroxymethylation. The effect of CR is an alteration in specific biochemical ways of the organism, such as gene expression and epigenetic alterations. Traditional laboratory systems that identify 5-hydroxymethylcytosine (5hmC) samples are expensive and time-consuming compared to other methods. To address this challenge, the paper proposed XGB5hmC, a machine learning algorithm based on a robust gradient boosting algorithm (XGBoost), with different residue based formulation methods to identify 5hmC samples. Their results were amalgamated, and six different frequency residue based encoding features were fused to form a hybrid vector in order to enhance model discrimination capabilities. In addition, the proposed model incorporates SHAP (Shapley Additive Explanations) based feature selection to demonstrate model interpretability by highlighting the high contributory features. Among the applied machine learning algorithms, the XGBoost ensemble model using the tenfold cross-validation test achieved improved results than existing state-of-the-art models. Our model reported an accuracy of 89.97%, sensitivity of 87.78%, specificity of 94.45%, F1-score of 0.8934%, and MCC of 0.8764%. This study highlights the potential to provide valuable insights for enhancing medical assessment and treatment protocols, representing a significant advancement in RNA modification analysis. [ABSTRACT FROM AUTHOR]

Early embryonic development is crucially important but also remarkably diverse among animal taxa. Axis formation and cell lineage specification occur due to both spatial and temporal control of gene expression. This complex system involves various signaling pathways and developmental genes such as transcription factors as well as other molecular interactants that maintain cellular states, including several types of epigenetic marks. 5mC DNA methylation, the chemical modification of cytosines in eukaryotes, represents one such mark. By influencing the compaction of chromatin (a high‐order DNA structure), DNA methylation can either repress or induce transcriptional activity. Mammals exhibit a reprogramming of DNA methylation from the parental genomes in the zygote following fertilization, and later in primordial germ cells (PGCs). Whether these periods of methylation reprogramming are evolutionarily conserved, or an innovation in mammals, is an emerging question. Looking into these processes in other vertebrate lineages is thus important, and teleost fish, with their extensive species richness, phenotypic diversity, and multiple rounds of whole genome duplication, provide the perfect research playground for answering such a question. This review aims to present a concise state of the art of DNA methylation reprogramming in early development in fish by summarizing findings from different research groups investigating methylation reprogramming patterns in teleosts, while keeping in mind the ramifications of the methodology used, then comparing those patterns to reprogramming patterns in mammals. [ABSTRACT FROM AUTHOR]

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

This study shows that Elastic Net (EN) DNA methylation (DNAme) clocks have low accuracy of predictions for individuals of the same age and a low resolution between healthy and disease cohorts; caveats inherent in applying linear model to non-linear processes. We found that change in methylation of cytosines with age is, interestingly, not the determinant for their selection into the clocks. Moreover, an EN clocks selected cytosines change when non-clock cytosines are removed from the training data; as expected from optimization in a machine learning (ML) context, but inconsistently with the identification of health markers in a biological context. To address these limitations, we moved from predictions to measurement of biological age, focusing on the cytosines that on average remain invariable in their methylation through lifespan, postulated to be homeostatically vital. We established that dysregulation of such cytosines, measured as the sums of standard deviations of their methylation values, quantifies biological noise, which in our hypothesis is a biomarker of aging and disease. We term this approach a noise barometer - the pressure of aging and disease on an organism. These noise-detecting cytosines are particularly important as sums of SD on the entire 450K DNAme array data yield a random pattern through chronology. Testing how many cytosines of the 450K arrays become noisier with age, we found that the paradigm of DNAme noise as a biomarker of aging and disease remarkably manifests in ~1/4 of the total. In that large set even the cytosines that have on average constant methylation through age show increased SDs and can be used as noise detectors of the barometer.

Huntington’s Disease (HD) is a rare neurodegenerative condition inherited in an autosomal dominant pattern, where Gamma Amino Butyric Acid-ergic (GABAergic) neurons in the basal ganglia progressively deteriorate. Symptoms include subcortical dementia, behavioural changes, midlife psychosis, and involuntary choreoathetosis movements. Imaging techniques like Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) show shrinkage of the caudate nucleus and putamen, alongside enlarged lateral ventricles. Diagnosis is confirmed through molecular analysis identifying the hallmark amplified Cytosine Adenine Guanine (CAG) triplet. The case report describes a 57-year-old female diagnosed with HD, presenting symptoms of accidental falls caused by involuntary jerky movements, along with behavioural changes and cognitive decline over three years. Initial Non Contrast Computed Tomography (NCCT) of the brain revealed bilateral basal ganglia and diffuse cerebral atrophy. Quantitative analysis indicated a decreased frontal horn width to intercaudate distance (FH/CC) ratio of 1.24 and an increased Intercaudate distance to inner table width (CC/IT) ratio of 0.30. Multivoxel Magnetic Resonance Spectroscopy (MRS) showed reduced N-Acetyl Alanine (NAA) and creatine (Cr) levels with an NAA/Cr ratio of 0.84. Magnetic Resonance Tractography and Diffusion Tensor Imaging (DTI) revealed elevated Fractional Anisotropy (FA) and diffusivity in the basal ganglia, and reduced FA with increased diffusivity in white matter tracts. Genetic analysis by Polymerase Chain Reaction (PCR) confirmed HD. Newer MRI like DTI allows early identification of disease and better assessment of disease progression and response to therapy. A multidisciplinary approach involving neurologists, psychiatrists, and other healthcare professionals is essential. Genetic counseling and psychosocial support are crucial for patients and their families. Early and accurate identification of HD is vital for timely interventions and improved symptom control.

Complete active space second-order perturbation theory (CASPT2) is useful for accurately predicting properties of complex electronic structures, but it is well known that it systematically underestimates excitation energies. The underestimation can be corrected using the ionization potential–electron affinity (IPEA) shift. In this study, analytic first-order derivatives of CASPT2 with the IPEA shift are developed. CASPT2-IPEA is not invariant with respect to rotations among active molecular orbitals, and two additional constraint conditions are necessary in the CASPT2 Lagrangian to formulate analytic derivatives. The method developed here is applied to methylpyrimidine derivatives and cytosine, and minimum energy structures and conical intersections are located. By comparing energies relative to the closed-shell ground state, we find that the agreement with experiments and high-level calculations is indeed improved by the inclusion of the IPEA shift. The agreement of geometrical parameters with high-level calculations may also be improved in some cases. [ABSTRACT FROM AUTHOR]

The application of iron oxide quantum dots (IOQDs) in this study led to the development of a straightforward, easy, and selective approach for cytosine sensing. To examine the capability of IOQDs in detecting nucleobases, attempts have been made to gain insight into the characteristics of IOQDs, including UV/Vis spectroscopic analysis, infrared spectroscopy, fluorescence spectroscopy, transmission electron microscopy, and powder‐XRD. We introduced IOQDs with a mean particle size distribution of 5.71 ± 0.08 nm for detecting cytosine by harnessing a turn‐on fluorescence phenomenon. The fostering fluorescence response of IOQDs was triggered in the presence of cytosine. In comparison, the presence of other nucleobases did not further induce the fluorescence signal of IOQDs. It was concluded that the hydrogen bonding bridging the carboxylate and hydroxyl groups on the surface of QDs with the amine groups of cytosine results in the elevation of IOQDs fluorescence. [ABSTRACT FROM AUTHOR]

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility. [ABSTRACT FROM AUTHOR]

Nucleobases are essential molecules for life, forming integral parts of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in all terrestrial life forms. Despite evidence of their abiotic synthesis in meteorites and laboratory simulations of interstellar medium (ISM) conditions, nucleobases have not been detected in the ISM. This study investigates the infrared spectra of uracil, cytosine, and thymine – pyrimidine nucleobases – embedded in an ice mixture simulating common volatiles found in protostellar discs. Our objective was to explore the feasibility of identifying unique infrared bands of pyrimidines in the ISM, despite significant overlapping absorption features from simpler, more abundant interstellar species such as H2O, CO, CH3OH, and NH3. Laboratory results revealed that although two common bands (1240 and 760 cm−1 in uracil; 1236 and 763 cm−1 in cytosine; and 1249 and 760 cm−1 in thymine) were identified, the detection of these bands in space is challenged by overlapping absorption features. Recent observations with the JWST have shown that interstellar organic species exhibit infrared signals within similar ranges, making it impossible to distinguish pyrimidine bands from these organics. Thus, detecting pyrimidines with current telescopes is infeasible, not due to sensitivity limitations or the need for more powerful instrumentation, but because of the intrinsic overlap in spectral features. This study complements previous research on purines by examining pyrimidines and including the impact of common ISM volatiles in the ice composition. The results highlight the significant challenges in detecting complex molecules in the ISM, underscoring the importance of understanding the spectral complexities and interactions to interpret astronomical observations accurately. [ABSTRACT FROM AUTHOR]

Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states. [ABSTRACT FROM AUTHOR]

Motivation The alignment of sequencing reads is a critical step in the characterization of ancient genomes. However, reference bias and spurious mappings pose a significant challenge, particularly as cutting-edge wet lab methods generate datasets that push the boundaries of alignment tools. Reference bias occurs when reference alleles are favoured over alternative alleles during mapping, whereas spurious mappings stem from either contamination or when endogenous reads fail to align to their correct position. Previous work has shown that these phenomena are correlated with read length but a more thorough investigation of reference bias and spurious mappings for ancient DNA has been lacking. Here, we use a range of empirical and simulated palaeogenomic datasets to investigate the impacts of mapping tools, quality thresholds, and reference genome on mismatch rates across read lengths. Results For these analyses, we introduce AMBER, a new bioinformatics tool for assessing the quality of ancient DNA mapping directly from BAM-files and informing on reference bias, read length cut-offs and reference selection. AMBER rapidly and simultaneously computes the sequence read mapping bias in the form of the mismatch rates per read length, cytosine deamination profiles at both CpG and non-CpG sites, fragment length distributions, and genomic breadth and depth of coverage. Using AMBER, we find that mapping algorithms and quality threshold choices dictate reference bias and rates of spurious alignment at different read lengths in a predictable manner, suggesting that optimized mapping parameters for each read length will be a key step in alleviating reference bias and spurious mappings. Availability and implementation AMBER is available for noncommercial use on GitHub (https://github.com/tvandervalk/AMBER.git). Scripts used to generate and analyse simulated datasets are available on Github (https://github.com/sdolenz/refbias_scripts). [ABSTRACT FROM AUTHOR]

Aberrant DNA methylation has been identified as biomarkers for breast cancer detection. Coiled-coil domain containing 12 gene (CCDC12) implicated in tumorigenesis. This study aims to investigate the potential of blood-based CCDC12 methylation for breast cancer detection. DNA methylation level of CpG sites (Cytosine-phosphate Guanine dinucleotides) in CCDC12 gene was measured by mass spectrometry in 255 breast cancer patients, 155 patients with benign breast nodules and 302 healthy controls. The association between CCDC12 methylation and breast cancer risk was evaluated by logistic regression and receiver operating characteristic curve analysis. A total of eleven CpG sites were analyzed. The CCDC12 methylation levels were higher in breast cancer patients. Compared to the lowest tertile of methylation level in CpG_6,7, CpG_10 and CpG_11, the highest quartile was associated with 82, 91 and 95% increased breast cancer risk, respectively. The CCDC12 methylation levels were associated with estrogen receptor (ER) and human epidermal growth factor 2 (HER2) status. In ER-negative and HER2-positive (ER-/HER2+) breast cancer subtype, the combination of four sites CpG_2, CpG_5, CpG_6,7 and CpG_11 methylation levels could distinguish ER−/HER2+ breast cancer from the controls (AUC = 0.727). The hypermethylation levels of CCDC12 in peripheral blood could be used for breast cancer detection. Breast cancer detection could be facilitated by novel blood-based DNA methylation biomarkers. The methylation levels of CpG sites in CCDC12 were higher in breast cancer than those in controls. The combination of four sites CpG_2, CpG_5, CpG_6,7 and CpG_11 methylation levels could distinguish ER-/HER2+ breast cancer subtype from the controls. [ABSTRACT FROM AUTHOR]

Owing to the highly targeted affinity of cytosine moieties toward the surface of cancer cells, cytosine-functionalized supramolecular nanogels undergo substantially enhanced specific uptake by cancer cells, which increases the cytotoxicity of the encapsulated anticancer drugs, thereby allowing safer, more effective induction of apoptosis in cancer cells. [Display omitted] • Supramolecular polymers containing cytosine groups (PECH-PEG-Cy) were developed. • PECH-PEG-Cy forms self-assembled nanogels with unique physical properties in water. • The nanogels with cytosine moieties exhibit high specificity for cancer cells. • Drug-loaded PECH-PEG-Cy selectively induces programmed cell death in cancer cells. • This new system has high potential for development of targeted cancer treatment. We demonstrate that cytosine moieties within physically cross-linked supramolecular polymers not only manipulate drug delivery and release, but also confer specific targeting of cancer cells to effectively enhance the safety and efficacy of chemotherapy—and thus hold significant potential as a new perspective for development of drug delivery systems. Herein, we successfully developed physically cross-linked supramolecular polymers (PECH-PEG-Cy) comprised of hydrogen-bonding cytosine pendant groups, hydrophilic poly(ethylene glycol) side chains, and a hydrophobic poly(epichlorohydrin) main chain. The polymers spontaneously self-assemble into a reversibly hydrogen-bonded network structure induced by cytosine and directly form spherical nanogels in aqueous solution. Nanogels with a high hydrogen-bond network density (i.e., a higher content of cytosine moieties) exhibit outstanding long-term structural stability in cell culture substrates containing serum, whereas nanogels with a relatively low hydrogen-bond network density cannot preserve their structural integrity. The nanogels also exhibit numerous unique physicochemical characteristics in aqueous solution, such as a desirable spherical size, high biocompatibility with normal and cancer cells, excellent drug encapsulation capacity, and controlled pH-responsive drug release properties. More importantly, in vitro experiments conclusively indicate the drug-loaded PECH-PEG-Cy nanogels can selectively induce cancer cell-specific apoptosis and cell death via cytosine receptor-mediated endocytosis, without significantly harming normal cells. In contrast, control drug-loaded PECH-PEG nanogels, which lack cytosine moieties in their structure, can only induce cell death in cancer cells through non-specific pathways, which significantly inhibits the induction of apoptosis. This work clearly demonstrates that the cytosine moieties in PECH-PEG-Cy nanogels confer selective affinity for the surface of cancer cells, which enhances their targeted cellular uptake, cytotoxicity, and subsequent induction of programmed cell death in cancer cells. [ABSTRACT FROM AUTHOR]

Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles in OsALS and creating synonymous mutations in OsSPL14 to resist OsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox. Base editors are a powerful but underexplored tool for genetic engineering and directed evolution of plants. Here, the authors investigate the editing efficiency and specificity of TadA-8e-derived cytosine base editors and dual base editor TadDE in rice and tomato. [ABSTRACT FROM AUTHOR]

Nucleotide base composition plays an influential role in the molecular mechanisms involved in gene function, phenotype, and amino acid composition. GC content (proportion of guanine and cytosine in DNA sequences) shows a high level of variation within and among species. Many studies measure GC content in a small number of genes, which may not be representative of genome-wide GC variation. One challenge when assembling extensive genomic data sets for these studies is the significant amount of resources (monetary and computational) associated with data processing, and many bioinformatic tools have not been optimized for resource efficiency. Using a high-performance computing (HPC) cluster, we manipulated resources provided to the targeted gene assembly program, automated target restricted assembly method (aTRAM), to determine an optimum way to run the program to maximize resource use. Using our optimum assembly approach, we assembled and measured GC content of all of the protein-coding genes of a diverse group of parasitic feather lice. Of the 499 426 genes assembled across 57 species, feather lice were GC-poor (mean GC = 42.96%) with a significant amount of variation within and between species (GC range = 19.57%-73.33%). We found a significant correlation between GC content and standard deviation per taxon for overall GC and GC3, which could indicate selection for G and C nucleotides in some species. Phylogenetic signal of GC content was detected in both GC and GC3. This research provides a large-scale investigation of GC content in parasitic lice laying the foundation for understanding the basis of variation in base composition across species. [ABSTRACT FROM AUTHOR]

Genomic aberrations are a critical impediment for the safe medical use of iPSCs and their origin and developmental mechanisms remain unknown. Here we find through WGS analysis of human and mouse iPSC lines that genomic mutations are de novo events and that, in addition to unmodified cytosine base prone to deamination, the DNA methylation sequence CpG represents a significant mutation-prone site. CGI and TSS regions show increased mutations in iPSCs and elevated mutations are observed in retrotransposons, especially in the AluY subfamily. Furthermore, increased cytosine to thymine mutations are observed in differentially methylated regions. These results indicate that in addition to deamination of cytosine, demethylation of methylated cytosine, which plays a central role in genome reprogramming, may act mutagenically during iPSC generation. Here the authors find that mutations detected in iPSCs are de novo by analyzing 135 lines. More than 10,000 point mutations were observed in some lines and, intriguingly, CpG site-specific C>Ts were frequently identified, implying a relationship between DNA demethylation and mutagenesis. [ABSTRACT FROM AUTHOR]

N4-acetylcysteine (ac4C) is a chemical modification in mRNAs that alters the structure and function of mRNA by adding an acetyl group to the N4 position of cytosine. Researchers have shown that ac4C is closely associated with the occurrence and development of various cancers. Therefore, accurate prediction of ac4C modification sites on human mRNA is crucial for revealing its role in diseases and developing new diagnostic and therapeutic strategies. However, existing deep learning models still have limitations in prediction accuracy and generalization ability, which restrict their effectiveness in handling complex biological sequence data. This paper introduces a deep learning-based model, STM-ac4C, for predicting ac4C modification sites on human mRNA. The model combines the advantages of selective kernel convolution, temporal convolutional networks, and multi-head self-attention mechanisms to effectively extract and integrate multi-level features of RNA sequences, thereby achieving high-precision prediction of ac4C sites. On the independent test dataset, STM-ac4C showed improvements of 1.81%, 3.5%, and 0.37% in accuracy, Matthews correlation coefficient, and area under the curve, respectively, compared to the existing state-of-the-art technologies. Moreover, its performance on additional balanced and imbalanced datasets also confirmed the model's robustness and generalization ability. Various experimental results indicate that STM-ac4C outperforms existing methods in predictive performance. In summary, STM-ac4C excels in predicting ac4C modification sites on human mRNA, providing a powerful new tool for a deeper understanding of the biological significance of mRNA modifications and cancer treatment. Additionally, the model reveals key sequence features that influence the prediction of ac4C sites through sequence region impact analysis, offering new perspectives for future research. [ABSTRACT FROM AUTHOR]

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors. Efficient base editors for direct thymine (T) editing are highly desirable. Here, authors develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by rounds of structure-informed mutagenesis on human DNA glycosylase UNG and further engineering. [ABSTRACT FROM AUTHOR]

DNA methylation, an epigenetic mechanism that alters gene expression without changing DNA sequence, is essential for organism development and key biological processes like genomic imprinting and X‐chromosome inactivation. Despite tremendous efforts in DNA methylation research, accurate quantification of cytosine methylation remains a challenge. Here, a single‐base methylation quantification approach is introduced by weighting methylation of consecutive CpG sites (Wemics) in genomic regions. Wemics quantification of DNA methylation better predicts its regulatory impact on gene transcription and identifies differentially methylated regions (DMRs) with more biological relevance. Most Wemics‐quantified DMRs in lung cancer are epigenetically conserved and recurrently occurred in other primary cancers from The Cancer Genome Atlas (TCGA), and their aberrant alterations can serve as promising pan‐cancer diagnostic markers. It is further revealed that these detected DMRs are enriched in transcription factor (TF) binding motifs, and methylation of these TF binding motifs and TF expression synergistically regulate target gene expression. Using Wemics on epigenomic‐transcriptomic data from the large lung cancer cohort, a dozen novel genes with oncogenic potential are discovered that are upregulated by hypomethylation but overlooked by other quantification methods. These findings increase the understanding of the epigenetic mechanism by which DNA methylation regulates gene expression. [ABSTRACT FROM AUTHOR]

In living cells, some reactions can be conducted by more than one enzyme and sometimes it is difficult to establish which enzyme is responsible. Such is the case with proteins from the TET family, capable of converting 5-methyl-2'-deoxycytidine (5- m d C ) in DNA to 5-(hydroxymethyl)-2'-deoxycytidine (5- h m d C ) and further to 5-formyl-2'-deoxycytidine (5- f d C ) and 5-carboxy-2'-deoxycytidine (5- c a d C ). The estimation of the efficiency of particular TETs in particular oxidative reactions and different cell types is important but experimentally difficult. Here, we propose an approach with mathematical modeling in which methylation and known deoxycytidine modification pathways are presented by 343 possible model versions with assumed different combinations of TET1, 2, and 3 activities in different pathways. Model parameters were calculated on the basis of 5- m d C , 5- h m d C , 5- f d C , 5- c a d C , and 5- h m d U levels experimentally assessed in five human cultured cell lines and previously published. Selection of the model versions that give in simulations the best average fit to experimental data suggested that not all TET proteins participate in all modification reactions and that TET3 activity may be especially important in the reaction of 5- f d C removal. [ABSTRACT FROM AUTHOR]

Huntington's Disease (HD) is a rare neurodegenerative condition inherited in an autosomal dominant pattern, where Gamma Amino Butyric Acid-ergic (GABAergic) neurons in the basal ganglia progressively deteriorate. Symptoms include subcortical dementia, behavioural changes, midlife psychosis, and involuntary choreoathetosis movements. Imaging techniques like Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) show shrinkage of the caudate nucleus and putamen, alongside enlarged lateral ventricles. Diagnosis is confirmed through molecular analysis identifying the hallmark amplified Cytosine Adenine Guanine (CAG) triplet. The case report describes a 57-year-old female diagnosed with HD, presenting symptoms of accidental falls caused by involuntary jerky movements, along with behavioural changes and cognitive decline over three years. Initial Non Contrast Computed Tomography (NCCT) of the brain revealed bilateral basal ganglia and diffuse cerebral atrophy. Quantitative analysis indicated a decreased frontal horn width to intercaudate distance (FH/CC) ratio of 1.24 and an increased Intercaudate distance to inner table width (CC/IT) ratio of 0.30. Multivoxel Magnetic Resonance Spectroscopy (MRS) showed reduced N-Acetyl Alanine (NAA) and creatine (Cr) levels with an NAA/Cr ratio of 0.84. Magnetic Resonance Tractography and Diffusion Tensor Imaging (DTI) revealed elevated Fractional Anisotropy (FA) and diffusivity in the basal ganglia, and reduced FA with increased diffusivity in white matter tracts. Genetic analysis by Polymerase Chain Reaction (PCR) confirmed HD. Newer MRI like DTI allows early identification of disease and better assessment of disease progression and response to therapy. A multidisciplinary approach involving neurologists, psychiatrists, and other healthcare professionals is essential. Genetic counseling and psychosocial support are crucial for patients and their families. Early and accurate identification of HD is vital for timely interventions and improved symptom control. [ABSTRACT FROM AUTHOR]

Single-nucleotide variants (SNVs) in segmental duplications (SDs) have not been systematically assessed because of the limitations of mapping short-read sequencing data1,2. Here we constructed 1:1 unambiguous alignments spanning high-identity SDs across 102 human haplotypes and compared the pattern of SNVs between unique and duplicated regions3,4. We find that human SNVs are elevated 60% in SDs compared to unique regions and estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with up to 4.3 megabase pairs of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of about 800 protein-coding genes. These include 171 genes that have 'relocated' on average 1.61 megabase pairs in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are slightly evolutionarily older when compared to unique sequences, probably owing to IGC. SNVs in SDs, however, show a distinct mutational spectrum: a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts and a 7.6% reduction in the frequency of CpG-associated mutations when compared to unique DNA. We reason that these distinct mutational properties help to maintain an overall higher GC content of SD DNA compared to that of unique DNA, probably driven by GC-biased conversion between paralogous sequences5,6.

We combined carbon dioxide (CO2)-responsive cytosine-containing rhodamine 6G (Cy-R6G) as a hydrophobic anticancer agent with hydrogen-bonded cytosine-functionalized polyethylene glycol (Cy-PEG) as a hydrophilic supramolecular carrier to construct a CO2-responsive drug delivery system, with the aim of enhancing the responsiveness of the system to the tumor microenvironment and thus the overall effectiveness of anticancer therapy. Due to self-complementary hydrogen bonding interactions between cytosine units, Cy-R6G and Cy-PEG co-assemble in water to form spherical-like nanogels, with Cy-R6G effectively encapsulated within the nanogels. The nanogels exhibit several distinctive physical features, such as widely tunable nanogel size and drug loading capacity for Cy-R6G, intriguing fluorescence properties, high co-assembled structural stability in normal aqueous environments, enhanced anti-hemolytic characteristics, sensitive dual CO2/pH-responsive behavior, and precise and easily controllable CO2-induced release of Cy-R6G. Cytotoxicity assays clearly indicated that, due to the presence of cytosine receptors on the surface of cancer cells, Cy-R6G-loaded nanogels exert selective cytotoxicity against cancer cells in pristine culture medium, but do not affect the viability of normal cells. Surprisingly, in CO2-rich culture medium, Cy-R6G-loaded nanogels exhibit a further significant enhancement in cytotoxicity against cancer cells, and remain non-cytotoxic to normal cells. More importantly, a series of in vitro experiments demonstrated that compared to pristine culture medium, CO2-rich culture medium promotes more rapid selective internalization of Cy-R6G-loaded nanogels into cancer cells through cytosine-mediated macropinocytosis and thus accelerates the induction of apoptosis. Therefore, this newly developed system provides novel avenues for the development of highly effective CO2-responsive drug delivery systems with potent anticancer capabilities.

The course of pathophysiological mechanisms involved in fragile X-associated tremor/ataxia syndrome (FXTAS) remains largely unknown. Previous proteomics and metabolomics studies conducted in blood samples collected from FMR1 premutation carriers with FXTAS reported abnormalities in energy metabolism, and precursors of gluconeogenesis showed significant changes in plasma expression levels in FMR1 premutation carriers who developed FXTAS. We conducted an analysis of postmortem human brain tissues from 44 donors, 25 brains with FXTAS, and 19 matched controls. We quantified the metabolite relative abundance in the inferior temporal gyrus and the cerebellum using untargeted mass spectrometry (MS)-based metabolomics. We investigated how the metabolite type and abundance relate to the number of cytosine-guanine-guanine (CGG) repeats, to markers of neurodegeneration, and to the symptoms of FXTAS. A metabolomic analysis identified 191 primary metabolites, the data were log-transformed and normalized prior to the analysis, and the relative abundance was compared between the groups. The changes in the relative abundance of a set of metabolites were region-specific with some overlapping results; 22 metabolites showed alterations in the inferior temporal gyrus, while 21 showed differences in the cerebellum. The relative abundance of cytidine was decreased in the inferior temporal gyrus, and a lower abundance was found in the cases with larger CGG expansions; oleamide was significantly decreased in the cerebellum. The abundance of 11 metabolites was influenced by changes in the CGG repeat number. A histological evaluation found an association between the presence of microhemorrhages in the inferior temporal gyrus and a lower abundance of 2,5-dihydroxypyrazine. Our study identified alterations in the metabolites involved in the oxidative-stress response and bioenergetics in the brains of individuals with FXTAS. Significant changes in the abundance of cytidine and oleamide suggest their potential as biomarkers and therapeutic targets for FXTAS.

Background: Direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platforms can produce reads covering up to full-length gene transcripts, while containing decipherable information about RNA base modifications and poly-A tail lengths. Although many published studies have been expanding the potential of dRNA-seq, its sequencing accuracy and error patterns remain understudied. Results: We present the first comprehensive evaluation of sequencing accuracy and characterisation of systematic errors in dRNA-seq data from diverse organisms and synthetic in vitro transcribed RNAs. We found that for sequencing kits SQK-RNA001 and SQK-RNA002, the median read accuracy ranged from 87% to 92% across species, and deletions significantly outnumbered mismatches and insertions. Due to their high abundance in the transcriptome, heteropolymers and short homopolymers were the major contributors to the overall sequencing errors. We also observed systematic biases across all species at the levels of single nucleotides and motifs. In general, cytosine/uracil-rich regions were more likely to be erroneous than guanines and adenines. By examining raw signal data, we identified the underlying signal-level features potentially associated with the error patterns and their dependency on sequence contexts. While read quality scores can be used to approximate error rates at base and read levels, failure to detect DNA adapters may be a source of errors and data loss. By comparing distinct basecallers, we reason that some sequencing errors are attributable to signal insufficiency rather than algorithmic (basecalling) artefacts. Lastly, we generated dRNA-seq data using the latest SQK-RNA004 sequencing kit released at the end of 2023 and found that although the overall read accuracy increased, the systematic errors remain largely identical compared to the previous kits. Conclusions: As the first systematic investigation of dRNA-seq errors, this study offers a comprehensive overview of reproducible error patterns across diverse datasets, identifies potential signal-level insufficiency, and lays the foundation for error correction methods. [ABSTRACT FROM AUTHOR]

Cancer is a multifaceted genetic disease characterized by the acquisition of several essential hallmarks. Notably, certain cancers exhibit horizontal transmissibility, observed across mammalian species and diverse bivalves, the latter referred to as hemic neoplasia. Within this complex landscape, epigenetic mechanisms such as histone modifications and cytosine methylation emerge as fundamental contributors to the pathogenesis of these transmissible cancers. Our study delves into the epigenetic landscape of Cerastoderma edule, focusing on whole-genome methylation and hydroxymethylation profiles in heathy specimens and transmissible neoplasias by means of Nanopore long-read sequencing. Our results unveiled a global hypomethylation in the neoplastic specimens compared to their healthy counterparts, emphasizing the role of DNA methylation in these tumorigenic processes. Furthermore, we verified that intragenic CpG methylation positively correlated with gene expression, emphasizing its role in modulating transcription in healthy and neoplastic cockles, as also highlighted by some up-methylated oncogenic genes. Hydroxymethylation levels were significantly more elevated in the neoplastic samples, particularly within satellites and complex repeats, likely related to structural functions. Additionally, our analysis also revealed distinct methylation and activity patterns in retrotransposons, providing additional insights into bivalve neoplastic processes. Altogether, these findings contribute to understanding the epigenetic dynamics of bivalve neoplasias and shed light on the roles of DNA methylation and hydroxymethylation in tumorigenesis. Understanding these epigenetic alterations holds promise for advancing our broader understanding of cancer epigenetics. [ABSTRACT FROM AUTHOR]

Introducing a rigid linear π‐conjugated acetylene linker into a supramolecular building block consisting of a hydrogen‐bond donor side and a hydrogen‐bond acceptor side yields a decrease in cooperativity in the resulting formed quartet. This follows from our Kohn‐Sham molecular orbital and Voronoi deformation density analyses of hydrogen‐bonded macrocycles based on guanine and cytosine nucleobases. The acetylene linker abstracts electron density from the hydrogen‐bond acceptor and donor, making the hydrogen‐bond acceptor more negatively charged and the hydrogen‐bond donor more positively charged and hence suppressing the donor–acceptor charge transfer interaction between the interacting fragments. This, ultimately, hampers the cooperativity in the hydrogen‐bonded macrocycle. We envision that these findings could open the door to new design principles for the development of novel hydrogen‐bond supramolecular macrocycles. [ABSTRACT FROM AUTHOR]

A promising de novo approach for the treatment of Castration-resistant prostate cancer (CRPC) exploits cell-mediated enzyme prodrug therapy comprising cytosine deaminase (CD) and fluorouracil (5-FC). The aim of this study was to determine the potential of bacterial CD-overexpressing hTERT-immortalized human adipose stem cells (hTERT-ADSC.CD) to suppress CRPC. A lentiviral vector encoding a bacterial CD gene was used to transfect and to generate the hTERT-ADSC.CD line. The ability of the cells to migrate selectively towards malignant cells was investigated in vitro. PC3 and hTERT-ADSC.CD cells were co-cultured. hTERT-ADSC.CD and 1 × 106 PC3 cells were administered to nude mice via intracardiac and subcutaneous injections, respectively, and 5-FC was given for 14 days. hTERT-ADSC.CD were successfully engineered. Enhanced in vitro hTERT-ADSC.CD cytotoxicity and suicide effect were evident following administration of 5 μM 5-FC. hTERT-ADSC.CD, together with 5-FC, augmented the numbers of PC3 cells undergoing apoptosis. In comparison to controls administered hTERT-ADSC.CD monotherapy, hTERT-ADSC.CD in combination with 5-FC demonstrated a greater suppressive effect on tumor. In CPRC-bearing mice, tumor suppression was enhanced by the combination of CD-overexpressing ADSC and the prodrug 5-FC. Stem cells exhibiting CD gene expression are a potential novel approach to treatment for CRPC. [ABSTRACT FROM AUTHOR]

Introduction: Rice blast disease caused by Magnaporthe oryzae has long been the main cause of rice (Oryza sativa L.) yield reduction worldwide. The quinone external inhibitor pyraclostrobin is widely used as a fungicide to effectively control the spread of pathogenic fungi, including M. oryzae. However, M. oryzae can develop resistance through multiple levels of mutation, such as target protein cytb mutation G143A/S, leading to a decrease in the effectiveness of the biocide after a period of application. Therefore, uncovering the possible mutational mechanisms from multiple perspectives will further provide feasible targets for drug development. Methods: In this work, we determined the gene expression changes in M. oryzae in response to pyraclostrobin stress and their relationship with DNA methylation by transcriptome and methylome. Results: The results showed that under pyraclostrobin treatment, endoplasmic reticulum (ER)-associated and ubiquitin-mediated proteolysis were enhanced, suggesting that more aberrant proteins may be generated that need to be cleared. DNA replication and repair processes were inhibited. Glutathione metabolism was enhanced, while lipid metabolism was impaired. The number of alternative splicing events increased. These changes may be related to the elevated methylation levels of cytosine and adenine in gene bodies. Both hypermethylation and hypomethylation of differentially methylated genes (DMGs) mainly occurred in exons and promoters. Some DMGs and differentially expressed genes (DEGs) were annotated to the same pathways by GO and KEGG, including protein processing in the ER, ubiquitin-mediated proteolysis, RNA transport and glutathione metabolism, suggesting that pyraclostrobin may affect gene expression by altering the methylation patterns of cytosine and adenine. Discussion: Our results revealed that 5mC and 6mA in the gene body are associated with gene expression and contribute to adversity adaptation in M. oryzae. This enriched the understanding for potential mechanism of quinone inhibitor resistance, which will facilitate the development of feasible strategies for maintaining the high efficacy of this kind of fungicide. [ABSTRACT FROM AUTHOR]

Intra-organism biodiversity is thought to arise from epigenetic modification of constituent genes and post-translational modifications of translated proteins. Here, we show that post-transcriptional modifications, like RNA editing, may also contribute. RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosine to uracil. RNAsee (RNA site editing evaluation) is a computational tool developed to predict the cytosines edited by these enzymes. We find that 4.5% of non-synonymous DNA single nucleotide polymorphisms that result in cytosine to uracil changes in RNA are probable sites for APOBEC3A/G RNA editing; the variant proteins created by such polymorphisms may also result from transient RNA editing. These polymorphisms are associated with over 20% of Medical Subject Headings across ten categories of disease, including nutritional and metabolic, neoplastic, cardiovascular, and nervous system diseases. Because RNA editing is transient and not organism-wide, future work is necessary to confirm the extent and effects of such editing in humans. A survey of known human DNA editing sites with an RNA editing site prediction algorithm suggests APOBEC-mediated RNA editing may produce some of the same protein variants, with the possibility of affecting multiple areas of health. [ABSTRACT FROM AUTHOR]

Cellular DNA and RNA are decorated with diverse chemical modifications, which add new layers to gene regulation and play crucial roles across development and disease progression. Interest in understanding the functions of DNA and RNA modifications, as well as the related molecular mechanisms, has been growing, driving progress in developing chemical and biochemical tools to detect specific modifications. New technologies are important not only for uncovering biological functions, but also for driving conceptual revolutions. In this review, we highlighted our recent advances in developing new chemical tools to detect DNA and RNA modifications in a direct, quantitative, and base‐resolution manner. These includes a novel borane reduction chemistry for DNA methylation sequencing; new cytosine modificaiton oxdation chemistry for enhanced DNA hydroxymethylation sequencing; and a novel bromoacrylamide cyclization chemistry for RNA pseudouridylation sequencing. We present a mechanistic overview of these tools and their applications in epigenetic and epitranscriptomic research. [ABSTRACT FROM AUTHOR]

Background: Glanzmann's thrombasthenia (GT) is a congenital platelet disorder affecting approximately 1:1 000 000 people globally and characterized by impaired platelet aggregation and clot retraction. Autosomal recessive, loss‐of‐function, variants in ITGA2B or ITGB3 of the αIIbβ3 receptor cause the disease in humans. A cat affected by Glanzmann's and macrothrombocytopenia was presented to the UC Davis VMTH. Hypothesis/Objectives: Severe thrombopathia in this cat has an underlying genetic etiology. Animals: A single affected patient, 2 age‐matched clinically healthy controls, and a geriatric population (n = 20) of normal cats. Methods: Physical examination and clinical pathology tests were performed on the patient. Flow cytometry and platelet aggregometry analyses for patient phenotyping were performed. Patient and validation cohort gDNA samples were extracted for Sanger sequencing of a previously identified ITGA2B (c.1986delC) variant. Reverse transcriptase PCR was performed on patient and healthy control PRP samples to verify ITGA2B variant consequence. Results: A novel c.1986_1987insCC autosomal recessive variant in ITGA2B was identified. This variant was absent in a population of 194 unrelated cats spanning 44 different breeds. Complete loss of ITGA2B transcript and protein expression was verified by RT‐PCR and flow cytometry, explaining the underlying etiology of GT, and likely macrothrombocytopenia, in this cat. Conclusions and Clinical Importance: This study emphasizes the role of precision medicine in cardiovascular disease of cats and identified yet another variant that may be of utility for screening in the feline population. This study provides a small‐volume, standardized, successful protocol for adequate platelet RNA isolation and subsequent molecular assessment of gene expression in cats. [ABSTRACT FROM AUTHOR]

Age estimates are essential for fisheries assessment and management, but deepwater (>200 m) fishes are often difficult to age using traditional techniques. Therefore, age-predictive epigenetic clocks were developed for a model deepwater reef fish, blackbelly rosefish Helicolenus dactylopterus, using two tissue types (fin clips and muscle; n = 61 individuals; 9−60 years) and Δ14C-validated consensus age estimates. The influence of biological information (length and sex) on epigenetic clock accuracy, and the potential for developing a multi-tissue clock, were also assessed. Bisulfite-converted restriction site-associated DNA sequencing (bsRADseq) was used to identify CpG sites (cytosines followed by guanines) exhibiting age-correlated DNA methylation, and epigenetic clocks showed strong agreement (R2 > 0.98) between predicted and consensus ages. Including length and sex data enhanced accuracy and precision (R2 > 0.99; mean absolute error < 1 year). Age-associated CpG sites were identified across tissues, but a multi-tissue clock performed poorly relative to single-tissue clocks. Overall, results demonstrate that accurate and precise epigenetic clocks can be developed for deepwater fishes, and the inclusion of biological information may enhance clock accuracy and precision. [ABSTRACT FROM AUTHOR]

The in vivo codon decoding preferences of tRNAs with an authentic adenosine residue at position 34 of the anticodon, the wobble position, are largely unexplored because very few unmodified A34 tRNA genes exist across the three domains of life. The expanded wobble rules suggest that unmodified adenosine pairs most strongly with uracil, modestly with cytosine, and weakly with guanosine and adenosine. Inosine, a modified adenosine, on the other hand, pairs strongly with both uracil and cytosine and to a lesser extent adenosine. Orthogonal pair directed sense codon reassignment experiments offer a tool with which to interrogate the translational activity of A34 tRNAs because the introduced tRNA can be engineered with any anticodon. Our fluorescencebased screen utilizes the absolute requirement of tyrosine at position 66 of superfolder GFP for autocatalytic fluorophore formation. The introduced orthogonal tRNA competes with the endogenous translation machinery to incorporate tyrosine in response to a codon typically assigned another meaning in the genetic code. We evaluated the codon reassignment efficiencies of 15 of the 16 possible orthogonal tRNAs with A34 anticodons. We examined the Sanger sequencing chromatograms for cDNAs from each of the reverse transcribed tRNAs for evidence of inosine modification. Despite several A34 tRNAs decoding closely-related C-ending codons, partial inosine modification was detected for only three species. These experiments employ a single tRNA body with a single attached amino acid to interrogate the behavior of different anticodons in the background of in vivo E. coli translation and greatly expand the set of experimental measurements of the in vivo function of A34 tRNAs in translation. For the most part, unmodified A34 tRNAs largely pair with only U3 codons as the original wobble rules suggest. In instances with GC pairs in the first two codon positions, unmodified A34 tRNAs decode the C- and G-ending codons as well as the expected U-ending codon. These observations support the "two-out-of-three" and "strong and weak" codon hypotheses. [ABSTRACT FROM AUTHOR]

Background: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. Results: To systematically investigate TALE-DdCBE architectures and editing rules, we established a β-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. Conclusions: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement. [ABSTRACT FROM AUTHOR]

Background: The Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes. Results: To provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate. Conclusions: FENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight. [ABSTRACT FROM AUTHOR]

In Trypanosoma brucei, gene expression is primarily regulated posttranscriptionally making RNA metabolism critical. T. brucei has an epitranscriptome containing modified RNA bases. Yet, the identity of the enzymes catalyzing modified RNA base addition and the functions of the enzymes and modifications remain unclear. Homology searches indicate the presence of numerous T. brucei cytosine RNA methyltransferase homologs. One such homolog, TbNop2 was studied in detail. TbNop2 contains the six highly conserved motifs found in cytosine RNA methyltransferases and is evolutionarily related to the Nop2 protein family required for rRNA modification and processing. RNAi experiments targeting TbNop2 resulted in reduced levels of TbNop2 RNA and protein, and a cessation of parasite growth. Next generation sequencing of bisulfite-treated RNA (BS-seq) detected the presence of two methylation sites in the large rRNA; yet TbNop2 RNAi did not result in a significant reduction of methylation. However, TbNop2 RNAi resulted in the retention of 28S internal transcribed spacer RNAs, indicating a role for TbNop2 in rRNA processing. [ABSTRACT FROM AUTHOR]

Mammalian genomes are regulated by epigenetic cytosine (C) modifications in palindromic CpG dyads. Including canonical cytosine 5‐methylation (mC), a total of four different 5‐modifications can theoretically co‐exist in the two strands of a CpG, giving rise to a complex array of combinatorial marks with unique regulatory potentials. While tailored readers for individual marks could serve as versatile tools to study their functions, it has been unclear whether a natural protein scaffold would allow selective recognition of marks that vastly differ from canonical, symmetrically methylated CpGs. We conduct directed evolution experiments to generate readers of 5‐carboxylcytosine (caC) dyads based on the methyl‐CpG‐binding domain (MBD), the widely conserved natural reader of mC. Despite the stark steric and chemical differences to mC, we discover highly selective, low nanomolar binders of symmetric and asymmetric caC‐dyads. Together with mutational and modelling studies, our findings reveal a striking evolutionary flexibility of the MBD scaffold, allowing it to completely abandon its conserved mC recognition mode in favour of noncanonical dyad recognition, highlighting its potential for epigenetic reader design. [ABSTRACT FROM AUTHOR]

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer. [ABSTRACT FROM AUTHOR]

The presence of higher order G-quadruplex-DNA (G4) structures in human cells in guanine-rich DNA sequences has been well established. In cases of G4 helicase impairment or G4 stabilization via association with G4 ligands, it has been determined that arrest and/or termination of DNA transcription/replication occurs because these structures act as physical barriers to both helicase and polymerase mobility along the DNA. Truncation of transcription or replication may result in the loss of genetic material expression leading to disorders. To ameliorate G4-induced barriers, agents that destabilize or "unwind" the quadraplex structures are sought. Earlier work has shown that the cytosine analogue phenylpyrrolocytosine has potential G4-destabilizing ability. It was hypothesized that the unwinding activity was related to its ability to base pair with guanosine. To facilitate the design and evaluation of the hydrogen bonding ability of cytosine analogues, solubility in aprotic, and low-polarity solvents, e.g., deutero-chloroform (CDCl3), is needed; then, determination of association constants (Ka) with guanine via 1H nuclear magnetic resonance (NMR) spectroscopy or isothermal titration calorimetry (ITC) will be possible. Herein, we report on the synthesis of three new chloroform-soluble, cytosine analogues: N1-cyclohexylmethyl-7-phenylimidazolocytosine, N1-cyclohexylmethyl-7-(6-(methoxycarbonyl)pyridin-2-yl)imidazolocytosine, and N1-cyclohexylmethyl-7-(6-carbamoylpyridin-2-yl)imidazolocytosine. We further report the binding interactions of each of these analogues with the guanosine derivative 2′,3′,5′-O-tris(tert-butyldimethylsilyl)guanosine through the use of both ITC and 1H NMR titration experiments in chloroform and compare the binding to persilyated ribo-cytidine. [ABSTRACT FROM AUTHOR]

DNA, or deoxyribonucleic acid, is found in every single cell and is the cell's primary information storage medium. DNA stores all an organism's genetic information, including the instructions it needs to grow, divide, and live. DNA is made up of four different building blocks called nucleotide bases: adenine (A), thymine (T), cytosine (C), and guanine (G). The genome is sequenced in vitro utilizing encoding strategies such as labelling one bond pair as 0 and the other as 1 to store digital information. In this study, the fractional differential order of double-chain DNA dynamical system was investigated, considering Atangana's conformable fractional derivative. The conformable sub-equation method was applied to the system. The analysis resulted in some interesting new exact solutions of the model. One-soliton kink solution, multiple-soliton solutions, and periodic-wave solutions are the three broad categories that may be used to describe the results. In order to better understanding the solutions found, we have visually investigated a few of them. Both solitary and anti-solitary waves of the DNA strands are seen, attesting to the nonlinear dynamics of the system. The gathered data might be used to conduct application evaluations and draw further scientific findings. [ABSTRACT FROM AUTHOR]

Over 70,000 DNA lesions occur in the cell every day, and the inability to properly repair them can lead to mutations and destabilize the genome, resulting in carcinogenesis. The base excision repair (BER) pathway is critical for maintaining genomic integrity by repairing small base lesions, abasic sites and single‐stranded breaks. Monofunctional and bifunctional glycosylases initiate the first step of BER by recognizing and excising specific base lesions, followed by DNA end processing, gap filling, and finally nick sealing. The Nei‐like 2 (NEIL2) enzyme is a critical bifunctional DNA glycosylase in BER that preferentially excises cytosine oxidation products and abasic sites from single‐stranded, double‐stranded, and bubble‐structured DNA. NEIL2 has been implicated to have important roles in several cellular functions, including genome maintenance, participation in active demethylation, and modulation of the immune response. Several germline and somatic variants of NEIL2 with altered expression and enzymatic activity have been reported in the literature linking them to cancers. In this review, we provide an overview of NEIL2 cellular functions and summarize current findings on NEIL2 variants and their relationship to cancer. [ABSTRACT FROM AUTHOR]