Production of a Cheese-Like Aroma via Fermentation of Plant Proteins and Coconut Oil with the Basidiomycetes Cyclocybe aegerita and Trametes versicolor.

Cheese is one of the most common dairy products and is characterized by its complex aroma. However, in times of climate change and resource scarcity, the possibility to mimic the characteristic cheese-like aroma from plant-based sources is in demand to offer alternatives to cheese. Accordingly, the production of a natural cheese-like aroma via fermentation of four plant-based proteins and coconut oil with basidiomycetes has been addressed. Mixtures of soy and sunflower protein with coconut oil (15 g/L) have shown the formation of a cheese-like aroma after 72 and 56 h after fermentation with Cyclocybe aegerita and Trametes versicolor, respectively. Isovaleric acid, butanoic acid, ethyl butanoate, 1-octen-3-ol, and various ketones were identified as the key odorants. Similarities to typical cheeses were observed by the principal component analysis. Overall, the finding offered an approach to a sustainable production of a natural cheese-like aroma from a plant source, thus contributing to the development of cheese alternatives.

Isolation of plastochromanol-8 from flaxseed oil by countercurrent separation methods.

The naturally occurring antioxidant plastochromanol-8 (PC-8) is a member of the tocochromanol (vitamin E) family which features eight unsaturated isoprene units in the side chain compared to three in the case of γ-tocotrienol. Due to the lack of a commercially available PC-8 standard, we developed a route to gain relevant amounts of highly pure PC-8. Specifically, ∼320 g flaxseed oil was saponified and the bulky PC-8 was enriched by gel permeation chromatography. It followed countercurrent chromatography using the solvent system n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). The final purification was achieved by centrifugal partition chromatography using the novel solvent system hexamethyldisiloxane/acetonitrile (1:1, v/v). This step provided ∼26 mg PC-8 (>99.5 %, according to HPLC, GC and NMR analysis). Two further, hitherto unknown minor tocochromanols (<1 % of PC-8) were detected and could be identified to be plastochromanol-7 (PC-7) and plastochromanol-9 (PC-9), i.e. tocochromanols with seven and nine unsaturated isoprene units, respectively, in the side chain.

IgE type multiple myeloma exhibits hypermutated phenotype and tumor reactive T cells.

Multiple myeloma (MM) is a hematological malignancy originating from malignant and clonally expanding plasma cells. MM can be molecularly stratified, and its clonal evolution deciphered based on the Ig heavy and light chains of the respective malignant plasma cell clone. Of all MM subtypes, IgE type MM accounts for only <0.1% of cases and is associated with an aggressive clinical course and consequentially dismal prognosis. In such malignancies, adoptive transfer of autologous lymphocytes specifically targeting presented (neo)epitopes encoded by either somatically mutated or specifically overexpressed genes has resulted in substantial objective clinical regressions even in relapsed/refractory disease. However, there are no data on the genetic and immunological characteristics of this rare and aggressive entity. Here, we comprehensively profiled IgE type kappa MM on a genomic and immune repertoire level by integrating DNA- and single-cell RNA sequencing and comparative profiling against non-IgE type MM samples. We demonstrate distinct pathophysiological mechanisms as well as novel opportunities for targeting IgE type MM. Our data further provides the rationale for patient-individualized neoepitope-targeting cell therapy in high tumor mutation burden MM.

Efficient DNA Repair Mitigates Replication Stress Resulting in Less Immunogenic Cytosolic DNA in Radioresistant Breast Cancer Stem Cells.

Cancer stem cells (CSCs) are a major cause of tumor therapy failure. This is mainly attributed to increased DNA repair capacity and immune escape. Recent studies have shown that functional DNA repair via homologous recombination (HR) prevents radiation-induced accumulation of DNA in the cytoplasm, thereby inhibiting the intracellular immune response. However, it is unclear whether CSCs can suppress radiation-induced cytoplasmic dsDNA formation. Here, we show that the increased radioresistance of ALDH1-positive breast cancer stem cells (BCSCs) in S phase is mediated by both enhanced DNA double-strand break repair and improved replication fork protection due to HR. Both HR-mediated processes lead to suppression of radiation-induced replication stress and consequently reduction of cytoplasmic dsDNA. The amount of cytoplasmic dsDNA correlated significantly with BCSC content (p=0.0002). This clearly indicates that HR-dependent avoidance of radiation-induced replication stress mediates radioresistance and contributes to its immune evasion. Consistent with this, enhancement of replication stress by inhibition of ataxia telangiectasia and RAD3 related (ATR) resulted in significant radiosensitization (SER37 increase 1.7-2.8 Gy, p<0.0001). Therefore, disruption of HR-mediated processes, particularly in replication, opens a CSC-specific radiosensitization option by enhancing their intracellular immune response.

Generation and Biological Evaluation of Fc Antigen Binding Fragment-Drug Conjugates as a Novel Antibody-Based Format for Targeted Drug Delivery.

Fragment crystallizable (Fc) antigen binding fragments (Fcabs) represent a novel antibody format comprising a homodimeric Fc region with an engineered antigen binding site. In contrast to their full-length antibody offspring, Fcabs combine Fc-domain-mediated and antigen binding functions at only one-third of the size. Their reduced size is accompanied by elevated tissue penetration capabilities, which is an attractive feature for the treatment of solid tumors. In the present study, we explored for the first time Fcabs as a novel scaffold for antibody-drug conjugates (ADCs). As model, various HER2-targeting Fcab variants coupled to a pH-sensitive dye were used in internalization experiments. A selective binding on HER2-expressing tumor cells and receptor-mediated endocytosis could be confirmed for selected variants, indicating that these Fcabs meet the basic prerequisite for an ADC approach. Subsequently, Fcabs were site-specifically coupled to cytotoxic monomethyl auristatin E yielding homogeneous conjugates. The conjugates retained HER2 and FcRn binding behavior of the parent Fcabs, showed a selective in vitro cell killing and conjugation site-dependent serum stability. Moreover, Fcab conjugates showed elevated penetration in a spheroid model, compared to their full-length antibody and Trastuzumab counterparts. Altogether, the presented results emphasize the potential of Fcabs as a novel scaffold for targeted drug delivery in solid cancers and pave the way for future in vivo translation.

Countercurrent chromatographic fractionation followed by gas chromatography/mass spectrometry identification of alkylresorcinols in rye.

Alkylresorcinols (5-alkyl-1,3-dihydroxybenzenes, ARs) are bioactive phenolic lipid compounds which are particularly abundant in rye and partly other cereals. In this study on ARs, whole rye grain extracts were gained with cyclohexane/ethyl acetate (46/54, w/w). Silylated extracts were used to develop a gas chromatography with mass spectrometry method in the selected ion monitoring mode (GC/MS-SIM) for the sensitive detection of conventional ARs along with keto-substituted (oxo-AR) and ring-methylated ARs (mAR) with 5-alkyl chain lengths of 14 to 27 carbon atoms and 0 to 4 double bonds in one run. Analysis was performed by countercurrent chromatographic (CCC) fractionation using the solvent system n-hexane/ethyl acetate/methanol/water (9/1/9/1, v/v/v/v). Subsequent GC/MS-(SIM) analysis of 80 silylated CCC fractions enabled the detection of 74 ARs in the sample. The CCC elution of the ARs followed the equivalent chain length (ECL) rule in which one double bond compensated the effect of two (additional) carbon atoms. Novel or rarely reported ARs were detected in virtually all classes, i.e. saturated AR (AR14:0), even-numbered monounsaturated AR isomers (AR16:1-AR26:1), triunsaturated ARs (AR25:3), oxo-ARs (AR17:0 oxo, AR19:1 oxo, AR21:2 oxo, AR23:2 oxo) and odd-numbered methyl-ARs (mAR15:0-mAR23:0). Positions of the double bonds of monounsaturated ARs and oxo-ARs were determined with the help of dimethyl disulfide (DMDS) derivatives. Graphical abstract.

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish.

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from (15)N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are alpha-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. (15)N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.

Imaging of an enterovaginal fistula with flexible colposcopy, an endoscopic retrograde cholangiopancreatography cannula and radiographic screening.

The diagnosis of a rectovaginal or enterovaginal fistula is mostly made by a careful historical of a faecal vaginal discharge. When a rectovaginal fistula is low, an examination will demonstrate it with ease. However, when there is a high and small fistula into the vault or the fornix of the vagina, traditional methods of small bowel enema and barium enema may not demonstrate the fistula. Ultrasound and magnetic resonance imaging may demonstrate a fistula but not the nature of the bowel involved. Vaginography has been the most successful imaging method to date. Demonstrating an enterovaginal fistula and substantiating whether or not the rectum, the small bowel alone or both are implicated are important for operation planning; a fistulogram through the vagina by endoscopic method is a simple and useful adjunct to the available methods of imaging enterovaginal fistulas. We report a case and discuss a technique of endoscopic fistulography through a small fistulous opening high in the vagina. We believe that this technique is accurate, simple and complementary to vaginography in demonstrating an enterovaginal fistula.

Three-dimensional head model simulation of transcranial magnetic stimulation.

This paper presents a finite element method used to evaluate the induced current density in a realistic model of the human head exposed to a time varying magnetic field. The tissue electric properties were varied to ascertain their influence on the induced currents. Current density magnitude and vector plots were generated throughout the tissue layers to determine the effects of tissue boundaries on the field. The current density magnitude correlated to the conductivity of the tissue in all the cases tested except where the tissue permittivity was raised to a level to allow for displacement currents. In this case, the permittivity of the tissue was the dominant factor. Current density components normal to the tissue interface were shown to exist in all solutions within the cortex contrary to the predictions of present models that rely on symmetrical geometries. Additionally, modifications in the cortical geometry were shown to perturb the field so that the site of activation could be altered in diseased patient populations. Finally, by varying the tissue permittivity values and the source frequency, we tested the effects of alpha dispersion theories on transcranial magnetic stimulation.