A Predictive Framework for Integrating Disparate Genomic Data Types Using Sample-Specific Gene Set Enrichment Analysis and Multi-Task Learning

Understanding the root molecular and genetic causes driving complex traits is a fundamental challenge in genomics and genetics. Numerous studies have used variation in gene expression to understand complex traits, but the underlying genomic variation that contributes to these expression changes is not well understood. In this study, we developed a framework to integrate gene expression and genotype data to identify biological differences between samples from opposing complex trait classes that are driven by expression changes and genotypic variation. This framework utilizes pathway analysis and multi-task learning to build a predictive model and discover pathways relevant to the complex trait of interest. We simulated expression and genotype data to test the predictive ability of our framework and to measure how well it uncovered pathways with genes both differentially expressed and genetically associated with a complex trait. We found that the predictive performance of the multi-task model was comparable to other similar methods. Also, methods like multi-task learning that considered enrichment analysis scores from both data sets found pathways with both genetic and expression differences related to the phenotype. We used our framework to analyze differences between estrogen receptor (ER) positive and negative breast cancer samples. An analysis of the top 15 gene sets from the multi-task model showed they were all related to estrogen, steroids, cell signaling, or the cell cycle. Although our study suggests that multi-task learning does not enhance predictive accuracy, the models generated by our framework do provide valuable biological pathway knowledge for complex traits.     

Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H₂O₂-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.     

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.     

Comparison between the effects of diallyl tetrasulfide on human retina pigment epithelial cells (ARPE-19) and HCT116 cells

Background

Diallyl mono- and polysulfanes from garlic are known to induce an adaptive cell response and the formation of antioxidants in cancer cells. In the case of a severe ER stress and a failure in the response, cancer cells eventually go into apoptosis. Only little is known about the response of normal cells upon treatment.

Methods

Normal ARPE-19 cells were treated with diallyl tetrasulfide to study their cellular response and the results were compared with those of HCT116 cancer cells. Cell viability was checked by an MTT assay and cytofluorimetry. The formation of superoxide radicals, H₂O₂ and thiols were determined and proteins involved in the ER stress response were also detected by Western blot analysis.

Results

We found that diallyl tetrasulfide induced reactive oxygen species (ROS) in normal cells similar to cancer cells in a time (0 to 60 min) and dose dependent manner (0 to 50 μM). The level of heme oxigenase-1 (HO-1) was up-regulated in both cell types. Initially, we found a decrease in the total thiol level in both cell types but in contrast to cancer cells, normal cells recovered from the decrease in the total thiol concentration within 60 min of treatment.

Conclusions

The recovery of the thiol concentration in normal cells treated with diallyl tetrasulfide seems to be responsible for the failure to induce the ER stress signalling pathway and finally apoptosis in normal cells.

General Significance

The difference in the recovery of the thiol status might be an explanation for the anti-carcinogenic effects of garlic compounds.     

A freshwater cyanophage whose genome indicates close relationships to photosynthetic marine cyanomyophages

Bacteriophage S-CRM01 has been isolated from a freshwater strain of Synechococcus and shown to be present in the upper Klamath River valley in northern California and Oregon. The genome of this lytic T4-like phage has a 178,563 bp circular genetic map with 297 predicted protein-coding genes and 33 tRNA genes that represent all 20-amino-acid specificities. Analyses based on gene sequence and gene content indicate a close phylogenetic relationship to the 'photosynthetic' marine cyanomyophages infecting Synechococcus and Prochlorococcus. Such relatedness suggests that freshwater and marine phages can draw on a common gene pool. The genome can be considered as being comprised of three regions. Region 1 is populated predominantly with structural genes, recognized as such by homology to other T4-like phages and by identification in a proteomic analysis of purified virions. Region 2 contains most of the genes with roles in replication, recombination, nucleotide metabolism and regulation of gene expression, as well as 5 of the 6 signature genes of the photosynthetic cyanomyophages (hli03, hsp20, mazG, phoH and psbA; cobS is present in Region 3). Much of Regions 1 and 2 are syntenic with marine cyanomyophage genomes, except that a segment encompassing Region 2 is inverted. Region 3 contains a high proportion (85%) of genes that are unique to S-CRM01, as well as most of the tRNA genes. Regions 1 and 2 contain many predicted late promoters, with a combination of CTAAATA and ATAAATA core sequences. Two predicted genes that are unusual in phage genomes are homologues of cellular spoT and nusG.     

Recovery of Cholinergic Phenotype in the Injured Rat Neostriatum: Roles for Endogenous and Exogenous Nerve Growth Factor

Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high-affinity 125I-rhNGF binding but not its receptor, and cross-reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, insulin-like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF-positive neurons in the rat neostriatum. The NGF-positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF- and glial fibrillary acidic protein-positive astrocytes. Enzyme-linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high-affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 microgram of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23-29% above intact neostriatal levels and by 53-65% relative to HACU at both survival times. Thus, lesion-induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.     

The production of haploid wheat plants from wheat x maize crosses

Summary Hybrid embryos from hexaploid wheat x maize crosses rapidly lose the maize chromosomes to produce haploid wheat embryos. Such embryos almost always aborted when left to develop on the plant, and only 1 was recovered from 2440 florets (0.17% of the expected number). Embryos had greater viability in spikelet culture, 47 (26.5% of the expected number) being recovered from 706 ovaries. Thirty-two of these embryos germinated to give green plants, 31 of which were haploid (21 wheat chromosomes) and 1 of which was euploid (42 wheat chromosomes). Spikelet culture enabled 17.1% of the expected number of embryos to be recovered as haploid plants, a 100-fold improvement on allowing embryos to develop in vivo. Ten haploid plants of Chinese Spring (kr1, kr2), 13 plants of Chinese Spring (Hope 5A) (kr1, Kr2), and 8 of Hope (Kr1, Kr2) were recovered. The potential of wheat x maize crosses for wheat haploid production and for gene transfer from maize to wheat is discussed.     

Global patterns of geographic range size in birds

Large-scale patterns of spatial variation in species geographic range size are central to many fundamental questions in macroecology and conservation biology. However, the global nature of these patterns has remained contentious, since previous studies have been geographically restricted and/or based on small taxonomic groups. Here, using a database on the breeding distributions of birds, we report the first (to our knowledge) global maps of variation in species range sizes for an entire taxonomic class. We show that range area does not follow a simple latitudinal pattern. Instead, the smallest range areas are attained on islands, in mountainous areas, and largely in the southern hemisphere. In contrast, bird species richness peaks around the equator, and towards higher latitudes. Despite these profoundly different latitudinal patterns, spatially explicit models reveal a weak tendency for areas with high species richness to house species with significantly smaller median range area. Taken together, these results show that for birds many spatial patterns in range size described in geographically restricted analyses do not reflect global rules. It remains to be discovered whether global patterns in geographic range size are best interpreted in terms of geographical variation in species assemblage packing, or in the rates of speciation, extinction, and dispersal that ultimately underlie biodiversity.     

Canopy interactions and physical stress gradients in subtidal communities

Species interactions are integral drivers of community structure and can change from competitive to facilitative with increasing environmental stress. In subtidal marine ecosystems, however, interactions along physical stress gradients have seldom been tested. We observed seaweed canopy interactions across depth and latitudinal gradients to test whether light and temperature stress structured interaction patterns. We also quantified interspecific and intraspecific interactions among nine subtidal canopy seaweed species across three continents to examine the general nature of interactions in subtidal systems under low consumer pressure. We reveal that positive and neutral interactions are widespread throughout global seaweed communities and the nature of interactions can change from competitive to facilitative with increasing light stress in shallow marine systems. These findings provide support for the stress gradient hypothesis within subtidal seaweed communities and highlight the importance of canopy interactions for the maintenance of subtidal marine habitats experiencing environmental stress.