Genetic context and mobilization of class 1 integrons in Pseudomonas aeruginosa : are plasmids redundant?

University of Technology, Sydney. Faculty of Science.Antibiotic resistance is a global problem with some predicting a return to the pre antibiotic era where a bacterial infection was commonly fatal. Pseudomonas aeruginosa is one example of this problem. This bacterium is a major cause of infection especially in cystic fibrosis sufferers and in burns victims. The rising rates of adverse outcomes are partly a consequence of strains commonly displaying multi-drug resistance (MDR) profiles. MDR is driven by a number of intrinsic mechanisms in P. aeruginosa clinical isolates as well as by the capture of diverse resistance-mediating genes by Lateral Gene Transfer (LGT). LGT and intrinsic factors often act cooperatively to generate complex MDR phenotypes. While these complex interactions have been examined in a small number of isolates there has not been a comprehensive survey of strains on a global scale. Thus it is not clear what mechanisms and genes may be important in influencing the evolution of MDR at regional or global levels. Also, in some isolates, resistance profiles cannot always be explained by identifying the common resistance determining pathways, suggesting that additional mechanisms of resistance may be emerging in P. aeruginosa. The focus of this project was to comprehensively study the major mechanisms responsible for antibiotic resistance in P. aeruginosa strains from diverse geographical areas. Pathogenic P. aeruginosa isolates from four countries (Australia and three South American countries) were characterized by PCR to identify mobile elements and their genetic context. Also, quantitative expression analysis for activity of several pathways that influence antibiotic resistance was assessed and culture experiments were conducted to test how random movement of mobile elements during growth may influence resistance to some antibiotics. Data presented in this thesis indicated that, in most strains, antibiotic resistance was being driven by changes in multiple pathways (including overexpression of AmpC and two efflux pumps) and by the presence or absence of genes acquired by Lateral Gene Transfer (LGT). Class 1 integrons, elements important in the spread of antibiotic resistance genes in Gram-negative bacteria, were most frequently recovered in South American countries. Many class 1 integrons were mapped to a specific location within the genome. Regardless of country of origin all these mapped integrons were found to be in the chromosome, often in Genomic islands, and not on a plasmid despite data in the literature implying the opposite. The association of class 1 integrons with genomic islands may be an important mechanism driving LGT in P. aeruginosa. Also, a newly emerging mechanism involving the insertion sequence IS26 was identified that is capable of mobilizing resistance and other genes. This IS26-mediated mechanism may allow phenotype switching in clonal lines in a way that is likely to further exacerbate the treatment of infections mediated by P. aeruginosa. Data presented here suggested that P. aeruginosa strains are evolving to become multidrug resistant in increasingly complex ways. This is occurring by single strains acquiring changes in numerous known pathways as well as by newly emerging resistance mechanisms in this species

Artificial light pollution influences behavioral and physiological traits in a keystone predator species, Concholepas concholepas

Artificial Light At Night (ALAN) is an increasing global problem that, despite being widely recognized in terrestrial systems, has been studied much less in marine habitats. In this study we investigated the effect of ALAN on behavioral and physiological traits of Concholepas concholepas, an important keystone species of the south-eastern Pacific coast. We used juveniles collected in intertidal habitats that had not previously been exposed to ALAN. In the laboratory we exposed them to two treatments: darkness and white LED (Lighting Emitting Diodes) to test for the impacts of ALAN on prey-searching behavior, self-righting time and metabolism. In the field, the distribution of juveniles was observed during daylight-hours to determine whether C. concholepas preferred shaded or illuminated microhabitats. Moreover, we compared the abundance of juveniles collected during day- and night-time hours. The laboratory experiments demonstrated that juveniles of C. concholepas seek out and choose their prey more efficiently in darkened areas. White LED illuminated conditions increased righting times and metabolism. Field surveys indicated that, during daylight hours, juveniles were more abundant in shaded micro-habitats than in illuminated ones. However, during darkness hours, individuals were not seen to aggregate in any particular microhabitats. We conclude that the exposure to ALAN might disrupt important behavioral and physiological traits of small juveniles in this species which, as a mechanism to avoid visual predators, are mainly active at night. It follows that ALAN in coastal areas might modify the entire community structure of intertidal habitats by altering the behavior of this keystone species

The Impact of Global Warming and Anoxia on Marine Benthic Community Dynamics: an Example from the Toarcian (Early Jurassic)

The Pliensbachian-Toarcian (Early Jurassic) fossil record is an archive of natural data of benthic community response to global warming and marine long-term hypoxia and anoxia. In the early Toarcian mean temperatures increased by the same order of magnitude as that predicted for the near future; laminated, organic-rich, black shales were deposited in many shallow water epicontinental basins; and a biotic crisis occurred in the marine realm, with the extinction of approximately 5% of families and 26% of genera. High-resolution quantitative abundance data of benthic invertebrates were collected from the Cleveland Basin (North Yorkshire, UK), and analysed with multivariate statistical methods to detect how the fauna responded to environmental changes during the early Toarcian. Twelve biofacies were identified. Their changes through time closely resemble the pattern of faunal degradation and recovery observed in modern habitats affected by anoxia. All four successional stages of community structure recorded in modern studies are recognised in the fossil data (i.e. Stage III: climax; II: transitional; I: pioneer; 0: highly disturbed). Two main faunal turnover events occurred: (i) at the onset of anoxia, with the extinction of most benthic species and the survival of a few adapted to thrive in low-oxygen conditions (Stages I to 0) and (ii) in the recovery, when newly evolved species colonized the re-oxygenated soft sediments and the path of recovery did not retrace of pattern of ecological degradation (Stages I to II). The ordination of samples coupled with sedimentological and palaeotemperature proxy data indicate that the onset of anoxia and the extinction horizon coincide with both a rise in temperature and sea level. Our study of how faunal associations co-vary with long and short term sea level and temperature changes has implications for predicting the long-term effects of “dead zones” in modern oceans

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

A missense TGFB2 variant p.(Arg320Cys) causes a paradoxical and striking increase in aortic TGFB1/2 expression.

Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder with a range of cardiovascular, skeletal, craniofacial and cutaneous manifestations. LDS type 4 is caused by mutations in TGFβ ligand 2 (TGFB2) and based on the family pedigrees described to date, appears to have a milder clinical phenotype, often presenting with isolated aortic disease. We sought to investigate its molecular basis in a new pedigree. We identified a missense variant p.(Arg320Cys) (NM_003238.3) in a highly evolutionary conserved region of TGFB2 in a new LDS type 4 pedigree with multiple cases of aortic aneurysms and dissections. There was striking upregulation of TGFB1 and TGFB2 expression on immunofluorescent staining, and western blotting of the aortic tissue from the index case confirming the functional importance of the variant. This case highlights the striking paradox of predicted loss-of-function mutations in TGFB2 causing enhanced TGFβ signaling in this emerging familial aortopathy.Raya Al Maskari has a PhD studentship funded by the Omani government.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ejhg.2016.14

The association of cold weather and all-cause and cause-specific mortality in the island of Ireland between 1984 and 2007

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.This article has been made available through the Brunel Open Access Publishing Fund.Background This study aimed to assess the relationship between cold temperature and daily mortality in the Republic of Ireland (ROI) and Northern Ireland (NI), and to explore any differences in the population responses between the two jurisdictions. Methods A time-stratified case-crossover approach was used to examine this relationship in two adult national populations, between 1984 and 2007. Daily mortality risk was examined in association with exposure to daily maximum temperatures on the same day and up to 6 weeks preceding death, during the winter (December-February) and cold period (October-March), using distributed lag models. Model stratification by age and gender assessed for modification of the cold weather-mortality relationship. Results In the ROI, the impact of cold weather in winter persisted up to 35 days, with a cumulative mortality increase for all-causes of 6.4% (95%CI=4.8%-7.9%) in relation to every 1oC drop in daily maximum temperature, similar increases for cardiovascular disease (CVD) and stroke, and twice as much for respiratory causes. In NI, these associations were less pronounced for CVD causes, and overall extended up to 28 days. Effects of cold weather on mortality increased with age in both jurisdictions, and some suggestive gender differences were observed. Conclusions The study findings indicated strong cold weather-mortality associations in the island of Ireland; these effects were less persistent, and for CVD mortality, smaller in NI than in the ROI. Together with suggestive differences in associations by age and gender between the two Irish jurisdictions, the findings suggest potential contribution of underlying societal differences, and require further exploration. The evidence provided here will hope to contribute to the current efforts to modify fuel policy and reduce winter mortality in Ireland

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

Flavor Violating Higgs Decays

We study a class of nonstandard interactions of the newly discovered 125 GeV Higgs-like resonance that are especially interesting probes of new physics: flavor violating Higgs couplings to leptons and quarks. These interaction can arise in many frameworks of new physics at the electroweak scale such as two Higgs doublet models, extra dimensions, or models of compositeness. We rederive constraints on flavor violating Higgs couplings using data on rare decays, electric and magnetic dipole moments, and meson oscillations. We confirm that flavor violating Higgs boson decays to leptons can be sizeable with, e.g., h -> tau mu and h -> tau e branching ratios of order 10% perfectly allowed by low energy constraints. We estimate the current LHC limits on h -> tau mu and h -> tau e decays by recasting existing searches for the SM Higgs in the tau-tau channel and find that these bounds are already stronger than those from rare tau decays. We also show that these limits can be improved significantly with dedicated searches and we outline a possible search strategy. Flavor violating Higgs decays therefore present an opportunity for discovery of new physics which in some cases may be easier to access experimentally than flavor conserving deviations from the Standard Model Higgs framework.Comment: 39 pages, 12 figures, 3 tables; v2: Improved referencing, updated mu -> 3e bounds to include large loop contributions, corrected single top constraints; conclusions unchanged; matches version to be published in JHEP; v3: included 2-loop contributions in mu -> e conversion, improved discussion of tau -> 3 mu and of EDM constraints on FV top-Higgs couplings; conclusions unchange