Of fingers, toes and penises

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62765/1/390029a0.pd

Intra-Organ Variation in Age-Related Mutation Accumulation in the Mouse

Using a transgenic mouse model harboring chromosomally integrated lacZ mutational target genes, we previously demonstrated that mutations accumulate with age much more rapidly in the small intestine than in the brain. Here it is shown that in the small intestine point mutations preferentially accumulate in epithelial cells of the mucosa scraped off the underlying serosa. The mucosal cells are the differentiated villus cells that have undergone multiple cell divisions. A smaller age-related increase, also involving genome rearrangements, was observed in the serosa, which consists mainly of the remaining crypts and non-dividing smooth muscle cells. In the brain we observed an accumulation of only point mutations in no other areas than hypothalamus and hippocampus. To directly test for cell division as the determining factor in the generation of point mutations we compared mutation induction between mitotically active and quiescent embryonic fibroblasts from the same lacZ mice, treated with either UV (a point mutagen) or hydrogen peroxide (a clastogen). The results indicate that while point mutations are highly replication-dependent, genome rearrangements are as easily induced in non-dividing cells as in mitotically active ones. This strongly suggests that the point mutations found to have accumulated in the mucosal part of the small intestine are the consequence of replication errors. The same is likely true for point mutations accumulating in hippocampus and hypothalamus of the brain since neurogenesis in these two areas continues throughout life. The observed intra-organ variation in mutation susceptibility as well as the variation in replication dependency of different types of mutations indicates the need to not only extend observations made on whole organs to their sub-structures but also take the type of mutations and mitotic activity of the cells into consideration. This should help elucidating the impact of genome instability and its consequences on aging and disease

High Preservation of CpG Cytosine Methylation Patterns at Imprinted Gene Loci in Liver and Brain of Aged Mice

A gradual loss of the correct patterning of 5-methyl cytosine marks in gene promoter regions has been implicated in aging and age-related diseases, most notably cancer. While a number of studies have examined DNA methylation in aging, there is no consensus on the magnitude of the effects, particularly at imprinted loci. Imprinted genes are likely candidate to undergo age-related changes because of their demonstrated plasticity in utero, for example, in response to environmental cues. Here we quantitatively analyzed a total of 100 individual CpG sites in promoter regions of 11 imprinted and non-imprinted genes in liver and cerebral cortex of young and old mice using mass spectrometry. The results indicate a remarkably high preservation of methylation marks during the aging process in both organs. To test if increased genotoxic stress associated with premature aging would destabilize DNA methylation we analyzed two DNA repair defective mouse models showing a host of premature aging symptoms in liver and brain. However, also in these animals, at the end of their life span, we found a similarly high preservation of DNA methylation marks. We conclude that patterns of DNA methylation in gene promoters of imprinted genes are surprisingly stable over time in normal, postmitotic tissues and that the

LIFE BEEF CARBON: a common framework for quantifying grass and corn based beef farms’ carbon footprints

peer-reviewedEurope’s roadmap to a low-carbon economy aims to cut greenhouse gas (GHG) emissions 80% below 1990 levels by 2050. Beef production is an important source of GHG emissions and is expected to increase as the world population grows. LIFE BEEF CARBON is a voluntary European initiative that aims to reduce GHG emissions per unit of beef (carbon footprint) by 15% over a 10-year period on 2172 farms in four large beef-producing countries. Changes in farms beef carbon footprint are normally estimated via simulation modelling, but the methods current models apply differ. Thus, our initial goal was to develop a common modelling framework to estimate beef farms carbon footprint. The framework was developed for a diverse set of Western Europe farms located in Ireland, Spain, Italy and France. Whole farm and life cycle assessment (LCA) models were selected to quantify emissions for the different production contexts and harmonized. Carbon Audit was chosen for Ireland, Bovid-CO2 for Spain and CAP’2ER for France and Italy. All models were tested using 20 case study farms, that is, 5 per country and quantified GHG emissions associated with on-farm live weight gain. The comparison showed the ranking of beef systems gross carbon footprint was consistent across the three models. Suckler to weaning or store systems generally had the highest carbon footprint followed by suckler to beef systems and fattening beef systems. When applied to the same farm, Carbon Audit’s footprint estimates were slightly lower than CAP’2ER, but marginally higher than Bovid-CO2. These differences occurred because the models were adapted to a specific region’s production circumstances, which meant their emission factors for key sources; that is, methane from enteric fermentation and GHG emissions from concentrates were less accurate when used outside their target region. Thus, for the common modelling framework, regionspecific LCA models were chosen to estimate beef carbon footprints instead of a single generic model. Additionally, the Carbon Audit and Bovid-CO2 models were updated to include carbon removal by soil and other environmental metrics included in CAP’2ER, for example, acidification. This allows all models to assess the effect carbon mitigation strategies have on other potential pollutants. Several options were identified to reduce beef farms carbon footprint, for example, improving genetic merit. These options were assessed for beef systems, and a mitigation plan was created by each nation. The cumulative mitigation effect of the LIFE BEEF CARBON plan was estimated to exceed the projects reduction target (−15%)

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes

Effect of Ku80 Deficiency on Mutation Frequencies and Spectra at a LacZ Reporter Locus in Mouse Tissues and Cells

Non-homologous end joining (NHEJ) is thought to be an important mechanism for preventing the adverse effects of DNA double strand breaks (DSBs) and its absence has been associated with premature aging. To investigate the effect of inactivated NHEJ on spontaneous mutation frequencies and spectra in vivo and in cultured cells, we crossed a Ku80-deficient mouse with mice harboring a lacZ-plasmid-based mutation reporter. We analyzed various organs and tissues, as well as cultured embryonic fibroblasts, for mutations at the lacZ locus. When comparing mutant with wild-type mice, we observed a significantly higher number of genome rearrangements in liver and spleen and a significantly lower number of point mutations in liver and brain. The reduced point mutation frequency was not due to a decrease in small deletion mutations thought to be a hallmark of NHEJ, but could be a consequence of increased cellular responses to unrepaired DSBs. Indeed, we found a substantial increase in persistent 53BP1 and γH2AX DNA damage foci in Ku80−/− as compared to wild-type liver. Treatment of cultured Ku80-deficient or wild-type embryonic fibroblasts, either proliferating or quiescent, with hydrogen peroxide or bleomycin showed no differences in the number or type of induced genome rearrangements. However, after such treatment, Ku80-deficient cells did show an increased number of persistent DNA damage foci. These results indicate that Ku80-dependent repair of DNA damage is predominantly error-free with the effect of alternative more error-prone pathways creating genome rearrangements only detectable after extended periods of time, i.e., in young adult animals. The observed premature aging likely results from a combination of increased cellular senescence and an increased load of stable, genome rearrangements

The progeroid phenotype of Ku80 deficiency Is dominant over DNA-PK CS deficiency

Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80-/- mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs -/- mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80-/-, dna-pkcs -/- and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver a

Retinoic Acid Excess Impairs Amelogenesis Inducing Enamel Defects.

Abnormalities of enamel matrix proteins deposition, mineralization, or degradation during tooth development are responsible for a spectrum of either genetic diseases termed Amelogenesis imperfecta or acquired enamel defects. To assess if environmental/nutritional factors can exacerbate enamel defects, we investigated the role of the active form of vitamin A, retinoic acid (RA). Robust expression of RA-degrading enzymes Cyp26b1 and Cyp26c1 in developing murine teeth suggested RA excess would reduce tooth hard tissue mineralization, adversely affecting enamel. We employed a protocol where RA was supplied to pregnant mice as a food supplement, at a concentration estimated to result in moderate elevations in serum RA levels. This supplementation led to severe enamel defects in adult mice born from pregnant dams, with most severe alterations observed for treatments from embryonic day (E)12.5 to E16.5. We identified the enamel matrix proteins enamelin (Enam), ameloblastin (Ambn), and odontogenic ameloblast-associated protein (Odam) as target genes affected by excess RA, exhibiting mRNA reductions of over 20-fold in lower incisors at E16.5. RA treatments also affected bone formation, reducing mineralization. Accordingly, craniofacial ossification was drastically reduced after 2 days of treatment (E14.5). Massive RNA-sequencing (RNA-seq) was performed on E14.5 and E16.5 lower incisors. Reductions in Runx2 (a key transcriptional regulator of bone and enamel differentiation) and its targets were observed at E14.5 in RA-exposed embryos. RNA-seq analysis further indicated that bone growth factors, extracellular matrix, and calcium homeostasis were perturbed. Genes mutated in human AI (ENAM, AMBN, AMELX, AMTN, KLK4) were reduced in expression at E16.5. Our observations support a model in which elevated RA signaling at fetal stages affects dental cell lineages. Thereafter enamel protein production is impaired, leading to permanent enamel alterations.journal article20162017 01 06importe

Reference procedures for the measurement of gaseous emissions from livestock houses and stores of animal manure.

In the ten years before the EMILI 2012 symposium, gaseous losses from animal farms became increasingly important in the m edia. The paradox of this tendency was the great number of publications, scientific or not, even though the emissions of most animal farms had never been measured. Therefor e, the development of reference tools to measure greenhouse gas and ammonia emissio ns was important. Such tools allow recognition and remuneration of the best pract ices and equipment. Accordingly, ADEME funded an international project associating several research and development organizations involved with the animal production chain. The project proposed an initial set of 18 procedures to measure ammonia and greenho use gas emissions from animal houses and manure stores. These were adapted to the diversity of animal farms found throughout the world. Some methods were compared duri ng a ?building? and a ?liquid manure? experiment. Results showed a high difference among methods (ca. 80%), much higher than the estimated uncertainty. Associat ing independent emission measurements, together with a mass balance of the system, is necessary for the reliability of further results. However, previously published references lack uncertainty estimates of measurements that conform to GUM 2008. In the coming years, this is one of the major concerns for measuring emission factor s. Uncertainty estimates should depend on the measurand (temporal: hourly, per batch, yearly; spatial: animal, house, national) and include the uncertainties associated with system representativity and temporal interpolation.Edited by Mélynda Hassouna and Nadine Guingand