Dosimetry for quantitative analysis of the effects of low-dose ionizing radiation in radiation therapy patients

We have developed and validated a practical approach to identifying the location on the skin surface that will receive a prespecified biopsy dose (ranging down to 1 cGy) in support of in vivo biological dosimetry in humans. This represents a significant technical challenge since the sites lie on the patient's surface outside the radiation fields. The PEREGRINE Monte Carlo simulation system was used to model radiation dose delivery, and TLDs were used for validation on phantoms and for confirmation during patient treatment. In the developmental studies, the Monte Carlo simulations consistently underestimated the dose at the biopsy site by approximately 15% (of the local dose) for a realistic treatment configuration, most likely due to lack of detail in the simulation of the linear accelerator outside the main beam line. Using a single, thickness-independent correction factor for the clinical calculations, the average of 36 measurements for the predicted 1-cGy point was 0.985 cGy (standard deviation: 0.110 cGy) despite patient breathing motion and other real-world challenges. Since the 10-cGy point is situated in the region of high-dose gradient at the edge of the field, patient motion had a greater effect, and the six measured points averaged 5.90 cGy (standard deviation: 1.01 cGy), a difference that is equivalent to approximately a 6-mm shift on the patient's surface.     

Platelet-activating factor receptor and innate immunity: uptake of gram-positive bacterial cell wall into host cells and cell-specific pathophysiology

The current model of innate immune recognition of Gram-positive bacteria suggests that the bacterial cell wall interacts with host recognition proteins such as TLRs and Nod proteins. We describe an additional recognition system mediated by the platelet-activating factor receptor (PAFr) and directed to the pathogen-associated molecular pattern phosphorylcholine that results in the uptake of bacterial components into host cells. Intravascular choline-containing cell walls bound to endothelial cells and caused rapid lethality in wild-type, Tlr2(-/-), and Nod2(-/-) mice but not in Pafr(-/-) mice. The cell wall exited the vasculature into the heart and brain, accumulating within endothelial cells, cardiomyocytes, and neurons in a PAFr-dependent way. Physiological consequences of the cell wall/PAFr interaction were cell specific, being noninflammatory in endothelial cells and neurons but causing a rapid loss of cardiomyocyte contractility that contributed to death. Thus, PAFr shepherds phosphorylcholine-containing bacterial components such as the cell wall into host cells from where the response ranges from quiescence to severe pathophysiology.     

Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance

Hepatitis C virus (HCV) clearance has been associated with reduced viral evolution in targeted cytotoxic T-lymphocyte (CTL) epitopes, suggesting that HCV clearers may mount CTL responses with a superior ability to recognize epitope variants and prevent viral immune escape. Here, 40 HCV-infected subjects were tested with 406 10-mer peptides covering the vast majority of the sequence diversity spanning a 197-residue region of the NS3 protein. HCV clearers mounted significantly broader CTL responses of higher functional avidity and with wider variant cross-recognition capacity than nonclearers. These observations have important implications for vaccine approaches that may need to induce high-avidity responses in vivo.     

An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments

Two cell recovery methods for IOB enrichments were evaluated for DNA extraction and further PCR-based 16S rRNA gene clone library creation. One was a published method consisting of heating plus oxalic acid treatment and the other one was a new method based on enzymatic agarose digestion (using beta-agarase I). The results indicated that the enzymatic method was much gentler on IOB cells and yielded an approximately 5000-fold higher DNA mass than the published method. The 16S rRNA gene clone library developed from the beta-agarase I treated IOB enrichments indicated a high IOB community diversity with sequences in alpha-, beta-, gamma-, delta-, epsilon-Proteobacteria, unclassified Proteobacteria, unclassified Bacteroidetes and unclassified Bacteria. In contrast, the published method resulted in mainly gamma-Proteobacterial clone sequences. In addition, only the cells recovered by agarase treatment were amenable to direct fluorescence in situ hybridization (FISH). Therefore, we propose that the agarase method is a better IOB cell recovery method, because it is simpler, faster, and retains more genetic diversity.     

Localized proton magnetic resonance spectroscopy of lipids in adipose tissue at high spatial resolution in mice in vivo

We describe a localized proton magnetic resonance spectroscopy ((1)H-MRS) method for in vivo measurement of lipid composition in very small voxels (1.5 mm x 1.5 mm x 1.5 mm) in adipose tissue in mice. The method uses localized point-resolved spectroscopy to collect (1)H spectra from voxels in intra-abdominal white adipose tissue (WAT) and brown adipose tissue (BAT) deposits. Nonlinear least-squares fits of the spectra in the frequency domain allow for accurate calculation of the relative amount of saturated, monounsaturated, and polyunsaturated fatty acids. All spectral data are corrected for spin-spin relaxation. The data show BAT of NMRI mice to be significantly different from BAT of NMRI nu/nu mice in all aspects except for the fraction of monounsaturated fatty acids (FM); for WAT, only the FM is different. BAT and WAT of NMRI mice differ in the amount of saturated and di-unsaturated fatty acids. This method provides a potential tool for studying lipid metabolism in small animal models of disease during the initiation, progression, and manifestation of obesity-related disorders in vivo. Our results clearly demonstrate that localized (1)H-MRS of adipose tissue in vivo is possible at high spatial resolution with voxel sizes down to 3.4 ml.     

Impact of cyclopentenone-oxylipins on the proteome of Arabidopsis thaliana

Both enzymatically and non-enzymatically generated oxylipins fulfill signalling functions in plant responses to biotic and oxidative stress on the cellular level. We studied the impact of two different exogenously applied cyclopentenone-oxylipins on the proteome of Arabidopsis thaliana leaves: the enzymatically formed 12-oxo-phytodienoic-acid, a member of the jasmonate family of mediators, and A(1)-phytoprostane generated by a free-radical mechanism upon biotic and oxidative stress. Infiltration of leaves with these oxylipins led to induction of classical stress proteins like chaperones as well as enzymes connected to the cellular redox and detoxification systems. A large proportion of the regulated proteins are localized in chloroplasts where these oxylipins are formed. Furthermore, we show that cyclopentenone-oxylipins spontaneously react with several proteins and glutathione in vitro and in vivo. Conjugation to the glutathione sulfhydryl group is a reversible process that is also catalysed by glutathione-S-transferases. In vitro, an oxidative stress inducible glutathione-S-transferase, GST6, localized both in plastids and the cytosol can be covalently modified and partially inactivated by several cyclopentenone-oxylipins.     

Recycling of apolipoprotein E is not associated with cholesterol efflux in neuronal cells

After receptor-mediated endocytosis of apolipoprotein E (apoE)-containing lipoproteins in hepatocytes, the isoform apoE3 is efficiently recycled in a process which is associated with cholesterol efflux. Recycling and cholesterol efflux are greatly reduced when apoE4 is the only isoform present. ApoE is the main apolipoprotein in cerebrospinal fluid, and it plays a pivotal role in maintaining cholesterol homeostasis in the brain. The isoform apoE4 is associated with an increased risk of Alzheimer's disease and it has been postulated that high intracellular cholesterol levels promote the amyloidogenic processing of amyloid precursor protein. Therefore we investigated the cellular processing of different apoE isoforms as well as the associated cholesterol efflux in the murine neuronal cell line HT-22. Uptake of apoE3-containing lipoproteins resulted in the expected recycling while, as seen in non-neuronal cells, recycling of apoE4 was significantly reduced. However, despite these differences in apoE recycling, there was no difference in rates of cholesterol efflux. Therefore we conclude that in this neuronal cell model the reduced recycling of apoE4 does not affect cellular cholesterol metabolism.     

Photoaffinity labelling of the human GM2-activator protein. Mechanistic insight into ganglioside GM2 degradation.

The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of ganglioside GM2 by lysosomal beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this glycoprotein cofactor, we synthesized the two photoaffinity labels [14C]C3-TPD-GM2 and [14C]C7-TPD-GM2. Incubation of GM2AP with these substrate analogues and subsequent irradiation led to covalent labelling of the protein. After separation of tryptic peptides by reverse-phase HPLC, the labelled peptide fractions were analysed by MALDI-TOF and sequenced by ESI-Q-TOF mass spectrometry. Both labels were found to be specifically photoincorporated into a part of the surface loop comprising residues V153-L163, a stretch of amino acids that was previously identified as the most flexible region in the crystal structure of the activator. Our results provide strong evidence that this loop constitutes the part of the activator protein that directly interacts with the ganglioside substrate, suggesting that the hydrophobicity and the great structural mobility of this element are crucial for the extraction of the membrane-embedded glycolipid, its stabilization inside the spacious cavity and its guidance to the enzyme's active site. This study demonstrates that the approach of photoaffinity labelling in conjunction with accurate mass measurements can provide insight into substrate binding interactions that complements structural information.     

Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing

miRNA-mediated gene silencing requires the GW182 proteins, which are characterized by an N-terminal domain that interacts with Argonaute proteins (AGOs), and a C-terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)-binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP-interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C-terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N-terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A-C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A-C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species-specific differences in the relative strength of the PABPC1-binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA-mediated silencing in animal cells.