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Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Functional cosmetics industries using skin microbiome screening and beneficial materials isolated from key microorganisms are receiving increasing attention. Since Epidermidibacterium keratini EPI-7T was first discovered in human skin, previous studies have confirmed that it can produce a new pyrimidine compound, 1,1′-biuracil, having anti-aging effects on human skin. Therefore, we conducted genomic analyses to judge the use value of E. keratini EPI-7T and provide up-to-date information. Whole-genome sequencing analysis of E. keratini EPI-7T was performed to generate new complete genome and annotation information. E. keratini EPI-7T genome was subjected to comparative genomic analysis with a group of closely-related strains and skin flora strains through bioinformatic analysis. Furthermore, based on annotation information, we explored metabolic pathways for valuable substances that can be used in functional cosmetics. In this study, the whole-genome sequencing (WGS) and annotation results of E. keratini EPI-7T were improved, and through comparative analysis, it was confirmed that the E. keratini EPI-7T has more metabolite-related genes than comparison strains. In addition, we annotated the vital genes for biosynthesis of 20 amino acids, orotic acid, riboflavin (B2) and chorismate. In particular, we were able to prospect that orotic acid could accumulate inside E. keratini EPI-7T under uracil-enriched conditions. Therefore, through a genomics approach, this study aims to provide genetic information for the hidden potential of E. keratini EPI-7T and the strain development and biotechnology utilization to be conducted in further studies.

The NPN compounds from dairy processing side-streams are a promising source for new products. In this study, the NPN profile of lactose production samples was screened using GC–MS and 1H NMR spectroscopy. These analytical platforms allowed the identification of 35 NPN compounds including, amino acids and derivatives, amino alcohols, organic acids, and other classes. Quantification of the NPN compounds revealed their attenuation by unit operations during a trial lactose production. Urea, ammonia, glycerophosphocholine, creatine, creatinine, orotic acid and choline were the most dominant compounds. Mother liquor concentrate had the highest concentration of NPN, whereas lactose powder had substantial relative amounts of N-acetylglucosamine, phosphocholine and orotic acid. The NPN compounds added up to 57–99% of the total nitrogen, depending on the sample type. The highest nitrogen recovery was found for the reverse osmosis retentate, mother liquid concentrate, wash water and reverse osmosis permeate, whereas the lowest was found for lactose powder.

Multicomponent crystal forms for drugs with poor solubility, such as orotic acid (OA), can potentially promote dissolution behavior and bioavailability. Herein an affinity prediction was carried ou...

A new hydrated molecular salt of GABA was crystallized, and its crystal structure was solved. This is the first example of a hydrated molecular salt of an achiral amino acid with L-tartaric acid and also the first communication on a hydrated molecular salt of GABA with a chiral molecule. The structure contains molecular bi-layers of L-tartaric acid anions connected together by R44(12) motifs based on GABA cations and water molecules. The role of water in the 3D hydrogen bonded network was analyzed. Structural similarity between the monohydrates of GABA with L-tartaric and orotic acid was found. We can suppose that the general R44(12) motif including 2 GABA cations and 2 water molecules would be common for other hydrated molecular salts of GABA with other co-formers, which will be obtained in the future. The stability on storage, thermal behavior and dehydration process for the novel molecular compound were investigated.

Binary orotic acid metal complexes of Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Hg(II), and two mixed metals complexes of (Co(II), Ni(II)) and (Ni(II), Cu(II)) were synthesized and characterized by elemental analysis, IR, electronic spectra, magnetic susceptibility, and ESR spectra. The Analysis proved that the ligand has different coordination modes and the complexes were of octahedral, tetrahedral, and trigonal bipyramidal geometries. Molecular modeling techniques and quantum chemical methods have been performed for orotic acid to calculate charges, bond lengths, bond angles, dihedral angles, electronegativity (χ), chemical potential (μ), global hardness (η), softness (σ) and the electrophilicity index (ω). The thermal decomposition of the complexes was monitored by TGA, DTA, and DSC techniques under the N2 atmosphere. The thermal decomposition mechanisms of the complexes were suggested. The biological activity of orotic acid and some of the complexes are tested against antibacterial and antifungal organisms.

Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6‐month‐old boy and a 5‐year‐old girl) and a healthy donor were transplanted into host mice (hemi‐, hetero‐OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi‐OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero‐OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi‐ and hetero‐patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient‐derived hepatocytes that would enable large scale and reproducible experiments using the same donor.

A 26-year-old male with a two-year history of FLC developed progressive somnolence and disorientation. Treatment history for FLC had included cytotoxic chemotherapy, lenvatinib, and immunotherapy. A CT scan confirmed extensive stage FLC with numerous liver, lung, and pelvic metastasis. Laboratory results showed bilirubin 0.3 mg/dL, creatinine 0.4 mg/dl, leukocytes of 9.5x109 /L, hemoglobin 11.2 g/dL, platelets 369x109 /L, and ammonia 247 µmol/L (reference range: 0-32 µmol/L). Plasma amino acid analysis revealed relatively low citrulline (14 µmol/L), arginine (32 µmol/L), and ornithine (35 µmol/L). Urinary orotic acid excretion was markedly elevated at 149 mmol/mol creat (reference range: 0.68-3.52 mmol/mol creat).

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations. A new safety switch allows for control of cell therapies by dietary uridine.

Orotic acid, a natural product, is involved in many biological processes. Human mesenchymal stem cells (hMSCs) have the potential of self-renewable and proliferation. They are commonly isolated from the bone marrow aspirates of large bones. The osteogenic potential of these stem cells has been extensively exploited by scientists in the past to evaluate the performance of synthetic scaffolds developed for tissue engineering. In this study, N-arylhydrazone derivatives of orotic acid have been synthesized, and their potential as stimulators of human mesenchymal stem cells has been evaluated. Some of the analogs exhibit well to moderate effect on the proliferation rate.

The control of pyrimidine nucleotide formation in the bacterium Pseudomonas aurantiaca ATCC 33663 by pyrimidines was studied. The activities of the pyrimidine biosynthetic pathway enzymes were investigated in P. aurantiaca ATCC 33663 cells and from cells of an auxotroph lacking orotate phosphoribosyltransferase activity under selected culture conditions. All activities of the pyrimidine biosynthetic pathway enzymes in ATCC 33663 cells were depressed by uracil addition to the minimal medium when succinate served as the carbon source. In contrast, all pyrimidine biosynthetic pathway enzyme activities in ATCC 33663 cells were depressed by orotic acid supplementation to the minimal medium when glucose served as the carbon source. The orotidine 5'-monophosphate decarboxylase activity in the phosphoribosyltransferase mutant strain increased by more than sixfold in succinate-grown cells and by more than 16-fold in glucose-grown cells after pyrimidine limitation showing possible repression of the decarboxylase by a pyrimidine-related compound. Inhibition by ATP, GTP, UTP and pyrophosphate of the in vitro activity of aspartate transcarbamoylase in ATCC 33663 was observed. The findings demonstrated control at the level of pyrimidine biosynthetic enzyme synthesis and activity for the P. aurantiaca transcarbamoylase. The control of pyrimidine synthesis in P. aurantiaca seemed to differ from what has been observed previously for the regulation of pyrimidine biosynthesis in related Pseudomonas species. This investigation could prove helpful to future work studying pseudomonad taxonomic analysis as well as to those exploring antifungal and antimicrobial agents produced by P. aurantiaca.

Post-transplantation nonalcoholic fatty liver disease (NAFLD) is common in liver transplant recipients. Changes in the expression levels and activities of drug-metabolizing enzymes and drug transporters have been reported in patients with NAFLD and relevant rodent models. Here, we evaluated whether the pharmacokinetics of mycophenolic acid (MPA), an immunosuppressant, would be altered in rats with NAFLD. NAFLD was induced by feeding a diet containing 1% (w/w) orotic acid for 20 days. The extent of hepatic glucuronidation of MPA to a major metabolite, mycophenolic acid-7-O-glucuronide (MPAG), did not differ between rats with NAFLD and controls. The expression levels of hepatic multidrug resistance-associated protein 2, responsible for biliary excretion of MPAG, were comparable in rats with NAFLD and controls; the biliary excretion of MPAG was also similar in the two groups. Compared with control rats, rats with NAFLD did not exhibit significant changes in the areas under the plasma concentration – time curves of MPA or MPAG after intravenous (5 mg/kg) or oral (10 mg/kg) administration of MPA. However, delayed oral absorption of MPA was observed in rats with NAFLD compared with controls; the MPA and MPAG peak plasma concentrations fell significantly and the times to achieve them were prolonged following oral administration of MPA.

The aim of this study was to develop a fast method of screening for orotic acid in untreated urine by capillary electrophoresis with using a combination of two capillaries with different internal diameters, 50 μm and 100 μm. Under the optimized conditions, 100 mM sodium phosphate buffer pH 2.2, voltage of 5 kV, hydrodynamic injection at 50 mbar for 5 s, and detection at 214 nm, the migration time of orotic acid was only 2.1 min. The validation of the method resulted in orotic acid recoveries of 96.5 ± 0.8%. We achieved good precision with RSD of migration times at less than 0.8% and peak areas lower than 3.1% intraday precision. We obtained a very good linearity in the validated range 1–100 μmol L−1, R2 0.9993, and a limit of detection of 2.4 μmol L−1. Finally, we proved that the method is robust, including the connection of capillaries, using the Youden test.

Fourier transform infrared spectral analysis is a cheap and fast method to predict milk composition. A not very well studied milk component is orotic acid. Orotic acid is an intermediate in the biosynthesis pathway of pyrimidine nucleotides and is an indicator for the metabolic cattle disorder deficiency of uridine monophosphate synthase. The function of orotic acid in milk and its effect on calf health, health of humans consuming milk or milk products, manufacturing properties of milk, and its potential as an indicator trait are largely unknown. The aims of this study were to determine if milk orotic acid can be predicted from infrared milk spectra and to perform a large-scale phenotypic and genetic analysis of infrared-predicted milk orotic acid. An infrared prediction model for orotic acid was built using a training population of 292 Danish Holstein and 299 Danish Jersey cows, and a validation population of 381 Danish Holstein cows. Milk orotic acid concentration was determined with nuclear magnetic resonance spectroscopy. For genetic analysis of infrared orotic acid, 3 study populations were used: 3,210 Danish Holstein cows, 3,360 Danish Jersey cows, and 1,349 Dutch Holstein Friesian cows. Using partial least square regression, a prediction model for orotic acid was built with 18 latent variables. The error of the prediction for the infrared model varied from 1.0 to 3.2 mg/L, and the accuracy varied from 0.68 to 0.86. Heritability of infrared orotic acid predicted with the standardized prediction model was 0.18 for Danish Holstein, 0.09 for Danish Jersey, and 0.37 for Dutch Holstein Friesian. We conclude that milk orotic acid can be predicted with moderate to good accuracy based on infrared milk spectra and that infrared-predicted orotic acid is heritable. The availability of a cheap and fast method to predict milk orotic acid opens up possibilities to study the largely unknown functions of milk orotic acid.