In vitro evidence that the vasorelaxant effects of 2-nitro-1-phenyl-1-propanol on rat coronary arteries involve cyclic nucleotide pathways.

The synthetic nitro-alcohol 2-nitro-1-phenyl-1-propanol (NPP) has endothelium-independent relaxing properties in isolated preparations of rat aorta and mesenteric artery. In this study, we investigated whether the vasodilator effects occur in coronary vessels and explored whether hyperpolarization is involved in the underlying mechanism of NPP-induced smooth muscle relaxation. The relaxing responses were studied in isolated preparations of the left anterior descending coronary (ADC) and the septal coronary (SC) arteries, which had been previously maintained under sustained contraction induced by the thromboxane A2 analogue U-46619. Administered cumulatively, NPP elicited concentration-dependent vasorelaxation with similar potency in both vessels. The relaxant effect remained unaffected by the nitric oxide synthase inhibitor L-NAME, the protein kinase C inhibitor bisindolylmaleimide IV and the Rho-associated protein kinase inhibitor Y-27632. However, it was significantly diminished by the adenylyl cyclase inhibitor MDL-12,330A, the guanylyl cyclase inhibitor ODQ, as well as the K+ channel inhibitors tetraethylammonium and CsCl. In ADC preparations impaled with intracellular micropipettes, NPP hyperpolarized the vascular preparation. When the isolated preparation was precontracted by 5-hydroxytryptamine or 80 mM KCl, NPP-induced relaxation with lower pharmacological potency compared to the vessels contracted by U-46619. In conclusion, NPP exhibits vasorelaxant effects on rat coronary arteries, likely involving pathways that include cyclic nucleotide production and membrane hyperpolarization.

Engineering Phi29-DNAP Variants for Customized DNA Hydrogel Materials.

DNA hydrogels, which hold potential for use in medicine, biosensors, and tissue engineering, can be produced through enzymatic rolling circle amplification (RCA) using phi29 DNA polymerase (DNAP). This paper introduces new DNAP variants designed for RCA-based DNA hydrogel production, featuring enzymes with modified DNA binding, enhanced thermostability, reduced exonuclease activity, and protein tags for fluorescence detection or specific immobilization. We evaluated these enzymes by quantifying DNA output via quantitative PCR (qPCR) and assessing hydrogel mechanical properties through micromechanical indentation. The results showed that most variants generated similar DNA amounts and hydrogels with comparable mechanical properties. Additionally, all variants successfully incorporated non-natural nucleotides, such as base-modified dGTP derivatives and 2'-fluoro-dGTP, during RCA. This study's robust analytical approach offers a strong foundation for selecting new enzymes and producing DNA hydrogels with tailored material properties.

The role of cyclic nucleotides in bacterial antimicrobial resistance and tolerance.

Nucleotide signalling molecules - mainly cyclic 3',5'-adenosine phosphate (cAMP), bis-(3',5')-cyclic diguanosine monophosphate (c-di-GMP), and bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) - contribute to the regulation of cellular pathways. Numerous recent works have focused on the involvement of these cyclic nucleotide phosphates (cNPs) in bacterial resistance and tolerance to antimicrobial treatment. Indeed, the rise of antimicrobial resistance (AMR) is a rising global threat to human health, while the rise of antimicrobial tolerance underlies the development of AMR and long-term infections, placing an additional burden on this problem. Here, we summarise the current understanding of cNP signalling in bacterial physiology with a focus on our understanding of how cNP signalling affects AMR and antimicrobial tolerance in different bacterial species. We also discuss additional cNP-related drug targets in bacterial pathogens that may have therapeutic potential.

Deciphering the Impact of Nucleosides and Nucleotides on Copper Ion and Dopamine Coordination Dynamics.

The mode of coordination of copper(II) ions with dopamine (DA, L) in the binary, as well as ternary systems with Ado, AMP, ADP, and ATP (L') as second ligands, was studied with the use of experimental-potentiometric and spectroscopic (VIS, EPR, NMR, IR)-methods and computational-molecular modeling and DFT-studies. In the Cu(II)/DA system, depending on the pH value, the active centers of the ligand involved in the coordination with copper(II) ions changed from nitrogen and oxygen atoms (CuH(DA)3+, Cu(DA)2+), via nitrogen atoms (CuH2(DA)24+), to oxygen atoms at strongly alkaline pH (Cu(DA)22+). The introduction of L' into this system changed the mode of interaction of dopamine from oxygen atoms to the nitrogen atom in the hydroxocomplexes formed at high pH values. In the ternary systems, the ML'-L (non-covalent interaction) and ML'HxL, ML'L, and ML'L(OH)x species were found. In the Cu(II)/DA/AMP or ATP systems, mixed forms were formed up to a pH of around 9.0; above this pH, only Cu(II)/DA complexes occurred. In contrast to systems with AMP and ATP, ternary species with Ado and ADP occurred in the whole pH range at a high concentration, and moreover, binary complexes of Cu(II) ions with dopamine did not form in the detectable concentration.

Effect of cooking methods on flavor profiles of Xuanwei Ham: Analytical insights into aromatic composition and sensory attributes.

To examine flavor variations in Xuanwei ham due to different cooking methods, we selected one-year cured Xuanwei ham and applied four techniques: dry frying (DF), baking (BA), steaming (ST), and boiling (BO). Organoleptic evaluation revealed ST received the highest overall sensory score. High-performance liquid chromatography (HPLC) revealed that the total nucleotide content was significantly different (P < 0.05), lactic acid predominated the measured organic acids. Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and chromatography-electronic nose (GC-E-Nose) data indicated that ST resulting in significantly higher total volatile compounds than the other methods (P < 0.05). SPME-GC-MS detected 55 volatile compounds, and 12 characteristic flavor substances were identified using orthogonal partial least squares discriminant analysis (OPLS-DA) (VIP > 1). This study aimed to support comprehensive research on the flavor characteristics of cooked Xuanwei ham and guide the selection of appropriate processing methods.

Cell-Permeable Nicotinamide Adenine Dinucleotides for Exploration of Cellular Protein ADP-Ribosylation.

Posttranslational modifications (PTMs) greatly enhance the functional diversity of proteins, surpassing the number of gene-encoded variations. One intriguing PTM is ADP-ribosylation, which utilizes nicotinamide adenine dinucleotide (NAD+) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell-permeable NAD+ analogs and demonstrate their utility for investigating cellular ADP-ribosylation. Using a desthiobiotin-labelled analog for affinity enrichment of proteins that are ADP-ribosylated in living cells under oxidative stress, we identified protein targets associated with host-virus interactions, DNA damage and repair, protein biosynthesis, and ribosome biogenesis. Most of these targets have been noted in various literature sources, highlighting the potential of our probes for cellular ADP-ribosylome studies.

A blend of bacillus fermented soybean meal, functional amino acids and nucleotides improves nutrient digestibility, bolsters immune response, reduces diarrhea and enhances growth performance in weaned piglets.

This study investigated the effects of a blend of bacillus-fermented soybean meal, functional amino acids, and nucleotides (Functional protein blend-FP Blend) as a replacement for animal protein sources in a weaner pig diet without antibiotic growth promoters on nutrient digestibility, blood profiles, intestinal morphology, diarrhea incidence and growth performance. A total of 288 crossbred weaned piglets [♂ Duroc x ♀ (Yorkshire x Landrace)] with an average body weight (BW) of 6.89 ± 0.71 kg were randomly allocated to 6 groups based on initial BW and sex (8 replicate pens per treatment; 3 gilts and 3 barrows/pen). The experiment lasted for 5 weeks. Dietary treatments included: PC [standard diet with 3% fish meal (FM) and 2% plasma protein (PP)], NC (non-animal protein -AP), T1 (3% FM replaced with 5% FP Blend), T2 (3% FM and 1% PP replaced with 5% FP Blend), T3 (2% PP replaced with FP Blend), and T4 (3% FM and 2% PP replaced with 5% FP Blend). Data were analyzed using Minitab version 17 software. Key results indicated that FP Blend improved the apparent ileal digestibility (AID) coefficient of dry matter, gross energy, lysine, and valine in T4 compared to NC treatment (P < 0.05), whereas AID coefficient of crude protein and other amino acids remained constant (P > 0.05). Compared to NC diet, the weaned pigs fed T4 diet reduced malondialdehyde, serum IL8, TNF-α, and increased IgG (P < 0.05), while showing no effect on serum IL6, IL10, white blood cells, IgA, and endotoxin (P > 0.05). Furthermore, FP Blend significantly increased villus height in the duodenum and ileum in T4 compared to NC (P < 0.05). The average daily gain (ADG) was highest in T4 (502.73 g/d), followed by T1 (477.96 g/d) and T2 (475.85 g/d), compared with PC (450.86 g/d) and NC (439.79 g/d). T4's ADG significantly differed from PC and NC (P < 0.001), while no significant differences were observed in T1, T2, and T3 (P > 0.05). The feed conversion ratio (FCR) was significantly lower in T4 (1.45) compared to PC (1.57) and NC (1.59) (P < 0.001), with no significant differences among other groups. In conclusion, FP Blend demonstrated efficacy in improving nutrient digestibility, optimizing intestinal morphology, bolstering immune responses, reducing diarrhea incidence, alleviating the adverse effects of weaning stress and enhancing growth performance of weaned piglets.

Uracil phosphoribosyltransferase is required to establish a functional cytochrome b6f complex.

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme and plants lacking this enzyme are strongly compromised in chloroplast function. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial to establish a fully functional photosynthesis as the RIESKE iron sulfur protein (PetC) is almost absent, leading to a block in photosynthetic electron transport. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis showed that protein homeostasis but not gene expression is most likely responsible for this observation and high light provoked an upregulation of protease levels, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery in UPP amiRNA lines. Strongly reduced PetC amounts were not only detected by immunoblot from mature plants but in addition in a de-etiolation experiment with young seedlings and are causing reduced high light-induced non-photochemical quenching Φ(NPQ) but increased unregulated energy dissipation Φ(NO). This impaired photosynthesis results in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline, and fumarate were found to be reduced. In sum, our work suggests that UPP assists in stabilization PetC during import, processing or targeting to the thylakoid membrane, or protects it against proteolytic degradation.

The P(III)-amidite based synthesis of stable isotope labeled mRNA-cap-structures enables their sensitive quantitation from brain tissue.

The 5' cap structure is crucial to mRNA function, with its diverse methylation patterns depending on the cellular state. Sensitive analytical methods are sought after to quantify this cap variety also referred to as cap epitranscriptome. To address a bottleneck for accurate and precise quantitation, we report a facile and fast access to high-quality synthetic standards via a new route, involving P(III)-amidite chemistry. A range of cap nucleotides and their stable heavy isotopic labeled analogues were derived from nucleoside diphosphates, which themselves were directly prepared in a one-step reaction sequence starting from unprotected nucleosides using a triphosphorylating reagent in combination with ethylenediamine. Considering a wider scope, the route also enables direct access to magic spot nucleotides and diphosphates of isoprenyl-alcohols. Stable-isotope labeled cap nucleotides derived from this route paved the way for the development of a highly sensitive LC-MS/MS method, applied to the characterization of mouse brain cap epitranscriptomes, which turned out to be very different from those of cultured cell lines of widespread use in the life sciences.

Application of Mass Spectrometry for Analysis of Nucleobases, Nucleosides and Nucleotides in Tea and Selected Herbs: A Critical Review of the Mass Spectrometric Data.

The main and most commonly known biological function of nucleobases, nucleosides, and nucleotides is usually associated with the fact that they are the building blocks of nucleic acids. However, these compounds also belong to plant secondary metabolites, although in that role they have attracted less attention than the others, e.g., terpenes, phenolics, or alkaloids. The former compounds are also important constituents of the human diet, e.g., as ingredients of tea and herbs, endowing them with specific taste qualities and pharmacological activities. Liquid chromatography-mass spectrometry seems to be the most important analytical method that permits the identification and determination of nucleobases, nucleosides, and nucleotides, along with the other metabolites. The main goal of this review is to discuss in detail the aspects of mass spectrometric detection of nucleobases, nucleosides, and nucleotides in tea and selected herbs. An important conclusion is that the identification of the compounds of interest should be performed not only on the basis of [M + H]+/[M - H]- ions but should also be confirmed by the respective product ions; however, as discussed in detail in this review, it may sometimes be problematic. It also clear that all difficulties that may be encountered when analyzing plant material are caused by the complexity of the analyzed samples and the need to analyze different classes of compounds, and this review absolutely does not debase any of the mentioned papers.

Dietary nucleotides drive changes in infant fecal microbiota in vitro and gut microbiota-gut-brain development in neonatal rats: A potential "nitrogen source" for early microbiota growth.

Various dietary factors in human milk are important nutrients for the formation of the infant gut microbiota (GM). While promoting the growth of the GM, some human milk components that are difficult to absorb and utilize will be broken down by the GM, and converted into nutrients that the baby can use, such as breast milk oligosaccharides-the 'carbon source' for infant GM. This study reveals that nucleotides (NTs), significant non-protein nitrogen sources in human milk, can enhance the abundance of beneficial microbial genera such as g_Bifidobacterium, g_Bacteroides, and g_Blautia in in vitro fecal fermentation fluids of infants at low doses (2 mg/mL). Conversely, high doses of NTs (20 mg/mL) increased the abundance of g_Escherichia-Shigella. Furthermore, low-dose NTs fermentation broth significantly enhanced the expression of neurodevelopmental marker genes such as Tuj1, Sox2, Dcx, and NeuN in NE-4C neural stem cells, whereas a single NTs digestion broth did not exhibit significant activity. However, in vivo studies using neonatal rats as a model demonstrated that both low-dose NTs fermentation broth and NTs digestive juices promoted behavioral development in neonatal rats (PND 20) and neuron maturation in the prefrontal cortex and hippocampus. Non-targeted metabolomics results indicate that low-dose dietary NTs promote the production of certain neuroregulatory metabolites in infant fecal fermentation, such as uridine, L-tyrosine, L-glutamic acid, and succinic acid. These findings suggest that NTs may serve as an important "nitrogen source" during GM formation in early life and have a dose effect in driving the development of the microbiota-gut-brain axis in early life.

Disruption of nucleotide biosynthesis reprograms mitochondrial metabolism to inhibit adipogenesis.

A key organismal response to overnutrition involves the development of new adipocytes through the process of adipogenesis. Preadipocytes sense changes in the systemic nutrient status and metabolites can directly modulate adipogenesis. We previously identified a role of de novo nucleotide biosynthesis in adipogenesis induction, whereby inhibition of nucleotide biosynthesis suppresses the expression of the transcriptional regulators PPARγ and C/EBPα. Here, we set out to identify the global transcriptomic changes associated with the inhibition of nucleotide biosynthesis. Through RNA sequencing (RNAseq), we discovered that mitochondrial signatures were the most altered in response to inhibition of nucleotide biosynthesis. Blocking nucleotide biosynthesis induced rounded mitochondrial morphology, and altered mitochondrial function, and metabolism, reducing levels of tricarboxylic acid cycle intermediates, and increasing fatty acid oxidation (FAO). The loss of mitochondrial function induced by suppression of nucleotide biosynthesis was rescued by exogenous expression of PPARγ. Moreover, inhibition of FAO restored PPARγ expression, mitochondrial protein expression, and adipogenesis in the presence of nucleotide biosynthesis inhibition, suggesting a regulatory role of nutrient oxidation in differentiation. Collectively, our studies shed light on the link between substrate oxidation and transcription in cell fate determination.

Identification of novel genetic factors that regulate c-di-AMP production in Staphylococcus aureus using a riboswitch-based biosensor.

Nucleotide secondary messengers regulate various processes in bacteria allowing them to rapidly respond to changes in environmental conditions. c-di-AMP is an essential second messenger required for the growth of the human pathogen Staphylococcus aureus, regulating potassium, osmolyte uptake, and beta-lactam resistance. Cellular concentrations of c-di-AMP are regulated by the activities of two enzymes, DacA and GdpP, which synthesize and hydrolyze c-di-AMP, respectively. Besides these, only a limited number of other factors are known to regulate c-di-AMP levels. Using a c-di-AMP biosensor consisting of the Bacillus subtilis c-di-AMP-binding kimA riboswitch and yfp, we were able to efficiently detect differences in cellular c-di-AMP levels in S. aureus. To identify novel factors that regulate c-di-AMP levels, we introduced the biosensor into a library of S. aureus transposon mutants. In this manner, we obtained mutants with increased c-di-AMP levels that contained insertions in gdpP coding for the c-di-AMP hydrolase and ybbR (cdaR) coding for a c-di-AMP cyclase regulator, thus validating our screen. We also identified two high c-di-AMP mutants with insertions upstream of the nrdIEF operon coding for the ribonucleotide reductase enzyme. Further analysis revealed that the insertion down-regulated nrdIEF expression, indicating that the enzyme is a negative regulator of c-di-AMP production. This negative regulation was dependent on rsh, encoding for the synthase of the endogenous GdpP inhibitor (p)ppGpp. The methods established in this work can be readily adapted for use in other bacteria to uncover genetic or environmental factors regulating c-di-AMP levels.IMPORTANCEc-di-AMP is an important secondary messenger, produced by many bacterial species including the opportunistic pathogen Staphylococcus aureus. In this bacterium, c-di-AMP controls cell wall homeostasis, cell size, and osmotic balance. In addition, it has been shown that strains with high c-di-AMP levels exhibit increased resistance to beta-lactam antibiotics. Here, we developed a biosensor-based method for the rapid detection of c-di-AMP levels in S. aureus. We utilized the biosensor in a genetic screen for the identification of novel factors that impact cellular c-di-AMP. In this manner, we identified the ribonucleotide reductase as a novel factor altering cellular c-di-AMP levels and showed that reducing its expression leads to increased cellular c-di-AMP levels. As methicillin-resistant S. aureus strains are considered as a global health threat, it is important to study processes that dictate cellular c-di-AMP levels, which are associated with antibiotic resistance.

Glutaminolysis provides nucleotides and amino acids to regulate osteoclast differentiation in mice.

Osteoclasts are bone resorbing cells that are essential to maintain skeletal integrity and function. While many of the growth factors and molecular signals that govern osteoclastogenesis are well studied, how the metabolome changes during osteoclastogenesis is unknown. Using a multifaceted approach, we identified a metabolomic signature of osteoclast differentiation consisting of increased amino acid and nucleotide metabolism. Maintenance of the osteoclast metabolic signature is governed by elevated glutaminolysis. Mechanistically, glutaminolysis provides amino acids and nucleotides which are essential for osteoclast differentiation and bone resorption in vitro. Genetic experiments in mice found that glutaminolysis is essential for osteoclastogenesis and bone resorption in vivo. Highlighting the therapeutic implications of these findings, inhibiting glutaminolysis using CB-839 prevented ovariectomy induced bone loss in mice. Collectively, our data provide strong genetic and pharmacological evidence that glutaminolysis is essential to regulate osteoclast metabolism, promote osteoclastogenesis and modulate bone resorption in mice.

Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates.

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.

Adenosine Triphosphate and Adenylate Energy Charge in Ready-to-Eat Food.

It is commonly accepted that dietary nucleotides should be considered as essential nutrients originating mainly but not exclusively from meat and fish dishes. Most research in food science related to nutrition nucleotides is focused on raw products, while the effects of thermal processing of ready-to-eat food on nucleotide content are largely overlooked by the scientific community. The aim of this study is to investigate the impact of thermal processing and cold storage on the content of dietary nucleotides in freshly prepared and canned ready-to-eat meat and fish food. The concentrations of ATP, ADP, AMP, IMP, Ino, and Hx were determined using NMR, HPLC, FPMLC, and ATP bioluminescence analytical techniques; freshness indices K and K1 and adenylate energy charge (AEC) values were estimated to assess the freshness status and confirm a newly unveiled phenomenon of the reappearance of adenylate nucleotides. It was found that in freshly prepared at 65 °C ≤ T ≤ +100 °C and canned food, the concentration of free nucleotides was in the range of 0.001-0.01 µmol/mL and remained unchanged for a long time during cold storage; the correct distribution of mole fractions of adenylates corresponding to 0 < AEC < 0.5 was observed compared to 0.2 < AEC < 1.0 in the original raw samples, with either a high or low content of residual adenylates. It could be assumed that heating at nonenzymatic temperatures T > 65 °C can rupture cell membranes and release residual intracell nucleotides in quite a meaningful concentration. These findings may lead to a conceptual change in the views on food preparation processes, taking into account the phenomenon of the free adenylates renaissance and AEC bioenergetics.

Effects of different thermal methods and degrees on the flavor of channel catfish (Ictalurus punctatus) fillets: Fatty acids, volatile flavor and taste compounds.

The effects of different thermal processing conditions on the flavor profiles of channel catfish were evaluated in terms of fatty acids, volatile flavor and taste compounds using steaming, boiling, roasting, and microwaving with different degrees. After thermal processing, 72 volatile organic compounds were detected, including 20 hydrocarbons, 5 ketones, 20 aldehydes, 7 heterocyclic compounds, 12 alcohols and others. Meanwhile, the contents of unsaturated fatty acids like oleic acid and linoleic showed a significant decline due to their heat-sensitive properties. With regard to taste compounds, thermal processing contributed to umami amino acids and free nucleotides conversion, with the initial glutamate and IMP contents of 15.87 and 164.91 mg/100 g in raw samples mainly increasing by 2.8-10.3 and 14.4-105.5 mg/100 g in processed ones. Compared to other methods, microwaving had limited effects on flavor compounds, and steaming and roasting had better performance to improve the flavor complexity of channel catfish.

SLC25A48 controls mitochondrial choline import and metabolism.

Choline is an essential nutrient for the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism with a critical step being its import into mitochondria. However, the underlying mechanisms and biological significance remain poorly understood. Here, we report that SLC25A48, a previously uncharacterized mitochondrial inner-membrane carrier protein, controls mitochondrial choline transport and the synthesis of choline-derived methyl donors. We found that SLC25A48 was required for brown fat thermogenesis, mitochondrial respiration, and mitochondrial membrane integrity. Choline uptake into the mitochondrial matrix via SLC25A48 facilitated the synthesis of betaine and purine nucleotides, whereas loss of SLC25A48 resulted in increased production of mitochondrial reactive oxygen species and imbalanced mitochondrial lipids. Notably, human cells carrying a single nucleotide polymorphism on the SLC25A48 gene and cancer cells lacking SLC25A48 exhibited decreased mitochondrial choline import, increased oxidative stress, and impaired cell proliferation. Together, this study demonstrates that SLC25A48 regulates mitochondrial choline catabolism, bioenergetics, and cell survival.

Structural Basis for C2'-methoxy Recognition by DNA Polymerases and Function Improvement.

DNA modified with C2'-methoxy (C2'-OMe) greatly enhances its resistance to nucleases, which is beneficial for the half-life of aptamers and DNA nanomaterials. Although the unnatural DNA polymerases capable of incorporating C2'-OMe modified nucleoside monophosphates (C2'-OMe-NMPs) were engineered via directed evolution, the detailed molecular mechanism by which an evolved DNA polymerase recognizes C2'-OMe-NTPs remains poorly understood. Here, we present the crystal structures of the evolved Stoffel fragment of Taq DNA polymerase SFM4-3 processing the C2'-OMe-GTP in different states. Our results reveal the structural basis for recognition of C2'-methoxy by SFM4-3. Based on the analysis of other mutated residues in SFM4-3, a new Stoffel fragment variant with faster catalytic rate and stronger inhibitor-resistance was obtained. In addition, the capture of a novel pre-insertion co-existing with template 5'-overhang stacking conformation provides insight into the catalytic mechanism of Taq DNA polymerase.

Evolving spectrum of adenosine deaminase (ADA) deficiency: Assessing genotype pathogenicity according to expressed ADA activity of 46 variants.

BACKGROUND: Deficiency of adenosine deaminase (ADA or ADA1) has broad clinical and genetic heterogeneity. Screening techniques can identify asymptomatic infants whose phenotype and prognosis are indeterminate, and who may carry ADA variants of unknown significance. OBJECTIVE: We systematically assessed the pathogenic potential of rare ADA missense variants to better define the relationship of genotype to red blood cell (RBC) total deoxyadenosine nucleotide (dAXP) content and to phenotype. METHODS: We expressed 46 ADA missense variants in the ADA-deficient SØ3834 strain of Escherichia coli and defined genotype categories (GCs) ranked I to IV by increasing expressed ADA activity. We assessed relationships among GC rank, RBC dAXP, and phenotype in 58 reference patients with 50 different genotypes. We used our GC ranking system to benchmark AlphaMissense for predicting variant pathogenicity, and we used a minigene assay to identify exonic splicing variants in ADA exon 9. RESULTS: The 46 missense variants expressed ∼0.001% to ∼70% of wild-type ADA activity (40% had <0.05% of wild-type ADA activity and 50% expressed >1%). RBC dAXP ranged from undetectable to >75% of total adenine nucleotides and correlated well with phenotype. Both RBC dAXP and clinical severity were inversely related to total ADA activity expressed by both inherited variants. Our GC scoring system performed better than AlphaMissense in assessing variant pathogenicity, particularly for less deleterious variants. CONCLUSION: For ADA deficiency, pathogenicity is a continuum and conditional, depending on the total ADA activity contributed by both inherited variants as indicated by GC rank. However, in patients with indeterminate phenotype identified by screening, RBC dAXP measured at diagnosis may have greater prognostic value than GC rank.