Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay – A methodological overview

There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results.     

Electrochemical characteristics of a hyperthermophilic enzyme in microdroplets stirred and heated by surface acoustic waves

Micro total analysis system (μTAS) is expected to be applied in various fields. In particular, since electrochemical measurement is inexpensive and easy, electrochemical measurement can be integrated with a microchannel. However, electrochemical detection sensitivity in a microchannel is lowered because the diffusion of the detection target is limited. In an ordinary electrochemical detection system, using a stirrer in a beaker can overcome limited diffusion. We previously proposed a new detection system that combines a microliquid solution agitation technology using surface acoustic waves (SAWs) with the μTAS. The SAWs function as microstirrers, thus making electrochemical detection possible by overcoming limited diffusion of the sample. However, when the solution is stirred by the SAWs, the temperature of the solution increases to 70°C due to vibrational energy. This leads to enzyme inactivation and impaired electrochemical response. Therefore, in this study, we used a hyperthermophile-derived enzyme. Temperature and electrochemical characteristics of the detection system using SAWs and a multi-copper oxidase (MCO) derived from the hyperthermophilic archaea Pyrobaculum aerophilum were studied. Laccase, which is an MCO derived from the thermophilic fungus Trametes versicolor, was also studied. We also characterized the enzyme-electrochemical reaction using SAWs by comparing the magnitude of the reduction current obtained using the two enzymes with different heat resistances. We observed an increase in the electrochemical response with the SAWs, without impaired enzyme activity. Thus, the use of a thermostable enzyme is suitable for the development of a biosensor that uses SAWs for agitation.     

Interorgan amino acid interchange in propionic acidemia: the missing key to understanding its physiopathology

Amino Acids - Propionic acidemia is an inborn error of metabolism caused by a deficiency in the mitochondrial enzyme propionyl-CoA carboxylase that converts the propionyl CoA to methyl malonyl CoA....     

Transgenerational effects of three global change drivers on an endemic Mediterranean plant

Plant populations are subjected to changes in their natural environment as a result of the incidence of simultaneous global change drivers. Despite the fact that these changes can largely affect early fitness components, information on the effects of simultaneous drivers of global change on offspring traits and performance is particularly scant. We analyzed the combined effect of three global change drivers of critical importance in Mediterranean ecosystems (habitat fragmentation, reductions in habitat quality and water availability) on germination and seedling performance of the gypsophile shrub Centaurea hyssopifolia. Seedlings from 39 mother plants from eight different environments (resulting from the combination of the three global change drivers) were sown and grown in a common garden. First, germination percentage, seedling size and seedling survival were monitored. Secondly, seedling performance and ecophysiological traits were measured under well and low‐watered conditions. Fragmentation showed the largest negative effect on germination and offspring performance. Seedlings of mothers from small fragments germinated more slowly, showed lower survival, died faster, and showed lower photosynthetic rates under well‐watered conditions compared to seedlings of mother plants from large populations. Seedlings of different maternal origins did not differ in their response to water stress or in their ability to survive to drought. Ninety‐five percent of the seedlings survived until soil water content was as low as 3%. Our study shows that global change can have not only immediate impacts on plant populations but also transgenerational effects, and highlights the importance of studies involving multiple drivers and a more integral understanding of global change.     

Tec1, a member of the TEA transcription factors family,is involved in virulence and basidiocarp development in Ustilago maydis

International Microbiology - The life cycle of Ustilago maydis involves alternation of a haploid saprophytic yeast-like stage and a dikaryotic hyphal virulent form. Under in vitro conditions,...     

Evolutionary aspects of urea cycle enzyme genes

The functions and expression pattern of urea cycle enzymes have undergone considerable changes during the course of evolution. Sequence analyses shows that urea cycle enzymes from mammals are homologous to microbial enzymes of the arginine-metabolic pathway. Recently, an unexpected relationship was found between argininosuccinate lyase (EC 4.3.2.1), the fourth enzyme of the cycle, and delta-crystallin, a lens structural protein of birds and reptiles.     

Sesamoids and Morphological Variation: a Hypothesis on the Origin of Rod-like Skeletal Elements in Aerial Mammals

Enigmatic rod-like skeletal structures that support compliant membranes (patagia) in aerial mammals have been often considered as neomorphic elements or as evolutionary novelties, and their origin has remained poorly understood. A potential source of skeletal plasticity and, probably, of morphofunctional innovations are sesamoids, which were recently demonstrated to have a common cellular origin with bone eminences. In this review, I compile information regarding anatomy, evolution, and development of rod-like skeletal elements in extant gliding and flying mammals and propose a working hypothesis on the origin of these structures. Rod-like skeletal elements, namely, the calcar in bats (Chiroptera), the unciform element in Anomaluridae (Rodentia), and the styliform cartilage in Pteromyini (Rodentia: Sciuridae), would derive from sesamoids, which, in turn, would have the same origin as eminences of long bones (or bones with a long-bone-like growth), i.e., calcaneus, ulna, and pisiform, respectively. Rod-like skeletal elements exhibit several features of sesamoids. However, further developmental data are needed to confirm this hypothesis, particularly whether these structures share a cellular origin and molecular developmental pathways with sesamoids and bone eminences. If this hypothesis were supported, a new role for sesamoids in generating morphofunctional innovations in mammals and, potentially, in other aerial amniotes, would be recognized. Rod-like skeletal elements, which are key in the evolution of aerial locomotion, might constitute an example of pre-existing traits that acquire novel functions through relatively little developmental plasticity.