Comparison of Rapid DNA Extraction Methods Applied to PCR Identification of Medicinal Mushroom Ganoderma spp.

Abstract

Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm‐broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high salt concentrations and low pH methods were the best for DNA extraction. The mycelia and sporoderm‐broken spore powder yielded higher and purer DNA. The method developed could effectively eliminate the influence of the secondary metabolites to DNA extraction. The DNA samples extracted from the developed method could be successfully used for PCR applications.     

Interaction of nucleoplasmin with core histones

Nucleoplasmin is one of the most abundant proteins in Xenopus laevis oocytes, and it has been involved in the chromatin remodeling that takes place immediately after fertilization. This molecule has been shown to be responsible for the removal of the sperm-specific proteins and deposition of somatic histones onto the male pronuclear chromatin. To better understand the latter process, we have used sedimentation velocity, sedimentation equilibrium, and sucrose gradient fractionation analysis to show that the pentameric form of nucleoplasmin binds to a histone octamer equivalent consisting of equal amounts of the four core histones, H2A, H2B, H3, and H4, without any noticeable preference for any of these proteins. Removal of the histone N-terminal "tail" domains or the major C-terminal polyglutamic tracts of nucleoplasmin did not alter these binding properties. These results indicate that interactions other than those electrostatic in nature (likely hydrophobic) also play a critical role in the formation of the complex between the negatively charged nucleoplasmin and positively charged histones. Although the association of histones with nucleoplasmin may involve some ionic interactions, the interaction process is not electrostatically driven.     

Quantum Dot Based Heterostructures for Unassisted Photoelectrochemical Hydrogen Generation

TiO₂ hollow nanowires (HNWs) and nanoparticles (NPs) constitute promising architectures for QDs sensitized photoanodes for H₂ generation. We sensitize these structures with CdS/CdSe quantum dots by two different methods (chemical bath deposition, CBD and succesive ionic layer adsorption and reaction, SILAR) and evaluate the performance of these photoelectrodes. Remarkable photocurrents of 4 mA·cm and 8 mA·cm^?2 and hydrogen generation rates of 40 ml·cm^?2·day^?1 and 80 ml·cm^?2·day^?1 have been obtained in a three electrode configuration with sacrificial hole scavengers (Na₂S and Na₂SO₃), for HNWs and NPs respectively, which is confirmed through gas analysis. More importantly, autonomous generation of H₂ (20 ml·cm^?2·day^?1 corresponding to 2 mA·cm^?2 photocurrent) is obtained in a two electrode configuration at short circuit under 100 mW·cm^?2 illumination, clearly showing that these photoanodes can produce hydrogen without the assistance of any external bias. To the best of the authors' knowledge, this is the highest unbiased solar H₂ generation rate reported for these of QDs based heterostructures. Impedance spectroscopy measurements show similar electron density of trap states below the TiO₂ conduction band while the recombination resistance was higher for HNWs, consistently with the much lower surface area compared to NPs. However, the conductivity of both structures is similar, in spite of the one dimensional character of HNWs, which leaves some room for improvement of these nanowired structures. The effect of the QDs deposition method is also evaluated. Both structures show remarkable stability without any appreciable photocurrent loss after 0.5 hour of operation. The findings of this study constitute a relevant step towards the feasibility of hydrogen generation with wide bandgap semiconductors/quantum dots based heterostructures.     

Nitrogen and carbon contents and δ15N and δ13C signatures in six bryophyte species: assessment of long‐term deposition changes (1980–2010) in Spanish beech forests

In this study we used recent (2010) and herbarium material (1980) of six bryophyte species to assess long‐term atmospheric deposition in natural forested areas in northern Spain. For this purpose, tissue nitrogen and carbon content, as well as δ¹³C and δ¹⁵N signatures of samples of Hypnum cupressiforme, Polytrichastrum formosum, Leucobryum juniperoideum, Rhytidiadelphus loreus, Homalothecium lutescens and Diplophyllum albicans were analysed and comparisons made between years and species. In addition, the usefulness of each of the six species was evaluated. The range of values observed was similar to that in other studies carried out in rural areas. Significantly lower values were found in 2010 for N (H. cupressiforme), δ¹⁵N (R. loreus and D. albicans), C (R. loreus) and δ¹³C (all except L. juniperoideum). Our natural areas are thus now less influenced by atmospheric pollutants than they were, most probably due to changes in some traditional local activities. Differences were observed between species for all the four parameters studied, so different species must not be analysed together. Finally, R. loreus and H. lutescens seem to be good bioindicators, sensitive even with a few samples, although further studies are needed to corroborate their usefulness.     

Extending ecological niche models to the past 120 000 years corroborates the lack of strong phylogeographic structure in the Crested Drongo (Dicrurus forficatus forficatus) on Madagascar

We conduct a phylogeographic study of the Crested Drongo (Dicrurus forficatus forficatus), a broadly distributed bird species on Madagascar. We first determined the demographic and spatial pattern inferred from mitochondrial and nuclear data, and then compared these results with predictions from a present to 0.120‐Myr‐old reconstruction of the spatial dynamics of the range of D. f. forficatus on Madagascar, enabling putative areas of stability (lineage persistence) to be detected. Weak genetic structure along an east–west pattern and comparatively low genetic diversity were recovered, with strong evidence of population expansion found at all ten loci sampled. The palaeoclimatic distribution models over the past 0.120 Myr suggest the presence of extensive areas of suitable climate in the east and west for the species since its colonization of Madagascar, a result in strong concordance with the spatial and genetic signal derived from our multilocus data set. © 2013 The Linnean Society of London     

Do sex ratio and development differ in sexually size‐dimorphic shiny cowbirds (Molothrus bonariensis) parasitizing smaller and larger hosts?

Two possible patterns of bias in primary sex ratio have been proposed for size‐dimorphic brood parasites that do not evict host chicks: (1) larger males should be laid at greater frequency in hosts larger than the parasite because they compete better (increasing their survival) than females with large host nest‐mates, and (2) more costly males (i.e. the larger sex) should be laid at greater frequency in hosts smaller than the parasite because, in these hosts, parasite nestlings are provisioned at a higher rate and grow faster than in larger hosts. We tested these hypotheses in two hosts of the sexually size‐dimorphic shiny cowbird, Molothrus bonariensis, one smaller (house wren, Troglodytes aedon) and one larger (chalk‐browed mockingbird, Mimus saturninus) than the parasite. We measured: (1) sex ratio at laying; (2) development of sexual differences in body mass during the nestling stage; and (3) chick survival and sex ratio of chicks before fledging. In both hosts, we found sexual differences in body mass of nestlings from 7 days of age onwards, although we did not find a bias in the sex ratio of eggs laid and chicks fledged. The results of the present study do not support the hypothesis that shiny cowbird females benefit from biasing the primary sex ratio depending on the size of the hosts they parasitize. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 442–448.     

Preservation of RNA and DNA from mammal samples under field conditions

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months.     

Vehiculization determines the endocytic internalization mechanism of Zn(II)-phthalocyanine

It is generally accepted that compounds of nanomolecular size penetrate into cells by different endocytic processes. The vehiculization strategy of a compound is a factor that could determine its uptake mechanism. Understanding the influence of the vehicle in the precise mechanism of drug penetration into cells makes possible to improve or modify the therapeutic effects. In this study, using human A-549 cells, we have characterized the possible internalization mechanism of the photosensitizer Zn(II)-phthalocyanine (ZnPc), either dissolved in dimethylformamide (ZnPc–DMF) or included in liposomes of dipalmitoyl-phosphatidyl-choline. Specific inhibitors involved in the main endocytic pathways were used. Co-incubation of cells with ZnPc–liposomes and dynasore (dinamin-mediated endocytosis inhibitor) resulted in a significant decrease of photodamage, whereas other inhibitors did not alter the photodynamic effect of ZnPc. On the contrary, cells treated with ZnPc–DMF in the presence of dynasore, genistein (caveolin-mediated endocytosis inhibitor) or cytochalasin D (macropinocytosis and caveolin-mediated endocytosis inhibitor) showed a significant decrease in ZnPc uptake and photodynamic damage. These results suggest that ZnPc–DMF penetrates into cells mainly by caveolin-mediated endocytosis, whereas ZnPc–liposomes are internalized into cells preferentially by clathrin-mediated endocytosis. We conclude that using different drug vehiculization systems, it is possible to modify the internalization mechanism of a therapeutic compound, which could be of great interest in clinical research.