Properties of triple helices formed by parallel-stranded hairpins containing 8-aminopurines

Parallel-stranded hairpins with a polypyrimidine sequence linked to a complementary purine carrying 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine bind polypyrimidine sequences complementary (in an antiparallel sense) to the purine part by a triple helix. The relative stabilities of triplexes were assessed by UV-absorption melting experiments as a function of pH and salt concentration. Hairpins carrying 8-aminopurines give very stable triple helical structures even at neutral pH, as confirmed by gel-shift experiments, circular dichroism and nuclear magnetic resonance spectroscopy. The modified hairpins may be redesigned to cope with small interruptions in the polypyrimidine target sequence.     

A yeast-based screen reveals that sulfasalazine inhibits tetrahydrobiopterin biosynthesis

We introduce an approach for detection of drug-protein interactions that combines a new yeast three-hybrid screening for identification of interactions with affinity chromatography for their unambiguous validation. We applied the methodology to the profiling of clinically approved drugs, resulting in the identification of previously known and unknown drug-protein interactions. In particular, we were able to identify off-targets for erlotinib and atorvastatin, as well as an enzyme target for the anti-inflammatory drug sulfasalazine. We demonstrate that sulfasalazine and its metabolites, sulfapyridine and mesalamine, are inhibitors of the enzyme catalyzing the final step in the biosynthesis of the cofactor tetrahydrobiopterin. The interference with tetrahydrobiopterin metabolism provides an explanation for some of the beneficial and deleterious properties of sulfasalazine and furthermore suggests new and improved therapies for the drug. This work thus establishes a powerful approach for drug profiling and provides new insights in the mechanism of action of clinically approved drugs.     

Vaccinia Virus Protein Complex F12/E2 Interacts with Kinesin Light Chain Isoform 2 to Engage the Kinesin-1 Motor Complex

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.     

The structure of the human LIM protein ACT gene and its expression in tumor cell lines

We describe the human ACT genomic and cDNA sequence which like its murine counterpart contains the defining secondary structure of the FHL (Four-and-a-Half LIM-domain) LIM-protein family. The coding region of the human ACT gene spans five exons. This distribution is very similar to the FHL1 gene and includes the arrangement of split codons across exon boundaries suggesting that these genes share a common ancestor. The human ACT gene was not detected by Northern analysis in the adult testis although this is the only known site of expression found with its murine counterpart. However, the human ACT gene was found to be expressed in a panel of human tumor cell lines derived from squamous cell carcinomas, melanomas, and leukemias. Interestingly, FHL1, FHL2, and FHL3 were also found to be expressed in some of these cell lines and the results suggest an important role for FHLs in tumor biology.     

SASI-Seq: sample assurance Spike-Ins,and highly differentiating 384 barcoding for Illumina sequencing

Background

A minor but significant fraction of samples subjected to next-generation sequencing methods are either mixed-up or cross-contaminated. These events can lead to false or inconclusive results. We have therefore developed SASI-Seq; a process whereby a set of uniquely barcoded DNA fragments are added to samples destined for sequencing. From the final sequencing data, one can verify that all the reads derive from the original sample(s) and not from contaminants or other samples.

Results

By adding a mixture of three uniquely barcoded amplicons, of different sizes spanning the range of insert sizes one would normally use for Illumina sequencing, at a spike-in level of approximately 0.1%, we demonstrate that these fragments remain intimately associated with the sample. They can be detected following even the tightest size selection regimes or exome enrichment and can report the occurrence of sample mix-ups and cross-contamination.As a consequence of this work, we have designed a set of 384 eleven-base Illumina barcode sequences that are at least 5 changes apart from each other, allowing for single-error correction and very low levels of barcode misallocation due to sequencing error.

Conclusion

SASI-Seq is a simple, inexpensive and flexible tool that enables sample assurance, allows deconvolution of sample mix-ups and reports levels of cross-contamination between samples throughout NGS workflows.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-110) contains supplementary material, which is available to authorized users.     

Enhancement of the inhibitory effect of an IL‐15 antagonist peptide by alanine scanning

IL‐15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including rheumatoid arthritis, where it had been proposed as a therapeutic target. We recently reported an IL‐15 antagonist peptide corresponding to sequence 36–45 of IL‐15 (KVTAMKCFLL) named P8, which specifically binds to IL‐15Rα and inhibits IL‐15 biological activity with a half maximal inhibitory concentration (IC50) of 130 µ m in CTLL‐2 proliferation assay. In order to improve binding of peptide P8 to the receptor IL‐15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide's antagonist effect. Here, we found that Phe and Cys are important for peptide binding to IL‐15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non‐charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 µm . We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from rheumatoid arthritis patients. The peptide [K6T]P8 described in this work is a new type of IL‐15 antagonist and constitutes a potential therapeutic agent for rheumatoid arthritis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

The use of specialisation indices to predict vulnerability of coral‐feeding butterflyfishes to environmental change

In the absence of detailed assessments of extinction risk, ecological specialisation is often used as a proxy of vulnerability to environmental disturbances and extinction risk. Numerous indices can be used to estimate specialisation; however, the utility of these different indices to predict vulnerability to future environmental change is unknown. Here we compare the performance of specialisation indices using coral‐feeding butterflyfishes as a model group. Our aims were to 1) quantify the dietary preferences of three butterflyfish species across habitats with differing levels of resource availability; 2) investigate how estimates of dietary specialisation vary with the use of different specialisation indices; 3) determine which specialisation indices best inform predictions of vulnerability to environmental change; and 4) assess the utility of resource selection functions to inform predictions of vulnerability to environmental change. The relative level of dietary specialisation estimated for all three species varied when different specialisation indices were used, indicating that the choice of index can have a considerable impact upon estimates of specialisation. Specialisation indices that do not consider resource abundance may fail to distinguish species that primarily use common resources from species that actively target resources disproportionately more than they are available. Resource selection functions provided the greatest insights into the potential response of species to changes in resource availability. Examination of resource selection functions, in addition to specialisation indices, indicated that Chaetodon trifascialis was the most specialised feeder, with highly conserved dietary preferences across all sites, suggesting that this species is highly vulnerable to the impacts of climate‐induced coral loss on reefs. Our results indicate that vulnerability assessments based on some specialisation indices may be misleading and the best estimates of dietary specialisation will be provided by indices which incorporate resource availability measures, as well as assessing responses of species to changes in resource availability.     

Psychological Distress, Depression, Anxiety, and Burnout among International Humanitarian Aid Workers: A Longitudinal Study

Background

International humanitarian aid workers providing care in emergencies are subjected to numerous chronic and traumatic stressors.

Objectives

To examine consequences of such experiences on aid workers'' mental health and how the impact is influenced by moderating variables.

Methodology

We conducted a longitudinal study in a sample of international non-governmental organizations. Study outcomes included anxiety, depression, burnout, and life and job satisfaction. We performed bivariate regression analyses at three time points. We fitted generalized estimating equation multivariable regression models for the longitudinal analyses.

Results

Study participants from 19 NGOs were assessed at three time points: 212 participated at pre-deployment; 169 (80%) post-deployment; and 154 (73%) within 3–6 months after deployment. Prior to deployment, 12 (3.8%) participants reported anxiety symptoms, compared to 20 (11.8%) at post-deployment (p = 0·0027); 22 (10.4%) reported depression symptoms, compared to 33 (19.5%) at post-deployment (p = 0·0117) and 31 (20.1%) at follow-up (p = .00083). History of mental illness (adjusted odds ratio [AOR] 4.2; 95% confidence interval [CI] 1·45–12·50) contributed to an increased risk for anxiety. The experience of extraordinary stress was a contributor to increased risk for burnout depersonalization (AOR 1.5; 95% CI 1.17–1.83). Higher levels of chronic stress exposure during deployment were contributors to an increased risk for depression (AOR 1·1; 95% CI 1·02–1.20) comparing post- versus pre-deployment, and increased risk for burnout emotional exhaustion (AOR 1.1; 95% CI 1.04–1.19). Social support was associated with lower levels of depression (AOR 0·9; 95% CI 0·84–0·95), psychological distress (AOR = 0.9; [CI] 0.85–0.97), burnout lack of personal accomplishment (AOR 0·95; 95% CI 0·91–0·98), and greater life satisfaction (p = 0.0213).

Conclusions

When recruiting and preparing aid workers for deployment, organizations should consider history of mental illness and take steps to decrease chronic stressors, and strengthen social support networks.     

Colon cancer associated genes exhibit signatures of positive selection at functionally significant positions

ABSTRACT: BACKGROUND: Cancer, much like most human disease, is routinely studied by utilizing model organisms. Of these model organisms, mice are often dominant. However, our assumptions of functional equivalence fail to consider the opportunity for divergence conferred by ~180 Million Years (MY) of independent evolution between these species. For a given set of human disease related genes, it is therefore important to determine if functional equivalency has been retained between species. In this study we test the hypothesis that cancer associated genes have different patterns of substitution akin to adaptive evolution in different mammal lineages. RESULTS: Our analysis of the current literature and colon cancer databases identified 22 genes exhibiting colon cancer associated germline mutations. We identified orthologs for these 22 genes across a set of high coverage (>6X) vertebrate genomes. Analysis of these orthologous datasets revealed significant levels of positive selection. Evidence of lineage-specific positive selection was identified in 14 genes in both ancestral and extant lineages. Lineage-specific positive selection was detected in the ancestral Euarchontoglires and Hominidae lineages for STK11, in the ancestral primate lineage for CDH1, in the ancestral Murinae lineage for both SDHC and MSH6 genes and the ancestral Muridae lineage for TSC1. CONCLUSION: Identifying positive selection in the primate, Hominidae, Muridae and Murinae lineages suggest an ancestral functional shift in these genes between the rodent and primate lineages. Analyses such as this, combining evolutionary theory and predictions - along with medically relevant data, can thus provide us with important clues for modeling human diseases.     

Dielectric relaxation measurements of 12 kbp plasmid DNA

The dielectric properties of 12 kbp plasmid DNA have been measured as a function of temperature in the range 5 degrees C to 40 degrees C. Time domain reflectometry was used to obtain dielectric data over the frequency range from 200 kHz to 3 GHz. Values of the frequency dependent polarisability per DNA macromolecule have been determined from the measurements. Possible mechanisms that could account for the dielectric dispersion are also discussed, in particular the counterion fluctuation model of Manning-Mandel-Oosawa.