Adaptive laboratory evolution (ALE) is a powerful strategy for exploring functional solution spaces, yet its pace is fundamentally constrained by low spontaneous mutation rates. Here, we present a broadly applicable genetic device that accelerates genome diversification in Gram-negative bacteria through the transient action of a dual base editor (ACBE3). This construct combines cytidine and adenosine deaminase activities with a single-stranded DNA-binding module, enabling efficient genome-wide mutagenesis without reliance on host-specific replication or repair systems. The expression of ACBE3 increased mutation rates by up to four orders of magnitude in Escherichia coli and Pseudomonas putida, outperforming classical random mutagenesis approaches such as UV irradiation and chemical treatment. Whole-genome sequencing revealed extensive and uniformly distributed mutations, predominantly consistent with deamination signatures, as well as a broader spectrum of nucleotide changes and small indels. The system displayed strong orthogonality, functioning across diverse bacterial taxa. As a proof of concept, coupling ACBE3-mediated diversification with selective growth conditions enabled the rapid evolution of Acinetobacter calcoaceticus P320, yielding mutant strains with enhanced growth on n-dodecane within only a few induction cycles. This approach decouples diversification from selection, enabling the controlled exploration of adaptive landscapes at unprecedented speeds. While high mutational loads impose cellular burdens and require tight regulatory control, the platform offers a versatile route to accelerate microbial evolution at both single-strain and community levels. Our results thus document dual base editing as a powerful framework for fostering evolution in microbial biotechnology.

The dysregulation of adenosine triphosphate (ATP) hydrolysis, a crucial process in energy metabolism, has been implicated in the complex landscape of tumor development and progression. This study aims to pinpoint crucial genes that characterize the ATP hydrolysis-driven molecular signature of hepatocellular carcinoma (HCC), with a view to examining their potential therapeutic applications in enhancing patient prognosis. Utilizing transcriptomic and clinical data from The Cancer Genome Atlas and Gene Expression Omnibus databases, a systematic framework was developed and validated for model construction and assessment. Univariate Cox regression analysis (P < .05) was performed on all 416 ATP hydrolysis-related genes (ARGs) to identify prognostically relevant genes. Following least absolute shrinkage and selection operator Cox regression with 10-fold cross-validation, an 11-gene signature was established, with the median training cohort risk score used as the cutoff for stratifying patients into high- and low-risk groups. Among 416 investigated ARGs, 85 displayed differential expression in HCC, with 179 demonstrating significant prognostic relevance (univariate Cox P < .05). Two distinct molecular subtypes, characterized by marked differences in prognosis and immune infiltration, were identified through unsupervised clustering. A refined 11-gene signature was constructed via least absolute shrinkage and selection operator Cox regression with 10-fold cross-validation (training area under the curve: 0.826/0.717/0.657 for 1/3/5 year; validation area under the curve: 0.608/0.603/0.621). These risk groups exhibited significantly divergent prognostic features, gene expression patterns, immune microenvironments, and drug sensitivities, suggesting the utility of this risk signature in predicting prognosis, drug responsiveness, and immune therapeutic outcomes. Ultimately, a nomogram incorporating the risk signature was devised, demonstrating favorable predictive performance in estimating HCC patient prognosis compared to other established prognostic factors. This study establishes a classification system and risk model grounded in ARGs, offering valuable insights into the prognosis and treatment responsiveness of HCC.

Feline infectious peritonitis (FIP) is responsive to treatment with the adenosine analogue GS-441524 (GS-44) and its prodrug, remdesivir (REM); both are now available on veterinary prescription in many countries. Therapeutic drug monitoring (TDM) of GS-44 has the potential to support dose selection for individual cats; however, TDM assays are currently only offered by one UK-based laboratory. This study describes a simple, cost-effective, and environmentally conscious high-performance liquid chromatography (HPLC) method for the quantification of GS-44 in feline plasma or serum. The method was validated in accordance with the International Council for Harmonisation M10 guidelines for bioanalytical methods. Calibration standards demonstrated linearity across a range of 2.59 to 5048.28 ng/mL, R[2] = 0.9880, with a lower limit of quantification of 2.59 ng/mL and upper limit of quantification of 5048 ng/mL; this is equivalent to an assay range of 0.06 to 208 µM. Precision, accuracy and spike recovery were within ±15% for 14 of 17 standard concentrations (±20% at the lowest three). Carry-over, dilution integrity, and analyte stability under common storage conditions all met the method's requirements. This is a simple, robust and accurate eco-friendly method suitable for adoption by diagnostic laboratories, enabling routine TDM for cats undergoing treatment for FIP with GS-44 and/or REM.

Background and Objectives: Liver regeneration following partial hepatectomy (PHx) is essential for restoring hepatic function. Polydeoxyribonucleotide (PDRN), an adenosine A2A receptor (A2AR) agonist, has demonstrated regenerative and proangiogenic properties in various tissues. However, its therapeutic potential for promoting liver regeneration after PHx has not been fully elucidated. The primary objective was to evaluate the effect of PDRN on liver regeneration after PHx, and the secondary objective was to examine associated changes in proliferation-related proteins and A2AR/cAMP, HGF/c-Met, and VEGF/VEGFR2 signaling. Materials and Methods: A rat model of 70% PHx was established, and PDRN (8 mg/kg) was administered intraperitoneally once daily for 5 consecutive days with or without the A2AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX; 8 mg/kg). Liver regeneration was evaluated using biochemical, histological, and molecular analyses. Results: PDRN enhanced liver regeneration following PHx, as demonstrated by reduced serum liver enzyme levels, enhanced recovery of liver mass, and improved histological architecture. PDRN also increased the expression of the proliferation-associated proteins proliferating cell nuclear antigen (PCNA) and cyclin D1. These effects were accompanied by increased A2AR expression and cyclic adenosine monophosphate (cAMP) levels, elevated hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) levels, and enhanced phosphorylation of c-Met and VEGFR2. Co-administration of DMPX attenuated the regenerative and molecular effects of PDRN, suggesting a potential involvement of A2AR-related signaling. Conclusions: Collectively, these findings suggest that PDRN may facilitate liver regeneration following PHx in association with increased expression of proliferation-associated proteins and changes in HGF/c-Met and VEGF/VEGFR2 signaling, supporting its potential as a therapeutic strategy following hepatic resection.

Background/Objectives: Rubidium-82 ([82]Rb) PET myocardial perfusion imaging (MPI) yields, in a single study, quantitative absolute myocardial blood flow (MBF, mL/min/g), myocardial flow reserve (MFR), and gated left ventricular (LV) function (LVEF, EDV, ESV, SV). These quantitative values depend on the tracer, scanner, kinetic model, software, and underlying population and have been characterised almost exclusively in North American and European cohorts. The Arabian Gulf, where Kuwait has among the highest age-standardised diabetes prevalence worldwide (25.6%), is essentially unstudied, so the distribution and behaviour of these parameters in such a real-world cardiometabolic population are unknown. To evaluate the distribution of the quantitative [82]Rb PET parameter set in a real-world Arabian Gulf cohort and, within a small clinically defined normal subgroup, to describe sex- and age-related patterns in absolute MBF, MFR, and LV function. Given the limited size of the normal subgroup, these values are presented as exploratory, hypothesis-generating observations rather than definitive population reference norms. Methods: Retrospective single-centre study of 330 consecutive [82]Rb PET/CT studies (analytic cohort n = 292 after exclusion of repeat studies and those with incomplete quantitative output; mean age 63.1 ± 12.1 years) who underwent adenosine-stress [82]Rb PET/CT MPI. The clinically defined normal subgroup, normal perfusion (C1), normal global MFR (≥2.0), normal resting LVEF, and no documented cardiac history, comprised 44 patients (25 female, 19 male). Reference values are reported as sex- and age-stratified centiles (5th, 25th, median, 95th percentiles), with the 5th percentile reported for each parameter. Sex differences used Mann-Whitney U with rank-biserial r and Cohen's d (95% CI); age was examined across broad bands. All analyses followed APA 7 standards with Bonferroni correction. Results: In the clinically defined normal subgroup, median global stress MBF was 2.94 mL/min/g (5th percentile 2.03) and median global MFR was 2.72 (5th percentile 2.06); every value satisfied the C1 definition (MFR ≥ 2.0) by construction, so these limits describe the preselected subgroup and cannot independently validate the 2.0 threshold. Women had higher resting MBF than men (median 1.07 vs. 0.90 mL/min/g; p = 0.007), with numerically lower global MFR that did not survive correction for multiple comparisons (2.57 vs. 2.98; p = 0.022); stress MBF did not differ by sex (3.00 vs. 2.91; p = 0.522). LV volumes were smaller in women, while LVEF was similar between sexes. The 5th percentile for stress LVEF was 54% (women) and 53% (men). These absolute-flow reference values are lower than those reported for Western low-risk cohorts (stress MBF ~3.25 mL/min/g; MFR ~3.18), consistent with the higher cardiometabolic burden of this population. Conclusions: In this exploratory single-centre evaluation, quantitative [82]Rb PET flow values in a small clinically defined normal Arabian Gulf subgroup were lower than Caucasian-derived values, while sex-related differences in resting flow were evident and age-related differences were numerically consistent with previously reported trends. Because the normal subgroup is small, these findings are hypothesis-generating and require confirmation in larger, prospectively screened cohorts before use as population reference values; they nonetheless indicate that population- and pipeline-specific calibration is needed when quantitative [82]Rb thresholds derived elsewhere are applied locally.

Background: Tuberculous pleural effusion (TPE) occasionally presents with polymorphonuclear leukocyte (PMN)-predominant pleural fluid (PF) and elevated serum C-reactive protein (CRP), mimicking parapneumonic effusion (PPE). We investigated factors distinguishing TPE from PPE in this diagnostically challenging setting. Methods: We retrospectively reviewed consecutive patients with confirmed TPE or PPE at a tertiary referral center (2010-2024). Patients with serum CRP ≥ 10 mg/dL were classified according to PF PMN-predominance. Independent discriminators between PMN-predominant TPE and PPE were identified using multivariable Firth logistic regression. Results: Among 414 patients with confirmed TPE, 108 (26%) had serum CRP ≥ 10 mg/dL, including 20 with PMN-predominance and 88 without. Anti-tuberculosis treatment was modestly delayed in the PMN-predominant group (p = 0.017). These 20 patients were compared with 335 PPE patients showing the same CRP/PMN profile. Pulmonary nodular lesions on chest computed tomography and PF adenosine deaminase (ADA) were independent discriminators. A composite two-tier rule-nodular lesions present or PF ADA > 80 IU/L if absent-achieved an AUROC of 0.925, 80.0% sensitivity, and 86.2% specificity. Conclusions: In patients with PMN-predominant pleural effusion and elevated serum CRP, this simple, readily available rule may help identify patients warranting early pleural biopsy and heightened suspicion for TPE before microbiological confirmation.

Background/Objectives: Polydeoxyribonucleotide (PDRN) has anti-inflammatory and tissue-protective properties associated with activation of the adenosine A2A receptor (A2AR). Although hepatoprotective actions of PDRN have previously been described, its potential association with high-mobility group box 1 (HMGB1)-related inflammatory responses and hepatic macrophage accumulation during acute liver injury (ALI) is not fully understood. This study examined whether PDRN affects HMGB1- and monocyte chemoattractant protein-1 (MCP-1)-associated responses together with hepatic macrophage accumulation in ALI. Methods: ALI was induced in ICR mice by carbon tetrachloride (CCl4) administration. PDRN was administered either alone or together with the selective A2AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were quantified. Histological alterations were evaluated using hematoxylin and eosin staining. Hepatic macrophage accumulation was assessed by F4/80 immunofluorescence staining. Enzyme-linked immunosorbent assays were used to quantify HMGB1, MCP-1, interleukin-10 (IL-10), cyclic adenosine monophosphate (cAMP), and A2AR. Results: PDRN significantly reduced serum markers of liver injury and attenuated histopathological damage following CCl4 administration. PDRN treatment also decreased F4/80-positive macrophage accumulation in the hepatic tissue. In addition, PDRN reduced HMGB1 and MCP-1 levels in both the serum and liver tissues while significantly increasing IL-10, cAMP, and A2AR levels. Conclusions: PDRN attenuated CCl4-induced ALI and was associated with reduced HMGB1 and MCP-1 levels, decreased hepatic macrophage accumulation, and increased A2AR/cAMP signaling. These findings suggest that modulation of macrophage-associated inflammatory responses may contribute to the hepatoprotective effects of PDRN.

This article describes the synthesis, structure confirmation, and evaluation of physico-chemical, pharmacological, and pharmacokinetic properties of new para-fluorophenylated dihydroimidazotriazinones. Based on the strategy of heteroannulation, and the concept of fluorine-hydrogen isosterism, an efficient and simple synthetic route was developed. The target heterobicycles (10-18) were obtained by the reaction of nucleophilic building blocks, i.e., 1-(R-phenyl)-2-hydrazinylideneimidazolidine hydroiodides (1-9), with an electrophilic two-carbon synthon, i.e., 2-(4-fluorophenyl)-2-oxoacetic acid. The structures of the new compounds were confirmed by spectroscopic data. The spectrum of anticancer activity, selectivity profile, and effect on caspase levels, as well as the haemolytic and antihaemolytic properties of the molecules were assessed in in vitro and ex vivo studies. Most compounds exhibited antiproliferative activity against human solid tumour and leukaemic cells (superior/comparable to anticancer drugs) with low toxicity to normal cells. Moreover, they revealed a broader spectrum of anticancer activity than previously obtained isosteres without fluorine substitution, suggesting that the isosteric replacement of hydrogen with fluorine was a fruitful modification. The most selective molecules were able to increase the levels of apoptotic caspases in lung, cervical, and breast cancer cells. Molecular docking results revealed that although all fluorophenylated fused triazinones exhibited affinity for the adenosine A2A and A2B receptors, ligands 13, 16, and 17 emerged as promising dual-target candidates due to their superior interaction profiles and stronger binding affinities for both receptor subtypes. All the compounds proved to be safe for red blood cells, as they did not induce any haemolytic effects. Additionally, most of them revealed protective effects on oxidatively stressed erythrocytes, and their antihaemolytic activity was better or comparable to that of antioxidant standards. In silico ADME (absorption, distribution, metabolism, excretion) profiling demonstrated favourable drug-like and pharmacokinetic properties of compounds. Lipophilicity of the molecules was measured by RP-HPLC on an ODS-2 column using an appropriately selected mobile phase. The retention factors, logs k, for most compounds showed a strong correlation with both in silico log P (logarithmic n-octanol/water partition coefficient) and log PHSA (logarithmic human serum albumin/water partition coefficient) values. In summary, among the nine original fluorophenylated fused 1,2,4-triazinones studied, five molecules (11, 13, 14, 15, and 17) seem to be the most promising anticancer drug candidates suitable for further development.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR1 and ADAR2, is an epitranscriptomic mechanism with emerging roles in cardiovascular disease. We investigated ADAR expression, clinical correlates, A-to-I RNA editing signatures, transcriptome and miRNA targetome in thoracic aortic aneurysm (TAA), focusing on Marfan syndrome (MFS). Immunohistochemical analysis of 58 TAA specimens (18 MFS-associated TAA, 19 tricuspid aortic valve-associated TAA (TAV), 21 bicuspid aortic valve-associated TAA (BAV)) showed the highest ADAR1 and ADAR2 in MFS. ADAR2 in vascular smooth muscle cells (vSMCs) correlated positively with aortic diameter across subtypes; ADAR1 inversely correlated with sinotubular junction dimension in MFS. Transcriptomic profiling of MFS-derived vSMC cultures identified 30 differentially expressed genes enriched in immune activation, impaired RTK signaling, and vSMC reprogramming. RNA editing analysis revealed 77 differentially edited sites across 65 genes, predominantly hypoedited in MFS, including four protein-coding recoding events at PPIL3, SLC12A6, CTSB, and VHL. miRNA seed-match analysis of 47 significant 3'UTR sites predicted 458 editing-dependent interactions across 36 genes. Only 14/36 genes showed concordant mRNA changes, suggesting that editing-dependent miRNA retargeting acts primarily at the translational level. These findings establish ADAR enzymes as candidates tissue biomarkers of TAA severity and ADAR1 as the principal driver of epitranscriptomic dysregulation in MFS aortopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the death of motor neurons leading to paralysis and death generally 3-5 years post-symptom onset. ALS is a cell- and non-cell-autonomous disease, with glia such as astrocytes influencing disease pathology and progression. Our laboratory has previously identified purine metabolism dysfunction in induced neural progenitor cell-derived astrocytes (iAstrocytes) from sporadic ALS (SALS) cases, driven by loss of the enzyme adenosine deaminase (ADA). Here, we have demonstrated that loss of ADA, along with changes to ecto-5'-nucleotidase and hypoxanthine-guanine phosphoribosyl transferase led to disruption in purine metabolite levels, linked to the level of the ADA enzyme. These alterations were recapitulated in SALS CSF and post-mortem tissue, with ageing and sex affecting purine metabolite levels downstream of ADA and positively correlating with disease progression. Loss of ADA led to reduced 53BP1-mediated DNA repair and increased P16 levels, which was recapitulated in control iAstrocytes via ADA inhibition. Our findings indicate that TDP43 dysfunction drives impairment of ADA-mediated purine metabolism in vitro, leading to downstream effects that include DNA damage, likely through inhibition of DNA repair mechanisms, and the induction of cellular senescence. Furthermore, these results suggest that therapeutic targeting of the ADA pathway may help slow ALS disease progression.

The pathophysiological mechanisms underlying diabetic lung disease (DLD) remain poorly understood. Beyond hyperglycemia, dysregulated molecules such as adenosine may contribute to pulmonary inflammation and fibrosis. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), has demonstrated anti-inflammatory and antifibrotic effects, including reductions in macrophage (MΦ) infiltration in animal models of diabetes-associated complications; however, its effects on DLD remain unexplored. Here, diabetic Sprague-Dawley rats were treated with MRS1754, and pulmonary inflammation, fibrosis, MΦ infiltration, and polarization were assessed in bronchoalveolar lavage fluid (BALF) and lung tissue using immunohistopathological and molecular approaches. According to data normality, one-way ANOVA/Tukey's test or Kruskal-Wallis/Dunn's test were employed, as appropriate; ordinal data were analyzed using the Mann-Whitney test with Bonferroni correction. Compared with controls, diabetic rats exhibited pulmonary inflammation and fibrosis, increased MΦ infiltration, and an M1/M2 polarization imbalance. MRS1754 attenuated these alterations, reducing Tumor necrosis factor-alpha (TNF-α) and Transforming growth factor-beta 1 (TGF-β1) levels, decreasing MΦ accumulation in BALF and lung tissue, and fully restoring the M1/M2 balance to control levels. BALF MΦ populations, particularly the M2 (Cluster of differentiation 163-positive; CD163[+]) subset, correlated strongly with TGF-β1 and fibronectin (FN1) levels. Reanalysis of human fibrotic lung single-cell transcriptomic data further identified MΦ as a major TGF-β1-expressing cell population. Collectively, these findings suggest that A2BAR antagonism partially mitigates inflammation, fibrosis, and MΦ infiltration while fully restoring MΦ polarization balance in DLD, supporting its potential as a therapeutic strategy.

Dietary nucleotides and nucleic-acid-derived compounds are considered bioactive nutrients, but their metabolic relevance remains uncertain. This narrative review, supported by a structured literature search, evaluates intestinal handling and evidence on glucose and lipid metabolism, redox homeostasis, immune function, gut barrier regulation, and microbiota. Direct supplementation studies are distinguished from nucleoside/nucleobase evidence, dietary nucleic acids or purine-rich foods, and endogenous extracellular purinergic signaling. Preclinical studies suggest defined nucleotide preparations may modulate AMP-activated protein kinase (AMPK), insulin receptor substrate 1 (IRS-1)/protein kinase B (AKT)/forkhead box protein O1 (FOXO1) signaling, lipid accumulation, and mitochondrial/redox-related endpoints. In contrast, extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine 5'-monophosphate (AMP) studies provide mechanistic context but do not show that oral supplementation modifies purinergic signaling in humans. Human intervention evidence is sparse and derived from older adults not selected for insulin resistance, type 2 diabetes, or metabolic-dysfunction-associated steatotic liver disease (MASLD). Reductions in homeostatic model assessment of insulin resistance (HOMA-IR) should be interpreted as changes in a surrogate estimate, not proof of therapeutic efficacy. The supplemental doses evaluated in available human intervention studies appear generally well-tolerated, whereas short-term high nucleotide intake can raise circulating uric acid. Dietary nucleotides therefore remain candidate, not established, metabolic or immune therapies.

Glucagon-Like Peptide-1 (GLP-1) is an incretin family peptide hormone with significant therapeutic applications. This study evaluated multiple functional methodologies for assessing GLP-1 agonist biological activity, encompassing in vitro cell-based bioassays and in vivo systems. We developed and comparatively analyzed cell-based bioassays measuring distinct cellular signaling events, such as β-arrestin recruitment, cyclic adenosine monophosphate (cAMP) elevation, and calcium (Ca[2+]) mobilization. Each assay was systematically evaluated for robustness and quality control applicability. All assay systems demonstrated functionality independent of the cellular mechanism employed. Real-time Ca[2+] flux measurements confirmed the cell-based assay endpoint data, while cross-verification analyses using the oral glucose tolerance test in mice confirmed the representativeness of the cAMP formats. Initial validation studies revealed inter-assay variations, leading to the selection of the cAMP HTRF assay as the preferred quality control methodology. Further assay refinement and subsequent validation studies confirmed the cAMP HTRF assay's suitability for quality control applications. This methodology has been successfully implemented in routine release testing of a commercially available GLP-1 agonist.

Hepatocellular carcinoma (HCC) is characterized by profound metabolic reprogramming and mitochondrial dysfunction, yet the molecular regulators underlying these alterations remain incompletely understood. AarF domain-containing kinase 1 (ADCK1) is an evolutionarily conserved protein associated with mitochondrial function, but its role in HCC bioenergetics has not been defined. In this study, we investigated the effects of ADCK1 on mitochondrial metabolism using CRISPR/Cas9-mediated ADCK1 knockout in HepG2 and SNU-449 HCC cells. Mitochondrial respiration, glycolytic activity, ATP production, lactate generation, mitochondrial membrane potential, and superoxide production were assessed following ADCK1 KO. ADCK1 KO resulted in marked reductions in basal and maximal mitochondrial respiration, ATP-linked respiration, glycolytic activity, intracellular ATP, and lactate production in both HCC cell models. ADCK1 KO also reduced mitochondrial membrane potential in a clone-dependent manner. Despite these profound bioenergetic defects, mitochondrial superoxide production was not consistently altered across the knockout clones. These findings indicate that ADCK1 supports both oxidative phosphorylation and glycolytic metabolism and is required for maintenance of bioenergetic homeostasis in HCC cells. Collectively, our results identify ADCK1 as a previously unrecognized regulator of HCC mitochondrial metabolism.