The Risky Business of Water Sensitive City Innovation : A Legal Analysis of Rick Allocation

The Risky Business of Water Sensitive City Innovation : A Legal Analysis of Rick Allocatio

Appendix A. Detailed methods, statistical analysis, figures, and references.

Detailed methods, statistical analysis, figures, and references

Covalent DNA–Protein Cross-Linking by Phosphoramide Mustard and Nornitrogen Mustard in Human Cells

<i>N</i>,<i>N</i>-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and <i>N</i>,<i>N</i>-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA–DNA and DNA–protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA–protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI⁺-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of <i>N</i>-[2-[cysteinyl]­ethyl]-<i>N</i>-[2-(guan-7-yl)­ethyl]­amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage

Results of gene-level rare variant association tests using various variant filters (MAF ≤ 1%).

<p>Results of gene-level rare variant association tests using various variant filters (MAF ≤ 1%).</p

Incorporating Non-Coding Annotations into Rare Variant Analysis

<div><p>Background</p><p>The success of collapsing methods which investigate the combined effect of rare variants on complex traits has so far been limited. The manner in which variants within a gene are selected prior to analysis has a crucial impact on this success, which has resulted in analyses conventionally filtering variants according to their consequence. This study investigates whether an alternative approach to filtering, using annotations from recently developed bioinformatics tools, can aid these types of analyses in comparison to conventional approaches.</p><p>Methods & Results</p><p>We conducted a candidate gene analysis using the UK10K sequence and lipids data, filtering according to functional annotations using the resource CADD (Combined Annotation-Dependent Depletion) and contrasting results with ‘nonsynonymous’ and ‘loss of function’ consequence analyses. Using CADD allowed the inclusion of potentially deleterious intronic variants, which was not possible when filtering by consequence. Overall, different filtering approaches provided similar evidence of association, although filtering according to CADD identified evidence of association between <i>ANGPTL4</i> and High Density Lipoproteins (P = 0.02, N = 3,210) which was not observed in the other analyses. We also undertook genome-wide analyses to determine how filtering in this manner compared to conventional approaches for gene regions. Results suggested that filtering by annotations according to CADD, as well as other tools known as FATHMM-MKL and DANN, identified association signals not detected when filtering by variant consequence and vice versa.</p><p>Conclusion</p><p>Incorporating variant annotations from non-coding bioinformatics tools should prove to be a valuable asset for rare variant analyses in the future. Filtering by variant consequence is only possible in coding regions of the genome, whereas utilising non-coding bioinformatics annotations provides an opportunity to discover unknown causal variants in non-coding regions as well. This should allow studies to uncover a greater number of causal variants for complex traits and help elucidate their functional role in disease.</p></div

Results of gene-level low frequency variant association tests using various variant filters (MAF ≤ 5%).

<p>Results of gene-level low frequency variant association tests using various variant filters (MAF ≤ 5%).</p

Additional file 1: of GTB – an online genome tolerance browser

High resolution versions of Figs. 1, 2, 3 and 4. (DOCX 823 kb

Healthy Animals, Healthy People: Zoonosis Risk from Animal Contact in Pet Shops, a Systematic Review of the Literature

<div><p>Background</p><p>Around 67 million pets are owned by households in the United Kingdom, and an increasing number of these are exotic animals. Approximately a third of pets are purchased through retail outlets or direct from breeders. A wide range of infections can be associated with companion animals.</p><p>Objectives</p><p>This study uses a systematic literature review to describe the transmission of zoonotic disease in humans associated with a pet shop or other location selling pets (incidents of rabies tracebacks and zoonoses from pet food were excluded).</p><p>Data sources</p><p>PubMed and EMBASE.</p><p>Results</p><p>Fifty seven separate case reports or incidents were described in the 82 papers that were identified by the systematic review. Summary information on each incident is included in this manuscript. The infections include bacterial, viral and fungal diseases and range in severity from mild to life threatening. Infections associated with birds and rodents were the most commonly reported. Over half of the reports describe incidents in the Americas, and three of these were outbreaks involving more than 50 cases. Many of the incidents identified relate to infections in pet shop employees.</p><p>Limitations</p><p>This review may have been subject to publication bias, where unusual and unexpected zoonotic infections may be over-represented in peer-reviewed publications. It was also restricted to English-language articles so that pathogens that are more common in non-Western countries, or in more exotic animals not common in Europe and the Americas, may have been under-represented.</p><p>Conclusions/implications</p><p>A wide spectrum of zoonotic infections are acquired from pet shops. Salmonellosis and psittacosis were the most commonly documented diseases, however more unusual infections such as tularemia also appeared in the review. Given their potential to spread zoonotic infection, it is important that pet shops act to minimise the risk as far as possible.</p></div

Hexbin plots representing gene-based SKAT analyses for all genes across the genome using a MAF cutoff of 1% with 4 lipid traits.

<p>The x-axis represents the–log10 transformed p-value from the analysis after filtering according to CADD annotations. The y-axis represents the–log10 transformed p-value from the analysis after filtering according to ‘nonsynonymous’ annotations. Only gene regions which had at least 2 variants in them after filtering by both methods were plotted.</p

Hexbin plots representing gene-based SKAT analyses for all genes across the genome using a MAF cutoff of 5% with 4 lipid traits.

<p>The x-axis represents the–log10 transformed p-value from the analysis after filtering according to CADD annotations. The y-axis represents the–log10 transformed p-value from the analysis after filtering according to ‘nonsynonymous’ annotations. Only gene regions which had at least 2 variants in them after filtering by both methods were plotted.</p