The protoplast two-way fusions and fusant characteristics of Antrodia cinnamomea and Cordyceps militaris

This study generated two fused protoplasts of Antrodia cinnamomea and Cordyceps militaris in two ways.The protoplasts of A.cinnamomea were inactivated by heat to inactivate biochemical processes and enzymatic activities in the cytoplasm,and the protoplasts of C.militaris were inactivated by UV radiation to invalidate their genome function,then they were fused under optimal conditions to get a fusion rate as(7.42±0.8)×10^(-6) fusants/mL;the new fusants were abbreviated as Ac-Cm.On the other hand,when A.cinnamomea and C.militaris were treated with heat and UV oppositely using similar experiments,the fusion rate was(9.70±0.68)×10^(-5) fusants/mL,and the new fusants were abbreviated as Cm-Ac.We selected each of two best-growing fused colonies Ac-Cm-1,Ac-Cm-2,Cm-Ac-1,and Cm-Ac-2,together with parental A.cinnamomea and C.militaris,and studied their morphology,growth antagonism tests,and genetic relationships by 18 S rRNA sequencing.In comparison with the initial cultures of 4 fusants,the yields of adenosine,biomass,cordycepic acid,cordycepin,total polysaccharide,and total triterpenoids were increased up 1.305-50.1563 times in the optimal medium conditions.For gene stability tests,those of the four fusants and their outputs were stabilized within 10 generations.

Research and development of Cordyceps in Taiwan

Cordyceps is treasured entomopathogenic fungi that have been used as antitumor,immunomodulating,antioxidant,and pro-sexual agent.Cordyceps,also called DongChongXiaCao in Chinese,Yartsa Gunbu(Tibetan),means winter worm-summer grass.Natural Cordyceps sinensis with parasitic hosts is difficult to be collected and the recent findings on its potential pharmacological functions,resulted in skyrocketing prices.Therefore,finding a mass-production method or an alternative for C.sinensis products is a top-priority task.In this review,we describe current status of Cordyceps research and its recent developments in Taiwan.The content and pharmacological activities of four major industrial species of Cordyceps(C.sinensis,Cordyceps militaris,Cordyceps cicadae and Cordyceps sobolifera)used in Taiwan,were reviewed.Moreover,we highlighted the effect of using different methods of fermentation and production on the morphology and chemical content of Cordyceps sp.Finally,we summarized the bottle-necks and challenges facing Cordyceps research as well as we proposed future road map for Cordyceps industry in Taiwan.

In silico assessment of human health risks caused by cyanotoxins from cyanobacteria

Harmful algal blooms(HABs)that are formed by cyanobacteria have become a serious issue worldwide in recent years.Cyanobacteria can release a type of secondary metabolites called cyanotoxins into aquatic systems which may indirectly or directly provide health risks to the environment and humans.Cyanotoxins provide some of the most powerful natural poisons including potent neurotoxins,hepatotoxins,cytotoxins,and endotoxins that may result in environmental health risks,and long-term morbidity and mortality to animals and humans.In this research,we used the chemcomputational tool Molinspiration for molecular property predictions,Pred-hERG 4.2 web software for cardiac toxicity prediction,and Pred-Skin 2.0 web software for predicting skin sensitization.We are predicting some toxicological aspects of cyanobacteria here using chemcomputational tools with the hypothesis that cyanotoxins are providing a risk to human health.We are using the tool Pred-hERG 4.2 to predict hERG channel blocking potential and the Pred-skin tool to predict skin sensitization due to cyanotoxins.The potential of anatoxin,ambigol,the microcystin group,and lyngbyatoxin A,lyngbyatoxin B,nodularin-R,and saxitoxin were predicted to cause skin sensitization in the final results(consensus model).Anatoxin-a and lyngbyatoxin were predicted to allow GI absorption and blood–brain barrier penetration.Among the 20 predicted cyanotoxins only aeruginosin 103-A,ambigol A,and ambigol were predicted by Pred-hERG 4.2 according to the applicability domain results as potential cardiotoxins with weak or moderate potency.Lyngbyatoxin shows activity through the GPCR ligand and protease,kinase,and enzyme inhibitor.

Evaluation of the Anti-Inflammatory Activities of 5,8,11-<i>cis</i>-Eicosatrienoic Acid

The main eicosanoids inflammatory mediators, prostaglandins and leukotrienes, are both generated from arachidonic acid (AA;20:4 n-6). AA is a member of polyunsaturated fatty acids (PUFAs). Numerous studies have demonstrated that various contents of PUFAs can modulate the inflammatory responses. However, fewer studies have examined n-9PUFAs and their effects on the inflammatory responses. In the present study, the role of 5,8,11-cis-eicosatrienoic acid (ETrA;20:3 n-9, also called Mead acid) in the inflammatory responses has been investigated. The anti-inflammatory activities of ETrA were examined using an in vitro macrophage system and the inhibitory effect was confirmed by western blot analysis for iNOS and COX-2 expressions. The interactions between ETrA and COX-2 protein were simulated to produce a computer modeling protein-ligand complexes and the results suggest a possible mechanism for the effects of ETrA. In this study, we described a significant inhibition of the inflammatory activities initiated by ETrA. Since ETrA is a substance presented in the tissues of young animals, we therefore anticipate that ETrA can be utilized as a natural therapeutic supplement to inhibit inflammatory activities.

Exploring the Environmental Physiology of the Indo-Pacific Reef Coral <em>Seriatopora hystrix</em>with Differential Proteomics

Although reef-building corals are threatened by a number of anthropogenic impacts, certain scleractinian-dinoflagellate (genus Symbiodinium) endosymbioses have proven markedly resilient to environmental change. For instance, corals from upwelling habitats of Southern Taiwan withstand both short- and long-term increases in temperature, potentially due to their routine exposure to highly variable temperature regimes in situ. To gain a greater understanding of the proteomic basis for such acclimatization to unstable environmental conditions, specimens of the Indo-Pacific reef-building coral Seriatopora hystrix Dana 1846 were sampled during a period of stable temperature conditions from 1) a site characterized by frequent upwelling events in Southern Taiwan and 2) a nearby, non-upwelling control site in the Taiwan Strait. Two-dimensional gel electrophoresis followed by sequencing of differentially concentrated proteins with mass spectrometry unveiled significantly more proteins involved in the cellular stress response in coral hosts of the upwelling site. Although such stress protein signatures could be indicative of sub-lethal levels of cellular stress, especially given the relatively higher sediment loads characteristic of the upwelling site, these proteins may, in contrast, have been constitutively maintained at high levels in preparation for large fluctuations in temperature and other abiotic parameters (e.g., nutrient levels) brought upon by upwelling events.

A synthetic biological secondary metabolite,Lycogen^TM,produced and extracted from Rhodobacter sphaeroides WL-APD911 in an optimizatioal scale-up strategy

The optimization of fermentation medium is important for synthetic biological secondary metabolite productions.The effect of rotation speed,inoculum amount,and medium supplements on the cell growth and Lycogen^TM secretion of photobacterium Rhodobacter sphaeroides WL-APD911 was evaluated.The results reveal that a higher rotational speed exhibit a higher cell density,and the increasing in the amount of inoculum amount show a slight augment on the growth of R.sphaeroides WL-APD911.In the case of nitrogen sources adding,Lycogen^TM production was achieved with a 0.5 mM l-lysine supplementation.Moreover,the attention of Tween 80 presented a tremendous increase in the secondary metabolite.Response surface methodology(RSM)exhibited the optimization of medium supplements for Lycogen^TM invention is accomplished at molasses concentration of 10 g/L,yeast extract concentration of 40 g/L,0.3%Tween 80 and NaCl concentration of 5 g/L,respectively.Further,the batch fermentation is carried out in both 5 L and 20 L fermentors to study the scale-up process factors to be adopted.At a 20 L fermentor,Lycogen^TM yields under the optimal culture condition are over 2 times than in the shake flask.The present results provide the Lycogen^TM optimal culture mediums,scale-up procedures and efficient extractions from R.sphaeroides WL-APD911.

Therapeutic effect of Guijiajiao (Colla Carapacis et Plastri) on bone regeneration in rats and zebrafish

OBJECTIVE:To investigate the effect of Guijiajiao(Colla Carapacis et Plastri, CCP), the glue of tortoise shell, on bone regeneration in zebrafish and male Wistar rats.METHODS: In this study, we applied in vitro and in vivo models, tissue section analysis, and quantitative polymerase chain reaction to assess the effects of CCP on bone repair. MG-63 cells were used in alkaline phosphatase(ALP) activity and mineralization assays. Zebrafish and male Wistar rats were used to evaluate the effects of CCP on bone repair in vivo.RESULTS: A simple preparation of CCP promoted osteogenesis in vivo and in vitro, and promoted MG-63 cell proliferation as well as ALP activity and mineralization. In addition, CCP activated Akt and extracellular signaling-regulated kinase pathways and significantly increased the expression of ossification-related genes and proteins such as runt-related transcription factor-2, osteocalcin, and osteopontin. The in vivo results revealed promotion of osteogenesis by immersing zebrafish in CCP for 72 h.An oral dose of 1.25 g/kg CCP significantly improved skull defects in rats, which was accompanied by an increase in serum ALP levels.CONCLUSION: One of the ingredients of Guilu Erxianjiao(tortoise shell and deer horn formula) pro-vides a practical alternative therapy for bone regeneration.

In-vitro study:Immunomodulatory and cytotoxicity effects of ethanolic leave extracts of Aegle marmelos and Ficus benghalensis

Objective:To assess the immunomodulatory and antibacterial properties of the coastal trees Aegle marmelos(A.marmelos)and Ficus benghalensis(F.benghalensis)by in-vitro methods.Methods:A.marmelos and F.benghalensis leaves were extracted with the solvents di-ethyl ether,ethanol and methanol.The extracts were investigated for antibacterial activity against human pathogenic bacteria and immunomodulatory activity against human peripheral blood mononuclear cells(PBMC),respectively.Results:F.benghalensis ethanol cold extract gave maximum inhibition zones against Escherichia coli NCIM 2931(B2)at 18 mm and enhanced the growth of human PBMC.It was non-toxic to human PBMC shown by the Tryphan blue dye exclusion method,sulforhodamine B and MTT assays.IR phyto-chemical analysis demonstrated the presence of the functional groups:NH,CH_(2),CH_(3),CO,OH,-C-C=O,and C-S.We confirmed the presence of quantifiable amounts of tannins and alkaloids with traces of phenolic compounds by thin layer chromatography analysis.Conclusions:Leaves of A.marmelos and F.benghalensis provide both antibacterial and immunomodulatory properties.

Dust mite allergen-specific immunotherapy increases IL4 DNA methylation and induces Der p-specific T cell tolerance in children with allergic asthma

Allergen-specific immunotherapy(allergen-SIT)is a highly effective treatment for children with allergic asthma(AA),an immune-mediated chronic disease leading to bronchial muscle hypertrophy and airway obstruction in response to specific allergens.T helper cells and secreted cytokines play important roles in the pathogenesis of asthma,and epigenetic modulation controls genes important for T cell development and cytokine expression.This study evaluated T helper cell-secreted cytokines and DNA methylation patterns in children treated with Dermatophagoides pteronyssinus(Der p)allergen-SIT.Our results showed that after Der p challenge,peripheral blood mononuclear cells(PBMCs)from the SIT group,compared with the non-SIT AA group,produced lower levels of IL-4,IL-5 and IL-2.The SIT group,compared with the AA group,exhibited decreased sensitivity to the Der p allergen,concurrent with IL-4 down-modulation due to increased promoter DNA methylation,as estimated in PBMCs.Our results showed that SIT decreased IL-4 and IL-5,and inhibited T cell proliferation,by inhibiting IL-2 production after the specific allergen challenge.These results suggest that decreased IL-2 production and increased IL-4 cytokine promoter methylation is a potential mechanism of Der p-specific allergen desensitization immunotherapy.

Increased susceptibility of algal symbionts to photo-inhibition resulting from the perturbation of coral gastrodermal membrane trafficking

The stability of cnidarian-dinoflagellate endosymbioses is dependent upon communication between the host gastrodermal cell and the symbionts housed within it.Although the molecular mechanisms remain to be elucidated,existing evidence suggests that the establishment of these endosymbioses may involve the sorting of membrane proteins.The present study examined the role of host gastrodermal membranes in regulating symbiont(genus Symbiodinium) photosynthesis in the stony coral Euphyllia glabrescens.In comparison with the photosynthetic behavior of Symbiodinium in culture,the Symbiodinium populations within isolated symbiotic gastrodermal cells(SGCs) exhibited a significant degree of photo-inhibition,as determined by a decrease in the photochemical efficiency of photosystem II(F v /F m).This photo-inhibition coincided with increases in plasma membrane perturbation and oxidative activity in the SGCs.Membrane trafficking in SGCs was examined using the metabolism of a fluorescent lipid analog,N-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-D-erythro-Sphingosylphosphorylcholine(BODIPY-Sphingomyelin or BODIPY-SM).Light irradiation altered both membrane distribution and trafficking of BODIPY-SM,resulting in metabolic changes.Cholesterol depletion of the SGC plasma membranes by methyl--cyclodextrin retarded BODIPY-SM degradation and further augmented Symbiodinium photo-inhibition.These results indicate that Symbiodinium photo-inhibition may be related to perturbation of the host gastrodermal membrane,providing evidence for the pivotal role of host membrane trafficking in the regulation of this environmentally important coral-dinoflagellate endosymbiosis.

CO_2 sequestration utilizing basic-oxygen furnace slag:Controlling factors,reaction mechanisms and V–Cr concerns

Basic-oxygen furnace slag（BOF-slag） contains 〉35%CaO,a potential component for CO_2sequestration.In this study,slag-water-CO_2 reaction experiments were conducted with the longest reaction duration extending to 96 hr under high CO_2 pressures of 100-300 kg/cm2 to optimize BOF-slag carbonation conditions,to address carbonation mechanisms,and to evaluate the extents of V and Cr release from slag carbonation.The slag carbonation degree generally reached the maximum values after 24 hr slag-water-CO_2 reaction and was controlled by slag particle size and reaction temperature.The maximum carbonation degree of 71%was produced from the experiment using fine slag of〈0.5 mm under 100℃and a CO_2 pressure of 250 kg/cm^2 with a water/slag ratio of 5.Vanadium release from the slag to water was significantly enhanced（generally 〉2 orders） by slag carbonation.In contrast,slag carbonation did not promote chromium release until the reaction duration exceeded 24 hr.However,the water chromium content was generally at least an order lower than the vanadium concentration,which decreased when the reaction duration exceeded 24 hr.Therefore,long reaction durations of 48-96 hr are proposed to reduce environmental impacts while keeping high carbonation degrees.Mineral textures and water compositions indicated that Mg-wustite,in addition to CaO-containing minerals,can also be carbonated.Consequently,the conventional expression that only considered carbonation of the CaO-containing minerals undervalued the CO_2 sequestration capability of the BOF-slag by^20%.Therefore,the BOF-slag is a better CO_2 storage medium than that previously recognized.