Comparison of the Structures of Triacylglycerols from Native and Transgenic Medium-Chain Fatty Acid-Enriched Rape Seed Oil by Liquid Chromatography–Atmospheric Pressure Chemical Ionization Ion-Trap Mass Spectrometry (LC–APCI-ITMS)

The sn position of fatty acids in seed oil lipids affects physiological function in pharmaceutical and dietary applications. In this study the composition of acyl-chain substituents in the sn positions of glycerol backbones in triacylglycerols (TAG) have been compared. TAG from native and transgenic medium-chain fatty acid-enriched rape seed oil were analyzed by reversed-phase high performance liquid chromatography coupled with online atmospheric-pressure chemical ionization ion-trap mass spectrometry. The transformation of summer rape with thioesterase and 3-ketoacyl-[ACP]-synthase genes of Cuphea lanceolata led to increased expression of 1.5% (w/w) caprylic acid (8:0), 6.7% (w/w) capric acid (10:0), 0.9% (w/w) lauric acid (12:0), and 0.2% (w/w) myristic acid (14:0). In contrast, linoleic (18:2n6) and alpha-linolenic acid (18:3n3) levels decreased compared with the original seed oil. The TAG sn position distribution of fatty acids was also modified. The original oil included eleven unique TAG species whereas the transgenic oil contained sixty. Twenty species were common to both oils. The transgenic oil included trioctadecenoyl-glycerol (18:1/18:1/18:1) and trioctadecatrienoyl-glycerol (18:3/18:3/18:3) whereas the native oil included only the latter. The transgenic TAG were dominated by combinations of caprylic, capric, lauric, myrisitic, palmitic (16:0), stearic (18:0), oleic (18:1n9), linoleic, arachidic (20:0), behenic (22:0), and lignoceric acids (24:0), which accounted for 52% of the total fat. In the original TAG palmitic, stearic, oleic, and linoleic acids accounted for 50% of the total fat. Medium-chain triacylglycerols with capric and lauric acids combined with stearic, oleic, linoleic, alpha-linolenic, arachidic, and gondoic acids (20:1n9) accounted for 25% of the transgenic oil. The medium-chain fatty acids were mainly integrated into the sn-1/3 position combined with the essential linoleic and alpha-linolenic acids at the sn-2 position. Eight species contained caprylic, capric, and lauric acids in the sn-2 position. The appearance of new TAG in the transgenic oil illustrates the extensive effect of genetic modification on fat metabolism by transformed plants and offers interesting possibilities for improved enteral applications.     

Resonance-mode electrochemical impedance measurements of silicon dioxide supported lipid bilayer formation and ion channel mediated charge transport

A single-chip electrochemical method based on impedance measurements in resonance mode has been employed to study lipid monolayer and bilayer formation on hydrophobic alkanethiolate and SiO(2) substrates, respectively. The processes were monitored by temporally resolving changes in interfacial capacitance and resistance, revealing information about the rate of formation, coverage, and defect density (quality) of the layers at saturation. The resonance-based impedance measurements were shown to reveal significant differences in the layer formation process of bilayers made from (i) positively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), (ii) neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on SiO(2), and (iii) monolayers made from POEPC on hydrophobic alkanethiolate substrates. The observed responses were represented with an equivalent circuit, suggesting that the differences primarily originate from the presence of a conductive aqueous layer between the lipid bilayers and the SiO(2). In addition, by adding the ion channel gramicidin D to bilayers supported on SiO(2), channel-mediated charge transport could be measured with high sensitivity (resolution around 1 pA).     

Excretion of Avenanthramides,Phenolic Acids and their Major Metabolites Following Intake of Oat Bran

1 Scope

Wholegrain has been associated with reduced chronic disease mortality, with oat intake particularly notable for lowering blood cholesterol and glycemia. To better understand the complex nutrient profile of oats, we studied urinary excretion of phenolic acids and avenanthramides after ingestion of oat bran in humans.

2 Methods and results

After a 2‐d (poly)phenol‐low diet, seven healthy men provided urine 12 h before and 48 h after consuming 60 g oat bran (7.8 μmol avenanthramides, 139.2 μmol phenolic acids) or a phenolic‐low (traces of phenolics) control in a crossover design. Analysis by ultra‐high performance liquid chromatography (UPLC)–MS/MS showed that oat bran intake resulted in an elevation in urinary excretion of 30 phenolics relative to the control, suggesting that they are oat bran‐derived. Mean excretion levels were elevated between 0–2 and 4–8 h, following oat bran intake, and amounted to a total of 33.7 ± 7.3 μmol total excretion (mean recovery: 22.9 ± 5.0%), relative to control. The predominant metabolites included: vanillic acid, 4‐ and 3‐hydroxyhippuric acids, and sulfate‐conjugates of benzoic and ferulic acids, which accounted collectively for two thirds of total excretion.

3 Conclusion

Oat bran phenolics follow a relatively rapid urinary excretion, with 30 metabolites excreted within 8 h of intake. These levels of excretion suggest that bound phenolics are, in part, rapidly released by the microbiota.     

Biochemical,Structural Analysis,and Docking Studies of Spiropyrazoline Derivatives

In this study, we evaluated the antiproliferative potential, DNA damage, crystal structures, and docking calculation of two spiropyrazoline derivatives. The main focus of the research was to evaluate the antiproliferative potential of synthesized compounds towards eight cancer cell lines. Compound I demonstrated promising antiproliferative properties, especially toward the HL60 cell line, for which IC₅₀ was equal to 9.4 µM/L. The analysis of DNA damage by the comet assay showed that compound II caused DNA damage to tumor lineage cells to a greater extent than compound I. The level of damage to tumor cells of the HEC-1-A lineage was 23%. The determination of apoptotic and necrotic cell fractions by fluorescence microscopy indicated that cells treated with spiropyrazoline-based analogues were entering the early phase of programmed cell death. Compounds I and II depolarized the mitochondrial membranes of cancer cells. Furthermore, we performed simple docking calculations, which indicated that the obtained compounds are able to bind to the PARP1 active site, at least theoretically (the free energy of binding values for compound I and II were −9.7 and 8.7 kcal mol⁻¹, respectively). In silico studies of the influence of the studied compounds on PARP1 were confirmed in vitro with the use of eight cancer cell lines. The degradation of the PARP1 enzyme was observed, with compound I characterized by a higher protein degradation activity.     

Mehr Nutzen als Schaden?

Background: Clinical research involving human subjects must be ethically legitimatised by being scientifically valid, satisfying legal norms, and adhering to basic ethical requirements such as informed consent and appropriate risk-benefit ratios. Autonomous institutional review boards (IRB) support researchers in meeting these demands. Methods: We propose and test a systematic approach to the ethical analysis of risks and potential benefits in clinical research involving human subjects. The scheme was applied on all study protocols from the year 2006 presented to the IRB of our medical faculty. Results: 46?% of the 206 analyzed protocols promise some potential direct benefit to study participants. 12?% of the planned research projects offer the chance of benefit for future patients with the same demographic and clinical characteristics as the study participants (??group-benefit??). The reminder of the protocols (42?%) reveal potential benefit only for medicine and science through gaining knowledge of clinical, social, or scientific value. More than minimal risks for research participants were identified in about 53?% of the studies. Our ethical analysis and evaluation resulted in 33 out of 206 protocols (16?%) with an unfavourable and hardly justifiable risk-benefit ratio. Conclusion: The developed taxonomy together with our conceptual framework for comparing and balancing potential research benefit and harm can increase the transparency and facilitate the communication between researchers and IRB members. Clear guidance for the IRBs supports the standardisation and harmonisation of ethical review, advice, and approval procedures.     

Vitamin D Metabolic Pathway Components in Orthopedic Patientes—Systematic Review

Vitamin D takes part in the functioning of many processes that ensure the homeostasis of the body. In orthopedics, it is indicated as an inseparable element ensuring proper bone growth and functioning, and its deficiencies are indicated in various diseases, mainly in the proper structure and function of the skeleton. In this review, we focus on the most important components of the vitamin D metabolic pathway, in correlation with selected orthopedic conditions. Records were obtained from the PubMed database in a timeline of 2010–2022. The keywords were as follows: vitamin D/cholesterol/vitamin D binding protein/ VDBP/Cytochrome/CYP24A1/CYP 27B1/Vitamin D receptor/VDR/ + diseases (ACL reconstruction, rotator cuff, arthroplasty knee/hip/shoulder). The recent original studies were analyzed, discussed, and the most important data were shown. The vast majority of articles concern the metabolite of vitamin D (25(OH)D), which is measured as a standard in diagnostic laboratories. Even though there is a lot of valuable information in the literature, we believe that the other elements of the vitamin D pathway also deserve attention and suggest their research in correlation with orthopedic disorders to supplement the missing knowledge on this topic.     

Foamed Propellants

Foamed propellants based on polymer bonded nitramines show high conversion rates due to their porous structure. The properties of the material can be varied in a wide range by using different explosive fillers, energetic binders and porous structures. Foamed propellants with high energy content and variable burning and material characteristics can be formulated. Due to this flexibility, these propellants can be adjusted to manifold applications. The burning characteristics of these porous charges show specific differences compared to standard gun propellants, e.g. the mass conversion rates are essentially above those obtained by combustion of compact materials. In addition, the burning behavior of foamed propellants depending on pressure deviates from that expected by a straightforward use of Vieille's law. This paper presents an overview on investigations carried out in the field of foamed propellants at Fraunhofer ICT concerning thermodynamical calculations, studies on burning behavior and vulnerability.     

Investigations on the allergenicity of gluten and hydrolyzed wheat proteins in wheat-dependent exercise-induced anaphylaxis

In the spectrum of wheat-related disorders, WDEIA is defined as immediate type-1 allergy. It is rare, but potentially life-threatening and caused by the exposition to wheat proteins and the presence of one or more cofactors. Patients suffer from anaphylactic reactions affecting the gastrointestinal and respiratory tracts, the skin and the cardiovascolar system with different severity. GPT, the storage proteins of wheat, are the most commonly reported WDEIA allergens. Thereby, the ω5-gliadins are known as major allergens and the HMW-GS as minor allergens. Reactions to other GPT and HWP are also reported. The golden standard to diagnose WDEIA is still an OFC, which puts patients at risk of anaphylaxis. The aim of this work was to further investigate the allergenicity of gluten and HWP in the context of WDEIA to contribute to a better understanding of the underlying mechanisms and expand diagnostic possibilities. First gluten and HWP were comparatively characterized (SDS-PAGE, content of free ammonium and RP-/GP-HPLC). Based on the results, products were selected from which ATS were prepared in order to investigate the BAT-FACS as a possible alternative diagnostic tool. Thereby, anti- CCR3-PE-mAb (BG identification marker) and anti-CD63-FITC-mAb (BG activation marker) were used. Second, patient studies with control subjects were carried out. The results showed that the BAT-FACS is very promising to supplement WDEIA routine diagnosis, as especially the ATS from ω5-gladins and sHWP showed very good test sensitivity and specificity. Individual senzitization profiles of the patients were generated after isolation of single GPT and their application as ATS in the BAT-FACS study. Gluten from a wheat/rye translocation line (G-ω5, 89% reduced content of ω5-gliadins in comparison to representative gluten G) was also applied in the BAT-FACS study to evaluate whether this could possibly be used as a hypoallergenic food. However, allergenic basophil activations in patients were documented, consequently this product is not recommended as hypoallergenic food for WDEIA patients. Our findings still leave a wheat-free diet and/or avoidance of cofactors as the only safe option for WDEIA patients. Further on, fast and easily preparable ATS, usable in clinical routine diagnosis where elaborate preparations are unwanted, were generated and sucessfully tested on patients and controls. Thereby, a detailed characterization of the contained proteins in the ATS was performed (SDS-PAGE, RP-/GP-HPLC and proteomics-based untargeted high-resolution UPLC-TripleTOF-MS). These results together with the determined BAT results indicated that non-gluten proteins are also relevant allergens in the context of WDEIA since high percentages of ATIs were determined in the ATS (G, G-ω5, sHWP). In addition, yet unidentified allergenic epitopes need to be present in the fast preparable ATS (G, G-ω5, sHWP, eHWP) because the identified proteins were searched for known WDEIA epitopes and only one epitope was found. Consequently, the known epitopes cannot explain the allergenic basophil activations triggered by these ATS in WDEIA patients. Innerhalb des Spektrums der weizenbedingten Erkrankungen wird die WDEIA als unmittelbare Typ-1- Allergie definiert. Sie ist selten, aber potenziell lebensbedrohlich und wird durch die Exposition gegenüber Weizenproteinen und das Vorhandensein eines oder mehrerer Kofaktoren verursacht. Die Patienten leiden unter anaphylaktischen Reaktionen, die den Magen-Darm-Trakt, die Atemwege, die Haut und das kardiovaskuläre System in unterschiedlichem Ausmaß betreffen. Glutenprotein Typen (GPT), die Speicherproteine des Weizens, sind die am häufigsten beschriebenen WDEIA-Allergene. Dabei sind die ω5-Gliadine als Hauptallergene und die HMW-GS als Nebenallergene bekannt. Es wird auch über Reaktionen auf andere GPT und hydrolysiertes Weizenprotein (HWP) berichtet. Der goldene Standard für die WDEIA Diagnose ist nach wie vor eine orale Provokationstestung mit dem verursachenden Lebensmittel oder Gluten, die die Patienten dem Risiko einer Anaphylaxie aussetzt. Ziel dieser Arbeit war es, die Allergenität von Gluten und HWP im Kontext der WDEIA zu untersuchen, um zu einem besseren Verständnis der zugrunde liegenden Mechanismen und Erweiterung der diagnostischen Möglichkeiten beizutragen. Zum einen wurden Gluten und HWP vergleichend charakterisiert (SDS-PAGE, Gehalt an freiem Ammonium und RP-/GP-HPLC). Auf dieser Grundlage wurden Produkte ausgewählt, aus denen allergene Testlösungen (ATS) hergestellt wurden, um den basophilen Aktivierungstest kombiniert mit fluoreszenz-aktivierter Zellsortierung (BATFACS) als alternative Diagnosemöglichkeit für WDEIA zu untersuchen. Dabei wurden Anti-CCR3-phycoerythrin- monoklonale Antikörper (Identifikationsmarker für Basophile Granulozyten) und Anti-CD63- Fluoresceinisothiocyanat-monoklonale Antikörper (Aktivierungsmarker für Basophile Granulozyten) verwendet. Es wurden anschließend Patientenstudien mit Kontrollpersonen durchgeführt. Die Ergebnisse zeigten, dass der BAT-FACS eine vielversprechende Ergänzung zur Routinediagnostik der WDEIA darstellt, da insbesondere die ATS aus ω5-Gladinen und je sHWP je eine sehr gute Testsensitivität und -spezifität aufwiesen. Durch die Isolierung einzelner GPT und deren Verwendung als ATS in der BAT-FACS-Studie wurden individuelle Sensibilisierungsprofile der Patienten erstellt. Gluten aus einer Weizen/Roggen-Translokationslinie (G-ω5, 89% reduzierter Gehalt an ω5-Gliadinen im Vergleich zu repräsentativem Gluten G) wurde ebenfalls in der BAT-FACS-Studie eingesetzt, um zu evaluieren, ob dies möglicherweise als hypoallergenes Lebensmittel verwendet werden könnte. Es wurden jedoch allergene basophile Aktivierungen bei Patienten dokumentiert, so dass dieses Produkt nicht als hypoallergenes Lebensmittel für WDEIA-Patienten zu empfehlen ist. Unsere Ergebnisse zeigen, dass eine weizenfreie Ernährung und/oder die Vermeidung von Kofaktoren die einzige sichere Option für WDEIA-Patienten ist. Weiterhin wurden schnelle und einfach zuzubereitende ATS generiert, die in der klinischen Routinediagnostik eingesetzt werden können, wo aufwendige Zubereitungen unerwünscht sind. Diese ATS wurden erfolgreich an Patienten und Kontrollen getestet. Dabei wurde eine detaillierte Charakterisierung der in den ATS enthaltenen Proteine durchgeführt (SDS-PAGE, RP- /GP-HPLC und Proteom-basierte nicht-zielgerichtete hochauflösende UPLC-TripleTOF-MS). Diese Ergebnisse deuten zusammen mit den ermittelten BAT-FACS Ergebnissen darauf hin, dass auch NichtGluten-Proteine relevante Allergene im Rahmen der WDEIA sind, da hohe Mengen an α-Amylase- Trypsin-Inhibitoren in den ATS bestimmt wurden (G, G-ω5, sHWP). Darüber hinaus zeigte sich, dass in den einfach-herstellbaren ATS (G, G-ω5, sHWP, eHWP) nicht identifizierte allergene Epitope vorhanden sein müssen, da die identifizierten Proteine auf bekannte WDEIA-Epitopen analysiert wurden und lediglich eines der bekannten Epitope gefunden wurde. Folglich können die allergenen basophilen Aktivierungen bei den WDEIA-Patienten nicht durch das Vorhandensein bekannter WDEIA- Epitope erklärt werden.