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Toxin–antitoxin (TA) systems are widespread in bacteria and implicated in genome stability, virulence, phage defense, and persistence. TA systems have diverse activities and cellular targets, but their physiological roles and regulatory mechanisms are often unclear. Here, we show that the NatR–NatT TA system, which is part of the core genome of the human pathogen Pseudomonas aeruginosa, generates drug-tolerant persisters by specifically depleting nicotinamide dinucleotides. While actively growing P. aeruginosa cells compensate for NatT-mediated NAD+ deficiency by inducing the NAD+ salvage pathway, NAD depletion generates drug-tolerant persisters under nutrient-limited conditions. Our structural and biochemical analyses propose a model for NatT toxin activation and autoregulation and indicate that NatT activity is subject to powerful metabolic feedback control by the NAD+ precursor nicotinamide. Based on the identification of natT gain-of-function alleles in patient isolates and on the observation that NatT increases P. aeruginosa virulence, we postulate that NatT modulates pathogen fitness during infections. These findings pave the way for detailed investigations into how a toxin–antitoxin system can promote pathogen persistence by disrupting essential metabolic pathways. Synopsis: The toxin-antitoxin system NatR-NatT promotes persister formation in Pseudomonas aeruginosa. This study shows that NatT acts by depleting the essential co-factors NAD and NADP and promotes drug tolerance in this human pathogen. The RES domain of NatT degrades NAD+ into nicotinamide and ADP-ribose-1"-phosphate. Actively growing bacteria can counteract NAD+ depletion by inducing the NAD+ salvage pathway. Under nutrient-limited conditions, bacteria are unable to compensate for NatT-mediated NAD+depletion, resulting in dormancy and persistence. NatT toxin activation is inhibited by the NAD+ precursor nicotinamide. The toxin NatT promotes drug tolerance in Pseudomonas aeruginosa under nutrient-limited conditions and is activated in clinical isolates. [ABSTRACT FROM AUTHOR]

In testing whether coronavirus defective viral genome 12.7 (DVG12.7) with transcription regulating sequence (TRS) can synthesize subgenomic mRNA (sgmRNA) in coronavirus-infected cells, it was unexpectedly found by Northern blot assay that not only sgmRNA (designated sgmDVG 12.7) but also an RNA fragment with a size less than sgmDVG 12.7 was identified. A subsequent study demonstrated that the identified RNA fragment (designated clvDVG) was a cleaved RNA product originating from DVG12.7, and the cleaved sites were located in the loop region of stem‒loop structure and after UU and UA dinucleotides. clvDVG was also identified in mock-infected HRT-18 cells transfected with DVG12.7 transcript, indicating that cellular endoribonuclease is responsible for the cleavage. In addition, the sequence and structure surrounding the cleavage sites can affect the cleavage efficiency of DVG12.7. The cleavage features are therefore consistent with the general criteria for RNA cleavage by cellular RNase L. Furthermore, both the cleavage of rRNA and the synthesis of clvDVG were also identified in A549 cells. Because (i) the cleavage sites occurred predominantly after single-stranded UA and UU dinucleotides, (ii) the sequence and structure surrounding the cleavage sites affected the cleavage efficiency, (iii) the cleavage of rRNA is an index of the activation of RNase L, and (iv) the cleavage of both rRNA and DVG12.7 was identified in A549 cells, the results together indicated that the preferential cleavage of DVG12.7 is correlated with cellular endoribonuclease with the characteristics of RNase L and such cleavage features have not been previously characterized in coronaviruses. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) is vital in the cytosolic DNA-sensing process and critical for initiating the innate immune response, which has important functions in host defense and contributes to the pathogenesis of inflammatory diseases. Zinc finger CCCH-type antiviral protein 1 (ZC3HAV1) specifically binds the CpG dinucleotides in the viral RNAs of multiple viruses and promotes their degradation. ZAPS (ZC3HAV1 short isoform) is a potent stimulator of retinoid acid-inducible gene I (RIG-I) signaling during the antiviral response. However, how ZC3HAV1 controls STING signaling is unclear. Here, we show that ZC3HAV1 specifically potentiates STING activation by associating with STING to promote its oligomerization and translocation from the endoplasmic reticulum (ER) to the Golgi, which facilitates activation of IRF3 and NF-κB pathway. Accordingly, Zc3hav1 deficiency protects mice against herpes simplex virus-1 (HSV-1) infection- or 5,6-dimethylxanthenone-4-acetic acid (DMXAA)-induced inflammation in a STING-dependent manner. These results indicate that ZC3HAV1 is a key regulator of STING signaling, which suggests its possible use as a therapeutic target for STING-dependent inflammation. ZC3HAV1 specifically enhances STING signaling responses to activate IRF3 and NF-κB pathway provides a potential ZC3HAV1-STING-targeting strategy for the clinical management of of STING-dependent inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: Studying the composition rules and evolution mechanisms of genome sequences are core issues in the post-genomic era, and k-mer spectrum analysis of genome sequences is an effective means to solve this problem. Result: We divided total 8-mers of genome sequences into 16 kinds of XY-type due to XY dinucleotides number in 8-mers. Previous works explored that the independent unimodal distributions observed only in three CG-type 8-mer spectra, while non-CG type 8-mer spectra have not the universal phenomenon from prokaryotes to eukaryotes. On this basis, we analyzed the distribution variation of non-CG type 8-mer spectra across 889 animal genome sequences. Following the evolutionary order of animals from primitive to more complex, we found that the spectrum distributions gradually transition from unimodal to tri-modal. The relative distance from the average frequency of each non-CG type 8-mers to the center frequency is different within a species and among different species. For the 8-mers contain CG dinucleotides, we further divided these into 16 subsets, where each 8-mer contains both CG and XY dinucleotides, called XY1_CG1 subsets. We found that the separability values of XY1_CG1 spectra are closely related to the evolution and specificity of animals. Considering the constraint of Chargaff's second parity rule, we finally obtained 10 separability values as the feature set to characterize the evolution state of genome sequences. In order to verify the rationality of the feature set, we used 14 common classification algorithms to perform binary classification tests. The results showed that the accuracy (Acc) ranged between 98.70% and 83.88% among birds, other vertebrates and mammals. Conclusion: We proposed a credible feature set to characterizes the evolution state of genomes and obtained satisfied results by the feature set on large scale classification of animals. [ABSTRACT FROM AUTHOR]

Petrocodon is a small genus in the family Gesneriaceae, which is special for its remarkable floral diversity, and has high ornamental value. In this study, the complete chloroplast genome sequence and genome characteristics of Petrocodon longitubus are first reported. The genome size is 152,958 bp, including a large single-copy region (LSC, 83,901 bp), a small single-copy region (SSC, 18,255 bp), and two inverted repeat sequences (IRs, 25,401 bp, each). The chloroplast genome of P. longitubus was analyzed, revealing a total GC content of 37.47%. A total of 131 genes were de novo assembled, consisting of 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. A comparative analysis was conducted between the chloroplast genome of P. longitubus and three other published species of Petrocodon. The chloroplast genome of four Petrocodon species was found to have a double-chain ring structure, with a size ranging from 152,958 to 153,292 bp. Chloroplast genome size had indistinguishable. Four Petrocodon species was ra elatively conserved sequence, with 87 or 88 protein-coding genes, and 8 rRNA were the most conserved, which contains 42 ~ 50 SSR sites, which are mainly mononucleotides and dinucleotides, 4 boundary transition regions, then trinucleotides, pentanucleotides and hexanucleotides have been not detected. The non-preferred codons of the chloroplast genome in the four Petrocodon species are those ending in A, C, G, or T. The chloroplast genomes of these four Petrocodon species are highly similar to each other and to several Primulina species. Phylogenetic trees indicate that P. longitubus and other Petrocodon species were grouped together in a clade, with P. longitubus form a single clade. The results support the scientific naming of P. Longitubusr based on horticultural traits and further clarify the systematic status using molecular information. [ABSTRACT FROM AUTHOR]

Formate oxidase (FOx), which contains 8-formyl flavin adenine dinucleotide (FAD), exhibits a distinct advantage in utilizing ambient oxygen molecules for the oxidation of formic acid compared to other glucose-methanol-choline (GMC) oxidoreductase enzymes that contain only the standard FAD cofactor. The FOx-mediated conversion of FAD to 8-formyl FAD results in an approximate 10-fold increase in formate oxidase activity. However, the mechanistic details underlying the autocatalytic formation of 8-formyl FAD are still not well understood, which impedes further utilization of FOx. In this study, we employ molecular dynamics simulation, QM/MM umbrella sampling simulation, enzyme activity assay, site-directed mutagenesis, and spectroscopic analysis to elucidate the oxidation mechanism of FAD to 8-formyl FAD. Our results reveal that a catalytic water molecule, rather than any catalytic amino acids, serves as a general base to deprotonate the C8 methyl group on FAD, thus facilitating the formation of a quinone-methide tautomer intermediate. An oxygen molecule subsequently oxidizes this intermediate, resulting in a C8 methyl hydroperoxide anion that is protonated and dissociated to form OHC-RP and OH−. During the oxidation of FAD to 8-formyl FAD, the energy barrier for the rate-limiting step is calculated to be 22.8 kcal/mol, which corresponds to the required 14-hour transformation time observed experimentally. Further, the elucidated oxidation mechanism reveals that the autocatalytic formation of 8-formyl FAD depends on the proximal arginine and serine residues, R87 and S94, respectively. Enzymatic activity assay validates that the mutation of R87 to lysine reduces the kcat value to 75% of the wild-type, while the mutation to histidine results in a complete loss of activity. Similarly, the mutant S94I also leads to the deactivation of enzyme. This dependency arises because the nucleophilic OH− group and the quinone-methide tautomer intermediate are stabilized through the noncovalent interaction provided by R87 and S94. These findings not only explain the mechanistic details of each reaction step but also clarify the functional role of R87 and S94 during the oxidative maturation of 8-formyl FAD, thereby providing crucial theoretical support for the development of novel flavoenzymes with enhanced redox properties. [ABSTRACT FROM AUTHOR]

Methylation of adenine at the N6 position is a crucial epigenetic modification that profoundly influences gene regulation and expression. Moreover, this modification intricately alters the excited state dynamics of adenine nucleobases. To explore the impact of N6‐methyladenine on the excited state dynamics within oligonucleotides, we conducted a comprehensive investigation of two dinucleotides containing N6‐methyladenosine, in conjunction with adenosine or guanosine. Using steady‐state and time‐resolved absorption and fluorescence spectroscopy techniques, we not only observed the customary monomer‐like and charge transfer emissive states, as reported in previous dinucleotides, but also identified an additional low‐energy emissive state. This unique state exhibits an extraordinary Stokes Shift exceeding 2.3 eV and has a relatively long lifetime of 4–5 ns. We propose that this state corresponds to a bonded exciplex state, governed by ground‐state geometries. [ABSTRACT FROM AUTHOR]

DNA methylation plays an important role in various biological processes, including cell differentiation, ageing, and cancer development. The most important methylation in mammals is 5-methylcytosine mostly occurring in the context of CpG dinucleotides. Sequencing methods such as whole-genome bisulfite sequencing successfully detect 5-methylcytosine DNA modifications. However, they suffer from the serious drawbacks of short read lengths and might introduce an amplification bias. Here we present Rockfish, a deep learning algorithm that significantly improves read-level 5-methylcytosine detection by using Nanopore sequencing. Rockfish is compared with other methods based on Nanopore sequencing on R9.4.1 and R10.4.1 datasets. There is an increase in the single-base accuracy and the F1 measure of up to 5 percentage points on R.9.4.1 datasets, and up to 0.82 percentage points on R10.4.1 datasets. Moreover, Rockfish shows a high correlation with whole-genome bisulfite sequencing, requires lower read depth, and achieves higher confidence in biologically important regions such as CpG-rich promoters while being computationally efficient. Its superior performance in human and mouse samples highlights its versatility for studying 5-methylcytosine methylation across varied organisms and diseases. Finally, its adaptable architecture ensures compatibility with new versions of pores and chemistry as well as modification types. DNA methylation is vital for various biological processes. Here, the authors present Rockfish, a deep learning algorithm that enhances 5-methylcytosine detection using Nanopore sequencing. Rockfish offers higher accuracy, lower read depth requirements, and greater confidence in crucial genomic regions. [ABSTRACT FROM AUTHOR]

Background: Autophagy and apoptosis are cellular processes that maintain cellular homeostasis and remove damaged or aged organelles or aggregated and misfolded proteins. Stress factors initiate the signaling pathways common to autophagy and apoptosis. An imbalance in the autophagy and apoptosis, led by cascade of molecular mechanism prior to both processes culminate into neurodegeneration. Objective: In present study, we urge to investigate the codon usage pattern of genes which are common before initiating autophagy and apoptosis. Methods: In the present study, we took up eleven genes (DAPK1, BECN1, PIK3C3 (VPS34), BCL2, MAPK8, BNIP3 L (NIX), PMAIP1, BAD, BID, BBC3, MCL1) that are part of molecular signaling mechanism prior to autophagy and apoptosis. We analyzed dinucleotide odds ratio, codon bias, usage, context, and rare codon analysis. Results: CpC and GpG dinucleotides were abundant, with the dominance of G/C ending codons as preferred codons. Clustering analysis revealed that MAPK8 had a distinct codon usage pattern compared to other envisaged genes. Both positive and negative contexts were observed, and GAG-GAG followed by CTG-GCC was the most abundant codon pair. Of the six synonymous arginine codons, two codons CGT and CGA were the rarest. Conclusions: The information presented in the study may be used to manipulate the process of autophagy and apoptosis and to check the pathophysiology associated with their dysregulation. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1–4, containing a methyl group at the ribose 2′-C and 3′-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1–4) have distinct conformational preferences. Biological evaluation of dinucleotides 1–4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2′-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization. [ABSTRACT FROM AUTHOR]

Dinucleoside 5',5'‐polyphosphates (NpnNs) play essential roles in various biological processes. Access to these high value‐added compounds, while avoiding the many drawbacks of solution phase synthesis, is a challenge that has been overcome thanks to mechanochemistry. A straightforward solid‐state synthesis based on activation of phosphate or pyrophosphate sodium salts to their diimidazolides, followed by coupling with commercially available nucleotides, under air conditions, gave symmetrical NpnNs. The scope ranged from Np3Ns to highly challenging Np6Ns. The methodology has also been implemented using medronic acid and imidodiphosphate to obtain NpnNs analogues, some of which are unprecedented. [ABSTRACT FROM AUTHOR]

Background: Methylation plays a crucial role in genome regulation, serving as an essential epigenetic mechanism. CpG islands, which are regions of DNA rich in CpG dinucleotides, are ubiquitous epigenetic regulatory features that can modulate the functional dynamics of genes, thereby influencing the pathophysiology of pathogenic organisms within a host. Objectives: In this study, we aimed to investigate the distribution of CpG islands on Papillomavirus genomes and assess their potential impact on the neoplastic progression of different Alphapapillomavirus genera within host cells. Methods: We conducted an analysis of dinucleotide frequencies in DNA sequences from various Alphapapillomavirus genera. The sequences for our analysis were sourced from the specialized HPV (human papillomavirus) database (pave.niaid.nih.gov). Following a comprehensive comparative examination of entire genomes from different HPV species, we specifically focused on 63 genomes belonging to the Alphapapillomavirus genus to identify internal CpG island profiles. These investigations were conducted using online bioinformatics software (DBCAT), and statistical analysis was performed using GraphPad Prism v. 8.0.1. Results: Our preliminary findings revealed that 76.1% of the viruses in our study had fewer than 3 CpG islands but multiple CpG sites, while fewer than 25% of the viruses possessed at least 3 CpG islands. High-risk viruses were identified in 68.42% of genotypes with fewer than 3 CpG islands, whereas low-risk genotypes were observed in 15.7% of cases. Notably, some oncogenic viruses lacked CpG islands entirely, and this pattern was also observed in viruses with a single CpG island, occurring in 50% of cases. Based on the absence of CpG islands, genotypes such as HPV 67, HPV 97, and HPV 34 could potentially be classified as carcinogenic. Additionally, the distribution of CpG islands across HPV genes did not appear to be random. Genes like L2 and E2 contained a CpG island in 50% of cases, whereas oncogenes E6 and E7 consistently had a CpG island in 100% of cases in high-risk genotypes. Conclusions: Our findings suggest that the loss of 1 or more CpG islands could potentially transform an HPV genotype into a high-risk genotype. This process may be accelerated in the presence of host-related risk factors. Further research is required to validate whether CpG island distribution can aid in identifying oncogenic viruses. [ABSTRACT FROM AUTHOR]

Here we present a systematic analysis of the synthesis of unsymmetrical disulphides employing 6‐thioguanosine and its mono‐ and dinucleotides by three of the most common strategies: a nucleophilic substitution of the thiol, a thiol exchange reaction with another disulphide and oxidative dehydrogenation coupling reactions of S−H bonds. The exchange reaction was found to be the most efficient approach and could be used not only for nucleosides but also for nucleotides. [ABSTRACT FROM AUTHOR]

Activation of stimulator of interferon genes (STING) by cyclic dinucleotides (CDNs) has been considered as a powerful immunotherapy strategy. While promising, the clinical translation of CDNs is still overwhelmed by its limited biostability and the resulting systemic immunotoxicity. Being differentiating from current application of exogenous CDNs to address these challenges, we herein developed one perylene STING agonist PDIC‐NS, which not only promotes the production of endogenous CDNs but also inhibits its hydrolysis. More significantly, PDIC‐NS can well reach lung‐selective enrichment, and thus mitigates the systemic immunotoxicity upon intravenous administration. As a result, PDIC‐NS had realized remarkable in vivo antitumor activity, and backward verified on STING knock out mice. Overall, this study states that PDIC‐NS can function as three‐in‐one small‐molecule STING agonist characterized by promoting the content and biostability of endogenous CDNs as well as possessing good tissue specificity, and hence presents an innovative strategy and platform for tumor chemo‐immunotherapy. [ABSTRACT FROM AUTHOR]

Epigenetic phenomena play a central role in cell regulatory processes and are important factors for understanding complex human disease. One of the best understood epigenetic mechanisms is DNA methylation. In the mammalian genome, cytosines (C) in CpG dinucleotides were long known to undergo methylation at the 5-position of the pyrimidine ring (mC). Later it was found that mC can be oxidized to 5-hydroxymethylcytosine (hmC) or even further to 5-formylcytosine (fC) and to 5-carboxylcytosine (caC) by the action of 2-oxoglutarate-dependent dioxygenases of the TET family. These findings unveiled a long elusive mechanism of active DNA demethylation and bolstered a wave of studies in the area of epigenetic regulation in mammals. This review is dedicated to critical assessment of recent data on biochemical and chemical aspects of the formation and conversion of hmC in DNA, analytical techniques used for detection and mapping of this nucleobase in mammalian genomes as well as epigenetic roles of hmC in DNA replication, transcription, cell differentiation and human disease. [ABSTRACT FROM AUTHOR]

Microsatellites or SSRs are small tandem repeats that are 1–6 bp long. They are usually highly polymorphic and form important portions of genomes. They have been extensively analyzed in humans, animals and model plants; however, information from non-flowering plants is generally lacking. Here, we examined 29 samples of Ophioglossaceae ferns, mainly from the genera Botrychium and Sceptridium. We analyzed the SSR distribution, density and composition in almost 400 nuclear exons and their flanking regions. We detected 45 SSRs in exons and 1475 SSRs in the flanking regions. In the exons, only di-, tri- and tetranucleotides were found, and all of them were 12 bp long. The annotation of the exons containing SSRs showed that they were related to various processes, such as metabolism, catalysis, transportation or plant growth. The flanking regions contained SSRs from all categories, with the most numerous being dinucleotides, followed by tetranucleotides. More than one-third of all the SSRs in the flanking regions were 12 bp long. The SSR densities in the exons were very low, ranging from 0 to 0.07 SSRs/kb, while those in the flanking regions ranged from 0.24 to 0.81 SSRs/kb; and those in the combined dataset ranged from 0.2 to 0.81 SSRs/kb. The majority of the detected SSRs in the flanking regions were polymorphic and present at the same loci across two or more samples but differing in the number of repeats. The SSRs detected here may serve as a basis for further population genetic, phylogenetic or evolutionary genetic studies, as well as for further studies focusing on SSRs in the genomes and their roles in adaptation, evolution and diseases. [ABSTRACT FROM AUTHOR]

Viruses represent a major threat to all animals, which defend themselves through induction of a large set of virus-stimulated genes that collectively control the infection. In vertebrates, these genes include interferons that play a critical role in the amplification of the response to infection. Virus- and interferon-stimulated genes include restriction factors targeting the different steps of the viral replication cycle, in addition to molecules associated with inflammation and adaptive immunity. Predictably, antiviral genes evolve dynamically in response to viral pressure. As a result, each animal has a unique arsenal of antiviral genes. Here, we exploit the capacity to experimentally activate the evolutionarily conserved stimulator of IFN genes (STING) signaling pathway by injection of the cyclic dinucleotide 2′3′-cyclic guanosine monophosphate-adenosine monophosphate into flies to define the repertoire of STING-regulated genes in 10 Drosophila species, spanning 40 million years of evolution. Our data reveal a set of conserved STING-regulated factors, including STING itself, a cGAS-like-receptor, the restriction factor pastel, and the antiviral protein Vago, but also 2 key components of the antiviral RNA interference pathway, Dicer-2, and Argonaute2. In addition, we identify unknown species- or lineage-specific genes that have not been previously associated with resistance to viruses. Our data provide insight into the core antiviral response in Drosophila flies and pave the way for the characterization of previously unknown antiviral effectors. [ABSTRACT FROM AUTHOR]

In the intensively developing field of synthetic biology, synonymous gene recoding is a leading method, which is not without drawbacks in its use. There are natural limitations in the use of codons in genes. Computer analysis in 30 genes of viruses, bacteria, archaea, and human beings was used to study the nucleotide composition, the translational code of the proteins encoded by them, and the composition of dinucleotides, dicodons, and tricodons. To identify limitations in gene coding, sequences of quantitative indicators of codons (complementarity indices (CIs) and dimension indices) were used. For each gene, limitations (particular) in the nucleotide composition, the translational code, and the composition of dinucleotides and dicodons were identified. A general limitation in gene coding is that the difference in the CIs of neighboring tricodons read with a frame shift of one codon, as happens when mRNA is translated on ribosomes, was no more than 2 in the vast majority of tricodons. Since in genes each codon is included in three sequentially read tricodons, with the exception of the first and last three codons, the CI of each codon is associated with the CIs of the two preceding and two subsequent codons. This leads to the recognition of the existence of a continuum of the codon connectivity in genes based on their CI values. [ABSTRACT FROM AUTHOR]

To improve the chemical regulation on the activity of cyclic dinucleotides (CDNs), we here designed a reduction-responsive dithioethanol (DTE)-based dCDN prodrug 9 (DTE-dCDN). Prodrug 9 improved the cell permeability with the intracellular levels peaking in 2 h in THP-1 cells. Under the reductive substance such as GSH or DTT, prodrug 9 could be quickly decomposed in 30 min to release the parent dCDN. In THP1-Lucia cells, prodrug 9 also retained a high bioactivity with the EC50 of 0.96 μM, which was 51-, 43-, and 3-fold more than the 2′,3′-cGAMP (EC50 = 48.6 μM), the parent compound 3′,3′-c-di-dAMP (EC50 = 41.3 μM), and ADU-S100 (EC50 = 2.9 μM). The high bioactivity of prodrug 9 was validated to be highly correlated with the activation of the STING signaling pathway. Furthermore, prodrug 9 could also improve the transcriptional expression levels of IFN-β, CXCL10, IL-6, and TNF-α in THP-1 cells. These results will be helpful to the development of chemically controllable CDN prodrugs with a high cellular permeability and potency. [ABSTRACT FROM AUTHOR]

A new synthesis of 3′‐deoxy‐3′‐fluoroadenosine for use as an important intermediate of antitumor‐active cyclic dinucleotide is disclosed. The synthesis started with the known 5‐O‐TBDPS‐D‐lyxofuranose 1,2‐acetonides, which was first transformed into a fluorinated compound after the DAST reaction. Desilylation and acidic methanolysis were then finished in one pot. After a sequence of triflation, displacement by OBz as well as benzoylation, perbenzoylated 3‐deoxy‐3‐fluoro‐D‐ribofuranoside was obtained, which would be transformed to 3′‐deoxy‐3′‐fluoroadenosine as the key intermediate of cyclic dinucleotide after ammonolysis. [ABSTRACT FROM AUTHOR]

Adenine base editors (ABEs) are genome-editing tools that have been harnessed to introduce precise A.T to G.C conversion. The discovery of split genes revealed that all introns contain two highly conserved dinucleotides, canonical "AG" (acceptor) and "GT" (donor) splice sites. ABE can directly edit splice acceptor sites of the adenine (A) base, leading to aberrant gene splicing, which may be further adopted to remodel splicing. However, spliced isoforms triggered with ABE have not been well explored. To address it, we initially generated a cell line harboring C-terminal enhanced GFP (eGFP)-tagged β-actin (ACTB), in which the eGFP signal can track endogenous β-actin expression. Expectedly, after the editing of splice acceptor sites, we observed a dramatical decrease in the percentage of eGFP-positive cells and generation of splicing products with the noncanonical splice site. Furthermore, we manipulated Peroxidasin in mouse embryos with ABE, in which a noncanonical acceptor was activated to remodel splicing, successfully generating a mouse disease model of anophthalmia and severely malformed microphthalmia. Collectively, we demonstrate that ABE-mediated splicing remodeling can activate a noncanonical acceptor to manipulate human and mouse genomes, which will facilitate the investigation of basic and translational medicine studies. [ABSTRACT FROM AUTHOR]

The CCCH-type zinc finger antiviral protein (ZAP) in humans, specifically isoforms ZAP-L and ZAP-S, is a crucial component of the cell's intrinsic immune response. ZAP acts as a post-transcriptional RNA restriction factor, exhibiting its activity during infections caused by retroviruses and alphaviruses. Its function involves binding to CpG (cytosine-phosphate-guanine) dinucleotide sequences present in viral RNA, thereby directing it towards degradation. Since vertebrate cells have a suppressed frequency of CpG dinucleotides, ZAP is capable of distinguishing foreign genetic elements. The expression of ZAP leads to the reduction of viral replication and impedes the assembly of new virus particles. However, the specific mechanisms underlying these effects have yet to be fully understood. Several questions regarding ZAP's mechanism of action remain unanswered, including the impact of CpG dinucleotide quantity on ZAP's activity, whether this sequence is solely required for the binding between ZAP and viral RNA, and whether the recruitment of cofactors is dependent on cell type, among others. This review aims to integrate the findings from studies that elucidate ZAP's antiviral role in various viral infections, discuss gaps that need to be filled through further studies, and shed light on new potential targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD) cofactor metabolism plays a significant role in cancer development. Tumor cells have an increased demand for NAD and ATP to support rapid growth and proliferation. Limiting the amount of available NAD by targeting critical NAD biosynthesis enzymes has emerged as a promising anticancer therapeutic approach. In mammals, the enzyme nicotinamide/nicotinic acid adenylyltransferase (NMNAT) catalyzes a crucial downstream reaction for all known NAD synthesis routes. Novel nicotinamide/nicotinic acid adenine dinucleotide (NAD/NaAD) analogues 1–4, containing a methyl group at the ribose 2′-C and 3′-C-position of the adenosine moiety, were synthesized as inhibitors of the three isoforms of human NMN-adenylyltransferase, named hNMNAT-1, hNMNAT-2, and hNMNAT-3. An NMR-based conformational analysis suggests that individual NAD-analogues (1–4) have distinct conformational preferences. Biological evaluation of dinucleotides 1–4 as inhibitors of hNMNAT isoforms revealed structural relationships between different conformations (North-anti and South-syn) and enzyme-inhibitory activity. Among the new series of NAD analogues synthesized and tested, the 2′-C-methyl-NAD analogue 1 (Ki = 15 and 21 µM towards NMN and ATP, respectively) emerged as the most potent and selective inhibitor of hNMNAT-2 reported so far. Finally, we rationalized the in vitro bioactivity and selectivity of methylated NAD analogues with in silico studies, helping to lay the groundwork for rational scaffold optimization.

Epigenetic drift or "disorder" increases across the mouse lifespan and is suggested to underlie epigenetic clock signals. While the role of epigenetic drift in determining maximum lifespan across species has been debated, robust tests of this hypothesis are lacking. Here, we test if epigenetic disorder at various levels of genomic resolution explains maximum lifespan across four mammal species. We show that epigenetic disorder increases with age in all species and at all levels of genomic resolution tested. The rate of disorder accumulation occurs faster in shorter lived species and corresponds to species adjusted maximum lifespan. While the density of cytosine-phosphate-guanine dinucleotides ("CpGs") is negatively associated with the rate of age-associated disorder accumulation, it does not fully explain differences across species. Our findings support the hypothesis that the rate of epigenetic drift explains maximum lifespan and provide partial support for the hypothesis that CpG density buffers against epigenetic drift. Epigenetic drift has been hypothesized to contribute to epigenetic clock signals and variation in lifespan across species. Here, the authors show that an empirical measure of epigenetic drift scales with maximum lifespan across four mammal species and accumulates in non-random genomic locations. [ABSTRACT FROM AUTHOR]

In most eukaryotes and prokaryotes TGA is used at a significantly higher frequency than TAG as termination codon of protein-coding genes. Although this phenomenon has been recognized several years ago, there is no generally accepted explanation for the TAG-TGA paradox. Our analyses of human mutation data revealed that out of the eighteen sense codons that can give rise to a nonsense codon by single base substitution, the CGA codon is exceptional: it gives rise to the TGA stop codon at an order of magnitude higher rate than the other codons. Here we propose that the TAG-TGA paradox is due to methylation and hypermutabilty of CpG dinucleotides. In harmony with this explanation, we show that the coding genomes of organisms with strong CpG methylation have a significant bias for TGA whereas those from organisms that lack CpG methylation use TGA and TAG termination codons with similar probability. [ABSTRACT FROM AUTHOR]

Background: Exploring evolution regularities of genome sequences and constructing more objective species evolution relationships at the genomic level are high-profile topics. Based on the evolution mechanism of genome sequences proposed in our previous research, we found that only the 8-mers containing CG or TA dinucleotides correlate directly with the evolution of genome sequences, and the relative frequency rather than the actual frequency of these 8-mers is more suitable to characterize the evolution of genome sequences. Result: Therefore, two types of feature sets were obtained, they are the relative frequency sets of CG1 + CG2 8-mers and TA1 + TA2 8-mers. The evolution relationships of mammals and reptiles were constructed by the relative frequency set of CG1 + CG2 8-mers, and two types of evolution relationships of insects were constructed by the relative frequency sets of CG1 + CG2 8-mers and TA1 + TA2 8-mers respectively. Through comparison and analysis, we found that evolution relationships are consistent with the known conclusions. According to the evolution mechanism, we considered that the evolution relationship constructed by CG1 + CG2 8-mers reflects the evolution state of genome sequences in current time, and the evolution relationship constructed by TA1 + TA2 8-mers reflects the evolution state in the early stage. Conclusion: Our study provides objective feature sets in constructing evolution relationships at the genomic level. [ABSTRACT FROM AUTHOR]

In recent years many scientists have begun to focus on the mRNA molecule's emeregence as a new type of drug. Its fast-moving and successful career as a vaccine technology cannot be underestimated. mRNA provides new opportunities and allows for the rapid preparation of effective drugs at low cost. These extensive possibilities stem from a number of factors, but the small cap structure located at the 5′ end of the mRNA is one contributing factor. Cap protects mRNA and ensures efficient recruitment to the biosynthesis machinery. Furthermore, it allows for the easy introduction of various modifications that influence the activity of the entire mRNA. Among the many different cap analogues that have been reported, those modified at the N2 position of guanosine have been systematically developed. N2-modified caps in the form of nucleoside monophosphates or dinucleotides show favorable biological properties, as well as a high capacity to inhibit the translation process in the cell-free RRL system. Modified N2 dinucleotides are efficiently incorporated into the structure of the mRNA transcript, and in specific circumstances with the correct orientation, making them an interesting alternative for ARCA-type analogues. Moreover, mRNA transcripts containing cap structures modified within the exocyclic amino group show very high translational activity. Therefore, analogues modified at the N2 position may have future applications as therapeutics against various manifestations of cancer and as desirable tools in RNA engineering. [ABSTRACT FROM AUTHOR]

Background: Epigenetic mechanisms are informational cellular processes instructing normal and diseased phenotypes. They are associated with DNA but without altering the DNA sequence. Whereas chemical processes like DNA methylation or histone modifications are well-accepted epigenetic mechanisms, we herein propose the existence of an additional quantum physics layer of epigenetics. Results: We base our hypothesis on theoretical and experimental studies showing quantum phenomena to be active in double-stranded DNA, even under ambient conditions. These phenomena include coherent charge transfer along overlapping pi-orbitals of DNA bases and chirality-induced spin selectivity. Charge transfer via quantum tunneling mediated by overlapping orbitals results in charge delocalization along several neighboring bases, which can even be extended by classical (non-quantum) electron hopping. Such charge transfer is interrupted by flipping base(s) out of the double-strand e.g., by DNA modifying enzymes. Charge delocalization can directly alter DNA recognition by proteins or indirectly by DNA structural changes e.g., kinking. Regarding sequence dependency, charge localization, shown to favor guanines, could influence or even direct epigenetic changes, e.g., modification of cytosines in CpG dinucleotides. Chirality-induced spin selectivity filters electrons for their spin along DNA and, thus, is not only an indicator for quantum coherence but can potentially affect DNA binding properties. Conclusions: Quantum effects in DNA are prone to triggering and manipulation by external means. By the hypothesis put forward here, we would like to foster research on "Quantum Epigenetics" at the interface of medicine, biology, biochemistry, and physics to investigate the potential epigenetic impact of quantum physical principles on (human) life. [ABSTRACT FROM AUTHOR]

Purine-containing nucleotide second messengers regulate diverse cellular activities. Cyclic di-pyrimidines mediate anti-phage functions in bacteria; however, the synthesis mechanism remains elusive. Here, we determine the high-resolution structures of cyclic di-pyrimidine-synthesizing cGAS/DncV-like nucleotidyltransferases (CD-NTases) in clade E (CdnE) in its apo, substrate-, and intermediate-bound states. A conserved (R/Q)xW motif controlling the pyrimidine specificity of donor nucleotide is identified. Mutation of Trp or Arg from the (R/Q)xW motif to Ala rewires its specificity to purine nucleotides, producing mixed purine-pyrimidine cyclic dinucleotides (CDNs). Preferential binding of uracil over cytosine bases explains the product specificity of cyclic di-pyrimidine-synthesizing CdnE to cyclic di-UMP (cUU). Based on the intermediate-bound structures, a synthetic pathway for cUU containing a unique 2'3'-phosphodiester linkage through intermediate pppU[3'−5']pU is deduced. Our results provide a framework for pyrimidine selection and establish the importance of conserved residues at the C-terminal loop for the specificity determination of CD-NTases. Here, the authors present structural and functional characterization of bacterial CD-NTases that synthesize cyclic dipyrimidines for phage resistance, revealing a (R/Q)xW motif dictating pyrimidine selection which suggests a sequential pathway for synthesizing 2'3'-cyclic di-UMP. [ABSTRACT FROM AUTHOR]

Simple Summary: Our study aims to understand mysterious parts of our DNA, known as ultra-conserved non-coding elements. Found in the genomes of mammals and other vertebrates, these long (300 nucleotides, on average) DNA fragments have remained unchanged for hundreds of millions of years, despite numerous mutations happening within them. We do not yet know the function of these elements, but their extraordinary evolutionary stability could help us devise new approaches to preventing harmful mutations that lead to cancer and genetic disorders. We sought to discover why ultra-conserved non-coding elements are so resistant to change by studying their smaller building blocks, called dinucleotides, which play a crucial role in the shape and structure of DNA. Using a bioinformatics approach, we compared the differences in arrangement of dinucleotides in the ultra-conserved non-coding elements to the rest of the human genome. Our study revealed unique patterns within the ultra-conserved non-coding elements sections that distinguish them from the rest of our genetic code. Long human ultra-conserved non-coding elements (UCNEs) do not have any sequence similarity to each other or other characteristics that make them unalterable during vertebrate evolution. We hypothesized that UCNEs have unique dinucleotide (DN) composition and arrangements compared to the rest of the genome. A total of 4272 human UCNE sequences were analyzed computationally and compared with the whole genomes of human, chicken, zebrafish, and fly. Statistical analysis was performed to assess the non-randomness in DN spacing arrangements within the entire human genome and within UCNEs. Significant non-randomness in DN spacing arrangements was observed in the entire human genome. Additionally, UCNEs exhibited distinct patterns in DN arrangements compared to the rest of the genome. Approximately 83% of all DN pairs within UCNEs showed significant (>10%) non-random genomic arrangements at short distances (2–6 nucleotides) relative to each other. At the extremes, non-randomness in DN spacing distances deviated up to 40% from expected values and were frequently associated with GpC, CpG, ApT, and GpG/CpC dinucleotides. The described peculiarities in DN arrangements have persisted for hundreds of millions of years in vertebrates. These distinctive patterns may suggest that UCNEs have specific DNA conformations. [ABSTRACT FROM AUTHOR]

Background: PRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While human PRPS1 (hPRPS1) and human PRPS2 (hPRPS2) are expressed in most tissues, human PRPS3 (hPRPS3) is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming filaments and cytoophidia is considered as a distinctive mechanism involving the polymerization of the protein. Previously we solved the filament structures of Escherichia coli PRPS (ecPRPS) using cryo-electron microscopy (cryo-EM) 1. Results: Order to investigate the function and molecular mechanism of hPRPS2 polymerization, here we solve the polymer structure of hPRPS2 at 3.08 Å resolution. hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity. Conclusion: Findings suggest that the regulation of hPRPS2 polymer is distinct from ecPRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis. [ABSTRACT FROM AUTHOR]

The genetic code conceals a 'code within the codons', which hints at biophysical interactions between amino acids and their cognate nucleotides. Yet, research over decades has failed to corroborate systematic biophysical interactions across the code. Using molecular dynamics simulations and NMR, we have analysed interactions between the 20 standard proteinogenic amino acids and 4 RNA mononucleotides in 3 charge states. Our simulations show that 50% of amino acids bind best with their anticodonic middle base in the −1 charge state common to the backbone of RNA, while 95% of amino acids interact most strongly with at least 1 of their codonic or anticodonic bases. Preference for the cognate anticodonic middle base was greater than 99% of randomised assignments. We verify a selection of our results using NMR, and highlight challenges with both techniques for interrogating large numbers of weak interactions. Finally, we extend our simulations to a range of amino acids and dinucleotides, and corroborate similar preferences for cognate nucleotides. Despite some discrepancies between the predicted patterns and those observed in biology, the existence of weak stereochemical interactions means that random RNA sequences could template non-random peptides. This offers a compelling explanation for the emergence of genetic information in biology. [ABSTRACT FROM AUTHOR]

Peronospora tabacina is considered the main limiting factor in tobacco production worldwide. In Mexico, information on the genetic diversity of this pathogen is scarce; therefore, the objective of this research was to evaluate 12 microsatellites in 20 isolates collected in the states of Nayarit, Chiapas, and Veracruz. PCR amplificationand sequencing of these microsatellites were performed; as well as the alignment and comparison of the sequences deposited in the GenBank database. A total of 19 isolates showed amplification for the 12 microsatellites evaluated, while in one of the isolates, the amplification of two microsatellites was not observed, it being possible to determine that P. tabacina isolates present in Nayarit, Chiapas, and Veracruz are genetically homogeneous. Regions of dinucleotides were observed, most corresponding to (GT)n repeat motifs or (TG)n variations, as well as (AC)n, (CA) n, (AT)n and (AG)n motifs. The isolates analyzed in this study can be considered products of clonal lines, therefore no genetic diversity was found in these isolates. [ABSTRACT FROM AUTHOR]

Summary Nanopore reads encode information on the methylation status of cytosines in CpG dinucleotides. The length of the reads makes it comparatively easy to look at patterns consisting of multiple loci; here, we exploit this property to search for regions where one can define subpopulations of molecules based on methylation patterns. As an example, we run our clustering algorithm on known imprinted genes; we also scan chromosome 15 looking for windows corresponding to heterogeneous methylation. Our software can also compute the covariance of methylation across these regions while keeping into account the mixture of different types of reads. Availability and implementation https://github.com/EmanueleRaineri/cvlr. Contact simon.heath@cnag.crg.eu Supplementary information Supplementary data are available at Bioinformatics Advances online. [ABSTRACT FROM AUTHOR]

CpG and its cytosine-methylated counterpart (5mCpG) are a unique reversible pair of sequences in regulating the expression of genes epigenetically. As DNA is the potential target of Pt-based anticancer metallodrugs, herein, we comparatively investigate the interactions of 5′-CpG and 5′-5mCpG with a photoactivatable anticancer Pt(IV) prodrug, trans,trans,trans-[PtIV(N3)2(OH)2(py)2] (1; py = pyridine), to explore the effects of methylation on the platination and ROS-induced oxidation of the CpG motif. Mono-platinated dinucleotides were demonstrated by ESI-MS to be the main products for both 5′-CpG and 5′-5mCpG with the bound Pt moiety as [PtII(N3)(py)2] generated by the photodecomposition of complex 1 under irradiation with blue light, accompanied by the formation of less abundant di-platinated adducts. G-N7 and C-N3/5mC-N3 were shown to be the major and minor platination sites, respectively, with G-N1 as the third and weakest platination site, in particular, in di-platinated products. Moreover, platinated dinucleotides associated with guanine and/or cytosine oxidation were also observed. Apart from 8-oxo-guanine (oxG) and N-formylamidoiminohydantoin (RedSp) reported previously, novel oxidation adducts 5-guanidinohydantoin (Gh) derived from guanine and 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (ImidCyt) derived from cytosine in CpG, and diimino imidazole (DIz) and 2,5-diaminoimidazol-4-one (imidazolone, Iz) derived from guanine and Imid5mCyt derived from 5mC in 5mCpG were proposed according to MS information. These results showed that methylation exerted little effects on the platination modes of CpG, but triggered distinct oxidation pathways of CpG, perhaps causing discriminated DNA damage to CpG-rich genes. This work provides novel insights into the role of the anticancer photoactivatable Pt(IV) prodrug through damaging the epigenetically modified DNA sequences. [ABSTRACT FROM AUTHOR]

The chemical nature of the DNA bases is an important factor in sequence‐mediated association of DNA molecules. Nucleotides are the fundamental DNA elements and the base identity impacts the molecular properties of nucleotide fragments. It is interesting to study the fundamental nature of nucleotides in DNA, on the basis of base‐specific interactions, association, and modes of standard atomic or molecular interactions. With all‐atom molecular dynamics simulations of model dinucleotide and tetranucleotide systems having single‐stranded dinucleotide or tetranucleotide fragments of varying sequences, we show how the base identity and interactions between the different bases as well as water may affect the clustering properties of nucleotides fragments in an ionic solution. Sequence‐dependent differential interactions between the nucleotide fragments, ionic concentration, and elevated temperature are found to influence the clustering properties and dynamics of association. Well‐known epigenetic modification of DNA, that is, cytosine methylation also promotes dinucleotide clustering in solution. These observations point to one possible chemical nature of the DNA bases, as well as the importance of the base pairing, base stacking, and ionic interactions in DNA structure formation, and DNA sequence‐mediated association. Sequence‐ and the ionic environment‐mediated self‐association properties of the dinucleotides indicate its great potential to develop biological nanomaterials for desired applications. [ABSTRACT FROM AUTHOR]

Dinucleotides are known as determinants for various structural and physiochemical properties of DNA and for binding affinities of proteins to DNA. These properties (e.g., stiffness) and bound proteins (e.g., transcription factors) are known to influence important biological functions, such as transcription regulation and 3D chromatin organization. Accordingly, the question arises of how the considerable variations in dinucleotide contents of eukaryotic chromosomes could still provide consistent DNA properties resulting in similar functions and 3D conformations. In this work, we investigate the hypothesis that coupled dinucleotide contents influence DNA properties in opposite directions to moderate each other's influences. Analyzing all 2478 chromosomes of 155 eukaryotic species, considering bias from coding sequences and enhancers, we found sets of correlated and anti-correlated dinucleotide contents. Using computational models, we estimated changes of DNA properties resulting from this coupling. We found that especially pure A/T dinucleotides (AA, TT, AT, TA), known to influence histone positioning and AC/GT contents, are relevant moderators and that, e.g., the Roll property, which is known to influence histone affinity of DNA, is preferably moderated. We conclude that dinucleotide contents might indirectly influence transcription and chromatin 3D conformation, via regulation of histone occupancy and/or other mechanisms. [ABSTRACT FROM AUTHOR]

The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host-guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5'-CTTBPPBBSSZZSAAG, 5'-CTTSSPBZPSZBBAAG and 5'-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, and B:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters of P:Z and B:S pairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further, Z can participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'. [ABSTRACT FROM AUTHOR]

Stimulator of interferon genes (STING) agonists can improve the anticancer efficacy of immune checkpoint blockade by amplifying tumor immunogenicity. However, the clinical translation of cyclic dinucleotides (CDNs) as STING agonists is hindered by their poor drug-like properties. In this study, we investigated the design criteria for DOTAP/cholesterol liposomes for the systemic delivery of ADU-S100 and delineated the impact of key formulation factors on the loading efficiency, serum stability, and STING agonistic activity of ADU-S100. Our findings demonstrate that the cationic liposomal formulation of ADU-S100 can be optimized to greatly potentiate STING activation in antigen-presenting cells. [ABSTRACT FROM AUTHOR]

Congenital factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by defects in the F5 gene associated with bleeding manifestations of variable severity. In this study we report molecular and functional characterization of a novel F5 variant that causes aberrant splicing and significantly reduces protein expression in a patient with severe FV deficiency. We performed F5 mutation screening and functional study in a proband (FV:C, 0.4%) with a history of gastrointestinal bleeding, post-traumatic bleeding, hematomas, ecchymoses, and discomfort in his ankle joints since infancy. Sequencing revealed a novel homozygous F5 gene variant NC_000001.10:169519985G>C (or NM_000130.5:c.1297 –8C>G). Bioinformatics sequence analysis predicted that this variant would lead to the acceptor site loss of the intron 8–exon 9 junction. However, mRNA analysis identified that it also activated an aberrant splice site located 7 nucleotides upstream of the normal one and was associated with the production of an anomalous F5 transcript with retention of seven nucleotides of intron 8 resulting in a premature stop codon. We revealed no traces of normal transcript in the patient. Our findings confirm that it is not only changes in canonical splicing dinucleotides that can significantly disrupt splicing sites and impair pre-mRNA processing. [ABSTRACT FROM AUTHOR]

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15. These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS). [ABSTRACT FROM AUTHOR]

The Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferons in response to sensing viral or bacterial cytosolic DNA. Cyclic dinucleotides (CDNs) are known to activate STING. Here, we describe the synthesis and biological evaluation of two new 3',3'‐CDN, in which the internucleotide linkages were replaced, respectively, by a 1,2,3‐triazole moiety (as more stable isosteres of phosphate linkers) and an unsaturated carbon chain (as flexibility can led to crucial binding affinity and specificity). Thus, CuAAC and macrocyclization by RCM are the two key steps. [ABSTRACT FROM AUTHOR]

The placenta undergoes many changes throughout gestation to support the evolving needs of the foetus. There is also a growing appreciation that male and female foetuses develop differently in utero, with unique epigenetic changes in placental tissue. Here, we report meta-analysed sexspecific associations between gestational age and placental DNA methylation from four cohorts in the National Institutes of Health (NIH) Environmental influences on Child Health Outcomes (ECHO) Programme (355 females/419 males, gestational ages 23-42 weeks). We identified 407 cytosineguanine dinucleotides (CpGs) in females and 794 in males where placental methylation levels were associated with gestational age. After cell-type adjustment, 55 CpGs in females and 826 in males were significant. These were enriched for biological processes critical to the immune system in females and transmembrane transport in males. Our findings are distinct between the sexes: in females, associations with gestational age are largely explained by differences in placental cellular composition, whereas in males, gestational age is directly associated with numerous alterations in methylation levels. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders with maturation and differentiation defects exhibiting morphological dysplasia in one or more hematopoietic cell lineages. They are associated with peripheral blood cytopenias and by increased risk for progression into acute myelogenous leukemia. Among their multifactorial pathogenesis, age-related epigenetic instability and the error-rate DNA methylation maintenance have been recognized as critical factors for both the initial steps of their pathogenesis and for disease progression. Although lower-risk MDS is associated with an inflammatory bone marrow microenvironment, higher-risk disease is delineated by immunosuppression and clonal expansion. "Epigenetics" is a multidimensional level of gene regulation that determines the specific gene networks expressed in tissues under physiological conditions and guides appropriate chromatin rearrangements upon influence of environmental stimulation. Regulation of this level consists of biochemical modifications in amino acid residues of the histone proteins' N-terminal tails and their concomitant effects on chromatin structure, DNA methylation patterns in CpG dinucleotides and the tissue-specific non-coding RNAs repertoire, which are directed against various gene targets. The role of epigenetic modifications is widely recognized as pivotal both in gene expression control and differential molecular response to drug therapies in humans. Insights to the potential of synergistic cooperations of epigenetic mechanisms provide new avenues for treatment development to comfort human diseases with a known epigenetic shift, such as MDS. Hypomethylating agents (HMAs), such as epigenetic modulating drugs, have been widely used in the past years as first line treatment for elderly higher-risk MDS patients; however, just half of them respond to therapy and are benefited. Rational outcome predictors following epigenetic therapy in MDS and biomarkers associated with disease relapse are of high importance to improve our efforts in developing patient-tailored clinical approaches. [ABSTRACT FROM AUTHOR]

Methylation microarray and bisulphite sequencing are often used to study 5'-methylcytosine (5'- mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustworthy results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulphite and/or the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5'-mC since it allows sequencing of native DNA molecules, whereas the long reads produced by this technology help to increase the resolution of those genomic regions. In this work, we show that nanopore sequencing with 10X coverage depth, using DNA from a human cell line, produces 5'-mC methylation frequencies consistent with those obtained by 450k microarray, digital restriction enzyme analysis of methylation, and reduced representation bisulphite sequencing. High correlation between methylation frequencies obtained by nanopore sequencing and the other methodologies was also noticeable in either low or high GC content regions, including CpG islands and transcription start sites. We also showed that a minimum of five reads per CpG yields strong correlations (>0.89) in replicate nanopore sequencing runs and an almost uniform linearity of the methylation frequency variation between zero and one. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns based on genomic annotations of CpG regions. These results demonstrate that nanopore sequencing is a fast, robust, and reliable approach to the study of 5'-mC in the human genome with low coverage depth. [ABSTRACT FROM AUTHOR]

Background: DNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences. Results: We identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising. Conclusions: In the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is an inflammatory disorder of the gastrointestinal tract. The present study attempted to understand the codon usage preferences in genes associated with IBD progression. Compositional analysis, codon usage bias (CUB), Relative synonymous codon usage (RSCU), RNA structure, and expression analysis were performed to obtain a comprehensive picture of codon usage in IBD genes. Compositional analysis of 62 IBD-associated genes revealed that G and T are the most and least abundant nucleotides, respectively. ApG, CpA, and TpG dinucleotides were overrepresented or randomly used, while ApC, CpG, GpT, and TpA dinucleotides were either underrepresented or randomly used in genes related to IBD. The codons influencing the codon usage the most in IBD genes were CGC and AGG. A comparison of codon usage between IBD, and pancreatitis (non-IBD inflammatory disease) indicated that only codon CTG codon usage was significantly different between IBD and pancreatitis. At the same time, there were codons ATA, ACA, CGT, CAA, GTA, CCT, ATT, GCT, CGG, TTG, and CAG for whom codon usage was significantly different for IBD and housekeeping gene sets. The results suggest similar codon usage in at least two inflammatory disorders, IBD and pancreatitis. The analysis helps understand the codon biology, factors affecting gene expression of IBD-associated genes, and the evolution of these genes. The study helps reveal the molecular patterns associated with IBD. [ABSTRACT FROM AUTHOR]

We report the absorption profile of isolated Flavin Adenine Dinucleotide (FAD) mono-anions recorded using photo-induced dissociation action spectroscopy. In this charge state, one of the phosphoric acid groups is deprotonated and the chromophore itself is in its neutral oxidized state. These measurements cover the first four optical transitions of FAD with excitation energies from 2.3 to 6.0 eV (210–550 nm). The S0 → S2 transition is strongly blue shifted relative to aqueous solution, supporting the view that this transition has a significant charge-transfer character. The remaining bands are close to their solution-phase positions. This confirms that the large discrepancy between quantum chemical calculations of vertical transition energies and solution-phase band maxima cannot be explained by solvent effects. We also report the luminescence spectrum of FAD mono-anions in vacuo. The gas-phase Stokes shift for S1 is 3000 cm−1, which is considerably larger than any previously reported for other molecular ions and consistent with a significant displacement of the ground and excited state potential energy surfaces. Consideration of the vibronic structure is thus essential for simulating the absorption and luminescence spectra of flavins. [ABSTRACT FROM AUTHOR]

In the brain, d-amino acid oxidase (DAAO) is a peroxisomal flavoenzyme. Through oxidative deamination by DAAO, d-serine, the main coagonist of synaptic N-methyl-d-aspartate receptors (NMDARs), is degraded into α-keto acids and ammonia; flavin adenine dinucleotide (FAD) is simultaneously reduced to dihydroflavine-adenine dinucleotide (FADH2), which is subsequently reoxidized to FAD, with hydrogen peroxide produced as a byproduct. NMDAR hypofunction is implicated in the pathogenesis of schizophrenia. In previous studies, compared with control subjects, patients with schizophrenia had lower d-serine levels in peripheral blood and cerebrospinal fluid but higher DAAO expression and activity in the brain. Inhibiting DAAO activity and slowing d-serine degradation by using DAAO inhibitors to enhance NMDAR function may be a new strategy for use in the treatment of schizophrenia. The aim of this leading article is to review the current research in DAAO inhibitors. [ABSTRACT FROM AUTHOR]

Diribonucleotides arise from two sources: turnover of RNA transcripts (rRNA, tRNA, mRNA, and others) and linearization of cyclic-di-nucleotide signaling molecules. In both cases, there appears to be a requirement for a dedicated set of enzymes that will cleave these diribonucleotides into mononucleotides. The first enzyme discovered to mediate this activity is oligoribonuclease (Orn) from Escherichia coli. In addition to being the enzyme that cleaves dinucleotides and potentially other short oligoribonucleotides, Orn is also the only known exoribonuclease enzyme that is essential for E. coli , suggesting that removal of the shortest RNAs is an essential cellular function. Organisms naturally lacking the orn gene encode other nanoRNases (nrn) that can complement the conditional E. coli orn mutant. This review covers the history and recent advances in our understanding of these enzymes and their substrates. In particular, we focus on (i) the sources of diribonucleotides; (ii) the discovery of exoribonucleases; (iii) the structural features of Orn, NrnA/NrnB, and NrnC; (iv) the enzymatic activity of these enzymes against diribonucleotides versus other substrates; (v) the known physiological consequences of accumulation of linear dinucleotides; and (vi) outstanding biological questions for diribonucleotides and diribonucleases. [ABSTRACT FROM AUTHOR]