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Ethnopharmacological Relevance: The prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species., Aim of the Study: We aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis., Materials and Methods: W. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration., Results: The anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2-8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo., Conclusion: Our results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Natural products from plants have been listed for hundreds of years as a source of biologically active molecules. In recent years, the marine environment has demonstrated its ability to provide new structural entities. More than 70% of our planet's surface is covered by oceans, and with the technical advances in diving and remotely operated vehicles, it is becoming easier to collect samples. Although the risk of rediscovery is significant, the discovery of silent gene clusters and innovative analytical techniques has renewed interest in natural product research. Different strategies have been proposed to activate these silent genes, including co-culture, or mixed fermentation, a cultivation-based approach. This review highlights the potential of co-culture of marine microorganisms to induce the production of new metabolites as well as to increase the yields of respective target metabolites with pharmacological potential, and moreover to indirectly improve the biological activity of a crude extract.

Background: Until now, the cost of allergy treatment in the insured public health care system and the non-insured self-financing private health care system in Indonesia has not been well documented and published, as well as the cost of allergy treatment with subcutaneous immunotherapy., Objective: To evaluate the clinical and cost benefits of allergic rhinitis treatment in children with subcutaneous immunotherapy in a non-insured self-financing private health care system., Methods: A retrospective cohort study conducted from 2015 until 2020 that compared the clinical improvement and health care costs over 18 months in newly diagnosed AR children who received SCIT versus matched AR control subjects who did not receive SCIT, with each group consisting of 1098 subjects., Results: A decrease in sp-HDM-IgE level (kU/mL) from 20.5 + 8.75 kU/mL to 12.1 + 3.07 kU/mL was observed in the SCIT group. To reduce the symptom score of allergic rhinitis by 1.0 with SCIT, it costs IDR 21,753,062.7 per child, and for non-SCIT, it costs IDR 104,147,878.0 per child. Meanwhile, to reduce the medication score (MS) by 1.0 with SCIT, it costs IDR 17,024,138.8, while with non-SCIT, it costs IDR 104,147,878.0. Meanwhile, to lower combination symptoms and medication score (CSMS) by 1.0, with SCIT, it costs IDR 9,550,126.6, while with non-SCIT, it costs IDR 52,073,938.9., Conclusions: In conclusion, this first Indonesia-based study demonstrates substantial health care cost savings associated with SCIT for children with AR in an uninsured private health care system and provides strong evidence for the clinical benefits and cost-savings benefits of AR treatment in children.

Polysaccharides from P. eryngii mushroom were selectively extracted using low-cost technologies (water at different conditions of temperature and pressure). Mannogalactan was the main polysaccharide in cold-water extracted fraction (CWEF), while a linear (1→6)-β-d-glucan was the main polymer in hot-water extracted fraction (HWEF). Autoclave-extracted fraction (AEF) contained a mixture of at least four different α- and β-glucans. The report of linear (1→6)-β-glucan and linear (1→3)-β-glucan is a new finding for P. eryngii fruiting bodies. The immunostimulatory properties of the fractions on THP-1 macrophages were studied. All fractions at 50, 250 and 500 μg/mL were not cytotoxic and produced different stimulus on NO, IL-1β and IL-10 secretion by the cells. Thus, our results showed that it is possible to concentrate different P. eryngii polysaccharides in selected fractions using a simple and low-cost procedure. Since biological effects depends on the polysaccharide structure, this technique allows the obtainment of fractions with distinct immunomodulatory activities., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Huaier extract, the main active constituent proteoglycan, has anti-tumor activity in various experimental and clinical settings. However, the potential anti-neuroblastoma and associated mechanisms have not been investigated. Therefore, in this study, we aimed to elucidate the potential role of Huaier extract in 3 human neuroblastoma cell lines. Our study demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability in a dose-dependent manner. Huaier extract induced cell cycle arrest at G0/G1 phase in neuroblastoma and decreased the cell cycle related protein expression of cyclin D3. Western blotting analysis also showed that Huaier extract induced neuroblastoma cell apoptosis and autophagy. Signaling analysis indicated that Huaier extract suppressed the MEK/ERK and mTOR signaling pathways simultaneously. In conclusion, we verify that Huaier extract causes cell proliferation inhibition, apoptosis, autophagy, and cell cycle arrest in G0/G1 phase via MEK/ERK and mTOR signaling. Huaier extract may act as a complementary agent for treating neuroblastoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

In the present work crude Agaricus bisporus extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, p -nitrophenol, o -nitrophenol, guaiacol, o -cresol, m -cresol, p -cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified A. bisporus tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10 -4 -1.0 × 10 -3 M l-tyrosine, 3.1 × 10 -6 -1.0 × 10 -4 M phenol, 5.4 × 10 -5 -1.0 × 10 -3 M catechol, 8.5 × 10 -5 -1.0 × 10 -3 M caffeic acid, 1.5 × 10 -4 -7.5 × 10 -4 M chlorogenic acid, 6.8 × 10 -5 -1.0 × 10 -3 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.

Three unusual polyketides with a 5/6/10-fused ring system, named colletotrichalactones A-Ca (1-3a), were isolated from cultures of the endophytic fungus, Colletotrichum sp. JS-0361, which was isolated from a leaf of Morus alba. Their structures, including their absolute stereochemistries, were completely established using extensive spectroscopic methods together with a chemical reaction utilizing competing enantioselective acylation coupled with LC/MS. Compounds possessing this ring skeleton were previously reported in three studies. Our rigorous chemical investigation revealed the complete configuration of this skeleton, which agreed with the results for glabramycin B with this ring skeleton established by computational chemistry and enantioselective synthesis in previous reports. 1 and 2 had unstable aldehyde groups that were easily converted to acetal groups in the presence of solvents. Meanwhile, compound 3a, with terminal acetal functionality, was deduced to be an artefact originating from compound 3 with a terminal aldehyde group. Compounds 1 and 3a displayed moderate-to-potent cytotoxic activities against MCF7 cells with IC 50 s of 35.06 and 25.20 µM, respectively., (Copyright © 2020 Elsevier Inc. All rights reserved.)

With the ever-increasing human lifespan, age-related affections have become a public health issue. The health sector is looking for new bioactive compounds to respond to this demand. The unexplored microbial biodiversity and its metabolites represent a major source of innovative bioactive molecules with health potential. Fermented foods, such as raw-milk cheese, have already been investigated for their rich microbial environment, especially for their organoleptic qualities. But studies remain limited regarding their effects on health and few metabolites of microbial origin have been identified. An efficient methodology was developed in this study to investigate the biological effect of raw-milk cheese, combining a chemical fractionation, to isolate the most metabolites from the cheese matrix, and an in vivo biological test using Caenorhabditis elegans. C. elegans was brought into contact with cheese extracts, obtained by means of chemical fractionation, and with freeze-dried whole cheese by supplementing the nematode growth medium. A longevity assay was performed to evaluate the effects of the extracts on the worms. Our results demonstrate the feasibility of the method developed to bring the worms into contact of the cheese extracts. The evaluation of the effects of the extracts on the longevity was possible. Some extracts showed a beneficial effect as extract W70 for example, obtained with water, which increases the mean lifespan by 16% and extends the longevity by 73% (p < 0.0001)., Competing Interests: The authors have read the journal's policies and the authors have the following competing interests: AC, ES and JP are paid employees of Dômes Pharma. Dômes Pharma also provided support in the form of research materials for this study. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The purpose of this study was to elucidate the antibacterial activity and possible mechanism of action of Amaranthus tricolor crude extract (ATCE) against Cronobacter sakazakii isolated from powdered infant formula (PIF). The antibacterial activity of ATCE was assessed by measuring the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The possible mechanism of action of ATCE was revealed by analyzing the effects of ATCE on growth curves and changes in cell membrane potential, intracellular pH, content of bacterial protein and genomic DNA, and cell morphology. Finally, ATCE was applied to the disinfection of C. sakazakii in biofilm on stainless steel tube. The results showed that the DIZ, MIC, and MBC of ATCE against C. sakazakii strains were from 14.35 ± 0.67 to 14.84 ± 0.67 mm, 20 mg/mL, and 40 mg/mL, respectively. Treatment with ATCE ended the logarithmic growth phase of C. sakazakii, and led to depolarization of the cell membranes, reducing intracellular pH and bacterial protein and genomic DNA contents, and resulting in cytoplasmic leakage and deformation. In addition, ATCE effectively inactivated C. sakazakii in biofilm, reducing viable bacteria by approximately 6.5 log cfu/mL bacterial count after treatment with 1 MIC (1 MIC = 20 mg/mL) of ATCE for 20 min at 25°C. Our findings showed that ATCE inactivated C. sakazakii strains isolated from PIF and has potential as a natural disinfectant to reduce the contamination of PIF by C. sakazakii., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Three pyrrole alkaloid derivatives were isolated from the edible mushroom Basidiomycetes-X (Echigoshirayukidake) by water extraction followed by ethyl acetate fractionation. The chemical structures determined by MS and NMR were 4-[2-formyl-5-(hydroxymethyl)-1 H -pyrrol-1-yl] butanoic acid (compound I), 4-[2-formyl-5-(hydroxymethyl)-1 H -pyrrol-1-yl] butanamide (compound II), and 5-(hydroxymethyl)-1 H -pyrrole-2-carboxaldehyde (compound III). Compound I was found to be the major component, followed by compound II, and compound III was the minor component. The dry powder of Basidiomycetes- X contained approximately 825 μg g -1 compound I and 484 μg g -1 compound II. Compound II was found to be a novel pyrrole aldehyde homologue not previously reported and thus is a specific component of this mushroom.

At present, diabetes and diabetic complications have become one of the serious diseases affecting human health. In this study, the inhibitory effects of Lentinus edodes mycelia polysaccharide (LMP) on α-glucosidase activity, the formation of advanced glycation end products (AGEs) and high glucose-induced human umbilical vein endothelial cells (HUVECs) damage were explored. The interaction between LMP and α-glucosidase and the inhibition against AGEs formation were investigated with spectroscopic techniques. The results revealed that LMP had a reversible inhibition on α-glucosidase activity in a mixed-type manner. When the concentration of LMP was 2.7 mM, the inhibition rate was 34.38 %. LMP quenched the fluorescence of α-glucosidase through the static quenching and formed the LMP-α-glucosidase complex. At 310 K, the number of binding sites (n) and binding constant (K b ) were 1.01 and 3.71 × 10 4 L mol -1 , respectively. In addition, LMP could inhibit the formation of AGEs. Compared with 40 mM glucose treatment group, treatment with 0.05 mM LMP for 48 h increased the cell viability from 70.17% to 91.14% and decreased ROS production from 3.33-fold to 1.21-fold. LMP inhibited high glucose-induced activation of MAPK signaling pathways. These findings may promote the application of LMP in the functional food industry., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Breast cancer is the leading cause of cancer death among women across the world. Trametes robiniophila Murr (Huaier), a traditional herbal medicine, has been used in China to protect human health for about 1600 years. Recent years, Huaier had been proven to be effective for multiple types of malignancies. This systematic review focused on breast cancer treatment, summarizing the curative function of Huaier aqueous extract and polysaccharides in preclinical researches. Huaier could markedly inhibit breast cancer progression with low toxicity, enhance immune response and increase the sensitivity to radiation and chemotherapy. The therapeutic effect of Huaier granule in clinical studies was also included. This review amalgamated the current studies and highlighted the promising role of Huaier and its polysaccharides as complementary alternative medicine in breast cancer treatment., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Trametes robiniophila Murr in the Treatment of Breast Cancer”. The authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritic and eczematous skin lesions. The skin of AD patients is generally in a dried condition. Therefore, it is important for AD patients to manage skin moisturization. In this study, we examined the effects of orally administered fermented barley extract P (FBEP), which is prepared from a supernatant of barley shochu distillery by-product, on stratum corneum (SC) hydration and transepidermal water loss (TEWL) in AD-like lesions induced in hairless mice using 2,4,6-trinitrochlorobenzene. Oral administration of FBEP increased SC hydration and decreased TEWL in the dorsal skin of this mouse model. Further fractionation of FBEP showed that a pyroglutamyl pentapeptide, pEQPFP comprising all -L-form amino acids, is responsible for these activities. These results suggested that this pyroglutamyl pentapeptide may serve as a modality for the treatment of AD.

A simple SFC/MS (Supercritical Fluid Chromatography/Mass Spectrometry) method set was developed to effectively screen separations of various crude synthetic peptide products of pharmaceutical interest. Additives to the modifier methanol which were successful for these separations were found to include TFA (trifluoroacetic acid) and ammonia mixed with TFA, each at 0.1 % (v/v) composition in methanol. A final screening column set consisted of 2-ethylpyride (2-EP), 4-ethylpyridine (4-EP) and cross-linked diol (Luna™ HILIC) stationary phases. Small, linear and macrocyclic peptides with fewer than ten residues could all be eluted with good performance under at least one of the method conditions comprising the final screening protocol. For larger peptides either the 4-EP and HILIC columns with the above additives provided a good initial screening method set without 2-EP. The gradient was slightly longer and shifted to higher polarity relative to the gradients for smaller peptides. This method was often successful to elute large, hydrophilic peptides up to a 41-mer with acceptable peak profiles although the largest peptides with most ionizable residues were most challenging. For these peptides generally HILIC column performance was better with TFA + ammonia in the modifier than with TFA, while 4-EP performance was usually improved with TFA relative to TFA + ammonia., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We studied activities of antioxidant system enzymes in tissues of rats with experimental allergic encephalomyelitis. It was shown that the development of pathology is accompanied by deformation of the neurons and axonal degeneration, intensification of free radical oxidation, exhaustion of the reduced glutathione pool, and multidirectional changes in activities of antioxidant enzymes in rat tissues. The observed imbalance in the antioxidant defense system can be associated with excessive glutathione utilization in the glutathione transferase reaction and different severity of the pathological process in the brain and spinal cord. The received data necessitate the search for compounds that can prevent inhibition of antioxidant system components in order to analyze the possibility of their use in the treatment of multiple sclerosis.

Sea cucumbers are bottom dwelling invertebrates, which are mostly found on subtropical and tropical sea grass beds, sandy reef flats, or reef slopes. Although constantly exposed to fouling communities in these habitats, many species are surprisingly free of invertebrate epibionts and microfouling algae such as diatoms. In our study, we investigated the anti-fouling (AF) activities of different crude extracts of tropical Indo-Pacific sea cucumber species against the fouling diatom Cylindrotheca closterium . Nine sea cucumber species from three genera (i.e., Holothuria , Bohadschia , Actinopyga ) were selected and extracted to assess their AF activities. To verify whether the sea cucumber characteristic triterpene glycosides were responsible for the observed potent AF activities, we tested purified fractions enriched in saponins isolated from Bohadschia argus , representing one of the most active anti-fouling extracts. Saponins were quantified by vanillin-sulfuric acid colorimetric assays and identified by LC-MS and LC-MS/MS analyses. We were able to demonstrate that AF activities in sea cucumber extracts were species-specific, and growth inhibition as well as attachment of the diatom to surfaces is dependent on the saponin concentration (i.e., Actinopyga contained the highest quantities), as well as on the molecular composition and structure of the present saponins (i.e., Bivittoside D derivative was the most bioactive compound). In conclusion, the here performed AF assay represents a promising and fast method for selecting the most promising bioactive organism as well as for identifying novel compounds with potent AF activities for the discovery of potentially novel pharmacologically active natural products.

Allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease‑rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50% glycerol in NS, 0.3% phenol in NS, or 0.3% phenol and 50% glycerol in NS. The extracts in different buffers were stored either at room temperature (18‑26˚C, RT) or refrigerated (2‑8˚C). Subsequently, the protein concentration and allergen content (Bla g 1 and Bla g 2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Bla g 1, 65.2% Bla g 2, and 91.4% potency remained after one year when 50% glycerol NS was added to the extract with refrigeration. However, less than 13.7% protein, 17.1% Bla g 1, 0% Bla g 2 and 32.5% potency were maintained after one year when 50% glycerol NS was not added to the extract and was maintained at RT. The addition of 0.3% phenol NS did not show significant effects on extract stability. The addition of 50% glycerol NS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease‑rich cockroach extract.

This study reported the genetic and oxidative effects of aqueous and methanol extracts from two edible mushrooms, Lepista nuda (Bull.) Cooke and Pleurotus ostreatus (Jacq.) P. Kummer, in cultured human lymphocytes. Chromosome aberration (CA) and micronucleus (MN) assays were used for genotoxic influences estimation. In addition, the changes of total antioxidant capacity (TAC) and total oxidative stress (TOS) in the cells were monitored. The fungal extracts at all applied concentrations did not indicate significant differences (p > 0.05) in CA and MN analyses. Furthermore, while the treatments with maximum concentration of aqueous extract of L. nuda statistically (p < 0.05) increased TAC especially, TOS levels in the cells were reduced by them in comparison with negative control. Based on TAC analysis, low IC50 values belonging to aqueous (5.43 mg/L) and methanol (10.88 mg/L) extracts of L. nuda were remarkable. Our data demonstrated that the extracts obtained from P. ostreatus and especially L. nuda can be a new resource for therapeutics with their nonmutagenic and antioxidant features.

The edible medicinal mushroom Flammulina velutipes (enokitake) has many applications as food and medicine, but its application in dentistry is unknown. This study aims to investigate the inhibitory effect of fruiting body extracts from F. velutipes on the growth and adhesion of Streptococcus mutans, the main cause of human caries, in vitro. Of the four extracts (named TG01 from water, TG02 from 95% ethanol, TG03 from 50% ethanol, and TG04 from ethyl acetate), TG03 had significant antibacterial activity (MIC = 10 mg/mL; MBC = 20 mg/mL). Planktonic growth and biofilm formation in S. mutans was repressed by TG03 at 5 mg/mL and above. Meanwhile, cytotoxicity analysis showed that TG03 was not toxic to human oral keratinocyte cells. HPLC-QQQ-MS analysis showed that the TG03 extract contained a large amount of arabitol, a sucrose substitute that reduces the development of caries. Thus, F. velutipes extracts can effectively inhibit the growth of the oral pathogen S. mutans without cytotoxicity against human oral keratinocytes. Therefore, F. velutipes is a good candidate for the development of oral hygiene agents to control dental caries.

The objective of this study was to explore the effect of magnesium acetate (MA) addition on the endo-polyphenol yield by Phellinus baumii and establish a feasible additive strategy. The optimal three-point MA addition strategy (0.05 g/L concentration of MA added at 0 h and 6 h, 0.9 g/L concentration of MA added at 12 h) was employed to obtain maximum endo-polyphenol yield. The maximum endo-polyphenol production was reached at 1.22 g/L, which was 1.39-fold higher than that of the control. Additionally, the endo-polyphenol showed stronger antioxidant activity in vitro compared with the control, including DPPH· scavenging capacity (78.76%) and Trolox equivalent antioxidant capacity (TEAC) (32.28 μmol Trolox/g sample). HPLC analysis showed that the endo-polyphenol production of the crude ethanol extracts was significantly higher than that of the control. Hispidin was isolated and identified from the ethanol extract of the culture mycelia from Ph. baumii with the three-point MA addition strategy. Hispidin showed a strong ability to scavenge DPPH free radicals and TEAC, equivalent to positive (vitamin C) value of 89.41% and 75.98%, respectively. Furthermore, hispidin protected H2O2-induced PC12 cells injured by decreased oxidative stress level. These results indicated that the MA multi-stage addition strategy was dependable, and could be used to develop new natural antioxidants for foods or medicines.

Melanoma is among the most aggressive and treatment-resistant human cancers. Phellinus baumii, a famous medicinal mushroom, has been used to treat different diseases, including cancer, in China and other east Asian countries. The purpose of this research was to explore its anticancer effects against melanoma, and the mechanisms that might be involved. CCK-8 assay exhibited that extracts of Ph. baumii (EPB) strongly inhibited cell viability of A375 melanoma cancer cell. Typical morphological changes of cell apoptosis were observed in EPB-treated A375 cells in Hoechst staining assay. Flow cytometry analysis indicated that EPB significantly induced A375 cells apoptosis and the cell cycle was disrupted in S phase. EPB increased the expression of Bax, and decreased Bcl-2 in A375 cells. EPB remarkably caused mitochondrial membrane potential collapse and induced a mitochondrial-dependent apoptosis in A375 cells evidenced by caspase-3 activation, followed by PARP cleavage. More importantly, EPB has shown a strong inhibitory effect on the migration and aggression of the A375 cells through the healing of the wound and transwell assay. In vivo, EPB was also found to strongly inhibit the growth of tumors in BALB/c nude mice. Our results indicated that Ph. baumii might be a natural therapeutic product for aggressive melanoma because it could induce apoptosis and inhibit metastasis in A375 cells.

Background: Commercially available antiviral drugs, when used in the treatment of viral infections, do not always result in success. This is an urgent problem currently that needs to be addressed because several viruses including influenza and paramyxoviruses are acquiring multi-drug resistance. A potential solution for this emerging issue is to create new antiviral drugs from available compounds of natural products. It is known that the majority of drugs have been developed using compounds derived from actinomycetes, which are naturally occurring gram-positive bacteria. The purpose of this study was to investigate the antiviral properties of extremophilic actinomycetes extracts from strains that were isolated from extreme environments in Kazakhstan., Methods: Five strains of extremophilic actinomycetes isolated from the unique ecosystems of Kazakhstan were extracted and tested for antiviral activity against influenza viruses (strains H7N1, H5N3, H1N1 and H3N2) and paramyxoviruses (Sendai Virus and Newcastle Disease Virus). The antiviral activity of these selected extracts was tested by checking their effect on hemagglutination and neuraminidase activities of the studied viruses. Additionally, actinomycetes extracts were compared with commercially available antiviral drugs and some plant preparations that have been shown to exhibit antiviral properties., Results: The main findings show that extracts from strains K-192, K-340, K-362, K-522 and K525 showed antiviral activities when tested using influenza viruses, Sendai Virus, and Newcastle Disease Virus. These activities were comparable to those shown by Rimantadine and Tamiflu drugs, and "Virospan" and "Flavovir" plant preparations., Conclusions: We identified several extracts with antiviral activities against several strains of influenza viruses and paramyxoviruses. Our research findings can be applied towards characterization and development of new antiviral drugs from the active actinomycetes extracts.

The aim of our study was to establish the effect of selenium and betaine on the growth of D. salina, accumulation of photosynthetic pigments and antioxidant activity of the hydrophobic fraction. This approach was an attempt to demonstrate 'microalgae biostimulant' effects, similar to 'plant biostimulant' effects, i.e. increased tolerance to abiotic stress and enhanced accumulation of bioactive compounds. A high-throughput assay was done in 24-well microplates, at 15% NaCl and different concentrations of sodium selenite (0, 0.5, 2 and 8 µM) or betaine (0, 5, 50 and 500 µM). Both selenium and betaine induced a slight delay in algae growth during the actively growing stage but the final density reached similar values to the control. Betaine significantly enhanced (50%-100%) carotenoids and chlorophyll a accumulation, in a concentration depending manner. Antioxidant activity increased almost 3-fold in extracts of algae treated with 50 µM betaine. Selenium had a much more discrete effect than betaine on pigments biosynthesis. The antioxidant activity of the extracts increased 2-fold in the presence of Se compared to the control. Our work proves that it is possible to enhance production and activity of bioactive compounds from microalgae by using ingredients, which already proved to act as plant biostimulants., (© FEMS 2020.)

Background: The occurrence of drug resistant infectious disease causing microbial pathogens was highly spreaded because of the wide level application of the commercially available antimicrobial agents. However, the eradication of the microbial pathogens was of huge demand. Although, many antimicrobial compounds were commercially available in the market however the spreading of the pathogens were hugely increased. Actinomycetes produce various secondary metabolites against pathogenic bacteria and fungi. The present investigation aimed to study the antimicrobial potential of the Streptomyces sp. towards infectious diseases causing pathogens., Methods: Culture dependable isolation techniques were followed for the isolation of the active actinomycetes isolates and the antimicrobial properties of the actinomcyetes were detected by primary screening techniques using modified starch casein agar medium. The active isolate was confirmed by various biochemical and morphological techniques., Results: In this study, 10 actinomycetes were isolated and later five were selected for secondary screening and noted significant activity against Enterobacter aerogenes and Proteus mirabilis. Among the selected Streptomyces sp., ES2 showed potent activity against selected microbes and was identified as Streptomyces sp. The studied isolates were resisitant towards streptomycin (10μg), ampicillin (50μg) and ciprofloxacin (5μg). The organic solvent extracts of the promising isolate ES2 prononunced comparatively better inhibitory properties towards the studied pathogenic bacteria., Conclusion: Overall, the present study evidenced that the actinomycetes were promising candidate for the eradication of the pathogenic strains., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The cyanobacterium Radiocystis fernandoi has been frequently identified in cyanobacterial blooms in Brazil. Recently, R. fernandoi strain R28, which produces microcystin (MC)-RR and MC-YR, was isolated from the Furnas reservoir, Minas Gerais, Brazil. The present study evaluated the hematological variables and erythrocyte antioxidant responses, lipid peroxidation (LPO), and genotoxicity in a neotropical fish (Hoplias malabaricus) after acute and subchronic exposure to a crude extract (CE) of R. fernandoi strain R28. Acute exposure (12 or 96 h) consisted of a single intraperitoneal (i.p.) CE injection, and subchronic exposure consisted of one i.p. CE injection every 72 h for 30 days. After acute exposure, fish exhibited macrocytic anemia (12 h post-injection) followed by normocytic anemia (96 h post-injection). The increased activity of superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and the glutathione level in the erythrocytes did not prevent oxidative stress, manifested as lipid peroxidation and elevated DNA damage after acute exposure. After subchronic exposure, the hematological variables recovered, and the absence of erythrocyte oxidative stress suggests possible modulation by other biological factors, including a possible decrease in MC uptake by the cells and/or increasing detoxification efficiency that precludes erythrocyte damage., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Inflammation and pain triggers several pathological illnesses. Synthetic drugs used for the controlling of inflammatory conditions convey significant toxic effects. Global scientific community continually attempt to improve effective, economic and harmless naturally derived remedies against inflammation and pain. The present study aimed to quantify the phytochemical constituents of the freshly cultivated Spirulina and targeted to examining the anti-inflammatory and analgesic activity of Spirulina extract (SE) derived from Arthrospira platensis., Methods: The anti-inflammatory effect of SE was evaluated in animal models including carrageenan-induced rat hind paw oedema, and cotton pellet-induced granuloma formation. Analgesic effects of SE were evaluated by acetic acid induced writhing response and hot plate test., Results: Phytochemical quantification guided to identify seven carbohydrates, thirteen amino acids, eleven fatty acids and polyphenolic compounds respectively. The results indicated that SE significantly attenuated carrageenan-induced hind paw oedema, and cotton pellet-induced granuloma. Preliminary molecular mechanistic studies established that SE decreased the productions of TNF-α, IL-1β, IL-6, PGE2 and NO, and suppressed the activities of COX-2 and iNOS., Conclusion: These results provide a strong scientific foundation for the anti-inflammatory and analgesic activities of SE against different studies in animal models., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Seaweeds have been used as a source of natural fertilizer and biostimulant in agriculture for centuries. However, their effects on soil and crop root microbiota remain unclear. Here, we used a commercially available Ascophyllum nodosum extract (ANE) to test its effect on bacterial and fungal communities of rhizospheric soils and roots of pepper and tomato plants in greenhouse trials. Two independent trials were conducted in a split-block design. We used amplicon sequencing targeting fungal ITS and bacterial 16S rRNA gene to determine microbial community structure changes. We find that productivity parameters of root, shoot and fruit biomass were positively and significantly influenced by the ANE amendment. In addition, a-diversity differed significantly between amended and control plants, but only in some of the experimental conditions. Species composition among sites (b-diversity) differed according to the amendment treatment in all four communities (fungal-root, fungal-soil, bacterial-root and bacterial-soil). Finally, we identified a number of candidate taxa most strongly correlated with crop yield increases. Further studies on isolation and characterization of these microbial taxa linked to the application of liquid seaweed extract may help to enhance crop yield in sustainable agro-ecosystems., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Closed-loop recycling dual-mode counter-current chromatography (CLR DM CCC) includes two separation stages: 1 - closed-loop recycling separation of solutes with mobile x-phase (CLR CCC); 2 - separation of solutes with the mobile y-phase in the opposite flow direction. Previous analysis of CLR DM CCC separations has been limited to the ideal recycling model, which neglects extra-column dispersion. In this study, the analysis of CLR CCC separations is based on the non-ideal recycling model, which takes into account the extra-column dispersion caused by the recycling system. This is of great practical importance, since by selecting the optimal parameters of the recycling system the separation can be significantly improved. Comparative analysis of CLR CCC and CLR DM CCC separations has shown that at low separations factors compounds with low partition coefficients can be separated by CLR CCC using recycling systems with a long recycling line; the separation of compounds with high partition coefficients and the separation of complex mixtures can be performed by CLR DM CCC. Simple equations for simulation and design of CLR DM CCC separations are developed. Several variants of the implementation of this separation method are discussed; examples of simulation are presented in "Mathcad" program., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Since Agaricus blazei Murill (AbM) extract reduced specific IgE and ameliorated a skewed Th1/Th2 balance in a mouse allergy model, it was tested in blood donors with self-reported, IgE-positive, birch pollen allergy and/or asthma. Sixty recruited donors were randomized in a placebo-controlled, double-blinded study with pre-seasonal, 7-week, oral supplementation with the AbM-based extract Andosan TM . Before and after the pollen season, questionnaires were answered for allergic rhino-conjunctivitis, asthma, and medication; serum IgE was measured, and Bet v 1-induced basophil activation was determined by CD63 expression. The reported general allergy and asthma symptoms and medication were significantly reduced in the AbM compared to the placebo group during pollen season. During the season, there was significant reduction in specific IgE anti-Bet v 1 and anti-t3 (birch pollen extract) levels in the AbM compared with the placebo group. While the maximal allergen concentrations needed for eliciting basophil activation before the season, changed significantly in the placebo group to lower concentrations (i.e., enhanced sensitization) after the season, these concentrations remained similar in the Andosan TM AbM extract group. Hence, the prophylactic effect of oral supplementation before the season with the AbM-based Andosan TM extract on aeroallergen-induced allergy was associated with reduced specific IgE levels during the season and basophils becoming less sensitive to allergen activation.

Tropical ecosystems hold an extremely diverse array of endophytic fungi, but their potential use still remains to be explored. In this study, we isolated an endophytic fungus from the leaves of Otoba gracilipes, a medicinal tree from a tropical rainforest in Colombia. Following isolation and cultivation, we evaluated its extracellular crude extract for antioxidant activity. Using traditional and molecular methods (ITS1, NL1 regions), the endophyte was identified as Fusarium oxysporum. Fresh spores from the fungal isolate were inoculated in liquid media (potato dextrose broth [PDB] and potato dextrose-yeast extract broth [PDYB]) and centrifuged for recovering extracellular polysaccharides from the exhausted medium after 30 days of cultivation. Crude extracts were recovered, purified, lyophilized, and evaluated for their ability to inactivate the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). The extracts obtained from PDB culture media had a 51.5% of scavenging effect on DPPH after 5 min of reaction compared with the extracts from PDBY (26.4%), which suggests a high antioxidant potential of these fungal extracts. Thus, our results suggest other fungi from tropical ecosystems should be explored as potential sources of novel enzymes and other metabolites with bioactivity., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Utilization of polyphenol oxidases (laccase, tyrosinase) in biosensor technology is an efficient approach towards phenol detection, which is significant in numerous fields such as environmental monitoring, food industry etc. The use of crude extract instead of pure enzyme eliminates the need for costly and laborious processes of enzyme separation and purification. This study employs polyphenol oxidase extract, biosynthesized by white-rot fungi Trametes pubescens (TP) for the development of amperometric biosensors for catechol detection. The catalytic activity of the crude extract was firstly used to induce the bio-synthesis of conducting polymer - polypyrrole (Ppy), resulting in the self-encapsulation of the enzyme extract within the conducting material. The viability and biological integrity of the enzyme extract was preserved after the synthesis and was able to efficiently detect phenolic compounds such as catechol. Comparative evaluations between the biosynthesized Ppy based biosensor (bio-Ppy) and the biosensor based on bio-PPy with additional enzyme extract (bio-Ppy-TP) were performed. Lastly, the performance of these two biosensors was compared with that of a third one, based on chemically synthesized Ppy with enzyme extract (chem-Ppy-TP). All three types of biosensors proved high efficiency for catechol detection at low concentration (1-60 μM) and were employed for real sample detection in fruit wines showing linear correlation with the spectrophotometric results obtained with the Folin-Ciocalteau standard test., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 μM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Nattokinase (NK, E.C. 3.4.21.62) is a serine protease produced by Bacillus subtilis natto that shows promise for the treatment of thrombotic disease. In this study, we assessed the effects of NK on the development of hepatocellular carcinoma (HCC), a principal malignancy of the liver that causes morbidity and mortality worldwide. Crude extracts of NK (NCE) were isolated from fermentation medium by centrifugation and separated into three fractions (<10 K, 100~30 K and >30K). Orthotopic HCC mouse models were established and NCE was administered by oral gavage. H&E staining was performed to examine the pathology of HCC livers. Immunohistochemistry and immunofluorescence were used to evaluate FOXM1, CD31, CD44 and vimentin expression in the liver. Compared to PBS groups, NCE increased the survival rates of HCC-bearing mice to 31% and decreased ascites. Low-intensity ultrasound imaging showed that the hypoechoic mass area was lower in NCE-treated mice and that tumor growth significantly decreased. IHC staining showed that the expression of FOXM1 was inhibited by NCE treatment. Immunofluorescence results revealed lower levels of CD31, CD44 and vimentin in the NCE groups. Taken together, these data demonstrate that NCE from Bacillus subtilis natto improves survival and inhibits tumor growth in HCC mice.

Phenolic (free, conjugated and bound) and carotenoid extracts from microalgae Nannochloropsis sp. and Spirulina sp. were investigated regarding their potential to mitigate contamination by Fusarium complex fungal pathogens. Free phenolic acid extracts from both microalgae were the most efficient, promoting the lowest mycelial growth rates of 0.51 cm day - 1 (Spirulina sp.) and 0.78 cm day - 1 (Nannochloropsis sp.). An experiment involving natural free phenolic acid extracts and synthetic solutions was carried out based on the natural phenolic acid profile. The results revealed that the synthetic mixtures of phenolic acids from both microalgae were less efficient than the natural extracts at inhibiting fungal growth, indicating that no purification is required. The half-maximal effective concentration (EC 50 ) values of 49.6 μg mL - 1 and 33.9 μg mL - 1 were determined for the Nannochloropsis and Spirulina phenolic acid extracts, respectively. The use of phenolic extracts represents a new perspective regarding the application of compounds produced by marine biotechnology to prevent Fusarium species contamination.

Aging is a natural process that accompanied with progressive cognitive deficits and functional decline in organisms. Selenium (Se), an essential trace element, exhibits antioxidative and anti-inflammatory abilities. Here, our study aimed to investigate the protective effects of aqueous extracts of Se-enriched Auricularia auricular (AESAA) on aging mice induced by d-galactose (D-gal) and explore its potential mechanism. d-gal was administered (100 mg/kg) subcutaneously for 12 weeks to establish an aging mouse model. Morris water maze (MWM) test was conducted to assess the cognitive deficits of mice. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities and malondialdehyde (MDA) level in hippocampus were measured to evaluate oxidative stress. The contents of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in hippocampus were determined by ELISA method. Further, hippocampal levels of RAGE, p-Erk, p-JNK, p-P38 and p-NF-κB were detected by western blot and the RAGE expression was confirmed by immunohistochemistry. We found that AESAA supplementation significantly decreased d-gal-induced cognitive deficits, as evidenced by better performance in the MWM test. Furthermore, AESAA treatment attenuated oxidative stress and decreased the contents of pro-inflammatory cytokines in hippocampus. Importantly, AESAA inhibited the up-regulation of RAGE, p-Erk, p-JNK, p-P38 in the hippocampus of d-gal treated mice. Moreover, the results also indicated that AESAA inhibited p-NF-κB and p-IκBα expression. In conclusion, our findings suggest that AESAA effectively decreases cognitive impairment, alleviates oxidative damage and neuroinflammation in mice through s RAGE/MAPK/NF-κB signaling pathway, which provides a potential therapy for delaying the aging process., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Vibrio diabolicus A1SM3 strain was isolated from a sediment sample from Manaure Solar Saltern in La Guajira and the produced crude extracts have shown antibacterial activity against methicillin-resistant Staphylococcus aureus and cytotoxic activity against human lung cell line. Thus, the aim of this research was to identify the main compound responsible for the biological activity observed and to systematically study how each carbon and nitrogen source in the growth media, and variation of the salinity, affect its production. For the characterization of the bioactive metabolites, 15 fractions obtained from Vibrio diabolicus A1SM3 crude extract were analyzed by HPLC-MS/MS and their activity was established. The bioactive fractions were dereplicated with Antibase and Marinlit databases, which combined with nuclear magnetic resonance (NMR) spectra and fragmentation by MS/MS, led to the identification of 2,2-di(3-indolyl)-3-indolone (isotrisindoline), an indole-derivative antibiotic, previously isolated from marine organisms. The influence of the variations of the culture media in isotrisindoline production was established by molecular network and MZmine showing that the media containing starch and peptone at 7% NaCl was the best culture media to produce it. Also, polyhydroxybutyrates (PHB) identification was established by MS/MS mainly in casamino acids media, contributing to the first report on PHB production by this strain.

Over the past three decades, many studies employing hollow-fiber liquid-phase microextraction (HF-LPME) bioanalytical methods have been published. The basic mechanism of extraction relies on the migration of the analytes through a liquid membrane sustained in the pores of the walls of a porous hollow fiber, and from there into an acceptor phase present in the lumen of the fiber. The mass transfer occurs by passive diffusion and it can be enhanced by using a carrier or applying an electrical potential across the phases. This type of extraction method presents many advantages over classical techniques, such as high preconcentration factor, clean extracts, and a green chemistry approach. Due to its advantages, and considering that no study systematically compiled the characteristics of the published methods in one single accessible source of information, the aim of this systematic review is to assess the data regarding bioanalytical methods, compile, and analyse the studies published until up to October of 2017. The data source used for the systematic review were Pubmed, Web of Science, and Science Direct, and 171 studies were included in the final review by two independent reviewers, resulting in a reliable and accessible source of information about bioanalytical methods employing HF-LPME., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The endoplasmic reticulum (ER) consists of morphologically distinct domains, including a polygonal network of tubules that is connected by three-way junctions. This network is found in all eukaryotic cells. Extracts from Xenopus laevis eggs contain stockpiles of components that allow the assembly of an ER network in vitro. Here we provide protocols for assembly of ER networks in extracts that are arrested at different stages of the cell cycle. Unfertilized Xenopus laevis eggs contain a cytostatic factor (CSF) that keeps them in the metaphase stage of the cell cycle. Disruption of the eggs by low-speed centrifugation releases calcium and the eggs cycle into interphase. This state can then be maintained by the addition of cycloheximide, which prevents the synthesis of cyclin B. CSF extracts can be also prepared in the presence of a calcium chelator, thus keeping the extract in metaphase. In this protocol, we outline procedures for the assembly of an ER network using either interphase- or metaphase-arrested Xenopus egg extracts. The network assembled is strikingly similar to the network observed in tissue culture cells. The extract allows easy biochemical manipulation, permitting the effects of purified proteins or small molecules, or the depletion of cytosolic components to be tested., (© 2019 Cold Spring Harbor Laboratory Press.)

Chromosome structure in both interphase and M-phase cells is strongly influenced by the action of the cohesin and condensin protein complexes. The cohesin complex tethers the identical copies of each chromosome, called sister chromatids, together following DNA replication and promotes normal interphase chromosome structure and gene expression. In contrast, condensin is active largely in M phase and promotes the compaction of individual chromosomes. The Xenopus egg extract system is uniquely suited to analyze the functions of both complexes. Egg extracts, in which the cell cycle state can be manipulated, contain stockpiles of nuclear proteins (including condensin and cohesin) sufficient for the assembly of thousands of nuclei per microliter. Extract prepared from unfertilized eggs is arrested by the presence of cytostatic factor (CSF) in a state with high levels of M-phase kinase activity, but can be stimulated to enter interphase, in which DNA replication occurs spontaneously. For cohesion assays, demembranated sperm nuclei are incubated in interphase extract, where they undergo rapid and synchronous DNA replication and cohesion establishment through the recruitment of proteins and other factors (e.g., nucleotides) from the extract. Sister chromatid cohesion is assessed by then driving the extract into M phase by the addition of fresh CSF-arrested extract. In contrast, because chromosome condensation occurs spontaneously in M-phase extracts, sperm nuclei are added directly to CSF extracts to assay condensation., (© 2019 Cold Spring Harbor Laboratory Press.)

Spent mushroom compost (SMC) is a residue generated in edible mushrooms production, such as Hypsizygus marmoreus. Its genome was recently sequenced, demonstrating cuticle-degrading protease genes. The present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae. The extraction of the crude extract directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ± 18.38 U g -1 of compound. Moreover, the zymogram and SDS-PAGE indicated the presence of two proteases with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, there was a significant reduction in the number of Panagrellus redivivus and L 3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively. A0A151VWY3 mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Xenopus laevis development is marked by accelerated cell division solely supported by the proteins maternally deposited in the egg. Oocytes mature to eggs with concomitant transcriptional silencing. The unique maternal chromatin state contributing to this silencing and subsequent zygotic activation is likely established by histone posttranslational modifications and histone variants. Therefore, tools for understanding the nature and function of maternal and embryonic histones are essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Here we describe protocols for isolating pronuclear sperm chromatin from Xenopus egg extracts and hydroxyapatite-based histone purification from this chromatin. The histones purified through this method can be directly assembled into chromatin through in vitro assembly reactions, providing a unique opportunity to biochemically dissect the effect of histone variants, histone modifications, and other factors in chromatin replication and assembly. We also describe how to isolate chromatin from staged embryos and analyze the proteins to reveal dynamic developmental histone modifications. Finally, we present protocols to measure chromatin assembly in extracts, including supercoiling and micrococcal nuclease assays. Using these approaches, analysis of maternal and zygotic histone posttranslational modifications concomitant with cell-cycle and developmental transitions can be tested., (© 2019 Cold Spring Harbor Laboratory Press.)

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). This protocol can be used to prepare embryonic tissues or yolk sac samples for PCR analysis, but it is also useful for adult tissues (such as tail) that do not lyse completely by boiling in SDS/NaOH. Nonionic detergents are substituted for SDS because they interfere less with the polymerase reaction., (© 2019 Cold Spring Harbor Laboratory Press.)

In this study, we aimed to investigate precise mechanism(s) of sphingolipid-imbalance and resulting ceramide-accumulation in COPD-emphysema. Where, human and murine emphysema lung tissues or human bronchial epithelial cells (Beas2b) were used for experimental analysis. We found that lungs of smokers and COPD-subjects with increasing emphysema severity demonstrate sphingolipid-imbalance, resulting in significant ceramide-accumulation and increased ceramide/sphingosine ratio, as compared to non-emphysema/non-smoker controls. Next, we found a substantial increase in emphysema chronicity-related ceramide-accumulation in murine (C57BL/6) lungs, while sphingosine levels only slightly increased. In accordance, the expression of the acid ceramidase decreased after CS-exposure. Moreover, CS-induced (sub-chronic) ceramide-accumulation was significantly (p < 0.05) reduced by treatment with TFEB/autophagy-inducing drug, gemfibrozil (GEM), suggesting that autophagy regulates CS-induced ceramide-accumulation. Next, we validated experimentally that autophagy/lipophagy-induction using an anti-oxidant, cysteamine, significantly (p < 0.05) reduces CS-extract (CSE)-mediated intracellular-ceramide-accumulation in p62 + aggresome-bodies. In addition to intracellular-accumulation, we found that CSE also induces membrane-ceramide-accumulation by ROS-dependent acid-sphingomyelinase (ASM) activation and plasma-membrane translocation, which was significantly controlled (p < 0.05) by cysteamine (an anti-oxidant) and amitriptyline (AMT, an inhibitor of ASM). Cysteamine-mediated and CSE-induced membrane-ceramide regulation was nullified by CFTR-inhibitor-172, demonstrating that CFTR controls redox impaired-autophagy dependent membrane-ceramide accumulation. In summary, our data shows that CS-mediated autophagy/lipophagy-dysfunction results in intracellular-ceramide-accumulation, while acquired CFTR-dysfunction-induced ASM causes membrane ceramide-accumulation. Thus, CS-exposure alters the sphingolipid-rheostat leading to the increased membrane- and intracellular- ceramide-accumulation inducing COPD-emphysema pathogenesis that is alleviated by treatment with cysteamine, a potent anti-oxidant with CFTR/autophagy-augmenting properties., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of the present study was to screen for mosquito larvicidal activity of methanol extract of a wild mushroom Psathyrella candolleana against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi. Larvae and eggs were exposed to four different concentrations (62.5, 125, 250, and 500 ppm) and mortality was assessed 24 and 120 h after treatment. The LC50 and LC90 values of the extract were calculated. Methanol crude extract of P. candolleana showed good larvicidal activity against the third instar larvae of Cx. quinquefasciatus with LC50 and LC90 values of 166.713 and 259.17 ppm, respectively, after 24 h of exposure. No larvicidal activity was observed against Ae. aegypti and An. stephensi. The methanol extract of P. candolleana presented 88% ovicidal activity against Cx. quinquefasciatus at 500 ppm concentration 120 h after treatment. The active methanol extract was tested for toxicity against non-target organism and found was to have no toxicity. Histopathological studies of the treated larvae revealed serious damages to the midgut cells. The mushroom species was confirmed by 5.8S rRNA sequence and phylogenetic tree was constructed. These results suggest that the methanol extract of P. candolleana could be used in Cx. quinquefasciatus control programs as a new eco-friendly larvicide.

Acacia honey, as a favored delicacy, was supplemented with extracts of distinctive Ganoderma lucidum (lingzhi or reishi) variants to enhance its biological value and lay the foundations for prospective functional food application. Health promoting effects of G. lucidum have already been documented and its prebiotic features have been verified. In spite of promising studies and its unambiguous nutritional value, G. lucidum mushroom has not gained extensive use in the food industry so far. Special honey-based foodstuffs fortified with G. lucidum extract were developed as novel food prototypes, and screened for their enhanced total phenolic content (TPC), antioxidant (FRAP), and antiradical activity (DPPH). The obtained variants were also investigated for the possibly increased prebiotic index by means of an in vitro digestion procedure. A concentration dependent positive correlation was observed for all the studied parameters in case of the investigated prototypes. Supplementation of acacia honey with 0.5% reishi mushroom extract significantly increased the TPC, the antioxidant activity, and the radical scavenging power. A statistically significant improvement in the prebiotic index was also observed for the developed prototypes when compared with pure acacia honey. The evidence gained from the conducted studies fully supports the conception of combining honey and reishi extract to obtain a functional food product of high biological potential.

Wild mushrooms, while largely explored for their ecological significance, have not been systematically studied for their medicinal properties. This is the first report of biological activities of mushrooms from Haida Gwaii, British Columbia, Canada. The 17 mushroom species in this study were collected from multiple locations on Haida Gwaii and were screened for antiproliferative, immunostimulatory, and anti-inflammatory activities. Prior to screening, mushrooms were genetically identified and then sequentially fractionated into four crude extracts using 80% ethanol, 50% methanol, water, and 5% sodium hydroxide. We report here the strong antiproliferative and antiinflammatory activities of Amanita augusta, Phellodon atratus, Guepinia helvelloides, Chroogomphus tomentosus, Laetiporus conifericola, and Inocybe sp. In addition, A. augusta, G. helvelloides, and Inocybe sp. showed potent immunostimulatory activity. Two other species (Ganoderma tsugae and Pleurotus ostreatus) displayed strong immunostimulatory activity consistent with previous reports by others, suggesting that similar constituents are present in the same species from Haida Gwaii. For nine species (Russula paludosa, Hygrophoropsis aurantiaca, Tricholomopsis rutilans, Tyromyces chioneus, Hydnum repandum, Hypholoma fasciculare, Clavulina cinerea, P. ostreatus and Ramaria cystidiophora), we describe antiproliferative, immunostimulatory, and/or anti-inflammatory activities that have never been reported before. The new findings serve as a platform for future investigations into the potentially novel bioactive constituents of these mushrooms, as well as an incentive to further study a wider array of wild mushrooms for medicinal properties.

Recently, mushroom species have been the focus of researchers' interest because of several bioactivities. The aim of this study was to investigate the chemical profile and biological activities of various extracts of two Stereum species (S. rugosum and S. sanguinolentum). Antioxidant activity was tested using β-carotene-linoleic acid, DPPH scavenging, ABTS·+ scavenging, cupric-reducing antioxidant capacity (CUPRAC), and metal chelating assays. The extracts were also tested for their enzyme inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). HPLC-DAD was applied for the analysis of phenolic compounds, and fatty acid compositions were determined using GC and GC-MS. When fumaric acid and catechin hydrate were found as the most abundant phenolic compounds in both Stereum species, oleic acid and palmitic acid were identified as major fatty acids. Both of the studied Stereum methanol extracts were determined as the most active in β-carotene-linoleic acid, DPPR, ABTS·+, and CUPRAC assays; the n-hexane extracts were found to be most active in metal chelating and AChE inhibitory activity assays. In addition, the methanol extract of S. sanguinolentum (IC50: 34.26 ± 0.31 μg/mL) showed higher ABTS·+ scavenging activity than α-tocopherol (IC50: 38.51 ± 0.54 μg/mL). The acetone extracts were found as potent inhibitors against BChE. These results suggest that Stereum species could be an antioxidant source and cholinesterase agent in pharmaceutic, food, and cosmetics industries.

Role of Trichoderma species is well documented as antagonists as well as plant growth enhancers. Presently, the fungicidal potential of three Trichoderma species namely, T. koningii (FCBP769) , T. viride (FCBP904) , and T. harzianum (FCBP1277) was assessed against Alternaria alternata that causes leaf necrotic spots of Syzygium cumini and broad range of other plants using 0, 15, 30, 45, 60 and 75% dilutions of filtrates. There was a significant reduction of around 40-95%, 22-86% and 52-91% in fungal biomass by T. koningii, T. viride and T. harzianum, respectively. In fractionation bioassays, Trichoderma metabolites were partitioned using organic solvents viz., n-butanol, n-hexane, chloroform and ethyl acetate. Antifungal activity at different concentrations (10, 20, 30, 40 and 50 ppm) was assessed against the pathogen. Ethyl acetate fraction of T. koningii extract displayed the most promising activity resulting in 10-90% suppression in biomass. In case of T. viride butanol fraction proved the most effective in retarding the growth of pathogen from 20 to 80%. While T. harzianum extract revealed 55-85% arrest in fungal biomass due to n-hexane fraction. Present study concludes that test Trichoderma species demonstrated a strong fungicidal activity against A. alternata. Current research offers the possibility of developing strategies for controlling pathogens with bioactive metabolites of Trichoderma.

The chaga medicinal mushroom (Inonotus obliquus) was traditionally used to treat various ailments. To establish the pharmacological properties of I. obliquus, studies were performed to show the antiulcer activity of the ethanolic extract. The ethanolic extract of I. obliquus was prepared. The antiulcer activity of I. obliquus was determined using gastric ulcerated rats (ulceration induced by ethanol). The ethanolic extract of I. obliquus (200 mg/kg) did not cause any sign of toxicity or sensitivity to rats when the extracts were administered by oral feed. Oral administration of ethanolic extract of I. obliquus exhibited antiulcer activity in all models used. The ethanolic extract of I. obliquus showed an effective antiulcer activity, which could be due to the presence of various biologically active compounds. This confirmed the traditional uses of I. obliquus in the treatment of ailments.

The present study evaluates the antiarthritic effect of hydroethanolic extract of Pleurotus ostreatus cv. Florida, which was tested against adjuvant induced arthritis in rat models. Arthritis was induced by administration of complete Freund's adjuvant into the subplantar surface of left paw of rats. The extract was given orally at doses 200 mg/ kg and 400 mg/kg and piroxicam was administered intraperitonially (4 mg/kg). In vitro testing on parameters including antiproteinestrase, albumin denaturation and heat induce hemolysis was also carried out. There was significant decrease (p < 0.001) in proteinase activity and membrane stabilization in vivo studies on cv. Florida extract treated rats showed a significant (p < 0.001) decrease in paw volume, joint diameter, and spontaneous change in body weight recorded for 21 days. The treatment also resulted in an increase in rats' gripping activity compared with arthritic control rats. X-ray examinations showed a decrease in joint swelling. Histopathological examination of the extract treated group showed a significant decrease in joint space. There was also an increase in antibody levels. The antioxidant parameters showed a significant (p < 0.001) increase in superoxide dismutase and catalase enzymatic activities. Thus P. ostreatus cv. Florida extract demonstrates a potent antioxidant activity in a rat model. It is concluded that the P. ostreatus cv. Florida extract contains medicinally important constituents that show antiarthritic activity in rats.