Identification of conjugated linoleic acid elongation and beta-oxidation products by coupled silver-ion HPLC APPI-MS

Atmospheric pressure photoionisation (APPI) was used in combination with silver-ion (Ag(+))-HPLC for detection of (conjugated) fatty acid methyl esters (FAME) by tandem-mass spectrometry. APPI-MS of methyl esters of conjugated linoleic acid showed an increase in signal-to-noise ratio by a factor of 40 compared to atmospheric pressure chemical ionization in the positive mode. It was possible to identify double bond position, configuration and chain length of FAME based on chromatographic separation and mass detection. The developed LC-MS method is useful for the analysis of CLA elongation and beta-oxidation products, especially with trans,trans-configuration, which are difficult to analyze by conventional GC-MS techniques.     

Diet selection by hares (Lepus europaeus) in arable land and its implications for habitat management

Populations of European hares (Lepus europaeus) have experienced a dramatic decline throughout Europe in recent decades. European hares are assumed to prefer weeds over arable crops, and weed abundance was reduced by the intensification of agriculture. Therefore, modern agriculture has been blamed as a major factor affecting European hare populations. However, it is questionable whether European hares select weeds at all, as previous studies had major methodological limitations. By comparing availability and use of plants with Chesson’s Electivity Index, we investigated whether the European hare actually feeds selectively on different plants in arable land. Food availability and use were dominated by cultivated crops (e.g. winter wheat, spring barley and sugar beet). Diet selection analysis revealed that in autumn and winter, European hares predominantly preferred cultivated crops (winter wheat) and food items provided by hunters (tubers of sugar beet and carrot). In spring and summer, apart from soy, only weeds (e.g. clover and corn poppy) were positively selected, especially after cereal crops were harvested. We suggest that the decline in European hare populations throughout Europe was facilitated by the decrease in weed abundance. Wildlife-friendly set-asides in arable land have the potential to reconcile the European Union’s Common Agricultural Policy with wildlife conservation.     

Statistics for proteomics: a review of tools for analyzing experimental data

Most proteomics experiments make use of 'high throughput' technologies such as 2-DE, MS or protein arrays to measure simultaneously the expression levels of thousands of proteins. Such experiments yield large, high-dimensional data sets which usually reflect not only the biological but also technical and experimental factors. Statistical tools are essential for evaluating these data and preventing false conclusions. Here, an overview is given of some typical statistical tools for proteomics experiments. In particular, we present methods for data preprocessing (e.g. calibration, missing values estimation and outlier detection), comparison of protein expression in different groups (e.g. detection of differentially expressed proteins or classification of new observations) as well as the detection of dependencies between proteins (e.g. protein clusters or networks). We also discuss questions of sample size planning for some of these methods.     

Proteases and chaperones are the most abundant proteins in the parasitophorous vacuole of Plasmodium falciparum-infected erythrocytes

After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole (PV) which forms an interface between the host cell cytosol and the parasite surface. This vacuole protects the parasite from potentially harmful substances, but allows access of essential nutrients to the parasite. Furthermore, the vacuole acts as a transit compartment for parasite proteins en route to the host cell cytoplasm. Recently we developed a strategy to biotin label soluble proteins of the PV. Here, we have paired this strategy with a high-throughput MALDI-TOF-MS analysis to identify 27 vacuolar proteins. These proteins fall into the following main classes: chaperones, proteases, and metabolic enzymes, consistent with the expected functions of the vacuole. These proteins are likely to be involved in several processes including nutrient acquisition from the host cytosol, protein sorting within the vacuole, and release of parasites at the end of the intraerythrocytic cycle.     

Sperm numbers vary between inter- and intra-population matings of the grasshopper Chorthippus parallelus

Comparing the reproductive output of intra- and inter-population matings is the most common way to assess whether post-mating reproductive isolation is caused by genetic incompatibilities. Such genetic incompatibility can however, only assume that the quantity of the post-mating signals involved does not differ between intra- and inter-population matings. This assumption may not be true because sexual selection predicts reduced mating effort towards low-quality mates and in many circumstances, allopatric partners are low-quality mates. Post-mating efforts may, therefore, be reduced in inter- compared to intra-population matings. Here, I test this crucial assumption by studying variation in one post-mating trait, sperm number, in crosses of two parapatric grasshopper populations. In both populations, males transferred fewer sperm to allopatric than sympatric females. If such plasticity with respect to population is common in other post-mating traits, differences between inter- and intra-population crosses may be more frequently caused by differences in sperm number rather than gamete incompatibility. Additionally, I found that sperm numbers declined less rapidly in the female storage organ of allopatric than sympatric females but its rate differed markedly between populations. This is discussed with respect to female adaptations to male traits.     

Grid technology in tissue-based diagnosis: fundamentals and potential developments

DNA fluorescence in situ hybridization (FISH) technology is used to study chromosomal and genomic changes in fixed cell suspensions and tissue block preparations. The technique is based on specific hybridization of small labeled DNA fragments, the probes, to complementary sequences in a target DNA molecule. Demand for FISH assays in formalin-fixed, paraffin-embedded tissues has been increasing, mainly in conditions in which diagnosis is not achieved in cell smears or tissue imprints, such as solid tumors. Moreover, the development of molecular targeted therapies in oncology has expanded the applicability of tests to predict sensitivity or resistance to these agents. The efficient use of tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) as therapeutical agents in advanced non-small cell lung cancer (NSCLC) depends on identification of patients likely to show clinical benefit from these specific treatments. The EGFR gene copy number determined by FISH has been demonstrated as an effective predictor of outcome from NSCLC patients to EGFR TKIs; however there are pending challenges for standardization of laboratory procedures and definition of the scoring system. This methodology article focuses on the EGFR FISH assay. It details the scoring system used in the studies conducted at the University of Colorado Cancer Center in which a significant association was found between increased EGFR copy numbers and clinical outcome to TKIs, and proposes interpretative guidelines for molecular stratification of NSCLC patients for TKI therapy.     

Spatiotemporal expression of connexin 39 and -43 during myoblast differentiation in cultured cells and in the mouse embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C(2)C(12) mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C(2)C(12) cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.     

Spatiotemporal Expression of Connexin 39 and -43 During Myoblast Differentiation in Cultured Cells and in the Mouse Embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C₂C₁₂ mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C₂C₁₂ cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.     

Interaction of liquid epicuticular hydrocarbons and tarsal adhesive secretion in Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)

Species of various insect orders possess specialised tarsal adhesive structures covered by a thin liquid film, which is deposited in the form of footprints. This adhesive liquid has been suggested to be chemically and physiologically related to the epicuticular lipid layer, which naturally covers the body of insects and acts as the prime barrier to environmental stresses, such as desiccation. The functional efficiency of the layer, however, is jeopardised by partial melting that may occur at physiological temperatures. In this study, light microscopic images of elytral prints show that the epicuticular lipid layer of the Colorado potato beetle Leptinotarsa decemlineata actually is partially liquid and chemical investigations reveal the high similarity of the epicuticular hydrocarbon pattern and the tarsal liquid. By means of chemical manipulation of the surface hydrocarbon composition of live beetles, the substance exchange between their tarsal adhesive hairs and the body surface is monitored. Histological sections of L. decemlineata tarsi, furthermore, reveal glandular cells connected to individual adhesive setae and departing from these results, an idea of a general mechanism of tarsal secretion is developed and discussed in a functional–ecological context.     

Clinical and immunological responses in metastatic melanoma patients vaccinated with a high-dose poly-epitope vaccine

Background

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.

Methods

Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-γ ELISPOT assays. Safety and clinical responses were monitored.

Results

Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit—one PR (24 months+), five SD (5 months+) and two mixed responses—seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.

Conclusions

DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.