Production of pro-inflammatory mediators stimulated by exposure of poultry house workers to airborne dust particulates.

INTRODUCTION AND OBJECTIVE: Poultry house employees spend a significant part of their work shift being exposed to airborne particulate pollutants. The aim of this study was to assess their exposure at different stages of chicken production cycle, based on quantification of pro-inflammatory mediators (IL-1ß, IL-6, IL-8, and TNFα) in nasal lavage (NAL) samples. MATERIAL AND METHODS: The concentrations of airborne dust at 3 different stages of the production cycle (i.e. empty poultry house, with 7- and 42-day-old chickens) were stationary measured using Grimm spectrometer, as well as CIS and Button samplers. The dust collected by the latter 2 samplers was analyzed for endotoxin and (1→3)-ß-D-glucan content. NAL samples were collected from employees after their work shift to determine the pro-inflammatory mediator levels. RESULTS: The maximum particulate aerosol, endotoxin, and (1→3)-ß-D-glucan concentrations at workplaces reached the levels of 4.12 mg/m3, 45.21 ng/m3, and 56.54 ng/m3, respectively. The IL-1ß, IL-6, and IL-8 concentrations in NAL samples ranged between 0.62-18.12 pg/mL, <0.70-25.37 pg/mL, and <3.50-259.5 pg/mL, respectively. All TNFα levels were below 4 pg/mL. There were no significant differences between these cytokine concentrations in NAL samples collected at different stages of chicken breeding in either 'winter' or 'summer' seasons. CONCLUSIONS: Inhalation stimulation with poultry dust containing endotoxins and (1→3)-ß-D-glucans resulted in the production of pro-inflammatory mediators, which proves the course of immunological processes in the exposed employees that may lead to adverse effects. The use of nasal lavage fluid in the control of such exposure confirms that NAL analysis is a reliable laboratory tool for assessing the impact of poultry dust on exposed farm workers.

Occupational Exposure to ß-d-Glucans, Mould Allergens, Endotoxins and Cultivable Fungi in Pig Farms.

Airborne concentrations of organic dust on animal farms are known to be very high. This dust is partly composed of microorganisms such as bacteria, fungi and their components [endotoxins, (1→3)-ß-d-glucans, mould allergens, mycotoxins], recognised as being responsible for numerous health effects. Several cross-sectional studies have measured levels of airborne bacteria, fungi and endotoxins on pig farms. However, the temporal dynamics of organic dust's components throughout the year have rarely been assessed, and airborne concentrations of (1→3)-ß-d-glucans and mould allergens remain poorly understood in these work environments. This longitudinal, four-season study measured cultivable fungi, endotoxins, (1→3)-ß-d-glucans, Aspergillus versicolor (AveX), Aspergillus fumigatus (Asp f1) and Alternaria sp (Alt a1) allergens on 31 pig farms in Switzerland. Results showed that exposure to AveX occurred in all four seasons. Total mean airborne concentration of endotoxins were between 3 and 4 times higher than the Swiss recommended limit value of 1000 EU m-3 and mean airborne concentrations of fungi were between 30 and 50 times higher than the Swiss recommended limit value of 1000 cfu m-3. Finally, accumulations of faecal matter on floors, humidity and dusty pathways were associated with increased concentrations of (1→3)-ß-d-glucans. In conclusion, pig farmers require better information about biological occupational risks, and measures to improve air quality should be implemented, especially in winter.

Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Effects of orally administered Euglena gracilis and its reserve polysaccharide, paramylon, on gastric dysplasia in A4gnt knockout mice.

Euglena gracilis is widely utilized as food or supplement to promote human and animal health, as it contains rich nutrients. In this study, we administered spray-dried powder of E. gracilis and paramylon, ß-glucan stored in E. gracilis cells, to A4gnt knockout (KO) mice. A4gnt KO mice are a mutant mouse model that spontaneously develops gastric cancer through hyperplasia-dysplasia-adenocarcinoma sequence in the antrum of the stomach, and we observed the effects of E. gracilis and paramylon on the early involvements of A4gnt KO mice. Male and female 10-week-old A4gnt KO mice and their age-matched wildtype C57BL/6J mice were orally administered with 50 mg of E. gracilis or paramylon suspended in saline or saline as a control. After 3-week administration, animals were euthanatized and the stomach was examined histopathologically and immunohistochemically. Gene expression patterns of the stomach, which have been reported to be altered with A4gnt KO, and IgA concentration in small intestine were also analyzed with real-time PCR and ELISA, respectively. Administration of Euglena significantly reduced the number of stimulated CD3-positive T-lymphocytes in pyloric mucosa of A4gnt KO mice and tend to reduce polymorphonuclear leukocytes infiltration. Euglena administration further downregulated the expression of Il11 and Cxcl1 of A4gnt KO mice. Euglena administration also affected IgA concentration in small intestinal contents of A4gnt KO mice. Paramylon administration reduced the number of CD3-positive lymphocytes in pyloric mucosa of A4gnt KO mice, and downregulated the expressions of Il11 and Ccl2 of A4gnt KO mice. Although we found no significant effects on gross and microscopic signs of gastric dysplasia and cell proliferation, the present study suggests that the administration of Euglena and paramylon may ameliorate the early involvements of A4gnt mice through the effects on inflammatory reactions in the gastric mucosa. The cancer-preventing effects should be studied with long-term experiments until actual gastric cancer formation.

Characterization of inhalable endotoxin, glucan, and dust exposures in Iowa farmers.

BACKGROUND: The observed deficit of lung cancer in farmers has been partly attributed to exposure to organic dusts and endotoxins based largely on surrogate metrics. To move beyond these surrogates for etiological studies, we characterized task-based and time-weighted average (TWA) exposure to inhalable endotoxin, (1 â†’ 3)-ß-D-glucan, and dust in Iowa farmers. METHODS: We collected 320 personal inhalable dust samples from 32 farmers during 69 sample days in 2015 and 2016. Samples were collected using Button aerosol samplers and analyzed for endotoxin using a kinetic chromogenic amebocyte lysate assay, and for (1 â†’ 3)-ß-D-glucan using a Limulus endpoint assay. We assessed relationships between bioaerosol concentrations and selected tasks and farm characteristics using linear mixed-effects models. RESULTS: Bedding work, hog handling, and working in barn/confinement buildings, grain bins, and grain elevators were associated with higher endotoxin exposure. We found a monotonic trend between higher endotoxin concentrations and increasing number of animals. Bedding work, cleaning, and feed/grain storage work were associated with higher (1 â†’ 3)-ß-D-glucan concentrations. The median concentrations by task spanned one order of magnitude for inhalable dust and two orders of magnitude for endotoxin and (1 â†’ 3)-ß-D-glucan. Pearson correlations between endotoxin and glucan concentrations were 0.22 for TWA exposure and 0.56 for task samples. CONCLUSIONS: This characterization of exposure factors that influence bioaerosol concentrations can support the development of refined bioaerosol exposure metrics for future etiologic analyses of cancer and other health outcomes in farmers.

Linkage and sequence analysis of neutral oligosaccharides by negative-ion MALDI tandem mass spectrometry with laser-induced dissociation.

Mass spectrometry (MS) has become the primary method for high-sensitivity structural determination of oligosaccharides. Fragmentation in the negative-ion MS can provide a wealth of structural information and these can be used for sequence determination. However, although negative-ion MS of neutral oligosaccharide using the deprotonated molecule [M-H]- as the precursor has been very successful for electrospray ionization (ESI), it has only limited success for matrix-assisted laser desorption/ionization (MALDI). In the present study, the features of negative-ion MALDI primary spectra were investigated in detail and the product-ion spectra using [M-H]- and [M+Cl]- as the precursors were carefully compared. The formation of [M-H]- was the main difficulty for MALDI while [M+Cl]- was proved to be useful as alternative precursor anion for MALDI-MS/MS to produce similar fragmentation for sequencing of neutral oligosaccharides. N-(1-naphthyl)ethylenediamine dihydrochloride was then used as both the matrix and the Cl- dopant to evaluate the extent of structural information that can be obtained by negative-ion fragmentation from [M+Cl]- using laser-induced dissociation (LID)-MS/MS for linkage assignment of gluco-oligosaccharides and for typing of blood-group ABO(H) and Lewis antigens on either type 1 or type 2 backbone-chains.

[Lafora disease: a review of the literature]. / Enfermedad de Lafora: revision de la bibliografia.

INTRODUCTION: Lafora disease is autosomal recessive progressive myoclonus epilepsy with late childhood-to teenage-onset caused by loss-of-function mutations in either EPM2A or EPM2B genes encoding laforin or malin, respectively. DEVELOPMENT: The main symptoms of Lafora disease, which worsen progressively, are: myoclonus, occipital seizures, generalized tonic-clonic seizures, cognitive decline, neuropsychiatric syptoms and ataxia with a fatal outcome. Pathologically, Lafora disease is characterized by the presence of polyglucosans deposits (named Lafora bodies), in the brain, liver, muscle and sweat glands. Diagnosis of Lafora disease is made through clinical, electrophysiological, histological and genetic findings. Currently, there is no treatment to cure or prevent the development of the disease. Traditionally, antiepileptic drugs are used for the management of myoclonus and seizures. However, patients become drug-resistant after the initial stage. CONCLUSIONS: Lafora disease is a rare pathology that has serious consequences for patients and their caregivers despite its low prevalence. Therefore, continuing research in order to clarify the underlying mechanisms and hopefully developing new palliative and curative treatments for the disease is necessary.

TITLE: Enfermedad de Lafora: revision de la bibliografia.Introduccion. La enfermedad de Lafora es una forma de epilepsia mioclonica progresiva de herencia autosomica recesiva, de inicio en la infancia tardia o en la adolescencia, y producida por mutaciones de perdida de funcion en los genes EPM2A o EPM2B, los cuales codifican para las proteinas laforina y malina, respectivamente. Desarrollo. Los principales sintomas de la enfermedad, que empeoran progresivamente, son mioclonias, crisis occipitales, crisis tonicoclonicas generalizadas, deterioro cognitivo, sintomas neuropsiquiatricos y ataxia. El curso es progresivo y fatal. Patologicamente, se caracteriza por la presencia de depositos de poliglucosanos (denominados cuerpos de Lafora) en el cerebro, el higado, el musculo y las glandulas sudoriparas. El diagnostico de enfermedad de Lafora se realiza mediante hallazgos clinicos, electrofisiologicos, histologicos y geneticos. En la actualidad no existe un tratamiento que erradique o prevenga su desarrollo. Tradicionalmente, se utilizan farmacos antiepilepticos para el tratamiento de las mioclonias y las convulsiones, aunque aparecen resistencias a estas. Conclusiones. La enfermedad de Lafora es una patologia rara que, pese a su baja prevalencia, supone graves consecuencias para los pacientes y sus cuidadores. Asi pues, resulta necesario continuar la investigacion para clarificar los mecanismos subyacentes y desarrollar nuevos tratamientos paliativos y curativos de la enfermedad.

Diagnostic Accuracy of ß-d-Glucan (Fungitell) Testing Among Patients With Hematologic Malignancies or Solid Organ Tumors: A Systematic Review and Meta-Analysis.

Objectives: To determine the accuracy of Fungitell, a ß-d-glucan (BDG) test, for the diagnosis of invasive fungal infection (IFI) among cancer patients. Methods: For this meta-analysis, MEDLINE and EMBASE were searched for references related to BDG testing. Study quality was evaluated using QUADAS-2. Statistical analysis was performed using Stata 14. Results: We screened 12,426 references and identified 189 studies for full-text review. Nineteen studies were included in the final meta-analysis. There was moderate heterogeneity between studies. Nine studies had a high risk of bias, which significantly elevated the overall specificity estimate. Restricting to only low-bias studies, the sensitivity and specificity were 80% and 63%, respectively. Conclusions: The overall sensitivity and specificity of Fungitell as a diagnostic test for IFI is moderate, and there is substantial heterogeneity between studies. Limiting studies to only low-bias risk reduced heterogeneity but also lowered the overall specificity estimate.

The contribution of cell wall composition in the expansion of Camellia sinensis seedlings roots in response to aluminum.

MAIN CONCLUSION: Treatment with aluminum triggers a unique response in tea seedlings resulting in biochemical modification of the cell wall, regulation of the activity of the loosening agents, and elongation of root. Unlike most terrestrial plants, tea (Camellia sinensis L.) responds to aluminum (Al) through the promotion of its root elongation; but the real mechanism(s) behind this phenomenon is not well understood. A plausible relationship between the modifications of the cell wall and the promotion of root elongation was examined in tea seedlings treated for 8 days with 400 µM Al. The mechanical properties of the cell wall, the composition of its polysaccharides and their capacity to absorb Al, the expression of genes, and the activities of the wall-modifying proteins were studied. With 6 h of the treatment, about 40% of the absorbed Al was bound to the cell wall; however, the amount did not increase thereafter. Meanwhile, the activity of pectin methylesterase, the level of pectin demethylation, the amounts and the average molecular mass of xyloglucan in the root apices significantly decreased upon exposure to Al, resulting in the reduction of Al binding sites. On the other hand, the activity and the gene expression of peroxidase decreased, whereas the activity and gene expression of xyloglucan-degrading enzymes, the expression of expansin A and the H +-ATPase4 genes increased in the Al-treated plants. Interestingly, it was accompanied by the increase of elastic and viscous extensibility of the root apices. From the results, it can be suggested that the biochemical modification of the cell walls reduces sites of Al binding to roots and triggers the activity of the loosening agents, thereby increasing the length of tea roots.

Occupational exposure and respiratory health problems among nutmeg production workers in Grenada, the Caribbean.

BACKGROUND: The nutmeg industry is a major contributor to the Grenadian economy. However, workers in the industry face many environmental and occupational health risks. OBJECTIVES: The goal of this study was to investigate respiratory health problems and possible related occupational exposures among nutmeg production workers. METHODS: A questionnaire, spirometry and allergen skin-prick test was given to 92 nutmeg workers. Samples for measurement of airborne dust, mold, and phosphine were also collected from work environments. RESULTS: Approximately half of the workers with lower respiratory symptoms such as dry cough (49.4%) and shortness of breath (42.9%) reported that their symptoms were work related. Spirometry results showed that 18.8% of workers had obstruction. Area geometric mean (GM) inhalable dust concentrations, GM personal concentrations, and total mold spores during work activities were found to be high. CONCLUSIONS: High prevalence of respiratory symptoms among workers in this facility was consistent with measured levels of dust and mold, and was widespread over all work areas.

Assessment of multi-contaminant exposure in a cancer treatment center: a 2-year monitoring of molds, mycotoxins, endotoxins, and glucans in bioaerosols.

Indoor air quality in health care facilities is a major public health concern, particularly for immunocompromised patients who may be exposed to microbiological contaminants such as molds, mycotoxins, endotoxins, and (1,3)-ß-D-glucans. Over 2 years, bioaerosols were collected on a monthly basis in a cancer treatment center (Centre F. Baclesse, Normandy, France), characterized from areas where there was no any particular air treatment. Results showed the complexity of mycoflora in bioaerosols with more than 100 fungal species identified. A list of major strains in hospital environments could be put forward due to the frequency, the concentration level, and/or the capacity to produce mycotoxins in vitro: Aspergillus fumigatus, Aspergillus melleus, Aspergillus niger, Aspergillus versicolor, Cladosporium herbarum, Purpureocillium lilacinum, and Penicillium brevicompactum. The mean levels of viable airborne fungal particles were less than 30.530 CFU per m3 of air and were correlated to the total number of 0.30 to 20 µm particles. Seasonal variations were observed with fungal particle peaks during the summer and autumn. Statistical analysis showed that airborne fungal particle levels depended on the relative humidity level which could be a useful indicator of fungal contamination. Finally, the exposure to airborne mycotoxins was very low (only 3 positive samples), and no mutagenic activity was found in bioaerosols. Nevertheless, some fungal strains such as Aspergillus versicolor or Penicillium brevicompactum showed toxigenic potential in vitro.

Effect of different cooking methods on nutritional value and antioxidant activity of cultivated mushrooms.

Influence of culinary treatments (boiling, microwaving, grilling, and deep frying) on proximate composition and antioxidant capacity of cultivated mushrooms (Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, and Pleurotus eryngii) was studied. Proximate composition was affected by the cooking method and the mushrooms species. Frying induced more severe losses in protein, ash, and carbohydrates content but increased the fat and energy. Boiling improved the total glucans content by enhancing the ß-glucans fraction. A significant decrease was detected in the antioxidant activity especially after boiling and frying, while grilled and microwaved mushrooms reached higher values of antioxidant activity. Maillard reaction products could be partially responsible, as supported by the absorbance values measured at 420 nm. Since cooking techniques clearly influence the nutritional attributes of mushrooms, the proper selection of treatments is a key factor to prevent/reduce nutritional losses. Microwaving and grilling were established as the best processes to maintain the nutritional profile of mushrooms.

Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis.

Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, ß-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and ß-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future.

Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response.

The aim of the study was to characterize exopolysaccharides (EPS) originated from Lactobacillus reuteri strain DSM 17938 (EPS-DSM17938) and L. reuteri strain L26 Biocenol™ (EPS-L26) and evaluate their influence on adherence of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells and proinflammatory gene expression. Both EPS were d-glucan polysaccharides with higher molecular weight (Mw), but differing in spatial conformation and elicited variable cytokine profile. EPS-DSM17938, relatively linear polysaccharide with (1→4) and (1→6) glycosidic linkages, increased IL-1ß gene expression (0.1mg/mL; P<0.05), while EPS-L26, more branched polysaccharide with (1→3) and (1→6) glycosidic linkages, exerted slight but statistically significant up-regulation of NF-κB, TNF-α and IL-6 mRNA (P<0.05). The most significant finding is that preincubation of IPEC-1 cells with both EPS followed by ETEC infection inhibit ETEC adhesion on IPEC-1 cells (P<0.01) and ETEC-induced gene expression of proinflammatory cytokine IL-1ß and IL-6 (P<0.01).

Water-Soluble Compounds from Lentinula edodes Influencing the HMG-CoA Reductase Activity and the Expression of Genes Involved in the Cholesterol Metabolism.

A water extract from Lentinula edodes (LWE) showed HMG-CoA reductase inhibitory activity but contained no statins. NMR indicated the presence of water-soluble α- and ß-glucans and fucomannogalactans. Fractions containing derivatives of these polysaccharides with molecular weight down to approximately 1 kDa still retained their inhibitory activity. Once digested LWE was applied to Caco2 in transport experiments, no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the Caco2 monolayer was applied to HepG2, some genes were modulated (after 24 h). LWE was also administrated to normo- and hypercholesterolemic mice, and no significant lowering of serum cholesterol levels was observed; but reduction of triglycerides in liver was observed. However, LWE supplementation modulated the transcriptional profile of some genes involved in the cholesterol metabolism similarly to simvastatin, suggesting that it could hold potential as a hypolipidemic/hypocholesterolemic extract, although further dose-dependent studies should be carried out.

Physiological and molecular analysis on root growth associated with the tolerance to aluminum and drought individual and combined in Tibetan wild and cultivated barley.

MAIN CONCLUSION: The drought-stimulated gene expression of NCED, SUS, and KS - DHN and ABA signal cross-talk with other phytohormones maintains barley root growth under drought stress at pH 4.0 plus polyethylene glycol plus aluminum. Aluminum (Al) toxicity and drought are two major factors that limit barley production. In this work, the individual and combined effects of Al/acid and polyethylene glycol (PEG 6000) induced drought stress that suppressed root growth and caused oxidative damage as characterized by increased H2O2 and O2(.-) accumulation. The wild-barley genotypes, XZ5 and XZ29, exhibited a higher tolerance than the two cultivars Dayton (Al tolerant) and Tadmor (drought tolerant) under combined stress (pH 4.0 + PEG + Al). The oxidative damage induced by PEG was more severe at pH 4.0 than at pH 6.0. In XZ29, the highest root secretion of malate and citrate was recorded, and the least Al uptake in the four genotypes. In XZ5, a peak accumulation of ABA and minor synthesis of zeatin riboside and ethylene were found being essential in maintaining primary root elongation and root hair development. PEG-induced drought stress repressed Al uptake in root tips, with a lower increase in callose formation and HvMATE (Hordeum vulgare multidrug and toxic compound exudation) expression compared to Al-induced callose production. Stress by pH 4.0 + PEG + Al up-regulated 9-cis-epoxycarotenoid dioxygenase (NCED) which is involved in ABA biosynthesis. Such treatment stimulated the regulation of ABA-dependent genes sucrose synthase (SUS) and KS-type dehydrin (KS-DHN) in root tips. Our results suggest that the tolerance ranking to pH 4.0 + PEG + Al stress in Tibetan wild barley by gene expression is closely correlated to physiological indices. The results show that acclimatisation to pH 4.0 + PEG + Al stress involves specific responses in XZ5 and XZ29. The present study provides insights into the effects of Al/acid and drought combined stress on the abundance of physiological indices in the roots of barley varieties.

Nutritional composition, antioxidant properties, and toxicology evaluation of the sclerotium of Tiger Milk Mushroom Lignosus tigris cultivar E.

The Tiger Milk Mushroom (Lignosus spp.) is an important medicinal mushroom in Southeast Asia and has been consumed frequently by the natives as a cure for a variety of illnesses. In this study, we hypothesized that Lignosus tigris (cultivar E) sclerotium may contain high nutritional value and antioxidant properties, is nontoxic and a potential candidate as a dietary supplement. The chemical and amino acid compositions of the sclerotium were evaluated and antioxidant activities of the sclerotial extracts were assessed using ferric reducing antioxidant power; 1,1-diphenyl-2-picrylhydrazyl; and superoxide anion radical scavenging assays. Acute toxicity of the L. tigris E sclerotium was assessed using a rat model study. The sclerotium was found to be rich in carbohydrate, protein, and dietary fibers with small amounts of fat, calories, and sugar. The amino acid composition of the protein contains all essential amino acids, with a protein score of 47. The sclerotial extracts contain phenolics, terpenoids, and glucan. The ferric reducing antioxidant power values of the various sclerotial extracts (hot water, cold water, and methanol) ranged from 0.008 to 0.015 mmol min(-1) g(-1) extract, while the 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radical scavenging activities ranged from 0.11 to 0.13, and -2.81 to 9.613 mmol Trolox equivalents g(-1) extract, respectively. Acute toxicity assessment indicated that L. tigris E sclerotial powder was not toxic at the dose of 2000 mg kg(-1). In conclusion, L. tigris E sclerotium has the potential to be developed into a functional food and nutraceutical.

Hericium erinaceus (Bull.: Fr.) Pers., a medicinal mushroom, activates peripheral nerve regeneration.

OBJECTIVE: To study the ability of aqueous extract of Hericium erinaceus mushroom in the treatment of nerve injury following peroneal nerve crush in Sprague-Dawley rats. METHODS: Aqueous extract of Hericium erinaceus was given by daily oral administration following peroneal nerve crush injury in Sprague-Dawley rats. The expression of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways; and c-Jun and c-Fos genes were studied in dorsal root ganglia (DRG) whereas the activity of protein synthesis was assessed in peroneal nerves by immunohistochemical method. RESULTS: Peripheral nerve injury leads to changes at the axonal site of injury and remotely located DRG containing cell bodies of sensory afferent neurons. Immunofluorescence studies showed that DRG neurons ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for Akt, MAPK, c-Jun and c-Fos as compared with negative control group (P <0.05). The intensity of nuclear ribonucleoprotein in the distal segments of crushed nerves of treated groups was significantly higher than in the negative control group (P <0.05). CONCLUSION: H. erinaceus is capable of promoting peripheral nerve regeneration after injury. Potential signaling pathways include Akt, MAPK, c-Jun, and c-Fos, and protein synthesis have been shown to be involved in its action.

Imaging callose at plasmodesmata using aniline blue: quantitative confocal microscopy.

Callose (ß-1,3-glucan) is both structural and functional component of plasmodesmata (Pd). The turnover of callose at Pd controls the cell-to-cell diffusion rate of molecules through Pd. An accurate assessment of changes in levels of Pd-associated callose has become a first-choice experimental approach in the research of intercellular communication in plants.Here we describe a detailed and easy-to-perform procedure for imaging and quantification of Pd-associated callose using fixed plant tissue stained with aniline blue. We also introduce an automated image analysis protocol for non-biased quantification of callose levels at Pd from fluorescence images using ImageJ. Two experimental examples of Pd-callose quantification using the automated method are provided as well.