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The development of two competitive real-time PCR assays for the quantitative detection of trace amounts of two major food allergens, peanut and soybean, is reported. In order to achieve very low detection levels for both allergens, we established PCR primers and probes targeting mitochondrial DNA sequences. We were able to demonstrate that this approach led to an increase in detection sensitivity in the range of at least 1 order of magnitude compared with published assays targeting nuclear DNA. Furthermore, we generated corresponding competitor molecules, which were used as internal standards to compete with matrix effects that are evident during DNA extraction and PCR amplification in heterogeneous analytical matrixes like food. According to the recently described competitive quantitative PCR method published by Holzhauser et al. (2014), we performed threshold calibration against milk powder spiked with 10 ppm peanut and soy. Matrix-independent quantitative determination of peanut and soy could be demonstrated for three different calibrated food matrix standards in a range between 1 and 100 ppm. The data presented indicate that both assay concepts are powerful analytical tools for the quantitative detection of trace amounts of peanut and soy in commercial food products.
Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipidbinding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the
N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the
binding sites of antibodies which will help to define a mode of action profile for their use in therapy.
Prenatal brain development is a complex and sensitive process, highly susceptible to environmental influences such as pollutants, stress, malnutrition, drugs, tobacco exposure, or ionizing radiation (IR). Disturbances in development may cause life-long disabilities and diseases, such as ADHD, childhood cancers, cognitive problems, depression, anxiety and more severe developmental disabilities. Due to increasing medical imaging, radiation therapy, natural terrestrial radiation, radioactive pollution and long-distance flights, humans are increasingly exposed to IR. However, data on impact of IR on very early human brain development are scarce, particularly in the very first weeks of gestation. Here we investigated the effects of low-dose X-ray IR (1 Gy) in a 3D early brain developmental model derived from human pluripotent stem cells. In this model very early neural stem cells, neuroectodermal progenitor cells (NEP), were exposed to low-dose IR and direct as well as delayed effects were investigated. Expression of 20 different marker genes crucial for normal neural development was determined 48 h and 9 days post IR (pIR). All but one of the analyzed marker genes were reduced 48 h after IR, and all but seven genes normalized their expression by day 9 pIR. Among the seven markers were genes involved in neurodevelopmental and growth abnormalities. Moreover, we could show that stemness of the NEP was reduced after IR. We were thus able to identify a significant impact of radiation in cells surviving low-dose IR, suggesting that low-dose IR could have a negative impact on the early developing human brain, with potential later detrimental effects.
Fungal stromata were recently discovered in association with charcoal and burnt soil aggregates during an archaeological survey in the Châtillon-sur-Seine forest massif. The wood and soil in the samples were dated to the medieval period (between 738 and 1411 AD). Light microscopy and scanning electron microscopy revealed that a few of the stromatal fragments still contained ascospores. Their macromorphological characters were described and secondary metabolite profiles were generated using high performance liquid chromatography with diode array and mass spectrometric detection (HPLC–DAD/MS). The combination of these two data lines then allowed species identification. Most of the fragments were assigned to Hypoxylon fragiforme, the type species of the Hypoxylaceae (Xylariales). Two further species whose stromata grew on the fossil charcoal could be tentatively identified as Jackrogersella cohaerens and (more tentatively) as Hypoxylon vogesiacum. These three species are still commonly encountered in the forests of Central Europe today. Furthermore, the HPLC-HRMS data of H. fragiforme suggested the presence of unknown azaphilone dimers and of further new pigments. These archaeological compounds were compared to fresh stromata of H. fragiforme collected in Germany and subjected to the same analytical protocol. While the major components in both samples were identified as the known mitorubrin type azaphilones and orsellinic acid, the chemical structures of seven novel complex azaphilone pigments, for which we propose the trivial names rutilins C-D and fragirubrins A-E, were elucidated using spectral methods (NMR and CD spectroscopy, high resolution mass spectrometry). It appears that these pigments had indeed persisted for millennia in the fossil stromata.
Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under
constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome
coverage and data quality because of advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine
learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a certain time point. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody in comparison to the
spatial proteome of an antibody-producing plasma cell–derived myeloma cell line. Both cell lines show colocalization of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However,
we report differences in the localization of proteins associated to vesicle-mediated transport, transcription, and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in
plasma cell–derived MPC-11 (Merwin plasma cell tumor-11) cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein–expressing cell lines. Data are available via ProteomeXchange with identifier
PXD029115.
Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits. Comparing the transcriptome, proteome, miRNome, surfaceome and secretome of both cell lines identified key differences including 392 potential overexpression targets for CHO cell engineering categorized in 15 functional classes like transcription factors, protein processing or secretory pathway. In addition, 3 protein classes including 209 potential knock-down/out targets for CHO engineering were determined likely to affect aggregation or proteolysis. For production phenotype engineering, several of these novel targets were successfully applied to transient and transposase mediated overexpression or knock-down strategies to efficiently improve productivity of CHO cells. Thus, substantial improvement of CHO productivity was achieved by taking nature as a blueprint for cell line engineering.
Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward
harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could
thus be a promising strategy for a transient and targeted
activation of the endogenous antioxidant machinery.
During the course of our ongoing work to discover new inhibitors of biofilm formation of Staphylococcus aureus from fungal sources, we observed biofilm inhibition by cytochalasans isolated from cultures of the ascomycete Hypoxylon fragiforme for the first time. Two new compounds were purified by a bioassay-guided fractionation procedure; their structures were elucidated subsequently by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). This unexpected finding prompted us to test further cytochalasans from other fungi and from commercial sources for comparison. Out of 21 cytochalasans, 13 showed significant inhibition of Staphylococcus aureus biofilm formation at subtoxic levels. These findings indicate the potential of cytochalasans as biofilm inhibitors for the first time, also because the minimum inhibitory concentrations (MIC) are independent of the anti-biofilm activities. However, cytochalasans are known to be inhibitors of actin, making some of them very toxic for eukaryotic cells. Since the chemical structures of the tested compounds were rather diverse, the inclusion of additional derivatives, as well as the evaluation of their selectivity against mammalian cells vs. the bacterium, will be necessary as next step in order to develop structure-activity relationships and identify the optimal candidates for development of an anti-biofilm agent.
