HPRT Research Articles

Lack of mutagenicity of TiO2 nanoparticles in vitro despite cellular and nuclear uptake.

The potential genotoxicity of titanium dioxide (TiO2) nanoparticles (NPs) is a conflictive topic because both positive and negative findings have been reported. To add clarity, we have carried out a study with two cell lines (V79-4 and A549) to evaluate the effects of TiO2 NPs (NM-101), with a diameter ranging from 15 to 60nm, at concentrations 1-75μg/cm2. Using two different dispersion procedures, cell uptake was determined by Transmission Electron Microscopy (TEM). Mutagenicity was evaluated using the Hprt gene mutation test, while genotoxicity was determined with the comet assay, detecting both DNA breaks and oxidized DNA bases (with formamidopyrimidine glycosylase - Fpg). Cell internalization, as determined by TEM, shows TiO2 NM-101 in cytoplasmic vesicles, as well as close to and inside the nucleus. Such internalization did not depend on the state of agglomeration, nor the dispersion used. In spite of such internalization, no cytotoxicity was detected in V79-4 cells (relative growth activity and plating efficiency assays) or in A549 cells (AlamarBlue assay) after exposure lasting for 24h. However, a significant decrease in the relative growth activity was detected at longer exposure times (48 and 72h) and at the highest concentration 75µg/cm2. When the modified enzyme-linked alkaline comet assay was performed on A549 cells, although no significant induction of DNA damage was detected, a positive concentration-effects relationship was observed (Spearman's correlation = 0.9, p0.0001). Furthermore, no significant increase of DNA oxidized purine bases was observed. When the frequency of Hprt gene mutants was determined in V79-4 cells, no increase was observed in the exposed cells, relative to the unexposed cultures. Our general conclusion is that, under our experimental conditions, TiO2 NM-101 exposure does not exert mutagenic effects despite the evidence of NP uptake by V79-4 cells.

Expression variation of OGG1 and HPRT gene and DNA damage in arsenic exposed industrial workers.

Arsenic exposure alters redox balance, induces DNA damage, and deregulates many genes. OGG1 gene involved in base repair mechanism, for excision of 8-oxoguanine (8-oxoG) from DNA formed as a result of accumulation of ROS in cell. HPRT gene encode transferase enzymes involved in purine recycling mechanism. The main focus of the study was to evaluate the expression variation in HPRT, OGG1 gene expression, and DNA damage of industrial workers. Blood samples of 300 occupational workers were collected from welding, brick kiln, furniture, pesticide, and paint industry (n = 60/industry) to evaluate the expression variation in HPRT, OGG1 gene expression, and DNA damage in blood cells by comet assay along with age and gender matched 300 control individuals. Blood arsenic content was higher (P<0.001) in an industrial group compared to the control. OGG1 and HPRT expression were (P<0.05) downregulated in exposed workers compared to controls. Spearman correlation analysis showed a significant positive correlation between HPRT vs OGG1 (P< 0.0001) in exposed workers compared to controls. Altered expression of both genes was observed between workers with <25years and >25years of age as well as between workers with <10years and >10year exposure. Reduced expression (P<0.05) of both genes and a high extent of DNA damage was evident in exposed smokers compared to respective non-smokers. DNA fragmentation was higher (P<0.05) in the furniture, welding and brick kiln group compared to control, and other industries. The present study suggests that altered expression of OGG1 and HPRT gene induce oxidative stress, showed a negative impact on the recycling of purines leading to DNA damage which increase the vulnerability of workers to carcinogenicity.

Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions.

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural proteins. In ectopic expression models, we observed that 6-TG increased the electrophoretic mobility of Spike from diverse betacoronaviruses, matching the effects of enzymatic removal of N-linked oligosaccharides from Spike in vitro. SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on production of lentiviruses pseudotyped with SARS-CoV-2 Spike, yielding pseudoviruses deficient in Spike and unable to infect ACE2-expressing cells. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. Using biochemical and genetic approaches we demonstrated that 6-TG is a pro-drug that must be converted to the nucleotide form by hypoxanthine phosphoribosyltransferase 1 (HPRT1) to achieve antiviral activity. This nucleotide form has been shown to inhibit small GTPases Rac1, RhoA, and CDC42; however, we observed that selective chemical inhibitors of these GTPases had no effect on Spike processing or accumulation. By contrast, the broad GTPase agonist ML099 countered the effects of 6-TG, suggesting that the antiviral activity of 6-TG requires the targeting of an unknown GTPase. Overall, these findings suggest that small GTPases are promising targets for host-targeted antivirals.

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23.

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

Modelling the HPRT-gene mutation induction of particle beams: systematic in vitro data collection, analysis and microdosimetric kinetic model implementation

Objective. Since the early years, particle therapy treatments have been associated with concerns for late toxicities, especially secondary cancer risk (SCR). Nowadays, this concern is related to patients for whom long-term survival is expected (e.g. breast cancer, lymphoma, paediatrics). In the aim to contribute to this research, we present a dedicated statistical and modelling analysis aiming at improving our understanding of the RBE for mutation induction () for different particle species. Approach. We built a new database based on a systematic collection of RBE data for mutation assays of the gene encoding for the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase from literature (105 entries, distributed among 3 cell lines and 16 particle species). The data were employed to perform statistical and modelling analysis. For the latter, we adapted the microdosimetric kinetic model (MKM) to describe the mutagenesis in analogy to lethal lesion induction. Main results. Correlation analysis between RBE for survival (RBES) and reveals significant correlation between these two quantities (ρ = 0.86, p < 0.05). The correlation gets stronger when looking at subsets of data based on cell line and particle species. We also show that the MKM can be successfully employed to describe obtaining comparably good agreement with the experimental data. Remarkably, to improve the agreement with experimental data the MKM requires, consistently in all the analysed cases, a reduced domain size for the description of mutation induction compared to that adopted for survival. Significance. We were able to show that RBES and are strongly related quantities. We also showed for the first time that the MKM could be successfully applied to the description of mutation induction, representing an endpoint different from the more traditional cell killing. In analogy to the RBES, can be implemented into treatment planning system evaluations.

Clinical and immunohistochemical evaluation of penile tumors in bulls

Fibropapilloma is the most common neoplasic formation associated with bovine papillomavirus type I. It occurs inside the penis of bulls and causes clinically adverse effects, such as phimosis, paraphimosis, urethral stricture, and urinary retention. This study aimed to conduct a clinical and immunohistochemical evaluation of penile tumors, which are associated with adverse outcomes with regard to breeding value and yield in bulls.A total of 40 bulls of different breeds aged between 10 months to 3 years were included in the study. Tumor tissue samples collected postoperatively were fixed in 10% buffered formaldehyde solution. The avidin–biotin–peroxidase method was used for immunohistochemical staining. Three separate areas were examined under 40X objective lens for each fibropapilloma tissue. The immunoreactivity was classified as none (-), mild (+), moderate (++), and severe (+++). Fibropapillomas ranged from 2 to 10 cm in diameter. Thirty-two papillomas were pedunculated and eight were sessile and attached to the body; 30 were solitary and 10 were multiple. There were 13 relapsed cases and 27 non-relapsed cases. Immunohistochemical examination revealed a statistically significant difference in the binary comparisons of proliferating cell nuclear antigen (PCNA) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) based on positive cell scoring between the relapsed and non-relapsed cases. As a result, it was concluded that the clinical data together with the cancer markers, which were used in the immunohistochemical evaluation and were of great importance in terms of the prognosis of penile tumors that caused significant economic losses, are useful parameters in evaluating the malignancy of penile tumors.

Differences in β-naphthylamine metabolism and toxicity in Chinese hamster ovary cell lines transfected with human CYP1A2 and NAT2*4, NAT2*5B or NAT2*7B N-acetyltransferase 2 haplotypes.

β-naphthylamine (BNA) is an important aromatic amine carcinogen. Current exposures derive primarily from cigarette smoking including e-cigarettes. Occupational and environmental exposure to BNA is associated with urinary bladder cancer which is the fourth most frequent cancer in the United States. N-acetyltransferase 2 (NAT2) is an important metabolizing enzyme for aromatic amines. Previous studies investigated mutagenicity and genotoxicity of BNA in bacteria and in rabbit or rat hepatocytes. However, the effects of human NAT2 genetic polymorphism on N-acetylation and genotoxicity induced by BNA still need to be clarified. We used nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells that were stably transfected with human CYP1A2 and NAT2 alleles: NAT2*4 (reference allele), NAT2*5B (variant slow acetylator allele common in Europe) or NAT2*7B (variant slow acetylator allele common in Asia). BNA N-acetylation was measured both in vitro and in situ via high-performance liquid chromatography (HPLC). Hypoxanthine phosphoribosyl transferase (HPRT) mutations, double-strand DNA breaks, and reactive oxygen species (ROS) were measured as indices of toxicity. NAT2*4 cells showed significantly higher BNA N-acetylation rates followed by NAT2*7B and NAT2*5B. BNA caused concentration-dependent increases in DNA damage and ROS levels. NAT2*7B showed significantly higher levels of HPRT mutants, DNA damage and ROS than NAT2*5B (p < 0.001, p < 0.0001, p < 0.0001 respectively) although both are slow alleles. Our findings suggest that BNA N-acetylation and toxicity are modified by NAT2 polymorphism. Furthermore, they confirm heterogeneity among slow acetylator alleles for BNA metabolism and toxicity supporting differential risk for individuals carrying NAT2*7B allele.

СИНДРОМ КЕЛЛИ–СИГМИЛЛЕРА: ОПИСАНИЕ КЛИНИЧЕСКОГО НАБЛЮДЕНИЯ И ОБЗОР ЛИТЕРАТУРЫ

Kelley-Seegmiller syndrome (KSS) is a hereditary recessive X-linked disease based on a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase that leads to impaired purine metabolism, which - in its turn - causes the development of gout in early childhood and the formation of neurological symptoms. The scientific literature represents an insufficient number of clinical cases of KSS described to date, most of which are characterized by the delayed diagnosis verification. The main reason for this may be the lack of information about this orphan disease. The article presents a rare clinical case observation of the development of KSS with mental retardation and the onset of gout and arthritis in early childhood and discusses the characteristic symptoms of the disease as well as the methods for its diagnosis and therapy.

Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages

BackgroundQuantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other.ResultsAs a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, β-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans.ConclusionsWe suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions.

Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland

In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (Ta) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.

Selection of stable reference genes for RT–qPCR in Salmo trutta

To identify suitable reference genes for Salmo trutta, real-time fluorescent quantitative PCR (RT–qPCR) technology was used to determine the expression of eight candidate genes in 11 tissues (heart, liver, spleen, head kidney, muscle, brain, gills, ovary, intestine, skin, testis) of S. trutta. Concurrently, expression of these eight reference genes was assessed in 1 d and 30 d S. trutta embryos and muscle tissue of 90 d and 180 d S. trutta. Finally, the eight candidate genes were comprehensively screened using four types of screening software (GeNorm, NormFinder, BestKeeper and RefFinder), and the stability of their expression in different tissues, in response to bacterial stress and during different developmental stages in S. trutta tissues was ranked. The results indicated that the eight candidate genes were specifically amplified in each tissue of S. trutta, with band sizes ranging from 73 bp to 150 bp. The stability ranking of the eight candidate genes in different tissues of healthy adult S. trutta was hprt1>β-actin>EF1a>RPL13a>RPS29>β2M>GAPDH>28 S rRNA. The ranking of the stability of expression in different tissues of S. trutta artificially infected with Aeromonas salmonicida was RPS29>hprt1>β2M>RPL13a>EF1a>β-actin>28 S rRNA>GAPDH. The ranking of the stability of expression in the tissues of S. trutta at different developmental stages was RPS29>GAPDH>hprt1>RPL13a>EF1a>28 S rRNA>β-actin>β2M. Comprehensive analysis revealed that expression of the hprt1 gene was relatively stable under different conditions. These results could guide the quantitative analysis of functional gene expression in S. trutta and calibrate the expression of major functional genes of S. trutta, facilitating the acquisition of more accurate, reliable and meaningful results.

Lesch-Nyhan Syndrome with Self-harming and Choreoathetosis after Improvement of Hyperuricemia: a case report

Lesch-Nyhan syndrome (LNS) is an inborn error of metabolism. Choreoathetosis, mental retardation and self-harming are the three symptoms. A genetic test that analyses the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) and blood tests showing hyperuricaemia support the diagnosis. However, the relevance of these test results to clinical symptoms is unclear. A 25 years-old man consulted our hospital for self-injury and choreoathetosis. He had epilepsy and mental retardation. He was also suffering from hyperuricemia and was prescribed allopurinol. These symptoms appeared with the improvement of hyperuricemia. He was suspected to have LNS. Metabolomic analysis of his urine before and after the allopurinol loading test was performed by gas chromatography-mass spectrometry (GC/MS) using dry filter paper urine samples as samples. The results showed that he was LNS.This case suggests that LNS is a condition that causes epileptic seizures and that rapid fluctuations in uric acid levels can cause choreoathetosis and self-harming. GC/MS using dry filter paper urine samples was a simple and valid test. This report also shows that genetic diagnosis opportunities are available to patients who have difficulty attending hospitals with advanced genetic analysis systems in urban areas.

Abnormalities of neural stem cells in Lesch–Nyhan disease

Lesch–Nyhan disease (LND) is a neurodevelopmental disorder caused by variants in the HPRT1 gene, which encodes the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGprt). HGprt deficiency provokes numerous metabolic changes which vary among different cell types, making it unclear which changes are most relevant for abnormal neural development. To begin to elucidate the consequences of HGprt deficiency for developing human neurons, neural stem cells (NSCs) were prepared from 6 induced pluripotent stem cell (iPSC) lines from individuals with LND and compared to 6 normal healthy controls. For all 12 lines, gene expression profiles were determined by RNA-seq and protein expression profiles were determined by shotgun proteomics. The LND lines revealed significant changes in expression of multiple genes and proteins. There was little overlap in findings between iPSCs and NSCs, confirming the impact of HGprt deficiency depends on cell type. For NSCs, gene expression studies pointed towards abnormalities in WNT signaling, which is known to play a role in neural development. Protein expression studies pointed to abnormalities in the mitochondrial F0F1 ATPase, which plays a role in maintaining cellular energy. These studies point to some mechanisms that may be responsible for abnormal neural development in LND.

In silico study, Synthesis and evaluation purine analogues as potential antimalarial agents

The challenges concerning the control of malaria remain due to the continuous emergence of drug resistant strains. Available treatments for this disease present several limitations such as lack of efficacy, toxic side effects and drug resistance. One of the most striking differences was found in the purine metabolism, because parasites are incapable of de novo purine biosynthesis in which enzyme HGPRT is central to the purine salvage pathway and whose activity is critical for the production of the nucleotides required for DNA/RNA synthesis within this protozoan parasite. Thus, new drugs are urgently needed. Herein we have designed, docked, synthesized and screened 9-alkyl purine derivatives for antimalarial activity against pfHGPRT. We have reported four compounds with promising activity against the highly artimisnin resistant P. falciparum namely: compounds 2,6-dichloro-9-(propan-2-yl)-9H-purine (1a), 9-Butyl-2,6-dichloro-9H-purine (1b), 2,6-dichloro-9-(2-methylbutyl)-9H-purine (1c), 2,6-dichloro-9-pentyl-9H-purine (1d) and binding energies to predictable antimalarial agents (-5.1, -5.3, -5.5 and -5.6 kcal/mole and rmsd 1.198, 1.238, 1.354 and 1.554 respectively). These analogs have acceptable binding interactions, Moreover, an in silico docking study revealed that P. falciparum and hypoxanthine guanine phosphoribosyltransferase enzymes could be the potential targets of those compounds. Our study identified novel, purine-based chemotypes that could be further optimized to generate potent and diversified anti-parasitic drugs against malaria.

Revisiting biocrystallization: purine crystalline inclusions are widespread in eukaryotes.

Despite the widespread occurrence of intracellular crystalline inclusions in unicellular eukaryotes, scant attention has been paid to their composition, functions, and evolutionary origins. Using Raman microscopy, we examined >200 species from all major eukaryotic supergroups. We detected cellular crystalline inclusions in 77% species out of which 80% is composed of purines, such as anhydrous guanine (62%), guanine monohydrate (2%), uric acid (12%) and xanthine (4%). Our findings shifts the paradigm assuming predominance of calcite and oxalates. Purine crystals emerge in microorganisms in all habitats, e.g., in freshwater algae, endosymbionts of reef-building corals, deadly parasites, anaerobes in termite guts, or slime molds. Hence, purine biocrystallization is a general and ancestral eukaryotic process likely present in the last eukaryotic common ancestor (LECA) and here we propose two proteins omnipresent in eukaryotes that are likely in charge of their metabolism: hypoxanthine-guanine phosphoribosyl transferase and equilibrative nucleoside transporter. Purine crystalline inclusions are multifunctional structures representing high-capacity and rapid-turnover reserves of nitrogen and optically active elements, e.g., used in light sensing. Thus, we anticipate our work to be a starting point for further studies spanning from cell biology to global ecology, with potential applications in biotechnologies, bio-optics, or in human medicine.

Establish an allele-specific real-time PCR for Leishmania species identification

BackgroundLeishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.MethodsA probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.ResultsThis new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.ConclusionsOur assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.Graphical

HGprt deficiency disrupts dopaminergic circuit development in a genetic mouse model of Lesch–Nyhan disease

In Lesch–Nyhan disease (LND), deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGprt) leads to a characteristic neurobehavioral phenotype dominated by dystonia, cognitive deficits and incapacitating self-injurious behavior. It has been known for decades that LND is associated with dysfunction of midbrain dopamine neurons, without overt structural brain abnormalities. Emerging post mortem and in vitro evidence supports the hypothesis that the dopaminergic dysfunction in LND is of developmental origin, but specific pathogenic mechanisms have not been revealed. In the current study, HGprt deficiency causes specific neurodevelopmental abnormalities in mice during embryogenesis, particularly affecting proliferation and migration of developing midbrain dopamine (mDA) neurons. In mutant embryos at E14.5, proliferation was increased, accompanied by a decrease in cell cycle exit and the distribution and orientation of dividing cells suggested a premature deviation from their migratory route. An abnormally structured radial glia-like scaffold supporting this mDA neuronal migration might lie at the basis of these abnormalities. Consequently, these abnormalities were associated with an increase in area occupied by TH+ cells and an abnormal mDA subpopulation organization at E18.5. Finally, dopaminergic innervation was disorganized in prefrontal and decreased in HGprt deficient primary motor and somatosensory cortices. These data provide direct in vivo evidence for a neurodevelopmental nature of the brain disorder in LND. Future studies should not only focus the specific molecular mechanisms underlying the reported neurodevelopmental abnormalities, but also on optimal timing of therapeutic interventions to rescue the DA neuron defects, which may also be relevant for other neurodevelopmental disorders.

Metabolism-Related Bioinformatics Analysis Reveals That HPRT1 Facilitates the Progression of Oral Squamous Cell Carcinoma In Vitro.

Objectives Many studies have shown that dysregulation of metabolism contributes to oncogenesis. However, the exact roles of metabolism-related genes (MRGs) in oral squamous cell carcinoma (OSCC) remain unclear. Thus, we aimed to identify a prognostic signature related to MRGs in OSCC. Methods The gene sequencing data of OSCC samples and the MRG set were downloaded from The Cancer Genome Atlas (TCGA) and the Molecular Signatures Database (MSigDB). The Wilcoxon rank-sum test was used to identify differentially expressed MRGs. Then, a prognostic signature was established by multivariate Cox regression analysis. Finally, prognosis-related MRGs were selected and further validated in OSCC tissues and cell lines. Results A prognostic signature that included 8 MRGs was constructed. Multiple survival analysis revealed that only HPRT1 might be an independent biomarker and indicator of poor overall survival in OSCC patients. The expression of HPRT1 was then found to be upregulated in OSCC tissues and cell lines, and suppression of HPRT1 gene expression by siRNA inhibited the proliferation, migration, and invasion of OSCC cells in vitro. Conclusions MRGs play an important role in the development of OSCC. Furthermore, HPRT1 might be an independent biomarker of OSCC and enhance OSCC proliferation, migration, and invasion in vitro; these results emphasize the potential utility of HPRT1 in OSCC therapy.

Description of the Molecular and Phenotypic Spectrum of Lesch-Nyhan Disease in Eight Chinese Patients.

Background: Lesch-Nyhan disease (LND) is a rare disorder involving pathogenic variants in the HPRT1 gene encoding the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) that result in hyperuricemia, intellectual disability, dystonic movement disorder, and compulsive self-mutilation. The purpose of the present study was to characterize the genetic basis of LND and describe its phenotypic heterogeneity by identifying the variation in the HPRT1 gene in a cohort of Chinese LND patients. Results: The median age at diagnosis was 31 mo (interquartile range (IQR): 7–76 mo), and the initial manifestations were mainly head control weakness and motor development delay. The median age of self-mutilation behavior onset was 19 mo (IQR: 17–24 mo), and all patients were required to travel in a wheelchair and fall into the predicament of compulsive self-harm behavior. There were two patients whose blood uric acid levels were normal for their high urinary acid excretion fraction without taking uric acid-lowering drugs. Seven different pathogenic variants of the HPRT1 gene were identified among eight independent pedigrees, including four novel mutations [c.299 (exon 3) T > A; loss (exon: 6) 84 bp; c.277_281delATTGC; c.468_470delGAT]. The pathogenic variant sites were mainly concentrated in exon 3, and truncating mutations (including frameshift mutations and nonsense mutations) were the most common genetic variant types (5/7, 71.4%). Conclusion: The present study described the phenotypic and molecular spectrum of LND in eight Chinese families, including four novel mutations, which expands our understanding of LND.

Identification of optimal reference genes for gene expression studies in a focal cerebral ischaemia model-Spatiotemporal effects.

A proper reference gene (RG) is required to reliably measure mRNA levels in biological samples via quantitative reverse transcription PCR (RT‐qPCR). Various experimental paradigms require specific and stable RGs. In studies using rodent models of brain ischaemia, a variety of genes, such as β‐actin (Actb), hypoxanthine phosphoribosyltransferase 1 (Hprt1), peptidyl‐propyl isomerase A (Ppia) and glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh), are used as RGs. However, most of these genes have not been validated in specific experimental settings. The aim of this study was to evaluate the time‐ and brain region‐dependent expression of RG candidates in a rat model of transient middle cerebral artery occlusion (tMCAO). The following genes were selected: Actb, Hprt1, Ppia, Gapdh, tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, zeta (Ywhaz) and beta‐2 microglobulin (B2m). Focal cerebral ischaemia was induced by 90 min of tMCAO in male Sprague‐Dawley rats. Expression was investigated at four time points (12 and 24 h; 3 and 7 days) and in three brain areas (the frontal cortex, hippocampus and dorsal striatum) within the ischaemic brain hemisphere. The RT‐qPCR results were analysed using variance analysis and the ΔCt, GeNorm, NormFinder and BestKeeper methods. Data from these algorithms were ranked using the geometric mean of ranks of each analysis. Ppia, Hprt1 and Ywhaz were the most stable genes across the analysed brain areas and time points. B2m and Actb exhibited the greatest fluctuations, and the results for Gapdh were ambiguous.