Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling

Musa (banana and plantain) is an important genus for the global export market and in local markets where it provides staple food for approximately 400 million people. Hybridization and polyploidization of several (sub)species, combined with vegetative propagation and human selection have produced a complex genetic history. We describe the application of the Ecotilling method for the discovery and characterization of nucleotide polymorphisms in diploid and polyploid accessions of Musa. We discovered over 800 novel alleles in 80 accessions. Sequencing and band evaluation shows Ecotilling to be a robust and accurate platform for the discovery of polymorphisms in homologous and homeologous gene targets. In the process of validating the method, we identified two single nucleotide polymorphisms that may be deleterious for the function of a gene putatively important for phototropism. Evaluation of heterozygous polymorphism and haplotype blocks revealed a high level of nucleotide diversity in Musa accessions. We further applied a strategy for the simultaneous discovery of heterozygous and homozygous polymorphisms in diploid accessions to rapidly evaluate nucleotide diversity in accessions of the same genome type. This strategy can be used to develop hypotheses for inheritance patterns of nucleotide polymorphisms within and between genome types. We conclude that Ecotilling is suitable for diversity studies in Musa, that it can be considered for functional genomics studies and as tool in selecting germplasm for traditional and mutation breeding approaches

Utility of arsenic-treated bird skins for DNA extraction

Background: Natural history museums receive a rapidly growing number of requests for tissue samples from preserved specimens for DNA-based studies. Traditionally, dried vertebrate specimens were treated with arsenic because of its toxicity and insect-repellent effect. Arsenic has negative effects on in vivo DNA repair enzymes and consequently may inhibit PCR performance. In bird collections, foot pad samples are often requested since the feet were not regularly treated with arsenic and because they are assumed to provide substantial amounts of DNA. However, the actual influence of arsenic on DNA analyses has never been tested. Findings: PCR success of both foot pad and body skin samples was significantly lower in arsenic-treated samples. In general, foot pads performed better than body skin samples. Moreover, PCR success depends on collection date in which younger samples yielded better results. While the addition of arsenic solution to the PCR mixture had a clear negative effect on PCR performance after the threshold of 5.4 μg/μl, such high doses of arsenic are highly unlikely to occur in dried zoological specimens. Conclusions: While lower PCR success in older samples might be due to age effects and/or DNA damage through arsenic treatment, our results show no inhibiting effect on DNA polymerase. We assume that DNA degradation proceeds more rapidly in thin tissue layers with low cell numbers that are susceptible to external abiotic influences. In contrast, in thicker parts of a specimen, such as foot pads, the outermost horny skin may act as an additional barrier. Since foot pads often performed better than body skin samples, the intention to preserve morphologically important structures of a specimen still conflicts with the aim to obtain optimal PCR success. Thus, body skin samples from recently collected specimens should be considered as alternative sources of DNA

Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach

Background: Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening. [br/] Methodology/Principal Findings: To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect. [br/] Conclusions/Significance: We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community

Whose Sense of Place? A Political Ecology of Amenity Development

Using a political ecology framework, this chapter examines the ways in which sense of place and amenity migration contribute to alternative residential development, which relies on uneven use of conservation subdivision features in the American West. Using case studies from Central Oregon, this chapter demonstrates how senses of place and developer decision-making are tied to wider political economic changes. It highlights the roles that amenity migrants and developers, two groups that are sometimes identical, play in landscape transformations that simultaneously draw on a particular sense of place and commodify landscapes in new ways

Challenges for Australia's Bio/Nanopharma Policies: trade deals, public goods and reference pricing in sustainable industrial renewal

Industrial renewal in the bio/nanopharma sector is important for the long term strength of the Australian economy and for the health of its citizens. A variety of factors, however, may have caused inadequate attention to focus on systematically promoting domestic generic and small biotechnology manufacturers in Australian health policy

The effect of long-term homocysteine-lowering on carotid intima-media thickness and flow-mediated vasodilation in stroke patients: a randomized controlled trial and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Experimental and epidemiological evidence suggests that homocysteine (tHcy) may be a causal risk factor for atherosclerosis. B-vitamin supplements reduce tHcy and improve endothelial function in short term trials, but the long-term effects of the treatment on vascular structure and function are unknown.</p> <p>Methods</p> <p>We conducted a sub-study of VITATOPS, a randomised, double-blind, placebo-controlled intervention trial designed to test the efficacy of long term B-vitamin supplementation (folic acid 2 mg, vitamin B₆25 mg and vitamin B₁₂0.5 mg) in the prevention of vascular events in patients with a history of stroke. We measured carotid intima-medial thickness (CIMT) and flow-mediated dilation (FMD) at least two years after randomisation in 162 VITATOPS participants. We also conducted a systematic review and meta-analysis of studies designed to test the effect of B-vitamin treatment on CIMT and FMD.</p> <p>Results</p> <p>After a mean treatment period of 3.9 ± 0.9 years, the vitamin-treated group had a significantly lower mean plasma homocysteine concentration than the placebo-treated group (7.9 μmol/L, 95% CI 7.5 to 8.4 versus 11.8 μmol/L, 95% CI 10.9 to 12.8, p < 0.001). Post-treatment CIMT (0.84 ± 0.17 mm vitamins versus 0.83 ± 0.18 mm placebo, p = 0.74) and FMD (median of 4.0%, IQR 0.9 to 7.2 vitamins versus 3.0%, IQR 0.6 to 6.6 placebo, p = 0.48) did not differ significantly between groups. A meta-analysis of published randomised data, including those from the current study, suggested that B-vitamin supplements should reduce CIMT (-0.10 mm, 95% CI -0.20 to -0.01 mm) and increase FMD (1.4%, 95% CI 0.7 to 2.1%). However, the improvement in endothelial function associated with homocysteine-lowering treatment was significant in short-term studies but not in longer trials.</p> <p>Conclusion</p> <p>Although short-term treatment with B-vitamins is associated with increased FMD, long-term homocysteine-lowering did not significantly improve FMD or CIMT in people with a history of stroke.</p> <p>Trial Registration</p> <p>Clinical Trial Registration URL: <url>http://www.actr.org.au/</url></p> <p>Trial Registration number: 12605000005651</p

Tuberculosis mortality and the male survival deficit in rural South Africa:An observational community cohort study

BACKGROUND: Women live on average five years longer than men, and the sex difference in longevity is typically lower in populations with high mortality. South Africa-a high mortality population with a large sex disparity-is an exception, but the causes of death that contribute to this difference are not well understood. METHODS: Using data from a demographic surveillance system in rural KwaZulu-Natal (2000-2014), we estimate differences between male and female adult life expectancy by HIV status. The contribution of causes of death to these life expectancy differences are computed with demographic decomposition techniques. Cause of death information comes from verbal autopsy interviews that are interpreted with the InSilicoVA tool. RESULTS: Adult women lived an average of 10.4 years (95% confidence Interval 9.0-11.6) longer than men. Sex differences in adult life expectancy were even larger when disaggregated by HIV status: 13.1 (95% confidence interval 10.7-15.3) and 11.2 (95% confidence interval 7.5-14.8) years among known HIV negatives and positives, respectively. Elevated male mortality from pulmonary tuberculosis (TB) and external injuries were responsible for 43% and 31% of the sex difference in life expectancy among the HIV negative population, and 81% and 16% of the difference among people living with HIV. CONCLUSIONS: The sex differences in adult life expectancy in rural KwaZulu-Natal are exceptionally large, atypical for an African population, and largely driven by high male mortality from pulmonary TB and injuries. This is the case for both HIV positive and HIV negative men and women, signalling a need to improve the engagement of men with health services, irrespective of their HIV status

Genomic and Proteomic Analysis of the Impact of Mitotic Quiescence on the Engraftment of Human CD34+ Cells

It is well established that in adults, long-term repopulating hematopoietic stem cells (HSC) are mitotically quiescent cells that reside in specialized bone marrow (BM) niches that maintain the dormancy of HSC. Our laboratory demonstrated that the engraftment potential of human HSC (CD34+ cells) from BM and mobilized peripheral blood (MPB) is restricted to cells in the G0 phase of cell cycle but that in the case of umbilical cord blood (UCB) -derived CD34+ cells, cell cycle status is not a determining factor in the ability of these cells to engraft and sustain hematopoiesis. We used this distinct in vivo behavior of CD34+ cells from these tissues to identify genes associated with the engraftment potential of human HSC. CD34+ cells from BM, MPB, and UCB were fractionated into G0 and G1 phases of cell cycle and subjected in parallel to microarray and proteomic analyses. A total of 484 target genes were identified to be associated with engraftment potential of HSC. System biology modeling indicated that the top four signaling pathways associated with these genes are Integrin signaling, p53 signaling, cytotoxic T lymphocyte-mediated apoptosis, and Myc mediated apoptosis signaling. Our data suggest that a continuum of functions of hematopoietic cells directly associated with cell cycle progression may play a major role in governing the engraftment potential of stem cells. While proteomic analysis identified a total of 646 proteins in analyzed samples, a very limited overlap between genomic and proteomic data was observed. These data provide a new insight into the genetic control of engraftment of human HSC from distinct tissues and suggest that mitotic quiescence may not be the requisite characteristic of engrafting stem cells, but instead may be the physiologic status conducive to the expression of genetic elements favoring engraftment

Identification of Y-Box Binding Protein 1 As a Core Regulator of MEK/ERK Pathway-Dependent Gene Signatures in Colorectal Cancer Cells

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties