A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation

BACKGROUND: Basement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm(2)) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations. RESULTS: Geltrex™ basement matrix at 5 μl/cm(2) in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix. CONCLUSIONS: We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users

Genetic variation exists for telomeric array organization within and among the genomes of normal, immortalized, and transformed chicken systems

This study investigated telomeric array organization of diverse chicken genotypes utilizing in vivo and in vitro cells having phenotypes with different proliferation potencies. Our experimental objective was to characterize the extent and nature of array variation present to explore the hypothesis that mega-telomeres are a universal and fixed feature of chicken genotypes. Four different genotypes were studied including normal (UCD 001, USDA-ADOL Line 0), immortalized (DF-1), and transformed (DT40) cells. Both cytogenetic and molecular approaches were utilized to develop an integrated view of telomeric array organization. It was determined that significant variation exists within and among chicken genotypes for chromosome-specific telomeric array organization and total genomic-telomeric sequence content. Although there was variation for mega-telomere number and distribution, two mega-telomere loci were in common among chicken genetic lines (GGA 9 and GGA W). The DF-1 cell line was discovered to maintain a complex derivative karyotype involving chromosome fusions in the homozygous and heterozygous condition. Also, the DF-1 cell line was found to contain the greatest amount of telomeric sequence per genome (17%) as compared to UCD 001 (5%) and DT40 (1.2%). The chicken is an excellent model for studying unique and universal features of vertebrate telomere biology, and characterization of the telomere length variation among genotypes will be useful in the exploration of mechanisms controlling telomere length maintenance in different cell types having unique phenotypes

FluTE, a Publicly Available Stochastic Influenza Epidemic Simulation Model

Mathematical and computer models of epidemics have contributed to our understanding of the spread of infectious disease and the measures needed to contain or mitigate them. To help prepare for future influenza seasonal epidemics or pandemics, we developed a new stochastic model of the spread of influenza across a large population. Individuals in this model have realistic social contact networks, and transmission and infections are based on the current state of knowledge of the natural history of influenza. The model has been calibrated so that outcomes are consistent with the 1957/1958 Asian A(H2N2) and 2009 pandemic A(H1N1) influenza viruses. We present examples of how this model can be used to study the dynamics of influenza epidemics in the United States and simulate how to mitigate or delay them using pharmaceutical interventions and social distancing measures. Computer simulation models play an essential role in informing public policy and evaluating pandemic preparedness plans. We have made the source code of this model publicly available to encourage its use and further development

Natural Killer Lysis Receptor (NKLR)/NKLR-Ligand Matching as a Novel Approach for Enhancing Anti-Tumor Activity of Allogeneic NK Cells

NK cells are key players in anti tumor immune response, which can be employed in cell-based therapeutic modalities. One of the suggested ways to amplify their anti tumor effect, especially in the field of stem cell transplantation, is by selecting donor/recipient mismatches in specific HLA, to reduce the inhibitory effect of killer Ig-like receptors (KIRs). Here we suggest an alternative approach for augmentation of anti tumor effect of allogeneic NK cells, which is founded on profile matching of donor NK lysis receptors (NKLR) phenotype with tumor lysis-ligands.We show that an NKLR-mediated killing directly correlates with the NKLR expression intensity on NK cells. Considerable donor variability in the expression of CD16, NKp46, NKG2D and NKp30 on circulating NK cells, combined with the stability of phenotype in several independently performed tests over two months, indicates that NKLR-guided selection of donors is feasible. As a proof of concept, we show that melanoma cells are dominantly recognized by three NKLRs: NKG2D, NKp30 and NKp44. Notably, the expression of NKp30 on circulating NK cells among metastatic melanoma patients was significantly decreased, which diminishes their ability to kill melanoma cells. Ex vivo expansion of NK cells results not only in increased amount of cells but also in a consistently superior and predictable expression of NKG2D, NKp30 and NKp44. Moreover, expanded NK cultures with high expression of NKG2D or NKp30 were mostly derived from the corresponding NKG2D(high) or NK30(high) donors. These NK cultures subsequently displayed an improved cytotoxic activity against melanoma in a HLA/KIR-ligand mismatched setup, which was NKLR-dependent, as demonstrated with blocking anti-NKG2D antibodies.NKLR/NKLR-ligand matching reproducibly elicits enhanced NK anti-tumor response. Common NKLR recognition patterns of tumors, as demonstrated here in melanoma, would allow implementation of this approach in solid malignancies and potentially in hematological malignancies, either independently or in adjunction to other modalities

A meta-analytic review of stand-alone interventions to improve body image

Objective Numerous stand-alone interventions to improve body image have been developed. The present review used meta-analysis to estimate the effectiveness of such interventions, and to identify the specific change techniques that lead to improvement in body image. Methods The inclusion criteria were that (a) the intervention was stand-alone (i.e., solely focused on improving body image), (b) a control group was used, (c) participants were randomly assigned to conditions, and (d) at least one pretest and one posttest measure of body image was taken. Effect sizes were meta-analysed and moderator analyses were conducted. A taxonomy of 48 change techniques used in interventions targeted at body image was developed; all interventions were coded using this taxonomy. Results The literature search identified 62 tests of interventions (N = 3,846). Interventions produced a small-to-medium improvement in body image (d+ = 0.38), a small-to-medium reduction in beauty ideal internalisation (d+ = -0.37), and a large reduction in social comparison tendencies (d+ = -0.72). However, the effect size for body image was inflated by bias both within and across studies, and was reliable but of small magnitude once corrections for bias were applied. Effect sizes for the other outcomes were no longer reliable once corrections for bias were applied. Several features of the sample, intervention, and methodology moderated intervention effects. Twelve change techniques were associated with improvements in body image, and three techniques were contra-indicated. Conclusions The findings show that interventions engender only small improvements in body image, and underline the need for large-scale, high-quality trials in this area. The review identifies effective techniques that could be deployed in future interventions

Screening for psychological distress in patients with lung cancer: results of a clinical audit evaluating the use of the patient Distress Thermometer

Patients with lung cancer frequently suffer psychological distress and guidelines in the United Kingdom recommend screening of all cancer patients for this problem. The audit investigated use of the Distress Thermometer in terms of staff adherence to locally developed guidelines, patient willingness to use the tool, its impact on referral rates to clinical psychology services and concordance between the tool and the clinical assessment. Use of the Distress Thermometer was audited over a 3-month period in one lung cancer outpatient clinic. Referrals to clinical psychology services in response to clearly delineated referral indicators were assessed. Patient-reported outcomes were compared with practitioner assessment of need during clinical consultations to see whether the tool was measuring distress effectively. Thirty three of 34 patients used the Distress Thermometer during the audit period. Ten reported distress levels above 4 in the emotional or family problems domains. On ten occasions, the clinical interview identified problems not elicited by the Distress Thermometer. Guidelines were adhered to by staff, and patients were offered information about local support services and referral to clinical psychology services where indicated. Whilst all patients were happy to receive written information about further sources of support, none wanted to be referred to psychological services at that time. The Distress Thermometer is acceptable to patients with lung cancer in outpatient settings but it did not increase referrals for psychological support. Staff found it to be a useful tool in opening up communication about patient issues although it should not replace a comprehensive clinical interview

Would school closure for the 2009 H1N1 influenza epidemic have been worth the cost?: a computational simulation of Pennsylvania

<p>Abstract</p> <p>Background</p> <p>During the 2009 H1N1 influenza epidemic, policy makers debated over whether, when, and how long to close schools. While closing schools could have reduced influenza transmission thereby preventing cases, deaths, and health care costs, it may also have incurred substantial costs from increased childcare needs and lost productivity by teachers and other school employees.</p> <p>Methods</p> <p>A combination of agent-based and Monte Carlo economic simulation modeling was used to determine the cost-benefit of closing schools (vs. not closing schools) for different durations (range: 1 to 8 weeks) and symptomatic case incidence triggers (range: 1 to 30) for the state of Pennsylvania during the 2009 H1N1 epidemic. Different scenarios varied the basic reproductive rate (R₀) from 1.2, 1.6, to 2.0 and used case-hospitalization and case-fatality rates from the 2009 epidemic. Additional analyses determined the cost per influenza case averted of implementing school closure.</p> <p>Results</p> <p>For all scenarios explored, closing schools resulted in substantially higher net costs than not closing schools. For R₀= 1.2, 1.6, and 2.0 epidemics, closing schools for 8 weeks would have resulted in median net costs of $21.0 billion (95$8.0 - $45.3 billion). The median cost per influenza case averted would have been$14,185 ($5,423 -$30,565) for R₀= 1.2, $25,253 ($9,501 - $53,461) for R₀= 1.6, and$23,483 ($8,870 -$50,926) for R₀= 2.0.</p> <p>Conclusions</p> <p>Our study suggests that closing schools during the 2009 H1N1 epidemic could have resulted in substantial costs to society as the potential costs of lost productivity and childcare could have far outweighed the cost savings in preventing influenza cases.</p