Detecting the coevolution of biosequences--an example of RNA interaction prediction

A probabilistic graphical model is proposed in order to detect the coevolution between different sites in biological sequences. The model extends the continuous-time Markov process of sequence substitution for single nucleic or amino acids and imposes general constraints regarding simultaneous changes on the substitution rate matrix. Given a multiple sequence alignment for each molecule of interest and a phylogenetic tree, the model can predict potential interactions within or between nucleic acids and proteins. Initial validation of the model is carried out using tRNA and 16S rRNA sequence data. The model accurately identifies the secondary interactions of tRNA as well as several known tertiary interactions. In addition, results on 16S rRNA data indicate this general and simple coevolutionary model outperforms several other parametric and nonparametric methods in predicting secondary interactions. Furthermore, the majority of the putative predictions exhibit either direct contact or proximity of the nucleotide pairs in the 3-dimensional structure of the Thermus thermophilus ribosomal small subunit. The results on RNA data suggest a general model of coevolution might be applied to other types of interactions between protein, DNA, and RNA molecules.     

Monosubstituted γ-lactam and conformationally constrained 1,3-diaminopropan-2-ol transition-state isostere inhibitors of β-secretase (BACE)

The synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented.     

Quantitative trait loci in a bacterially induced model of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are complex disorders caused by a combination of environmental, microbial, and genetic factors. Genome-wide association studies in humans have successfully identified multiple genes and loci associated with disease susceptibility, but the mechanisms by which these loci interact with each other and/or with environmental factors (i.e., intestinal microbiota) to cause disease are poorly understood. Helicobacter hepaticus-induced intestinal inflammation in mice is an ideal model system for elucidating the genetic basis of IBD susceptibility in a bacterially induced system, as there are significant differences in H. hepaticus-induced disease susceptibility among inbred mouse strains. Infected A/J mice develop acute overexpression of proinflammatory cytokines followed 2?C3?months later by chronic cecal inflammation, whereas infected C57BL/6 mice fail to develop cecal inflammation or increased cytokine expression. The goal of this project was to use quantitative trait locus (QTL) mapping to evaluate genetic factors that contribute to the differential disease susceptibility between these two mouse strains. Using acute cecal IL-12/23p40 expression as a biomarker for disease susceptibility, QTL analysis of H. hepaticus-infected F₂ mice revealed involvement of multiple loci. The loci with the strongest association were located on Chromosome 3 and Chromosome 17, with logarithm of odds (LOD) scores of 6.89 and 3.09, respectively. Cecal expression of IL-12/23p40 in H. hepaticus-infected C57BL/6J-Chr3^A/J/NaJ chromosome substitution mice had an intermediate phenotype, significantly higher than in resistant C57BL/6 but lower than in susceptible A/J mice, confirming the importance of this locus to the immune response to H. hepaticus infection.     

The environmental basis of North American species richness patterns among Epicauta (Coleoptera: Meloidae)

Understanding regional variability in species richness is necessary for conservation efforts to succeed in the face of large-scale environmental deterioration. Several analyses of North American vertebrates have shown that climatic energy provides the best explanation of contemporary species richness patterns. The paucity of analyses of insect diversity patterns, however, remains a serious obstacle to a general hypothesis of spatial variation in diversity. We collected species distribution data on a North American beetle genus, Epicauta (Coleoptera: Meloidae) and tested several major diversity hypotheses. These beetles are generally grasshopper egg predators as larvae, and angiosperm herbivores as adults. Epicauta richness is highest in the hot, dry American southwest, and decreases north and east, consistent with the species richness-energy hypothesis. Potential evapotranspiration, which is also the best predictor of richness patterns among North American vertebrates, explains 80.2% of the variability in Epicauta species richness. Net primary productivity and variables measuring climatic heat energy only (such as PET) are not generally comparable, though they are sometimes treated as if they were equivalent. We conclude that the species richness-energy hypothesis currently provides a better overall explanation for Epicauta species richness patterns in North America than other major diversity hypotheses. The observed relationship between climatic energy and regional species richness may provide significant insight into the response of ecological communities to climate change.     

Distant segments of Saccharomyces cerevisiae scR1 RNA promote assembly and function of the signal recognition particle

The conserved signal recognition particle targets ribosomes synthesizing presecretory proteins to the endoplasmic reticulum membrane. Key to the activity of SRP is its ability to bind the ribosome at distant locations, the signal sequence exit and elongation factor-binding sites. These contacts are made by the S and Alu domains of SRP, respectively. We tested earlier secondary structure predictions of the Saccharomyces cerevisiae SRP RNA, scR1, and provide and test a consensus structure. The structure contains four non-conserved insertions, helices 9-12, into the core SRP RNA fold, and an extended helix 7. Using a series of scR1 mutants lacking part or all of these structural elements, we find that they are important for the RNA in both function and assembly of the RNP. About 20% of the RNA, corresponding to the outer regions of these helices, is dispensable for function. Further, we examined the role of several features within the S-domain section of the core, helix 5, and find that its length and flexibility are important for proper SRP function and become essential in the absence of helix 10, 11 and/or 7 regions. Overall, the genetic data indicate that regions of scR1 distant in both primary sequence and secondary structure have interrelated roles in the function of the complex, and possibly mediate communication between Alu and S domains during targeting.     

Rapid evolution of spermathecal duct length in the Allonemobius socius complex of crickets: species, population and Wolbachia effects

The three species in the Allonemobius socius complex of crickets have recently diverged and radiated across North America. Interestingly, the only barriers to gene flow between these species in zones of secondary contact appear to be associated with fertilization traits - e.g., conspecific sperm precedence and the ability of males to induce females to lay eggs. Other traits, such as the length of female's reproductive tract, may also influence fertilization success and be associated with species boundaries. However, the underlying variation in this duct has not been assessed across populations and species. Moreover, the effects of reproductive parasites like Wolbachia on these morphological features have yet to be addressed, even though its infections are concentrated in reproductive tissues. I evaluated both the natural variation in and the effects of Wolbachia infection on spermathecal duct length among several populations of two species in the Allonemobius socius complex. My results suggest the following: (1) spermathecal duct length varies between species and is associated with species boundaries, (2) there is considerable variation among populations within species, (3) there is a Wolbachia infection-by-population interaction effect on the length of the spermathecal duct, and (4) experimental curing of Wolbachia recovers the uninfected morphology. These findings suggest the following hypotheses: (1) spermathecal duct length, like other fertilization traits in Allonemobius, is evolving rapidly and influences reproductive isolation and (2) Wolbachia-induced modifications of this duct could influence the dynamics of male-female coevolution. Further experiments are needed, however, to explicitly test these latter two hypotheses.     

Metabonomics in pharmaceutical R&D

This minireview is based on a lecture given at the First Maga Circe Conference on metabolomics held at Sabaudia, Italy, in March 2006 in which the analytical and statistical techniques used in metabonomics, efforts at standardization and some of the major applications to pharmaceutical research and development are reviewed. Metabonomics involves the determination of multiple metabolites simultaneously in biofluids, tissues and tissue extracts. Applications to preclinical drug safety studies are illustrated by the Consortium for Metabonomic Toxicology, a collaboration involving several major pharmaceutical companies. This consortium was able, through the measurement of a dataset of NMR spectra of rodent urine and serum samples, to build a predictive expert system for liver and kidney toxicity. A secondary benefit was the elucidation of the endogenous biochemicals responsible for the classification. The use of metabonomics in disease diagnosis and therapy monitoring is discussed with an exemplification from coronary artery disease, and the concept of pharmaco-metabonomics as a way of predicting an individual's response to treatment is exemplified. Finally, some advantages and perceived difficulties of the metabonomics approach are summarized.     

Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma

Background

Arginase overexpression contributes to airways hyperresponsiveness (AHR) in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR.

Methods

To investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA) and challenged with nebulized PBS (OVA/PBS) or OVA (OVA/OVA) for three consecutive days (sub-acute model) or 12 weeks (chronic model), which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O₃), or HEPA-filtered air (FA), for 4 hours. After the CAP+O₃ exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC) or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent^®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization.

Results

Compared to FA, arginase activity was significantly augmented in the lungs of CAP+O₃-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O₃-induced increase in AHR in both models.

Conclusions

This study demonstrates that arginase is upregulated following environmental exposures in murine models of asthma, and contributes to the pollution-induced exacerbation of airways responsiveness. Thus arginase may be a therapeutic target to protect susceptible populations against the adverse health effects of air pollution, such as fine particles and ozone, which are two of the major contributors to smog.     

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.     

The use of whole genome amplification in the study of human disease

The availability of large amounts of genomic DNA is of critical importance for many of the molecular biology assays used in the analysis of human disease. However, since the amount of patient tissue available is often limited and as particular foci of interest may consist of only a few hundred cells, the yield of DNA is often insufficient for extensive analysis. To address this problem, several whole genome amplification (WGA) methodologies have been developed. Initial WGA approaches were based on the polymerase chain reaction (PCR). However, recent reports have described the use of non-PCR-based linear amplification protocols for WGA. Using these methods, it is possible to generate microgram quantities of DNA starting with as little as 1mg of genomic DNA. This review will provide an overview of WGA approaches and summarize some of the uses for amplified DNA in various high-throughput genetic applications.