The Role of XPG in Processing (CAG)n/(CTG)n DNA Hairpins

BACKGROUND: During DNA replication or repair, disease-associated (CAG)n/(CTG)n expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Recent studies identified a nick-directed (CAG)n/(CTG)n hairpin repair (HPR) system that removes (CAG)n/(CTG)n hairpins from human cells via endonucleolytic incisions. Because the process is highly similar to the mechanism by which XPG and XPF endonucleases remove bulky DNA lesions during nucleotide excision repair, we assessed the potential role of XPG in conducting (CAG)n/(CTG)n HPR. RESULTS: To determine if the XPG endonuclease is involved in (CAG)n/(CTG)n hairpin removal, two XPG-deficient cell lines (GM16024 and AG08802) were examined for their ability to process (CAG)n/(CTG)n hairpins in vitro. We demonstrated that the GM16024 cell line processes all hairpin substrates as efficiently as HeLa cells, and that the AG08802 cell line is partially defective in HPR. Analysis of repair intermediates revealed that nuclear extracts from both XPG-deficient lines remove CAG/CTG hairpins via incisions, but the incision products are distinct from those generated in HeLa extracts. We also show that purified recombinant XPG protein greatly stimulates HPR in XPG-deficient extracts by promoting an incision 5\u27 to the hairpin. CONCLUSIONS: Our results strongly suggest that 1) human cells possess multiple pathways to remove (CAG)n/(CTG)n hairpins located in newly synthesized (or nicked) DNA strand; and 2) XPG, although not essential for (CAG)n/(CTG)n hairpin removal, stimulates HPR by facilitating a 5\u27 incision to the hairpin. This study reveals a novel role for XPG in genome-maintenance and implicates XPG in diseases caused by trinucleotide repeat expansion

Arsenic Inhibits DNA Mismatch Repair by Promoting EGFR Expression and PCNA Phosphorylation

Both genotoxic and non-genotoxic chemicals can act as carcinogens. However, while genotoxic compounds lead directly to mutations that promote unregulated cell growth, the mechanism by which non-genotoxic carcinogens lead to cellular transformation is poorly understood. Using a model non-genotoxic carcinogen, arsenic, we show here that exposure to arsenic inhibits mismatch repair (MMR) in human cells, possibly through its ability to stimulate epidermal growth factor receptor (EGFR)-dependent tyrosine phosphorylation of proliferating cellular nuclear antigen (PCNA). HeLa cells exposed to exogenous arsenic demonstrate a dose- and time-dependent increase in the levels of EGFR and tyrosine 211-phosphorylated PCNA. Cell extracts derived from arsenic-treated HeLa cells are defective in MMR, and unphosphorylated recombinant PCNA restores normal MMR activity to these extracts. These results suggest a model in which arsenic induces expression of EGFR, which in turn phosphorylates PCNA, and phosphorylated PCNA then inhibits MMR, leading to increased susceptibility to carcinogenesis. This study suggests a putative novel mechanism of action for arsenic and other non-genotoxic carcinogens

Glucitol-core containing gallotannins-enriched red maple (Acer rubrum) leaves extract alleviated obesity via modulating short-chain fatty acid production in high-fat diet-fed mice

Glucitol-core containing gallotannins (GCGs) are characteristic constituents of the red maple (Acer rubrum) species. To pursue the development of red maple for nutraceutical applications, GCGs-enriched red maple leaves extract (MLE) was evaluated for its effects on obesity, gut dysbiosis and short chain fatty acids (SCFAs) production. Our results demonstrated that MLE alleviated high-fat diet-induced obesity, reduced body weight gain and fat mass, improved liver steatosis and insulin resistance, and mitigated adipose hypertrophy and inflammation. Additionally, MLE increased total SCFAs, acetic acid and n-butyric acid content, but exerted no impact on propionic acid production. Moreover, MLE modulated gut microbiota community structure and certain bacteria relative abundance, including Prevotella and Eubacterium. Our work firstly reports a potential association between colon-derived SCFAs production and metabolic improvement due to GCGs-enriched red maple leaves extract administration, and highlights the utilization of red maple gallotannins as a dietary ingredient for preventing obesity and related metabolic diseases

Human DNA Exonuclease TREX1 Is Also an Exoribonuclease That Acts on Single-Stranded RNA

3\u27 repair exonuclease 1 (TREX1) is a known DNA exonuclease involved in autoimmune disorders and the antiviral response. In this work, we show that TREX1 is also a RNA exonuclease. Purified TREX1 displays robust exoribonuclease activity that degrades single-stranded, but not double-stranded, RNA. TREX1-D200N, an Aicardi-Goutieres syndrome disease-causing mutant, is defective in degrading RNA. TREX1 activity is strongly inhibited by a stretch of pyrimidine residues as is a bacterial homolog, RNase T. Kinetic measurements indicate that the apparent Km of TREX1 for RNA is higher than that for DNA. Like RNase T, human TREX1 is active in degrading native tRNA substrates. Previously reported TREX1 crystal structures have revealed that the substrate binding sites are open enough to accommodate the extra hydroxyl group in RNA, further supporting our conclusion that TREX1 acts on RNA. These findings indicate that its RNase activity needs to be taken into account when evaluating the physiological role of TREX1

Tissue-specific inactivation of p53 tumor suppression in the mouse.

The p53 gene is the most frequent target of structural and functional genetic mutations in human cancer. Thus, considerable effort has been devoted to mapping the functional domains of p53 with regard to their impact on tumorigenesis in vivo. Studies have shown that the carboxy-terminal domain of p53 is sufficient for transformation in vitro. To determine whether a transdominant-negative p53 protein could be used to elicit a tissue-specific p53-null effect in vivo, we tested whether a carboxy-terminal p53 fragment (amino acids 302-390) could abolish p53-dependent apoptosis in an established tumor progression model. We showed previously that loss of p53-dependent apoptosis accelerates brain tumorigenesis in a transgenic mouse model. Here, we show that the same effect can be elicited by expressing a dominant-negative p53 protein tissue specifically in the presence of wild-type p53. Transgenic mice in which pRb function has been disrupted and that coexpress a p53 carboxy-terminal dominant-negative fragment (p53DD) develop aggressive brain tumors mimicking genetic loss of p53 in this model. Inactivation of endogenous p53, which we show to be complexed with p53DD, results in a reduction in apoptosis and acceleration of tumorigenesis. These studies establish a mechanism for tissue-specific knock out of p53 function in vivo

Identification and Characterization of \u3cem\u3eOGG1\u3c/em\u3e Mutations in Patients with Alzheimer\u27s Disease

Patients with Alzheimer\u27s disease (AD) exhibit higher levels of 8-oxo-guanine (8-oxoG) DNA lesions in their brain, suggesting a reduced or defective 8-oxoG repair. To test this hypothesis, this study investigated 14 AD patients and 10 age-matched controls for mutations of the major 8-oxoG removal gene OGG1. Whereas no alterations were detected in any control samples, four AD patients exhibited mutations in OGG1, two carried a common single base (C796) deletion that alters the carboxyl terminal sequence of OGG1, and the other two had nucleotide alterations leading to single amino acid substitutions. In vitro biochemical assays revealed that the protein encoded by the C796-deleted OGG1 completely lost its 8-oxoG glycosylase activity, and that the two single residue-substituted OGG1 proteins showed a significant reduction in the glycosylase activity. These results were consistent with the fact that nuclear extracts derived from a limited number of AD patients with OGG1 mutations exhibited greatly reduced 8-oxoG glycosylase activity compared with age-matched controls and AD patients without OGG1 alterations. Our findings suggest that defects in OGG1 may be important in the pathogenesis of AD in a significant fraction of AD patients and provide new insight into the molecular basis for the disease

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits

Modèles déterministe, stochastique et multicritère pour l'équilibrage de lignes d'assemblage

Our work considers the problem of balancing the assembly line (ALBP). This is a combinatorial optimization problem which consists in assigning operations to stations of the assembly line while respecting various constraints in order to optimize a criterion of efficiency. Two types of problems are defined, based on the objective to minimize. The type I problem (SALBP-1) minimizes the number of stations in a given cycle time. And the problem of type II (SALBP-2) minimizes the cycle time given by the largest time station with a given number of stations. Problems of type II are only considered in our work. Firstly, a method for determining the lower bound of the cycle time which respects the number of stations is determined. This method combines the Lagrangian relaxation and the columns generation method. The Lagrangian relaxation is used to relax the constraints of precedence. The problem of the Lagrangian relaxation is given by the columns generation. Then, a new heuristic is proposed for solving the SALBP-2 problem. This heuristic consists of two steps. In the first step, an initial solution is produced by an heuristic based on a weight corresponding to the position of operations and a threshold defined for each station. The, in the second step, the solution is improved by a process of transfer and exchange. Finally, meta-heuristics are used to solve deterministic and stochastic problems.Two main meta-heuristics are considered: the electromagnetism-like mechanism algorithm (EM) and the estimated distribution (ED). The performance of our method is confirmed via simulation and it is compared with simulated annealing (SA). Due to its better performance, EM was chosen to balance the stochastic lines with random operation times. In this case, the cycle time is minimized under constraint of the reliability of the line which must be greater than a given value. EM is also used to solve multi-objective problems such as minimizing the cycle time and maximizing the reliability of the line by determining a set of Pareto-optimal solutionsDans nos travaux, nous travaillons sur le problème de l?équilibrage de la ligne d?assemblage (ALBP). C?est un problème d?optimisation combinatoire qui permet de définir la répartition des opérations et leur affectation aux stations qui constituent la ligne d?assemblage tout en respectant différentes contraintes de façon à optimiser un critère d?efficacité donné. Deux types de problèmes sont définis d?après l?objectif à minimiser. Le problème de type I (SALBP-1) minimise le nombre de stations sous un temps de cycle donné. Et le problème de type II (SALBP-2) minimise le temps de cycle déterminé par le temps de station le plus grand avec un nombre donné de stations. Nous considérons dans nos travaux uniquement des problèmes de type II. Nous proposons d?abord une méthode pour déterminer la borne inférieure du temps de cycle qui assure de respecter le nombre donné de stations. Cette méthode combine la relaxation lagrangienne et la génération de colonnes. La relaxation lagrangienne est utilisée pour relaxer les contraintes de précédence. Le problème lagrangien de la relaxation lagrangienne est résolu par la génération de colonnes. Ensuite, nous proposons un heuristique pour résoudre les problèmes SALBP-2. L?heuristique proposé se compose de deux phases. Dans la première phase, une solution initiale est produite par un heuristique basé sur un poids correspondant à la position des opérations et d'un certain seuil défini selon les poids pour chaque station. La solution est ensuite améliorée par un procédé de transfert et d?échange dans la deuxième phase. Enfin, les méta-heuristiques sont utilisés pour résoudre le problème déterministe ainsi que le problème stochastique. Ainsi deux méthodes basées sur la génération sont utilisées : l?algorithme de electromagnetism-like mechanism (EM) et l?estimation de distribution (ED). Les résultats de simulation sont comparés avec ceux du recuit simulé (SA). De part sa meilleure performance, EM est choisi pour équilibrer les lignes stochastiques qui considèrent les temps des opérationsomme aléatoires. Dans ce cas, les temps de cycle sont minimisés de façon à assurer que la fiabilité de la ligne est supérieure à une valeur donnée. EM est aussi utilisé pour résoudre les problèmes de type multi-objectif avec minimisation du temps de cycle et maximisation de la fiabilité de la ligne en déterminant un ensemble de solutions Pareto-optimale