Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and have only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ~17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene knockout strategy, and it has promising application potential.

S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P5 has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P5 in renal inflammation and fibrosis. Male S1P5 knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P5 knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P5 knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P5 might be a promising goal for the pharmacological treatment of kidney diseases.

Riboswitches are cis-acting regulatory RNA elements prevalently located in the leader sequences of bacterial mRNA. An adenine sensing riboswitch cis-regulates adeninosine deaminase gene (add) in Vibrio vulnificus. The structural mechanism regulating its conformational changes upon ligand binding mostly remains to be elucidated. In this open framework it has been suggested that the ligand stabilizes the interaction of the distal "kissing loop" complex. Using accurate full-atom molecular dynamics with explicit solvent in combination with enhanced sampling techniques and advanced analysis methods it could be possible to provide a more detailed perspective on the formation of these tertiary contacts.

Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studying of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility.

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A-rule. In this study, we present X-ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non-instructive lesions by DNA polymerases at least from this sequence family.

Polyamines, such as putrescine, spermidine and spermine, have indirectly been linked with the regulation of gene expression, and their concentrations are typically increased in cancer cells. Although effects on transcription factor binding to cognate DNA targets have been demonstrated, the mechanisms of the biological action of polyamines is poorly understood. Employing uranyl photo-probing we now demonstrate that polyamines at submillimolar concentrations bind preferentially to bent adenine tracts in double-stranded DNA. These results provide the first clear evidence for the sequence-specific binding of polyamines to DNA, and thereby suggest a mechanism by which the cellular effects of polyamines in terms of differential gene transcriptional activity could, at least partly, be a direct consequence of sequence-specific interactions of polyamines with promoters at the DNA sequence level.

The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2-19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas9 nuclease (7-320, 160.7 on average). In vivo off-target deamination is further validated through target site deep sequencing. Moreover, we demonstrated that six different ABE-gRNA complexes could be examined in a single EndoV-seq assay. Our study presents the first detection method to evaluate genome-wide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas9 nuclease.

Nicotinamide adenine dinucleotide (NAD) is a universal electron carrier that participates in important intracellular metabolic reactions and signaling events. Interestingly, emerging evidence in animals indicates that cellular NAD can be actively or passively released into the extracellular space, where it is processed or perceived by ectoenzymes or cell-surface receptors. We have recently shown in Arabidopsis thaliana that exogenous NAD induces defense responses, that pathogen infection leads to release of NAD into the extracellular space at concentrations sufficient for defense activation, and that depletion of extracellular NAD (eNAD) by transgenic expression of the human NAD-hydrolyzing ectoenzyme CD38 inhibits plant immunity. We therefore hypothesize that, during plant-microbe interactions, NAD is released from dead or dying cells into the extracellular space where it interacts with adjacent naïve cells' surface receptors, which in turn activate downstream immune signaling. However, it is currently unknown whether eNAD signaling is unique to Arabidopsis or the Brassicaceae family. In this study, we treated citrus plants with exogenous NAD+ and tested NAD+-induced transcriptional changes and disease resistance. Our results show that NAD+ induces profound transcriptome changes and strong resistance to citrus canker, a serious citrus disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). Furthermore, NAD+-induced resistance persists in new flushes emerging after removal of the tissues previously treated with NAD+. Finally, NAD+ treatment primes citrus tissues, resulting in a faster and stronger induction of multiple salicylic acid pathway genes upon subsequent Xcc infection. Taken together, these results indicate that exogenous NAD+ is able to induce immune responses in citrus and suggest that eNAD may also be an elicitor in this woody plant species.

N6-methyldeoxyadenine (6mA) has recently been reported as a prevalent DNA modification in eukaryotes. The Tetrahymena thermophila MTA1 complex consisting of four subunits, namely MTA1, MTA9, p1, and p2, is the first identified eukaryotic 6mA methyltransferase (MTase) complex. Unlike the prokaryotic 6mA MTases which have been biochemically and structurally characterized, the operation mode of the MTA1 complex remains largely elusive. Here, we report the cryogenic electron microscopy structures of the quaternary MTA1 complex in S-adenosyl methionine (SAM)-bound (2.6 Å) and S-adenosyl homocysteine (SAH)-bound (2.8 Å) states. Using an AI-empowered integrative approach based on AlphaFold prediction and chemical cross-linking mass spectrometry, we further modeled a near-complete structure of the quaternary complex. Coupled with biochemical characterization, we revealed that MTA1 serves as the catalytic core, MTA1, MTA9, and p1 likely accommodate the substrate DNA, and p2 may facilitate the stabilization of MTA1. These results together offer insights into the molecular mechanism underpinning methylation by the MTA1 complex and the potential diversification of MTases for N6-adenine methylation.

The useful concepts of reticular chemistry, rigid and predictable metal nodes together with strong and manageable covalent interactions between metal centers and organic linkers, have made the so-called metal-organic frameworks (MOFs) a flourishing area of enormous applicability. In this work, the extension of similar strategies to supramolecularly assembled metal-organic materials has allowed us to obtain a family of isoreticular compounds of the general formula [Cu7(μ-adeninato-κN3:κN9)6(μ3-OH)6(μ-OH2)6](OOC-R-COO)·nH2O (R: ethylene-, acetylene-, naphthalene-, or biphenyl-group) in which the rigid copper-adeninato entities and the organic dicarboxylate anions are held together not by covalent interactions but by a robust and flexible network of synergic hydrogen bonds and π-π stacking interactions based on well-known supramolecular synthons (SMOFs). All compounds are isoreticular, highly insoluble, and water-stable and show a porous crystalline structure with a pcu topology containing a two-dimensional (2D) network of channels, whose dimensions and degree of porosity of the supramolecular network are tailored by the length of the dicarboxylate anion. The partial loss of the crystallization water molecules upon removal from the mother liquor produces a shrinkage of the unit cell and porosity, which leads to a color change of the compounds (from blue to olive green) if complete dehydration is achieved by means of gentle heating or vacuuming. However, the supramolecular network of noncovalent interactions is robust and flexible enough to reverse to the expanded unit cell and color after exposure to a humid atmosphere. This humidity-driven breathing behavior has been used to design a sensor in which the electrical resistance varies reversibly with the degree of humidity, very similar to the water vapor adsorption isotherm of the SMOF. The in-solution adsorption properties were explored for the uptake and release of the widely employed 5-fluorouracil, 4-aminosalycilic acid, 5-aminosalycilic acid, and allopurinol drugs. In addition, cytotoxicity activity assays were completed for the pristine and 5-fluorouracil-loaded samples.

Metabolism is dynamically regulated to accompany immune cell function, and altered immunometabolism can result in impaired immune responses. Concomitantly, the pharmacological manipulation of metabolic processes offers an opportunity for therapeutic intervention in inflammatory disorders. The nicotinamide adenine dinucleotide (NAD+ ) is a critical metabolic intermediate that serves as enzyme cofactor in redox reactions, and is also used as a co-substrate by many enzymes such as sirtuins, adenosine diphosphate ribose transferases and synthases. Through these activities, NAD+ metabolism regulates a broad spectrum of cellular functions such as energy metabolism, DNA repair, regulation of the epigenetic landscape and inflammation. Thus, the manipulation of NAD+ availability using pharmacological compounds such as NAD+ precursors can have immune-modulatory properties in inflammation. Here, we discuss how the NAD+ metabolism contributes to the immune response and inflammatory conditions, with a special focus on multiple sclerosis, inflammatory bowel diseases and inflammageing. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Comparative genomic sequencing of laboratory-derived vancomycin-intermediate Staphylococcusaureus (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in apt and ssaA6), and MM66-4 (in apt and walK), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (ssaA6 and walK), the same mutation in apt which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 apt mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that apt mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.

Alterations in microbiota are known to affect kidney disease conditions. We have previously shown that germ-free conditions exacerbated adenine-induced kidney damage in mice; however, the mechanism by which this occurs has not been elucidated. To explore this mechanism, we examined the influence of germ-free conditions on purine metabolism and renal immune responses involved in the kidney damage. Germ-free mice showed higher expression levels of purine-metabolizing enzymes such as xanthine dehydrogenase, which converts adenine to a nephrotoxic byproduct 2,8-dihydroxyadenine (2,8-DHA). The germ-free mice also showed increased urinary excretion of allantoin, indicating enhanced purine metabolism. Metabolome analysis demonstrated marked differences in the purine metabolite levels in the feces of germ-free mice and mice with microbiota. Furthermore, unlike the germ-free condition, antibiotic treatment did not increase the expression of purine-metabolizing enzymes or exacerbate adenine-induced kidney damage. Considering renal immune responses, the germ-free mice displayed an absence of renal IL-17A expression. However, the adenine-induced kidney damage in wild-type mice was comparable to that in IL-17A-deficient mice, suggesting that IL-17A does not play a major role in the disease condition. Our results suggest that the enhanced host purine metabolism in the germ-free mice potentially promotes the conversion of the administered adenine into 2,8-DHA, resulting in exacerbated kidney damage. This further suggests a role of the microbiota in regulating host purine metabolism.

Chronic renal failure (CRF) is a major public health problem worldwide. In this work, we investigated the effects of a purified Laminaria japonica polysaccharide (LJP61A) on renal function using an adenine-induced CRF mice model. Results exhibited that adenine treatment caused serious renal pathological damages and elevation of serum creatinine and blood urea nitrogen of mice. However, these changes could be significantly reversed by the administration of LJP61A in a dose-dependent manner. Additionally, LJP61A could dramatically reduce weight loss, improve the urine biochemical index, and regulate the electrolyte disturbance of CRF mice. These results suggest that the renal function of adenine-induced CRF mice can be improved by LJP61A, which might be developed into a potential therapeutic agent for CRF patients.

Aim. Chronic kidney disease (CKD) represents endothelial dysfunction. Monocyte adhesion is recognized as the initial step of arteriosclerosis. Indoxyl sulfate (IS) is considered to be a risk factor for arteriosclerosis in CKD. Oral adsorbent AST-120 retards deterioration of renal function, reducing accumulation of IS. In the present study, we determined the monocyte adhesion in the adenine-induced uremic rats in vivo and effects of AST-120 on the adhesion molecules. Methods. Twenty-four rats were divided into control, control+AST-120, adenine, and adenine+AST-120 groups. The number of monocytes adherent to the endothelium of thoracic aorta by imaging the entire endothelial surface and the mRNA expressions of adhesion and atherosclerosis-related molecules were examined on day 49. The mRNA expressions of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells were also examined. Results. Adenine increased the number of adherent monocytes, and AST-120 suppressed the increase. The monocyte adhesion was related to serum creatinine and IS in sera. Overexpression of VCAM-1 and TGF- β 1 mRNA in the arterial walls was observed in uremic rats. IS induced increase of the ICAM-1 and VCAM-1 mRNA expressions in vitro. Conclusion. It appears that uremic condition introduces the monocyte adhesion to arterial wall and AST-120 might inhibit increasing of the monocyte adherence with CKD progression.

Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine - induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered.

The adenine-thymine tautomer (A*-T*) has previously been discounted as a spontaneous mutagenesis mechanism due to the energetic instability of the tautomeric configuration. We study the stability of A*-T* while the nucleobases undergo DNA strand separation. Our calculations indicate an increase in the stability of A*-T* as the DNA strands unzip and the hydrogen bonds between the bases stretch. Molecular Dynamics simulations reveal the time scales and dynamics of DNA strand separation and the statistical ensemble of opening angles present in a biological environment. Our results demonstrate that the unwinding of DNA, an inherently out-of-equilibrium process facilitated by helicase, will change the energy landscape of the adenine-thymine tautomerization reaction. We propose that DNA strand separation allows the stable tautomerization of adenine-thymine, providing a feasible pathway for genetic point mutations via proton transfer between the A-T bases.

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

Expression of Salmonella enterica loci harboring undermethylated GATC sites at promoters or regulatory regions was monitored by single cell analysis. Cell-to-cell differences in expression were detected in ten such loci (carA, dgoR, holA, nanA, ssaN, STM1290, STM3276, STM5308, gtr and opvAB), with concomitant formation of ON and OFF subpopulations. The ON and OFF subpopulation sizes varied depending on the growth conditions, suggesting that the population structure can be modulated by environmental control. All the loci under study except STM5308 displayed altered patterns of expression in strains lacking or overproducing Dam methylase, thereby confirming control by Dam methylation. Bioinformatic analysis identified potential binding sites for transcription factors OxyR, CRP and Fur, and analysis of expression in mutant backgrounds confirmed transcriptional control by one or more of such factors. Surveys of gene expression in pairwise combinations of Dam methylation-dependent loci revealed independent switching, thus predicting the formation of a high number of cell variants. This study expands the list of S. enterica loci under transcriptional control by Dam methylation, and underscores the relevance of the DNA adenine methylome as a source of phenotypic heterogeneity.

Adenine nucleotide translocase 1 (ANT1) transfers ATP and ADP over the mitochondrial inner membrane and thus supplies the cell with energy. This study analyzed the role of ANT1 in the immune response of ischemic heart tissue. Ischemic ANT1 overexpressing hearts experienced a shift toward an anti-inflammatory immune response. The shift was characterized by low interleukin (IL)-1β expression and M1 macrophage infiltration, whereas M2 macrophage infiltration and levels of IL-10, IL-4, and transforming growth factor (TGFβ) were increased. The modulated immune response correlated with high mitochondrial integrity, reduced oxidative stress, low left ventricular end-diastolic heart pressure, and a high survival rate. Isolated ANT1-transgenic (ANT1-TG) cardiomyocytes expressed low levels of pro-inflammatory cytokines such as IL-1α, tumor necrosis factor α, and TGFβ. However, they showed increased expression and cellular release of anti-inflammatory immunomodulators such as vascular endothelial growth factor. The secretome from ANT1-TG cardiomyocytes initiated stress resistance when applied to ischemic wild-type cardiomyocytes and endothelial cells. It additionally prevented macrophages from expressing pro-inflammatory cytokines. Additionally, ANT1 expression correlated with genes that are related to cytokine and growth factor pathways in hearts of patients with ischemic cardiomyopathy. In conclusion, ANT1-TG cardiomyocytes secrete soluble factors that influence ischemic cardiac cells and initiate an anti-inflammatory immune response in ischemic hearts.