Toward using an oxidatively damaged plasmid as an intra- and inter-laboratory standard for ancient DNA studies

The following paper was originally presented by Dr Thomas H. Loy at the 6th International Conference on Ancient DNA and Associated Biomolecules held in Israel, July 2002. It is included here with editorial and formatting changes with the intention of demonstrating the passion and lateral thinking that underpinned Tomï½s approach to the field of Molecular Archaeology. The paper represents research from three honours projects conducted during the late 1990s and early 2000s. Building a modern model for ancient DNA that could be used during routine procedures was a concept that Tom had long held as an important step forward for the burgeoning discipline. With the equipment and technology that was available at the time, the Damaged Plasmid Model concept was completely viable and worthy of detailed validation. As with all historical accounts, an understanding of more recent developments in molecular techniques and equipment will highlight the need for considerable optimisation of the model before it can be used as an interlaboratory standard for ancient DN

Expanding the parameters of academia

This paper draws on qualitative data gathered from two studies funded by the UK Leadership Foundation for Higher Education to examine the expansion of academic identities in higher education. It builds on Whitchurch’s earlier work, which focused primarily on professional staff, to suggest that the emergence of broadly based projects such as widening participation, learning support and community partnership is also impacting on academic identities. Thus, academic as well as professional staff are increasingly likely to work in multi-professional teams across a variety of constituencies, as well as with external partners, and the binary distinction between ‘academic’ and ‘non-academic’ roles and activities is no longer clear-cut. Moreover, there is evidence from the studies of an intentionality about deviations from mainstream academic career routes among respondents who could have gone either way. Consideration is therefore given to factors that influence individuals to work in more project-oriented areas, as well as to variables that affect ways in which these roles and identities develop. Finally, three models of academically oriented project activity are identified, and the implications of an expansion of academic identities are reviewed

Calpains, Cleaved Mini-Dysferlin(C72), and L-Type Channels Underpin Calcium-Dependent Muscle Membrane Repair

Dysferlin is proposed as a key mediator of calcium-dependent muscle membrane repair, although its precise role has remained elusive. Dysferlin interacts with a new membrane repair protein, mitsugumin 53 (MG53), an E3 ubiquitin ligase that shows rapid recruitment to injury sites. Using a novel ballistics assay in primary human myotubes, we show it is not full-length dysferlin recruited to sites of membrane injury but an injury-specific calpain-cleavage product, mini-dysferlin(C72). Mini-dysferlin(C72)-rich vesicles are rapidly recruited to injury sites and fuse with plasma membrane compartments decorated by MG53 in a process coordinated by L-type calcium channels. Collective interplay between activated calpains, dysferlin, and L-type channels explains how muscle cells sense a membrane injury and mount a specialized response in the unique local environment of a membrane injury. Mini-dysferlin(C72) and MG53 form an intricate lattice that intensely labels exposed phospholipids of injury sites, then infiltrates and stabilizes the membrane lesion during repair. Our results extend functional parallels between ferlins and synaptotagmins. Whereas otoferlin exists as long and short splice isoforms, dysferlin is subject to enzymatic cleavage releasing a synaptotagmin-like fragment with a specialized protein-or phospholipid-binding role for muscle membrane repair

Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production

© 2015 Australian Society for Parasitology Inc. Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-eastern Asia

Multiple Holins Contribute to Extracellular DNA Release in Pseudomonas aeruginosa Biofilms

Bacterial biofilms are comprised of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms

Bacterial membrane vesicles transport their DNA cargo into host cells

© 2017 The Author(s). Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines

Pfsec13 is an unusual chromatin-associated nucleoporin of plasmodium falciparum that is essential for parasite proliferation in human erythrocytes

In Plasmodium falciparum, the deadliest form of human malaria, the nuclear periphery has drawn much attention due to its role as a subnuclear compartment involved in virulence gene expression. Recent data have implicated components of the nuclear envelope in regulating gene expression in several eukaryotes. Special attention has been given to nucleoporins that compose the nuclear pore complex (NPC). However, very little is known about components of the nuclear envelope in Plasmodium parasites. Here we characterize PfSec13, an unusual nucleoporin of P. falciparum, which shows unique structural similarities suggesting that it is a fusion between Sec13 and Nup145C of yeast. Using super resolution fluorescence microscopy (3D-SIM) and in vivo imaging, we show that the dynamiclocalization of PfSec13 during parasites' intra-erythrocytic development corresponds with that of the NPCs and that these dynamics are associated with microtubules rather than with F-actin. In addition, PfSec13 does not co-localize with the heterochormatin markers HP1 and H3K9me3, suggesting euchromatic location of the NPCs. The proteins associated with PfSec13 indicate that this unusual Nup is involved in several cellular processes. Indeed, ultrastructural and chromatin immunoprecipitation analyses revealed that, in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin. Finally, we used peptide nucleic acids (PNA) to downregulate PfSec13 and show that it is essential for parasite proliferation in human erythrocytes. © 2013. Published by The Company of Biologists Ltd

Self-organization of bacterial biofilms is facilitated by extracellular DNA

Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the "bulldozer" aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia

© 2015 Smout et al. Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world

Understanding the everyday designer in organisations

This paper builds upon the existing concept of an everyday designer as a non-expert designer who carries out design activities using available resources in a given environment. It does so by examining the design activities undertaken by non-expert, informal, designers in organisations who make use of the formal and informal technology already in use in organisations while designing to direct, influence, change or transform the practices of people in the organisation. These people represent a cohort of designers who are given little attention in the literature on information systems, despite their central role in the formation of practice and enactment of technology in organisations. The paper describes the experiences of 18 everyday designers in an academic setting using three concepts: everyday designer in an organisation, empathy through design and experiencing an awareness gap. These concepts were constructed through the analysis of in-depth interviews with the participants. The paper concludes with a call for tool support for everyday designers in organisations to enable them to better understand the audience for whom they are designing and the role technology plays in the organisation