Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

Potential of gas chromatography–atmospheric pressure chemical ionization–time-of-flight mass spectrometry for the determination of sterols in human plasma

The application of Gas Chromatography (GC)–Atmospheric Pressure Chemical Ionization (APCI)–Time-of-Flight Mass Spectrometry (TOF-MS) is presented for sterol analysis in human plasma. A commercial APCI interface was modified to ensure a well-defined humidity which is essential for controlled ionization. In the first step, optimization regarding flow rates of auxiliary gases was performed by using a mixture of model analytes. Secondly, the qualitative and quantitative analysis of sterols including oxysterols, cholesterol precursors, and plant sterols as trimethylsilyl-derivatives was successfully carried out. The characteristics of APCI together with the very good mass accuracy of TOF-MS data enable the reliable identification of relevant sterols in complex matrices. Linear calibration lines and plausible results for healthy volunteers and patients could be obtained whereas all mass signals were extracted with an extraction width of 20 ppm from the full mass data set. One advantage of high mass accuracy can be seen in the fact that from one recorded run any search for m/z can be performed.     

The human lambda L chain Ig locus. Recharacterization of JC lambda 6 and identification of a functional JC lambda 7

The human lambda L chain Ig gene complex consists of multiple JC gene segments. A seventh human lambda C region gene segment, C lambda 7, was found 2.7 kb downstream of C lambda 6 in this gene complex. A J lambda gene segment, J lambda 7, was found 1.2 kb upstream of C lambda 7 and contains potentially functional nonamer and heptamer recombination sites, an RNA splice site and J coding region. C lambda 7 maintains an open reading frame and encodes a new lambda isotype. C lambda 7 encodes Kern+ and Oz- determinants, but does not encode any of the Kern+Oz- myeloma proteins published to date. Nevertheless, we present evidence that JC lambda 7 is transcribed in normal lymphocytes and is functional. In contrast, we present new data that the C lambda 6 gene segment, reported by others to encode the Kern+Oz- protein, is non-functional due to a 4-bp insertion in our cosmid clone. The 4-bp insertion was characterized further in 32 genomic DNA samples by producing a distinctive restriction fragment length and verified by the DNA sequences of the polymerase chain reaction products of two different cell lines. We discuss the possibility that the Kern+Oz- myeloma proteins do not define an isotype and are not encoded by JC lambda 7 nor other non-allelic genes, and we discuss the level of expression of JC lamba 7 as compared to that of JC lambda 2 and JC lambda 3.     

Localization of a Cell Adhesion Site on Collagen XIV (Undulin)

Cell adhesion to collagen XIV is implied to be mediated by proteoglycans as cellular receptors (T. Ehniset al.,1996,Exp. Cell Res.229, 388–397). In order to define the cell binding region(s), fusion proteins expressed inEscherichia coliand covering the large noncollagenous domain NC3 of collagen XIV were used as substrates for the adhesion of skin fibroblasts. A prominent cell binding site could be localized in the N-terminal fibronectin type III repeat of collagen XIV and its immediate C-terminal extension. Since this region also mediates the binding of the small chondroitin/dermatan sulfate proteoglycan decorin (T. Ehniset al.,1997,J. Biol. Chem.272, 20414–20419), our finding could provide the molecular basis for the observation that decorin serves as inhibitor and potential modulator of cellular interactions with collagen XIV.     

Morphological diversity of Ethiopian barley (Hordeum vulgare L.) in relation to geographic regions and altitudes

A total of 199 germplasm accessions collected from 10 administrative regions of Ethiopia and four released cultivars, which were used for estimating of error variance, of barley in Ethiopia were field evaluated for nine agronomic traits at Holetta and Bekoji Agricultural Research Centers of Ethiopia during the 2006 main cropping season using non-replicated augmented design plots consisting of four incomplete blocks. The objectives were to assess the extent and pattern of morphological variation in the barley accessions with respect to regions and altitude of collection, to classify the genotypes tested into relatively homogenous groups and to identify the major traits contributing to the overall observed diversity in the germplasm. Genotype variance estimate of regions and altitudes indicated wide variation among accessions depending on the traits involved. The presence of high morphological variation within regions and altitudes particularly above 2000 m a.s.l. indicated the potential of each region and high altitude zones for barley improvement and conservation in the country. The clustering of accessions did not show grouping on the basis of regions of origin. Traits like thousand kernel weight, plant height, days to head and days to maturity accounted for most of the gross variance among the barley accessions and played role in differentiating accessions collected from different regions and altitude classes into principal components. In general because of environmental factors on the observed morphological variation future germplasm collection should consider to explore wide geographical and climatic differences within the country.     

<Emphasis Type="Italic">Mycosphaerella podagrariae</Emphasis>—a necrotrophic phytopathogen forming a special cellular interaction with its host <Emphasis Type="Italic">Aegopodium podagraria</Emphasis>

We present a new kind of cellular interaction found between Mycosphaerella podagrariae and Aegopodium podagraria, which is remarkably different to the interaction type of the obligate biotrophic fungus Cymadothea trifolii, another member of the Mycosphaerellaceae (Capnodiales, Dothideomycetes, Ascomycota) which we have described earlier. Observations are based on both conventional and cryofixed material and show that some features of this particular interaction are better discernable after chemical fixation. We were also able to generate sequences for nuclear ribosomal DNA (complete SSU, 5.8 S and flanking ITS-regions, D1–D3 region of the LSU) confirming the position of M. podagrariae within Mycosphaerellaceae.     

Review and classification of variability analysis techniques with clinical applications

Background

Representation of independent biophysical sources using Fourier analysis can be inefficient because the basis is sinusoidal and general. When complex fractionated atrial electrograms (CFAE) are acquired during atrial fibrillation (AF), the electrogram morphology depends on the mix of distinct nonsinusoidal generators. Identification of these generators using efficient methods of representation and comparison would be useful for targeting catheter ablation sites to prevent arrhythmia reinduction.

Method

A data-driven basis and transform is described which utilizes the ensemble average of signal segments to identify and distinguish CFAE morphologic components and frequencies. Calculation of the dominant frequency (DF) of actual CFAE, and identification of simulated independent generator frequencies and morphologies embedded in CFAE, is done using a total of 216 recordings from 10 paroxysmal and 10 persistent AF patients. The transform is tested versus Fourier analysis to detect spectral components in the presence of phase noise and interference. Correspondence is shown between ensemble basis vectors of highest power and corresponding synthetic drivers embedded in CFAE.

Results

The ensemble basis is orthogonal, and efficient for representation of CFAE components as compared with Fourier analysis (p ≤ 0.002). When three synthetic drivers with additive phase noise and interference were decomposed, the top three peaks in the ensemble power spectrum corresponded to the driver frequencies more closely as compared with top Fourier power spectrum peaks (p ≤ 0.005). The synthesized drivers with phase noise and interference were extractable from their corresponding ensemble basis with a mean error of less than 10%.

Conclusions

The new transform is able to efficiently identify CFAE features using DF calculation and by discerning morphologic differences. Unlike the Fourier transform method, it does not distort CFAE signals prior to analysis, and is relatively robust to jitter in periodic events. Thus the ensemble method can provide a useful alternative for quantitative characterization of CFAE during clinical study.