Regulation of enzymes involved in C4 photosynthesis and the antioxidant metabolism by UV-B radiation in Egeria densa,a submersed aquatic species

Egeria densa, a submersed aquatic species, was exposed to different treatments under UV-B radiation, and the response of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) was determined. Exposure to UV-B radiation for 4 h per day over 7–16 days caused an increase in both enzymes, together with an increase in the activity of some isoforms of several enzymes involved in the antioxidant metabolism, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD). The content of chlorophylls and carotenoids was considerably decreased, suggesting that degradation or repression of the synthesis of these molecules may be occurring after UV-B exposure. Reactive oxygen species (ROS) were also required for UV-B induction of PEPC and NADP-ME, as the addition of ascorbic acid before UV-B treatment prevented the induction of these enzymes, while salicylic acid was not effective in inducing NADP-ME but increased the expression of the lower molecular mass isoform of PEPC. On the other hand, damage to the photosynthetic machinery may be occurring after exposure to UV-B radiation for 8 per day over 1–2 days, as indicated by a decrease in the levels of Rubisco, PEPC and NADP-ME. Some of the enzymes involved in the antioxidant metabolism, such as CAT and APX, were also sensitive to continuous exposure, evidenced by a decrease in their activity. In this way, in E. densa, several enzymes involved in different metabolic pathways showed a distinct response, depending on the UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quantitative evaluation of siRNA delivery in vivo

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.     

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.     

Effect of Organic Residues and Liming Materials on Metal Extraction from a Mining-Contaminated Soil

Incubation tests were used to assess the effectiveness of three different organic residues and three different liming materials, alone or in combination, in the remediation of a mine contaminated soil. The organic residues tested were sewage sludge from a municipal wastewater treatment plant (SS), compost from the organic fraction of unsorted municipal solid waste (MSWC), and garden waste compost (GWC), applied at 100 and 200 Mg ha^{? 1}. The liming materials tested were agriculture limestone (6.4 Mg ha^{? 1}), calcium oxide (3.7 Mg ha^{? 1}), and sugar beet sludge (12.2 Mg ha^{? 1}) from the sugar manufacturing process. The soil and mixtures of soil and amendments were adjusted to 70% of the maximum water holding capacity and incubated for 28 days in a controlled-temperature room at 20°C ± 1°C. At the end of the incubation, samples were analyzed for pH, electrical conductivity, organic matter content, CaCl₂-extractable, and' NH4Ac/HAc+ EDTA–extractable metal fractions (Cu, Zn, and Pb). Correlations among the variables and/or similarities among the treatments were identified by principal component analysis and hierarchical cluster analysis. The amendments tested decreased the CaCl₂-extractable Cu and Zn fractions, considered as mobile metal fractions, to below analytical detectable limits, providing organic matter to the soil with levels between 1% and 2% at the end of the experiment, significantly different relatively to the original soil. pH and electrical conductivity reached high values when using 200 Mg ha^{? 1} SS or 200 Mg ha^{? 1} MSWC, with any of the liming materials tested, making these application rates excessive for this particular situation. Furthermore, the treatments using MSWC increased the NH4Ac/HAc+ EDTA–extractable Cu, Pb, and Zn fractions, considered as mobilizable metal fractions, as did the 200 Mg ha^{? 1} SS for Pb and Zn. Considering the overall results, the compost made from garden waste decreased metal solubility in the soil and increased soil pH and organic matter content, without the addition of large amounts of soluble salts, and without increasing the mobilizable metal content. Of the organic materials tested, this was the only one that can be considered adequate for remediation of the contaminated soil under study, at the application rates tested.     

Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.     

Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples

Our goal was to establish a quantitative real-time PCR (QRT-PCR) method to detect Bacteroides fragilis group and related organisms from clinical specimens. Compared to conventional anaerobic culture, QRT-PCR can provide accurate and more rapid detection and identification of B.?fragilis group and similar species. B.?fragilis group and related organisms are the most frequently isolated anaerobic pathogens from clinical samples. However, culture and phenotypic identification is quite time-consuming. We designed specific primers and probes based on the 16S rRNA gene sequences of Bacteroides caccae, Bacteroides eggerthii, B.?fragilis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Odoribacter splanchnicus (Bacteroides splanchnicus), Parabacteroides distasonis (Bacteroides distasonis) and Parabacteroides merdae (Bacteroides merdae), and detected these species by means of QRT-PCR in 400 human surgical wound infection samples or closed abscesses. The target bacteria were detected from 31 samples (8%) by culture, but from 132 samples (33%) by QRT-PCR (p-value?相似文献   

Replication study of 34 common SNPs associated with prostate cancer in the Romanian population

Prostate cancer is the third‐most common form of cancer in men in Romania. The Romanian unscreened population represents a good sample to study common genetic risk variants. However, a comprehensive analysis has not been conducted yet. Here, we report our replication efforts in a Romanian population of 979 cases and 1027 controls, for potential association of 34 literature‐reported single nucleotide polymorphisms (SNPs) with prostate cancer. We also examined whether any SNP was differentially associated with tumour grade or stage at diagnosis, with disease aggressiveness, and with the levels of PSA (prostate specific antigen). In the allelic analysis, we replicated the previously reported risk for 19 loci on 4q24, 6q25.3, 7p15.2, 8q24.21, 10q11.23, 10q26.13, 11p15.5, 11q13.2, 11q13.3. Statistically significant associations were replicated for other six SNPs only with a particular disease phenotype: low‐grade tumour and low PSA levels (rs1512268), high PSA levels (rs401681 and rs11649743), less aggressive cancers (rs1465618, rs721048, rs17021918). The strongest association of our tested SNP's with PSA in controls was for rs2735839, with 29% increase for each copy of the major allele G, consistent with previous results. Our results suggest that rs4962416, previously associated only with prostate cancer, is also associated with PSA levels, with 12% increase for each copy of the minor allele C. The study enabled the replication of the effect for the majority of previously reported genetic variants in a set of clinically relevant prostate cancers. This is the first replication study on these loci, known to associate with prostate cancer, in a Romanian population.     

Influence of drift and admixture on population structure of American black bears (Ursus americanus) in the Central Interior Highlands,USA, 50 years after translocation

Bottlenecks, founder events, and genetic drift often result in decreased genetic diversity and increased population differentiation. These events may follow abundance declines due to natural or anthropogenic perturbations, where translocations may be an effective conservation strategy to increase population size. American black bears (Ursus americanus) were nearly extirpated from the Central Interior Highlands, USA by 1920. In an effort to restore bears, 254 individuals were translocated from Minnesota, USA, and Manitoba, Canada, into the Ouachita and Ozark Mountains from 1958 to 1968. Using 15 microsatellites and mitochondrial haplotypes, we observed contemporary genetic diversity and differentiation between the source and supplemented populations. We inferred four genetic clusters: Source, Ouachitas, Ozarks, and a cluster in Missouri where no individuals were translocated. Coalescent models using approximate Bayesian computation identified an admixture model as having the highest posterior probability (0.942) over models where the translocation was unsuccessful or acted as a founder event. Nuclear genetic diversity was highest in the source (A_R = 9.11) and significantly lower in the translocated populations (A_R = 7.07–7.34; P = 0.004). The Missouri cluster had the lowest genetic diversity (A_R = 5.48) and served as a natural experiment showing the utility of translocations to increase genetic diversity following demographic bottlenecks. Differentiation was greater between the two admixed populations than either compared to the source, suggesting that genetic drift acted strongly over the eight generations since the translocation. The Ouachitas and Missouri were previously hypothesized to be remnant lineages. We observed a pretranslocation remnant signature in Missouri but not in the Ouachitas.