Identification of Conserved Splicing Motifs in Mutually Exclusive Exons of 15 Insect Species

Background: During alternative splicing, the inclusion of an exon in the final mRNA molecule is determined by nuclear proteins that bind cis-regulatory sequences in a target pre-mRNA molecule. A recent study suggested that the regulatory codes of individual RNA-binding proteins may be nearly immutable between very diverse species such as mammals and insects. The model system Drosophila melanogaster therefore presents an excellent opportunity for the study of alternative splicing due to the availability of quality EST annotations in FlyBase. Methods: In this paper, we describe an in silico analysis pipeline to extract putative exonic splicing regulatory sequences from a multiple alignment of 15 species of insects. Our method, ESTs-to-ESRs (E2E), uses graph analysis of EST splicing graphs to identify mutually exclusive (ME) exons and combines phylogenetic measures, a sliding window approach along the multiple alignment and the Welch’s t statistic to extract conserved ESR motifs. Results: The most frequent 100% conserved word of length 5 bp in different insect exons was “ATGGA”. We identified 799 statistically significant “spike” hexamers, 218 motifs with either a left or right FDR corrected spike magnitude p-value < 0.05 and 83 with both left and right uncorrected p < 0.01. 11 genes were identified with highly significant motifs in one ME exon but not in the other, suggesting regulation of ME exon splicing through these highly conserved hexamers. The majority of these genes have been shown to have regulated spatiotemporal expression. 10 elements were found to match three mammalian splicing regulator databases. A putative ESR motif, GATGCAG, was identified in the ME-13b but not in the ME-13a of Drosophila N-Cadherin, a gene that has been shown to have a distinct spatiotemporal expression pattern of spliced isoforms in a recent study. Conclusions: Analysis of phylogenetic relationships and variability of sequence conservation as implemented in the E2E spikes method may lead to improved identification of ESRs. We found that approximately half of the putative ESRs in common between insects and mammals have a high statistical support (p < 0.01). Several Drosophila genes with spatiotemporal expression patterns were identified to contain putative ESRs located in one exon of the ME exon pairs but not in the other.

The epidemiology of prostate cancer among multiethnic men

The research goal was to document differences in the epidemiology of prostate cancer among multicultural men [non-Hispanic White (NHW), Hispanic (H), non-Hispanic Black (NHB)], and Black subgroups, particularly among NHB subgroups [US-born (USB) and Caribbean-born (CBB)]. Study findings will be useful in supporting further research into Black subgroups. Aim 1 explored changes over time in reported prostate cancer prevalence, by race/ethnicity and by birthplace (within the Black subgroups). Aim 2 investigated relationships between observed and latent variables. The analytical approaches included confirmatory factor analysis (CFA for measurement models) and structural equation modeling (SEM for regression models). National Center for Health Statistics, National Health Interview Survey (NHIS) data from 1999–2008 were used. The study sample included men aged 18 and older, grouped by race/ethnicity. Among the CBB group, survey respondents were limited to the English-speaking Caribbean. Prostate cancer prevalence, by race showed a higher trend among NHB men than NHW men overall, however differences over time were not significant. CBB men reported a higher proportion of prostate cancer among cancers diagnosed than USB men overall. Due to small sample sizes, stable prostate cancer prevalence trends could not be assessed over time nor could trends in the receipt of a PSA exam among NHB men when stratified by birthplace. USB and CBB men differ significantly in their screening behavior. The effect of SES on PSA screening adjusted for risk factors was statistically significant while latent variable lifestyle was not. Among risk factors, family history of cancer exhibited a consistent positive effect on PSA screening for both USB and CBB men. Among the CBB men, the number of years lived in the US did not significantly affect PSA screening behavior. When NHB men are stratified by birthplace, CBB men had a higher overall prevalence of prostate cancer diagnoses than USB men although not statistically significant. USB men were 2 to 3 times more likely to have had a PSA exam compared to CBB men, but among CBB men birthplace did not make a significant difference in screening behavior. Latent variable SES, but not lifestyle, significantly affected the likelihood of a PSA exam.

Better primer design for metagenomics applications by increasing taxonomic distinguishability

Current methods of understanding microbiome composition and structure rely on accurately estimating the number of distinct species and their relative abundance. Most of these methods require an efficient PCR whose forward and reverse primers bind well to the same, large number of identifiable species, and produce amplicons that are unique. It is therefore not surprising that currently used universal primers designed many years ago are not as efficient and fail to bind to recently cataloged species. We propose an automated general method of designing PCR primer pairs that abide by primer design rules and uses current sequence database as input. Since the method is automated, primers can be designed for targeted microbial species or updated as species are added or deleted from the database. In silico experiments and laboratory experiments confirm the efficacy of the newly designed primers for metagenomics applications.

Mechanisms of Arsenic Detoxification and Resistance

Arsenic is a ubiquitous environmental toxic substance. As a consequence of continual exposure to arsenic, nearly every organism, from Escherichia coli to humans have evolved arsenic detoxification pathways. One of the pathways is extrusion of arsenic from inside the cells, thereby conferring resistance. The R773 arsRDABC operon in E. coli encodes an ArsAB efflux pump that confers resistance to arsenite. ArsA is the catalytic subunit of the pump, while ArsB forms the oxyanion conducting pathway. ArsD is an arsenite metallochaperone that binds arsenite and transfers it to ArsA. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. The interaction between ArsA ATPase and ArsD metallochaperone was examined. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. Mutations in ArsA that propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface also affect their interactions. The second objective was to examine the mechanism of arsenite resistance through methylation and subsequent volatilization. Microbial ArsM (As(III) S-adenosylmethyltransferase) catalyzes the formation of trimethylarsine as the volatile end product. The net result is loss of arsenic from cells. The gene for CrArsM from the eukaryotic green alga Chlamydomonas reinhardtii was chemically synthesized and expressed in E. coli. The purified protein catalyzed the methylation of arsenite into methyl-, dimethyl- and trimethyl products. Synthetic purified CrArsM was crystallized in an unliganded form. Biochemical and biophysical studies conducted on CrArsM sheds new light on the pathways of biomethylation. While in microbes ArsM detoxifies arsenic, the human homolog, hAS3MT, converts inorganic arsenic into more toxic and carcinogenic forms. An understanding of the enzymatic mechanism of ArsM will be critical in deciphering its parallel roles in arsenic detoxification and carcinogenesis.