Vibrio cholerae O1 Inhabit Intestines and Spleens of Fish in Aquaculture Ponds.

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that spreads locally and globally in epidemics and pandemics. Although it was discovered that fish harbor V. cholerae strains in their intestines, most investigations revealed non-toxic V. cholerae serogroups in fish. Due to the rarity of toxigenic V. cholerae serogroups, it is difficult to cultivate these strains from environmental samples. Hence, here we aimed to uncover evidence of the occurrence of toxigenic V. cholerae in the intestines and spleens of various fish species. By using molecular detection tools, we show that V. cholerae O1 and strains positive for the cholera toxin inhabit both healthy and diseased fish intestines and spleens, suggesting that fish may serve as intermediate vectors of toxigenic V. cholerae. No significant differences were found between the abundance of toxigenic V. cholerae (either O1 or cholera toxin positive strains) in the healthy and the diseased fish intestines or spleens. In conclusion, a variety of fish species may serve as potential vectors and reservoirs of toxigenic V. cholerae as they form a link between the other reservoirs of V. cholerae (chironomids, copepods, and waterbirds). Similarly, they may aid in the spread of this bacterium between water bodies.

The COP9 signalosome mediates the Spt23 regulated fatty acid desaturation and ergosterol biosynthesis.

The COP9 signalosome (CSN) is a conserved eukaryotic complex, essential for vitality in all multicellular organisms and critical for the turnover of key cellular proteins through catalytic and non-catalytic activities. Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of the CSN complex, since it includes a conserved enzymatic core but lacks non-catalytic activities, probably explaining its non-essentiality for life. A previous transcriptomic analysis of an S. cerevisiae strain deleted in the CSN5/RRI1 gene, encoding to the CSN catalytic subunit, revealed a downregulation of genes involved in lipid metabolism. We now show that the S. cerevisiae CSN holocomplex is essential for cellular lipid homeostasis. Defects in CSN assembly or activity lead to decreased quantities of ergosterol and unsaturated fatty acids (UFA); vacuole defects; diminished lipid droplets (LDs) size; and to accumulation of endoplasmic reticulum (ER) stress. The molecular mechanism behind these findings depends on CSN involvement in upregulating mRNA expression of SPT23. Spt23 is a novel activator of lipid desaturation and ergosterol biosynthesis. Our data reveal for the first time a functional link between the CSN holocomplex and Spt23. Moreover, CSN-dependent upregulation of SPT23 transcription is necessary for the fine-tuning of lipid homeostasis and for cellular health.

Free circulating active elastase contributes to chronic inflammation in patients on hemodialysis.

Atherosclerosis and cardiovascular complications are prevalent among patients undergoing chronic hemodialysis (HD). In this population, peripheral polymorphonuclear leukocytes (PMNLs) are primed, releasing proinflammatory mediators such as elastase. Elastase is normally inhibited by a specific inhibitor, avoiding undesirable degradation of cellular and extracellular components. This study tested the hypothesis that in states of noninfectious inflammation, elastase is released by PMNLs and acts in an uncontrolled manner to inflict vascular damage. Blood was collected from patients undergoing HD and healthy controls (HC). PMNL intracellular and surface expressions of elastase were determined by quantitative real-time PCR, Western blotting, and flow cytometry. The elastase activity was evaluated using a fluorescent substrate. The levels of serum α1-antitrypsin (α1-AT), the natural elastase inhibitor, were determined by Western blot. Free active elastase was elevated in HD sera, whereas the levels of α1-AT were decreased compared with HC. The levels of the intracellular elastase enzyme and its activity were lower in HD PMNLs despite similar expression levels of elastase mRNA. Elastase binding to PMNL cell surface was higher in HD compared with HC. The increased circulating levels of free active elastase released from primed HD PMNLs together with the higher cell surface-bound enzymes and the lower levels of α1-AT result in the higher elastase activity in HD sera. This exacerbated elastase activity could lead to the endothelial dysfunction, as hypothesized. In addition, it suggests that free circulating elastase can serve as a new biomarker and therapeutic target to reduce inflammation and vascular complications in patients on hemodialysis.

Investigation of phycobilisome subunit interaction interfaces by coupled cross-linking and mass spectrometry.

The phycobilisome (PBS) is an extremely large light-harvesting complex, common in cyanobacteria and red algae, composed of rods and core substructures. These substructures are assembled from chromophore-bearing phycocyanin and allophycocyanin subunits, nonpigmented linker proteins and in some cases additional subunits. To date, despite the determination of crystal structures of isolated PBS components, critical questions regarding the interaction and energy flow between rods and core are still unresolved. Additionally, the arrangement of minor PBS components located inside the core cylinders is unknown. Different models of the general architecture of the PBS have been proposed, based on low resolution images from electron microscopy or high resolution crystal structures of isolated components. This work presents a model of the assembly of the rods onto the core arrangement and for the positions of inner core components, based on cross-linking and mass spectrometry analysis of isolated, functional intact Thermosynechococcus vulcanus PBS, as well as functional cross-linked adducts. The experimental results were utilized to predict potential docking interactions of different protein pairs. Combining modeling and cross-linking results, we identify specific interactions within the PBS subcomponents that enable us to suggest possible functional interactions between the chromophores of the rods and the core and improve our understanding of the assembly, structure, and function of PBS.

Bacterial detoxification of plant defence secondary metabolites mediates the interaction between a shrub and frugivorous birds.

Many plants produce fleshy fruits, attracting fruit-eating animals that disperse the seeds in their droppings. Such seed dispersal results in a conflict between the plant and the animal, as digestion of seeds can be highly beneficial to the animal but reduces plant fitness. The plant Ochradenus baccatus uses the myrosinase-glucosinolates system to protect its seeds. We show that hydrolysis of the O. baccatus fruit glucosinolates by the myrosinase enzyme inhibited digestive enzymes and hampered digestion in naïve individuals of the bird Pycnonotus xanthopygos. However, digestion in birds regularly feeding on O. baccatus fruits was unaffected. We find that Pantoea bacteria, dominating the gut of these experienced birds as well as the fruits, thrive on glucosinolates hydrolysis products in culture. Augmentation of Pantoea protects both naïve birds and plant seedlings from the effects of glucosinolates hydrolysis products. Our findings demonstrate a tripartite interaction, where the plant-bird mutually beneficial interactions are mediated by a communal bacterial tenant.

Direct Evidence That Sunbirds' Gut Microbiota Degrades Floral Nectar's Toxic Alkaloids.

Orange-tufted sunbirds (Cinnyris osea) feed on the nectar of the tobacco tree (Nicotiana glauca) which contains toxic pyridine alkaloids characterized by high concentrations of anabasine and much lower concentrations of nicotine. We aimed at determining whether the gut microbiota of sunbirds harbors bacterial species that enable the birds to cope with these toxic alkaloids. An in vivo experiment that included 12 birds showed that inducing dysbiosis in sunbirds' guts by the addition of sulfamethoxazole and trimethoprim, significantly reduced the birds' ability to degrade anabasine (n = 3) compared to control birds (n = 3) with undisturbed microbiota. Sunbirds whose gut bacterial communities were altered by the antibacterial agents and who were fed with added nicotine, also showed a lower percentage of nicotine degradation (n = 3) in their excreta compared to the sunbirds with undisturbed microbiota (n = 3), though this difference was not significant. In an in vitro experiment, we studied the ability of Lactococcus lactis, Enterobacter hormaechei, Chryseobacterium gleum, Kocuria palustris, and Methylorubrum populi that were isolated from sunbirds' excreta, to degrade anabasine and nicotine. By using gas chromatography-mass spectrometry (GC-MS) analysis, we successfully demonstrated, for the first time, the ability of these species to degrade the focal secondary metabolites. Our findings demonstrate the role of gut bacteria in detoxifying toxic secondary metabolites found in the N. glauca nectar. The degradation products may supply the birds with nitrogen which is scarce in nectar-rich diets. These findings support another role of bacteria in mediating the interactions between plants and their pollinators.

Investigation of glucosinolates in the desert plant Ochradenus baccatus (Brassicales: Resedaceae). Unveiling glucoochradenin, a new arabinosylated glucosinolate.

Here we describe the structure elucidation and quantification of six glucosinolates (GSLs) from the roots of the desert plant Ochradenus baccatus, Delile 1813 (family Resedaceae; order Brassicales). The structure elucidation was established on the corresponding enzymatically desulfated derivatives of the native GSLs of the plant. Among these GSLs we describe the previously undescribed 2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate (1a), for which we propose the name glucoochradenin. The other five glucosinolates (2a-6a) were (2S)-2-hydroxy-2-phenylethylglucosinolate (2a; glucobarbarin), 2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate (3a), benzylglucosinolate (4a; glucotropaeolin), indol-3-ylmethylglucosinolate (5a; glucobrassicin) and phenethylglucosinolate (6a; gluconasturtiin), all elucidated as their desulfo-derivatives, 2b-6b respectively). Structures were elucidated by MS and 1D and 2D-NMR techniques, the identity of the arabinose verified by ion chromatography, and the absolute configuration of the sugar units determined by hydrolysis, coupling to cysteine methyl-ester and phenyl isothiocyanate followed by HPLC-MS analysis of the resulted diastereomers. Response factors were generated for desulfo-2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate and for desulfo-2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate and all six GSLs were quantified, indicating that the root of O. baccatus is rich in GSLs (Avg. 61.3 ± 10.0 µmol/g DW and up to 337.2 µmol/g DW).

Chemical Composition and Medicinal Value of the New Ganoderma tsugae var. jannieae CBS-120304 Medicinal Higher Basidiomycete Mushroom.

In this research, the chemical composition and anticancer and antioxidant activity of the new medicinal mushroom Ganoderma tsugae var. jannieae CBS-120304 were evaluated. The chemical composition assay includes amounts of total carbohydrates and proteins, amino acids, fatty acids, micro- and macroelements, and vitamins. The investigated medicinal mushroom seemed to be a rich source of nutritional components. Mycelium accumulated more than 2-fold more total protein compared with the fruiting body and reached 37% and 16% of dry weight, respectively. Carbohydrate content in the fruiting body seemed to be conspicuously higher than in the mycelium (50% of dry weight) and reached 80% of dry weight. Quantification of the identified fatty acids indicated that, in general, palmitic acid, oleic acid, and linoleic acid were the major fatty acids. Toxic elements, such as silver, arsenic, cadmium, and mercury, were found only in trace amounts in mycelium and were not detected in the fruiting body. Furthermore, the 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay was used to evaluate antioxidant activity. The highest radical scavenging activity was 9.0 mg/mL (65.9%) by ethanol extract. In addition, mycelial extracts were tested to inhibit MCF7 breast cancer cells. Ganoderma tsugae var. jannieae ethyl acetate extract (GTEAE) extract showed high potential by inhibiting reporter activity by more than 70%. Results demonstrated that GTEAE had a strong effect on inhibitory protein κΒα level in the higher concentration used (200 gg/mL), which could be compared with the effect of parthenolide. Furthermore, GTEAE demonstrated strong inhibition of IκΒα phosphorylation.

Taste and physiological responses to glucosinolates: seed predator versus seed disperser.

In contrast to most other plant tissues, fleshy fruits are meant to be eaten in order to facilitate seed dispersal. Although fleshy fruits attract consumers, they may also contain toxic secondary metabolites. However, studies that link the effect of fruit toxins with seed dispersal and predation are scarce. Glucosinolates (GLSs) are a family of bitter-tasting compounds. The fleshy fruit pulp of Ochradenus baccatus was previously found to harbor high concentrations of GLSs, whereas the myrosinase enzyme, which breaks down GLSs to produce foul tasting chemicals, was found only in the seeds. Here we show the differential behavioral and physiological responses of three rodent species to high dose (80%) Ochradenus' fruits diets. Acomys russatus, a predator of Ochradenus' seeds, was the least sensitive to the taste of the fruit and the only rodent to exhibit taste-related physiological adaptations to deal with the fruits' toxins. In contrast, Acomys cahirinus, an Ochradenus seed disperser, was more sensitive to a diet containing the hydrolyzed products of the GLSs. A third rodent (Mus musculus) was deterred from Ochradenus fruits consumption by the GLSs and their hydrolyzed products. We were able to alter M. musculus avoidance of whole fruit consumption by soaking Ochradenus fruits in a water solution containing 1% adenosine monophosphate, which blocks the bitter taste receptor in mice. The observed differential responses of these three rodent species may be due to evolutionary pressures that have enhanced or reduced their sensitivity to the taste of GLSs.

Intraspecific directed deterrence by the mustard oil bomb in a desert plant.

Plant secondary metabolites (SMs) acting as defensive chemicals in reproductive organs such as fruit tissues play roles in both mutualistic and antagonistic interactions between plants and seed dispersers/predators. The directed-deterrence hypothesis states that SMs in ripe fruits deter seed predators but have little or no effect on seed dispersers. Indeed, studies have demonstrated that birds are able to cope with fruit SMs whereas rodents are deterred by them. However, this mechanism was only demonstrated at the class level, i.e., between birds and mammals, based on differences in the vanilloid receptors. Here we present experimental and behavioral data demonstrating the use of the broad-range, class-independent "mustard oil bomb" mechanism in Ochradenus baccatus fruits to force a behavioral change at an ecological timescale, converting rodents from seed predators to seed dispersers. This is achieved by a unique compartmentalization of the mustard oil bomb, causing activation of the system only upon seed and pulp coconsumption, encouraging seed dispersal via seed spitting by rodents. Our findings demonstrate the power of SMs to shift the animal-plant relationship from predation to mutualism and provide support for the directed-deterrence hypothesis at the intraspecific level, in addition to the interspecific level.