Differential expression profiling of components associated with exoskeletal hardening in crustaceans

<p>Abstract</p> <p>Background</p> <p>Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from <it>Portunus pelagicus </it>were used to identify genes possibly associated with the activation pathways involved in these processes.</p> <p>Results</p> <p>Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt.</p> <p>Conclusion</p> <p>Cuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.</p

Differentiation of meat species of raw and processed meat based on polar metabolites using 1H NMR spectroscopy combined with multivariate data analysis

Meat species of raw meat and processed meat products were investigated by $^1$H NMR spectroscopy with subsequent multivariate data analysis. Sample preparation was based on aqueous extraction combined with ultrafiltration in order to reduce macromolecular components in the extracts. $^1$H NMR data was analyzed by using a non—targeted approach followed by principal component analysis (PCA), linear discrimination analysis (LDA), and cross-validation (CV) embedded in a Monte Carlo (MC) resampling approach. A total of 379 raw meat samples (pork, beef, poultry, and lamb) and 81 processed meat samples (pork, beef, poultry) were collected between the years 2018 and 2021. A 99% correct prediction rate was achieved if the raw meat samples were classified according to meat species. Predicting processed meat products was slightly less successful (93 %) with this approach. Furthermore, identification of spectral regions that are relevant for the classification via polar chemical markers was performed. Finally, data on polar metabolites were fused with previously published $^1$H NMR data on non-polar metabolites in order to build a broader classification model and to improve prediction accuracy

¹H NMR spectroscopy combined with multivariate data analysis for authentication of “Swabian–Hall Quality Pork” with protected geographical indication

1H NMR spectroscopy was applied to analyse samples of “Swabian–Hall Quality Pork” with protected geographical indication (PGI). To obtain maximum chemical information sample preparation was based on both polar extraction and non-polar extraction. A non-targeted approach was used to analyse the 1H NMR data followed by principal component analysis (PCA), linear discriminant analysis (LDA), and cross-validation (CV) embedded in a Monte Carlo (MC) resampling approach. A total of 275 raw pork samples were collected in the years 2018 to 2021. The correct prediction rate of “Swabian–Hall Quality Pork” was about 92% on average for both models based on either the polar or non-polar metabolites. In addition, 1H NMR data describing the polar and non-polar metabolites were combined in a classification model to improve the prediction accuracy. By performing a mid-level data fusion, a correct prediction rate of 98% was achieved. Furthermore, spectral regions in the NMR spectra of the polar and non-polar metabolites that are relevant for the classification of the pork samples were identified to describe potential chemical marker compounds

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton

Das E6-Onkoprotein humaner Papillomviren und seine Wechselwirkung mit E6AP als Zielstruktur für eine molekulare Therapie zervikaler Karzinome

Bestimmte Typen humaner Papillomviren (´high risk´ HPVs) spielen bei der Entstehung des Zervixkarzinoms eine kausale Rolle. Dabei scheint die Expression der viralen Proteine E6 und E7 in allen Stadien der Karzinogenese essentiell zu sein. E6 besitzt anti-apoptotisches Potential, das vermutlich zur Kompensation einer E7-vermittelten Apoptose-Sensitivität infizierter Zellen benötigt wird. Für E6 sind potentielle Wechselwirkungen mit einer Vielzahl von zellulären Proteinen beschrieben, wobei die physiologische Relevanz für die meisten Interaktionen weitestgehend unbekannt ist. In dieser Arbeit wurden mutierte Formen von E6 durch gezielte Mutagenese generiert und jeweils in ihren Wechselwirkungen mit dem Tumorsuppressor p53, der Protein-Ubiquitin-Ligase E6AP und den PDZ-Proteinen Scribble und Dlg charakterisiert. Dabei wurden Beobachtungen gemacht, die auf einen E6AP-vermittelten Abbau von Dlg durch E6 hinweisen und somit im Widerspruch zu publizierten Daten stehen. Dies ist aber vermutlich dadurch zu erklären, dass in vorherigen Studien ein Abbau von Dlg durch Formen von E6, die sich von ´low risk´ E6 Proteinen ableiten, beobachtet wurde. Da diese E6 Formen nicht in der Lage sind, E6AP in vitro zu kopräzipitieren, wurde geschlussfolgert, dass sie nicht mit E6AP interagieren. Durch die Verwendung von E6AP -/- Mausfibroblasten konnten in dieser Arbeit allerdings deutliche Hinweise für eine Interaktion von E6AP mit dem E6 Protein des ´low risk´ HPV-Typen 11 gefunden werden. Zusätzlich wurde das onkogene Potential der generierten E6 Mutanten in Zellkulturstudien untersucht. Vorläufige Ergebnisse zeigen an, dass dem extremen C-Terminus von ´high risk´ E6 Proteinen eine wichtige Rolle bei der Immortalisierung von Zellen zukommt. Um diesen Einfluss des C-Terminus näher zu spezifizieren, ist die Generierung weiterer E6 Mutanten notwendig. Um zu untersuchen, ob die Interaktion von E6 mit E6AP eine potentielle Zielstruktur für molekulare Ansätze bei der Behandlung des Zervixkarzinoms darstellt, wurde die Methode der Proteintransduktion etabliert. Dazu wurde das von E6AP-abgeleitete E6-Bindepeptid e6bp1 an ein zellpenetrierendes Peptid (im vorliegenden Fall ´ARG7´) fusioniert und es konnten alle wesentlichen Eigenschaften von zellpenetrierenden Peptiden beobachtet werden. Allerdings wurden alle Untersuchungen unter Bedingungen durchgeführt, für die kürzlich die Möglichkeit der Erzeugung von Artefakten beschrieben wurde. Daher sind weitere Untersuchungen nötig, um Aussagen über einen Einfluss von e6bp1 auf das Wachstum von HPV-positiven Zellen treffen zu können

Determination of Diethyl Phthalate and Polyhexamethylene Guanidine in Surrogate Alcohol from Russia

Analytical methods based on spectroscopic techniques were developed and validated for the determination of diethyl phthalate (DEP) and polyhexamethylene guanidine (PHMG), which may occur in unrecorded alcohol. Analysis for PHMG was based on UV-VIS spectrophotometry after derivatization with Eosin Y and 1H NMR spectroscopy of the DMSO extract. Analysis of DEP was performed with direct UV-VIS and 1H NMR methods. Multivariate curve resolution and spectra computation methods were used to confirm the presence of PHMG and DEP in the investigated beverages. Of 22 analysed alcohol samples, two contained DEP or PHMG. 1H NMR analysis also revealed the presence of signals of hawthorn extract in three medicinal alcohols used as surrogate alcohol. The simple and cheap UV-VIS methods can be used for rapid screening of surrogate alcohol samples for impurities, while 1H NMR is recommended for specific confirmatory analysis if required

Determination of atmospheric mercury during the North Sea experiment

Total gaseous mercury (TGM) and rainwater were collected on board of two research vessels (F. S. ALKOR and R.V. BELGICA) positioned 200 km apart in the center of the North Sea during the North Sea Experiment, September 1991. On the F. S. ALKOR (up-wind ship) TGM concentrations ranged from 0.7 to 2.6 ng.m(-3) with an average of 1.5 ng.m(-3) and on the R. V. BELGICA (down-wind ship) TGM ranged from 0.7 to 1.9 ng.m(-3) with an average of 1.2 ng.m(-3). An average 20% decrease is observed from the up-wind to the downwind ship. which may largely be affected by entrainment into the free troposphere. An overall removal (entrainment) velocity of 0.95 cm.s(-1) was calculated for the whole experiment. The average removal velocity was 0.5 cm.s(-1) for dry periods and varied between 1 to 5 cm.s(-1) during rain events. Rainwater concentrations varied between 5 and 25 ng.l(-1). Based on these data an annual wet deposition flux of 1.08 ng Hg cm(-2) yr(1-) was estimated for the North Sea

Determination of the biologically active flavour substances thujone and camphor in foods and medicines containing sage (Salvia officinalis L.)

BACKGROUND: The sage plant Salvia officinalis L. is used as ingredient in foods and beverages as well as in herbal medicinal products. A major use is in the form of aqueous infusions as sage tea, which is legal to be sold as either food or medicine. Sage may contain two health relevant substances, thujone and camphor. The aim of this study was to develop and validate an analytical methodology to determine these active principles of sage and give a first overview of their concentrations in a wide variety of sage foods and medicines. RESULTS: A GC/MS procedure was applied for the analysis of α- and β-thujone and camphor with cyclodecanone as internal standard. The precision was between 0.8 and 12.6%, linearity was obtained from 0.1 - 80 mg/L. The recoveries of spiked samples were between 93.7 and 104.0% (average 99.1%). The time of infusion had a considerable influence on the content of analytes found in the teas. During the brewing time, thujone and camphor show an increase up to about 5 min, after which saturation is reached. No effect was found for preparation with or without a lid on the pot used for brewing the infusion. Compared to extracts with ethanol (60% vol), which provide a maximum yield, an average of 30% thujone are recovered in the aqueous tea preparations. The average thujone and camphor contents were 4.4 mg/L and 16.7 mg/L in food tea infusions and 11.3 mg/L and 25.4 mg/L in medicinal tea infusions. CONCLUSIONS: The developed methodology allows the efficient determination of thujone and camphor in a wide variety of sage food and medicine matrices and can be applied to conduct surveys for exposure assessment. The current results suggest that on average between 3 and 6 cups of sage tea could be daily consumed without reaching toxicological thresholds