Effects of biodegradable and conventional microplastics on the intestine, intestinal community composition, and metabolic levels in tilapia (Oreochromis mossambicus).

Despite growing interest in conventional microplastics (CMPs) and their toxicological effects on aquatic species, little is known about biodegradable microplastics (BMPs) and their corresponding implications for aquatic life. Here, tilapia (Oreochromis mossambicus) were semi-statically exposed for 14 days to the bio-based plastic polylactic acid (PLA, 100 µg/L, 2.52 ± 0.46 µm) and the petroleum-based plastic polyvinyl chloride (PVC, 100 µg/L, 1.58 ± 0.36 µm). The results showed that ingesting the above two types of microplastics (MPs) led to oxidative stress in the fish gut, and damage to gut tissues and organelles, and PLA resulted in more obvious gut tissue edema than PVC. Furthermore, PLA caused increased levels of gut microbiota dysbiosis and a decrease in the abundance of the genus Cetobacterium, which is linked to vitamin B-12 synthesis, whereas an opposite relationship was observed on PVC. Metabolomic analysis indicated that PVC caused a significant down-regulation of orotic acid, co-metabolite of folic acid with vitamin B-12, while PLA did not affect orotic acid, which may lead to the accumulation of folic acid in fish. The joint analysis found that MPs disturbed gut metabolism homeostasis, implying that abnormal gut microbiota metabolites may be a key mechanism for MPs to induce tissue damage and oxidative stress in the gut. Overall, this study systematically illustrates the differential toxic effects of BMPs and CMPs on tilapia through gut microbiota and metabolite interactions, which will contribute to assessing the risks of BMPs to organismal health.

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells.

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10-250 µM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 µM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.

Combination of Secondary Plant Metabolites and Micronutrients Improves Mitochondrial Function in a Cell Model of Early Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by excessive formation of beta-amyloid peptides (Aß), mitochondrial dysfunction, enhanced production of reactive oxygen species (ROS), and altered glycolysis. Since the disease is currently not curable, preventive and supportive approaches are in the focus of science. Based on studies of promising single substances, the present study used a mixture (cocktail, SC) of compounds consisting of hesperetin (HstP), magnesium-orotate (MgOr), and folic acid (Fol), as well as the combination (KCC) of caffeine (Cof), kahweol (KW) and cafestol (CF). For all compounds, we showed positive results in SH-SY5Y-APP695 cells-a model of early AD. Thus, SH-SY5Y-APP695 cells were incubated with SC and the activity of the mitochondrial respiration chain complexes were measured, as well as levels of ATP, Aß, ROS, lactate and pyruvate. Incubation of SH-SY5Y-APP695 cells with SC significantly increased the endogenous respiration of mitochondria and ATP levels, while Aß1-40 levels were significantly decreased. Incubation with SC showed no significant effects on oxidative stress and glycolysis. In summary, this combination of compounds with proven effects on mitochondrial parameters has the potential to improve mitochondrial dysfunction in a cellular model of AD.

In vitro to in vivo extrapolation and high-content imaging for simultaneous characterization of chemically induced liver steatosis and markers of hepatotoxicity.

Chemically induced steatosis is characterized by lipid accumulation associated with mitochondrial dysfunction, oxidative stress and nucleus distortion. New approach methods integrating in vitro and in silico models are needed to identify chemicals that may induce these cellular events as potential risk factors for steatosis and associated hepatotoxicity. In this study we used high-content imaging for the simultaneous quantification of four cellular markers as sentinels for hepatotoxicity and steatosis in chemically exposed human liver cells in vitro. Furthermore, we evaluated the results with a computational model for the extrapolation of human oral equivalent doses (OED). First, we tested 16 reference chemicals with known capacities to induce cellular alterations in nuclear morphology, lipid accumulation, mitochondrial membrane potential and oxidative stress. Then, using physiologically based pharmacokinetic modeling and reverse dosimetry, OEDs were extrapolated from data of any stimulated individual sentinel response. The extrapolated OEDs were confirmed to be within biologically relevant exposure ranges for the reference chemicals. Next, we tested 14 chemicals found in food, selected from thousands of putative chemicals on the basis of structure-based prediction for nuclear receptor activation. Amongst these, orotic acid had an extrapolated OED overlapping with realistic exposure ranges. Thus, we were able to characterize known steatosis-inducing chemicals as well as data-scarce food-related chemicals, amongst which we confirmed orotic acid to induce hepatotoxicity. This strategy addresses needs of next generation risk assessment and can be used as a first chemical prioritization hazard screening step in a tiered approach to identify chemical risk factors for steatosis and hepatotoxicity-associated events.

Physico-chemical, microbiological, and sensory characteristics of yogurt as affected by various ingredients.

l-Glutamine, quercetin, slippery elm bark, marshmallow root, N-acetyl-d-glucosamine, licorice root, maitake mushrooms, and zinc orotate have been reported to help treat leaky gut. The purpose of this research was to explore the impact of these functional ingredients on the physico-chemical, microbiological, and sensory properties of yogurt. The milk from same source was equally divided into 9 pails and the 8 ingredients were randomly assigned to the 8 pails. The control had no ingredient. Milk was fermented to yogurt. The pH, titratable acidity, syneresis, viscosity, color (L*, a*, b*, C*, and h*), Streptococcus thermophilus counts, and Lactobacillus delbrueckii spp. bulgaricus counts of yogurts were determined on d 1, 7, 14, 21, 28, 35, and 42, whereas coliform counts, yeast and mold counts, and rheological characteristics were determined on d 1 and 42. The sensory study was performed on d 3 and particle size of the functional ingredients (powder form) was also determined. When compared with control, the incorporation of slippery elm bark into yogurts led to less syneresis. l-Glutamine increased pH and n' values (relaxation exponent derived from G') and lowered titratable acidity values. N-Acetyl-d-glucosamine incorporation resulted in higher n' and lower titratable acidity values, whereas maitake mushroom led to lower n' values. Incorporating quercetin increased the growth of L. bulgaricus. Adding maitake mushrooms increased the growth of S. thermophilus but lowered apparent viscosity values, whereas quercetin decreased its S. thermophilus counts. Quercetin decreased L* and a* values but increased b* values, and maitake mushroom increased a* values. Thixotropic behavior increased with the addition of licorice root and quercetin. Adding slippery elm bark, N-acetyl-d-glucosamine, licorice root, maitake mushrooms, and zinc orotate into yogurt did not affect the sensory properties, whereas yogurts with quercetin had the lowest sensory scores. Overall, most of these ingredients did not cause major changes to yogurt properties.

A Facile Fluorometric Assay of Orotate Phosphoribosyltransferase Activity Using a Selective Fluorogenic Reaction for Orotic Acid.

Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells. The technique utilizes 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent, which produces selective fluorescence for orotic acid. To perform the OPRT reaction, orotic acid was added to HeLa cell lysate, and a portion of the enzyme reaction mixture was heated at 80 °C for 4 min in the presence of 4-TFMBAO under basic conditions. The resulting fluorescence was measured using a spectrofluorometer, which reflects the consumption of orotic acid by the OPRT. After optimization of the reaction conditions, the OPRT activity was successfully determined in 15 min of enzyme reaction time without further procedures such as purification of OPRT or deproteination for the analysis. The activity obtained was compatible with the value measured by the radiometric method with [3H]-5-FU as the substrate. The present method provides a reliable and facile measurement of OPRT activity and could be useful for a variety of research fields targeting pyrimidine metabolism.

Enzyme-substrate interactions in orotate-mimetic OPRT inhibitor complexes: a QM/MM analysis.

Orotate phosphoribosyltransferase (OPRT) catalyses the reversible phosphoribosyl transfer from α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to orotic acid (OA) to yield orotidine 5'-monophosphate (OMP) during the de novo synthesis of nucleotides. Numerous studies have reported the inhibition of this reaction as a strategy to check diseases like tuberculosis, malaria and cancer. Insight into the inhibition of this reaction is, therefore, of urgent interest. In this study, we implemented a QM/MM framework on OPRT derived from Saccharomyces cerevisiae to obtain insights into the competitive binding of OA and OA-mimetic inhibitors by quantifying their interactions with OPRT. 4-Hydroxy-6-methylpyridin-2(1H) one showed the best inhibiting activity among the structurally similar OA-mimetic inhibitors, as quantified from the binding energetics. Our analysis of protein-ligand interactions unveiled the association of this inhibitory ligand with a strong network of hydrogen bonds, a large contribution of hydrophobic contacts, and bridging water molecules in the binding site. The ortho-substituted CH3 group in the compound resulted in a large population of π-electrons in the aromatic ring of this inhibitor, supporting the ligand binding further.

Screening of non-protein nitrogen compounds in lactose refining streams from industrial whey permeate processing.

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.

Evaluation of cytotoxicity and genotoxicity of a novel oxovanadium complex with orotate.

The development of new drugs based on metal complexes requires a detailed analysis of their biological endpoints. In this study, we report the genotoxic profile and influence on cell proliferation and death of the oxovanadium(IV) complex with orotic acid ([VO(C5H4N2O4)2], VO(oro)). Human hepatocellular carcinoma cells (HepG2) were the most sensitive tumor cells to VO(oro), which interfered with the integrity of cell membranes and proliferative capacity in a dose-dependent manner, inducing cell death by apoptosis. Regarding genotoxicity, VO(oro) did not induce considerable levels of DNA damage in HepG2 cells (comet test) and gene mutations (Ames test). However, it caused a statistically significant increase in the frequency of micronuclei at the highest concentration tested (12.5 µmol.L-1), indicating aneuploidy and clastogenicity. The data presented here provide information on various biological aspects of the VO(oro) complex, which may allow the elucidation of its mechanism of action as a possible therapeutic agent.

Sea Cucumber Saponins Derivatives Alleviate Hepatic Lipid Accumulation Effectively in Fatty Acids-Induced HepG2 Cells and Orotic Acid-Induced Rats.

The sulfated echinoside A (EA) and holothurin A (HA) are two prominent saponins in sea cucumber with high hemolytic activity but also superior lipid-lowering activity. Deglycosylated derivatives EA2 and HA2 exhibit low hemolysis compared to EA and HA, but their efficacies on lipid metabolism regulation remains unknown. In this study, fatty acids-treated HepG2 cells and orotic acid-treated rats were used to investigate the lipid-lowering effects of sea cucumber saponin derivatives. Both the saponin and derivatives could effectively alleviate lipid accumulation in HepG2 model, especially EA and EA2. Moreover, though the lipid-lowering effect of EA2 was not equal with EA at the same dosage of 0.05% in diet, 0.15% dosage of EA2 significantly reduced hepatic steatosis rate, liver TC and TG contents by 76%, 41.5%, and 63.7%, respectively, compared to control and reversed liver histopathological features to normal degree according to H&E stained sections. Possible mechanisms mainly included enhancement of fatty acids ß-oxidation and cholesterol catabolism through bile acids synthesis and excretion, suppression of lipogenesis and cholesterol uptake. It revealed that the efficacy of EA2 on lipid metabolism regulation was dose-dependent, and 0.15% dosage of EA2 possessed better efficacy with lower toxicity compared to 0.05% dosage of EA.

Milk metabolome reveals pyrimidine and its degradation products as the discriminant markers of different corn silage-based nutritional strategies.

The purpose of this study was to evaluate the effect of 6 different feeding systems (based on corn silage as the main ingredient) on the chemical composition of milk and to highlight the potential of untargeted metabolomics to find discriminant marker compounds of different nutritional strategies. Interestingly, the multivariate statistical analysis discriminated milk samples mainly according to the high-moisture ear corn (HMC) included in the diet formulation. Overall, the most discriminant compounds, identified as a function of the HMC, belonged to AA (10 compounds), peptides (71 compounds), pyrimidines (38 compounds), purines (15 compounds), and pyridines (14 compounds). The discriminant milk metabolites were found to significantly explain the metabolic pathways of pyrimidines and vitamin B6. Interestingly, pathway analyses revealed that the inclusion of HMC in the diet formulation strongly affected the pyrimidine metabolism in milk, determining a significant up-accumulation of pyrimidine degradation products, such as 3-ureidopropionic acid, 3-ureidoisobutyric acid, and 3-aminoisobutyric acid. Also, some pyrimidine intermediates (such as l-aspartic acid, N-carbamoyl-l-aspartic acid, and orotic acid) were found to possess a high discrimination degree. Additionally, our findings suggested that the inclusion of alfalfa silage in the diet formulation was potentially correlated with the vitamin B6 metabolism in milk, being 4-pyridoxic acid (a pyridoxal phosphate degradation product) the most significant and up-accumulated compound. Taken together, the accumulation trends of different marker compounds revealed that both pyrimidine intermediates and degradation products are potential marker compounds of HMC-based diets, likely involving a complex metabolism of microbial nitrogen based on total splanchnic fluxes from the rumen to mammary gland in dairy cows. Also, our findings highlight the potential of untargeted metabolomics in both foodomics and foodomics-based studies involving dairy products.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity.

The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to combat the highly infectious variants. The main protease (Mpro) is an promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of potent non-covalent non-peptide Mpro inhibitors featuring a 1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent hit MCULE-5948770040 by structure-based rational design combined with multi-site binding and privileged structure assembly strategies. The optimized compound GC-14 inhibits Mpro with high potency (IC50 = 0.40 µM) and displays excellent antiviral activity (EC50 = 1.1 µM), being more potent than Remdesivir. Notably, GC-14 exhibits low cytotoxicity (CC50 > 100 µM) and excellent target selectivity for SARS-CoV-2 Mpro (IC50 > 50 µM for cathepsins B, F, K, L, and caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple subpockets by critical non-covalent interactions. These studies may provide a basis for developing a more efficient and safer therapy for COVID-19.

Proximal urea cycle defects are challenging to detect with newborn screening: Results of a prospective pilot study using post-analytical tools.

The purpose of this pilot project was to evaluate the efficacy of the Collaborative Integrated Laboratory Reports (CLIR) postanalytical tools from Mayo Clinic for detection of newborns with proximal urea cycle disorders (PUCD) in the Georgia newborn screening program that uses the underivatized Neobase2 kit (Perkin Elmer). We evaluated 138,560 newborn screening (NBS) samples (between 125,000 and 130,000 children) and used the CLIR result interpretation guidelines to stratify results. Children at higher risk of having a PUCD received follow-up services including confirmatory lab testing (ammonia, plasma amino acids, urine orotic acid) or a repeat NBS sample. We made multiple adjustments to our CLIR PUCD tool and to our follow-up algorithms in order to reduce false positives. Regardless, a high number of NBS samples resulted with false positives in part due to the glutamine peak also containing lysine. No children were diagnosed with a PUCD during our study period, and the Emory Genetics Metabolic Center is unaware of any children diagnosed outside of the NBS system during that time. Based on our experience, PUCD is not suitable for statewide NBS using Neobase2 and CLIR. Other methodologies that can separate glutamine from other amino acids may have better performance.

Magnesium Orotate and the Microbiome-Gut-Brain Axis Modulation: New Approaches in Psychological Comorbidities of Gastrointestinal Functional Disorders.

Magnesium orotate has been cited in the medical literature for the past three years as a possible adjuvant in some pediatric and adult gastroenterological disorders associated with dysbiosis. Studies also focus on the possibility of adding magnesium orotate in psychiatric disorders' treatment, such as major depression and anxiety. The most relevant element in these studies is the efficiency of magnesium orotate therapy in cases with both gastroenterological and psychiatric symptoms. This article proposes a literature review, focused on the studies published in the last three years, targeting magnesium orotate treatment and probiotic supplementation in patients with both digestive and psychiatric symptoms. Moreover, this review will compare the efficiency of magnesium orotate and probiotics within both the pediatric and adult communities, focusing on the possibility of gut-brain axis modulation and its involvement in the clinical evolution of these patients.

A Multi-Center, Double-Blind Randomized Controlled Phase III Clinical Trial to Evaluate the Antiviral Activity and Safety of DA-2802 (Tenofovir Disoproxil Orotate) and Viread (Tenofovir Disoproxil Fumarate) in Chronic Hepatitis B Patients.

BACKGROUND: Tenofovir disoproxil fumarate (TDF, Viread®) had been used as a standard treatment option of chronic hepatitis B (CHB). This clinical trial was conducted to evaluate the efficacy and safety of DA-2802 (tenofovir disoproxil orotate) compared to TDF. METHODS: The present study was a double blind randomized controlled trial. Patients with CHB were recruited from 25 hospitals in Korea and given DA-2802 at a dose of 319 mg once daily or Viread® at a dose of 300 mg once daily for 48 weeks from March 2017 to January 2019. Change in hepatitis B virus (HBV) DNA level at week 48 after dosing compared to baseline was the primary efficacy endpoint. Secondary efficacy endpoints were proportions of subjects with undetectable HBV DNA, those with normal alanine aminotransferase (ALT) levels, and those with loss of hepatitis B envelop antigen (HBeAg), those with loss of hepatitis B surface antigen (HBsAg). Adverse events (AEs) were also investigated. RESULTS: A total of 122 patients (DA-2802 group: n = 61, Viread® group: n = 61) were used as full analysis set for efficacy analysis. Mean age, proportion of males, laboratory results and virologic characteristics were not different between the two groups. The change in HBV DNA level at week 48 from baseline was -5.13 ± 1.40 in the DA-2802 group and -4.97 ± 1.40 log10 copies/mL in the Viread® group. The analysis of primary endpoint using the nonparametric analysis of covariance showed statistically significant results (P < 0.001), which confirmed non-inferiority of DA-2802 to Viread® by a prespecified noninferiority margin of 1. The proportion of undetectable HBV DNA was 78.7% in the DA-2802 group and 75.4% in the Viread® group (P = 0.698). The proportion of subjects who had normal ALT levels was 75.4% in the DA-2802 group and 73.3% in the Viread® group (P = 0.795). The proportion of those with HBeAg loss was 8.1% in the DA-2802 group and 10.8% in the Viread® group (P = 1.000). No subject showed HBsAg loss. The frequency of AEs during treatment was similar between the two groups. Most AEs were mild to moderate in severity. CONCLUSION: DA-2802 is considered an effective and safe treatment for patients with CHB. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02967939.

A Plausible Prebiotic One-Pot Synthesis of Orotate and Pyruvate Suggestive of Common Protometabolic Pathways.

A reaction between two prebiotically plausible building blocks, hydantoin and glyoxylate, generates both the nucleobase orotate, a precursor of biological pyrimidines, and pyruvate, a core metabolite in the citric acid cycle and amino acid biosynthesis. The reaction proceeds in water to provide significant yields of the two widely divergent chemical motifs. Additionally, the reaction of thiohydantoin and glyoxylate produces thioorotate in high yield under neutral aqueous conditions. The use of an open-chain thiohydantoin derivative also enables the potential pre-positioning of a nucleosidic bond prior to the synthesis of an orotate nucleoside. The observation that diverse building blocks of modern metabolism can be produced in a single reaction pot, from common reactants under mild conditions, supports the plausibility of orthogonal chemistries operating at the origins of chemical evolution.

High-Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Noncovalent Inhibitor.

Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel noncovalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) by employing a scalable high-throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this Mpro inhibitor with an inhibition constant (Ki) of 2.9 µM (95% CI 2.2, 4.0). Furthermore, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple µs-time scale molecular dynamics (MD) simulations and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design.

Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine.

Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5'-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

Protein-Ribofuranosyl Interactions Activate Orotidine 5'-Monophosphate Decarboxylase for Catalysis.

The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5'-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5'-monophosphate (OMP) is examined in experiments that focus on the interactions between OMPDC and the ribosyl hydroxyl groups of OMP. The D37 and T100' side chains of OMPDC interact, respectively, with the C-3' and C-2' hydroxyl groups of enzyme-bound OMP. D37G and T100'A substitutions result in 1.4 kcal/mol increases in the activation barrier ΔG⧧ for catalysis of decarboxylation of the phosphodianion-truncated substrate 1-(ß-d-erythrofuranosyl)orotic acid (EO) but result in larger 2.1-2.9 kcal/mol increases in ΔG⧧ for decarboxylation of OMP and for phosphite dianion-activated decarboxylation of EO. This shows that these substitutions reduce transition-state stabilization by the Q215, Y217, and R235 side chains at the dianion binding site. The D37G and T100'A substitutions result in <1.0 kcal/mol increases in ΔG⧧ for activation of OMPDC-catalyzed decarboxylation of the phosphoribofuranosyl-truncated substrate FO by phosphite dianions. Experiments to probe the effect of D37 and T100' substitutions on the kinetic parameters for d-glycerol 3-phosphate and d-erythritol 4-phosphate activators of OMPDC-catalyzed decarboxylation of FO show that ΔG⧧ for sugar phosphate-activated reactions is increased by ca. 2.5 kcal/mol for each -OH interaction eliminated by D37G or T100'A substitutions. We conclude that the interactions between the D37 and T100' side chains and ribosyl or ribosyl-like hydroxyl groups are utilized to activate OMPDC for catalysis of decarboxylation of OMP, EO, and FO.

Orotic acid protects pancreatic ß cell by p53 inactivation in diabetic mouse model.

Impairment of pancreatic ß cells is a principal driver of the development of diabetes. Restoring normal insulin release from the ß cells depends on the ATP produced by the intracellular mitochondria. In maintaining mitochondrial function, the tumor suppressor p53 has emerged as a novel regulator of metabolic homeostasis and participates in adaptations to nutritional changes. In this study, we used orotic acid, an intermediate in the pathway for de novo synthesis of the pyrimidine nucleotide, to reduce genotoxicity. Administration of orotic acid reduced p53 activation of MIN6 ß cells and subsequently reduced ß cell death in the db/db mouse. Orotic acid intake helped to maintain the islet size, number of ß cells, and protected insulin secretion in the db/db mouse. In conclusion, orotic acid treatment maintained ß cell function and reduced cell death, and may therefore, be a future therapeutic strategy for the prevention and treatment of diabetes.