Regulation of endothelial-specific transgene expression by the LacI repressor protein in vivo

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R), and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R) protein.CEH and KIM acknowledge funding support from NH&MRC Project Grant #471421

FVB/N mice are highly resistant to primary infection with Nippostrongylus brasiliensis

Nippostrongylus brasiliensis larvae are particularly susceptible to immunological attack during the pre-lung stage of primary and secondary infections in mice. Whilst most of the common laboratory strains of mice are permissive hosts for the parasite, in this study we report for the first time, the strong resistance of naive FVB/N mice to N. brasiliensis. Damage to larvae is evident within the first 24 h of infection and this may be critical to later larval development and reproductive success. Inflammatory responses in the skin, and larval escape from this tissue were comparable in susceptible CBA/Ca and resistant FVB/N mice, with most larvae exiting within 4 h of a primary infection. Lung larval burdens were also similar between strains, but larvae recovered from FVB/N mice were smaller and less motile. In FVB/N mice, larval colonization of the gut was impaired and worms produced very few eggs. However FVB/N mice did not show enhanced resistance to Heligmosomoides bakeri (also known as Heligmosomoides polygyrus), a nematode largely restricted to the gut. Damage done in the pre-lung or lung stages of infection with N. brasiliensis is likely to contribute to ongoing developmental and functional abnormalities, which are profoundly evident in the gut phase of infection.M. L. Knott, S. P. Hogan, H. Wang, K. I. Matthaei and L. A. Den

Multipoint Schur algorithm and orthogonal rational functions: convergence properties, I

Classical Schur analysis is intimately connected to the theory of orthogonal polynomials on the circle [Simon, 2005]. We investigate here the connection between multipoint Schur analysis and orthogonal rational functions. Specifically, we study the convergence of the Wall rational functions via the development of a rational analogue to the Szeg\H o theory, in the case where the interpolation points may accumulate on the unit circle. This leads us to generalize results from [Khrushchev,2001], [Bultheel et al., 1999], and yields asymptotics of a novel type.Comment: a preliminary version, 39 pages; some changes in the Introduction, Section 5 (Szeg\H o type asymptotics) is extende

Bactericidal activity of human eosinophilic granulocytes against Escherichia coli

Eosinophils participate in allergic inflammation and may have roles in the bodys defense against helminthic infestation. Even under noninflammatory conditions, eosinophils are present in the mucosa of the large intestine, where large numbers of gram-negative bacteria reside. Therefore, roles for eosinophils in host defenses against bacterial invasion are possible. In a system for bacterial viable counts, the bactericidal activity of eosinophils and the contribution of different cellular antibacterial systems against Escherichia coli were investigated. Eosinophils showed a rapid and efficient killing of E. coli under aerobic conditions, whereas under anaerobic conditions bacterial killing decreased dramatically. In addition, diphenylene iodonium chloride (DPI), an inhibitor of the NADPH oxidase and thereby of superoxide production, also significantly inhibited bacterial killing. The inhibitor of nitric oxide (NO) production L-N5-(1-iminoethyl)-ornithine dihydrochloride did not affect the killing efficiency, suggesting that NO or derivatives thereof are of minor importance under the experimental conditions used. To investigate the involvement of superoxide and eosinophil peroxidase (EPO) in bacterial killing, EPO was blocked by azide. The rate of E. coli killing decreased significantly in the presence of azide, whereas addition of DPI did not further decrease the killing, suggesting that superoxide acts in conjunction with EPO. Bactericidal activity was seen in eosinophil extracts containing granule proteins, indicating that oxygen-independent killing may be of importance as well. The findings suggest that eosinophils can participate in host defense against gram-negative bacterial invasion and that oxygen-dependent killing, i.e., superoxide acting in conjunction with EPO, may be the most important bactericidal effector function of these cells

Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease

<p>Abstract</p> <p>Background</p> <p>Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.</p> <p>Materials and methods</p> <p>We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.</p> <p>Results</p> <p>We found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/<it>neu </it>status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).</p> <p>Conclusion</p> <p>The detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.</p

Corporate governance for sustainability : Statement

The current model of corporate governance needs reform. There is mounting evidence that the practices of shareholder primacy drive company directors and executives to adopt the same short time horizon as financial markets. Pressure to meet the demands of the financial markets drives stock buybacks, excessive dividends and a failure to invest in productive capabilities. The result is a ‘tragedy of the horizon’, with corporations and their shareholders failing to consider environmental, social or even their own, long-term, economic sustainability. With less than a decade left to address the threat of climate change, and with consensus emerging that businesses need to be held accountable for their contribution, it is time to act and reform corporate governance in the EU. The statement puts forward specific recommendations to clarify the obligations of company boards and directors and make corporate governance practice significantly more sustainable and focused on the long term

Is the ADA/EASD algorithm for the management of type 2 diabetes (January 2009) based on evidence or opinion? A critical analysis

The ADA and the EASD recently published a consensus statement for the medical management of hyperglycaemia in patients with type 2 diabetes. The authors advocate initial treatment with metformin monotherapy and lifestyle modification, followed by addition of basal insulin or a sulfonylurea if glycaemic goals are not met (tier 1 recommendations). All other glucose-lowering therapies are relegated to a secondary (tier 2) status and only recommended for selected clinical settings. In our view, this algorithm does not offer physicians and patients the appropriate selection of options to individualise and optimise care with a view to sustained control of blood glucose and reduction both of diabetes complications and cardiovascular risk. This paper critically assesses the basis of the ADA/EASD algorithm and the resulting tiers of treatment options

How freshwater biomonitoring tools vary sub‐seasonally reflects temporary river flow regimes

Characterizing temporary river ecosystem responses to flow regimes is vital for conserving their biodiversity and the services they provide to society. However, freshwater biomonitoring tools rarely reflect community responses to hydrological variations or flow cessation events, and those available have not been widely tested within temporary rivers. This study examines two invertebrate biomonitoring tools characterizing community responses to different flow‐related properties: the “Drought Effect of Habitat Loss on Invertebrates” (DEHLI) and “Lotic‐invertebrate Index for Flow Evaluation” (LIFE), which, respectively reflect community responses to habitat and hydraulic properties associated with changing flow conditions. Sub‐seasonal (monthly) variations of LIFE and DEHLI were explored within two groundwater‐fed intermittent rivers, one dries sporadically (a flashy, karstic hydrology—River Lathkill) and the other dries seasonally (a highly buffered flow regime—South Winterbourne). Biomonitoring tools were highly sensitive to channel drying and also responded to reduced discharges in permanently flowing reaches. Biomonitoring tools captured ecological recovery patterns in the Lathkill following a supra‐seasonal drought. Some unexpected results were observed in the South Winterbourne where LIFE and DEHLI indicated relatively high‐flow conditions despite low discharges occurring during some summer months. This probably reflected macrophyte encroachment, which benefitted certain invertebrates (e.g., marginal‐dwelling taxa) and highlights the importance of considering instream habitat conditions when interpreting flow regime influences on biomonitoring tools. Although LIFE and DEHLI were positively correlated, the latter responded more clearly to drying events, highlighting that communities respond strongly to the disconnection of instream habitats as flows recede. The results highlighted short‐term ecological responses to hydrological variations and the value in adopting sub‐seasonal sampling strategies within temporary rivers. Findings from this study indicate the importance of establishing flow response guilds which group taxa that respond comparably to flow cessation events. Such information could be adopted within biomonitoring practices to better characterize temporary river ecosystem responses to hydrological variations