HPRT Research Articles

Establishment and characterization of Lesch-Nyhan syndrome rabbit model.

Lesch-Nyhan syndrome (LNS) is a congenital defect disease that results in defective purine metabolism. It is caused by pathogenic variants of the HPRT gene. Its clinical symptoms mainly include high uric acid levels, gout, and kidney stones and damage. The mechanism of LNS has not been fully elucidated, and no cure exists. Animal models have always played an important role in exploring causative mechanisms and new therapies. This study combined CRISPR/Cas9 and microinjection to knock out the HPRT gene to create an LNS rabbit model. A sgRNA targeting exon 3 of HPRT gene was designed. Subsequently, Cas9 mRNA and sgRNA were injected into rabbit zygotes, and injected embryos were transferred to the uterus. The genotype and phenotype of rabbits were analyzed after birth. Four infant rabbits (named R1, R2, R3 and R4), which showed varying levels of gene modification, were born. The gene-editing efficiency was 100%. No wild-type sequences at the target HPRT gene were detected in R4 rabbit. Next, 6-thioguanine drug testing confirmed that HPRT enzymatic activity was deficient in R4 infant rabbit. HE staining revealed kidney abnormalities in all infant rabbits. Overall, an sgRNA capable of knocking out the HPRT gene in rabbits was successfully designed, and HPRT gene-modified rabbits were successfully constructed by using CRISPR/Cas9 technology and microinjection. This study provides a new nonrodent animal model for studying LNS syndrome.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats.

To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats' retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Xanthine oxidoreductase inhibition ameliorates high glucose-induced glomerular endothelial injury by activating AMPK through the purine salvage pathway

Xanthine oxidoreductase (XOR) contributes to reactive oxygen species production. We investigated the cytoprotective mechanisms of XOR inhibition against high glucose (HG)-induced glomerular endothelial injury, which involves activation of the AMP-activated protein kinase (AMPK). Human glomerular endothelial cells (GECs) exposed to HG were subjected to febuxostat treatment for 48 h and the expressions of AMPK and its associated signaling pathways were evaluated. HG-treated GECs were increased xanthine oxidase/xanthine dehydrogenase levels and decreased intracellular AMP/ATP ratio, and these effects were reversed by febuxostat treatment. Febuxostat enhanced the phosphorylation of AMPK, the activation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1α and PPAR-α and suppressed the phosphorylation of forkhead box O (FoxO)3a in HG-treated GECs. Febuxostat also decreased nicotinamide adenine dinucleotide phosphate oxidase (Nox)1, Nox2, and Nox4 expressions; enhanced superoxide dismutase activity; and decreased malondialdehyde levels in HG-treated GECs. The knockdown of AMPK inhibited PGC-1α–FoxO3a signaling and negated the antioxidant effects of febuxostat in HG-treated GECs. Despite febuxostat administration, the knockdown of hypoxanthine phosphoribosyl transferase 1 (HPRT1) also inhibited AMPK–PGC-1α–FoxO3a in HG-treated GECs. XOR inhibition alleviates oxidative stress by activating AMPK–PGC-1α–FoxO3a signaling through the HPRT1-dependent purine salvage pathway in GECs exposed to HG conditions.

Metabolic and neurobehavioral disturbances induced by purine recycling deficiency in Drosophila.

Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch-Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in Aprt-deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or N6-methyladenosine (m6A) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt deficiency. Overall, our results suggest that Drosophila could be used in different ways to better understand LND and seek a cure for this dramatic disease.

Metabolic and neurobehavioral disturbances induced by purine recycling deficiency in Drosophila

Adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) are two structurally related enzymes involved in purine recycling in humans. Inherited mutations that suppress HGPRT activity are associated with Lesch–Nyhan disease (LND), a rare X-linked metabolic and neurological disorder in children, characterized by hyperuricemia, dystonia, and compulsive self-injury. To date, no treatment is available for these neurological defects and no animal model recapitulates all symptoms of LND patients. Here, we studied LND-related mechanisms in the fruit fly. By combining enzymatic assays and phylogenetic analysis, we confirm that no HGPRT activity is expressed in Drosophila melanogaster, making the APRT homolog (Aprt) the only purine-recycling enzyme in this organism. Whereas APRT deficiency does not trigger neurological defects in humans, we observed that Drosophila Aprt mutants show both metabolic and neurobehavioral disturbances, including increased uric acid levels, locomotor impairments, sleep alterations, seizure-like behavior, reduced lifespan, and reduction of adenosine signaling and content. Locomotor defects could be rescued by Aprt re-expression in neurons and reproduced by knocking down Aprt selectively in the protocerebral anterior medial (PAM) dopaminergic neurons, the mushroom bodies, or glia subsets. Ingestion of allopurinol rescued uric acid levels in Aprt-deficient mutants but not neurological defects, as is the case in LND patients, while feeding adenosine or N6-methyladenosine (m6A) during development fully rescued the epileptic behavior. Intriguingly, pan-neuronal expression of an LND-associated mutant form of human HGPRT (I42T), but not the wild-type enzyme, resulted in early locomotor defects and seizure in flies, similar to Aprt deficiency. Overall, our results suggest that Drosophila could be used in different ways to better understand LND and seek a cure for this dramatic disease.

Co-administration of a Hepatitis B vaccine with CpG-ODN 2395 induces stronger immune response in BALB/c mice

Background Proof of effects of Cytosine Phosphoguanine Oligodeoxynucleotides (CpG ODNs), adjuvanted Hepatitis B Virus (HBV) vaccine on immune response is limited. This study aimed to assess the effect of five CpG ODNs in HBV Vaccine-immunized BALB/c mice and to identify the most effective CpG ODN adjuvant. Methods This laboratory-based experimental study was conducted using a total of 36 female BALB/c mice, which were clustered into 12 groups and immunized intramuscularly. Group 1 was immunized with CpG ODN 18281-1 alone, group 2 with vaccine plus CpG ODN 18281-1, group 3 with CpG ODN 18281-2 alone, group 4 with CpG ODN 18281-2 plus vaccine, group 5 CpG ODN 18289 alone, group 6 with CpG ODN 18289 plus vaccine, group 7 CpG ODN 1826 S alone, group 8 with CpG ODN 1826 S plus vaccine, group 9 CpG ODN 2395 alone, group 10 with CpG ODN 2395 plus vaccine, group 11 with vaccine alone and group 12 with Phosphate Buffer Saline (PBS). All the groups were observed for 14 and 28 days after immunization. Results In the vaccinated groups, those receiving supplementation with CpG 2395 exhibited a significant 4.4-fold elevation, resulting in a signal-to-noise ratio (S/N) value of 14.1 compared to the vaccine group only (S/N = 3.22) by day 28 (p-value < 0.0001). For mice immunized with the vaccine plus CpG ODNs, cytokine profiling using real-time quantitative polymerase chain reaction revealed increased IL-6 expression levels and decreased TNF-α levels compared to the untreated group, normalized with the housekeeping gene HPRT 1. However, the expression of IL-6 and TNF-α was not statistically significant between the treated groups (p-value ≥ 0.2). Parameters for toxicity were within the normal range in all treatment groups. Conclusion Based on these results the co-administration of the HBV vaccine with CpG ODN 2395 induces high immune responses in comparison to HBV vaccine alone.

Alleviation of doxorubicin adverse effects via loading into various drug-delivery systems: a comparative study.

Aim: Drug resistance is still a significant barrier to effective hepatocellular carcinoma therapy. Address the issue of doxorubicin resistance and inter-receptor crosstalk various doxorubicin formulations were investigated. Methods: Hepatocellular carcinoma was carried out using 3-methylechloroanthrene. Animals were then treated with doxorubicin, liposomal doxorubicin, titanium-loaded doxorubicin (TiO2-Dox), lactoferrin-doxorubicin and PEGylated doxorubicin. Biochemical and molecular analyses were assessed. Results: Results have declared a significant alternation of both sodium and potassium concentrations upon 3-methylechloroanthrene administration. Arginase-I and α-L-Fucodinase tumor biomarkers were significantly elevated. C-myc, Hprt-1 and EGFR gene expression were over-expressed. Treatment with the aforementioned treatment regimens significantly modulated all measured parameters. Conclusion: TiO2-Dox, doxorubicin-lactoferrin and PEGylated doxorubicin could be a promising regimen in hepatocellular carcinoma and overcoming the problem ofdrug resistance.

Purine salvage promotes treatment resistance in H3K27M-mutant diffuse midline glioma

BackgroundDiffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT) but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain.MethodsWe performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and our models, quantified purine synthesis using stable isotope tracing, and characterized the in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT.ResultsDMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo synthesis and apparent lower activity of purine salvage demonstrated via stable isotope tracing of key metabolites in purine synthesis and by lower expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the rate-limiting enzyme of purine salvage into IMP and GMP. Inhibition of de novo guanylate synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells upregulated HGPRT expression and hypoxanthine-derived guanylate salvage but maintained high levels of guanine-derived salvage. Exogenous guanine supplementation decreased radiosensitization in cells treated with combination RT and de novo purine synthesis inhibition. Silencing HGPRT combined with RT markedly suppressed DMG-H3K27M tumor growth in vivo.ConclusionsOur results indicate that DMG-H3K27M cells rely on highly active purine synthesis, both from the de novo and salvage synthesis pathways. However, highly active salvage of free purine bases into mature guanylates can bypass inhibition of the de novo synthetic pathway. We conclude that inhibiting purine salvage may be a promising strategy to overcome treatment resistance in DMG-H3K27M tumors.

Very Early Levodopa May Prevent Self-Injury in Lesch-Nyhan Disease

BackgroundIn Lesch-Nyhan disease (LND), early dopamine deficiency is thought to contribute to dystonia and self-injury, gradually developing over the first years of life. Previous attempts to restore dopamine levels in older patients have been unsuccessful. Based on the hypothesis that very early dopamine replacement can prevent full phenotypic development, we treated three patients with LND from infancy with levodopa. MethodsLevodopa/carbidopa (4:1) was started at age 11 to 13 months, aiming at escalating to 5 to 6 mg/kg levodopa per day. Follow-up focused on dystonia severity and whether self-injury occurred. In addition, the literature was reviewed to delineate the age at onset of self-injury for all reported cases to date. ResultsDuring long-term follow-up, self-injury appears to have been prevented in two patients (now aged 14 and 15.5 years), as their HPRT1 gene mutations had been invariably associated with self-injury before. Future self-injury is unlikely, as only 1.1% of 264 published cases had self-injury onset later in life than these patients’ current ages. The third patient started self-injury at age 1.5 years, while on a substantially lower levodopa dose. A clear effect of levodopa on dystonia could not be determined. ConclusionsOur observations suggest that levodopa, given early enough and sufficiently dosed, might be able to prevent self-injury in LND. Therefore, levodopa could be considered in patients with LND as early as possible, at least before the self-injury appears. Further research is needed to establish very early levodopa as an effective treatment strategy in LND, and to optimize timing and dosing.

Comprehensive blood metabolomics profiling of Parkinson’s disease reveals coordinated alterations in xanthine metabolism

Parkinson’s disease (PD) is a highly heterogeneous disorder influenced by several environmental and genetic factors. Effective disease-modifying therapies and robust early-stage biomarkers are still lacking, and an improved understanding of the molecular changes in PD could help to reveal new diagnostic markers and pharmaceutical targets. Here, we report results from a cohort-wide blood plasma metabolic profiling of PD patients and controls in the Luxembourg Parkinson’s Study to detect disease-associated alterations at the level of systemic cellular process and network alterations. We identified statistically significant changes in both individual metabolite levels and global pathway activities in PD vs. controls and significant correlations with motor impairment scores. As a primary observation when investigating shared molecular sub-network alterations, we detect pronounced and coordinated increased metabolite abundances in xanthine metabolism in de novo patients, which are consistent with previous PD case/control transcriptomics data from an independent cohort in terms of known enzyme-metabolite network relationships. From the integrated metabolomics and transcriptomics network analysis, the enzyme hypoxanthine phosphoribosyltransferase 1 (HPRT1) is determined as a potential key regulator controlling the shared changes in xanthine metabolism and linking them to a mechanism that may contribute to pathological loss of cellular adenosine triphosphate (ATP) in PD. Overall, the investigations revealed significant PD-associated metabolome alterations, including pronounced changes in xanthine metabolism that are mechanistically congruent with alterations observed in independent transcriptomics data. The enzyme HPRT1 may merit further investigation as a main regulator of these network alterations and as a potential therapeutic target to address downstream molecular pathology in PD.

Prognostic significance and immunological role of HPRT1 in human cancers.

Hypoxanthine phosphoribosyl transferase 1 (HPRT1), once considered a housekeeping gene, has been identified as playing an important role in several tumors. Its role in pan-cancer, however, has not been systematically studied. This study evaluates the relationship between HPRT1 and clinical parameters, survival prognosis, and tumor immunity based on multi omics data from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Drug sensitivity analysis screened 16 effective drugs against HPRT1, exploring the interactions with chemicals and genes. The significance of HPRT1 in tumor immunotherapy has also been investigated. Immunohistochemistry confirmed significant differences in the expression of HPRT1 between five tumor types (colon adenocarcinoma [COAD], head-neck squamous cell carcinoma [HNSC], lung adenocarcinoma [LUAD], thyroid carcinoma [THCA], and uterine corpus endometrial carcinoma [UCEC]) and adjacent normal tissues (P < 0.05). HPRT1 competitive endogenous RNA network was constructed in HNSC. Through cytological experiments, it was verified that HPRT1 plays a carcinogenic role in HNSC and is associated with tumor cell proliferation, migration, invasion, and apoptosis. In addition, there was a significant positive correlation between HPRT1 and programmed cell death-1 (PD-1) expression in HNSC (P < 0.05). These findings suggest that HPRT1 may be a potential biomarker for predicting and treating cancer.

Abstract A052: ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell

Abstract Objective: Ovulation is a major risk factor of epithelial ovarian cancer (EOC), presumably acts from the cancer initiation stage. High-grade serous carcinoma (HGSC) is the most prevalent and fatal EOC, mainly arising from fallopian tube fimbrial epithelium (FTE). TP53 missense mutations are considered the earliest event of carcinogenesis since they form “p53 signatures” in FTE and were found in virtually all HGSC. Previously, we discovered that a subset of ovulatory follicular fluids (FF) harbored high levels of ROS, which were both mutagenic and oncogenic, causing DNA double-strand breaks (DSB) and tumorigenesis. However, DSB is unlikely the cause of TP53 mutations which are mostly CG: TA base substitutions involving deamination. We hypothesized that during ovulation FF exposes the fallopian tube fimbria and causes TP53 mutations by activating the activation-induced cytidine deaminase (AID). Materials and Methods: By conducting an HPRT gene mutation assay, we determined the mutagenic activity of FF on FTE cells with or without AID-knockdown. AID gene activation and protein nuclear translocation were analyzed in human FTE tissues and cells with different degrees of transformation, as well as in the oviducts of mice under controlled ovulation. The binding of AID to the TP53 gene and the subsequent deamination were determined by ChIP-PCR and uracil-qPCR, respectively. Results: After FF exposure, the mutation frequency of HPRT in FTE cells increased by 4.1 folds, at levels 40% higher than the ethyl methane sulfonate control and could be diminished by antioxidants or knockdown of the AID gene. AID activation, including mRNA and protein inductions and nuclear translocation, was evident in FTE cells and tissue after FF exposure and in mouse oviducts after ovulation. Two waves of AID activations were noted, peaking at 45 minutes (early activity) and 8.5 hours (late activity) after ovulation or FF exposure. TNF-α and ROS were found to be responsible for the activities in FF. Mechanistically, the early activity worked through the MAPK, while the late activity worked through the NF-κB pathway. The induced AID in both activities physically binds to and biochemically deaminates the TP53 gene irrelative to the known mutational hotspots of HGSC. However, it did not act on PTEN, the other prevalent tumor suppressor gene in HGSC. Meanwhile, ovulatory FF also activates the putative tumor-initiating and stemness gene LGR6 in FTE at locations where AID was also activated. Conclusion: The serial studies characterized the mutagenic activities of ovulatory FF, which acts on the LGR6-expressing cells in the fallopian tube fimbria and targets the TP53 gene. The study also identified ROS as the mutagen and pointed to strategies of ovulation inhibition and antioxidation for the prevention of EOC. Citation Format: Tang-Yuan Chu, Sanchana Subramani, Hsuan-Shun Huang. ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A052.

Reference Gene Validation in the Embryonic and Postnatal Brain in the Rat Hyperhomocysteinemia Model.

Maternal hyperhomocysteinemia (HCY) induced by genetic defects in methionine cycle enzymes or vitamin imbalance is known to be a pathologic factor that can impair embryonal brain development and cause long-term consequences in the postnatal brain development as well as changes in the expression of neuronal genes. Studies of the gene expression on this model requires the selection of optimal housekeeping genes. This work aimed to analyze the expression stability of housekeeping genes in offspring brain. Pregnant female Wistar rats were treated daily with a 0.15% L-methionine solution in the period starting on the 4th day of pregnancy until delivery, to cause the increase in the homocysteine level in fetus blood and brain. Housekeeping gene expression was assessed by RT-qPCR on whole embryonic brain and selected rat brain areas at P20 and P90. The amplification curves were analyzed, and raw means Cq data were imported to the RefFinder online tool to assess the reference genes stability. Most of the analyzed genes showed high stability of mRNA expression in the fetal brain at both periods of analysis (E14 and E20). However, the most stably expressed genes at different age points differed. Actb, Ppia, Rpl13a are the most stably expressed on E14, Ywhaz, Pgk1, Hprt1 - on E20 and P20, Hprt1, Actb, and Pgk1 - on P90. Gapdh gene used as a reference in various studies demonstrates high stability only in the hippocampus and cannot be recommended as the optimal reference gene on HCY model. Hprt1 and Pgk1 genes were found to be the most stably expressed in the brain of rat subjected to HCY. These two genes showed high stability in the brain on E20 and in various areas of the brain on the P20 and P90. On E14, the preferred genes for normalization are Actb, Ppia, Rpl13a.

Evaluation of purine-nucleoside degrading ability and in vivo uric acid lowering of Streptococcus thermophilus IDCC 2201, a novel antiuricemia strain.

This study evaluated 15 lactic acid bacteria with a focus on their ability to degrade inosine and hypo-xanthine-which are the intermediates in purine metabolism-for the management of hyperuricemia and gout. After a preliminary screening based on HPLC, Lactiplantibacillus plantarum CR1 and Lactiplantibacillus pentosus GZ1 were found to have the highest nucleoside degrading rates, and they were therefore selected for further characterization. S. thermophilus IDCC 2201, which possessed the hpt gene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and exhibited purine degradation, was also selected for further characterization. These three selected strains were examined in terms of their probiotic effect on lowering serum uric acid in a Sprague-Dawley (SD) rat model of potassium oxonate (PO)-induced hyperuricemia. Among these three strains, the level of serum uric acid was most reduced by S. thermophilus IDCC 2201 (p < 0.05). Further, analysis of the microbiome showed that administration of S. thermophlilus IDCC 2201 led to a significant difference in gut microbiota composition compared to that in the group administered with PO-induced hyperuricemia. Moreover, intestinal short-chain fatty acids (SCFAs) were found to be significantly increased. Altogether, the results of this work indicate that S. thermophilus IDCC 2201 lowers uric acid levels by degrading purine-nucleosides and also restores intestinal flora and SCFAs, ultimately suggesting that S. thermophilus IDCC 2201 is a promising candidate for use as an adjuvant treatment in patients with hyperuricemia.

Collaboration of Hprt/K-RAS/c-Myc mutation in the oncogenesis of T-lymphocytic leukemia: acomparative study.

Aim: Leukemia is a malignant clonal illness stem from the mutations of hematopoietic cells. Acute lymphoblastic leukemia is one of the utmost prevalent kinds of leukemia, is brought on by atypical lymphoid progenitor cell division in the bone marrow. Materials & methods: A comparative study between, titanium Nanoparticle-loadeddoxorubicin or cisplatin and lactoferrin-loaded doxorubicin or cisplatin, on 7,12-dimethylbenz[a]-anthracene (DMBA)-induced leukemiawas investigated and confirming the hypothesis that messenger RNA of Hprt/K-RAS/c-Myc/SAT-2/P53/JAK-2 is a forthcoming signaling pathways in leukemia. Results: A significant alteration in Hprt, K-RAS, C-Myc, P53, JAK-2 and SAT-2 genes was observed post DMBA intoxication the aforementioned Nanodrugs modulated these signaling pathways. Conclusion: The carrier-loaded drugs triggered cytotoxicity of cancer cells via enhancing drug efficacy and bio-availability.

In vivo selection of anti-HIV-1 gene-modified human hematopoietic stem/progenitor cells to enhance engraftment and HIV-1 inhibition

Hematopoietic stem/progenitor cell (HSPC)-based anti-HIV-1 gene therapy holds great promise to eradicate HIV-1 or to provide long-term remission through a continuous supply of anti-HIV-1 gene-modified cells without ongoing antiretroviral therapy. However, achieving sufficient engraftment levels of anti-HIV gene-modified HSPC to provide therapeutic efficacy has been a major limitation. Here we report an in vivo selection strategy for anti-HIV-1 gene-modified HSPC by introducing 6-thioguanine (6TG) chemo-resistance through knocking down hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression using RNA interference (RNAi). We developed a lentiviral vector capable of co-expressing short hairpin RNA (shRNA) against HPRT alongside two anti-HIV-1 genes: shRNA targeting HIV-1 co-receptor CCR5 and a membrane anchored HIV-1 fusion inhibitor, C46, for efficient in vivo selection of anti-HIV-1 gene-modified human HSPC. 6TG-mediated preconditioning and in vivo selection significantly enhanced engraftment of HPRT-knockdown anti-HIV-1 gene-modified cells (>2 fold, p<0.005) in humanized bone marrow/liver/thymus (huBLT) mice. Viral load was significantly reduced (>1 log fold, p<0.001) in 6TG-treated HIV-1 infected huBLT mice compared to 6TG-untreated mice. We demonstrated that 6TG-mediated preconditioning and in vivo selection considerably improved engraftment of HPRT-knockdown anti-HIV-1 gene-modified HSPC and repopulation of anti-HIV-1 gene-modified hematopoietic cells in huBLT mice, allowing for efficient HIV-1 inhibition.

Selection of suitable reference genes for gene expression studies in HMC3 cell line by quantitative real-time RT-PCR

Microglia represent the primary immune defense system within the central nervous system and play a role in the inflammatory processes occurring in numerous disorders, such as Parkinson’s disease (PD). PD onset and progression are associated with factors considered possible causes of neuroinflammation, i.e. genetic mutations. In vitro models of microglial cells were established to identify specific molecular targets in PD through the analysis of gene expression data. Recently, the Human Microglial Clone 3 cell line (HMC3) has been characterized and a new human microglia model has emerged. Here we perform RT-qPCR analyses to evaluate the expression of ten reference genes in HMC3, untreated or stimulated to a pro-inflammatory status. The comparative ∆CT method, BestKeeper, Normfinder, geNorm and RefFinder algorithms were used to assess the stability of the candidate genes. The results showed that the most suitable internal controls are HPRT1, RPS18 and B2M genes. In addition, the most stable and unstable reference genes were used to normalize the expression of a gene of interest in HMC3, resulting in a difference in the statistical significance in cells treated with Rotenone. This is the first reference gene validation study in HMC3 cell line in pro-inflammatory status and can contribute to more reliable gene expression analysis in the field of neurodegenerative and neuroinflammatory research.

Validation of Endogenous Control Genes by Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction for Acute Leukemia Gene Expression Studies.

Reference genes are used as internal reaction controls for gene expression analysis, and for this reason, they are considered reliable and must meet several important criteria. In view of the absence of studies regarding the best reference gene for the analysis of acute leukemia patients, a panel of genes commonly used as endogenous controls was selected from the literature for stability analysis: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Abelson murine leukemia viral oncogene human homolog 1 (ABL), Hypoxanthine phosphoribosyl-transferase 1 (HPRT1), Ribosomal protein lateral stalk subunit P0 (RPLP0), β-actin (ACTB) and TATA box binding protein (TBP). The stability of candidate reference genes was analyzed according to three statistical methods of assessment, namely, NormFinder, GeNorm and R software (version 4.0.3). From this study's analysis, it was possible to identify that the endogenous set composed of ACTB, ABL, TBP and RPLP0 demonstrated good performances and stable expressions between the analyzed groups. In addition to that, the GAPDH and HPRT genes could not be classified as good reference genes, considering that they presented a high standard deviation and great variability between groups, indicating low stability. Given these findings, this study suggests the main endogenous gene set for use as a control/reference for the gene expression in peripheral blood and bone marrow samples from patients with acute leukemias is composed of the ACTB, ABL, TBP and RPLP0 genes. Researchers may choose two to three of these housekeeping genes to perform data normalization.

Analysis of Polyphenol Extract from Hazel Leaf and Ameliorative Efficacy and Mechanism against Hyperuricemia Zebrafish Model via Network Pharmacology and Molecular Docking.

Hazel leaf, a by-product of hazelnuts, is commonly used in traditional folk medicine in Portugal, Sweden, Iran and other regions for properties such as vascular protection, anti-bleeding, anti-edema, anti-infection, and pain relief. Based on our previous studies, the polyphenol extract from hazel leaf was identified and quantified via HPLC fingerprint. The contents of nine compounds including kaempferol, chlorogenic acid, myricetin, caffeic acid, p-coumaric acid, resveratrol, luteolin, gallic acid and ellagic acid in hazel leaf polyphenol extract (ZP) were preliminary calculated, among which kaempferol was the highest with 221.99 mg/g, followed by chlorogenic acid with 8.23 mg/g. The inhibition of ZP on α-glucosidase and xanthine oxidase activities was determined via the chemical method, and the inhibition on xanthine oxidase was better. Then, the effect of ZP on hyperuricemia zebrafish was investigated. It was found that ZP obviously reduced the levels of uric acid, xanthine oxidase, urea nitrogen and creatinine, and up-regulated the expression ofOAT1 and HPRT genes in hyperuricemia zebrafish. Finally, the targeted network pharmacological analysis and molecular docking of nine polyphenol compounds were performed to search for relevant mechanisms for alleviating hyperuricemia. These results will provide a valuable basis for the development and application of hazel leaf polyphenols as functional ingredients.

A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts

BackgroundLesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine–guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5ʹ-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells.MethodsWe prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts.ResultsLND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND.ConclusionsA complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.