RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection

Early decrements in bone density after completion of neoadjuvant chemotherapy in pediatric bone sarcoma patients

<p>Abstract</p> <p>Background</p> <p>Bone mineral density (BMD) accrual during childhood and adolescence is important for attaining peak bone mass. BMD decrements have been reported in survivors of childhood bone sarcomas. However, little is known about the onset and development of bone loss during cancer treatment. The objective of this cross-sectional study was to evaluate BMD in newly diagnosed Ewing's and osteosarcoma patients by means of dual-energy x-ray absorptiometry (DXA) after completion of neoadjuvant chemotherapy.</p> <p>Methods</p> <p>DXA measurements of the lumbar spine (L2-4), both femora and calcanei were performed perioperatively in 46 children and adolescents (mean age: 14.3 years, range: 8.6-21.5 years). Mean <it>Z</it>-scores, areal BMD (g/cm²), calculated volumetric BMD (g/cm³) and bone mineral content (BMC, g) were determined.</p> <p>Results</p> <p>Lumbar spine mean Z-score was -0.14 (95% CI: -0.46 to 0.18), areal BMD was 1.016 g/cm²(95% CI: 0.950 to 1.082) and volumetric BMD was 0.330 g/cm³(95% CI: 0.314 to 0.347) which is comparable to healthy peers. For patients with a lower extremity tumor (n = 36), the difference between the affected and non-affected femoral neck was 12.1% (95% CI: -16.3 to -7.9) in areal BMD. The reduction of BMD was more pronounced in the calcaneus with a difference between the affected and contralateral side of 21.7% (95% CI: -29.3 to -14.0) for areal BMD. Furthermore, significant correlations for femoral and calcaneal DXA measurements were found with Spearman-rho coefficients ranging from ρ = 0.55 to ρ = 0.80.</p> <p>Conclusions</p> <p>The tumor disease located in the lower extremity in combination with offloading recommendations induced diminished BMD values, indicating local osteopenia conditions. However, the results revealed no significant decrements of lumbar spine BMD in pediatric sarcoma patients after completion of neoadjuvant chemotherapy. Nevertheless, it has to be taken into account that bone tumor patients may experience BMD decrements or secondary osteoporosis in later life. Furthermore, the peripheral assessment of BMD in the calcaneus via DXA is a feasible approach to quantify bone loss in the lower extremity in bone sarcoma patients and may serve as an alternative procedure, when the established assessment of femoral BMD is not practicable due to endoprosthetic replacements.</p

Interlaboratory comparison of methodologies for measuring the angle of incidence dependence of solar cells

The aim of this work is to compare angle of incidence (AOI) measurement setups for solar cells between laboratories with such capability. For the first time, we compare relative light transmission measurements among eight laboratories, whose measurement techniques include indoor and outdoor methods. We present the relative transmission measurements on three 156 mm x 156 mm crystalline-Si (c-Si) samples with different surface textures. The measurements are compared using the expanded uncertainties provided by each laboratory. Five of the eight labs showed an agreement better than ±2% to the weighted mean between AOIs from -75° to 70°. At AOIs of ±80° and ±85°, the same five labs showed a worst case deviation to the weighted mean of -3% to 5% and 0% to 18%, respectively. When measurement uncertainty is considered, the results show that measurements at the highest incidence angle of ±85° are problematic, as measurements from four out of the six labs reporting uncertainty were found non-comparable within their stated uncertainties. At 85° AOI a high to low range of up to 75% was observed between all eight laboratories

An infection-relevant transcriptomic compendium for Salmonella enterica Serovar Typhimurium.

Bacterial transcriptional networks consist of hundreds of transcription factors and thousands of promoters. However, the true complexity of transcription in a bacterial pathogen and the effect of the environments encountered during infection remain to be established. We present a simplified approach for global promoter identification in bacteria using RNA-seq-based transcriptomic analyses of 22 distinct infection-relevant environmental conditions. Individual RNA samples were combined to identify most of the 3,838 Salmonella enterica serovar Typhimurium promoters in just two RNA-seq runs. Individual in vitro conditions stimulated characteristic transcriptional signatures, and the suite of 22 conditions induced transcription of 86% of all S. Typhimurium genes. We highlight the environmental conditions that induce the Salmonella pathogenicity islands and present a small RNA expression landscape of 280 sRNAs. This publicly available compendium of environmentally controlled expression of every transcriptional feature of S. Typhimurium constitutes a useful resource for the bacterial research community

Broad scale redistribution of mRNA abundance and transcriptional machinery in response to growth rate in Salmonella enterica serovar Typhimurium

We have investigated the connection between the four-dimensional architecture of the bacterial nucleoid and the organism\u27s global gene expression programme. By localizing the transcription machinery and the transcriptional outputs across the genome of the model bacterium Salmonella enterica serovar Typhimurium at different stages of the growth cycle, a surprising disconnection between gene dosage and transcriptional output was revealed. During exponential growth, gene output occurred chiefly in the Ori (origin), Ter (terminus) and NSL (non-structured left) domains, whereas the Left macrodomain remained transcriptionally quiescent at all stages of growth. The apparently high transcriptional output in Ter was correlated with an enhanced stability of the RNA expressed there during exponential growth, suggesting that longer mRNA half-lives compensate for low gene dosage. During exponential growth, RNA polymerase (RNAP) was detected everywhere, whereas in stationary phase cells, RNAP was concentrated in the Ter macrodomain. The alternative sigma factors RpoE, RpoH and RpoN were not required to drive transcription in these growth conditions, consistent with their observed binding to regions away from RNAP and regions of active transcription. Specifically, these alternative sigma factors were found in the Ter macrodomain during exponential growth, whereas they were localized at the Ori macrodomain in stationary phase

Diffusion in diamond-like carbon

The diffusion of carbon and five other elements in amorphous carbon (a-C) films was studied. One set of samples were sp(2) dominated a-C and the other set of samples were sp(3) dominated tetrahedral amorphous carbon (ta-C). The films were deposited using mass separated ion beam deposition under UHV conditions. The diffusion of C-13 as well as that of hydrogen and deuterium was studied using high resolution elastic recoil detection analysis. No apparent self-diffusion could be detected using this technique. The diffusion of hydrogen was found to start at temperatures between 600 and 800 degreesC. For deuterium, activation energies of 3.34(5) and 3.39(5) eV were found for diffusion in ta-C and a-C, respectively. Tungsten, copper and silver were used to study the diffusion of metals in ta-C. Up to annealing temperatures of 1000 degreesC no diffusion took place in the samples. During annealing at 1200 degreesC the ta-C is converted into graphite, making diffusion into the carbon matrix possible. The fact that there is no diffusion of copper in ta-C at temperatures below 1200 degreesC shows that ta-C is a possible diffusion barrier between copper and silicon. (C) 2003 Elsevier Science B.V. All rights reserved