Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

Time-dependent analysis of length of stay and mortality due urinary tract infections in ten developing countries : INICC findings

Catheter associated urinary tract infections (CAUTI) are a worldwide problem that may lead to increased patient morbidity, cost and mortality.1e3 The literature is divided on whether there are real effects from CAUTI on length of stay or mortality. Platt4 found the costs and mortality risks to be largeyetGraves et al found the opposite.5 A reviewof the published estimates of the extra length of stay showed results between zero and 30 days.6 The differences in estimates may have been caused by the different epidemiological methods applied. Accurately estimating the effects of CAUTI is difficult because it is a time-dependent exposure. This means that standard statistical techniques, such asmatched case-control studies, tend to overestimate the increased hospital stay and mortality risk due to infection. The aim of the study was to estimate excess length of stay andmortality in an intensive care unit (ICU) due to a CAUTI, using a statistical model that accounts for the timing of infection. Data collected from ICU units in lower and middle income countries were used for this analysis.7,8 There has been little research for these settings, hence the need for this paper.

Social networks in arid Australia : a review of concepts and evidence

Arid systems are markedly different from non-arid systems. This distinctiveness extends to arid-social networks, by which we mean social networks which are influenced by the suite of factors driving arid and semi-arid regions. Neither the process of how aridity interacts with social structure, nor what happens as a result of this interaction, is adequately understood. This paper postulates three relative characteristics which make arid-social networks distinct: that they are tightly bound, are hierarchical in structure and, hence, prone to power abuses, and contain a relatively higher proportion of weak links, making them reactive to crisis. These ideas were modified from workshop discussions during 2006. Although they are neither tested nor presented as strong beliefs, they are based on the anecdotal observations of arid-system scientists with many years of experience. This paper does not test the ideas, but rather examines them in the context of five arid-social network case studies with the aim of hypotheses building. Our cases are networks related to pastoralism, Aboriginal outstations, the ‘Far West Coast Aboriginal Enterprise Network’ and natural resources in both the Lake-Eyre basin and the Murray–Darling catchment. Our cases highlight that (1) social networks do not have clear boundaries, and that how participants perceive their network boundaries may differ from what network data imply, (2) although network structures are important determinants of system behaviour, the role of participants as individuals is still pivotal, (3) and while in certain arid cases weak links are engaged in crisis, the exact structure of all weak links in terms of how they place participants in relation to other communities is what matters.

Growth medium selection and its economic impact on plasmid DNA production

Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100-1000 μg in 2002 to 500-5000 μg in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5α harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 μg/ml and 6.3 μg/mg dry cell mass respectively) and the lowest was for LB (2.8 μg/ml and 3.3 μg/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and specific yields of 23.8 μg/ml and 11.2 μg/mg dry cell mass respectively. However, it is the economic advantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production.

In pursuit of some appreciation: Assessment and group membership in children’s second stories

Group membership is central to social interaction. Within peer groups, social hierarchies and affiliations are matters to which members seriously attend (Corsaro, 2014). Studies of peer groups highlight how status is achieved through oppositional actions. This paper examines the way in which competition and collaboration in a children’s peer group accomplishes status during the production and management of “second stories” (Sacks 1992). We present analysis of the interaction of young boys in a preparatory year playground who are engaged in a single instance of storytelling “rounds”. Analysis highlights the pivotal role of members’ contributions, assessments and receipts in a series of second stories that enact a simultaneously competitive and collaborative local order.

A confirmatory factor analysis of the Toronto Alexithymia Scale (TAS-20) in an alcohol-dependent sample

Confirmatory factor analyses were conducted to evaluate the factorial validity of the Toronto Alexithymia Scale in an alcohol-dependent sample. Several factor models were examined, but all models were rejected given their poor fit. A revision of the TAS-20 in alcohol-dependent populations may be needed.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

A confirmatory factor analysis of the Observer Alexithymia Scale in treatment seeking alcohol-dependent patients

Confirmatory factor analyses evaluated the factorial validity of the Observer Alexithymia Scale (OAS) in an alcohol-dependent sample. Observation was conducted by clinical psychologists. All models examined were rejected, given their poor fit. Given the psychometric limitations of the OAS shown in this study, the OAS may not be the most appropriate measure to use early in treatment among alcohol-dependent individuals.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

Shrinkage empirical likelihood estimator in longitudinal analysis with time-dependent covariates-application to modeling the health of Filipino children

The method of generalized estimating equations (GEE) is a popular tool for analysing longitudinal (panel) data. Often, the covariates collected are time-dependent in nature, for example, age, relapse status, monthly income. When using GEE to analyse longitudinal data with time-dependent covariates, crucial assumptions about the covariates are necessary for valid inferences to be drawn. When those assumptions do not hold or cannot be verified, Pepe and Anderson (1994, Communications in Statistics, Simulations and Computation 23, 939–951) advocated using an independence working correlation assumption in the GEE model as a robust approach. However, using GEE with the independence correlation assumption may lead to significant efficiency loss (Fitzmaurice, 1995, Biometrics 51, 309–317). In this article, we propose a method that extracts additional information from the estimating equations that are excluded by the independence assumption. The method always includes the estimating equations under the independence assumption and the contribution from the remaining estimating equations is weighted according to the likelihood of each equation being a consistent estimating equation and the information it carries. We apply the method to a longitudinal study of the health of a group of Filipino children.

Burn healing is dependent on burn site : a quantitative analysis from a porcine burn model

This retrospective review examines healing in different sites on a porcine burn model; 24 pairs of burns on 18 pigs from other animal trials were selected for analysis. Each pair of burns was located on the either the cranial or the caudal part of the thoracic ribs region, on the same side of the animal. The burns were 40-50 cm(2) in size and of uniform deep-dermal partial thickness. Caudal burns healed significantly better than cranial burns, demonstrated by earlier closure of wounds, less scar formation and better cosmesis. To our knowledge, this is the first detailed study reporting that burn healing is affected by location on a porcine burn model. We recommend that similar symmetrical burns should be used for future comparative assessments of burn healing.