Linear amplification for deep sequencing

Linear amplification for deep sequencing (LADS) is an amplification method that produces representative libraries for Illumina next-generation sequencing within 2 d. The method relies on attaching two different sequencing adapters to blunt-end repaired and A-tailed DNA fragments, wherein one of the adapters is extended with the sequence for the T7 RNA polymerase promoter. Ligated and size-selected DNA fragments are transcribed in vitro with high RNA yields. Subsequent cDNA synthesis is initiated from a primer complementary to the first adapter, ensuring that the library will only contain full-length fragments with two distinct adapters. Contrary to the severely biased representation of AT- or GC-rich fragments in standard PCR-amplified libraries, the sequence coverage in T7-amplified libraries is indistinguishable from that of nonamplified libraries. Moreover, in contrast to amplification-free methods, LADS can generate sequencing libraries from a few nanograms of DNA, which is essential for all applications in which the starting material is limited.     

Increased Iron Sequestration in Alveolar Macrophages in Chronic Obtructive Pulmonary Disease

Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer’s and Parkinson’s diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2–3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1–3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV₁) and the ratio between FEV₁ and forced vital capacity (FEV₁/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Futhermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.     

Micromere lineages in the glossiphoniid leech Helobdella

In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c"' and dm' clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b" and b"' (2b and 3b in traditional nomenclature) die in normal development; the b" clone can be rescued to assume the normal c" fate if micromere c" or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b" does not normally die, and contributes a subset of the cells arising exclusively from c" in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c"' makes no definitive contribution, whereas micromere dm' gives rise to cells equivalent to those arising from c"' and dm' in H. robusta (Sacramento).     

Computational identification of RNA functional determinants by three-dimensional quantitative structure–activity relationships

Anti-infection drugs target vital functions of infectious agents, including their ribosome and other essential non-coding RNAs. One of the reasons infectious agents become resistant to drugs is due to mutations that eliminate drug-binding affinity while maintaining vital elements. Identifying these elements is based on the determination of viable and lethal mutants and associated structures. However, determining the structure of enough mutants at high resolution is not always possible. Here, we introduce a new computational method, MC-3DQSAR, to determine the vital elements of target RNA structure from mutagenesis and available high-resolution data. We applied the method to further characterize the structural determinants of the bacterial 23S ribosomal RNA sarcin–ricin loop (SRL), as well as those of the lead-activated and hammerhead ribozymes. The method was accurate in confirming experimentally determined essential structural elements and predicting the viability of new SRL variants, which were either observed in bacteria or validated in bacterial growth assays. Our results indicate that MC-3DQSAR could be used systematically to evaluate the drug-target potentials of any RNA sites using current high-resolution structural data.     

Analysis of sibling cannibalism among pike,Esox lucius,juveniles reared under semi-natural conditions

Synopsis Sibling cannibalism in pike, Esox lucius, larvae and juveniles living in outdoor rearing ponds was studied using stomach contents analysis. For the two initial densities tested (6 and 18 larvae m^–2, equivalent to 12 and 36 larvae m^–3), cannibalism was non-existent during the larval period (13 to 35 mm total length) and was observed only during the juvenile stages. Initial density of larvae influenced both the date of first detection of cannibalistic individuals and the rate of development of cannibalism in the population. At initial stocking densities of 18 larvae m^–2 (36 larvae m^–3), cannibalism was observed from 21 days after the start of exogenous feeding (mean total length: 60 mm) onwards. At a mean total length of 100 mm and for initial stocking densities of 6 and 18 larvae m^–2, (12 and 36 larvae m^–3), the average proportions of cannibals in the populations of juveniles were 7.8% and 41.3% and the cannibals accounted for 15.5% and 65.9% of the total pike biomass, respectively. In stomachs of cannibals, young pike were the dominant prey in terms of weight. Dry weights of invertebrate-prey were lower in cannibals than in non-cannibals of similar size. Cannibalism among pike juveniles was characterized by the prey being swallowed whole and head first in the vast majority of cases. There was a strong positive correlation between predator and prey size and the mouth size of a cannibal was found to be an important constraint determining maximum victim size. The overall mean ratio of pike prey length to pike cannibal length was 66.2% and the average ratio of prey head depth to predator mouth width amounted to 87.6%. Prey size selection could be demonstrated for several length-groups of cannibals. These results are compared with the characteristics of early cannibalism in other fish species.     

Structure of a legume lectin from the bark of Robinia pseudoacacia and its complex with N-acetylgalactosamine

The structure of the bark lectin RPbAI (isoform A4) from Robinia pseudoacacia has been determined by protein crystallography both in the free form and complexed with N-acetylgalactosamine. The free form is refined at 1.80 A resolution to an R-factor of 18.9% whereas the complexed structure has an R-factor of 19.7% at 2.05 A resolution. Both structures are compared to each other and to other available legume lectin structures. The polypeptide chains of the two structures exhibit the characteristic legume lectin tertiary fold. The quaternary structure resembles that of the Phaseolus vulgaris lectin, the soybean agglutinin, and the Dolichos biflorus lectin, but displays some unique features leading to the extreme stability of this lectin.