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In household washing machines, opportunistic pathogens such as

Rhizobia are Gram-negative bacteria that are able to establish a nitrogenfixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids., Puede accederse a los datos con los que se realizó este trabajo haciendo clic en "Documentos relacionados"., Instituto de Biotecnología y Biología Molecular

OBJECTIVES: We aimed to assess the relationship of left atrial appendage (LAA) fibrosis with atrial fibrillation (AF) and postoperative events in patients receiving coronary artery bypass graft surgery (CABG).; BACKGROUND: Increased atrial fibrosis has been associated with AF and worse outcome following catheter ablation. Only limited data exists focusing on the impact of LAA fibrosis on AF after CABG.; METHODS: LAA tissue from 164 CABG-patients was stained with Masson-Goldner trichrome. The histological landscape was scanned and segmented into superpixels for software analysis (QuPath). A classification algorithm was extensively trained to detect fibrotic superpixels for quantification. In 43 propensity score matched pairs with AF or sinus rhythm (SR), LAA fibrosis was compared. Moreover, subgroups of mitral valve regurgitation (MR) were analyzed as follows: SR, SR+MR, AF and AF+MR. The predictive value of LAA fibrosis postoperative stroke, postoperative AF and mortality was assessed.; RESULTS: Fibrotic remodeling (%) showed no significant difference for the total cohort between the SR and AF group (SR: 30.8±11.4% and AF: 33.8±16.0%, respectively, p=.32). However, significant fibrotic remodeling was observed for SR and AF subgroups (SR: 27.2±12.2% vs. AF: 35.3±13.7%; respectively, p=.049) and between SR and SR+MR subgroups (SR: 27.2±12.2% vs. SR+MR: 34.9±9.1%, respectively, p=.027). LAA fibrosis was not significantly associated with postoperative stroke, postoperative AF or overall mortality (all p>.05).; CONCLUSION: LAA fibrosis may contribute to an individual arrhythmia substrate for AF in patients with AF but also in those with SR and coincidence of MR. LAA fibrosis was not found to be predictive for clinical events in patients after CABG. © 2022 The Authors. Clinical Cardiology published by Wiley Periodicals LLC.

Cisplatin is a widely used chemotherapeutic agent. However, it is causing nephrotoxic side effects including a reduced glomerular filtration rate and acute kidney injury. Although kidneys can recover to an extent from the treatment, long-term damage is possible. While a lot of research is focusing on short-term effects, little is known about adverse metabolic effects in the process of recovery. In this study, male Han Wistar rats were dosed with a single intraperitoneal injection of 3 mg/kg cisplatin. Urine and kidney samples were harvested 3, 8 and 26 days after administration. Tubular injury was demonstrated through urinary biomarkers. Complementing this, mass spectrometry imaging gives insight on molecular alterations on a spatial level, thus making it well suited to analyze short- and long-term disturbances. Various metabolic pathways seem to be affected, as changes in a wide range of metabolites were observed between treated and control animals. Besides previously reported early changes in kidney metabolism, unprecedented long-term effects were detected including deviation in nucleotides, antioxidants, and phospholipids. Copyright © 2020 Elsevier B.V. All rights reserved.

The Coxsackie- and adenovirus receptor (CAR) mediates homophilic cell-cell contacts and susceptibility to both human pathogenic viruses through its membrane-distal immunoglobulin domain. In the present study, we screened five missense variants of the human CAR gene for their influence on adenovector or Coxsackievirus entry into Chinese hamster ovary cells. The CAR variants facilitated virus internalisation to a similar extent as wild type CAR. This underlines CAR's presumed invariance and essential physiological role in embryogenesis. Copyright © 2020 Elsevier Inc. All rights reserved.

Purpose Most cancer-related deaths worldwide are associated with lung cancer. Subtyping of non-small cell lung cancer (NSCLC) into adenocarcinoma (AC) and squamous cell carcinoma (SqCC) is of importance, as therapy regimes differ. However, conventional staining and immunohistochemistry have their limitations. Therefore, a spatial metabolomics approach was aimed to detect differences between subtypes and to discriminate tumor and stroma regions in tissues. Methods Fresh-frozen NSCLC tissues (n = 35) were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of small molecules ( Results Histology-guided spatial metabolomics revealed differences between AC and SqCC and between NSCLC tumor and tumor microenvironment. A diagnostic ability of 0.95 was achieved for the discrimination of AC and SqCC. Metabolomic contrast to the tumor microenvironment was revealed with an area under the curve of 0.96 due to differences in phospholipid profile. Furthermore, the detection of NSCLC with rarely arising mutations of the isocitrate dehydrogenase (IDH) gene was demonstrated through 45 times enhanced oncometabolite levels. Conclusion MALDI-MSI of small molecules can contribute to NSCLC subtyping. Measurements can be performed intraoperatively on a single tissue section to support currently available approaches. Moreover, the technique can be beneficial in screening of IDH-mutants for the characterization of these seldom cases promoting the development of treatment strategies.

In this study we used Medicago truncatula, to identify and analyze the expression of small GTP-binding proteins (Arf1, Arl1, Sar1, Rabs, Rop/Rac) and their interacting partners in the infection process in the roots and nodules. A real-time polymerase chain reaction analysis was carried out and our results showed that Arf1 (AtArfB1c-like), MtSar1, AtRabA1e-like, AtRabC1-like, MsRab11-like and AtRop7-like genes were highly expressed in the nodules of rhizobium inoculated plants compared to the non-inoculated ones. On the contrary, AtRabA3 like, AtRab5c and MsRac1-like genes were highly expressed in non-infected nitrogen supplied roots of M. truncatula. Other Rab genes (AtRabA4a, AtRabA4c and AtRabG3a-like genes) were nearly equally expressed in both treatments. Interestingly, RbohB (a respiratory burst NADPH oxidase homologue) was more highly expressed in rhizobium infected than in non-infected roots and nodules. Our data show a differential expression pattern of small GTP-binding proteins in roots and nodules of the plants. This study demonstrates an important role of small GTP-binding proteins in symbiosome biogenesis and root nodule development in legumes.

Flavin-dependent halogenases selectively introduce halogen substituents into (hetero-)aromatic substrates and require only molecular oxygen and halide salts for this regioselective oxidative CH-functionalization. Genomic analysis of Xanthomonas campestris pv. campestris B100 identified three novel putative members of this enzyme class. They were shown to introduce halogen substituents into, e. g., substituted indoles, while preferring bromide over chloride.

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with a mortality rate of about 50%. It represents the sixth most common type of malignant tumor in the world. Risk factors include tobacco and alcohol usage and infection with the human papilloma virus (HPV). HPV-negative HNSCCs frequently display areas of cornification evident by keratin pearls in the tumor tissue. Cornification represents a natural differentiation path of keratinocytes in the normal epidermis and oral mucosa. To investigate the mechanisms of HNSCC cell differentiation we have established spheroid cell cultures from patient-derived HNSCC and parotid gland adenoid cystic carcinoma (ACC) tissue that grow in suspension under serum-free conditions. The use of a specific differentiation medium induced striking adhesion, loss of proliferation, and differentiation in tumor cells. Spheroid cells grew as single cell clones under serum-free conditions with a cloning efficiency of 40-60%, which was fully diminished under differentiating conditions. HNSCC cells cornified as indicated by the formation of lamellar bodies in the cytoplasm of adherent cells and an upregulation of cornification markers SPRR3 and involucrin. ACC cells upregulated parotid gland differentiation markers including α-amylase. RNA-seq analysis in HNSCC cells confirmed an upregulation of signaling pathways associated with cornification and epithelial cell differentiation. Conversely, pathways regulating the three-dimensional organization of the genome were downregulated upon differentiation. This was accompanied by the formation of ATRX-positive heterochromatin foci in the nucleus of differentiated ACC and HNSCC cells resembling those previously described to arise during therapy-induced senescence. Moreover, gas chromatography mass spectrometry analysis revealed a lack of essential amino acids including leucine to be implicated in the differentiation process. Altogether, our spheroid model of HNSCC and ACC cells is suitable to analyze the mechanisms underlying tumor cell differentiation and might lead to new therapeutic approaches that can drive long-term repopulating HNSCC and ACC cells into differentiation. Citation Format: Felix Oppel, Senyao Shao, Sarah Gendreizig, Philipp Kühnel, Vivien Przybycin, Carsten Hain, Pascal Schmidt, Matthias Schürmann, Peter Goon, Karsten Niehaus, Jörn Kalinowski, Holger Sudhoff. Head and neck cancer cells can differentiate and resemble their tissue of origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 111.

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

Background Plant growth-promoting rhizobacteria (PGPR) are known to improve plant growth and are used as biofertilizers, thanks to their numerous benefits to agriculture such as phosphorus solubilization and phytohormone production. In this paper, four rhizospheric bacteria (Phyllobacterium sp., Bacillus sp., Agrobacterium sp., and Rhizobium sp.) isolated from surface-sterilized root nodules of Acacia cyanophylla were tested for their ability to solubilize inorganic phosphate and to produce indole-3-acetic acid (IAA) under laboratory conditions. Then, the best IAA producer (Rhizobium sp.) was selected to test optimized conditions for IAA production. Finally, the effect of the four strains on plant growth for A. cyanophylla was evaluated in vivo. Results The results showed that the totality of the tested isolates had solubilized inorganic phosphate (P) in both NBRIP (National Botanical Research Institute Phosphate) and PVK (Pikovskaya) media. Bacillus sp. was a high P-solubilizer and showed maximum solubilization in PVK (519 μg ml-1) and NBRIP (782 μg ml-1). The optimization of maximum phosphate solubilization was done using different sources of carbon (1%) and nitrogen (0.1%). Glucose and ammonium sulfate were selected to be the best carbon and nitrogen source for phosphate solubilization by all tested strains, except for Phyllobacterium sp., which recorded the highest phosphate solubilization with ammonium nitrate. The IAA production by the tested strains indicated that Rhizobium sp. produced the highest amount of IAA (90.21 μg ml-1) in culture media supplemented with L-tryptophan. The best production was observed with L-Trp concentration of 0.2% (116.42 μg ml-1) and at an initial pH of 9 (116.07 μg ml-1). The effect of NaCl on IAA production was tested at concentrations of 0 to 5% and the maximum production of 89.43 μg ml-1 was found at 2% NaCl. The extraction of crude IAA from this strain was done and purity was confirmed with Thin Layer Chromatography (TLC) analysis. A specific spot from the extracted IAA production was found to correspond with a standard spot of IAA with the same Rf value. Finally, the tested PGPR demonstrated growth stimulatory effects on Acacia cyanophylla seedlings in vivo, with a great increase of shoots’ and roots’ dry weights, and shoot length compared to control. The rhizobacterial isolates were identified by 16S rDNA sequence analysis as Agrobacterium sp. NA11001, Phyllobacterium sp. C65, Bacillus sp. CS14, and Rhizobium sp. V3E1. Conclusion This study highlights the importance of the use of phosphate solubilizing and IAA producer microorganisms as biofertilizers to increase crop yields. The studied strains showed a significant phosphate solubilization potential and IAA production. The use of selected strains as inoculants would be interesting, in particular with a view of promoting sustainable agriculture. However, further studies to verify the efficacy of the best isolates in situ is certainly required.

Summary Nitrogen‐fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen‐activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress‐induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well‐known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP‐tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP‐tagged SIMK led to increase of biomass in above‐ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.

Training systems are an option to handle the pronounced apical dominance of grapevines and to influence diverse traits of the corresponding wine. However, it is still unclear if different training systems generate signatures in the metabolome of the wine. By an untargeted metabolomics approach using (SPME) GC-MS wine (volatiles) and leaves were evaluated. Different training directions such as vertical shoot positioning systems, hanging shoot systems, and minimal pruning systems were distinguishable in wine. It was shown, that different training systems generate a metabolomic signature in the wine which was more pronounced than in leaves. Moreover, the sensory analysis showed some changes in the aroma of the different training systems. Thus, the influence of the training system ranges from the leaf metabolome to the wine metabolome. Copyright © 2021. Published by Elsevier Ltd.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor. High infiltration rates and poor therapy responses make it the deadliest glioma. The tumor metabolism is known to differ from normal one and is influenced through various factors which can lead to longer survival. Metabolites are small molecules (

The E -proteobacterium pound Xanthomonas campestris pv. campestris (Xcc) is the producer of the biopolymer xanthan, a polysaccharide which is used as a thickener in numerous industrial applications. In this study, we present a global transcriptome profiling of two Xcc strain B100 cultures obtained from fermentation during the growth phase and the subsequent stationary phase associated with xanthan biosynthesis. During the xanthan production phase, highly abundant transcripts belonged to genes encoding for small RNAs, glycogen biosynthesis, and xanthan export. A total of 1850 (40%) genes were differentially transcribed during the stationary phase where 924 were transcriptionally up-regulated and 926 genes were down-regulated. An overview of differentially transcribed genes includes a significant down-regulation of genes involved in transcription, translation, and amino acid biosynthesis pathways. A group of up-regulated genes was involved in cellular response against oxidative stress, such as those coding for superoxide dismutase and catalase. Genes encoding enzymes involved in nucleotide sugar precursor synthesis of xanthan biosynthesis, such as xanA, galU, and ugd, exhibited a transcription pattern that did not change during the growth and stationary phase. Regarding the transcription pattern of the gum gene cluster that govern xanthan biosynthesis, a significant up-regulation of the genes gumB, gumC, and gumD was observed, while the transcript pools of the genes gumG, gumH, gumI, and gumJ were reduced and those of genes gumE, gumF, gumK, gumL, and gumM remained un-changed during the stationary phase compared to the growth phase. The obtained data represents the first analysis of gene expression patterns under xanthan production conditions and provides the bases for future studies aiming at enhancing xanthan yield.

Rice is known to contain limiting amino acids. Synthesis of GABA in plants is an adaptive response by initiating glutamic acid. A higher rate of GABA production was observed in samples enriched with glutamic acid and vacuum impregnation (VI) with longer germination time. Heat map profiles classified GABA and essential amino acids into 1) small increments consisting of Arg, His and Met, 2) moderate increments consisting of GABA, Trp, Lys, Phe and Thr, and 3) large increments consisting of Ile, Leu and Val. In Jasmine rice, highest essential amino acids were found in samples soaked with water, enriched with glutamic acid, and germinated for 72–96 h. Highest GABA (44.8 mg/100 g) was noticed after VI for 20–40 min and germinated for 72–96 h. In Riceberry, highest GABA (74.2 mg/100 g) and essential amino acids were associated with samples treated with VI for 20–40 min and germinated for 96 h.

The combined occurrence of salt stress and hypoxia leads to increased growth reduction and severe toxic effects compared to salt stress alone. In the present work, we analyzed the metabolic response of sugar beet (Beta vulgaris L.) to salt stress combined with hypoxia in roots as well as in young and mature leaves. B. vulgaris plants were grown in a hydroponic culture under low and high salt concentrations combined with normoxic and hypoxic conditions. A non-targeted metabolic approach was used to identify the biochemical pathways underlying the metabolic and physiological adaptation mechanisms. Young and mature leaves showed a similar metabolic response to salt stress alone and combined stresses, accumulating sugar compounds. Osmoprotectants such as proline and pinitol were accumulated under combined stress. Roots exposed to hypoxic conditions showed increased TCA (tricarboxylic acid cycle) intermediates levels such as succinate, fumarate and malate. During hypoxia, the concentration of free amino acids as well as intermediates of the GABA (gamma-aminobutyric acid) shunt increased in roots as well as in leaves. The combination of salt stress and hypoxia results in a severe stress response in roots and leaves. A partial flux of the TCA cycle linked with the GABA shunt might be activated during hypoxia to regain reduction equivalents. Copyright © 2021 Elsevier GmbH. All rights reserved.

The original version of this article unfortunately contained a mistake. Due to the submission of Table 1 in a separate file, the following citations were missing in the references, leading to an incorrect linking in the table caption. The missing reference are: (A) Sheldon CA, Clayman RV, Gonzalez R, Williams RD, Fraley EE. Malignant urachal lesions. J Urol. 131, 1–8 (1984). (B) Ashley RA, Inman BA, Sebo TJ, Leibovich BC, Blute ML, Kwon ED, et al. Urachal carcinoma: clinicopathologic features and longterm outcomes of an aggressive malignancy. Cancer. 107, 712–720 (2006). (C) Brierley J, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumors. In: Brierley J, Gospodarowicz MK, Wittekind C, editors. Chichester, West Sussex, UK, Hoboken, NJ: John Wiley & Sons Inc; 2017. The authors apologize for the mistake. The original article has been corrected.

Micro-organisms populate on rapeseed after harvest during storage depending on the growing conditions. The composition of the bacterial colonization is unknown, although its contribution to the profile of volatile aroma-active compounds determines the sensory quality of virgin cold-pressed rapeseed oil.; Results: From four rapeseed samples, 46 bacterial strains were isolated. By DNA-sequencing, the identification of four bacteria species and 17 bacteria genera was possible. In total, 22 strains were selected, based on their typical off-flavors resembling those of virgin sensory bad cold-pressed rapeseed oils. The cultivation of these strains on rapeseed meal agar and examination of volatile compounds by solid phase microextraction-gas chromatography-mass spectrometry allowed the identification of 29 different compounds, mainly degradation products of fatty acids such as alkanes, alkenes, aldehydes, ketones and alcohols and, in addition, sulfur-containing compounds, including one terpene and three pyrazines. From these compounds, 19 are described as aroma-active in the literature.; Conclusion: Micro-organisms populating on rapeseed during storage may strongly influence the sensory quality of virgin rapeseed oil as a result of the development of volatile aroma-active metabolic products. It can be assumed that occurrence of off-flavor of virgin rapeseed oils on the market are the result of metabolic degradation products produced by micro-organisms populating on rapeseed during storage. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Alkaloids are common secondary metabolites found also in plants of the large family of Solanaceae, and they contribute frequently to the economic importance of these plants as food, spices, drugs, or pharmaceuticals. Matrix-assisted laser desorption/ionization mass spectrometry imaging was applied to visualize the spatial localization of the main steroidal alkaloids in diverse plant tissues of Lycopersicon esculentum, Solanum nigrum, and Solanum dulcamara. Among others, the basic aglycons, tomatidenol, tomatidine, solasodine, and soladulcine, along with their corresponding glycoalkaloids, were identified with distinct distributions within plant tissue structures and plant parts and with respect to the degree of ripeness. The alkaloids are identified by their mass and fragmentation pattern. Multivariate unsupervised principal compound analysis and the k-means clustering analysis were calculated on the basis of all peaks, automatically picked from all selected regions, with total ion count normalization resulting in characterization of the tissues and organs with respect to their chemical similarity. It can therefore be concluded that the tissue-specific localizations of alkaloids in nightshades depend upon the ripeness status and the developmental stage of the plants.

The nitrogen-fixing root nodule is generally derived through a successful symbiotic interaction between legume plants and bacteria of the genusRhizobium. A root nodule shelter hundreds ofRhizobia, which are thought to invade into the plant cells through an endocytosis-like process despite the existence of turgor pressure. Each invadingRhizobiumis surrounded by the peribacteroid membrane to form the symbiosome, which results in the higher acquisition of host membrane materials. In this study, we show the localization of Rab11F, a RabA6b homolog with the large Rab-GTPase family, which was highly expressed in root nodules ofMedicago sativaandM. truncatula. Rab11F-labeled organelles accumulated the membrane specific dye FM4-64 and were sensitive to Brefeldin A by forming aggregates after treatment with this drug. By co-localization with the cis-Golgi marker, GmMan1-mCherry, Rab11F-organelles formed tri-colored organelles, whereby Rab11F was located to the opposite side of GmMan1-mCherry indicating that Rab11F-labeled structures were localized within the trans-Golgi network (TGN). In root nodules, Rab11F was localized transiently at the infection thread-covering membrane on the side of infection droplets and the peribacteroid membranes. The symbiosome acquires Rab11F during the entry process and differentiation. However, the symbiosome did not recruit Rab11F after cessation of division. In conclusion, thelegumeplant seemed to use a specialized secretion pathway from the TGN, which was marked by Rab11F, to proliferate the symbiosome membrane.

Automated matrix deposition for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is crucial for producing reproducible analyte ion signals. Here we report an innovative method employing an automated immersion apparatus, which enables a robust matrix deposition within 5 minutes and with scalable throughput by using MAPS matrix and non-polar solvents. MSI results received from mouse heart and rat brain tissues were qualitatively similar to those from nozzle sprayed samples with respect to peak number and quality of the ion images. Overall, the immersion-method enables a fast and careful matrix deposition and has the future potential for implementation in clinical tissue diagnostics. This article is protected by copyright. All rights reserved.

At a molecular level, the regulation of many important cellular processes is still obscure in xanthomonads, a bacterial group of outstanding relevance as world-wide plant pathogens and important for biotechnology as producers of the polysaccharide xanthan. Transcriptome analysis indicated a sucrose-dependent regulation of 18 genes in Xanthomonas campestris pv. campestris (Xcc) B100. The expression of 12 of these genes was clearly increased in the presence of sucrose. Only part of these genes was obviously involved in sucrose utilization. To identify regulatory proteins involved in transcriptional regulation, a DNA fragment-specific pull-down approach was established for Xcc. Putative promoter regions were identified and used to isolate DNA-binding proteins, which were separated by SDS PAGE and identified by MALDI-TOF mass spectrometry. This led to the identification of four transcriptional regulators, among them the global transcriptional regulator Clp and a previously identified regulator of sucrose utilization, SuxR, plus a third DNA-binding transcriptional regulator encoded by xcc-b100_2861 and recently shown to interact with a cyclic di-GMP-binding protein. The fourth regulatory protein was encoded by xcc-b100_2791. These results indicate DNA fragment-specific pull-down experiments as promising approaches to screen for specific DNA-binding regulatory proteins in Xcc. (C) 2016 Elsevier B.V. All rights reserved.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and MALDI MS imaging are ubiquitous analytical methods in medical, pharmaceutical, biological, and environmental research. Currently, there is a strong interest in the investigation of low molecular weight compounds (LMWCs), especially to trace and understand metabolic pathways, requiring the development of new matrix systems that have favorable optical properties and a high ionization efficiency and that are MALDI silent in the LMWC area. In this paper, five conjugated polymers, poly{[ N, N'-bis(2-octyldodecyl)-naphtalene-1,4,5,8-bis(dicarboximide)-2,6-diyl]- alt-5,5'(2,2'-bithiophene)} (PNDI(T2)), poly(3-dodecylthiophene-2,5-diyl) (P3DDT), poly{[2,3-bis(3-octyloxyphenyl)quinoxaline-5,8-diyl]- alt-(thiophene-2,5-diyl)} (PTQ1), poly{[ N, N'-bis(2-octyldodecyl)-isoindigo-5,5'-diyl] -alt-5,5'(2,2'-bithiophene)} (PII(T2)), and poly(9,9-di- n-octylfluorenyl-2,7-diyl) (P9OFl) are investigated as matrices. The polymers have a strong optical absorption, are solution processable, and can be coated into thin films, allowing a vast reduction in the amount of matrix used. All investigated polymers function as matrices in both positive and negative mode MALDI, classifying them as rare dual-mode matrices, and show a very good analyte ionization ability in both modes. PNDI(T2), P3DDT, PTQ1, and PII(T2) are MALDI silent in the full measurement range (m/ z = 150k), except at high laser intensities. In MALDI MS experiments of single analytes and a complex biological sample, the performance of the polymers was found to be as good as two commonly used matrices (2,5-DHB for positive and 9AA for negative mode measurements). The detection limit of two standard analytes was determined as being below 164 pmol for reserpine and below 245 pmol for cholic acid. Additionally P3DDT was used successfully in first MALDI MS imaging experiments allowing the visualization of the tissue morphology of rat brain sections.

Salt stress impairs global agricultural crop production by reducing vegetative growth and yield. Despite this importance, a number of gaps exist in our knowledge about very early metabolic responses that ensue minutes after plants experience salt stress. Surprisingly, this early phase remains almost as a black box. Therefore, systematic studies focussing on very early plant physiological responses to salt stress (in this case NaCl) may enhance our understanding on strategies to develop crop plants with a better performance under saline conditions. In the present study, hydroponically grown Vicia faba L. plants were exposed to 90 min of NaCl stress, whereby every 15 min samples were taken for analyzing short-term physiologic responses. Gas chromatography-mass spectrometry-based metabolite profiles were analysed by calculating a principal component analysis followed by multiple contrast tests. Follow-up experiments were run to analyze downstream effects of the metabolic changes on the physiological level. The novelty of this study is the demonstration of complex stress-induced metabolic changes at the very beginning of a moderate salt stress in V. faba, information that are very scant for this early stage. This study reports for the first that the proline analogue trans-4-hydroxy-L-proline, known to inhibit cell elongation, was increasingly synthesized after NaCl-stress initiation. Leaf metabolites associated with the generation or scavenging of reactive oxygen species (ROS) were affected in leaves that showed a synchronized increase in ROS formation. A reduced glutamine synthetase activity indicated that disturbances in the nitrogen assimilation occur earlier than it was previously thought under salt stress. (C) 2015 Elsevier Masson SAS. All rights reserved.

A strain of a species of the genusCorynebacterium, designated AJ 3170T, was isolated during the 1980s from putrefied bananas. Since then, there have been no further updates on the description of the strain or its phylogenetic classification. However, phylogenetic analysis of this strain using 16S rRNA andin silicoDNA–DNA hybridization has confirmed that it is a member of the genusCorynebacteriumand that strain AJ 3170Tclusters withCorynebacterium variabileDSM 44702T,Corynebacterium terpenotabidumY-11TandCorynebacterium nurukiS6-4Tin one subgroup. Furthermore, a combination of enzymatic, chemical, and morphological characterization techniques was applied in order to describe strain AJ 3170Tfurther. The strain grew well at pH values of 6–10 and at temperatures of 30–41 °C. The major fatty acids were C16 : 0(42.15 %), C18 : 1ω9c(41.6 %) and C18 : 010-methyl (TBSA) (8.56 %). The whole-cell sugars were determined to comprise galactose, arabinose and ribose. On the basis of this phenotypic, chemotaxonomic and phylogenetic characterization, it is proposed that strain AJ 3170Trepresents a novel species, for which the nameCorynebacterium glyciniphilumsp. nov. is proposed; the type strain is AJ 3170T( = DSM 45795T = ATCC 21341T).

CO2 is known as a major attractant for many arthropod pests which can be exploited for pest control within novel attract-and-kill strategies. This study reports on the development of a slow-release system for CO2 based on calcium alginate beads containing granular corn starch, amyloglucosidase and Saccharomyces cerevisiae. Our aim was to evaluate the conditions which influence the CO2 release and to clarify the biochemical reactions taking place within the beads. The amyloglucosidase was immobilized with a high encapsulation efficiency of 87% in Ca-alginate beads supplemented with corn starch and S. cerevisiae biomass. The CO2 release from the beads was shown to be significantly affected by the concentration of amyloglucosidase and corn starch within the beads as well as by the incubation temperature. Beads prepared with 0.1 amyloglucosidase units/g matrix solution led to a long-lasting CO2 emission at temperatures between 6 and 25 °C. Starch degradation data correlated well with the CO2 release from beads during incubation and scanning electron microscopy micrographs visualized the degradation of corn starch granules by the co-encapsulated amyloglucosidase. By implementing MALDI-ToF mass spectrometry imaging for the analysis of Ca-alginate beads, we verified that the encapsulated amyloglucosidase converts starch into glucose which is immediately consumed by S. cerevisiae cells. When applied into the soil, the beads increased the CO2 concentration in soil significantly. Finally, we demonstrated that dried beads showed a CO2 production in soil comparable to the moist beads. The long-lasting CO2- releasing beads will pave the way towards novel attract-and-kill strategies in pest control.

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Background Dissolved oxygen tension (DOT) is hardly constant and homogenously distributed in a bioreactor, which can have a negative impact in the metabolism and product synthesis. However, the effects of DOT on plasmid DNA (pDNA) production and quality have not been thoroughly investigated. In the present study, the effects of aerobic (DOT ≥30% air sat.), microaerobic (constant DOT = 3% air sat.) and oscillatory DOT (from 0 to 100% air sat.) conditions on pDNA production, quality and host performance were characterized. Results Microaerobic conditions had little effect on pDNA production, supercoiled fraction and sequence fidelity. By contrast, oscillatory DOT caused a 22% decrease in pDNA production compared with aerobic cultures. Although in aerobic cultures the pDNA supercoiled fraction was 98%, it decreased to 80% under heterogeneous DOT conditions. The different oxygen availabilities had no effect on the fidelity of the produced pDNA. The estimated metabolic fluxes indicated substantial differences at the level of the pentose phosphate pathway and TCA cycle under different conditions. Cyclic changes in fermentative pathway fluxes, as well as fast shifts in the fluxes through cytochromes, were also estimated. Model-based genetic modifications that can potentially improve the process performance are suggested. Conclusions DOT heterogeneities strongly affected cell performance, pDNA production and topology. This should be considered when operating or scaling-up a bioreactor with deficient mixing. Constant microaerobic conditions affected the bacterial metabolism but not the amount or quality of pDNA. Therefore, pDNA production in microaerobic cultures may be an alternative for bioreactor operation at higher oxygen transfer rates. Electronic supplementary material The online version of this article (doi:10.1186/s12896-017-0378-x) contains supplementary material, which is available to authorized users.

The prevailing stigmatization of synthetic antioxidants and the inefficiency of endogenous antioxidants like tocopherols during high temperature processing of edible oils necessitate the search for effective natural antioxidants. Here, polyphenolic extracts from chokecherry and hawthorn fruits were screened for possible antioxidative application in fats/oils. Extracts were successively partitioned on sephadex columns and fractions were screened for radical scavenging activity using DPPH and beta-carotene assays. Furthermore, sunflower oil fortified with extracts was assessed for stability using accelerated storage at 65 degrees C, Rancimat at 120 degrees C. and frying at 180 degrees C. Phenolic extracts showed significantly high radical scavenging and antioxidant activity in the oil. At the end of storage, hydroperoxide formation in sunflower oil was reduced by up to 50%, and the induction period significantly increased in the presence of extracts. Similarly, a significantly high frying stability was observed for the fortified samples, suggesting that the phenolic extracts can offer effective natural alternative to synthetic antioxidants during frying. (C) 2014 Elsevier Ltd. All rights reserved.

Mass spectrometry imaging (MSI) opens the unique possibility to localize a broad number of small molecules in entire plant organs or in cryosections. In tomato the antioxidant flavonoid quercetin was localized in the outer parts of the fruit while the alkaloid α-tomatine was distributed all over the tissues. In germinating barley seedlings the antifungal hordatines and their glycol conjugates could be localized. The combination of MSI and liquid chromatography-mass spectrometry was ideal in the identification of novel compounds.

Mass spectrometry imaging was applied on germinated barley for the detailed localization of metabolites in longitudinal and transversal seed sections. Among others, 20 m/z signals occurred in three regular peak clusters with specific, distinct localizations in embryo tissues. High resolution FT-ICR MS, MALDI-TOF MS/MS, and UHPLC-ESI MS/MS served for the identification and structural characterization of these compounds. Only five metabolites were published in their structures, namely the antifungal compounds hordatine A and B in non-glycosylated and glycosylated forms. All other non-identified cluster compounds were of hordatine-like structure and differed by systematic O-methylations, hydroxylations, and glycosylations. These differences in molecular structures correlated to distinct localization patterns within the embryo and might serve for the regulation of antifungal properties. Based on the structural investigations by mass spectrometry, an array of different hordatines that comprises the five published hordatines, 15 novel hordatine derivates and their six precursors could be localized in the embryo of germinated barley. Implications for the biosynthetic pathway and transport processes are discussed.

BACKGROUND: Identification of biomarkers capable of distinguishing organic and conventional products would be highly welcome to improve the strength of food quality assurance. Metabolite profiling was used for biomarker search in organic and conventional wheat grain (Triticum aestivum L.) of 11 different old and new bread wheat cultivars grown in the DOK system comparison trial. Metabolites were extracted using methanol and analysed by gas chromatography-mass spectrometry. RESULTS: Altogether 48 metabolites and 245 non-identified metabolites (TAGs) were detected in the cultivar Runal. Principal component analysis showed a sample clustering according to farming systems and significant differences in peak areas between the farming systems for 10 Runal metabolites. Results obtained from all 11 cultivars indicated a greater influence of the cultivar than the farming system on metabolite concentrations. Nevertheless, a t-test on data of all cultivars still detected 5 metabolites and 11 TAGs with significant differences between the farming systems. CONCLUSION: Based on individual cultivars, metabolite profiling showed promising results for the categorization of organic and conventional wheat. Further investigations are necessary with wheat from more growing seasons and locations before definite conclusions can be drawn concerning the feasibility to evolve a combined set of biomarkers for organically grown wheat using metabolite profiles. © 2014 Society of Chemical Industry.

Motivation: Comprehensive 2D gas chromatography-mass spectrometry is an established method for the analysis of complex mixtures in analytical chemistry and metabolomics. It produces large amounts of data that require semiautomatic, but preferably automatic handling. This involves the location of significant signals (peaks) and their matching and alignment across different measurements. To date, there exist only a few openly available algorithms for the retention time alignment of peaks originating from such experiments that scale well with increasing sample and peak numbers, while providing reliable alignment results. Results: We describe BiPACE 2D, an automated algorithm for retention time alignment of peaks from 2D gas chromatography-mass spectrometry experiments and evaluate it on three previously published datasets against the mSPA, SWPA and Guineu algorithms. We also provide a fourth dataset from an experiment studying the H2 production of two different strains of Chlamydomonas reinhardtii that is available from the MetaboLights database together with the experimental protocol, peak-detection results and manually curated multiple peak alignment for future comparability with newly developed algorithms. Availability and implementation: BiPACE 2D is contained in the freely available Maltcms framework, version 1.3, hosted at http://maltcms.sf.net, under the terms of the L-GPL v3 or Eclipse Open Source licenses. The software used for the evaluation along with the underlying datasets is available at the same location. The C.reinhardtii dataset is freely available at http://www.ebi.ac.uk/metabolights/MTBLS37. Contact: nils.hoffmann@cebitec.uni-bielefeld.de or jens.stoye@uni-bielefeld.de Supplementary information: Supplementary data are available at Bioinformatics online.

The coxsackie and adenovirus receptor (CAR) mediates the entry of coxsackievirus B (CVB) and adenovirus into host cells and is, therefore, a key determinant for the molecular pathogenesis of viral diseases such as myocarditis. The aim was to investigate the influence of HMG-CoA reductase inhibitor lovastatin on CAR expression in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were exposed to different concentrations of lovastatin (0.05-5 mu mol/l) for up to 48 h. Alterations in CAR expression were examined by quantitative real-time PCR (qRT-PCR) and flow cytometry. In addition, after treatment with 1 mu mol/l lovastatin for 48 h, HUVECs were infected for 8 h with CVB3 and virus replication was detected by qRT-PCR using viral-specific TaqMan probes. We found that lovastatin decreases CAR mRNA expression by up to 80 % (p < 0.01) and CAR protein expression by up to 19 % (p < 0.01), in a concentration-dependent manner. Moreover, virus replication of CVB3 was significantly inhibited after lovastatin treatment (p < 0.05). The signaling mechanism of CAR down-regulation by lovastatin depends on the Rac1/Cdc42 pathway. This study shows for the first time that lovastatin reduces the expression of CAR and subsequently the replication of CVB3 in HUVECs.

The γ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) B100 synthesizes the polysaccharide xanthan, a commercially important viscosifier. Since the complete genome of Xcc B100 is available, systems biology tools were applied to obtain a deeper understanding of the metabolism involved in xanthan biosynthesis. A large-scale metabolic network was reconstructed and manually curated. The reconstructed network included 352 genes, 437 biochemical reactions, 10 transport reactions, and 338 internal metabolites. To use this network for flux balance analysis, the biomass composition of Xcc B100 was determined. The comprehensive model obtained was applied for in silico analyses to predict biomass generation and gene essentiality. Predictions were extensively validated by analyzing batch culture performance and by carbon balancing including xanthan production. Single gene deletion mutants causing deficiencies in the central carbohydrate metabolism were constructed to enforce major flux redistributions. The impact of xanthan production was studied in vivo and in silico, comparing the physiology of a gumD mutant, negative in xanthan production, with the original strain. The results indicate a redistribution of resources from xanthan to biomass, rather than a reduction in carbon uptake. With this high quality metabolic model, both systems biology analyses and synthetic biology reengineering of Xcc gained an important tool.

Summary Xanthomonas translucens pv. graminis (Xtg) is a gammaproteobacterium that causes bacterial wilt on a wide range of forage grasses. To gain insight into the host–pathogen interaction and to identify the virulence factors of Xtg, we compared a draft genome sequence of one isolate (Xtg29) with other Xanthomonas spp. with sequenced genomes. The type III secretion system (T3SS) encoding a protein transport system for type III effector (T3E) proteins represents one of the most important virulence factors of Xanthomonas spp. In contrast with other Xanthomonas spp. assigned to clade 1 on the basis of phylogenetic analyses, we identified an hrp (hypersensitive response and pathogenicity) gene cluster encoding T3SS components and a representative set of 35 genes encoding putative T3Es in the genome of Xtg29. The T3SS was shown to be divergent from the hrp gene clusters of other sequenced Xanthomonas spp. Xtg mutants deficient in T3SS regulating and structural genes were constructed to clarify the role of the T3SS in forage grass colonization. Italian ryegrass infection with these mutants led to significantly reduced symptoms (P < 0.05) relative to plants infected with the wild-type strain. This showed that the T3SS is required for symptom evocation. In planta multiplication of the T3SS mutants was not impaired significantly relative to the wild-type, indicating that the T3SS is not required for survival until 14 days post-infection. This study represents the first major step to understanding the bacterial colonization strategies deployed by Xtg and may assist in the identification of resistance (R) genes in forage grasses.

Wortmannin is a widely used pharmaceutical compound which is employed to define vesicular trafficking routes of particular proteins or cellular compounds. It targets phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinases in a dose-dependent manner leading to the inhibition of protein vacuolar sorting and endocytosis. Combined proteomics and cell biological approaches have been used in this study to explore the effects of wortmannin on Arabidopsis root cells, especially on proteome and endomembrane trafficking. On the subcellular level, wortmannin caused clustering, fusion, and swelling of trans-Golgi network (TGN) vesicles and multivesicular bodies (MVBs) leading to the formation of wortmannin-induced multivesicular compartments. Appearance of wortmannin-induced compartments was associated with depletion of TGN as revealed by electron microscopy. On the proteome level, wortmannin induced massive changes in protein abundance profiles. Wortmannin-sensitive proteins belonged to various functional classes. An inhibition of vacuolar trafficking by wortmannin was related to the downregulation of proteins targeted to the vacuole, as showed for vacuolar proteases. A small GTPase, RabA1d, which regulates vesicular trafficking at TGN, was identified as a new protein negatively affected by wortmannin. In addition, Sec14 was upregulated and PLD1 alpha was downregulated by wortmannin.

Burkholderia cenocepacia is an opportunistic respiratory pathogen that can cause severe infections in immune-compromised individuals and is associated with poor prognosis for patients suffering from cystic fibrosis. The second messenger cyclic diguanosine monophosphate (c-di-GMP) has been shown to control a wide range of functions in bacteria, but little is known about these regulatory mechanisms in B. cenocepacia. Here we investigated the role that c-di-GMP plays in the regulation of biofilm formation and virulence in B. cenocepacia. Elevated intracellular levels of c-di-GMP promoted wrinkly colony, pellicle and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to elevated levels of c-di-GMP led to the identification of the mutant bcam1349 that did not display increased biofilm and pellicle formation with excessive c-di-GMP levels, and displayed a biofilm defect with physiological c-di-GMP levels. The bcam1349 gene is predicted to encode a transcriptional regulator of the CRP/FNR superfamily. Analyses of purified Bcam1349 protein and truncations demonstrated that it binds c-di-GMP in vitro. The Bcam1349 protein was shown to regulate the production of a number of components, including cellulose and fimbriae. It was demonstrated that the Bcam1349 protein binds to the promoter region of the cellulose synthase genes, and that this binding is enhanced by the presence of c-di-GMP. The bcam1349 mutant showed reduced virulence in a Galleria mellonella wax moth larvae infection model. Taken together, these findings suggest that the Bcam1349 protein is a transcriptional regulator that binds c-di-GMP and regulates biofilm formation and virulence in B. cenocepacia in response to the level of c-di-GMP.

Listeria monocytogenes causes a life-threatening food-borne disease known as Listeriosis. Elderly,immunocompromised, and pregnant women are primarily the victims of this facultative intracellular Gram-positive pathogen. Since the bacteria survive intracellularly within the human host cells they are protected against the immune system and poorly accessed by many antibiotics. In order to screen pharmaceutical substances for their ability to interfere with the infection, persistence and release of L. monocytogenes a high content as say is required. We established a high content screen (HCS) using the RAW 264.7 mouse macrophage cell line seeded into 96-well glass bottom microplates. Cells were infected with GFP-expressing L. monocytogenes and stained thereafter with Hoechst 33342.Automated image acquisition was carried out by the Scan(R) screening station. We have developed an algorithm that automatically grades cells in microscopy images of fluorescent-tagged Listeria for the severity of infection. The grading accuracy of this newly developed algorithm is 97.1% as compared to a 74.3%grading accuracy we obtained using the commercial Olympus Scan(R) software.

The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.

The growing importance of vesicular trafficking and cytoskeleton dynamic reorganization during plant development requires the exploitation of novel experimental approaches. Several genetic and cell biological studies have used diverse pharmaceutical drugs that inhibit vesicular trafficking and secretion to study these phenomena. Here, proteomic and cell biology approaches were applied to study effects of brefeldin A (BFA), an inhibitor of vesicle recycling and secretion, in Arabidopsis roots. The main aim of this study was to obtain an overview of proteins affected by BFA, but especially to identify new proteins involved in the vesicular trafficking and its cross-talk to the actin cytoskeleton. The results showed that BFA altered vesicular trafficking and caused the formation of BFA-compartments which was accompanied by differential expression of several proteins in root cells. Some of the BFA-up-regulated proteins belong to the class of the vesicular trafficking proteins, such as V-ATPase and reversibly glycosylated polypeptide, while others, such as profilin 2 and elongation factor 1 alpha, are rather involved in the remodeling of the actin cytoskeleton. Upregulation of profilin 2 by BFA was verified by immunoblot and live imaging at subcellular level. The latter approach also revealed that profilin 2 accumulated in BFA-compartments which was accompanied by remodeling of the actin cytoskeleton in BFA-treated root cells. Thus, profilin 2 seems to be involved in the cross-talk between vesicular trafficking and the actin cytoskeleton, in a BFA-dependent manner.

Proanthocyanidins (PAs) are a class of flavonoids with numerous functions in plant ecology and development, including protection against microbial infection, animal foraging and damage by UV light. PAs are also beneficial in the human diet and livestock farming, preventing diseases of the cardiovascular system and lowering the risk of cancer, asthma and diabetes. Apples (Malus x domestica Borkh.) are naturally rich in flavonoids, but the flavonoid content and composition varies significantly between cultivars. In this work, we applied knowledge from the model plant Arabidopsis thaliana, for which the main features of flavonoid biosynthesis have been elucidated, to investigate PA accumulation in apple. We identified functional homologues of the Multidrug And Toxic compound Extrusion (MATE) gene TRANSPARENT TESTA12 from A. thaliana using a comparative genomics approach. MdMATE1 and MdMATE2 were differentially expressed, and the function of the encoded proteins was verified by complementation of the respective A. thaliana mutant. In addition, MdMATE genes have a different gene structure in comparison to homologues from other species. Based on our findings, we propose that MdMATE1 and MdMATE2 are vacuolar flavonoid/H(+) -antiporters, active in PA accumulating cells of apple fruit. The identification of these flavonoid transporter genes expands our understanding of secondary metabolite biosynthesis and transport in apple, and is a prerequisite to improve the nutritional value of apples and apple-derived beverages.

In addition to establishing methods for proteome analysis of cell wall proteins (CWPs) for the model plant Medicago truncatula, this work highlights the presence of several protein classes in cell culture. Using a combination of two-dimensional gel electrophoresis (2D-PAGE) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS), we established the proteome reference map of M. truncatula cell wall proteins. CWPs extracted from purified cell wall fragments resulted in the identification of 46 (2D-PAGE) and 65 (LC-MS/MS) proteins, respectively, with a total of 111 proteins. The identified proteins are involved in various processes, including cell wall modifications, signaling, defense mechanisms, membrane transport, protein synthesis and processing. Further, we conducted comparative proteome analysis to identify changes in protein composition during interaction of M. truncatula cell suspension culture with a pathogen-derived yeast elicitor (YE) and suppressor using Sinorhizobium meliloti LPS. 2D-PAGE analysis for the CWPs after YE and LPS treatment resembled the proteome map of YE alone, with a few up-regulated proteins involved in defense, and in the case of the LPS-treated cell wall proteome, there was no significant difference observed. Using this approach, proteins involved in defense, such as L-ascorbate peroxidase, specifically targeted proteins to the cell wall during defense, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and proteins that play an important role during growth and development were identified. Also, some defense-related proteins were absent in the same gel after YE treatment, suggesting that oxidant protection is regulated by these proteins. (C) 2010 Elsevier GmbH. All rights reserved.

Methylation of protein arginines represents an important post-translational modification mechanism, which has so far primarily been characterized in mammalian cells. In this work, we successfully identified and characterized arginine methylation as a crucial type of post-translational modification in the activity regulation of the cytosolic translation repressor protein NAB1 in the plant model organism Chlamydomonas reinhardtii. NAB1 represses the cytosolic translation of light-harvesting protein encoding mRNAs by sequestration into translationally silent messenger ribonucleoprotein complexes (mRNPs). Protein arginine methylation of NAB1 could be demonstrated by PRMT1 catalyzed methylation of recombinant NAB1 in vitro, and by immunodetection of methylated NAB1 arginines in vivo. Mass spectrometric analyses of NAB1 purified from C. reinhardtii revealed the asymmetric dimethylation of Arg90 and Arg92 within GAR motif I. Inhibition of arginine methylation by either adenosine-2'-3'-dialdehyde (AdOx) or 7,7'-carbonylbis(azanediyl)bis(4-hydroxynaphthalene-2-sulfonic acid) sodium salt hydrate (AMI-1) caused a dark-green phenotype characterized by the increased accumulation of light-harvesting complex proteins, and indicating a reduced translation repressor activity of NAB1. The extent of NAB1 arginine methylation depends on the growth conditions, with phototrophic growth causing a high methylation state and heterotrophic growth resulting in lowered methylation of the protein. In addition, we could show that NAB1 activity regulation by arginine methylation operates independently from cysteine-based redox control, which has previously been shown to control the activity of NAB1.

In viral myocarditis, adeno- and enteroviruses have most commonly been implicated as causes of infection. Both viruses require the human coxsackie-adenovirus receptor (CAR) to infect the myocardium. Due to its crucial role for viral entry, CAR-downregulation may lead to novel approaches for treatment for viral myocarditis. In this study, we report on pharmaceutical drug influences on CAR levels in human umbilical vein endothelial cells (HUVEC) and cervical carcinoma cells (HeLa) detected by immunoblotting, quantitative real time-PCR and cellular susceptibility to the cardiotropic coxsackie-B3 virus strain Nancy (CVB3). Our results indicate, for the first time, a dose-dependent CAR mRNA and protein downregulation upon Valsartan and Bosentan treatment. Most interestingly, drug-induced CAR diminution significantly reduced the viral load in CVB3-infected HUVEC. In order to assess the regulatory effects of both drugs in detail, we knocked down their protein targets, the G-protein coupled receptors angiotensin-II type-1 receptor (AT1R) and endothelin-1 type-A and -B receptors (ETAR/ETBR) in HUVEC. Receptor-specific gene silencing indicates that CAR gene expression is regulated by agonistic and antagonistic binding to ETBR, but not ETAR. In addition, neither stimulation nor inhibition of AT1R seemed to be involved in CAR gene regulatory processes. Our study indicates that Valsartan and Bosentan protected human endothelial cells from CVB3-infection. Therefore, besides their well-known anti-hypertensive effects these drugs may also protect the myocardium and other tissues from coxsackie- and adenoviral infection.

*† SUMMARY The polarization of sterol-enriched lipid microdomains has been linked to morphogenesis and cell movement in diverse cell types. Recent biochemical evidence has confirmed the presence of lipid microdomains in plant cells; however, direct evidence for a functional link between these microdomains and plant cell growth is still lacking. Here, we reported the involvement of lipid microdomains in NADPH oxidase (NOX)-dependent reactive oxygen species (ROS) signaling in Picea meyeri pollen tube growth. Staining with di-4-ANEPPDHQ or filipin revealed that sterol-enriched microdomains were polarized to the growing tip of the pollen tube. Sterol sequestration with filipin disrupted membrane microdomain polarization, depressed tip-based ROS formation, dissipated tip-focused cytosolic Ca 2+ gradient and thereby arrested tip growth. NOX clustered at the growing tip, and corresponded with the ordered membrane domains. Immunoblot analysis and native gel assays demonstrated that NOX was partially associated with detergent-resistant membranes and, furthermore, that NOX in a sterol-dependent fashion depends on membrane microdomains for its enzymatic activity. In addition, in vivo time-lapse imaging revealed the coexistence of a steep tip-high apical ROS gradient and subapical ROS production, highlighting the reported signaling role for ROS in polar cell growth. Our results suggest that the polarization of lipid microdomains to the apical plasma membrane, and the inclusion of NOX into these domains, contribute, at least in part, to the ability to grow in a highly polarized manner to form pollen tubes.

A comprehensive proteomic approach was applied to investigate molecular events occurring upon inoculation of Medicago truncatula cell-suspension cultures with the oomycete root pathogen Aphanomyces euteiches. Establishment of an inoculation assay in the cell cultures allowed a direct comparison between proteins induced by elicitation with a crude culture extract of the oomycete and by inoculation with A. euteiches zoospores representing the natural infection carrier. Oxidative burst assays revealed responsiveness of the cell cultures for perception of elicitation and inoculation signals. The plant “elicitation proteome” resembles the “inoculation proteome” in early incubation stages and includes proteins induced following initial oxidative burst and defense reactions, but also proteins involved in the antioxidative system. However, approximately 2 days after incubation, the inoculation proteome differs drastically from the proteome of elicited cultures, where a cessation of responses assignable to A. euteiches elicitation occurred. The specific protein induction patterns of zoospore-inoculated cells appeared consistent with the protein induction identified in recent studies for an A. euteiches infection in planta and consist of three functional groups: i) pathogenesis-related proteins, ii) proteins associated with secondary phenylpropanoid or phytoalexin metabolism, and, particularly, iii) proteins assigned to carbohydrate metabolism and energy-related cellular processes. Phosphoproteomic analyses revealed consistent and specific activation of these defense-related pathways already at very early timepoints of inoculation, providing evidence that the identified protein profiles are representative for an established A. euteiches infection of M. truncatula.