Martina Marchetti-Deschmann

Evaluation of matrix-assisted laser desorption/ionization (MALDI) preparation techniques for surface characterization of intact Fusarium spores by MALDI linear time-of-flight mass spectrometry

Unambiguous identification of mycotoxin-producing fungal species as Fusarium is of great relevance to agriculture and the food-producing industry as well as in medicine. Protein profiles of intact fungal spores, such as Penicillium, Aspergillus and Trichoderma, derived from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were shown to provide a rapid and straightforward method for species identification and characterization. In this study, we applied this approach to five different Fusarium spp. strains which are known to affect the growth of different grain plants. To obtain a suitable MALDI matrix system and sample preparation method, thin-layer, dried-droplet and sandwich methods and several MALDI matrices, namely CHCA, DHB, FA, SA and THAP dissolved in various solvent mixtures (organic solvents such as ACN, MeOH, EtOH and iPrOH and for the aqueous phase water and 0.1% TFA), were evaluated in terms of mass spectrometric pattern and signal intensities. The most significant peptide/protein profiles were obtained with 10 mg ferulic acid (FA) in 1 mL ACN/0.1% TFA (7:3, v/v) used as matrix system. Mixing the spores with the matrix solution directly on the MALDI target (dried-droplet technique) resulted in an evenly distributed spores/matrix crystal layer, yielding highly reproducible peptide/protein profiles from the spore surfaces. Numerous abundant ions throughout the investigated m/z range (m/z 1500-15 000) could be detected. Differences in the obtained mass spectral patterns allowed the differentiation of spores of various Fusarium species.

Development of a MALDI two-layer volume sample preparation technique for analysis of colored conidia spores of Fusarium by MALDI linear TOF mass spectrometry

AbstractMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been proved to be a powerful tool for the identification and characterization of microorganisms based on their surface peptide/protein pattern. Because of the complexity of microorganisms, there are no standardized protocols to acquire reproducible peptide/protein profiles for a broad range of microorganisms and for fungi in particular. Small variations during MALDI MS sample preparation affect the quality of mass spectra quite often. In this study, we were aiming to develop a sample preparation method for the analysis of colored, a quite often observed phenomenon, and mycotoxin-producing Fusarium conidia spores using MALDI–TOF MS. Different washing solvent systems for light- and deep-colored (from slightly orange to red-brown) conidia spores and connected sample deposition techniques were evaluated based on MS reproducibility and number and intensities of peaks. As a method of choice for generation of reproducible and characteristic MALDI–TOF mass spectra, the use of a washing process for colored Fusarium conidia spores with acetonitrile/0.5% formic acid (7/3) was found and subsequently combined with two-layer volume technique (spores/matrix (ferulic acid) solution was deposited onto a MALDI target, and after solvent evaporation, a second matrix layer was deposited). With the application of this sample preparation method, for deep-colored Fusarium species, 19 abundant molecular ions in the m/z range 2,000–10,000 were always detected with an S/N ratio of 3:1 or better. Finally this optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level. FigureIntact spore mass spectrometry - from fungal conidia spores to optimal MALDI TOF mass spectrum for species differentiation and identification.

Biodegradable, thermoplastic polyurethane grafts for small diameter vascular replacements

Biodegradable vascular grafts with sufficient in vivo performance would be more advantageous than permanent non-degradable prostheses. These constructs would be continuously replaced by host tissue, leading to an endogenous functional implant which would adapt to the need of the patient and exhibit only limited risk of microbiological graft contamination. Adequate biomechanical strength and a wall structure which promotes rapid host remodeling are prerequisites for biodegradable approaches. Current approaches often reveal limited tensile strength and therefore require thicker or reinforced graft walls. In this study we investigated the in vitro and in vivo biocompatibility of thin host-vessel-matched grafts (n=34) formed from hard-block biodegradable thermoplastic polyurethane (TPU). Expanded polytetrafluoroethylene (ePTFE) conduits (n=34) served as control grafts. Grafts were analyzed by various techniques after retrieval at different time points (1 week; 1, 6, 12 months). TPU grafts showed significantly increased endothelial cell proliferation in vitro (P<0.001). Population by host cells increased significantly in the TPU conduits within 1 month of implantation (P=0.01). After long-term implantation, TPU implants showed 100% patency (ePTFE: 93%) with no signs of aneurysmal dilatation. Substantial remodeling of the degradable grafts was observed but varied between subjects. Intimal hyperplasia was limited to ePTFE conduits (29%). Thin-walled TPU grafts offer a new and desirable form of biodegradable vascular implant. Degradable grafts showed equivalent long-term performance characteristics compared to the clinically used, non-degradable material with improvements in intimal hyperplasia and ingrowth of host cells.

Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment

ABSTRACT Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.

Application of gold thin-films for internal standardization in LA-ICP-MS imaging experiments

LA-ICP-MS imaging experiments are of growing interest within the field of biosciences. Revealing the distributions of major components as well as trace elements in biological samples can help to understand fundamental biological processes. However, highly variable sample conditions and changing instrumental parameters during measurement time aggravate reliable quantification especially in biological tissues. Normally matrix matched standards used for calibration are scarcely available and the manufacturing process thereof is rather complicated. Thus most experiments reported in the literature only delivered qualitative information on the analyte distributions. The use of appropriate internal standards facilitates the preparation of calibrations even without the utilization of matrix-matched standards. In the presented work an approach for providing reliable quantitative bio-images is proposed using gold thin-layers as an internal standard and patterns printed with commercially available inkjet printers as standards. The method development is based on copper from blue ink as the element of interest. It could be shown that gold standardization compensates instrumental drifts, matrix related ablation differences and day-to-day signal changes. Not only was the quality of the obtained images improved by gold standardization; while the relative standard deviation of the measurements was around 15% before standardization it could be decreased to less than 5% by gold standardization. Also quantitative information could be obtained for samples with unknown analyte concentrations. Depending on the used beam diameter limits of detection in the range of some hundreds ng g(-1) were achieved. The presented method is a promising and easy-to-handle alternative to matrix matched standards for signal quantification.

A proteomic study reveals unspecific apoptosis induction and reduction of glycolytic enzymes by the phosphorothioate antisense oligonucleotide oblimersen in human melanoma cells

The question of specificity and the elucidation of the exact molecular mechanism of action of post-transcriptional gene silencing agents are two major challenges for their establishment as therapeutics. A proteomic off-target effect study (2-DE with MS) in combination with DIGE comparing the phosphorothioate antisense oligonucleotide oblimersen (Genasense, G3139) to a Bcl-2-targeting siRNA-sequence on human melanoma cells showed that additional off-target effects contribute to the apoptotic effect of oblimersen. When both oligonucleotides were transfected with lipofectamine 2000, only oblimersen increased apoptosis as determined by annexin staining and caspase activity measurement. In contrast to the highly specific siRNA, the expression level of a number of proteins was found to be altered after oblimersen treatment. Several proteins linked to apoptosis and stress response, among those galectin-1, cofilin-1, GRP78, HSP60, nucleophosmin, and peroxiredoxins, were identified and found to be down-regulated after oblimersen treatment. A down-regulation of enolase-1 and three other glycolytic enzymes indicates a reversion of the cancer-related Warburg effect. The observed effects may be caused by a phosphorothioate mediated blockage of the mitochondrial voltage dependent anion channel (VDAC).

Tyrosine Kinase 2 Controls IL-1β Production at the Translational Level

IL-1β is an important proinflammatory cytokine with a major role in several inflammatory diseases. Expression of IL-1β is tightly regulated at the level of transcription, mRNA stability, and proteolytic processing. In this study, we report that IL-1β expression in response to LPS is also regulated at the translational level. LPS-induced IL-1β protein levels in macrophages derived from murine bone marrow are markedly increased in the absence of tyrosine kinase 2 (Tyk2). Increased IL-1β is found intra- and extracellularly, irrespective of the efficiency of IL-1β processing. We show that the absence of Tyk2 results both in higher translational rates and in enhanced association of IL-1β mRNA with polysomes. Induction and stability of IL-1β mRNA are not affected by the lack of Tyk2. We show further that the Tyk2-dependent translational inhibition is mediated by autocrine/paracrine type I IFN signaling and requires signal transducer and activator of transcription 1. Enhanced IL-1β production in Tyk2- and IFN receptor 1-deficient macrophages is also observed following Listeria monocytogenes infection. Taken together, the data describe a novel mechanism for the control of IL-1β synthesis.

Combining light microscopy, dielectric spectroscopy, MALDI intact cell mass spectrometry, FTIR spectromicroscopy and multivariate data mining for morphological and physiological bioprocess characterization of filamentous organisms

Along with productivity and physiology, morphological growth behavior is the key parameter in bioprocess design for filamentous fungi. Lacking tools for fast, reliable and efficient analysis however, fungal morphology is still commonly tackled by empirical trial-and-error techniques during strain selection and process development procedures. Bridging the gap, this work presents a comprehensive analytical approach for morphological analysis combining automated high-throughput microscopy, multi-frequency dielectric spectroscopy, MALDI intact cell mass spectrometry and FTIR spectromicroscopy. Industrial fed-batch production processes were investigated in fully instrumented, automated bioreactors using the model system Penicillium chrysogenum. Physiological process characterization was based on the determination of specific conversion rates as scale-independent parameters. Conventional light microscopic morphological analysis was based on holistic determination of time series for more than 30 morphological parameters and their frequency distributions over the respective parameter range by automated high-throughput light microscopy. Characteristic protein patterns enriched in specific morphological and physiological states were further obtained by MALDI intact cell mass spectrometry. Spatial resolution of molecular biomass composition was facilitated by FTIR spectromicroscopy. Real-time in situ monitoring of morphological process behavior was achieved by linking multi-frequency dielectric spectroscopy with above outlined off-line methods. Data integration of complementing orthogonal techniques for morphological and physiological analysis together with multivariate modeling of interdependencies between morphology, physiology and process parameters facilitated complete bioprocess characterization. The suggested approach will thus help understanding morphological and physiological behavior and, in turn, allow to control and optimize those complex processes.

Lectin bioconjugates trigger urothelial cytoinvasion--a glycotargeted approach for improved intravesical drug delivery.

A unique structural and functional configuration renders the human urothelium, one of the hardest to overcome biological barriers, and accounts for critical shortcomings in the adjuvant localized therapy of bladder cancer and other severe medical conditions. Strategies to improve intravesical drug absorption are urgently sought, but so far have hardly adopted biorecognitive delivery vectors that are more specifically tailored to the natural characteristics of the target site. The efficient cytoinvasion of uropathogenic bacteria, mediated via a mannose-directed FimH lectin adhesin, and malignancy-dependent differences in bladder cell glycosylation point to considerable unrealized potential of lectins as targeting vectors on the molecular/functional and recognitive level. Here, we outline the ability of wheat germ agglutinin (WGA) to induce endocytosis of conjugated payload in human urothelial SV-HUC-1 cells after stable adhesion to internalizing receptors. A panel of model bioconjugates was prepared by covalently coupling one to six WGA units to fluorescein-labeled bovine serum albumin (fBSA). Cytoadhesive capacity was found to directly correlate to the degree of modification up to a critical threshold of on average three targeting ligands per conjugate. The highly specific, glycan-triggered interaction proved essential for endosomal sorting and was followed by rapid (<60min) and extensive (>40%) internalization. fBSA/WGA bioconjugates were processed analogously to the free lectin, irrespective of the significantly higher molecular weight (100-300kD). Durable entrapment of conjugates in acidic, perinuclear compartments without kiss-and-run recycling to the plasma membrane was found in both single cells and monolayers. Our results assign promising potential to glycotargeted delivery concepts in the intravesical setting and offer new perspectives for the application of complex biologicals in the urinary tract.

Ammodytagin, a heterodimeric metalloproteinase from Vipera ammodytes ammodytes venom with strong hemorrhagic activity

Ammodytagin, a hemorrhagic Zn(2+)-dependent metalloproteinase from Vipera ammodytes ammodytes (Vaa) venom, is a glycosylated heterodimer of 108 kDa, as determined by MALDI mass spectrometry. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences belonging to metalloproteinase, disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allows classification into the P-IIIc group of snake venom metalloproteinases (SVMPs). Only few members of that group have been described so far. Ammodytagin possesses potent azocaseinolytic activity which can be inhibited by Na(2)EDTA, Zn(2+) and DTT. It cleaves insulin B-chain, hydrolysing it at positions Gln(4)-His(5), His(10)-Leu(11) and Tyr(16)-Leu(17). Furthermore, ammodytagin acts as a strong hemorrhagin in both rats and mice. Investigation of a substrate specificity revealed that the hemorrhagic activity of the novel SVMP might be the result of its involvement in cleavage of basal membrane components and depletion of fibrinogen, prothrombin and factor X in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective Vaa specific antivenom should be directed.