Interactive Effects of Selenium, Zinc, and Iron on the Uptake of Selenium in Mycelia of the Culinary-Medicinal Winter Mushroom Flammulina velutipes (Agaricomycetes).

The present study for the first time addressed whether the simultaneous presence of selenium, zinc and iron may have effects on the selenium uptake in the mycelia of the winter mushroom (also known as enoki), Flammulina velutipes. Response surface methodology was used to optimize concentrations of selenium, zinc and iron in the range of 0 to 120 mg L-1. The findings showed that application of selenium, zinc and iron (singly, in pairs, or triads) significantly enhanced the selenium accumulation in the mycelia. The highest amount of the selenium accumulation was observed when selenium (60 mg L-1) and zinc (120 mg L-1) were applied into submerged culture media, concurrently, leading to an 85-fold and 88-fold increase in the selenium content of the mycelia compared to that of the mycelia treated with selenium only and untreated mycelia, respectively. In addition, accumulation of selenium into the mycelia had no deteriorative effects on the mycelial biomass. The findings presented in this study may have implications for daily nutrition and industrial bioproduction of mushroom mycelia enriched with selenium.

Antibacterial Activity of Culinary-Medicinal Polypore Mushroom Lentinus tigrinus (Agaricomycetes).

Medicinal mushrooms belonging to Lentinus spp. exhibit significant antibacterial activities, but little attention has been paid to their efficacy against the food-borne pathogen, Bacillus cereus. The present study for the first time quantitatively evaluated the antibacterial activity of different extracts from fruiting bodies of a well-authenticated Iranian native strain of medicinal mushroom, Lentinus tigrinus, against Gram-positive spore-forming bacterium B. cereus. The findings revealed that the acetone extract inhibited the growth of B. cereus at concentrations as low as 31.25 µg/ML, while it had no effect against Escherichia coli and Staphylococcus aureus even at 10,000 µg/ML. The rest of the bacteria were also susceptible to the acetone extract at concentrations greater than 5 mg/ML. Antibacterial activities of the methanol-ethyl acetate extract and the hot water extract were significantly weaker than that of the acetone extract, which contained high amounts of total phenols (5.83 ± 0.08 mg GAE/g, dw), while Fourier transform infrared spectroscopy (FT-IR) confirmed the presence of functional groups, such as hydroxyl, carbonyl, amide, and amine. Further studies by scanning electron microscopy (SEM) confirmed obvious changes in the morphology of B. cereus in response to the acetone extract of L. tigrinus. This study may suggest that L. tigrinus could be a good natural source for isolating and purifying antibacterial compounds against B. cereus.

Lentinan and ß-glucan extract from shiitake mushroom, Lentinula edodes, alleviate acute LPS-induced hematological changes in mice.

Objectives: Immunomodulatory activity of ß-glucans of shiitake mushroom (Lentinula edodes) has been known. We investigated whether ß-glucans from L. edodes would attenuate the acute effects of lipopolysaccharides (LPS) on peripheral hematological parameters in mice. Materials and Methods: An in-house ß-glucans extract (BG) prepared from fruiting bodies of shiitake mushroom L. edodes was chemically measured and characterized using spectrophotometry and HPLC. Male BALB/c mice directly inhaled aerosolized LPS of 3 mg/ml and were treated with BG or commercial ß-glucan (known as lentinan; LNT) (10 mg/kg bw) at 1 hr before or 6 hr after LPS inhalation. The blood samples were collected by cardiac puncture from euthanized mice at 16 hr post-treatment. Results: The results showed a significant reduction in levels of blood parameters, including red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), and platelets (PLT); and a significant increase in blood lymphocyte counts in LPS-treated mice as compared with the control mice (P≤0.05). Total white blood cells, neutrophils, and monocyte counts did not show any significant difference among the groups. Treatment of LPS-challenged mice with LNT or BG significantly increased the levels of RBC, HGB, HCT, and PLT; and reduced blood lymphocyte counts as compared with LPS-treated mice (P≤0.05). Conclusion: These findings suggest that ß-glucans from L. edodes might be effective in attenuating the effects of inhaled LPS on peripheral blood parameters. Thus, these findings might be useful in acute inflammatory diseases particularly pulmonary infectious diseases in which the hematological parameters would be affected.

Improvement and Characterization of Naturally Occurring Folate Compounds in Fruiting Bodies of Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

Edible mushrooms are among food sources containing natural folate compounds. However, little is known about how the content of folates in edible mushrooms may be improved. This study aimed to enrich Flammulina velutipes with higher levels of folates and to characterize patterns of the bioconversion of folates in the fruiting bodies. A convenient method was developed to treat a lignocellulosic substrate with synthetic folic acid. Folate compounds in the fruiting bodies grown in folic acid-treated substrates were evaluated relative to those in untreated substrates. Reverse phase high-performance liquid chromatography revealed different patterns of changes in levels of unsubstituted and substituted folates. While there was an approximately 15-fold and 8-fold increase in 5-formyl tetrahydrofolate and 5-methyl tetrahydrofolate, respectively, the most inconsistency was seen in the tetrahydrofolate content. There were also differences in the level of folate derivatives between commercial and native F. velutipes mushrooms. Since F. velutipes mushrooms can be consumed raw, the enriched mushrooms can be used as a dietary source to meet adult requirements for the daily uptake of natural folates.

The overview and perspectives of biosensors and Mycobacterium tuberculosis: A systematic review.

Tuberculosis (TB) is referred to as a "consumption" or phthisis, which has been a fatal human disease for thousands of years. Mycobacterium tuberculosis (M. tb) might have been responsible for the death of more humans than any other bacterial pathogens. Therefore, the rapid diagnosis of this bacterial infection plays a pivotal role in the timely and appropriate treatment of the patients, as well as the prevention of disease spread. More than 98% of TB cases are reported in developing countries, and due to the lack of well-equipped and specialized diagnostic laboratories, development of effective diagnostic methods based on biosensors is essential for this bacterium. In this review, original articles published in English were retrieved from multiple databases, such as PubMed, Scopus, Google Scholar, Science Direct, and Cochrane Library during January 2010-October 2019. In addition, the reference lists of the articles were also searched. Among 109 electronically searched citations, 42 articles met the inclusion criteria. The highest potential and wide usage of biosensors for the diagnosis of M. tb and its drug resistance belonged to DNA electrochemical biosensors (isoniazid and rifampin strains). Use of biosensors is expanding for the detection of resistant strains of anti-TB antibiotics with high sensitivity and accuracy, while the speed of these sensory methods is considered essential as well. Furthermore, the lowest limit of detection (0.9 fg/ml) from an electrochemical DNA biosensor was based on graphene-modified iron-oxide chitosan hybrid deposited on fluorine tin oxide for the MPT64 antigen target. According to the results, the most common methods used for M. tb detection include acid-fast staining, cultivation, and polymerase chain reaction (PCR). Although molecular techniques (e.g., PCR and real-time PCR) are rapid and sensitive, they require sophisticated laboratory and apparatuses, as well as skilled personnel and expertise in the commentary of the results. Biosensors are fast, valid, and cost-efficient diagnostic method, and the improvement of their quality is of paramount importance in resource-constrained settings.

Rapid and label-free electrochemical DNA biosensor based on a facile one-step electrochemical synthesis of rGO-PPy-(L-Cys)-AuNPs nanocomposite for the HTLV-1 oligonucleotide detection.

Human T cell leukemia virus type 1 (HTLV-1) as the first human retrovirus is currently a serious endemic health challenge. Despite the use of assorted molecular or serological assays for HTLV-1 detection, there are several limitations due to the lack of a confirmatory test that may affect the accuracy of the results. Herein, a novel label-free biosensor for the detection of HTLV-1 Tax gene has been reported. An electrochemical facile ecofriendly synthesis method has been demonstrated based on a synthesis of nanocomposite of reduced graphene oxide, polypyrrole, and gold nanoparticles (rGO-PPy-(l-Cys)-AuNPs) deposited on the surface of screen-printed carbon electrode. Electrochemical techniques were used to characterize and study the electrochemical behavior of the rGO-PPy-(l-Cys)-AuNPs, which exhibited a stable reference peak at 0.21 V associated with hybridization forms by applying the differential pulse voltammetry. The designed DNA biosensor presented a wide linear range from 0.1 fM to 100 µM and a low detection limit of 20 atto-molar. The proposed biosensor presented in this study provides outstanding selectivity, sensitivity, repeatability, and reproducibility.

Anticancer potentiality and mode of action of low-carbohydrate proteins and peptides from mushrooms.

Severe side effects of chemotherapy as well as drug resistance highlight the ongoing need to discover novel natural bioactive compounds with anticancer potentiality. Mushroom-derived proteins are among the naturally occurring compounds that have been the subject of a body of research on their potentiality in cancer therapy. The greatest attention in relevant review articles has been paid to well-known mushroom-derived glycoproteins such as lectins and protein-bound polysaccharide complexes such as polysaccharide-K (PSK) or krestin and polysaccharopeptide (PSP), which contain substantial amounts of carbohydrates (50-90%). These complex compounds exert their anticancer activity mainly by binding to cell membranes leading to extrinsic (death receptor) apoptosis or intrinsic (mitochondrial) apoptotic pathways. However, several other research studies have reported pure, well-characterized, proteins or peptides from mushrooms, which are carbohydrate-free or have very low amounts of carbohydrate. These proteins may fall into four categories including fungal immunomodulatory proteins, ubiquitin-like proteins, enzymes, and unclassified proteins. Well-defined chemical structure, elucidated full amino acid or N-terminal sequences, purity, and having some distinct and specific pathways compared to glycoproteins have made these low-carbohydrate proteins attractive for cancer research. The aim of this review was therefore to improve the current understanding of mushroom-derived low-carbohydrate proteins and to consolidate the existing knowledge of the most promising mushroom species from which low-carbohydrate proteins have been derived, characterized, and examined for their anticancer activity. In addition, molecular targets and mechanisms of action of these proteins have been discussed. Key points • Mushroom-derived low-carbohydrate proteins lack or have low carbohydrate. • Low-carbohydrate proteins show potent anticancer activities in vitro and in vivo. • There are specific pathways for low-carbohydrate proteins to inhibit cancer cells.

Toxicity and Nutritional Assessment of Extracts of Medicinal Tiger Sawgill Mushroom, Lentinus tigrinus (Agaricomycetes), a Newly Domesticated in Iran.

Information on the biosafety of tiger sawgill mushroom, Lentinus tigrinus, is limited and controversial. In the present study, the toxicity of a native L. tigrinus strain was evaluated in both cell cultures and mice. In addition to proximate analysis, the amino acid composition and the substrate-dependent uptake of elements were also evaluated. The mushroom (dry weight) had 7.53 ± 0.11% ash, 4.23 ± 0.23% lipid, 13.4 ± 0.04% fiber, 74.84 ± 0.38% total carbohydrate, and 4.16 ± 0.08 (mg GAEs/g) total phenol. Lysine was found at the highest amount among the L-amino acids determined in the L. tigrinus soluble protein. Comparison of the elemental profile of L. tigrinus with that of the substrate demonstrated a great capacity for absorption of K, P, S, and Mg elements, while the Na uptake was low. Neither the substrate nor the mushroom contained toxic elements beyond the standards of the joint FAO/WHO. The viability of normal human and mouse-derived cells was not influenced by the extract up to 250 µg/mL, where 70% of cancerous PC3 and MCF-7 cells were killed. Selectivity index analysis suggested that the L. tigrinus extract was selective only against MCF-7 cells. The extract also did not affect mice treated orally or via i.p. injection, while i.v. injection caused some mortality in mice with an LD50 of 47.19 mg/mL. In conclusion, L. tigrinus may be considered a source of macronutrients and micronutrients with a selective anticancer activity, while it is much less likely to have detrimental effects on humans at low serving levels.

Biosynthesis and antibiotic activity of silver nanoparticles using different sources: Glass industrial sewage-adapted Bacillus sp. and herbaceous Amaranthus sp.

Synergistic effects of metallic nanoparticles (NPs) with commonly used antibiotics have encouraged the exploration of novel biological entities, including bacteria and weed plants. The present study for the first time reports the capability of an extracellular fraction of Bacillus sp. isolated from effluents of a glass-manufacturing unit to biosynthesis silver nanoparticles (AgNPs) without hazardous materials. Besides, the biosynthesis of AgNPs using an aqueous extract of herbaceous weed plant (Amaranthus sp.), as a low-cost natural source, has been addressed in this study. Our findings confirmed the fabrication of microbial and plant-sourced AgNPs, being thoroughly characterized by UV-vis, transmission electron microscopy, X-ray diffraction, dynamic light scattering, energy dispersive X-ray spectroscopy, and zeta potential measurements. Further, biological activities of the plant- and bacterium-derived AgNPs were investigated against several pathogenic bacteria, in combination with streptomycin. The antibacterial effectiveness of the antibiotic coated with 400 µg/disk of AgNPs increased over 50% toward all the pathogenic bacteria. The data presented here demonstrate that both industrial wastewater-adapted Bacillus sp. and wild-growing Amaranthus sp. are efficient natural sources with excellent capabilities for creating biologically active AgNPs, which would be of considerable interest for circumventing bacterial resistance to current antibiotics.

The Use of Microalgae for Coupling Wastewater Treatment With CO2 Biofixation.

Production and emission of CO2 from different sources have caused significant changes in the climate, which is the major concern related to global warming. Among other CO2 removal approaches, microalgae can efficiently remove CO2 through the rapid production of algal biomass. In addition, microalgae have the potential to be used in wastewater treatment. Although, wastewater treatment and CO2 removal by microalgae have been studied separately for a long time, there is no detailed information available on combining both processes. In this review article, microalgae-based CO2 biofixation, various microalgae cultivation systems,¯ and microalgae-derived wastewater treatment are separately discussed, followed by the concept of integration of CO2 biofixation process and wastewater treatment. In each section, details of energy efficiency and differences across microalgae species are also given.

Agronomic and environmental factors affecting cultivation of the winter mushroom or Enokitake: achievements and prospects.

The global interest in production of the winter mushroom or Enokitake (Flammulina filiformis previously known as Flammulina velutipes) is increasing owing to its nutritional and medicinally important bioactive compounds along with a marketable texture and flavor. This review presents the state of knowledge on achievements in solid-state cultivation and submerged cultures of Enokitake and how they are influenced by environmental factors and agronomic characteristics. A wide range of basic lignocellulosic substrates and supplementations have been reviewed in order to formulate an efficient and locally available substrate. Domestication of wild types of Enokitake and its economic and research implications are also discussed. Besides, the influence of environmental and agronomic factors on production and efficacy of the most important biologically active metabolites of Enokitake in both solid-state cultivation and submerged cultures has been discussed. Some of shortcomings of studies reporting cultivation of Enokitake are described and their contribution to future prospects is also discussed in this review.

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region.

BACKGROUND: The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions. OBJECTIVES: The aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it. METHODS: Specific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequenced. RESULTS: Quantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture. CONCLUSIONS: Real-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines.

Antioxidant capacity of several Iranian, wild and cultivated strains of the button mushroom.

The white button mushroom, Agaricus bisporus, is the most commonly grown mushroom in Iran; however, there is a significant shortage of research on its antioxidant activity and other medicinal properties. The aim of this study was to evaluate antioxidant capacity of the methanolic extracts from four cultivated strains and four Internal Transcribed Spacer (ITS)-identified, Iranian wild isolates of A. bisporus. Evaluations were made for total phenols, flavonoids and anthocyanins, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Overall, results showed that all the wild isolates exhibited significantly lower DPPH-derived EC50, compared to the cultivated strains (p < 0.05). A relatively high relationship was observed between total phenols and flavonoids or anthocyanins (r(2) > 0.60). However, these constituents could not statistically differentiate the group of wild samples from the cultivated ones, and there was low correlation with the DPPH-derived EC50s (r(2) < 0.40). In conclusion, comparisons showed that wild isolate 4 and cultivated strains A15 and H1 had higher antioxidant capacity than the others (p < 0.05). This result identifies these mushrooms as good candidates for further investigation.

Antioxidant capacity of several Iranian, wild and cultivated strains of the button mushroom

The white button mushroom, Agaricus bisporus, is the most commonly grown mushroom in Iran; however, there is a significant shortage of research on its antioxidant activity and other medicinal properties. The aim of this study was to evaluate antioxidant capacity of the methanolic extracts from four cultivated strains and four Internal Transcribed Spacer (ITS)-identified, Iranian wild isolates of A. bisporus. Evaluations were made for total phenols, flavonoids and anthocyanins, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. Overall, results showed that all the wild isolates exhibited significantly lower DPPH-derived EC₅₀, compared to the cultivated strains (p < 0.05). A relatively high relationship was observed between total phenols and flavonoids or anthocyanins (r² > 0.60). However, these constituents could not statistically differentiate the group of wild samples from the cultivated ones, and there was low correlation with the DPPH-derived EC₅₀s (r² < 0.40). In conclusion, comparisons showed that wild isolate 4 and cultivated strains A15 and H1 had higher antioxidant capacity than the others (p < 0.05). This result identifies these mushrooms as good candidates for further investigation.

Development of antiviral agents toward enterovirus 71 infection.

Enterovirus 71 (EV71) infection remains a public health problem at a global level, particularly in the Asia-Pacific region. The infection normally manifests as hand-foot-mouth disease; however, it is capable of developing into potentially fatal neurological complications. There is currently no approved vaccine or antiviral substance available for the prevention or treatment of EV71 infection. This paper, thus, reviews efforts to develop or discover synthetic as well as naturally occurring compounds directed against EV71 infection. The recent achievements in cellular receptors of EV71 are also highlighted, and their contribution to the development of antiviral drugs against EV71 is discussed in this article.

Initial evidence on differences among Enterovirus 71, Coxsackievirus A16 and Coxsackievirus B4 in binding to cell surface heparan sulphate.

Cell surface heparan sulphate (HS) mediates infection for many viruses from diverse families. We demonstrated significant antiviral potencies for a number of HS mimetics against a cloned strain of Enterovirus 71 (EV71) in a previous study. Thus, the involvement of HS in mediating viral infection of isolates of human enteroviruses was investigated in Vero and human neural cells in the present work. In both cell lines, heparin and pentosan polysulphate significantly inhibited both infection and attachment of low passage clinical isolates of EV71 and Coxsackievirus A16 (CVA16) but showed no affect on Coxsackievirus B4 (CVB4) (p < 0.05). In addition, enzymatic removal of cell surface HS by heparinase I prevented binding of the clinical EV71 by nearly 50 % but failed to significantly inhibit CVA16 or CVB4 binding in Vero cells. Overall, the findings of this study provides evidence that whilst highly sulphated domains of HS serve as an essential attachment co-receptor for EV71, HS might be used as an alternative attachment receptor by the other member of Human Enterovirus group A, CVA16. In addition, HS may not mediate early infection in CVB4.

Global impact of heparin on gene expression profiles in neural cells infected by enterovirus 71.

OBJECTIVES: Heparan sulphate mimetics, particularly heparin (Hep), have previously been shown to considerably inhibit infection of enterovirus 71 (EV71) in Vero cells. Therefore, in this study, a genome-wide DNA microarray was performed to gain insight into the mechanism(s) of action of Hep against infection of a human neural cell line, SK-N-SH, with a clinical strain of EV71. METHODS: This study focused on a selection of EV71-induced genes whose expression profiling was exclusively affected by the antiviral activity of Hep. The selection procedure was performed through a statistical multi-level comparison with the following controls: negative control cells, compound control (cells treated with Hep only), virus control (cells treated with virus only) and treatment control (EV71-infected cells treated with Hep). RESULTS: Overall, of more than 30,000 genes studied, 14 well-known annotated genes were selected that may be targets for the antiviral activity of Hep against EV71 infection in neural cells. For most of these genes, Hep appeared to modulate the impact of EV71 infection on the expression pattern of the genes. CONCLUSIONS: The findings of this research may provide initial assistance in new directions for studies to design molecular drug targets against EV71 infection.

In vitro evaluation of the antiviral activity of heparan sulfate mimetic compounds against Enterovirus 71.

Enterovirus 71 (EV71) is the causative agent of hand foot and mouth disease (HFMD) and can also cause fatal neurological complications for which currently there is no vaccine or approved antiviral drug. Despite suggestions that heparan sulfate (HS)-like compounds are effective antivirals against various viruses, no research has been undertaken to examine their effects upon EV71. Therefore, this study aimed to investigate the in vitro anti-EV71 effect of HS mimetics (heparin, heparan sulfate, and pentosan polysulfate). The results revealed that all of the compounds exhibited significant antiviral actions (p<0.05) against EV71 at concentrations less than 250 µg/mL, compared to virus control and positive control, ribavirin. Among the compounds, heparin exhibited the most potent antiviral activity, as 7.81 µg/mL of it prevented the infection by more than 90% (p<0.05). Assays to reveal the mode of action revealed that all of the compounds were capable of exerting antiviral activity through hindrance of viral attachment to the cells. In addition, some of the compounds could inhibit viral replication when added to cells 1h before infection, but none significantly reduced viral penetration. Overall, this research revealed that HS mimetic compounds could inhibit EV71 infection, and that HS may be involved in EV71-host cell interactions, as the virus binding to the host cells was significantly hindered by the HS-like compounds but not by ribavirin. Thus, further investigations to discover the molecular mechanisms underlying the anti-EV71 action of HS-like compounds are warranted.

A colorimetric-based accurate method for the determination of enterovirus 71 titer.

The 50 % tissue culture infectious dose (TCID50) is still one of the most commonly used techniques for estimating virus titers. However, the traditional TCID50 assay is time consuming, susceptible to subjective errors and generates only quantal data. Here, we describe a colorimetric-based approach for the titration of Enterovirus 71 (EV71) using a modified method for making virus dilutions. In summary, the titration of EV71 using MTT or MTS staining with a modified virus dilution method decreased the time of the assay and eliminated the subjectivity of observational results, improving accuracy, reproducibility and reliability of virus titration, in comparison with the conventional TCID50 approach (p < 0.01). In addition, the results provided evidence that there was better correlation between a plaquing assay and our approach when compared to the traditional TCID50 approach. This increased accuracy also improved the ability to predict the number of virus plaque forming units present in a solution. These improvements could be of use for any virological experimentation, where a quick accurate titration of a virus capable of causing cell destruction is required or a sensible estimation of the number of viral plaques based on TCID50 of a virus is desired.