A computational and experimental approach toward a priori identification of alternatively spliced exons

Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data.     

Genome sequencing reveals fine scale diversification and reticulation history during speciation in Sus

Background

Elucidating the process of speciation requires an in-depth understanding of the evolutionary history of the species in question. Studies that rely upon a limited number of genetic loci do not always reveal actual evolutionary history, and often confuse inferences related to phylogeny and speciation. Whole-genome data, however, can overcome this issue by providing a nearly unbiased window into the patterns and processes of speciation. In order to reveal the complexity of the speciation process, we sequenced and analyzed the genomes of 10 wild pigs, representing morphologically or geographically well-defined species and subspecies of the genus Sus from insular and mainland Southeast Asia, and one African common warthog.

Results

Our data highlight the importance of past cyclical climatic fluctuations in facilitating the dispersal and isolation of populations, thus leading to the diversification of suids in one of the most species-rich regions of the world. Moreover, admixture analyses revealed extensive, intra- and inter-specific gene-flow that explains previous conflicting results obtained from a limited number of loci. We show that these multiple episodes of gene-flow resulted from both natural and human-mediated dispersal.

Conclusions

Our results demonstrate the importance of past climatic fluctuations and human mediated translocations in driving and complicating the process of speciation in island Southeast Asia. This case study demonstrates that genomics is a powerful tool to decipher the evolutionary history of a genus, and reveals the complexity of the process of speciation.     

Local Knowledge and Conservation of Seagrasses in the Tamil Nadu State of India

Local knowledge systems are not considered in the conservation of fragile seagrass marine ecosystems. In fact, little is known about the utility of seagrasses in local coastal communities. This is intriguing given that some local communities rely on seagrasses to sustain their livelihoods and have relocated their villages to areas with a rich diversity and abundance of seagrasses. The purpose of this study is to assist in conservation efforts regarding seagrasses through identifying Traditional Ecological Knowledge (TEK) from local knowledge systems of seagrasses from 40 coastal communities along the eastern coast of India. We explore the assemblage of scientific and local traditional knowledge concerning the 1. classification of seagrasses (comparing scientific and traditional classification systems), 2. utility of seagrasses, 3. Traditional Ecological Knowledge (TEK) of seagrasses, and 4. current conservation efforts for seagrass ecosystems. Our results indicate that local knowledge systems consist of a complex classification of seagrass diversity that considers the role of seagrasses in the marine ecosystem. This fine-scaled ethno-classification gives rise to five times the number of taxa (10 species = 50 local ethnotaxa), each with a unique role in the ecosystem and utility within coastal communities, including the use of seagrasses for medicine (e.g., treatment of heart conditions, seasickness, etc.), food (nutritious seeds), fertilizer (nutrient rich biomass) and livestock feed (goats and sheep). Local communities are concerned about the loss of seagrass diversity and have considerable local knowledge that is valuable for conservation and restoration plans. This study serves as a case study example of the depth and breadth of local knowledge systems for a particular ecosystem that is in peril.     

A predictive model for respiratory syncytial virus (RSV) hospitalisation of premature infants born at 33–35 weeks of gestational age,based on data from the Spanish FLIP study

Background

The aim of this study, conducted in Europe, was to develop a validated risk factor based model to predict RSV-related hospitalisation in premature infants born 33–35 weeks'' gestational age (GA).

Methods

The predictive model was developed using risk factors captured in the Spanish FLIP dataset, a case-control study of 183 premature infants born between 33–35 weeks'' GA who were hospitalised with RSV, and 371 age-matched controls. The model was validated internally by 100-fold bootstrapping. Discriminant function analysis was used to analyse combinations of risk factors to predict RSV hospitalisation. Successive models were chosen that had the highest probability for discriminating between hospitalised and non-hospitalised infants. Receiver operating characteristic (ROC) curves were plotted.

Results

An initial 15 variable model was produced with a discriminant function of 72% and an area under the ROC curve of 0.795. A step-wise reduction exercise, alongside recalculations of some variables, produced a final model consisting of 7 variables: birth ± 10 weeks of start of season, birth weight, breast feeding for ≤ 2 months, siblings ≥ 2 years, family members with atopy, family members with wheeze, and gender. The discrimination of this model was 71% and the area under the ROC curve was 0.791. At the 0.75 sensitivity intercept, the false positive fraction was 0.33. The 100-fold bootstrapping resulted in a mean discriminant function of 72% (standard deviation: 2.18) and a median area under the ROC curve of 0.785 (range: 0.768–0.790), indicating a good internal validation. The calculated NNT for intervention to treat all at risk patients with a 75% level of protection was 11.7 (95% confidence interval: 9.5–13.6).

Conclusion

A robust model based on seven risk factors was developed, which is able to predict which premature infants born between 33–35 weeks'' GA are at highest risk of hospitalisation from RSV. The model could be used to optimise prophylaxis with palivizumab across Europe.     

Diacylglycerols interact with the L2 lipidation site in TRPC3 to induce a sensitized channel state

Coordination of lipids within transient receptor potential canonical channels (TRPCs) is essential for their Ca²⁺ signaling function. Single particle cryo‐EM studies identified two lipid interaction sites, designated L1 and L2, which are proposed to accommodate diacylglycerols (DAGs). To explore the role of L1 and L2 in TRPC3 function, we combined structure‐guided mutagenesis and electrophysiological recording with molecular dynamics (MD) simulations. MD simulations indicate rapid DAG accumulation within both L1 and L2 upon its availability within the plasma membrane. Electrophysiological experiments using a photoswitchable DAG‐probe reveal potentiation of TRPC3 currents during repetitive activation by DAG. Importantly, initial DAG exposure generates a subsequently sensitized channel state that is associated with significantly faster activation kinetics. TRPC3 sensitization is specifically promoted by mutations within L2, with G652A exhibiting sensitization at very low levels of active DAG. We demonstrate the ability of TRPC3 to adopt a closed state conformation that features partial lipidation of L2 sites by DAG and enables fast activation of the channel by the phospholipase C‐DAG pathway.     

Field measurement of nitrogen mineralization using soil core incubation and acetylene inhibition of nitrification

A technique for measuring net rates of mineralization under field conditions is described. Soil cores were incubated in the field in sealed containers with acetylene to inhibit nitrification and thereby minimize losses of N through denitrification. Mineralization was estimated as the difference between the mineral N content after a 14-d incubation and that determined from soil samples taken at the start of incubation. Mineralization in the spring and summer in unfertilized plots in the field amounted to 90 and 70 kg N ha⁻¹ in S.E. England under grass and grass/clover swards, respectively, and 40 kg N ha⁻¹ under a grass sward in S.W. England. Daily rates of mineralization ranged from 0.02 to 1.90 kg N ha⁻¹, with peak values related to re-wetting of the soil after dry weather. Laboratory incubation of soil showed that neither the low concentration of acetylene (2% v/v) adopted for field incubation, nor the accumulation of mineral N during incubation was likely to affect the total measurement, but that frequent and regular soil sampling was necessary to minimize the effects of changes in soil water content. Estimates for mineralization over the whole growing season (180 d) were obtained for two years from extrapolation of the early season field measurements and were, on average, 50% higher than predictions based on a chemical extraction index of potentially mineralizable N.     

Fractionation and characterization of histones from barley (Hordeum vulgare) leaves

Histones were extracted from purified nuclei isolated from four cereals,viz. barley (Hordeum vulgare), wheat(Triticum aestivum), Aegilops squarrosa and corn (Zea mais), and from tobacco (Nicotiana tabacum). Analysis of the histones on SDS gels showed complex electrophoretic patterns with one species of both H3 and H4, one to three species of H1 and two to five species of H2. Judged from the electrophoretic patterns, the histones from barley, wheat and Aegilops are identical but different from those of corn with respect to H2. Like tobacco, corn showed two H2 components, whereas barley, wheat and Aegilops showed five H2 components. SDS gel electrophoresis of histones extracted from buds and roots of germinating seeds at different steps of germination and from different parts of ten-day-old leaves revealed that the existence in barley of multiple histone 2 variants is not restricted to any particular stage of differentiation of barley. Histones from barley leaves were resolved into four fractions by Biogel P-100 gel filtration and histones 2 were further fractionated by their differential solubility in HCl-ethanol. Each of these five fractions (H1, H3, H4, H2A and H2B, respectively) were characterized by electrophoresis on SDS or Triton-acid-urea gels and by their amino acid compositions as compared with the homologous histones of calf thymus and chicken erythrocytes. This revealed the following:

1. H3 and H4 are strictly analogous to their animal counterparts. However, H4 has an unexplained lower electrophoretic mobility in Triton-containing acid-urea gels.
2. H1 contains three components with lower electrophoretic mobilities than H1 from erythrocytes, contains more alanine than lysine and has a lower ratio of basic to acidic residues.
3. Both H2A and H2B contain at least four variants each, with higher molecular weights than in animals and higher lysine to arginine ratios. H2A variants comigrate in acid-urea-Triton gels with chicken erythrocytes H2A, whereas H2B migrate much slower.

It was concluded that the presence of multiple major variants of H2A and H2B is a frequent but not universal feature in cereals. The existence of these variants is not restricted to the embryonic stage as previously suggested for wheat (31).