A Novel Robust Screening Assay Identifies Pseudomonas Strains as Reliable Antagonists of the Root-Knot Nematode Meloidogyne incognita.

Forty-four bacterial strains isolated from greenhouse soil and beetroots were tested for their antagonistic activity against the plant-parasitic root-knot nematode (RKN) Meloidogyne incognita, which causes significant yield losses in a number of important crops worldwide. Through a novel combination of in vitro and on planta screening assays, Pseudomonas spp. 105 and 108 were identified as the most promising bacterial isolates. Both strains were evaluated for their potential to control different RKN population densities and as root protectants against nematode infestation. Regardless of the application method, both strains significantly reduced root galling caused by M. incognita. These two strains were subjected to whole genome sequencing and de novo genome assembly as a basis for phylogenetic and future functional characterization. Phylogenetic analysis revealed that both Pseudomonas strains cluster within the Pseudomonas fluorescens clade among previously characterized RKN antagonists and Pseudomonas-based biocontrol agents of plant diseases.

Tomato Sterol 22-desaturase Gene CYP710A11: Its Roles in Meloidogyne incognita Infection and Plant Stigmasterol Alteration.

Sterols are isoprenoid-derived lipids that play essential structural and functional roles in eukaryotic cells. Plants produce a complex mixture of sterols, and changes in plant sterol profiles have been linked to plant-pathogen interactions. ß-Sitosterol and stigmasterol, in particular, have been associated with plant defense. As nematodes have lost the ability to synthesize sterols de novo, they require sterols from the host. Tomato (Solanum lycopersicum) plants infected by the plant parasitic nematode Meloidogyne incognita show a reduced level of stigmasterol and a repression of the gene CYP710A11, encoding the sterol C-22 desaturase that is responsible for the conversion of ß-sitosterol to stigmasterol. In this study, we investigated the role of the tomato sterol C-22 desaturase gene CYP710A11 in the response to infection by M. incognita. We explored the plant-nematode interaction over time by analyzing the plant sterol composition and CYP710A11 gene regulation in S. lycopersicum after M. incognita infection. The temporal gene expression analysis showed that 3 days after inoculation with M. incognita, the CYP710A11 expression was significantly suppressed in the tomato roots, while a significant decrease in the stigmasterol content was observed after 14 days. A cyp710a11 knockout mutant tomato line lacking stigmasterol was analyzed to better understand the role of CYP710A11 in nematode development. M. incognita grown in the mutant line showed reduced egg mass counts, presumably due to the impaired growth of the mutant. However, the nematodes developed as well as they did in the wild-type line. Thus, while the suppression of CYP710A11 expression during nematode development may be a defense response of the plant against the nematode, the lack of stigmasterol did not seem to affect the nematode. This study contributes to the understanding of the role of stigmasterol in the interaction between M. incognita and tomato plants and shows that the sterol C-22 desaturase is not essential for the success of M. incognita.

Facial Type, Sex and Skin Color of Pediatric Patients Are Associated with Dental Students' Empathy and Self-Confidence During Dental Care.

Purpose: To assess the levels of empathy and self-confidence of pre- and post-doctoral dental students during the dental care of children of different facial types, sex and skin color.
Methods: A cross-sectional survey was conducted with 170 Brazilian dental students. Photos of four mesofacial pediatric patients of different sex (male, female) and skin color (white, black) were digitally altered to dolichofacial and brachyfacial facial types. The 12 photos obtained (four originals and eight digitally altered) were projected during lectures and students reported their level of empathy (scores from zero to 10) and self-confidence (not confident/confident) using a structured questionnaire, based exclusively on children's appearance. Bivariate analysis and Poisson robust regression were performed (P <0.05).
Results: Students' lower empathy was observed among brachyfacial children (preva- lence ratio [PR] = 0.940; 95 percent confidence interval [95 percent CI] = 0.904 to 0.978). Female (PR=1.202; 95 percent CI=1.162 to 1.243) and black children (PR=1.068; 95 percent CI=1.035 to 1.103) received students' higher empathy scores. Higher percentages of self-confidence were associated with mesofacial (P =0.049), female (P <0.001), and black (P =0.001) children. Students' lower self-confidence was associated with brachyfacial (PR=1.106; 95 percent CI=1.066 to 1.147), male (PR=1.202; 95 percent CI=1.165 to 1.239) and white (PR=1.056; 95 percent CI =1.025 to 1.088) pediatric patients.
Conclusions: Brachyfacial children were associated with dental students' lower levels of empathy and self-confidence while female and black children received higher levels of empathy and self-confidence. However, dental students felt less confident regarding the dental care of white children.

The Impact of Management Strategies on the Development and Status of Potato Cyst Nematode Populations in Switzerland: An Overview from 1958 to Present.

Globodera rostochiensis and G. pallida are some of the most successful and highly specialized plant parasitic nematodes and among the most regulated quarantine pests globally. In Switzerland, they have been monitored by annual surveys since their first detection in Swiss soil in 1958. The dataset created was reviewed to produce an overview of the development and actual status of potato cyst nematodes (PCNs) in Switzerland. Positive fields represent 0.2% of all the samples analyzed, and their distribution is limited to central-west and western Switzerland, suggesting that new introduction of PCNs and the spread of the initial introduced PCN populations did not occur. In this way, the integrated management used in Switzerland appears to be effective. However, the increasing availability of potato varieties with resistance to G. rostochiensis and the limited availability of varieties with resistance to G. pallida, together with other biotic and abiotic factors, have promoted changes in the dominance of either species. Consequently, an extended monitoring program is of interest to Swiss farmers, to avoid favoring virulent traits that could be present in Swiss Globodera populations.

Prebiotic potential of mushroom d-glucans: implications of physicochemical properties and structural features.

Mushroom d-glucans are recognized as dietary fibers and as biologically active natural polysaccharides, with the advantages of being quite inexpensive for production, tolerable, and having a range of possible structures and physicochemical properties. The prebiotic potential of mushroom d-glucans has been explored in recent years, but the relationship between their various structural features and activity is poorly understood. This review focuses on comprehensively evaluating the prebiotic potential of mushroom d-glucans in face of their structural variations. Overall, mushroom d-glucans provide a unique set of different structures and physicochemical properties with prebiotic potential, where linkage type and solubility degree seem to be associated with prebiotic activity outcomes. The understanding of the effects of distinct structures and physicochemical properties in mushroom d-glucans on the gut microbiota contributes to the design and selection of new prebiotics in a more predictable way.

Cytotoxic effect of crude and purified pectins from Campomanesia xanthocarpa Berg on human glioblastoma cells.

A new source of pectin with a cytotoxic effect on glioblastoma cells is presented. A homogeneous GWP-FP-S fraction (Mw of 29,170 g mol-1) was obtained by fractionating the crude pectin extract (GW) from Campomanesia xanthocarpa pulp. According to the monosaccharide composition, the GWP-FP-S was composed of galacturonic acid (58.8%), arabinose (28.5%), galactose (11.3%) and rhamnose (1.1%), comprising 57.7% of homogalacturonans (HG) and 42.0% of type I rhamnogalacturonans (RG-I). These structures were characterized by chromatographic and spectroscopic methods; GW and GWP-FP-S fractions were evaluated by MTT and crystal violet assays for their cytotoxic effects. Both fractions induced cytotoxicity (15.55-37.65%) with concomitant increase in the cellular ROS levels in human glioblastoma cells at 25-400 µg mL-1, after 48 h of treatment, whereas no cytotoxicity was observed for normal NIH 3T3 cells. This is the first report of in vitro bioactivity and the first investigation of the antitumor potential of gabiroba pectins.

Agaricus bisporus and its by-products as a source of valuable extracts and bioactive compounds.

Edible mushrooms constitute an appreciated nutritional source for humans due to their low caloric intake and their high content in carbohydrates, proteins, dietary fibre, phenolic compounds, polyunsaturated fatty acids, vitamins and minerals. It has been also demonstrated that mushrooms have health-promoting benefits. Cultivation of mushrooms, especially of the most common species Agaricus bisporus, represents an increasingly important food industry in Europe, but with a direct consequence in the increasing amount of by-products from their industrial production. This review focuses on collecting and critically investigating the current data on the bioactive properties of Agaricus bisporus as well as the recent research for the extraction of valuable functional molecules from this species and its by-products obtained after industrial processing. The state of the art regarding the antimicrobial, antioxidant, anti-allergenic and dietary compounds will be discussed for novel applications such as nutraceuticals, additives for food or cleaning products.

Pectins from the pulp of gabiroba (Campomanesia xanthocarpa Berg): Structural characterization and rheological behavior.

The pulp of gabiroba fruits was submitted to a hot water extraction, giving rise to a crude pectin named GW. GW was shown to be composed mainly of arabinose (54.5%), galacturonic acid (33.5%), galactose (7.6%), and rhamnose (1.6%). GW was characterized by chromatographic and spectroscopic methods indicating the presence of homogalacturonans (HG) with a degree of methyl-esterification (DM) of 60% and rhamnogalacturonans I (RG-I). HG domain represents 31.9% and RG-I domain 65.3%. Furthermore, GW was submitted to sequential fractionation methods, giving rise to GWP-TEP fraction, structurally characterized by the predominance of HG regions, and confirmed by NMR analysis. The rheological behavior of GW was analyzed at 1%, 3%, and 5% (w/v) concentration with 0.1 mol L-1 NaCl. All samples showed shear thinning behavior. In the oscillatory measurements, the 1% GW showed a liquid-like behavior, while the 3% presented a concentrated solution behavior and the 5% GW a gel behavior.

Chemical structure of a partially 3-O-methylated mannofucogalactan from edible mushroom Grifola frondosa.

An unusual heteropolysaccharide was isolated from the fruiting bodies of the medicinal mushroom Grifola frondosa, via successive cold aqueous extraction, followed by fractionation through freeze-thawing, precipitation with Fehling solution and dialysis using a membrane with a size exclusion cut-off of 500 kDa. Its chemical structure was determined based on total acid hydrolysis, methylation analysis and NMR studies. The mannofucogalactan had a molar mass of 15.9 × 103 g mol-1, which was determinate by HPSEC-MALLS. This heteropolymer showed to have a main chain of (1 → 6)-linked α-d-Galp partially substituted at O-2 by 3-O-α-d-mannopyranosyl-α-l-fucopyranosyl groups and in a minor proportion with α-l-Fucp single-unit side chains. Moreover, the presence of 3-O-Me-Galp units could also be observed in the main chain of the G. frondosa mannofucogalactan.

Partially methylated galactans containing different proportions of 3-O-methyl-galactose from Pleurotus citrinopileatus.

Pleurotus citrinopileatus, popularly known as "golden oyster mushroom" have medicinal properties, which are attributed mainly to the presence of bioactive polysaccharides. In this work, two partially 3-O-methylated galactans were isolated from the fruiting bodies of this fungus, via successive aqueous extraction, followed by fractionation by freeze-thawing, and precipitation of soluble material with Fehling solution. The structural assignments were carried out using mono- and bidimensional NMR spectroscopy, monosaccharide composition, and methylation analyses. The polysaccharides were characterized as linear, partially 3-O-methylated (1 → 6)-linked α-galactopyranans, containing only Gal and 3-O-Me-Gal, in 2:1 and 1:1 molar ratios, with molar masses of 37.6 × 103 g/mol and 28.5 × 103 g/mol, respectively. Similar structures have been described for other Pleurotus spp., but showing a lower content of 3-O-Me-Gal.

In vitro fermentation of Cookeina speciosa glucans stimulates the growth of the butyrogenic Clostridium cluster XIVa in a targeted way.

Dietary fiber chemical and physical structures may be critical to the comprehension of how they may modulate gut bacterial composition. We purified insoluble polymers from Cookeina speciosa, and investigated its fermentation profile in an in vitro human fecal fermentation model. Two glucans, characterized as a (1 → 3),(1 → 6)-linked and a (1→3)-linked ß-D-glucans were obtained. Both glucans were highly butyrogenic and propiogenic, with low gas production, during in vitro fecal fermentation and led to distinct bacterial shifts if compared to fructooligosaccharides. Specific increases in Bacteroides uniformis and genera from the Clostridium cluster XIVa, such as butyrogenic Anaerostipes and Roseburia were observed. The (1 → 3)-linked ß-D-glucan presented a faster fermentation profile compared to the branched (1 → 3),(1 → 6)-linked ß-D-glucan. Our findings support the view that depending on its fine chemical structure, and likely its insoluble nature, these dietary fibers can be utilized to direct a targeted promotion of the intestinal microbiota to butyrogenic Clostridium cluster XIVa bacteria.

ß-(1→6)-D-glucan secreted during the optimised production of exopolysaccharides by Paecilomyces variotii has immunostimulatory activity.

Paecilomyces variotii is a filamentous fungus that occurs worldwide in soil and decaying vegetation. Optimization of the fermentation process for exopolysaccharide (EPS) production from the fungus P. variotii, structure determination and immuno-stimulating activity of EPS were performed. Response surface methodology (RSM) coupled with central composite design (CCD) was used to optimize the physical and chemical factors required to produce EPS in submerged fermentation. Preliminary investigations to choose the three factors for the present work were made using a factorial experimental design. Glucose, ammonium nitrate (NH4NO3) and pH were used as variables for which, with constant temperature of 28 °C and agitation of 90 rpm, the optimal process parameters were determined as glucose values of 0.96%, NH4NO3 0.26% and pH 8.0. The three parameters presented significant effects. In this condition of culture, the main composition of the isolated EPS was a linear ß-(1 â†’ 6)-linked-D-glucan, as determined by Nuclear Magnetic Resonance (NMR) and methylation analysis. This polysaccharide is a very unusual as an EPS from fungi, especially a filamentous fungus such as P. variotii. Murine peritoneal macrophages cultivated with ß-glucan for 6 and 48 h showed an increase in TNF-α, IL-6 and nitric oxide release with increased polysaccharide concentrations. Therefore, we conclude that the ß-(1 â†’ 6)-linked-D-glucan produced in optimised conditions of P. variotii cultivation has an immune-stimulatory activity on murine macrophages.

Extraction, purification and structural characterization of a galactoglucomannan from the gabiroba fruit (Campomanesia xanthocarpa Berg), Myrtaceae family.

In this study, we isolated and structurally characterized, for the first time, a galactoglucomannan (GGM) from the pulp of gabiroba, a Myrtaceae family species. The HPSEC-MALLS-RI analysis showed a homogeneous polysaccharide with molar mass of 25,340gmol-1. The monosaccharide composition showed that the GGM consisted of Man:Glc:Gal in a molar ratio of 1:1:0.6. Methylation and 1D and 2D NMR analyses suggested that the main chain of the GGM consisted of ß-d-Glcp and ß-d-Manp units (1→4)-linked. The α-d-Galp substitutions occur mainly at O-6 position of ß-d-Manp units. The glycosidic linkages of the GGM were evident by the presence of the characteristic signals of 4-O-substituted residues at δ 78.6/3.69 for both ß-d-Glcp and ß-d-Manp. Furthermore, the O-6 substitutions for both ß-d-Glcp and ß-d-Manp units were confirmed by signals at δ 67.1/4.00 and 3.93. The interglycosidic correlations, obtained through the analysis of the HMBC spectrum, further confirm the structure.

Cytotoxic effect of a mannogalactoglucan extracted from Agaricus bisporus on HepG2 cells.

A mannogalactoglucan (RK2-Ab; Mw 1.8×104gmol-1) composed by Man (27.3%), Gal (24.4%) and Glc (48.3%) was extracted and characterized from Agaricus bisporus, and its biological activity was evaluated on human hepatocarcinoma cells (HepG2). The partially-O-methylated alditol acetates together with the NMR data suggest the main chain to be composed of α-d-Galp (32.8%) and ß-d-Glcp (37.0%) units (1→6)-linked, with ß-d-Manp (14.6%), as non-reducing end units, substituting the side chains at O-2 (α-d-Galp units; 3.3%) and O-2 and O-4 (ß-d-Glcp units; 3.6%). (1→2)-linked ß-d-Glcp (2.7%) and ß-d-Manp (6.0%) can also be observed. RK2-Ab reduced cellular viability of HepG2 cells, by both, the MTT and lactate dehydrogenase release assays, promoted the increase of cytochrome c release and decrease of ATP content. Suggesting that the mannogalactoglucan from A. bisporus may have antitumor activity by inducing apoptosis by the mitochondrial death pathway, and could be used in cancer therapy.

Reciprocal regulation of the cholinic phenotype and epithelial-mesenchymal transition in glioblastoma cells.

Glioblastoma (GBM) is the most malignant brain tumor with very limited therapeutic options. Standard multimodal treatments, including surgical resection and combined radio-chemotherapy do not target the most aggressive subtype of glioma cells, brain tumor stem cells (BTSCs). BTSCs are thought to be responsible for tumor initiation, progression, and relapse. Furthermore, they have been associated with the expression of mesenchymal features as a result of epithelial-mesenchymal transition (EMT) thereby inducing tumor dissemination and chemo resistance. Using high resolution proton nuclear magnetic resonance spectroscopy (1H NMR) on GBM cell cultures we provide evidence that the expression of well-known EMT activators of the ZEB, TWIST and SNAI families and EMT target genes N-cadherin and VIMENTIN is associated with aberrant choline metabolism. The cholinic phenotype is characterized by high intracellular levels of phosphocholine and total choline derivatives and was associated with malignancy in various cancers. Both genetic and pharmacological inhibition of the cardinal choline metabolism regulator choline kinase alpha (CHKα) significantly reduces the cell viability, invasiveness, clonogenicity, and expression of EMT associated genes in GBM cells. Moreover, in some cell lines synergetic cytotoxic effects were observed when combining the standard of care chemotherapeutic temozolomide with the CHKα inhibitor V-11-0711. Taken together, specific inhibition of the enzymatic activity of CHKα is a powerful strategy to suppress EMT which opens the possibility to target chemo-resistant BTSCs through impairing their mesenchymal transdifferentiation. Moreover, the newly identified EMT-oncometabolic network may be helpful to monitor the invasive properties of glioblastomas and the success of anti-EMT therapy.

Insulin-like growth factor-1 receptor (IGF-1R) inhibition promotes expansion of human NK cells which maintain their potent antitumor activity against Ewing sarcoma cells.

BACKGROUND: Patients with primary metastatic or relapsed Ewing sarcomas (EwS) have a poor prognosis. While inhibitory insulin-like growth factor 1 receptor (IGF-1R)-specific antibodies have shown single agent activity in some patients with refractory disease, effective therapeutic targeting will rely on optimal combinations with conventional or innovative therapies. Specifically, combination of inhibitory IGF-1R antibodies with adoptive transfer of activated natural killer (NK) cells may have therapeutic benefit in EwS without adding toxicity. PROCEDURE: We investigated the in vitro effects of IGF-1R targeting on the immunological profile of EwS cells and on the survival and tumor targeting capacity of K-562-activated NK cells. RESULTS: IGF-1R inhibition reliably reduced EwS cell viability without affecting expression of immune-modulatory and MHC molecules. In NK cells, we observed a significant superior expansion following in vitro activation in the presence of IGF-1R-specific antibodies, while expression of differentiation markers and activating receptors remained unaffected. Activated NK cells coincubated with EwS cells showed potent degranulation responses unaffected by IGF-1R inhibition. These findings were reproducible in a stimulator cell-free NK cell expansion system, suggesting that direct effects of IGF-R1 antibodies on the IGF-R1 pathway in NK cells induce their activation and expansion. CONCLUSIONS: Activated human NK cells respond to IGF-1R inhibition with superior expansion kinetics while maintaining potent antitumor responses against EwS. Combination of adoptive NK cell transfer with IGF-1R targeting may be an efficient means to eliminate minimal residual disease after conventional therapy and thereby rescue patients at the highest risk of relapse.

Exopolysaccharide produced by Pleurotus sajor-caju: its chemical structure and anti-inflammatory activity.

Edible mushrooms are high nutritional value foods, which contain proteins, fibers, minerals, vitamins, and carbohydrates. Among their carbohydrates are some polysaccharides with recognized therapeutic effects. It was reported in this manuscript the structural characterization and antinociceptive and anti-inflammatory activities of an exopolysaccharide (EPS) produced by Pleurotus sajor-caju. The purified EPS was a mannogalactan (PEIsR), which was composed by mannose (37.0%), galactose (39.7%), and 3-O-methyl-galactose (23.3%). The polysaccharide was purified by freeze-thawing and dialysis, and it was characterized by GC-MS analysis and NMR spectroscopy. The mannogalactan is constituted by a main chain of (1 → 6)-linked α-D-Galp and 3-O-methyl-α-D-Galp units. Some of the α-D-Galp units were substituted at O-2 by non-reducing end units of ß-D-Manp. According to the literature review conducted, this is the first time that a methylated polysaccharide was observed on EPS of P. sajor-caju. The mannogalactan was able to reduce the nociception, in vivo, in the writhing and formalin tests and also reduced the carrageenan-induced paw edema, which indicates that it could be an effective antinociceptive and anti-inflammatory agent.

Handheld reflectance confocal microscopy for the diagnosis of conjunctival tumors.

PURPOSE: To evaluate whether the handheld in vivo reflectance confocal microscopy that has been recently developed for the study of skin tumors is suitable for the diagnosis of conjunctival tumors. DESIGN: Prospective study, observational case series. METHODS: We prospectively evaluated the reflectance confocal microscopy features of 53 conjunctival lesions clinically suspicious for tumors of 46 patients referred to the University Hospital of Saint-Etienne (France) by using the handheld device. Twenty-three lesions were excised (3 nevi, 10 melanomas, 5 squamous cell carcinoma, 2 lymphomas, and 3 pinguecula/pterygium) while the other 30, presenting no reflectance confocal microscopy malignant features, were under follow-up for at least 1 year. Clinical reflectance confocal microscopy and histologic diagnosis were compared. RESULTS: In vivo reflectance confocal microscopy diagnosis was in agreement with the histologic diagnosis in all cases and none of the lesions that were not excised show any clinical progression under follow-up. CONCLUSION: In vivo reflectance confocal microscopy with a handheld dermatology-dedicated microscope can play a role in the noninvasive diagnosis of conjunctival lesions. Further studies should be performed to better define the diagnostic ability of this technique.

D-glucans from edible mushrooms: a review on the extraction, purification and chemical characterization approaches.

D-Glucans from edible mushrooms present diversified chemical structures. The most common type consists of a backbone of ß-D-glucose (1→3)-linked frequently branched at O-6 by ß-D-glucose residues as side chains. However it is possible to distinguish α-, ß- and mixed D-glucans. Further discrimination could be made on the basis of glycosidic bond position in a pyranoid ring, distribution of specific glycosidic bonds along the chain, branching and molecular weight. The present manuscript reviews the processes of extraction, purification and chemical characterization of D-glucans, such as NMR studies, methylation analysis, Smith degradation, and some other methodologies employed in carbohydrate chemistry characterization. In addition, these polysaccharides are important because they can provide many therapeutic benefits related to their biological activity in animals and humans, either immunostimulatory activity, inhibiting tumor growth, as well as exerting antinociceptive and anti-inflammatory action, among others, which are usually attached to their structure, molecular weight and degree of branching.