Bacterial adenine cross-feeding stems from a purine salvage bottleneck.

Diverse ecosystems host microbial relationships that are stabilized by nutrient cross-feeding. Cross-feeding can involve metabolites that should hold value for the producer. Externalization of such communally valuable metabolites is often unexpected and difficult to predict. Previously, we discovered purine externalization by Rhodopseudomonas palustris by its ability to rescue an Escherichia coli purine auxotroph. Here we found that an E. coli purine auxotroph can stably coexist with R. palustris due to purine cross-feeding. We identified the cross-fed purine as adenine. Adenine was externalized by R. palustris under diverse growth conditions. Computational modeling suggested that adenine externalization occurs via diffusion across the cytoplasmic membrane. RNAseq analysis led us to hypothesize that adenine accumulation and externalization stem from a salvage pathway bottleneck at the enzyme encoded by apt. Ectopic expression of apt eliminated adenine externalization, supporting our hypothesis. A comparison of 49 R. palustris strains suggested that purine externalization is relatively common, with 16 strains exhibiting the trait. Purine externalization was correlated with the genomic orientation of apt, but apt orientation alone could not always explain purine externalization. Our results provide a mechanistic understanding of how a communally valuable metabolite can participate in cross-feeding. Our findings also highlight the challenge in identifying genetic signatures for metabolite externalization.

Identification of AtALKBH1A and AtALKBH1D as DNA N6-adenine demethylases in Arabidopsis thaliana.

DNA N6-methyladenine (6 mA) has recently been discovered as a novel DNA modification in animals and plants. In mammals, AlkB homolog 1 (ALKBH1) has been identified as a DNA 6 mA demethylase. ALKBH1 tightly controls the DNA 6 mA methylation level of mammalian genomes and plays important role in regulating gene expression. DNA 6 mA methylation has also been reported to exist in plant genomes, however, the plant DNA 6 mA demethylases and their function remain largely unknown. Here we identify homologs of ALKBH1 as DNA 6 mA demethylases in Arabidopsis. We discover that there are four homologs of ALKBH1, AtALKBH1A, AtALKBH1B, AtALKBH1C and AtALKBH1D, in Arabidopsis. In vitro enzymatic activity studies reveal that AtALKBH1A and 1D can efficiently erase DNA 6 mA methylation. Loss of function of AtALKBH1A and AtALKBH1D causes elevated DNA 6 mA methylation levels in vivo. atalkbh1a/1d mutant displays delayed seed gemination. Based on our RNA-seq data, we find some regulators of seed gemination are dysregulated in atalkbh1a/1d, and the dysregulation is correlated with changes of DNA 6 mA methylation levels. This study identifies plant DNA 6 mA demethylases and reports the function of DNA 6 mA methylation in regulating seed germination.

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation.

KEY MESSAGE: Barley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5' terminal nucleotide of small RNAs, demonstrating functional conservation and divergence. The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5' adenine residue, while also accepting 5' guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5' adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Recovery of Water Homeostasis in Adenine-Induced Kidney Disease Is Mediated by Increased AQP2 Membrane Targeting.

Chronic kidney disease (CKD) represents a major public health burden with increasing prevalence. Current therapies focus on delaying CKD progression, underscoring the need for innovative treatments. This necessitates animal models that accurately reflect human kidney pathologies, particularly for studying potential reversibility and regenerative mechanisms, which are often hindered by the progressive and irreversible nature of most CKD models. In this study, CKD was induced in mice using a 0.2% adenine-enriched diet for 4 weeks, followed by a recovery period of 1 or 2 weeks. The aim was to characterize the impact of adenine feeding on kidney function and injury as well as water and salt homeostasis throughout disease progression and recovery. The adenine diet induced CKD is characterized by impaired renal function, tubular injury, inflammation, and fibrosis. A significant decrease in urine osmolality, coupled with diminished aquaporin-2 (AQP2) expression and membrane targeting, was observed after adenine treatment. Intriguingly, these parameters exhibited a substantial increase after a two-week recovery period. Despite these functional improvements, only partial reversal of inflammation, tubular damage, and fibrosis were observed after the recovery period, indicating that the inclusion of the molecular and structural parameters is needed for a more complete monitoring of kidney status.

Optimization and validation of a UPLC-MS/MS assay for simultaneous quantification of 2,8-dihydroxyadenine, adenine, allopurinol, oxypurinol and febuxostat in human plasma.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare , hereditary disorder characterized by renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stone formation and chronic kidney disease (CKD). Treatment with a xanthine oxidoreductase inhibitor, allopurinol or febuxostat, reduces urinary DHA excretion and slows the progression of CKD. The method currently used for therapeutic monitoring of APRT deficiency lacks specificity and thus, a more reliable measurement technique is needed. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma was optimized and validated. Plasma samples were prepared with protein precipitation using acetonitrile followed by evaporation. The chemometric approach design of experiments was implemented to optimize gradient steepness, amount of organic solvent, flow rate, column temperature, cone voltage, desolvation temperature and desolvation flow rate. Experimental screening was conducted using fractional factorial design with addition of complementary experiments at the axial points for optimization of peak area, peak resolution and peak width. The assay was validated according to the US Food and Drug Administration guidelines for bioanalytical method validation over the concentration range of 50 to 5000 ng/mL for DHA, allopurinol and febuxostat, 100 to 5000 ng/mL for adenine and 50 to 12,000 ng/mL for oxypurinol, with r2 ≥ 0.99. The analytical assay achieved acceptable performance of accuracy (-10.8 to 8.3 %) and precision (CV < 15 %). DHA, adenine, allopurinol, oxypurinol and febuxostat were stable in plasma samples after five freeze-thaw cycles at -80 °C and after storage at -80 °C for 12 months. The assay was evaluated for quantification of the five analytes in clinical plasma samples from six APRT deficiency patients and proved to be both efficient and accurate. The proposed assay will be valuable for guiding pharmacotherapy and thereby contribute to improved and more personalized care for patients with APRT deficiency.

Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope.

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first use domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibit shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expand the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We then use these enhancements to introduce therapeutically relevant edits in a variety of cell types. Finally, we validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

The solute carrier SLC25A17 sustains peroxisomal redox homeostasis in diverse mammalian cell lines.

Despite the crucial role of peroxisomes in cellular redox maintenance, little is known about how these organelles transport redox metabolites across their membrane. In this study, we sought to assess potential associations between the cellular redox landscape and the human peroxisomal solute carrier SLC25A17, also known as PMP34. This carrier has been reported to function as a counter-exchanger of adenine-containing cofactors such as coenzyme A (CoA), dephospho-CoA, flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD+), adenosine 3',5'-diphosphate, flavin mononucleotide, and adenosine monophosphate. We found that inactivation of SLC25A17 resulted in a shift toward a more reductive state in the glutathione redox couple (GSSG/GSH) across HEK-293 cells, HeLa cells, and SV40-transformed mouse embryonic fibroblasts, with variable impact on the NADPH levels and the NAD+/NADH redox couple. This phenotype could be rescued by the expression of Candida boidinii Pmp47, a putative SLC25A17 orthologue reported to be essential for the metabolism of medium-chain fatty acids in yeast peroxisomes. In addition, we provide evidence that the alterations in the redox state are not caused by changes in peroxisomal antioxidant enzyme expression, catalase activity, H2O2 membrane permeability, or mitochondrial fitness. Furthermore, treating control and ΔSLC25A17 cells with dehydroepiandrosterone, a commonly used glucose-6-phosphate dehydrogenase inhibitor affecting NADPH regeneration, revealed a kinetic disconnection between the peroxisomal and cytosolic glutathione pools. Additionally, these experiments underscored the impact of SLC25A17 loss on peroxisomal NADPH metabolism. The relevance of these findings is discussed in the context of the still ambiguous substrate specificity of SLC25A17 and the recent observation that the mammalian peroxisomal membrane is readily permeable to both GSH and GSSG.

Adenine is an anti-inflammatory metabolite found to be more abundant in M-CSF over GM-CSF-differentiated human macrophages.

Immunometabolism has been unveiled in the last decade to play a major role in controlling macrophage metabolism and inflammation. There has been a constant effort to understand the immunomodulating properties of regulated metabolites during inflammation with the aim of controlling and re-wiring aberrant macrophages in inflammatory diseases. M-CSF and GM-CSF-differentiated macrophages play a key role in mounting successful innate immune responses. When a resolution phase is not achieved however, GM-CSF macrophages contribute substantially more towards an adverse inflammatory milieu than M-CSF macrophages, consequently driving disease progression. Whether there are specific immunometabolites that determine the homoeostatic or inflammatory nature of M-CSF and GM-CSF-differentiated macrophages is still unknown. As such, we performed metabolomics analysis on LPS and IL-4-stimulated M-CSF and GM-CSF-differentiated human macrophages to identify differentially accumulating metabolites. Adenine was distinguished as a metabolite significantly higher in M-CSF-differentiated macrophages after both LPS or IL-4 stimulation. Human macrophages treated with adenine before LPS stimulation showed a reduction in inflammatory gene expression, cytokine secretion and surface marker expression. Adenine caused macrophages to become more quiescent by lowering glycolysis and OXPHOS which resulted in reduced ATP production. Moreover, typical metabolite changes seen during LPS-induced macrophage metabolic reprogramming were absent in the presence of adenine. Phosphorylation of metabolic signalling proteins AMPK, p38 MAPK and AKT were not responsible for the suppressed metabolic activity of adenine-treated macrophages. Altogether, in this study we highlight the immunomodulating capacity of adenine in human macrophages and its function in driving cellular quiescence.

Role of RNA Modifications, Especially m6A, in Aflatoxin Biosynthesis of Aspergillus flavus.

RNA modifications play key roles in eukaryotes, but the functions in Aspergillus flavus are still unknown. Temperature has been reported previously to be a critical environmental factor that regulates the aflatoxin production of A. flavus, but much remains to be learned about the molecular networks. Here, we demonstrated that 12 kinds of RNA modifications in A. flavus were significantly changed under 29 °C compared to 37 °C incubation; among them, m6A was further verified by a colorimetric method. Then, the transcriptome-wide m6A methylome and m6A-altered genes were comprehensively illuminated through methylated RNA immunoprecipitation sequencing and RNA sequencing, from which 22 differentially methylated and expressed transcripts under 29 °C were screened out. It is especially notable that AFCA_009549, an aflatoxin biosynthetic pathway gene (aflQ), and the m6A methylation of its 332nd adenine in the mRNA significantly affect aflatoxin biosynthesis in A. flavus both on media and crop kernels. The content of sterigmatocystin in both ΔaflQ and aflQA332C strains was significantly higher than that in the WT strain. Together, these findings reveal that RNA modifications are associated with secondary metabolite biosynthesis of A. flavus.

Efficient gene editing in induced pluripotent stem cells enabled by an inducible adenine base editor with tunable expression.

The preferred method for disease modeling using induced pluripotent stem cells (iPSCs) is to generate isogenic cell lines by correcting or introducing pathogenic mutations. Base editing enables the precise installation of point mutations at specific genomic locations without the need for deleterious double-strand breaks used in the CRISPR-Cas9 gene editing methods. We created a bulk population of iPSCs that homogeneously express ABE8e adenine base editor enzyme under a doxycycline-inducible expression system at the AAVS1 safe harbor locus. These cells enabled fast, efficient and inducible gene editing at targeted genomic regions, eliminating the need for single-cell cloning and screening to identify those with homozygous mutations. We could achieve multiplex genomic editing by creating homozygous mutations in very high efficiencies at four independent genomic loci simultaneously in AAVS1-iABE8e iPSCs, which is highly challenging with previously described methods. The inducible ABE8e expression system allows editing of the genes of interest within a specific time window, enabling temporal control of gene editing to study the cell or lineage-specific functions of genes and their molecular pathways. In summary, the inducible ABE8e system provides a fast, efficient and versatile gene-editing tool for disease modeling and functional genomic studies.

m6A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3.

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m6A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m6A level of polysomal and cytoplasmic mRNAs with m6A-LAIC-seq and m6A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m6A-methylated, whereas those enriched in P-body are hyper-m6A-methylated. Downregulation of the m6A writer METTL14 enhances translation by switching originally hyper-m6A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m6A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m6A-dependent manner.

METTL14-m6A-FOXO3a axis regulates autophagy and inflammation in ankylosing spondylitis.

The role of m6A in ankylosing spondylitis (AS) remains largely obscure. In this study, we found that m6A modification was decreased in T cells of AS, and the abnormal m6A modification was attributed to the downregulation of methyltransferase-like 14 (METTL14). METTL14 exerted a critical role in regulating autophagy activity and inflammation via targeting Forkhead box O3a (FOXO3a). Mechanistically, the loss of METTL14 decreased the expression of FOXO3a, leading to the damage of autophagic flux and the aggravation of inflammation. Inversely, the forced expression of METTL14 upregulated the expression of FOXO3a, thereby activating autophagy and alleviating inflammation. Furthermore, our results revealed that METTL14 targeted FOXO3a mRNA and regulated its expression and stability in a m6A-dependent manner. These findings uncovered the functional importance of m6A methylation mechanisms in the regulation of autophagy and inflammation, which expanded our understanding of this interaction and was critical for the development of therapeutic strategies for AS.

Decreasing predictable DNA off-target effects and narrowing editing windows of adenine base editors by fusing human Rad18 protein variant.

Adenine base editors, enabling targeted A-to-G conversion in genomic DNA, have enormous potential in therapeutic applications. However, the currently used adenine base editors are limited by wide editing windows and off-target effects in genetic therapy. Here, we report human e18 protein, a RING type E3 ubiquitin ligase variant, fusing with adenine base editors can significantly improve the preciseness and narrow the editing windows compared with ABEmax and ABE8e by diminishing the abundance of base editor protein. As a proof of concept, ABEmax-e18 and ABE8e-e18 dramatically decrease Cas9-dependent and Cas9-independent off-target effects than traditional adenine base editors. Moreover, we utilized ABEmax-e18 to establish syndactyly mouse models and achieve accurate base conversion at human PCSK9 locus in HepG2 cells which exhibited its potential in genetic therapy. Furthermore, a truncated version of base editors-RING (ABEmax-RING or AncBE4max-RING), which fusing the 63 amino acids of e18 protein RING domain to the C terminal of ABEmax or AncBE4max, exhibited similar effect compared to ABEmax-e18 or AncBE4max-e18.In summary, the e18 or RING protein fused with base editors strengthens the precise toolbox in gene modification and maybe works well with various base editing tools with a more applicable to precise genetic therapies in the future.

N6-Methyladenine Progressively Accumulates in Mitochondrial DNA during Aging.

N6-methyladenine (6mA) in the DNA is a conserved epigenetic mark with various cellular, physiological and developmental functions. Although the presence of 6mA was discovered a few years ago in the nuclear genome of distantly related animal taxa and just recently in mammalian mitochondrial DNA (mtDNA), accumulating evidence at present seriously questions the presence of N6-adenine methylation in these genetic systems, attributing it to methodological errors. In this paper, we present a reliable, PCR-based method to determine accurately the relative 6mA levels in the mtDNA of Caenorhabditis elegans, Drosophila melanogaster and dogs, and show that these levels gradually increase with age. Furthermore, daf-2(-)-mutant worms, which are defective for insulin/IGF-1 (insulin-like growth factor) signaling and live twice as long as the wild type, display a half rate at which 6mA progressively accumulates in the mtDNA as compared to normal values. Together, these results suggest a fundamental role for mtDNA N6-adenine methylation in aging and reveal an efficient diagnostic technique to determine age using DNA.

Identification of m6A suppressor EIF4A3 as a novel cancer prognostic and immunotherapy biomarker through bladder cancer clinical data validation and pan-cancer analysis.

EIF4A3 represents a novel m6A suppressor that exerts control over the global m6A mRNA modification level, therefore influencing gene destiny. Despite increasing evidence that highlights a pivotal role of EIF4A3 in tumor progression and immunity, a comprehensive pan-cancer analysis of EIF4A3 has yet to be conducted, in order to ascertain whether EIF4A3 could be a viable biomarker for cancer screening, prediction of prognosis, and to facilitate accurate therapy design in various human malignancies. We analyzed the expression levels of EIF4A3 in bladder cancer compared to para-cancer tissue. Subsequently survival analysis was conducted to ascertain the potential association between EIF4A3 expression and patient prognosis. To further corroborate this evidence, we conducted an extensive data mining process of several publicly available databases, including UCSC Xena database, TCGA, and GTEx. Raw data from the UCSC Xena database was processed using online tools to obtain results that could be subjected to further analysis. Our study unveiled a considerable increase in the expression levels of EIF4A3 in bladder cancer compared to para-cancer tissue. Subsequent validation experiments confirmed that bladder cancer patients exhibiting higher levels of EIF4A3 expression have significantly worse prognostic outcomes. Next, our pan-cancer analysis found that the expression level of EIF4A3 is significantly higher in most cancers. Notably, high expression levels of EIF4A3 were negatively associated with patient prognosis across various cancer types. Furthermore, as a novel m6A suppressor, EIF4A3 was found to be correlated with numerous RNA modification genes in multiple cancer types. Meanwhile, analysis of publicly available databases revealed that EIF4A3 expression was significantly related to immune score and immune cell levels in most cancer types. Interestingly, EIF4A3 was also identified as a superior immunotherapy biomarker when compared to several traditional immunotherapy biomarkers. Lastly, genetic alterations analysis revealed that amplification was the most frequently occurring abnormality in the EIF4A3 gene. EIF4A3 emerges as a promising biomarker with the potential to significantly enhance tumor screening, prognostic evaluation, and the design of individualized treatment strategies across a diverse array of malignancies.

Effect of P-glycoprotein and Cotreatment with Sofosbuvir on the Intestinal Permeation of Tenofovir Disoproxil Fumarate and Tenofovir Alafenamide Fumarate.

PURPOSE: We aimed to compare the effects of P-glycoprotein (ABCB1) on the intestinal uptake of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), and metabolites, tenofovir isoproxil monoester (TEM) and tenofovir (TFV), and to study the molecular mechanism of drug-drug interaction (DDI) between sofosbuvir (SOF) and TDF/TAF. METHODS: Bidirectional transport experiments in Caco-2 cells and accumulation studies in precision-cut intestinal slices prepared from the ileal segment of rodent (rPCIS) and human (hPCIS) intestines were performed. RESULTS: TDF and TAF were extensively metabolised but TAF exhibited greater stability. ABCB1 significantly reduced the intestinal transepithelial transfer and uptake of the TFV(TDF) and TFV(TAF)-equivalents. However, TDF and TAF were absorbed more efficiently than TFV and TEM. SOF did not inhibit intestinal efflux of TDF and TAF or affect intestinal accumulation of TFV(TDF) and TFV(TAF)-equivalents but did significantly increase the proportion of absorbed TDF. CONCLUSIONS: TDF and TAF likely produce comparable concentrations of TFV-equivalents in the portal vein and the extent of permeation is reduced by the activity of ABCB1. DDI on ABCB1 can thus potentially affect TDF and TAF absorption. SOF does not inhibit ABCB1-mediated transport of TDF and TAF but does stabilise TDF, albeit without affecting the quantity of TFV(TDF)-equivalents crossing the intestinal barrier. Our data thus suggest that reported increases in the TFV plasma concentrations in patients treated with SOF and TDF result either from a DDI between SOF and TDF that does not involve ABCB1 or from a DDI involving another drug used in combination therapy.

Impaired renal ischemia reperfusion recovery after bilateral renal artery ligation in rats treated with adenine: role of renal mitochondria.

Vascular calcification (VC) and ischemia reperfusion (IR) injury is characterised to have mitochondrial dysfunction. However, the impact of dysfunctional mitochondria associated with vascular calcified rat kidney challenged to IR is not explored and is addressed in the present study. Male Wistar rats were treated with adenine for 20 days to induce chronic kidney dysfunction and VC. After 63 days, renal IR protocol was performed with subsequent recovery for 24 h and 7 days. Various mitochondrial parameters and biochemical assays were performed to assess kidney function, IR injury and its recovery. Adenine-induced rats with VC, decreased creatinine clearance (CrCl), and severe tissue injury demonstrated an increase in renal tissue damage and decreased CrCl after 24 h of IR (CrCl in ml: IR-0.220.02, VC-IR-0.050.01). Incidentally, the 24 h IR pathology in kidney was similar in both VC-IR and normal rat IR. But, the magnitude of dysfunction was higher with VC-IR due to pre-existing basal tissue alterations. We found severed deterioration in mitochondrial quantity and quality supported by low bioenergetic function in both VC basal tissue and IR challenged sample. However, post 7 days of IR, unlike normal rat IR, VC rat IR did not improve CrCl and corresponding mitochondrial damage in terms of quantity and its function were observed. Based on the above findings, we conclude that IR in VC rat adversely affect the post-surgical recovery, mainly due to the ineffective renal mitochondrial functional restoration from the surgery.

Modular and Flexible Molecular Device for Simultaneous Cytosine and Adenine Base Editing at Random Genomic Loci in Filamentous Fungi.

Random base editing is regarded as a fundamental method for accelerating the genomic evolution in both scientific research and industrial applications. In this study, we designed a modular interaction-based dual base editor (MIDBE) that assembled a DNA helicase and various base editors through dockerin/cohesin-mediated protein-protein interactions, resulting in a self-assembled MIDBE complex capable of editing bases at any locus in the genome. The base editing type of MIDBE can be readily controlled by the induction of cytidine or/and adenine deaminase gene expression. MIDBE exhibited the highest editing efficiency 2.3 × 103 times greater than the native genomic mutation rate. To evaluate the potential of MIDBE in genomic evolution, we developed a removable plasmid-based MIDBE tool, which led to a remarkable 977.1% increase of lovastatin production in Monascus purpureus HJ11. MIDBE represents the first biological tool for generating and accumulating base mutations in Monascus chromosome and also offers a bottom-up strategy for designing the base editor.

Genome- and transcriptome-wide off-target analyses of a high-efficiency adenine base editor in tomato.

Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)-expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)-dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA-level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato.