Characterization of Different Inflammatory Skin Conditions in a Mouse Model of DNCB-Induced Atopic Dermatitis.

The mouse model of 2,4-dinitrochlorbenzene (DNCB)-induced human-like atopic dermatitis (hlAD) has been widely used to test novel treatment strategies and compounds. However, the study designs and methods are highly diverse, presenting different hlAD disease patterns that occur after sensitization and repeated challenge with DNCB on dorsal skin. In addition, there is a lack of information about the progression of the disease during the experiment and the achieved pheno- and endotypes, especially at the timepoint when therapeutic treatment is initiated. We here examine hlAD in a DNCB-induced BALB/cJRj model at different timepoints: (i) before starting treatment with dexamethasone, representing a standard drug control (day 12) and (ii) at the end of the experiment (day 22). Both timepoints display typical AD-associated characteristics: skin thickening, spongiosis, hyper- and parakeratosis, altered cytokine and gene expression, increased lipid mediator formation, barrier protein and antimicrobial peptide abnormalities, as well as lymphoid organ hypertrophy. Increased mast cell infiltration into the skin and elevated immunoglobulin E plasma concentrations indicate a type I allergy response. The DNCB-treated skin showed an extrinsic moderate sub-acute hlAD lesion at day 12 and an extrinsic mild sub-acute to chronic pheno- and endotype at day 22 with a dominating Th2 response. A dependency of the filaggrin formation and expression in correlation to the disease severity in the DNCB-treated skin was found. In conclusion, our study reveals a detailed classification of a hlAD at two timepoints with different inflammatory skin conditions and pheno- and endotypes, thereby providing a better understanding of the DNCB-induced hlAD model in BALB/cJRj mice.

Influence of NOD2 risk variants on hepatic encephalopathy and association with inflammation, bacterial translocation and immune activation.

BACKGROUND: Nucleotide-binding oligomerization domain containing 2 (NOD2) risk variants lead to impaired mucosal barrier function, increased bacterial translocation (BT), and systemic inflammation. AIM: To evaluate the association between the presence of NOD2 risk variants, BT, inflammation, and hepatic encephalopathy (HE). PATIENTS AND METHODS: This prospective multicenter study included patients with cirrhosis and testing for NOD2 risk variants (p.R702W, p.G908R, c.3020insC, N289S, and c.-958T>C). Patients were evaluated for covert (C) and overt (O) HE. Markers of systemic inflammation (leukocytes, CRP, IL-6, LBP) and immune activation (soluble CD14) as well as bacterial endotoxin (hTRL4 activation) were determined in serum. RESULTS: Overall, 172 patients (70% men; median age 60 [IQR 54-66] years; MELD 12 [IQR 9-16]; 72% ascites) were included, of whom 53 (31%) carried a NOD2 risk variant. In this cohort, 11% presented with OHE and 27% and CHE. Presence and severity of HE and surrogates of inflammation, BT, and immune activation did not differ between patients with and without a NOD2 risk variant, also not after adjustment for MELD. HE was associated with increased ammonia and systemic inflammation, as indicated by elevated CRP (w/o HE: 7.2 [2.7-16.7]; with HE 12.6 [4.5-29.7] mg/dL; p < 0.001) and elevated soluble CD14 (w/o HE 2592 [2275-3033]; with HE 2755 [2410-3456] ng/mL; p = 0.025). CONCLUSIONS: The presence of NOD2 risk variants in patients with cirrhosis is not associated with HE and has no marked impact on inflammation, BT, or immune activation. In contrast, the presence of HE was linked to ammonia, the acute phase response, and myeloid cell activation.

Alternative transcript splicing regulates UDP-glucosyltransferase-catalyzed detoxification of DIMBOA in the fall armyworm (Spodoptera frugiperda).

Herbivorous insects often possess the ability to detoxify chemical defenses from their host plants. The fall armyworm (Spodoptera frugiperda), which feeds principally on maize, detoxifies the maize benzoxazinoid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) by stereoselective re-glucosylation using a UDP-glucosyltransferase, SfUGT33F28. SfUGT33F28 activity is induced by feeding on a DIMBOA-containing diet, but how this induction is regulated is unknown. In the present work, we describe the alternative splicing of the SfUGT33F28 transcript. Variant transcripts are differentially expressed in response to DIMBOA, and this transcriptional response is mediated by an insect aryl hydrocarbon receptor. These variants have large deletions leading to the production of truncated proteins that have no intrinsic UGT activity with DIMBOA but interact with the full-length enzyme to raise or lower its activity. Therefore, the formation of SfUGT33F28 splice variants induces DIMBOA-conjugating UGT activity when DIMBOA is present in the insect diet and represses activity in the absence of this plant defense compound.

Protein extract of Bromelia karatas L. rich in cysteine proteases (ananain- and bromelain-like) has antibacterial activity against foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium.

Bromelia karatas L. is a plant species from the Americas. The presence of proteases in fruits of B. karatas has been reported but scarcely studied in detail. Proteolytic enzymes from Ananas comosus have displayed antifungal and antibacterial activity. Thus, novel proteases present in B. karatas may be useful as a source of compounds against microorganisms in medicine and food production. In this work, the protein extract from the fruits of B. karatas was characterized and its antibacterial activity against Salmonella Typhimurium and Listeria monocytogenes was determined for the first time. Proteins highly similar to ananain and the fruit bromelain from A. comosus were identified as the main proteases in B. karatas fruits using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The soluble protein extract (SPE) at a concentration of 2.0 mg/mL displayed up to 80% of antibacterial activity against S. Typhimurium. Complete inhibition of L. monocytogenes was reached with up to 1.65 mg/mL of SPE. Plant protease extract containing ananain-like enzyme inhibited up to 90% against S. Typhimurium and up to 85% against L. monocytogenes using only 10 µg/mL of the partial-purified enzyme.

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases.

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the ß-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle's energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

An Integrated-Omics/Chemistry Approach Unravels Enzymatic and Spontaneous Steps to Form Flavoalkaloidal Nudicaulin Pigments in Flowers of Papaver nudicaule L.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions.

Enzymatic Bioweathering and Metal Mobilization From Black Slate by the Basidiomycete Schizophyllum commune.

Schizophyllum commune is a filamentous basidiomycete causing white-rot in many wood species with the help of a broad range of enzymes including multicopper oxidases such as laccases and laccase-like oxidases. Since these enzymes exhibit a broad substrate range, their ability to oxidatively degrade black slate was investigated. Both haploid monokaryotic, and mated dikaryotic strains were able to grow on black slate rich in organic carbon as sole carbon source. On defined media, only the monokaryon showed growth promotion by addition of slate. At the same time, metals were released from the slate and, after reaching a threshold concentration, inhibited further growth of the fungus. The proteome during decomposition of the black slate showed induction of proteins potentially involved in rock degradation and stress resistance, and the gene for laccase-like oxidase mco2 was up-regulated. Specifically in the dikaryon, the laccase gene lcc1 was induced, while lcc2 as well as mco1, mco3, and mco4 expression levels remained similar. Spectrophotometric analysis revealed that both life forms were able to degrade the rock and produce smaller particles.

Plant Defensive ß-Glucosidases Resist Digestion and Sustain Activity in the Gut of a Lepidopteran Herbivore.

Two-component activated chemical defenses are a major part of many plants' strategies to disrupt herbivory. The activation step is often the ß-glucosidase-catalyzed removal of a glucose moiety from a pro-toxin, leading to an unstable and toxic aglycone. While some ß-glucosidases have been well studied, several aspects of their roles in vivo, such as their precise sites of enzymatic activity during and after ingestion, and the importance of particular isoforms in plant defense are still not fully understood. Here, plant defensive ß-glucosidases from maize, white mustard and almonds were shown to resist digestion by larvae of the generalist lepidopteran Spodoptera littoralis, and the majority of the ingested activities toward both general and plant pro-toxic substrates was recovered in the frass. Among other proteins potentially involved in defense, we identified specific plant ß-glucosidases and a maize ß-glucosidase aggregating factor in frass from plant-fed insects using proteomic methods. We therefore found that, while S. littoralis larvae efficiently degraded bulk food protein during digestion, ß-glucosidases were among a small number of plant defensive proteins that resist insect digestive proteolysis. These enzymes remain intact in the gut lumen and frass and can therefore further catalyze the activation of plant defenses after ingestion, especially in pH-neutral regions of the digestive system. As most of the ingested enzymatic activity persists in the frass, and only particular ß-glucosidases were detected via proteomic analyses, our data support the involvement of specific isoforms (maize ZmGlu1 and S. alba MA1 myrosinase) in defense in vivo.

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum.

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,ß,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.

Morphology and histochemistry of the aesthetasc-associated epidermal glands in terrestrial hermit crabs of the genus Coenobita (Decapoda: Paguroidea).

Crustaceans have successfully adapted to a variety of environments including fresh- and saltwater as well as land. Transition from an aquatic to a terrestrial lifestyle required adaptations of the sensory equipment of an animal, particularly in olfaction, where the stimulus itself changes from hydrophilic to mainly hydrophobic, air-borne molecules. Hermit crabs Coenobita spp. (Anomura, Coenobitidae) have adapted to a fully terrestrial lifestyle as adults and have been shown to rely on olfaction in order to detect distant food items. We observed that the specialized olfactory sensilla in Coenobita, named aesthetascs, are immersed in a layer of mucous-like substance. We hypothesized that the mucous is produced by antennal glands and affects functioning of the aesthetascs. Using various microscopic and histochemical techniques we proved that the mucous is produced by aesthetasc-associated epidermal glands, which we consider to be modified rosette-type aesthetasc tegumental glands known from aquatic decapods. These epidermal glands in Coenobita are multicellular exocrine organs of the recto-canal type with tubulo-acinar arrangement of the secretory cells. Two distinct populations of secretory cells were clearly distinguishable with light and electron microscopy. At least part of the secretory cells contains specific enzymes, CUB-serine proteases, which are likely to be secreted on the surface of the aesthetasc pad and take part in antimicrobial defense. Proteomic analysis of the glandular tissue corroborates the idea that the secretions of the aesthetasc-associated epidermal glands are involved in immune responses. We propose that the mucous covering the aesthetascs in Coenobita takes part in antimicrobial defense and at the same time provides the moisture essential for odor perception in terrestrial hermit crabs. We conclude that the morphological modifications of the aesthetasc-associated epidermal glands as well as the functional characteristics of their secretions are important adaptations to a terrestrial lifestyle.

Cervimycin C resistance in Bacillus subtilis is due to a promoter up-mutation and increased mRNA stability of the constitutive ABC-transporter gene bmrA.

Two independent cervimycin C (CmC)-resistant clones of Bacillus subtilis were identified, each carrying two mutations in the intergenic region preceding the ABC transporter gene bmrA. In the double mutant, real-time PCR revealed an increased amount of bmrA mRNA with increased stability. Accordingly, isolation of membrane proteins yielded a strong band at 64 kDa corresponding to BmrA. Analyses showed that one mutation optimized the -35 box sequence conferring resistance to 3 µM CmC, while the +6 mutation alone had no effect, but increased the potential of the strain harboring the -35 mutation to grow at 5 µM CmC. Transcriptional fusions revealed an elevated bmrA promoter activity for the double mutant. Electrophoretic mobility shift assays (EMSAs) confirmed a 30-fold higher binding affinity of RNA polymerase for this mutant compared with the wild type, and the effect was due to the -35 box alteration of the bmrA promoter. In vitro transcription experiments substantiated the results of the EMSA. EMSAs in the presence of heparin indicated that the mutations did not influence the formation and/or the stability of open complexes. Half-life measurements demonstrated that the +6 mutation stabilized bmrA mRNA ≈ 2-fold. Overall, we found that an ABC transporter confers antibiotic resistance by the cumulative effects of two mutations in the promoter region.