The use of dna sequencing (ITS and trnL-F), AFLP, and fluorescent in situ hybridization to study allopolyploid Miscanthus (Poaceae)

Two clones of Miscanthus, grown under the names M. ×giganteus and M. sacchariflorus, have been used in biomass trials in Europe, but neither the identity of these clones nor their origin has been established. DNA sequencing, amplified fragment length polymorphism (AFLP), and chromosome studies confirm that M. ×giganteus is an allotriploid (2n = 3x = 57) combining genomes from M. sinensis (2n = 2x = 38) and M. sacchariflorus (2n = 38 or 76). Two alleles of the internal transcribed spacer of 18S-25S nuclear ribosomal DNA (ITS) were discovered in polymerase chain reaction products of M. ×giganteus. Cloning of these revealed that one matched M. sinensis and the other M. sacchariflorus. Plastid trnL intron and trnL-F spacer sequences showed that the maternal lineage of M. ×giganteus was M. sacchariflorus. Fluorescent in situ hybridization, FISH, was used to investigate genome organization in Miscanthus but was unable to differentiate between the different parental genomes present in M. ×giganteus, indicating that two parental genomes are still extremely similar at the repetitive DNA level. This study is an example in which rDNA sequences and AFLP fingerprints permit identification of the parental genomes in a hybrid, but FISH methods, at the repetitive DNA level (including genomic in situ hybridization, GISH), were unable to do so because their sequences remain too similar.     

Selective Amplification of Classical and Atypical Prions Using Modified Protein Misfolding Cyclic Amplification

With the development of protein misfolding cyclic amplification (PMCA), the topic of faithful propagation of prion strain-specific structures has been constantly debated. Here we show that by subjecting brain material of a synthetic strain consisting of a mixture of self-replicating states to PMCAb, selective amplification of PrP^Sc could be achieved, and that PMCAb mimicked the evolutionary trend observed during serial transmission in animals. On the other hand, using modified PMCAb conditions that employ partially deglycosylated PrP^C (dgPMCAb), an alternative transmissible state referred to as atypical protease-resistant form of the prion protein (atypical PrPres) was selectively amplified from a mixture. Surprisingly, when hamster-adapted strains (263K and Hyper) were subjected to dgPMCAb, their proteinase K digestion profile underwent a dramatic transformation, suggesting that a mixture of atypical PrPres and PrP^Sc might be present in brain-derived materials. However, detailed analysis revealed that the proteinase K-resistant profile of PrP^Sc changed in response to dgPMCAb. Despite these changes, the 263K strain-specific disease phenotype was preserved after passage through dgPMCAb. This study revealed that the change in PrP^Sc biochemical phenotype does not always represent an irreversible transformation of a strain, but rather demonstrated the existence of a wide range of variation for strain-specific physical features in response to a change in prion replication environment. The current work introduced a new PMCA technique for amplification of atypical PrPres and raised a number of questions about the need for a clever distinction between actual strain mutation and variation of strain-specific features in response to a change in the replication environment.     

Predictive mapping of human risk for West Nile virus (WNV) based on environmental and socioeconomic factors

A West Nile virus (WNV) human risk map was developed for Suffolk County, New York utilizing a case-control approach to explore the association between the risk of vector-borne WNV and habitat, landscape, virus activity, and socioeconomic variables derived from publically available datasets. Results of logistic regression modeling for the time period between 2000 and 2004 revealed that higher proportion of population with college education, increased habitat fragmentation, and proximity to WNV positive mosquito pools were strongly associated with WNV human risk. Similar to previous investigations from north-central US, this study identified middle class suburban neighborhoods as the areas with the highest WNV human risk. These results contrast with similar studies from the southern and western US, where the highest WNV risk was associated with low income areas. This discrepancy may be due to regional differences in vector ecology, urban environment, or human behavior. Geographic Information Systems (GIS) analytical tools were used to integrate the risk factors in the 2000-2004 logistic regression model generating WNV human risk map. In 2005-2010, 41 out of 46 (89%) of WNV human cases occurred either inside of (30 cases) or in close proximity (11 cases) to the WNV high risk areas predicted by the 2000-2004 model. The novel approach employed by this study may be implemented by other municipal, local, or state public health agencies to improve geographic risk estimates for vector-borne diseases based on a small number of acute human cases.     

The cellular form of the prion protein is involved in controlling cell cycle dynamics,self‐renewal,and the fate of human embryonic stem cell differentiation

Prion protein (PrP^C), is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrP^C in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline‐regulated lentiviral vectors that up‐regulate or suppresses PrP^C expression. Here, we show that expression of PrP^C in pluripotent hESCs cultured under self‐renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrP^C in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, over‐expression of PrP^C in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrP^C is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self‐renewal and differentiation, and determining the fate of hESCs differentiation. This study suggests that PrP^C is at the crossroads of several signaling pathways that regulate the switch between preservation of or departure from the self‐renewal state, control cell proliferation activity, and define stem cell fate.     

CHILD: a new tool for detecting low-abundance insertions and deletions in standard sequence traces

Several methods have been proposed for detecting insertion/deletions (indels) from chromatograms generated by Sanger sequencing. However, most such methods are unsuitable when the mutated and normal variants occur at unequal ratios, such as is expected to be the case in cancer, with organellar DNA or with alternatively spliced RNAs. In addition, the current methods do not provide robust estimates of the statistical confidence of their results, and the sensitivity of this approach has not been rigorously evaluated. Here, we present CHILD, a tool specifically designed for indel detection in mixtures where one variant is rare. CHILD makes use of standard sequence alignment statistics to evaluate the significance of the results. The sensitivity of CHILD was tested by sequencing controlled mixtures of deleted and undeleted plasmids at various ratios. Our results indicate that CHILD can identify deleted molecules present as just 5% of the mixture. Notably, the results were plasmid/primer-specific; for some primers and/or plasmids, the deleted molecule was only detected when it comprised 10% or more of the mixture. The false positive rate was estimated to be lower than 0.4%. CHILD was implemented as a user-oriented web site, providing a sensitive and experimentally validated method for the detection of rare indel-carrying molecules in common Sanger sequence reads.     

Self-assembly of square-lattice copper sheets displaying intra-ferromagnetism

A homoleptic copper(II) complex supported by methyliminodiacetate [ = MIDA] has been prepared and characterized by elemental, infra-red, X-ray diffraction and magnetic methods. The complex assembles as a two-dimensional lattice in which the copper centers are arranged in a square grid, bridged by the carboxylate moieties of the ligand. [Cu^II₄MIDA₄]_∞ crystallizes in the tetragonal system P-4 2₁ c (a = 9.8943(5), b = 9.8943(5), c = 14.4687(7) Å, Z = 8, R = 0.0495). Each Cu^II atom displays severely distorted square pyramidal geometry (τ = 0.44) and is bridged to four additional copper centers by a syn-anti arrangement of the carboxylate groups. Within the sheet, the copper centers are separated by approximately 4.96 Å. The sheets are layered by arranging the copper squares directly on top of one another, resulting in parallel channels that extend throughout the material. Variable temperature magnetic susceptibility measurements reveal the presence of ferromagnetic exchange between the spin carriers within each two-dimensional sheet and antiferromagnetic exchange across the layers.     

Amyloid features and neuronal toxicity of mature prion fibrils are highly sensitive to high pressure

Prion proteins (PrP) can aggregate into toxic and possibly infectious amyloid fibrils. This particular macrostructure confers on them an extreme and still unexplained stability. To provide mechanistic insights into this self-assembly process, we used high pressure as a thermodynamic tool for perturbing the structure of mature amyloid fibrils that were prepared from recombinant full-length mouse PrP. Application of high pressure led to irreversible loss of several specific amyloid features, such as thioflavin T and 8-anilino-1-naphthalene sulfonate binding, alteration of the characteristic proteinase K digestion pattern, and a significant decrease in the β-sheet structure and cytotoxicity of amyloid fibrils. Partial disaggregation of the mature fibrils into monomeric soluble PrP was observed. The remaining amyloid fibrils underwent a change in secondary structure that led to morphologically different fibrils composed of a reduced number of proto-filaments. The kinetics of these reactions was studied by recording the pressure-induced dissociation of thioflavin T from the amyloid fibrils. Analysis of the pressure and temperature dependence of the relaxation rates revealed partly unstructured and hydrated kinetic transition states and highlighted the importance of collapsing and hydrating inter- and intramolecular cavities to overcome the high free energy barrier that stabilizes amyloid fibrils.