Antibacterial and antibiofilm efficacy of the preferred fractions and compounds from Euphorbia humifusa (herba euphorbiae humifusae) against Staphylococcus aureus.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia humifusa Willd., known as Di-Jin-Cao in Chinese, has long been utilized as a traditional herb for the treatment of furuncles and carbuncles mainly caused by Staphylococcus aureus infection. Despite extensive chemical and pharmacological studies reported previously for E. humifusa, the antibacterial and antibiofilm activities against S. aureus as well as the related mechanism of action (MoA) remain largely obscure. AIM OF THE STUDY: To investigate the antibacterial and antibiofilm activities of the preferred fractions and compounds from E. humifusa against S. aureus and assess the associated MoA. MATERIALS AND METHODS: The bioactive fractions and compounds were obtained from the 75% ethanol extract of E. humifusa (75%-EEEH) with the assistance of the related antibacterial and antibiofilm screening. Their antibacterial activities were determined using the broth microdilution method, whilst the inhibition of biofilm formation and the disruption of preformed biofilm were assessed by crystal violet staining and confocal laser scanning microscopy (CLSM). To achieve more effective therapies, the combinatory effects of different components were also studied. The biofilm metabolic activities of isolated compounds were evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. The scanning electron microscopy (SEM) and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to explore the antibiofilm mechanism. RESULTS: Fractions DJC06 and DJC07 collected from the ethyl acetate extract of the 75%-EEEH exhibited antibacterial activity (MIC = 256 µg/mL) against S. aureus and further separation of these two fractions led to the isolation and characterization of 22 compounds. Among the isolates, luteolin (LU), quercetin (QU), and kaempferol (KA) are the verified components associated with the antibacterial and antibiofilm activities by displaying individual or combinational MIC values of 8-128 µg/mL and 70.9-99.7% inhibition for biofilm formation. Importantly, QU and KA can work in synergy with LU to significantly enhance the efficacy via destroying cell integrity, increasing membrane permeability, and down-regulating the biofilm-related gene expression. CONCLUSIONS: The preferred fractions and compounds from E. humifusa exerted desired antibacterial and antibiofilm efficacy against S. aureus via a MoA involving cell morphology disruption and altered genes expression. The findings herein not only support its traditional use in the treatment of furuncles and carbuncles, but reveal E. humifusa is a potential source for producing promising antibiofilm alternatives against S. aureus and highlight the isolated components (LU, QU, KA) that can potentiate the efficacy when used in synergy.

The adjuvant effects of rosin and coconut oil on nicosulfuron and mesotrione to control weeds.

Natural adjuvants are novel options to reduce the doses of chemical herbicides. The aim of the current study was to examine the compositions and adjuvant effects of rosin and coconut oil on herbicides using a combination of indoor experiment and field trial. The GC-MS results showed that the main component of rosin was abietic acid (40.02%), and the main components of coconut oil were 2-pentanone, 4-hydroxy-4-methyl- (21.45%) and dodecanoic acid (14.59%). In greenhouse experiment, rosin showed a significant adjuvant effect on nicosulfuron against Digitaria sanguinalis and Amaranthus retroflexus, with the GR50 ratios of 1.47 and 1.69, respectively. The GR50 values of nicosulfuron in the present of coconut oil were 3.99 and 10.13 g a.i./hm2 against D. sanguinalis and A. retroflexus, lower than that of individual application. The adjuvant effect of rosin and coconut oil on mesotrione was also found. In field trial, the fresh weight control efficiency of nicosulfuron (45 g a.i./hm2) and mesotrione (112.5 g a.i./hm2) was significantly improved after the addition of rosin and coconut oil, similar with that of recommended dose. Rosin and coconut oil could reduce the contact angle of nicosulfuron, with the results of 56.68° and 53.90°, respectively, lower than that of individual application. Furthermore, rosin and coconut oil could decrease the surface tension, wetting and penetration time; and increase the spreading diameter and maximum retention. Both rosin and coconut oil have adjuvant effects on herbicides in the lab & field with multiple mechanisms. Thus, they have the potential to be developed into natural adjuvants for herbicide formulation to control weeds.

Transcriptome analysis associated with polysaccharide synthesis and their antioxidant activity in Cyclocarya paliurus leaves of different developmental stages.

BACKGROUND: Cyclocarya paliurus (Batal.) Iljinskaja is a common endemic tree species and used as a Chinese medicine. The main active components in the leaves of this plant are polysaccharides. However, the temporal patterns of gene expression underlying the synthesis of polysaccharides in C. paliurus at different leaf developmental stages and its relationship with the polysaccharide content and antioxidant activities has not been reported to date. METHODS: RNA-seq was used to investigate the biosynthesis pathway of polysaccharides at the four developmental stages of C. paliurus leaves. The content and the antioxidant activities of polysaccharides were measured with typical biochemical methods and the identified correlations were statistically evaluated. RESULTS: Sixty-nine differentially expressed genes were found in the leaves during different developmental stages of C. paliurus. These are associated with glycosyltransferases and belong to 18 families. During different developmental stages of C. paliurus, the polysaccharide content first increased and then decreased, and the UDP-glucose 4-epimerase gene was found to be significantly positively correlated with the polysaccharide content. The clearance rates of DPPH radicals, superoxide anion radicals, hydroxyl radicals, and the reducing power of polysaccharides in the leaves of C. paliurus at different developmental stages showed a dose-dependent relationship with the concentration of polysaccharides. CONCLUSIONS: The smallest fully expanded leaves are suitable for high-quality tea, and leaves with sizes below the largest fully expanded leaves are suitable for industrial production of polysaccharides.

Laser Mutagenesis of Phellinus igniarius Protoplasts for the Selective Breeding of Strains with High Laccase Activity.

Phellinus igniarius is a medicinal fungus that utilizes lignin as a nutrient substrate. This fungus has a weak lignin degradation ability and, as a result, a slow growth rate. Laccases are crucial enzymes for lignin degradation in P. igniarius, and thus, the cultivation of strains with high laccase activity is expected to increase the growth rate of P. igniarius. To generate P. igniarius strains with high laccase activity, we performed laser mutagenesis of P. igniarius protoplasts and screened for mutants with high laccase activity. Our results showed that the laser power density and P. igniarius protoplast survival rate exhibited a power-function relationship. The power density threshold value between lethality and growth promotion was 0.24 mW/mm2. Mutagenesis was carried out using a laser beam diameter of 3 mm and an irradiation period of 40 min. After five generations of selection, we identified a high laccase activity strain, termed SJZ2. The laccase activity in SJZ2 during 4 h of fermentation was increased by 36.84% in comparison with the control and ranged from 0.20216 to 0.27664 U. The Km and Vmax of the laccase produced by SJZ2 were 0.21 mmol/mL and 0.53 mmol/L/min, respectively. This study demonstrated the feasibility of laser mutagenesis of P. igniarius protoplasts for the selection of high laccase activity. This study characterized the key factors in the laser mutagenesis process of P. igniarius protoplasts and provided a reference for the application of lasers in biological mutagenesis. Future studies should evaluate the bioactive functionality and stability of this novel strain of P. igniarius, particularly the organoleptic and medical characteristics of the fruiting bodies.

Network pharmacology-based study of the mechanisms of action of anti-diabetic triterpenoids from Cyclocarya paliurus.

Diabetes is a complex illness requiring long-term therapy. Cyclocarya paliurus, a recently confirmed new food resource, shows significant hypoglycemic and hypolipidemic effects in type II diabetes. Triterpenoid saponins are considered as the effective medicinal components of C. paliurus and are useful for the treatment of diabetes mellitus. However, little is known regarding their specific mechanism of actions. In this study, we used active ingredient screening and target prediction techniques to determine the components of C. paliurus responsible for its anti-diabetic effects as well as their targets. In addition, we used bioinformatics technology and molecular docking analysis to determine the mechanisms underlying their anti-diabetic effects. A total of 39 triterpenes were identified through a literature search and 1 triterpene compound by experiments. In all, 33 potential target proteins associated with 36 pathways were predicted to be related to diabetes. Finally, 7 compounds, 15 target proteins, and 15 signaling pathways were found to play important roles in the therapeutic effects of C. paliurus against diabetes. These results provide a theoretical framework for the use of C. paliurus against diabetes. Moreover, molecular docking verification showed that more than 90% of the active ingredients had binding activity when tested against key target proteins, and a literature search showed that the active ingredients identified had anti-diabetic effects, indicating that the results were highly reliable.

Rapid visual detection of lily mottle virus using a loop-mediated isothermal amplification method.

Lily mottle virus (LMoV; genus Potyvirus, family Potyviridae) infects plants of the genus Lilium, causing a reduction in flower and bulb quality. A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed to detect the coat protein gene of LMoV. This LAMP method was highly specific for LMoV, with no cross-reaction with other lily viruses. The sensitivity of LMoV using the LAMP assay was 100 times more sensitive than that using conventional polymerase chain reaction. A reverse transcription LAMP (RT-LAMP) was then successfully applied to detect LMoV RNA. The newly established LAMP and one-step RT-LAMP provide an alternative method for detecting LMoV in lily plants.

A new approach for breeding low-temperature-resistant Volvariella volvacea strains: Genome shuffling in edible fungi.

Volvariella volvacea is difficult to store fresh because of the lack of low-temperature resistance. Many traditional mutagenic strategies have been applied in order to select out strains resistant to low temperature, but few commercially efficient strains have been produced. In order to break through the bottleneck of traditional breeding and significantly improve low-temperature resistance of the edible fungus V. volvacea, strains resistant to low temperature were constructed by genome shuffling. The optimum conditions of V. volvacea strain mutation, protoplast regeneration, and fusion were determined. After protoplasts were treated with 1% (v/v) ethylmethylsulfonate (EMS), 40 Sec of ultraviolet (UV) irradiation, 600 Gy electron beam implantation, and 750 Gy60 Co-γ irradiation, separately, the lethality was within 70%-80%, which favored generating protoplasts being used in following forward mutation. Under these conditions, 16 strains of V. volvacea mutated by EMS, electron beam, UV irradiation, and 60 Co-γ irradiation were obtained. The 16 mutated protoplasts were selected to serve as the shuffling pool based on their excellent low-temperature resistance. After four rounds of genome shuffling and low-temperature resistance testing, three strains (VF1 , VF2 , and VF3 ) with high genetic stability were screened. VF1 , VF2 , and VF3 significantly enhanced fruit body shelf life to 20, 28, and 28 H at 10 °C, respectively, which exceeded 25%, 75%, and 75%, respectively, compared with the storage time of V23, the most low-temperature-resistant strain. Genome shuffling greatly improved the low-temperature resistance of V. volvacea, and shortened the course of screening required to generate desirable strains. To our knowledge, this is the first paper to apply genome shuffling to breeding new varieties of mushroom, and offers a new approach for breeding edible fungi with optimized phenotype.

ABC50 modulates sensitivity of HL60 leukemic cells to endoplasmic reticulum (ER) stress-induced cell death.

ABC50 (aka ABCF1) is a member of the ATP Binding Cassette protein family. ABC50 stimulates complex formation between eIF2, GTP and Met-tRNA implicating it in translation initiation. Econazole (Ec) is an imidazole anti-fungal that induces endoplasmic reticulum (ER) stress in mammalian cells by promoting ER Ca(2+) depletion and sustained protein synthesis inhibition. HL60 cells selected for Ec resistance were found to exhibit a multi-drug resistance phenotype associated specifically with ER stress. Differential Display was used to identify ABC50 as an overexpressed gene in resistant cells. ABC50 knockdown (KD) in Ec-resistant HL60 cells partially restored Ec sensitivity. In parental HL60 cells, ABC50 KD increased sensitivity to Ec, thapsigargin and tunicamycin but not to serum withdrawal or etoposide. ABC50 overexpression (OE) partially and specifically decreased sensitivity to ER stress agents. ABC50 KD or OE had no effect on ROS generation by Ec, ER Ca(2+) stores or thapsigargin-stimulated influx. Increased eIF2α phosphorylation in response to ER stress was observed in the KD cells while decreased phosphorylation was observed in the OE cells. Ribosomal content was reduced in ABC50 KD cells and increased in OE cells. Knockdown suppressed protein synthesis while OE increased it. Protein synthesis was sustained in ABC50 OE cells exposed to Ec. ABC50 OE promoted ER stress resistance and increased antibody production in the hybridoma GK1.5 suggesting it may be useful for the overproduction of specific proteins. Taken together, these results indicate that ABC50 modulates sensitivity to Ec and other ER stress agents primarily through its effects on protein synthesis.

Chronic exposure to sub-lethal beta-amyloid (Abeta) inhibits the import of nuclear-encoded proteins to mitochondria in differentiated PC12 cells.

Studies on amyloid beta (Abeta|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in Abeta-mediated neurotoxicity. We used differentiated PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N'-terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub-lethal Abeta on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal Abeta(25-35) (10 mumol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 +/- 4%. Concomitant with the decline in mtGFP, cytoplasmic mtGFP increased significantly while mtGFP expression and intramitochondrial mtGFP turnover were unchanged. Sub-lethal Abeta(1-42) inhibited mtGFP import and increased cytoplasmic mtGFP but only after 96 h. The import of two endogenous nuclear-encoded mitochondrial proteins, mortalin/mtHsp70 and Tom20 also declined. Prior to the decline in import, mitochondrial membrane potential (mmp), and reactive oxygen species levels were unchanged in Abeta-treated cells versus reverse phase controls. Sustained periods of decreased import were associated with decreased mmp, increased reactive oxygen species, increased vulnerability to oxygen-glucose deprivation and altered mitochondrial morphology. These findings suggest that an Abeta-mediated inhibition of mitochondrial protein import, and the consequent mitochondrial impairment, may contribute to Alzheimer's disease.

[Separation and regeneration of protoplast from Phellinus igniarius].

OBJECTIVE: To study the conditions on separation and regeneration of protoplast from Phellinus igniarius. METHOD: The effects of enzymolysis conditions of P. igniarius mycelia on yield of protoplast and culturing conditons on regeneration ratio of protoplast were investigated. RESULT: When the 8 days-old mycelia was hydrolysed by 1.5% of lywallzyme adding to driselase of 0. 5% and at 30 degrees C for 3 h and enzymolysis was stablized by sucrose as a stablisher of osmotic pressure, higher yield of P. igniarius protoplast was obtained. If 10 days-old mycelia was used as raw material of enzymolysis and manntol was selected as stablisher of osmotic pressure of enzymolysis, higher regeneration ratio of P. igniarius protoplast also would be obtained in following regeneration step at same time keeping higher yield. For the regeneration processing, it was beneficial for the regeneration of P. igniarius protoplast that PDA plusing mulberry ramulus was used as the culture medium of regeneration and manntol was selected as the osmotic pressure establisher of regeneration culture medium. CONCLUSION: The method and conditions to keep both higher yield and regeneration ratio of P. igniarius protoplast were obtained.

Inhibition of Ca2+ influx is required for mitochondrial reactive oxygen species-induced endoplasmic reticulum Ca2+ depletion and cell death in leukemia cells.

Disturbances of endoplasmic reticulum (ER) Ca2+ homeostasis or protein processing can lead to ER stress-induced cell death. Increasing evidence suggests that oxidative stress (OS) plays an important role in a variety of cell death mechanisms. To investigate the role of OS in ER stress, we measured OS in response to three ER stress agents: econazole (Ec), which stimulates ER Ca2+ release and blocks Ca2+ influx; thapsigargin (Tg), a sarco(endo)plasmic reticulum Ca2+ ATPase inhibitor that releases ER Ca2+ and stimulates Ca2+ influx; and tunicamycin (Tu), a glycosylation inhibitor that causes protein accumulation in the ER. Ec, but not Tg or Tu, caused a rapid increase in OS. Reactive oxygen species (ROS) generation was observed within mitochondria immediately after exposure to Ec. Furthermore, Ec hyperpolarized the mitochondrial membrane and inhibited adenine nucleotide transport in cell-free mitochondria, suggesting a mitochondrial target. Antimycin A, an inhibitor of complex III in electron transport, reversed mitochondrial hyperpolarization, OS generation, ER Ca2+ depletion, and cell death by Ec, suggesting complex III dependence for these effects. Antioxidants butylated hydroxytoluene and N-Acetyl-L-cysteine prevented ER Ca2+ depletion and cell death by Ec. However, inhibition of Ca2+ influx by Ec was unaffected by either antimycin A or the antioxidants, suggesting that this target is distinct from the mitochondrial target of Ec. Atractyloside, an adenine nucleotide transport inhibitor, generated ROS and stimulated ER Ca2+ release, but it did not block Ca2+ influx, deplete the ER or induce cell death. Taken together, these results demonstrate that combined mitochondrial ROS generation and Ca2+ influx blockade by Ec is required for cell death.

[Study on liver targeted drug delivery system of the effective anticancer component from Bolbstemma paniculatum].

OBJECTIVE: To study the liver targeted drug delivery system of TBMS--the effective anticancer component from Bolbstemma paniculatum, and to discuss the system's function of decreasing toxicity. METHOD: BCA was used as carrier material. The preparation through overall feedback dynamic techniques. The properties of preparation and toxicology were also technology of nanoparticles was optimized studied. Thenanoparticles' targeting in mice vivo was observed with transmission electron microscopy. The function of decreasing toxicity was researched by the XXTX-2000 automatic quantitative analysis management system. RESULT: D50 was 0.68 microm. Drug-loading rate and entrapment rate were 37.3% and 88.6% respectively. The release in vitro accorded with Weibull equation. The reaching release balance time and the t 1/2 extended 26 times and 19 times respectively comparing with injection. Nanoparticles mainly distributed in liver tissue. Their toxicity to lung and liver was evidently lower than injection. Nanoparticles' LD50 exceeded injection's by 13.5% and their stimulus was much lower than injection. CONCLUSION: The TBMS can be targeted to liver by liver targeted drug delivery system. At the same time, the problem about the toxicity hindering clinical application could be solved, which lays the foundation for the further studies on TBMS.

Flow cytometry and GFP: a novel assay for measuring the import and turnover of nuclear-encoded mitochondrial proteins in live PC12 cells.

BACKGROUND: Mitochondrial protein import is typically measured by adding radiolabeled precursor proteins to isolated mitochondria. We have developed a novel, high-throughput method for measuring protein import in live differentiated PC12 cells using a tetracycline (Tet) regulated, nuclear encoded, mitochondrially-targeted GFP fusion protein and flow cytometry. METHODS: We generated a PC12 cell line stably transfected with an inducible GFP fusion protein (GFPmt) targeted to mitochondria. GFPmt PC12 cells were treated with NGF for one week to induce neuronal differentiation in the presence of Tet to silence GFP expression. On day seven GFPmt expression was induced by removal of Tet and these "GFP-on" cells were exposed to sublethal levels of CCCP (2 microM) for 24 h. At 24 h, the cells were harvested in Ca(++)-free PBS and the GFPmt signal in live intact cells was measured using flow cytometry. Since GFPmt is not fluorescent prior to being imported into mitochondria, the GFPmt signal reflected only GFPmt imported to mitochondria. PI was used to gate out contributions from dead cells. Turnover of GFPmt in mitochondria was also assessed; in this case, Tet was added to arrest GFPmt expression in GFP-on cells, and the subsequent decline of the fluorescent signal, in the absence of any new GFP synthesis, was measured by flow cytometry. RESULTS: Exposure to 2 microM CCCP for 24 h caused a 61% +/- 0.4 decline in GFPmt fluorescence compared to controls. This decline corresponded to a 30% +/- 7 decrease in GFPmt protein levels measured by Western blot of mitochondrial fractions, and a 72% +/- 5 decline in the import of newly synthesized GFPmt to mitochondria over a 1 h period 24-h after addition of 2 microM CCCP measured by autoradiography. CCCP partially depolarized mitochondria but was not lethal for up to five days. CONCLUSIONS: This novel GFP-based flow cytometry assay is a rapid and sensitive technique for quantifying protein import to mitochondria in live neuronal cells.

Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle.

Calcineurin mediates repression of plasma membrane Ca2+-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which 45Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca2+ concentrations ([Ca2+]i) increased twofold, and real-time RT-PCR and Western blot revealed a approximately 40% decrease in PMCA4 expression levels from G0 to G1/S in the cell cycle, where PMCA4 constituted approximately 20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G0 to G1/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G1/S. By contrast, Ca2+-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G0 to G1/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G1/S-associated repression of PMCA4. These data show that G1/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca2+]i through alternate targets.