p53 and p21 regulate error-prone DNA repair to yield a lower mutation load

Regulation of mutation rates is critical for maintaining genome stability and controlling cancer risk. A special challenge to this regulation is the presence of multiple mutagenic DNA polymerases in mammals. These polymerases function in translesion DNA synthesis (TLS), an error-prone DNA repair process that involves DNA synthesis across DNA lesions. We found that in mammalian cells TLS is controlled by the tumor suppressor p53, and by the cell cycle inhibitor p21 via its PCNA-interacting domain, to maintain a low mutagenic load at the price of reduced repair efficiency. This regulation may be mediated by binding of p21 to PCNA and via DNA damage-induced ubiquitination of PCNA, which is stimulated by p53 and p21. Loss of this regulation by inactivation of p53 or p21 causes an out of control lesion-bypass activity, which increases the mutational load and might therefore play a role in pathogenic processes caused by genetic instability.     

Work to rest durations ratios exceeding unity are a risk factor for low back disorder; a feline model.

Low back disorders are prominent among the work force engaged in static anterior flexion during the workday. As a continuing part of a long-term research aimed to identify the biomechanical and physiological processes and corresponding risk factors leading to such cumulative trauma disorder (CTD), we ventured to assess the effect of rest and the work-to-rest duration ratios that may prevent CTD. Three groups of the feline model were subjected to three load/rest paradigms: two 30 min loading periods spaced by 10 min rest in Group I, two 30 min loading period spaced by 30 min rest in Group II and one 60 min loading period for Group III. The cumulative loading duration in the three groups was 60 min. Each of the groups were allowed 7h of rest while monitoring EMG and lumbar viscoelastic tissue creep each hour. The results demonstrate that for two 30 min load periods with a 30 min in between rest, an acute neuromuscular disorder was not present whereas for two 30 min loading with a 10 min rest it was. Similarly, for a 60 min loading with long-term rest, the disorder was present. Post hoc Fisher analysis demonstrated significant differences in the delayed hyperexcitability between the first and second group (P<0.0001) and the third and second (P<0.0001) group. Statistical difference in the displacement data of the three groups was not present. ANOVA showed a significant effect of time post-loading (P<0.0001 and different rest durations (P<0.0001) on the EMG data during the 7h recovery. The new data allow us to conclude that a work-to-rest duration ratio of 1:1 can prevent the development of CTD as long as the work periods are not too long (<60 min). Longer static flexion durations do not respond favorably to rest even if it is of equal or longer duration. It is suggested that appropriate durations of rest may be a viable tool to avert CTD in a certain range whereas long static flexion durations should be avoided at all cost.     

DNA Methylation, Behavior and Early Life Adversity

The impact of early physical and social environments on life-long phenotypes is well known. Moreover, we have documented evidence for gene–environment interactions where identical gene variants are associated with different phenotypes that are dependent on early life adversity. What are the mechanisms that embed these early life experiences in the genome? DNA methylation is an enzymatically-catalyzed modification of DNA that serves as a mechanism by which similar sequences acquire cell type identity during cellular differentiation and embryogenesis in the same individual. The hypothesis that will be discussed here proposes that the same mechanism confers environmental-exposure specific identity upon DNA providing a mechanism for embedding environmental experiences in the genome, thus affecting long-term phenotypes. Particularly important is the environment early in life including both the prenatal and postnatal social environments.     

A statistical pattern recognition approach for determining cellular viability and lineage phenotype in cultured cells and murine bone marrow.

BACKGROUND: Cellular binding of annexin V and membrane permeability to 7-aminoactinomycin D (7AAD) are important tools for studying apoptosis and cell death by flow cytometry. Combining viability markers with cell surface marker expression is routinely used to study various cell lineages. Current classification methods using strict thresholds, or "gates," on the fluorescent intensity of these markers are subjective in nature and may not fully describe the phenotypes of interest. We have developed objective criteria for phenotypic boundary recognition through the application of statistical pattern recognition. This task was achieved using artificial neural networks (ANNs) that were trained to recognize subsets of cells with known phenotypes, and then used to determine decision boundaries based on statistical measures of similarity. This approach was then used to test the hypothesis that erythropoietin (EPO) inhibits apoptosis and cell death in erythroid precursor cells in murine bone marrow. METHODS: Our method was developed for classification of viability using an in vitro cell system and then applied to an ex vivo analysis of murine late-stage erythroid progenitors. To induce apoptosis and cell death in vitro, an EPO-dependent human leukemic cell line, UT-7(EPO) cells were incubated without recombinant human erythropoietin (rhEPO) for 72 h. Five different ANNs were trained to recognize live, apoptotic, and dead cells using a "known" subset of the data for training, and a K-fold cross validation procedure for error estimation. The ANNs developed with the in vitro system were then applied to classify cells from an ex vivo study of rhEPO treated mice. Tg197 (human tumor necrosis-alpha transgenic mice, a model of anemia of chronic disease) received a single s.c. dose of 10,000 U/kg rhEPO and femoral bone marrow was collected 1, 2, 4, and 8 days after dosing. Femoral bone marrow cells were stained with TER-119 PE, CD71 APC enable identification of erythroid precursors, and annexin V FITC and 7AAD to identify the apoptotic and dead cells. During classification forward and side angle light scatter were also input to all pattern recognition systems. RESULTS: Similar decision boundaries between live, apoptotic, and dead cells were consistently identified by the neural networks. The best performing network was a radial basis function multi-perceptron that produced an estimated average error rate of 4.5% +/- 0.9%. Using these boundaries, the following results were reached: depriving UT-7(EPO) cells of rhEPO induced apoptosis and cell death while the addition of rhEPO rescued the cells in a dose-dependent manner. In vivo, treatment with rhEPO resulted in an increase of live erythroid cells in the bone marrow to 119.8% +/- 9.8% of control at the 8 day time point. However, a statistically significant transient increase in TER-119(+) CD71(+) 7AAD(+) dead erythroid precursors was observed at the 1 and 2 day time points with a corresponding decrease in TER-119(+) CD71(+) 7AAD(-) Annexin V(-) live erythroid precursors, and no change in the number of TER-119(+) CD71(+) annexin V(+) 7AAD(-) apoptotic erythroid precursors in the bone marrow. CONCLUSIONS: A statistical pattern recognition approach to viability classification provides an objective rationale for setting decision boundaries between "positive" and "negative" intensity measures in cytometric data. Using this approach we have confirmed that rhEPO inhibits apoptosis and cell death in an EPO dependent cell line in vitro, but failed to do so in vivo, suggesting EPO may not act as a simple antiapoptotic agent in the bone marrow. Rather, homeostatic mechanisms may regulate the pharmacodynamic response to rhEPO.     

Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells

Vascular calcification is recognized as an independent predictor of cardiovascular mortality, particularly in subjects with chronic kidney disease. However, the pathways by which dysregulation of lipid and mineral metabolism simultaneously occur in this particular population remain unclear. We have shown that activation of the farnesoid X receptor (FXR) blocks mineralization of bovine calcifying vascular cells (CVCs) and in ApoE knock-out mice with 5/6 nephrectomy. In contrast to FXR, this study showed that liver X receptor (LXR) activation by LXR agonists and adenovirus-mediated LXR overexpression by VP16-LXRα and VP16-LXRβ accelerated mineralization of CVCs. Conversely, LXR inhibition by dominant negative (DN) forms of LXRα and LXRβ reduced calcium content in CVCs. The regulation of mineralization by FXR and LXR agonists was highly correlated with changes in lipid accumulation, fatty acid synthesis, and the expression of sterol regulatory element binding protein-1 (SREBP-1). The rate of lipogenesis in CVCs through the SREBP-1c dependent pathway was reduced by FXR activation, but increased by LXR activation. SREBP-1c overexpression augmented mineralization in CVCs, whereas SREBP-1c DN inhibited alkaline phosphatase activity and mineralization induced by LXR agonists. LXR and SREBP-1c activations increased, whereas FXR activation decreased, saturated and monounsaturated fatty acids derived from lipogenesis. In addition, we found that stearate markedly promoted mineralization of CVCs as compared with other fatty acids. Furthermore, inhibition of either acetyl-CoA carboxylase or acyl-CoA synthetase reduced mineralization of CVCs, whereas inhibition of stearoyl-CoA desaturase induced mineralization. Therefore, a stearate metabolite derived from lipogenesis might be a risk factor for the development of vascular calcification.     

Potentiometric redox measurements in the environment: a novel perspective on method powers and limitations

Biogeochemistry - The South China Sea (SCS) is one of the largest marginal seas in the world, but the processes that control the silicon cycle are not well understood. Here, we analyse the factors...     

Characterization of a novel bifunctional dihydropteroate synthase/dihydropteroate reductase enzyme from Helicobacter pylori

Tetrahydrofolate is a ubiquitous C(1) carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl tRNA(fMet), which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence of Helicobacter pylori failed to reveal any sequence that encodes DHFR. Previous studies demonstrated that the H. pylori dihydropteroate synthase gene folP can complement an Escherichia coli strain in which folA and folM, encoding two distinct DHFRs, are deleted. It was also shown that H. pylori FolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated that H. pylori FolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzyme-bound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.     

Ezetimibe: A biomarker for efficacy of liver directed UGT1A1 gene therapy for inherited hyperbilirubinemia

As recently demonstrated in patients with factor IX deficiency, adeno-associated virus (AAV)-mediated liver-directed therapy is a viable option for inherited metabolic liver disorders. Our aim is to treat Crigler-Najjar syndrome type I (CN I), an inherited severe unconjugated hyperbilirubinemia, as a rare recessive inherited disorder. Because the number of patients eligible for this approach is small, the efficacy can only be demonstrated by a beneficial effect on the pathophysiology in individual patients. Serum bilirubin levels in potential candidates have been monitored since birth, providing an indication of their pathophysiology. Adjuvant phototherapy to prevent brain damage reduces serum unconjugated bilirubin (UCB) levels in CN I patients to the level seen in the milder form of the disease, CN type II. This therapy increases the excretion of UCB, thereby complicating the use of UCB and conjugated bilirubin levels in serum as biomarkers for the gene therapy we try to develop. Therefore, a suitable biomarker that is not affected by phototherapy is currently needed. To this end, we have investigated whether estradiol, ethinylestradiol or ezetimibe could be used as markers for uridine 5'-di-phospho-glucuronosyltransferase isoform 1A1 (UGT1A1) activity restored by AAV gene therapy in Gunn rats, a relevant animal model for CN I. Of these compounds, ezetimibe appeared most suitable because its glucuronidation rate in untreated control Gunn rats is low. Subsequently, ezetimibe glucuronidation was studied in both untreated and AAV-treated Gunn rats and the results suggest that it may serve as a useful serum marker for restored hepatic UGT1A1 activity.