Optimization of Phenolic Compounds Extraction and Antioxidant Activity from Inonotus hispidus Using Ultrasound-Assisted Extraction Technology.

The use of ultrasound-assisted extraction (UAE) of bioactive compounds has been increasing because it is a good alternative to the conventional extraction methods. UAE was used to maximize total polyphenol content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity, and ferric reducing antioxidant power (FRAP) of the mushroom Inonotus hispidus using response surface methodology (RSM). Firstly, the effect of 40% (v/v) ethanol and 80% (v/v) methanol on the TPC, DPPH scavenging capacity, and FRAP was evaluated. The ethanolic extracts showed a significantly higher (p < 0.0001) TPC, DPPH scavenging capacity, and FRAP than the methanolic extracts. The best condition to produce an extract with the higher TPC and antioxidant activity was achieved when using 40% (v/v) ethanol, a ratio of 75 mL/g, and an extraction time of 20 min. The chromatographic profile of the extract obtained in the optimized condition revealed that hispidin is the main polyphenol present in the extracts of I. hispidus, representing, together with hispidin-like compounds, the majority of the phenolic compounds (159.56 µg/g DW out of 219.01 µg/g DW). The model allowed us to optimize the conditions to maximize the extraction of phenolic compounds with antioxidant activity from I. hispidus, demonstrating its potential as a source of antioxidant compounds, with possible industrial, pharmaceutical, and food applications.

Chickpea (Cicer arietinum L.) Seeds as a Reservoir of Endophytic Plant Growth-Promoting Bacteria.

The seed microbiome, the primary source of inoculum for plants, may play an important role in plant growth, health and productivity. However, the structure and function of chickpea seed endophytes are poorly characterized. Bacteria with beneficial characteristics can be selected by the plant and transmitted vertically via the seed to benefit the next generation. Studying the diversity and multifunctionality of seed microbial communities can provide innovative opportunities in the field of plant-microbe interaction. This study aimed to isolate, identify and characterize culturable endophytic bacteria from chickpea (Cicer arietinum L.) seeds. Phylogenetic analysis based on 16S rDNA showed that the endophytic bacteria belong to the genera Mesorhizobium, Burkholderia, Bacillus, Priestia, Paenibacillus, Alcaligenes, Acinetobacter, Rahnella, Enterobacter, Tsukamurella, and Microbacterium. The most frequently observed genus was Bacillus; however, rhizobia typically associated with chickpea roots were also found, which is a novel finding of this study. Siderophore production and phosphorus solubilization were the most widespread plant growth-promoting features, while hydrogen cyanide production was relatively rare among the isolates. Most of the isolates possess two or more plant growth-promoting features; however, only Bacillus thuringiensis Y2B, a well-known entomopathogenic bacteria, exhibited the presence of all plant growth-promoting traits evaluated. Results suggest that endophytic bacteria such as Bacillus, Mesorhizobium, and Burkholderia may be vertically transferred from inoculated plants to seeds to benefit the next generation.

Effects of Dietary Incorporation of Grape Stalks Untreated and Fungi-Treated in Growing Rabbits: A Preliminary Study.

This study aimed to evaluate the effect of the incorporation of untreated grape stalks (UGS) and fungi-treated grape stalks (Lentinula edodes, TGS) in rabbits' diets. The control group was fed with a control diet without grape stalks (C), two experimental groups were fed on diets with 5% and 10% incorporation of UGS (5UGS and 10UGS), and two with 5% and 10% incorporation of TGS (5TGS and 10TGS). Rabbits fed with TGS diets showed higher daily weight gain (p = 0.034), feed conversion rate (p = 0.002), carcass weight (p = 0.038), and reference carcass weight (p = 0.03) when compared to the control diet. Moreover, animals fed with TGS diets showed an increase in the caecum (p = 0.015) and small intestine (p = 0.021) lengths and in the total volatile fatty acid content (p = 0.005) compared to animals fed UGS diets. Blood triglyceride levels were lower in animals fed with TGS diets compared to UGS (p = 0.005) and C (p ≤ 0.001) diets (12% and 19% lower, respectively), and a trend to lower cholesterol levels was observed (p = 0.071). Meat from rabbits fed with TGS diets had higher levels of linoleic acid, γ-linolenic, ∑ω-6, ∑PUFA, and ∑PUFA/∑SFA ratio compared to rabbits fed with the C diet. Results indicated that grape stalks (UGS and TGS) could be effectively used as an alternative raw material in rabbits' diets without compromising animal performance.

Effect of Soil Chemical Properties on the Occurrence and Distribution of Entomopathogenic Fungi in Portuguese Grapevine Fields.

Entomopathogenic fungi (EPF) contribute to different ecosystem services. However, factors affecting their natural occurrences in soil remain poorly understood. In a previous study, 81 soil samples were subjected to insect baiting using Galleria mellonella and Tenebrio molitor to isolate EPF from Portuguese vine farms. Here, soils yielding any of the four common EPF, i.e., Beauveria bassiana, Purpureocillium lilacinum, Metarhizium robertsii, and Clonostachys rosea f. rosea, were correlated with their chemical properties. Beauveria bassiana was negatively affected by higher available P (p = 0.02), exchangeable K-ions (p = 0.016) and positively affected by higher soil pH_H2O (p = 0.021). High exchangeable K-ions inhibited P. lilacinum (p = 0.011) and promoted C. rosea f. rosea (p = 0.03). Moreover, high available K also suppressed P. lilacinum (p = 0.027). Metarhizium robertsii was inhibited by higher organic matter content (p = 0.009), higher C:N (p = 0.017), total N (p = 0.007), and exchangeable Mg-ions (p = 0.026), and promoted by higher exchangeable Na-ions (p = 0.003). Nonetheless, mean comparisons and principal component analysis suggested that higher soil pH and exchangeable Ca-ions have contrasting effects on EPF occurrences, as they promote B. bassiana and inhibit M. robertsii. Herbicides did not seem to affect EPF presence. Overall, this study is among the first reports on the effects of soil chemistry on EPF other than Metarhizium, and will facilitate biological pest management approaches.

Antimicrobial, Antibiofilm, and Antioxidant Properties of Boletus edulis and Neoboletus luridiformis Against Multidrug-Resistant ESKAPE Pathogens.

Multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become the most recurrent global cause of skin and soft-tissue infections, belonging to the WHO priority pathogens list. Successful therapy remains challenging and entails the assessment of novel and successful antibiotics. In this study, mushrooms are considered a valuable and unique source of natural antimicrobial compounds. Therefore, this study aimed to evaluate the antimicrobial and antibiofilm properties of Boletus edulis (B. edulis) and Neoboletus luridiformis (N. luridiformis) aqueous and methanolic extracts against ESKAPE isolates from clinical wound infections. Disk diffusion and microdilution methods were used to assess the antimicrobial activity. Phytochemical characterization was achieved by analysis of total phenols, orthodiphenols content, and antioxidant activity as well as by high-performance liquid chromatography-diode array detector (HPLC-DAD). Human foreskin fibroblasts-1 (HFF-1) cell viability was performed by the MTT assay. Aqueous and methanolic extracts of B. edulis and N. luridiformis showed antimicrobial and antibiofilm properties against multidrug-resistant bacteria, although with different efficacy rates. The results showed that there is a convincing relation between the content of phenolic compounds, antioxidant activity, and antimicrobial activity suggesting that the presence of phenolic compounds may explain the biological effects. HPLC analysis revealed high levels of protocatechuic acid, homogentisic acid, pyrogallol, gallic acid, p-catechin, and dihydroxybenzoic acid in the aqueous extract of B. edulis, explaining the highest antimicrobial and antibiofilm properties. Importantly, the mushrooms extracts were non-cytotoxic at all the tested concentrations. Overall, the tested mushrooms extracts are good candidates to further explore its use in the prevention of wound infection, particularly by multidrug-resistant pathogens.

Advances in Entomopathogen Isolation: A Case of Bacteria and Fungi.

Entomopathogenic bacteria and fungi are quite frequently found in soils and insect cadavers. The first step in utilizing these microbes as biopesticides is to isolate them, and several culture media and insect baiting procedures have been tested in this direction. In this work, the authors review the current techniques that have been developed so far, in the last five decades, and display brief protocols which can be adopted for the isolations of these entomopathogens. Among bacteria, this review focuses on Serratia spp. and bacteria from the class Bacilli. Among fungi, the review focuses those from the order Hypocreales, for example, genera Beauveria, Clonostachys, Lecanicillium, Metarhizium, and Purpureocillium. The authors chose these groups of entomopathogenic bacteria and fungi based on their importance in the microbial biopesticide market.

The effects of granulocyte colony-stimulating factors (G-CSFs) in leucocytes.

Granulocyte colony-stimulating factors may be responsible for a set of laboratory findings in the peripheral blood film that may be confused with those associated with important diseases like myeloproliferative neoplasms, infection, or megaloblastic anaemia. We present a case report of a 29-year-old woman where these laboratory findings are described.

Severe malaria.

Malaria represents a medical emergency. Without rapid diagnosis and treatment, it can progress and lead to severe complications and, eventually, death. Severe malaria is almost always caused by Plasmodium falciparum. Here, we present an image showing a set of hematological findings associated with severe malaria, highlighting the importance of a correct morphological diagnosis.

Cantharellus cibarius branched mannans inhibits colon cancer cells growth by interfering with signals transduction in NF-ĸB pathway.

In order to evaluate the therapeutic potential of polysaccharides obtained from the widely consumed Cantharellus cibarius mushroom on NF-ĸB pathway involved in cancer cells proliferation, survival, and metastasis, their antiproliferative and cytotoxic properties in human colon cancer cells LS180 and human colon epithelial cells CCD841 CoN were studied. BrdU and LDH assays results show that a branched mannan isolated from C. cibarius (CC2a) was selective against colon cancer cells, suppressing their proliferation and destroying membrane integrity but at the same time not adversely affecting colon epithelial cells. Results of Western blotting, immunofluorescence, RealTime PCR revealed that CC2a anticancer abilities were accompanied by disorders in signals transduction in NF-ĸB pathway in particular inhibition of IκBα degradation, attenuation of activated NF-κB phosphorylation and the subsequent decrease of NF-κB nuclear level as well as significant down-regulation of NF-κB target genes BAX, BCL2, CCND1, MMP9, MYC, BIRC5 and the corresponding proteins (except Bax). Furthermore, CC2a treatment of LS180 cells resulted in perturbation in G0/G and S phases of the cell cycle also associated with a marked increase of DNA fragmentation as well as inhibition of cancer cells motility. Obtained results indicated C. cibarius branched mannans as a new option in fighting with colon cancer.

Mushroom small RNAs as potential anticancer agents: a closer look at Cantharellus cibarius proapoptotic and antiproliferative effects in colon cancer cells.

Screening aimed at the evaluation of the presence of small RNAs with anticancer properties in three mushrooms species, besides Boletus edulis, namely Boletus spretus (current name Baorangia emilei), Boletus pinophilus and Cantharellus cibarius, was conducted. All mushrooms yielded an ethanol insoluble and water soluble small RNA fraction purified from co-extracted polysaccharides by anion-exchange chromatography. Small RNAs from B. spretus and C. cibarius showed strong antiproliferative activity against human colon adenocarcinoma cell lines (IC50 of 5.6 µg mL-1 and 11.1 µg mL-1 for LS180 and 1.9 µg mL-1 and 12.6 µg mL-1 for HT-29 cell lines, respectively) while those isolated from B. pinophilus showed a much lower antiproliferative activity in these cells. All RNA fractions were nontoxic against CCD841 CoTr human colon epithelial cells. A detailed study of the anticancer mechanism of C. cibarius small RNAs showed that their antiproliferative activity was due to p53-dependent cell cycle arrest mediated by p21, while the proapoptotic effect was mostly dependent on the enhancement of p53 expression. Overall, small RNA fractions isolated from some edible mushrooms, namely C. cibarius, show potent antiproliferative activity without cytotoxicity to normal cells, being a potential new anticancer agent naturally present in mushrooms that we eat.

Comparative study of plant growth-promoting bacteria on the physiology, growth and fruit quality of strawberry.

BACKGROUND: The strawberry (Fragaria × ananassa Duch.) is, among small fruits, the most cultivated and commercialized in Portugal. Recent studies have evidenced the positive effect of plant growth-promoting bacteria (PGPB) inoculation on strawberry production and, at the same time, provided an alternative strategy to reduce the use of fertilizers. In this study the effects of root inoculation with three PGPB strains (Pedobacter sp. CC1, Bacillus safensis B106 and Bacillus subtilis B167A) on the physiology, growth, fruit production and quality of strawberry cv. Camarosa are presented. RESULTS: PGPB inoculation significantly accelerated crop maturation, with inoculated plants fruiting about 2 weeks earlier than non-inoculated plants. Inoculated plants with Pedobacter sp. CC1 and Bacillus safensis B106 influenced the gas exchange parameters of strawberry plants. The contents of total phenolics and flavonoids in strawberry leaves were found to be greater with Pedobacter sp. CC1, when compared with non-inoculated plants. Furthermore, plants inoculated with the same bacterial strain showed enhancement in the dimensions of fruits, especially fruit length, and shape as well as in the total soluble solids content (°Brix). CONCLUSIONS: The results showed that the PGPB Pedobacter sp. CC1 improved performance of strawberry plants, suggesting that it could be a potential biofertilizer for strawberry plant nutrition. © 2019 Society of Chemical Industry.

MYH9 Disorders (May-Hegglin Anomaly) the Role of the Blood Smear.

MYH9 disorders are associated with sensorineural hearing loss, glomerular nephropathy, and presenile cataract. With this case report, we show the important role of the blood smear in its identification.

Soil Chemical Properties Barely Perturb the Abundance of Entomopathogenic Fusarium oxysporum: A Case Study Using a Generalized Linear Mixed Model for Microbial Pathogen Occurrence Count Data.

Fusarium oxysporum exhibits insect pathogenicity-however, generalized concerns of releasing phytopathogens within agroecosystems marred its entomopathogenicity-related investigations. In a previous study, soils were sampled from Douro vineyards and adjacent hedgerows. In this study, 80 of those soils were analyzed for their chemical properties and were subsequently co-related with the abundance of entomopathogenic F. oxysporum, after insect baiting of soils with Galleria mellonella and Tenebrio molitor larvae. The soil chemical properties studied were organic matter content; total organic carbon; total nitrogen; available potassium; available phosphorus; exchangeable cations, such as K⁺, Na⁺, Ca2+, and Mg2+; pH; total acidity; degree of base saturation; and effective cation exchange capacity. Entomopathogenic F. oxysporum was found in 48 soils, i.e., 60% ± 5.47%, of the total soil samples. Out of the 1280 insect larvae used, 93, i.e., 7.26% ± 0.72%, were found dead by entomopathogenic F. oxysporum. Stepwise deletion of non-significant variables using a generalized linear model was followed by a generalized linear mixed model (GLMM). A higher C:N (logarithmized) (p < 0.001) and lower exchangeable K⁺ (logarithmized) (p = 0.008) were found significant for higher fungal abundance. Overall, this study suggests that entomopathogenic F. oxysporum is robust with regard to agricultural changes, and GLMM is a useful statistical tool for count data in ecology.

Fusarium, an Entomopathogen-A Myth or Reality?

The Fusarium species has diverse ecological functions ranging from saprophytes, endophytes, and animal and plant pathogens. Occasionally, they are isolated from dead and alive insects. However, research on fusaria-insect associations is very limited as fusaria are generalized as opportunistic insect-pathogens. Additionally, their phytopathogenicity raises concerns in their use as commercial biopesticides. Insect biocontrol potential of Fusarium is favored by their excellent soil survivability as saprophytes, and sometimes, insect-pathogenic strains do not exhibit phytopathogenicity. In addition, a small group of fusaria, those belonging to the Fusarium solani species complex, act as insect mutualists assisting in host growth and fecundity. In this review, we summarize mutualism and pathogenicity among fusaria and insects. Furthermore, we assert on Fusarium entomopathogenicity by analyzing previous studies clearly demonstrating their natural insect-pathogenicity in fields, and their presence in soils. We also review the presence and/or production of a well-known insecticidal metabolite beauvericin by different Fusarium species. Lastly, some proof-of-concept studies are also summarized, which demonstrate the histological as well as immunological changes that a larva undergoes during Fusarium oxysporum pathogenesis. These reports highlight the insecticidal properties of some Fusarium spp., and emphasize the need of robust techniques, which can distinguish phytopathogenic, mutualistic and entomopathogenic fusaria.

Entomopathogenic fungi in Portuguese vineyards soils: suggesting a 'Galleria-Tenebrio-bait method' as bait-insects Galleria and Tenebrio significantly underestimate the respective recoveries of Metarhizium (robertsii) and Beauveria (bassiana).

Entomopathogenic fungi (EPF) are the natural enemies of insect-pests. However, EPF recoveries can be influenced by the soil habitat-type(s) incorporated and/or the bait-insect(s) used. Galleriamellonella (GM) as bait-insect, i.e. 'Galleria-bait', is arguably the most common methodology, which is sometimes used solely, to isolate EPF from soils. Insect baiting using Tenebriomolitor (TM) has also been employed occasionally. Here 183 soils were used to estimate the functional diversity of EPF in Portuguese Douro vineyards (cultivated habitat) and adjacent hedgerows (semi-natural habitat), using the TM bait method. Moreover, to study the effect of insect baiting on EPF recovery, 81 of these 183 soil samples were also tested for EPF occurrences using the GM bait method. Twelve species were found in 44.26% ± 3.67% of the total of 183 soils. Clonostachysroseaf.rosea was found in maximum soils (30.05% ± 3.38%), followed by Beauveriabassiana (12.57% ± 2.37%), Purpureocilliumlilacinum (9.29% ± 2.14%) and Metarhiziumrobertsii (6.01% ± 1.75%). Beauveriapseudobassiana (P < 0.001), C.roseaf.rosea (P = 0.006) and Cordycepscicadae (P=0.023) were isolated significantly more from hedgerows, highlighting their sensitivities towards agricultural disturbances. Beauveriabassiana (P = 0.038) and M.robertsii (P = 0.003) were isolated significantly more using GM and TM, respectively. Principal component analysis revealed that M.robertsii was associated both with TM baiting and cultivated habitats, however, B.bassiana was slightly linked with GM baiting only. Ecological profiles of B.bassiana and P.lilacinum were quite similar while M.robertsii and C.roseaf.rosea were relatively distant and distinct. To us, this is the first report on (a) C.cicadae isolation from Mediterranean soils, (b) Purpureocilliumlavendulum as an EPF worldwide; and (c) significant recoveries of M.robertsii using TM over GM. Overall, a 'Galleria-Tenebrio-bait method' is advocated to study the functional diversity of EPF in agroecosystems.

Neuroprotective properties of Cantharellus cibarius polysaccharide fractions in different in vitro models of neurodegeneration.

The percentage of people suffering from neurodegenerative diseases is constantly increasing, because of that searching for substances able to prevent or inhibit neuronal death sseems to be reasonable. Because of the high popularity the search of new neuroprotective agents we started from Cantharellus cibarius. Neuroprotective properties of C. cibarius polysaccharides fractions was investigated in different models of neurodegeneration including trophic stress, excitotoxicity and andoxidative stress. Fractions influence on neurons viability was examined using Neurite Outgrowth Staining, MTT and LDH tests, while antioxidant capacity was determined by commercial antioxidant assays. Performed studies revealed beneficial effect of C. cibarius fractions (CC2a, CC3) on neurons viability and neurite outgrowth in normal and different stress conditions. Both tested fractions have shown antioxidant capacity and effectively neutralize the negative changes induced by glutamatergic system activators. Discovered neuroprotective properties of investigated compounds suggested the their use for developing effective and safety therapeutic strategy for neurodegenerative diseases.