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) chemoresistance 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 < 0.0001) 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.

When Tautomers Matter: UV-Vis Absorption Spectra of Hypoxanthine in Aqueous Solution from Fully Atomistic Simulations.

The UV-Vis spectrum of the solvated purine derivative Hypoxanthine (HYX) is investigated using the Quantum Mechanics/Fluctuating Charges (QM/FQ) multiscale approach combined with a sampling of configurations through atomistic Molecular Dynamics (MD) simulations. Keto 1H7H and 1H9H tautomeric forms of HYX are the most stable in aqueous solution and form different stable complexes with the surrounding water molecules, ultimately affecting the electronic absorption spectra. The final simulated spectrum resulting from the combination of the individual spectra of tautomers agrees very well with most of the characteristics in the measured spectrum. The importance of considering the effect of the solute tautomers and, in parallel, the contribution of the different solvent arrangements around the solute when modeling spectral properties, is highlighted. In addition, the high quality of the computed spectra leads to suggesting an alternative way for acquiring tautomeric populations from combined computational/experimental spectra.

Identification of the Reference Genes for Relative qRT-PCR Assay in Two Experimental Models of Rabbit and Horse Subcutaneous ASCs.

Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study.

Oocytes and hypoxanthine orchestrate the G2-M switch mechanism in ovarian granulosa cells.

In mammalian growing follicles, oocytes are arrested at the diplotene stage (which resembles the G2/M boundary in mitosis), while the granulosa cells (GCs) continue to proliferate during follicular development, reflecting a cell cycle asynchrony between oocytes and GCs. Hypoxanthine (Hx), a purine present in the follicular fluid, has been shown to induce oocytes meiotic arrest, although its role in GC proliferation remains ill-defined. Here, we demonstrate that Hx indiscriminately prevents G2-to-M phase transition in porcine GCs. However, oocyte-derived paracrine factors (ODPFs), particularly GDF9 and BMP15, maintain the proliferation of GCs, partly by activating the ERK1/2 signaling and enabling the G2/M transition that is suppressed by Hx. Interestingly, GCs with lower expression of GDF9/BMP15 receptors appear to be more sensitive to Hx-induced G2/M arrest and become easily detached from the follicular wall. Importantly, Hx-mediated inhibition of G2/M progression instigates GC apoptosis, which is ameliorated in the presence of GDF9 and/or BMP15. Therefore, our data indicate that the counterbalance of intrafollicular factors, particularly Hx and oocyte-derived GDF9/BMP15, fine-tunes the development of porcine follicles by regulating the cell cycle progression of GCs.

Structural differences between hypoxanthine phosphoribosyltransferase family members highlight opportunities for antiparasitic drug design in neglected diseases.

Approaches to identify novel druggable targets for treating neglected diseases include computational studies that predict possible interactions of drugs and their molecular targets. Hypoxanthine phosphoribosyltransferase (HPRT) plays a central role in the purine salvage pathway. This enzyme is essential for the survival of the protozoan parasite T. cruzi, the causal agent of Chagas disease, and other parasites related to neglected diseases. Here we found dissimilar functional behaviours between TcHPRT and the human homologue, HsHPRT, in the presence of substrate analogues that can lie in differences in their oligomeric assemblies and structural features. To shed light on this issue, we carried out a comparative structural analysis between both enzymes. Our results show that HsHPRT is considerably more resistant to controlled proteolysis than TcHPRT. Moreover, we observed a variation in the length of two key loops depending on the structural arrangement of each protein (groups D1T1 and D1T1'). Such variations might be involved in inter-subunit communication or influencing the oligomeric state. Besides, to understand the molecular basis that govern D1T1 and D1T1' folding groups, we explored the distribution of charges on the interaction surfaces of TcHPRT and HsHPRT, respectively. To know whether the rigidity degree bears effect on the active site, we studied the flexibility of both proteins. The analysis performed here illuminates the underlying reasons and significance behind each protein's preference for one or the other quaternary arrangement that can be exploited for therapeutic approaches.

Corneal metabolic biomarkers for moderate and high myopia in human.

This study aimed to identify the corneal metabolic biomarkers for moderate and high myopia in human. We enrolled 221 eyes from 221 subjects with myopia to perform the femtosecond laser small incision lenticule extraction (SMILE) surgery. Among these, 71 eyes of 71 subjects were enrolled in the low myopic group, 75 eyes of 75 subjects in the moderate myopic group and 75 eyes of 75 subjects in the high myopic group. The untargeted metabolomics analysis was performed to analyze the corneal tissues extracted during the SMILE surgery using an ultra-high-performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometry (MS). The one-way analysis of variance (ANOVA) was used to identify the different metabolites among the three myopic groups, the orthogonal partial least-squares discriminant analysis (OPLS-DA) model was used to reveal the different metabolites between moderate myopia and low myopia, and between high myopia and low myopia. The Venn gram was used to find the overlapped metabolites of the three datasets of the different metabolites. The stepwise multiple linear regression analysis was used to determine the metabolic molecules associated with manifest refractive spherical equivalents (MRSE). The Receiver Operating Characteristics (ROC) analysis was performed to reveal the corneal biomarkers for moderate and high myopia. The hub biomarker was further selected by the networks among different metabolites created by the Cytoscape software. A total of 1594 metabolites were identified in myopic corneas. 321 metabolites were different among the three myopic groups, 106 metabolites were different between high myopic corneas and low myopic corneas, 104 metabolites were different between moderate myopic corneas and low myopic corneas, and 30 metabolic molecules overlapped among the three datasets. The multivariate linear regression analysis revealed the myopic degree was significantly influenced by the corneal levels of azelaic acid, arginine-proline (Arg-Pro), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, and hypoxanthine. The ROC curve analysis showed that azelaic acid, Arg-Pro and hypoxanthine were effective in discriminating low myopia from moderate to high myopia with the area under the curve (AUC) values as 0.982, 0.991 and 0.982 for azelaic acid, Arg-Pro and hypoxanthine respectively. The network analysis suggested that Arg-Pro had the maximum connections among these three biomarkers. Thus, this study identified azelaic acid, Arg-Pro and hypoxanthine as corneal biomarkers to discriminate low myopia from moderate to high myopia, with Arg-Pro serving as the hub biomarker for moderate and high myopia.

Oxygenation of the newborn. The impact of one molecule on newborn lives.

Hypoxanthine is a purine metabolite which increases during hypoxia and therefore is an indicator of this condition. Further, when hypoxanthine is oxidized to uric acid in the presence of xanthine oxidase, oxygen radicals are generated. This was the theoretical basis for suggesting and studying, beginning in the 1990s, resuscitation of newborn infants with air instead of the traditional 100% O2. These studies demonstrated a 30% reduction in mortality when resuscitation of term and near term infants was carried out with air compared to pure oxygen. The mechanism for this is not fully understood, however the hypoxanthine -xanthine oxidase system increases oxidative stress and plays a role in regulation of the perinatal circulation. Further, hyperoxic resuscitation inhibits mitochondrial function, and one reason may be that genes involved in ATP production are down-regulated. Thus, the study of one single molecule, hypoxanthine, has contributed to the global prevention of an estimated 2-500,000 annual infant deaths.

Xanthine Oxidoreductase Inhibitors Suppress the Onset of Exercise-Induced AKI in High HPRT Activity Urat1-Uox Double Knockout Mice.

BACKGROUND: Hereditary renal hypouricemia type 1 (RHUC1) is caused by URAT1/SLC22A12 dysfunction, resulting in urolithiasis and exercise-induced AKI (EIAKI). However, because there is no useful experimental RHUC1 animal model, the precise pathophysiologic mechanisms underlying EIAKI have yet to be elucidated. We established a high HPRT activity Urat1-Uox double knockout (DKO) mouse as a novel RHUC1 animal model for investigating the cause of EIAKI and the potential therapeutic effect of xanthine oxidoreductase inhibitors (XOIs). METHODS: The novel Urat1-Uox DKO mice were used in a forced swimming test as loading exercise to explore the onset mechanism of EIAKI and evaluate related purine metabolism and renal injury parameters. RESULTS: Urat1-Uox DKO mice had uricosuric effects and elevated levels of plasma creatinine and BUN as renal injury markers, and decreased creatinine clearance observed in a forced swimming test. In addition, Urat1-Uox DKO mice had increased NLRP3 inflammasome activity and downregulated levels of Na+-K+-ATPase protein in the kidney, as Western blot analysis showed. Finally, we demonstrated that topiroxostat and allopurinol, XOIs, improved renal injury and functional parameters of EIAKI. CONCLUSIONS: Urat1-Uox DKO mice are a useful experimental animal model for human RHUC1. The pathogenic mechanism of EIAKI was found to be due to increased levels of IL-1ß via NLRP3 inflammasome signaling and Na+-K+-ATPase dysfunction associated with excessive urinary urate excretion. In addition, XOIs appear to be a promising therapeutic agent for the treatment of EIAKI.

Metabolic Signature of Energy Metabolism Alterations and Excess Nitric Oxide Production in Culture Media Correlate with Low Human Embryo Quality and Unsuccessful Pregnancy.

Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those in the highest class give rise to successful pregnancy. Previous studies highlighted the potentiality of the analysis of metabolites in human embryo culture media, useful for the selection of embryos for implantation. In the present study, we analyzed in blind 66 human embryo culture media at 5 days after in vitro fertilization with the aim of quantifying compounds released by cell metabolism that were not present as normal constituents of the human embryo growth media, including purines, pyrimidines, nitrite, and nitrate. Only some purines were detectable (hypoxanthine and uric acid) in the majority of samples, while nitrite and nitrate were always detectable. When matching biochemical results with morphological evaluation, it was found that low grade embryos (n = 12) had significantly higher levels of all the compounds of interest. Moreover, when matching biochemical results according to successful (n = 17) or unsuccessful (n = 25) pregnancy, it was found that human embryos from the latter group released higher concentrations of hypoxanthine, uric acid, nitrite, and nitrate in the culture media. Additionally, those embryos that developed into successful pregnancies were all associated with the birth of healthy newborns. These results, although carried out on a relatively low number of samples, indicate that the analysis of the aforementioned compounds in the culture media of human embryos is a potentially useful tool for the selection of embryos for implantation, possibly leading to an increase in the overall rate of ART.

Simultaneous Determination of Xanthine and Hypoxanthine Using Polyglycine/rGO-Modified Glassy Carbon Electrode.

A novel electrochemical sensor was developed for selective and sensitive determination of xanthine (XT) and hypoxanthine (HX) based on polyglycine (p-Gly) and reduced graphene oxide (rGO) modified glassy carbon electrode (GCE). A mixed dispersion of 7 µL of 5 mM glycine and 1 mg/mL GO was dropped on GCE for the fabrication of p-Gly/rGO/GCE, followed by cyclic voltammetric sweeping in 0.1 M phosphate buffer solution within -0.45~1.85 V at a scanning rate of 100 mV·s-1. The morphological and electrochemical features of p-Gly/rGO/GCE were investigated by scanning electron microscopy and cyclic voltammetry. Under optimal conditions, the linear relationship was acquired for the simultaneous determination of XT and HX in 1-100 µM. The preparation of the electrode was simple and efficient. Additionally, the sensor combined the excellent conductivity of rGO and the polymerization of Gly, demonstrating satisfying simultaneous sensing performance to both XT and HX.

The biochemistry of the vitreous humour in estimating the post-mortem interval-a review of the literature, and use in forensic practice in Galicia (northwestern Spain).

The K+ and hypoxanthine (Hx) concentrations of the vitreous humour (VH) rise gradually after death, providing a means of estimating the post-mortem interval (PMI). The correlation between these analytes and the PMI is good since the vitreous chamber is partially isolated from autolytic events occurring elsewhere; the [K +] and [Hx] recorded is thus the result of changes within the eye. The present work provides a systematic review, following PRISMA recommendations, of 36 articles (3 reviews and 33 retrospective cohort studies) discussing the many procedures and regression models that have been developed for improving PMI estimates involving VH analytes. The results of a descriptive study are also provided, highlighting the causes and distribution of mortality as registered in medico-legal autopsies performed in 2019 in Galicia (northwestern Spain), and revealing the use of these PMI estimation methods in real forensic practice. Great heterogeneity was detected in the collection of VH samples, the treatments to which they were subjected before examination, and in their conservation and analysis. A lack of reproducibility in the analytical methods employed to estimate [K +] and [Hx] was noted, as well as an absence of external validation for most of the regression formulae used to determine the PMI from analyte values. The use of methods based on high-performance liquid chromatography, focal electrophoresis, or thermogravimetric/chemometric procedures might solve the problems encountered with traditional analytical techniques, offering reliable results more quickly and effectively (even when samples are contaminated). This study recommends using flexible multiple regression models that combine physical and chemical variables, and that population databases be constructed so that models can be properly validated.

IPMICALC: an Integrated Post-mortem Interval Calculator.

Correctly estimating the post-mortem interval (PMI) is essential in forensic practice. In recent decades, the measurement of vitreous humor analyte concentrations - especially of hypoxanthine and potassium - and their correlation with the PMI have returned good results. However, calculating the PMI from the data collected can be quite complex. The present paper describes a web resource ( http://modestya.usc.es:3838/Forensic/ ) that simplifies the procedure. The PMI is determined (with its 95% confidence interval) in a rapid, easy, and reliable manner based on the use of mathematical models, the biochemistry of the vitreous humor, and physical variables such as the ambient temperature, the rectal temperature, and bodyweight. The application is entirely free to use.

A bacterial riboswitch class senses xanthine and uric acid to regulate genes associated with purine oxidation.

Dozens of candidate orphan riboswitch classes have been discovered previously by using comparative sequence analysis algorithms to search bacterial genomic sequence databases. Each orphan is classified by the presence of distinct conserved nucleotide sequences and secondary structure features, and by its association with particular types of genes. One previously reported orphan riboswitch candidate is the "NMT1 motif," which forms a hairpin structure with an internal bulge that includes numerous highly conserved nucleotides. This motif associates with genes annotated to encode various dioxygenase enzymes, transporters, or proteins that have roles associated with thiamin or histidine metabolism. Biochemical evaluation of numerous ligand candidates revealed that NMT1 motif RNA constructs most tightly bind 8-azaxanthine, xanthine, and uric acid, whereas most other closely related compounds are strongly rejected. Genetic assays revealed that NMT1 motif RNAs function to turn off gene expression upon ligand binding, likely by regulating translation initiation. These results suggest that NMT1 motif RNAs function as aptamer domains for a riboswitch class that specifically responds to high concentrations of oxidized purines. Members of this "xanthine riboswitch" class appear to regulate genes predominantly related to purine transport and oxidation, thus avoiding the effects of overproduction of these common purine derivatives.

Overexpression and surface localization of HPRT in prostate cancer provides a potential target for cancer specific antibody mediated cellular cytotoxicity.

We chose to evaluate Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) as a possible biomarker for prostate cancer due to its involvement in nucleotide synthesis and cell cycle progression. We utilized two prostate cancer cell lines (PC3 and DU145) along with patient tissue and knockdowns to evaluate overall HPRT expression. The surface localization of HPRT was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy followed by ADCC to evaluate targeting potential. We found significant upregulation of HPRT within malignant samples with approximately 47% of patients had elevated levels of HPRT compared to normal controls. We also observed a significant association between HPRT and the plasma membrane of DU145 cells (p = 0.0004), but found no presence on PC3 cells (p = 0.14). This was confirmed with scanning electron microscopy and confocal microscopy. ADCC experiments were performed to determine whether HPRT could be used as a target antigen for selective cell-mediated killing. We found that DU145 cells treated with HPRT antibodies had a significantly higher incidence of cell death than both isotype treated samples and PC3 cells treated with the same concentrations of HPRT antibody. Finally, we determined that p53 had a significant impact on HPRT expression both internally and on the surface of cancer cells. These results suggest HPRT as a possible biomarker target for the treatment of patients with prostate cancer.

Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.

Purine metabolism is essential for all known living creatures, including humans in whom elevated serum concentration of purine break-down product uric acid (UA) is probably an independent risk factor for mortality, type 2 diabetes and cardiovascular events. An automated multiplex assay that measures several purine metabolites could therefore prove useful in many areas of medical, veterinary and biological research. The aim of the present work was to develop a sensitive LC-MS/MS method for simultaneous quantitation of xanthine, hypoxanthine, UA, allantoin, and creatinine in biobanked urine samples. This article describes details and performance of the new method studied in 55 samples of human urine. Archival sample preparation and effect of storage conditions on stability of the analytes are addressed. The intra-day and inter-day coefficients of variation were small for all the analytes, not exceeding 1% and 10%, respectively. Measurements of UA and creatinine in biobanked urine showed good agreement with values obtained using routine enzymatic assays on fresh urine. Spearman's correlation coefficients were 0.869 (p < .001) for creatinine and 0.964 (p < .001) for UA. Conclusion: the newly developed LC-MS/MS method allows reliable quantitative assessment of xanthine, hypoxanthine, allantoin, UA and creatinine. The proposed pre-analytical processing makes the method suitable for both fresh and biobanked urine stored frozen at -80 °C for at least 5.5 years.

Co(OH)2/MXene-Ti3C2 nanocomposites with triple-enzyme mimic activities as hydrogel sensing platform for on-site detection of hypoxanthine.

Novel Co(OH)2/MXene-Ti3C2 nanocomposites with oxidase (OXD)-mimic, peroxidase (POD)-mimic, and catalase (CAT)-mimic activities were prepared by a simple two-step method. The Co(OH)2/MXene-Ti3C2 nanocomposites with triple-enzyme mimic activities were embedded into sodium alginate (SA) gels for the first time to fabricate a target-responsive hydrogel-assisted assay. The catalytic mechanism and steady-state kinetics of Co(OH)2/MXene-Ti3C2 nanocomposites were investigated. Subsequently, hypoxanthine (Hx) was catalyzed by xanthine oxidase (XOD) to form H2O2, which reacts with 3,3',5,5'-tetramethyl-benzidine (TMB) in the presence of Co(OH)2/MXene-Ti3C2 nanocomposites to form a blue oxide (ox-TMB) in the hydrogel. The visible color change of the hydrogel with the increase of Hx concentration can be recognized through a smartphone App to transfer the red (R), green (G), and blue (B) values for the quantitative determination of  Hx, with a detection range from 5 to 250 µM, and detection limit of 0.2 µM. The method was applied to the determination of Hx content in different aquatic products. The spiked recoveries of the aquatic products were from 94.1 to 106.4%, and the relative standard deviations (RSD) were less than 5.4%. Our results show that the Co(OH)2/MXene-Ti3C2 nanocomposites hydrogel-assisted colorimetric biosensor is cost-effective, sensitive, and selective and has excellent application prospects for in-the-field determination of Hx.

Dissipative Photochemical Abiogenesis of the Purines.

We have proposed that the abiogenesis of life around the beginning of the Archean may have been an example of "spontaneous" microscopic dissipative structuring of UV-C pigments under the prevailing surface ultraviolet solar spectrum. The thermodynamic function of these Archean pigments (the "fundamental molecules of life"), as for the visible pigments of today, was to dissipate the incident solar light into heat. We have previously described the non-equilibrium thermodynamics and the photochemical mechanisms which may have been involved in the dissipative structuring of the purines adenine and hypoxanthine from the common precursor molecules of hydrogen cyanide and water under this UV light. In this article, we extend our analysis to include the production of the other two important purines, guanine and xanthine. The photochemical reactions are presumed to occur within a fatty acid vesicle floating on a hot (∼80 ∘C) neutral pH ocean surface exposed to the prevailing UV-C light. Reaction-diffusion equations are resolved under different environmental conditions. Significant amounts of adenine (∼10-5 M) and guanine (∼10-6 M) are obtained within 60 Archean days, starting from realistic concentrations of the precursors hydrogen cyanide and cyanogen (∼10-5 M).

Structural Basis for The Recognition of Deaminated Nucleobases by An Archaeal DNA Polymerase.

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5'-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.

Recombinant protein production-associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response.

A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.

Gene expressing human artificial chromosome vectors: Advantages and challenges for gene therapy.

After the construction of genomic libraries with yeast artificial chromosomes in the late 1980's for gene isolation and expression studies in cells, human artificial chromosomes were then a natural development in the 1990's, based on the same principles of formation requiring centromeric sequences for generating functional artificial chromosomes. Over the past twenty years, they became a useful research tool for understanding human chromosome structure and organization, and important vectors for expression of large genes and gene loci and the regulatory regions for full expression. Now they are being modified and developed for gene therapy both ex vivo and in vivo. The advantages of using HAC vectors are that they remain autonomous and behave as a normal chromosome. They are attractive for therapy studies without the harmful consequences of integration of exogenous DNA into host chromosomes. HAC vectors are also the only autonomous stable vectors that accommodate large sequences (>100 kb) compared to other vectors. The challenges of manipulating these vectors for efficient delivery of genes into human cells is still ongoing, but we have made advances in transfer of gene expressing HAC vectors using the helper free (HF) amplicon vector technology for generating de novo HAC in human cells. Efficient multigene delivery was successfully achieved following simultaneous infection with two HF amplicons in a single treatment and the input DNA recombined to form a de novo HAC. Potentially several amplicons containing gene expressing HAC vectors could be transduced simultaneously which would increase the gene loading capacity of the vectors for delivery and studying full expression in human cells.