UMP Synthase Research Articles

Distribution of Recessive Genetic Defect Carriers in Holstein Friesian Cattle: A Polish Perspective

Genetic disorders are caused by a hereditary change in the structure of DNA that may hurt the health and life of animals. Several recessive haplotypes and a few causative mutations are known in Holstein Friesian cattle: CDH (Holstein cholesterol deficiency), haplotypes with a homozygous deficiency in Holstein (HH1, HH3, HH4, HH5, HH6, HH7), BLAD (bovine leukocyte adhesion deficiency), DUMPS (deficiency of uridine monophosphate synthase), FXI (factor XI deficiency), HHM (mule foot, syndactyly), and BC (citrullinaemia). From a breeding point of view, these genetic diseases have highly negative effects and are a significant problem for breeders, exposing them to economic losses and hurting animal welfare. This study aimed to characterize the Polish population of Holstein Friesian dairy cattle, considering the carrier status of twelve selected genetic defects. This study was based on genotype data collected from 78,884 cows and 691 bulls of the Holstein Friesian variety. The studies were performed using Illumina Infinium microarrays. Among both bulls and cows, the highest numbers of carriers were detected for HH5 (appropriately 6.7% and 5.4%). The lowest numbers of carriers were detected for DUMPS, factor XI, and HHM. The study revealed one calf suffering from cholesterol deficiency.

Selected Monogenic Genetic Diseases in Holstein Cattle-A Review.

Genetic disorders arise from alterations in the hereditary information encoded in DNA, leading to potential detrimental effects on the well-being and vitality of organisms. Within the bovine population, genetic conditions inherited in an autosomal recessive manner are frequently associated with particular breeds. In recent years, several recessive haplotypes and a few causative mutations have been discovered in Holstein cattle: CDH (Holstein cholesterol deficiency), haplotypes with a homozygous deficiency in Holstein (HH1, HH3, HH4, HH5, HH6 and HH7), BLAD (bovine leukocyte adhesion deficiency) and DUMPS (deficiency of uridine monophosphate synthase). All of these diseases are inherited in an autosomal recessive manner. From a breeding perspective, recessive mutations specifically exhibit considerable detrimental effects and are a significant problem for breeders, exposing them to economic losses. Individual mutations can cause embryo death at any stage of pregnancy. Only genetic research and conscious selection of animals for mating will lead to a reduction in the number of carriers and elimination of mutations from the population.

Optimal conditions of algal breeding using neutral beam and applying it to breed Euglena gracilis strains with improved lipid accumulation

Microalgae are considered to be more useful and effective to use in biomass production than other photosynthesis organisms. However, microalgae need to be altered to acquire more desirable traits for the relevant purpose. Although neutron radiation is known to induce DNA mutations, there have been few studies on its application to microalgae, and the optimal relationship between irradiation intensity and mutation occurrence has not been established. In this study, using the unicellular red alga Cyanidioschyzon merolae as a model, we analyzed the relationship between the absorbed dose of two types of neutrons, high-energy (above 1 MeV) and thermal (around 25 meV) neutrons, and mutation occurrence while monitoring mutations in URA5.3 gene encoding UMP synthase. As a result, the highest mutational occurrence was observed when the cells were irradiated with 20 Gy of high-energy neutrons and 13 Gy of thermal neutrons. Using these optimal neutron irradiation conditions, we next attempted to improve the lipid accumulation of Euglena gracilis, which is a candidate strain for biofuel feedstock production. As a result, we obtained several strains with a maximum 1.3-fold increase in lipid accumulation compared with the wild-type. These results indicate that microalgae breeding by neutron irradiation is effective.

P53 activation enhances the sensitivity of non-small cell lung cancer to the combination of SH003 and docetaxel by inhibiting de novo pyrimidine synthesis

BackgroundIdentifying molecular biomarkers for predicting responses to anti-cancer drugs can enhance treatment precision and minimize side effects. This study investigated the novel cancer-targeting mechanism of combining SH003, an herbal medicine, with docetaxel in non-small cell lung cancer (NSCLC) cells. Also, the present study aimed to identify the genetic characteristics of cancer cells susceptible to this combination.MethodsCell viability was analyzed by WST-8 assay. Apoptosis induction, BrdU incorporation, and cell cycle analysis were performed using flow cytometry. Metabolites were measured by LC–MS/MS analysis. Real-time PCR and western blotting evaluated RNA and protein expression. DNA damage was quantified through immunofluorescence. cBioPortal and GEPIA data were utilized to explore the mutual co-occurrence of TP53 and UMPS and UMPS gene expression in NSCLC.ResultsThe combination treatment suppressed de novo pyrimidine nucleotide biosynthesis by reducing the expression of related enzymes. This blockade of pyrimidine metabolism led to DNA damage and subsequent apoptosis, revealing a novel mechanism for inducing lung cancer cell death with this combination. However, some lung cancer cells exhibited distinct responses to the combination treatment that inhibited pyrimidine metabolism. The differences in sensitivity in lung cancer cells were determined by the TP53 gene status. TP53 wild-type lung cancer cells were effectively inhibited by the combination treatment through p53 activation, while TP53 mutant- or null-type cells exhibited lower sensitivity.ConclusionsThis study, for the first time, established a link between cancer cell genetic features and treatment response to simultaneous SH003 and docetaxel treatment. It highlights the significance of p53 as a predictive factor for susceptibility to this combination treatment. These findings also suggest that p53 status could serve as a crucial criterion in selecting appropriate therapeutic strategies for targeting pyrimidine metabolism in lung cancer.

Structural and functional properties of uridine 5′-monophosphate synthase from Coffea arabica

In higher eukaryotes and plants, the last two sequential steps in the de novo biosynthesis of uridine 5′-monophosphate (UMP) are catalyzed by a bifunctional natural chimeric protein called UMP synthase (UMPS). In higher plants, UMPS consists of two naturally fused enzymes: orotate phosphoribosyltransferase (OPRTase) at N-terminal and orotidine-5′-monophosphate decarboxylase (ODCase) at C-terminal. In this work, we obtained the full functional recombinant protein UMPS from Coffea arabica (CaUMPS) and studied its structure-function relationships. A biochemical and structural characterization of a plant UMPS with its two functional domains is described together with the presentation of the first crystal structure of a plant ODCase at 1.4 Å resolution. The kinetic parameters measured of CaOPRTase and CaODCase domains were comparable to those reported. The crystallographic structure revealed that CaODCase is a dimer that conserves the typical fold observed in other ODCases from prokaryote and eukaryote with a 1-deoxy-ribofuranose-5′-phosphate molecule bound in the active site of one subunit induced a closed conformation. Our results add to the knowledge of one of the key enzymes of the de novo biosynthesis of pyrimidines in plant metabolism and open the door to future applications.

Analysis of deficiency of uridine monophosphate synthase syndrome and complex vertebral malformation in repeat breeding and anoestrous cattle using PCR-RFLP

Analysis of deficiency of uridine monophosphate synthase syndrome and complex vertebral malformation in repeat breeding and anoestrous cattle using PCR-RFLP

Investigation of Some Autosomal Recessive Inherited Diseases (BLAD, DUMPS, CVM, and FXID) in Holstein Cattle

Genetic disorders are often transmitted via autosomal recessive inheritance, which negatively affect health, welfare, and yield traits in farm animals such as cattle. In this study, a total of 80 animals belonging to Holstein Friesian (HF) reared in the dairy farm of Akdeniz University were investigated in terms of Bovine Leukocyte Adhesion Deficieny (BLAD), Deficiency of Uridine Monophosphate Synthase (DUMPS), Factor XI Deficiency (FXID), and Complex Vertebral Malformation (CVM) via three molecular genotyping methods such as Polymerase Chain Reaction (PCR), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), and Allele-Sprcific PCR (AS-PCR). BLAD and DUMPS were analysed by PCR-RFLP, while PCR and AS-PCR were utilized to investigate FXID and CVM disorders, respectively. Based on fragment patterns on agarose gel electrophoresis, animals were genotyped as normal, mutant, and carrier. In this study, no carrier or mutant animals were detected for BLAD, CVM, DUMPS, and FXID diseases in HF cattle, since these animals were previously imported from disease-free dairy farms located in different provinces of Türkiye. Nevertheless, this population should be periodically checked for autosomal genetic disorders, since inherited diseases may be observed in the next generations due to causative mutations in the related genomic regions.

Towards Automated Engineering of Donor-Derived T Lymphocytes into CRISPR/Cas9-Mediated CAR T Cells in a Closed-System

Chimeric antigen receptor (CAR)-expressing T cells have revolutionized immunotherapy of cancer. However, some patients derive no benefit from approved CARTcell therapies due to limitations such as toxicity and use of autologous cells which are sometimes unfit and risk manufacturing failures. Additional or more effective CARTcell therapies are needed. Harnessing the endogenous homology-directed DNA repair pathway via precision genome editing based on Cas9RNP/rAAV6, we and others have demonstrated the feasibility of targeting transgene(s) integration into specific gene site(s) including their incorporation under endogenous regulatory elements. Using this strategy, we aimed to engineer healthy donor-derived CAR T cells from αβ+ T lymphocytes. For enhanced safety, we metabolically reprogrammed the CAR T cells by inactivating the UMPS gene needed for uridine biosynthesis. The UMPS-/-CAR T cells become dependent on exogenous uridine thereby serving as a cell control mechanism. We hypothesized that donor-derived αβ+ T lymphocytes can be engineered using the Cas9RNP-rAAV6 strategy in an automated closed-system into controllable CAR T cells. We inactivated TRAC and UMPS gene loci in αβ+T lymphocytes using Lonza4D nucleofector. Electroporated cells were simultaneously transduced with CD19CAR-containing rAAV6 DNA vector. We obtained TRAC gene knockout up to 97% with knock-in up to 89%. Disruption of the UMPS gene would transform the cells to being auxotrophic and depend on uridine supply for viability and growth. To evaluate the CART cells' antitumor potency, we cocultured the CD19CART cells with GFP-expressing Nalm6 leukemia cell lines. The CD19CART cells eradicated the leukemia cells within 72 hours. To mimic relapse, we re-challenged cocultured CART with Nalm6 cells and the tumor cells were again depleted within 72 hours. Expectedly, IL-2 production was elevated in CD19CART cells challenged with Nalm6. Next, we tested the ability to control the cytotoxicity of the CD19CART cells by coculturing them with Nalm6 cells in separate groups with/without exogenous uridine. Interestingly, the CD19CART cells cocultured without uridine eradicated the target cells within 72 hours but lost their potency upon subsequent challenge (Figure 1). In contrast, CD19CAR T cells supplied with uridine remained potent and repeatedly eradicated target cells indicating the ability to turn off the potency of CART cells with uridine supply/withdrawal. To establish an automated closed-system for CART cells, we adopted the Miltenyi CliniMACS Prodigy with in-line electroporator (Elpo) for process engineering. First, we determined optimized parameters for electroporating αβ+ T lymphocytes on Elpo(Table1). Cells electroporated with these settings were (>80% viability) comparable to untreated cells. We first engineered CART cells with Elpo/Miltenyi reagents in an open system(i.e.cultivated Elpo-electroporated cells in culture vessels/incubators). We obtained 95% of TRAC and UMPS gene knockouts and up to 90% CD19CAR transgene knock-in to the TRAC locus - an efficiency slightly higher than using Lonza 4D nucleofector. To scale up the Cas9-based T cell engineering into a single automated process on the CliniMACS Prodigy, the Cas9 and guide RNA reagents would be scaled up 1000fold. To make the process cost-effective, we tested AZD7468, a small molecule DNAPKcs inhibitor. We optimized/reduced the amounts of reagents upto 90% less of Cas9RNP used without any significant reduction in gene editing outcomes. AZD7648 reduced the MOI needed for viral transduction by 50% while maintaining 80% or higher levels of transgene knock-in efficiency. Next, we commenced pilot studies to automate the CAR T cell engineering process in closed-system. Currently, we have achieved an automated TRAC gene knockout of over 90% and CAR transgene knock-in of slightly >10%. Further optimization studies are underway to improve automated gene knock-in efficiency. We have engineered donor-derived αβ+ T lymphocytes into potent/controllable CD19CART cells. The UMPS-deficient CD19CART cells lose antileukemia activity without uridine. We show that metabolic reprogramming can be combined with precision genome editing to generate controllable CART cells. Finally, our data demonstrate the feasibility of automating CRISPR/Cas9-mediated engineering of CAR T cells within closed-system.

Drug repurposing screen identifies vidofludimus calcium and pyrazofurin as novel chemical entities for the development of hepatitis E interventions

Hepatitis E virus (HEV) infection can cause severe complications and high mortality, particularly in pregnant women, organ transplant recipients, individuals with pre-existing liver disease and immunosuppressed patients. However, there are still unmet needs for treating chronic HEV infections. Herein, we screened a best-in-class drug repurposing library consisting of 262 drugs/compounds. Upon screening, we identified vidofludimus calcium and pyrazofurin as novel anti-HEV entities. Vidofludimus calcium is the next-generation dihydroorotate dehydrogenase (DHODH) inhibitor in the phase 3 pipeline to treat autoimmune diseases or SARS-CoV-2 infection. Pyrazofurin selectively targets uridine monophosphate synthetase (UMPS). Their anti-HEV effects were further investigated in a range of cell culture models and human liver organoids models with wild type HEV strains and ribavirin treatment failure-associated HEV strains. Encouragingly, both drugs exhibited a sizeable therapeutic window against HEV. For instance, the IC50 value of vidofludimus calcium is 4.6–7.6-fold lower than the current therapeutic doses in patients. Mechanistically, their anti-HEV mode of action depends on the blockage of pyrimidine synthesis. Notably, two drugs robustly inhibited ribavirin treatment failure-associated HEV mutants (Y1320H, G1634R). Their combination with IFN-α resulted in synergistic antiviral activity. In conclusion, we identified vidofludimus calcium and pyrazofurin as potent candidates for the treatment of HEV infections. Based on their antiviral potency, and also the favorable safety profile identified in clinical studies, our study supports the initiation of clinical studies to repurpose these drugs for treating chronic hepatitis E.

Mobilization of a novel diatom Mutator-Like Element (MULE) transposon inactivates the uridine monophosphate synthase (UMPS) locus in Phaeodactylum tricornutum.

Diatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic diversity, which often result in peculiar biochemical and biological traits. Transposable elements (TE) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic diversity and centrally contribute to genome evolution. Here, through long-read whole genome sequencing, we identified a novel Mutator-Like Element (MULE) in the model diatom Phaeodactylum tricornutum, and we report the direct observation of its mobilization within the course of a single laboratory experiment. Under selective conditions, this novel TE inactivated the uridine monophosphate synthase (UMPS) gene of P. tricornutum, one of the few endogenous genetic loci currently targeted for selectable auxotrophy for functional genetics and genome editing applications. We report the observation of a recently mobilized transposon in diatoms with unique features. These include the combined presence of a MULE transposase with Zinc finger, SWIM-type domains, and of a diatom-specific E3 ubiquitin ligase of the zinc finger UBR type, which indicate a novel mobilization mechanism. Our findings provide new elements for the understanding of the role of TEs in diatom genome evolution and in the enrichment of intraspecific genetic variability.

De novo pyrimidine biosynthetic complexes support cancer cell proliferation and ferroptosis defence.

De novo pyrimidine biosynthesis is achieved by cytosolic carbamoyl-phosphate synthetase II, aspartate transcarbamylase and dihydroorotase (CAD) and uridine 5'-monophosphate synthase (UMPS), and mitochondrial dihydroorotate dehydrogenase (DHODH). However, how these enzymes are orchestrated remains enigmatical. Here we show that cytosolic glutamate oxaloacetate transaminase 1 clusters with CAD and UMPS, and this complex then connects with DHODH, which is mediated by the mitochondrial outer membrane protein voltage-dependent anion-selective channel protein 3. Therefore, these proteins form a multi-enzyme complex, named 'pyrimidinosome', involving AMP-activated protein kinase (AMPK) as a regulator. Activated AMPK dissociates from the complex to enhance pyrimidinosome assembly but inactivated UMPS, which promotes DHODH-mediated ferroptosis defence. Meanwhile, cancer cells with lower expression of AMPK are more reliant on pyrimidinosome-mediated UMP biosynthesis and more vulnerable to its inhibition. Our findings reveal the role of pyrimidinosome in regulating pyrimidine flux and ferroptosis, and suggest a pharmaceutical strategy of targeting pyrimidinosome in cancer treatment.

N-carbamoyl aspartate reduced body weight by stimulating the thermogenesis of iBAT

Uridine has formerly been shown to alleviate obesity and hepatic lipid accumulation. N-carbamoyl aspartate (NCA) provides carbon atoms to uridine in de novo pyrimidine biosynthesis pathway. However, whether NCA is involved in the lipid metabolism remains elusive. Here we showed that NCA supplementation significantly decreased (P < 0.05) serum cholesterol (CHOL), high-density lipoprotein (HDL), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) levels of mice, and significantly increased (P < 0.05) relative mRNA expression of genes related to the synthesis of pyrimidine nucleotides and polyunsaturated fatty acids. Besides, supplemented with NCA significantly decreased body weight and area under the curve (AUC), and increased body temperature in the high-fat diet fed mice. For further, relative protein expression of uridine monophosphate synthase (UMPS), sterol regulatory element-binding protein 1(SREBP-1) and phosphorylated hormone-sensitive triglyceride lipase (P-HSL) in the liver, and uncoupling protein 1 (UCP-1) in interscapular brown adipose tissue (iBAT) also showed upregulated in the high-fat diet fed mice. Thus, NCA promoted de novo synthesis of pyrimidine and polyunsaturated fatty acid, and reduced body weight by stimulating high-fat diet-induced thermogenesis of iBAT.

Hereditary orotic aciduria identified by newborn screening.

Introduction: Hereditary orotic aciduria is an extremely rare, autosomal recessive disease caused by deficiency of uridine monophosphate synthase. Untreated, affected individuals may develop refractory megaloblastic anemia, neurodevelopmental disabilities, and crystalluria. Newborn screening has the potential to identify and enable treatment of affected individuals before they become significantly ill. Methods: Measuring orotic acid as part of expanded newborn screening using flow injection analysis tandem mass spectrometry. Results: Since the addition of orotic acid measurement to the Israeli routine newborn screening program, 1,492,439 neonates have been screened. The screen has identified ten Muslim Arab newborns that remain asymptomatic so far, with DBS orotic acid elevated up to 10 times the upper reference limit. Urine organic acid testing confirmed the presence of orotic aciduria along with homozygous variations in the UMPS gene. Conclusion: Newborn screening measuring of orotic acid, now integrated into the routine tandem mass spectrometry panel, is capable of identifying neonates with hereditary orotic aciduria.

Identification of an immune-related metabolic gene signature to predict possible prognosis in endometrial cancer and reveals immune landscape feature

The immunotherapy of endometrial cancer (EC) has gradually attracted attention and metabolic reprogramming is associate with tumor immune infiltration. Our goal was to use proteome analysis to examine the role of immune-related metabolic genes (IRMGs) in EC. Data-independent acquisition mass spectrometry (DIA-MS) was performed on 20 EC patients, consisting of 10 high-grade and 10 low-grade cancer tissues. IRMGs were screened using Spearman correlation, and an immune-related metabolic prognosis signature (IRMPS) was constructed based on the Cancer Genome Atlas-Uterine Corpus Endometrioid Carcinoma (TCGA-UCEC) cohort using the least absolute shrinkage and selection operator (LASSO) regression analysis. We also investigated differences between different risk groups in terms of prognostic value, clinical potency, immune characteristics and therapy response. In total, 285 differentially expressed genes (DEGs) were acquired via DIA-MS. Subsequently, metabolic-DEGs and immune-DEGs were analyzed by Spearman correlation to identify 41 IRMGs. Finally, seven IRMGs, including NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 2 (NDUFA2), AMPK-alpha2 (PRKAA2), syntaxin binding protein 1 (STXBP1), NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 9 (NDUFB9), ribosomal protein S27-like (RPS27L), lysolecithin acyltransferase 2 (LPCAT2) and uridine monophosphate synthetase (UMPS) were identified to establish a prognosis signature. The risk score was determined as an independent prognostic indicator, and patients in the IRMPS-high group was strong linked with adverse prognosis for EC. Additionally, IRMPS was closely related with tumor immune infiltration. Notably, the IRMPS-low group had better immune checkpoint inhibitors (ICI) treatment response and more sensitive to chemotherapy drugs. In conclusion, IRMPS can serve as a precise prognostic tool to guide the personalized treatment of EC patients.

Identification of Dihydroorotate Dehydrogenase Inhibitors Using the Cell Painting Assay.

Profiling approaches have been increasingly employed for the characterization of disease-relevant phenotypes or compound perturbation as they provide a broad, unbiased view on impaired cellular states. We report that morphological profiling using the cell painting assay (CPA) can detect modulators of de novo pyrimidine biosynthesis and of dihydroorotate dehydrogenase (DHODH) in particular. The CPA can differentiate between impairment of pyrimidine and folate metabolism, which both affect cellular nucleotide pools. The identified morphological signature is shared by inhibitors of DHODH and the functionally tightly coupled complex III of the mitochondrial respiratory chain as well as by UMP synthase, which is downstream of DHODH. The CPA appears to be particularly suited for the detection of DHODH inhibitors at the site of their action in cells. As DHODH is a validated therapeutic target, the CPA will enable unbiased identification of DHODH inhibitors and inhibitors of de novo pyrimidine biosynthesis for biological research and drug discovery.

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

Uricase sensitizes hepatocellular carcinoma cells to 5-fluorouracil through uricase-uric acid-UMP synthase axis.

Conventional chemotherapy plays a key role in hepatocellular carcinoma (HCC) treatment, however, with intrinsic or acquired chemoresistance being a major constraint. Here, we aimed to identify potential target to reverse such chemoresistance. In the present study, we found significant difference in uridine monophosphate synthetase (UMPS) expression between 5-FU resistant and sensitive HCC cell lines and the overexpression or downregulation of UMPS impacted 5-FU response in HCC cells. We further found that inhibition of UMPS activity with uric acid at concentration present in human plasma decreased the 5-FU sensitivity of HCC cells, while reduction of uric acid levels with uricase improved the 5-FU sensitivity of HCC cells as well as colorectal cancer cells. In vivo studies also suggested that modulation of uric acid levels did affect 5-FU sensitivity of tumors. These data indicated that UMPS was correlated with the 5-FU resistance in HCC cells and uricase sensitized cancer cells to 5-FU through uricase-uric acid-UMP synthase axis, which provided a potential strategy to improve the efficacy of 5-FU-based chemotherapy for human cancers.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Frequency of genotypic markers for genetic disorders, colour, polledness, and major genes in Blanco Orejinegro cattle.

The purpose of this work was to establish DNA marker frequencies for genetic disorders, colour, horned/polled trait, and major genes of importance for productive and reproductive traits in Blanco Orejinegro (BON) cattle. The Blanco Orejinegro breed is a Colombian creole breed characterized by a white hair coat on black skin with black ears, black hair on the middle part of the legs, and absence of horns. We genotyped 420 animals of Colombia, 70 with the GGP-Bovine 150K chip and 350 with the GGP-Bovine 50K chip. The markers were associated with 50 genetic diseases, 52 major gene variants related to productive traits, and 12 variants related to coat coloration, presence of horns, and adaptation, selected from the information contained in the chips. Genotype frequencies were estimated using the R statistical program. Genetic disorder annotations were derived using the Online Mendelian Inheritance in Animals tool (OMIA) and the average inbreeding coefficient (F) (n = 7799) using the MTDFNRM program. Carriers were found for 16 of the genetic disorders evaluated but with low frequencies (0.24 to 2.46%); no homozygous animals were found for the disorders. Carriers were associated with disorders such as bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), syndactyly, and epidermolysis bullosa (EB). The F was 4.41%. Concerning the genes associated with colour (TYR, MC1R, and PMEL), alleles related to black pigmentation, the absence of horns (polledness), and slick coat (an adaptive trait) were highly frequent (> 81.90%). Markers associated with milk production and quality, yellow fat, and fertility showed variable frequencies, indicating selection potential. Allele frequency of genetic disorders in BON cattle was low, suggesting few genetic disorder problems, with syndactyly being the most frequent condition. The markers associated with colour and polledness were almost fixed, with a frequency at or near 100%. Production and reproduction markers showed variability for selection.

HEREDITARY OROTIC ACIDURIA WITH UNUSUAL PRESENTATION- A RARE CASE REPORT

Hereditary orotic aciduria (HOA) is an extremely rare inborn error of pyrimidine metabolism. It results from a defect in the uridine-5-monophosphate synthase (UMPS) gene. It can result in megaloblastic anemia, developmental delay and crystalluria. Molecular genetics is the conrm diagnostic modality. Uridine triacetate is the only established treatment. We thus report a case of 13 month male child having developmental delay with regression, dysmorphic facies, nystagmus, skeletal abnormalities and massive hepatosplenomegaly with past history of repeated LRTI. After clinical evaluation, an impression of storage disorder was presumed. Bicytopenia was also obtained in CBC with high MCV. After detailed evaluation with haematological, bone marrow, TMS and GCMS of urine, a diagnosis of hereditary orotic aciduria was established. Although extremely rare, hereditary orotic aciduria should be suspected in any child with megaloblastic bone marrow, immunodeciency or when developmental delay and anemia coexist