Orthogonal projections to latent structures as a strategy for microarray data normalization

Background  

During generation of microarray data, various forms of systematic biases are frequently introduced which limits accuracy and precision of the results. In order to properly estimate biological effects, these biases must be identified and discarded.     

Exonuclease Degradation of DNA Studied by Fluorescence Correlation Spectroscopy

Abstract

Here we developed an accurate method for kinetic analysis of enzymatic degradation processes of double and/or single-stranded DNA/oligonucleotides using fluorescent reporter dyes. 217-bp DNA fragments were produced by polymerase chain reaction and cleaved by the 3′ to 5′ exonuclease activity of T7-DNA polymerase. The analysis of the products was performed by Fluorescence Correlation Spectroscopy measuring autocorrelation amplitudes and diffusion times. We give proof of (i) complete enzymatic degradation, (ii) retardation of complete enzymatic degradation by internally labelled Rhodamine-4-nucleotides and Cy5-nucleotides, respectively. Data evaluation by global analysis indicated first-order reaction kinetics with full-length DNA and free fluorescent nucleotides in the time window of measurements used.     

Mast cell accumulation in glioblastoma with a potential role for stem cell factor and chemokine CXCL12

Glioblastoma multiforme (GBM) is the most common and malignant form of glioma with high mortality and no cure. Many human cancers maintain a complex inflammatory program triggering rapid recruitment of inflammatory cells, including mast cells (MCs), to the tumor site. However, the potential contribution of MCs in glioma has not been addressed previously. Here we report for the first time that MCs infiltrate KRas+Akt-induced gliomas, using the RCAS/TV-a system, where KRas and Akt are transduced by RCAS into the brains of neonatal Gtv-a- or Ntv-a transgenic mice lacking Ink4a or Arf. The most abundant MC infiltration was observed in high-grade gliomas of Arf-/- mice. MC accumulation could be localized to the vicinity of glioma-associated vessels but also within the tumor mass. Importantly, proliferating MCs were detected, suggesting that the MC accumulation was caused by local expansion of the MC population. In line with these findings, strong expression of stem cell factor (SCF), i.e. the main MC growth factor, was detected, in particular around tumor blood vessels. Further, glioma cells expressed the MC chemotaxin CXCL12 and MCs expressed the corresponding receptor, i.e. CXCR4, suggesting that MCs could be attracted to the tumor through the CXCL12/CXCR4 axis. Supporting a role for MCs in glioma, strong MC infiltration was detected in human glioma, where GBMs contained significantly higher MC numbers than grade II tumors did. Moreover, human GBMs were positive for CXCL12 and the infiltrating MCs were positive for CXCR4. In conclusion, we provide the first evidence for a role for MCs in glioma.     

Born to cope with climate change? Experimentally manipulated hatching time does not affect duckling survival in the mallard <Emphasis Type="Italic">Anas platyrhynchos</Emphasis>

Two main hypotheses proposed to explain the seasonal decline in reproductive performance in birds are (1) deterioration of environmental conditions and (2) lower parental quality of late breeders. Previous experimental work addressing these hypotheses generally have problematic biases pertaining to delay of hatching, costs of re-laying and incubation, as well as variation in the quality of eggs, territories, offspring and parental traits. We address these biases in an experimental test of the timing hypothesis (i.e. (1) above) in a precocial bird. Using a 2-year cross-over design and game-farm mallard (Anas platyrhynchos) eggs originating from a number of hens and a standardised delay procedure, we introduced early and late broods with a foster female onto boreal oligotrophic lakes and monitored subsequent duckling survival. Standardised invertebrate sampling was done concurrently to get a measure of lake-level abundance of aquatic prey, a likely causative agent of putative seasonal difference in duckling survival. Survival data and covariates (duckling age; days) were analysed by an information theoretic approach. There was no effect of treatment (i.e. manipulation of hatching date) on duckling survival, which was higher in 2005 than in 2004. In contrast to observational studies from more seasonal wetlands, our experiment demonstrates that duckling survival on boreal lakes was not affected by a 12-day delay in hatching date. Since we did not find any consistent trends in abundance of aquatic prey, i.e. neither clear peaks nor differences between treatment periods, we hypothesise that moderate climate change has minor effects on resource abundance and hence also on mallard duckling survival in boreal environments.     

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

Acrylic microspheres in vivo. X. Elimination of circulating cells by active targeting using specific monoclonal antibodies bound to microparticles

The elimination from the blood of 51Cr-labelled mouse erythrocytes modified with trinitrophenyl (TNP) groups was followed in mice. After 24 hours, when a stable concentration of the labelled erythrocytes has been attained, monoclonal anti-TNP-antibodies were given intravenously, either in free, soluble form, or bound to microparticles containing immobilized protein A. The anti-TNP-antibodies induced a rapid elimination of the TNP- and 51Cr-labelled erythrocytes. Over the 8-hours time period studied, the elimination rate was significantly faster when the antibodies were administered bound to the particles. After the elimination of the target cells, the radioactivity was found in the liver, spleen and bone marrow. These results and relevant control experiments indicate that a solid carrier 1. can be directed to a specific target cell with a specific antibody and 2. can induce a rapid elimination of the target cell from the circulation.     

Biosurfactant yields and nutrient consumption of Pseudomonas fluorescens 378 studied in a microcomputer controlled multifermentation system

Production of biosurfactant AP-6 and consumption of carbon (succinic acid) and nitrogen (ammonium ions) by Pseudomonas fluorescens 378 were studied under different growth conditions. The study was performed in a microcomputer controlled multibatch fermentation system which enabled simultaneous running of 10 fermentors. The fermentors were mantled glass vessels, temperature controlled by circulated water, and mixing was arranged by magnetic stirrers. They were connected to the computer system (pH measurement and control) via signal conditioning cards. The microcomputer had a 128 kbytes RAM, two 800-kbyte floppy disc drives, a graphic terminal, and expansion cards. Biosurfactant production was independent of the carbon-to-nitrogen ratio and the phosphorus content in the medium. Omitting the Fe(III) supplement to the medium increased the product yield by 120%. Changes in oxygen transfer rate and pH in the iron deficient cultures did not have any effect on the product yield. Iron deficiency increased the cell consumption of carbon source. Consumption of carbon source in relation to nitrogen uptake (carbon/nitrogen quotient) increased with increasing quotient in the growth medium. The uptake of carbon and nitrogen changed in the intervals of 1.2-1.5 g/g biomass and 0.09-0.16 g/g biomass, respectively. The consumption of carbon increased from 1.5 g/g biomass to 2.0 g/g biomass when the medium concentration of phosphorus was decreased from 0.18 to 0.027 g/L.