Development of a Real-Time TaqMan PCR Method for Absolute Quantification of the Biocontrol Agent Esteya vermicola.

Esteya vermicola has been used as an effective biocontrol agent for the management of the pinewood nematode, Bursaphelenchus xylophilus. Tools for monitoring the colonization and parasitism patterns of E. vermicola are required for the development of highly effective biocontrol strategies. Because the TaqMan PCR technique is effective for quantification of species in environmental samples, a real-time PCR-based methodology was developed for absolute quantification of E. vermicola via internal standard addition and extrapolation of DNA quantity to hyphal length. Primers and a probe for the 28S ribosomal RNA gene of E. vermicola were designed, and nested TaqMan real-time PCR-based quantification was performed. In addition, internal standard-based yield measurement was correlated to the absolute quantity of target genomic DNA. Moreover, an extrapolation curve obtained by optical microscopy and image analysis of the mycelia was constructed for the measurement of fungal hyphal length. The absolute quantification method developed in the present study provides a sensitive and accurate technique to quantify fungal density in either wood or other substrate samples and can be used as an effective tool for future studies of biocontrol agents.

A pine wood sample preparation method for high target and quality DNA extraction for detection of Esteya vermicola by PCR from living pine.

Esteya vermicola is an endophytic fungus of pine wood nematode with demonstrated biocontrol efficacy. At present, the methods for detection of this fungus from pine wood is still inconvenient and inefficient such as thin-section microscopic observations. In the present study, a simple protocol was developed for wood sample preparation for effective extraction of fungal DNA from wood samples for PCR detection. The protocol of preparation of the sample involves washing in sterile water overnight on a shaking table followed by filtration and centrifugation to obtain the extracted DNA. The result indicates that with this sample preparation protocol, any proper DNA extraction method can be effectively used for the rapid and reliable detection of E. vermicola from pine wood. This method can provide valuable support for follow-up studies with practical applications, such as investigation of the growth rate of E. vermicola and how long it remains viable inside a pine tree.

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde.

BACKGROUND: Plant parasitic nematodes are harmful to agricultural crops and plants, and may cause severe yield losses. Cinnamaldehyde, a volatile, yellow liquid commonly used as a flavoring or food additive, is increasingly becoming a popular natural nematicide because of its high nematicidal activity and, there is a high demand for the development of a biological platform to produce cinnamaldehyde. RESULTS: We engineered Escherichia coli as an eco-friendly biological platform for the production of cinnamaldehyde. In E. coli, cinnamaldehyde can be synthesized from intracellular L-phenylalanine, which requires the activities of three enzymes: phenylalanine-ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and cinnamoyl-CoA reductase (CCR). For the efficient production of cinnamaldehyde in E. coli, we first examined the activities of enzymes from different sources and a gene expression system for the selected enzymes was constructed. Next, the metabolic pathway for L-phenylalanine biosynthesis was engineered to increase the intracellular pool of L-phenylalanine, which is a main precursor of cinnamaldehyde. Finally, we tried to produce cinnamaldehyde with the engineered E. coli. According to this result, cinnamaldehyde production as high as 75 mg/L could be achieved, which was about 35-fold higher compared with that in the parental E. coli W3110 harboring a plasmid for cinnamaldehyde biosynthesis. We also confirmed that cinnamaldehyde produced by our engineered E. coli had a nematicidal activity similar to the activity of commercial cinnamaldehyde by nematicidal assays against Bursaphelenchus xylophilus. CONCLUSION: As a potential natural pesticide, cinnamaldehyde was successfully produced in E. coli by construction of the biosynthesis pathway and, its production titer was also significantly increased by engineering the metabolic pathway of L-phenylalanine.

In vivo hair growth-stimulating effect of medicinal plant extract on BALB/c nude mice.

CONTEXT: Chrysanthemum zawadskii var. latilobum (Asteraceae) (CZ) and Polygonum multiflorum Thunb. (Polygonaceae) (PM) have been used traditionally to treat different systemic diseases and acclaimed for various biological activities including hair growth. OBJECTIVE: This study investigates the hair restoration efficacy of selected medicinal plant extracts on nude mice. MATERIALS AND METHODS: Nude mice genetically predisposed to pattern balding were used in this study. Topical methanol extracts of CZ and PM (10 mg/mouse/d) with standardized vehicle formulation, only vehicle (propylene glycol:ethanol:dimethyl sulfoxide, 67:30:3% v/v) and Minoxidil (2%) were applied daily for 40 consecutive days. RESULTS: In our study, the maximum hair score (2.5 ± 0.29) was obtained in the CZ-treated group. Histological observation revealed a significant increase (p < 0.001) in the number of hair follicles (HF) in CZ-treated mice (58.66 ± 3.72) and Minoxidil-treated mice (40 ± 2.71). Subsequently, immunohistochemical analysis also confirmed the follicular keratinocyte proliferation by detection of BrdU-labeling, S-phase cells in Minoxidil and CZ-treated mouse follicular bulb and outer root sheaths. CONCLUSION: Our study revealed the underlying mechanism of stimulating hair growth in athymic nude mice by repair the nu/nu follicular keratin differentiation defect. Thus, the topical application of CZ may represent a novel strategy for the management and therapy of certain forms of alopecia.

The multi-herbal formula Chong-Myung-Tang improves spatial memory and increases cell genesis in the dentate gyrus of aged mice.

Chong-Myung-Tang (CMT) is a multi-herbal formula that has been used to improve memory. However, the potential mechanism remains unknown. The present study investigated the effects of CMT (50, 100, and 200 mg/kg) on spatial memory of aged mice. The behavioral training tests indicated that 200 mg/kg CMT treatment can significantly improve spatial memory of aged mice in the Morris water maze. Moreover, cell survival was examined by injecting bromodeoxyuridine (BrdU) on the first three days. The result showed that 200 mg/kg CMT treatment significantly increased cell survival in the dentate gyrus. Cell proliferation was determined by injecting BrdU 2 h before the mice were killed. The result suggested that CMT treatments had no influence on cell proliferation in the dentate gyrus. Thus, an increase in cell survival in the dentate gyrus stimulated by CMT may be involved in the effect of CMT on spatial memory improvement.

Aqueous extract of red deer antler promotes hair growth by regulating the hair cycle and cell proliferation in hair follicles.

Deer antlers are the only mammalian appendage capable of regeneration. We aimed to investigate the effect of red deer antler extract in regulating hair growth, using a mouse model. The backs of male mice were shaved at eight weeks of age. Crude aqueous extracts of deer antler were prepared at either 4 °C or 100 °C and injected subcutaneously to two separate groups of mice (n = 9) at 1 mL/day for 10 consecutive days, with water as a vehicle control group. The mice skin quantitative hair growth parameters were measured and 5-bromo-2-deoxyuridine was used to identify label-retaining cells. We found that, in both the 4 °C and the 100 °C deer antler aqueous extract-injection groups, the anagen phase was extended, while the number of BrdU-incorporated cells was dramatically increased. These results indicate that deer antler aqueous extract promotes hair growth by extending the anagen phase and regulating cell proliferation in the hair follicle region.

Optimization of storage condition for maintaining long-term viability of nematophagous fungus Esteya vermicola as biocontrol agent against pinewood nematode.

The fungus, Esteya vermicola has been proposed as biocontrol agent against pine wilting disease caused by Bursaphelenchus xylophilus. In this study, we reported the effects of temperature and different additives on the viability and biocontrol efficacy of E. vermicola formulated by alginate-clay. The viability of the E. vermicola formulation was determined for six consecutive months at temperature ranged from -70 to 25 °C. The fresh conidia without any treatment were used as control. Under the optimal storage conditions with E. vermicola alginate-clay formulation, the results suggested that E. vermicola alginate-clay formulation with a long shelf life could be a non-vacuum-packed formulation that contains 2 % sodium alginate and 5 % clay at 4 °C. Three conidial formulations prepared with additives of 15 % glycerol, 0.5 % yeast extract and 0.5 % herbal extraction, respectively significantly improved the shelf life. In addition, these tested formulations retained the same biocontrol efficacy as the fresh conidial against pinewood nematode. This study provided a tractable and low-cost method to preserve the shelf life of E. vermicola.

Compound K is able to ameliorate the impaired cognitive function and hippocampal neurogenesis following chemotherapy treatment.

Chemotherapy frequently results in neurocognitive deficits that include impaired learning and memory. Thus, it is important to prevent or ameliorate the persistence of cognitive impairment. Compound K was employed to examine the ameliorating effect on chronic treatment with cyclophosphamide. Eight week-old ICR mice were given 80 mg/kg cyclophosphamide, cyclophosphamide combined with compound K (2.5, 5 and 10 mg/kg) or saline injections once per week for 4 weeks. Passive avoidance test and Y maze were used to evaluate memory and learning ability. Immunohistochemical staining for progenitor cell and immature neurons was used to assess changes in neurogenesis. Compound K (10 mg/kg) is able to ameliorate the decrease of neurogenesis in the hippocampus caused by cyclophosphamide. These results suggest that compound K might be a potential strategy to ameliorate or repair the disrupted hippocampal neurogenesis induced by the side effect of chemotherapy agent.

Encapsulated fat necrosis mimicking subcutaneous liposarcoma: radiologic findings on MR, PET-CT, and US imaging.

We present a case of a subcutaneous encapsulated fat necrosis of the upper extremity that mimicked subcutaneous liposarcoma because of heterogeneous signal intensity, its relatively large size, and pronounced enhancement on magnetic resonance imaging (MRI). For this case, we present the radiologic findings including MRI, positron emission tomography-computed tomography (PET-CT), ultrasonography, and radiography. We emphasize the imaging features of this lesion, which has a fibrous capsule and briefly discuss its nomenclature and pathophysiology.

Ginsenosides Rb1 and Rd regulate proliferation of mature keratinocytes through induction of p63 expression in hair follicles.

Ginsenosides Rb1 and Rd are the two main types of ginsenosides in Panax ginseng and have been used as an additive to against alopecia. However, the mechanisms involved are largely unknown. To determine how ginsenosides prevent hair loss, we topically applied protopanaxadiol-type ginsenosides Rb1 and Rd over the shaved skin of B57CL/6 mice, and monitored and assessed them for 35 days. We then investigated the effects of ginsenosides on cell genesis in different phases of adult hair follicles (HFs), using 5-bromo-2'-deoxyuridine as a marker for dividing cells. Moreover, p63, a specific marker and a major regulator of keratinocyte progenitor cells of the multi-layered epithelia, was detected in epidermis. Results indicated that treatment with ginsenosides Rb1 and Rd increased cell proliferation in both anagen and telogen of HFs. However, it had no significant effect on the survival of cells in the bulge and upper follicle region. Investigation of p63 demonstrated that up-regulation of p63 expression in the matrix and outer root sheath might be one of the mechanisms by which ginsenosides Rb1 and Rd promote cell proliferation in HFs. Our study reveals a novel mechanism by which ginsenoside promotes hair growth through p63 induction in follicular keratinocytes and indicates that ginsenosides Rb1 and Rd might be developed as a therapeutic agent for the prevention of hair loss.

Comparison between conidia and blastospores of Esteya vermicola, an endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus.

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively.

Correction to: Effect of red and black ginseng on cholinergic markers, presynaptic markers, and neurotrophins in the brain of aged mice.

[This corrects the article DOI: 10.1007/s10068-017-0235-7.].

2-(2,5-Dimeth-oxy-phen-yl)-N-[2-(4-hy-droxy-phen-yl)eth-yl]acetamide.

In the title compound, C(18)H(21)NO(4), the dihedral angles between the acetamide group and the meth-oxy- and hy-droxy-substitured benzene rings are 80.81 (5) and 8.19 (12)°, respectively. The benzene rings are twisted with respect to each other, making a dihedral angle of 72.89 (5)°. In the crystal, N-H⋯O and O-H⋯O hydrogen bonds link the mol-ecules into a three-dimensional network.

1-(3-Hy-droxy-phen-yl)-3-(3-meth-oxy-phenyl)thio-urea.

In the title compound, C(14)H(14)N(2)O(2)S, the dihedral angles between the thio-urea group and the methoxyphenyl and hydroxyphenyl rings are 61.91 (4) and 76.90 (4)°, respectively. The benzene rings are twisted with respect to each other, making a dihedral angle of 71.03 (4)°. The H atoms of the thio-urea NH groups are positioned anti to each other. In the crystal, inter-molecular N-H⋯S, N-H⋯O and O-H⋯S hydrogen bonds link the mol-ecules into a three-dimensional network.

Growth of Esteya vermicola in media amended with nitrogen sources yields conidia with increased predacity and resistance to environmental stress.

Esteya vermicola , an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study to enhance the sporulation, predacity, and environmental resistance of E. vermicola, various nitrogen sources, such as glycine, L-leucine, and ammonium nitrate, were tested. The supplement of glycine and L-leucine had a significant influence on the growth rate of the colony, enhancing colony dry mass by 5-fold more than did ammonium nitrate or the control. Of the nitrogen sources tested, ammonium nitrate and L-leucine promoted sporulation, yielding more than 6 × 10(6) CFU/g, while glycine enhanced the proportion of lunate spores. Meanwhile, the supplement of nitrogen sources had a significant influence on adhesive rate and mortality rate against Bursaphelenchus xylophilus . Moreover, the supplement of glycine enhanced the survival rate against heat stress by more than 3-fold that of L-leucine, ammonium nitrate, and control. The spores produced in media amended with glycine, L-leucine, and ammonium nitrate had slightly but not significantly higher UV resistance and drought resistance than spores produced without nitrogen sources. These results suggested that the addition of glycine resulted in the production of E. vermicola conidia with increased predacity and resistance to environmental stress that may be more suitable for control of pine wilt disease.

Variabilities of two Drechslerella dactyloides isolates in Korea and high predacity against Bursaphelenchus xylophilus.

Drechslerella dactyloides generates elongate ellipsoid conidia and three-celled rings. Recently, Drechslerella dactyloides CNU 091025 and CNU 091026 were isolated in Korea, which generated elongate ellipsoid, Y-shaped and reaphook-shaped conidia, three-celled rings and fishhook-shaped traps. Therefore, molecular phylogenetic analysis, morphological variability, and nematode capturing ability were tested in this study. CNU 091025 generated two-celled or three-celled Y-shaped conidia, reaphook-shaped conidia, and elongate ellipsoid conidia, 17.2, 40.9, and 41.9%, respectively. Some fishhook-shaped rings connecting together formed two-dimensional web. Both fungi showed high trap-forming and nematode-capturing ability; especially CNU 091026 captured 100% Bursaphelenchus xylophilus within 24 h after inoculation.

1-[3-(Hy-droxy-meth-yl)phen-yl]-3-phenyl-urea.

In the title compound, C(14)H(14)N(2)O(2), the dihedral angle between the benzene rings is 23.6 (1)°. The H atoms of the urea NH groups are positioned syn to each other. In the crystal, inter-molecular N-H⋯O and O-H⋯O hydrogen bonds link the mol-ecules into a three-dimensional network.

1-(2-Hy-droxy-2-phenyl-eth-yl)-3-(4-meth-oxy-phen-yl)urea.

In the title compound, C(16)H(18)N(2)O(3), the dihedral angle between the 4-meth-oxy-phenyl ring and the urea group is 35.6 (2) °. The H atoms of the urea NH groups are positioned syn to each other. In the crystal, inter-molecular N-H⋯O and O-H⋯O hydrogen bonds link the mol-ecules into a two-dimensional array in the ac plane; the carbonyl-O atom is trifurcated.

N-(4-Hy-droxy-phen-yl)-3,4,5-trimeth-oxy-benzamide.

In the title amide compound, C(16)H(17)NO(5), the dihedral angle between the benzene rings is 71.59 (4)°. In the crystal, inter-molecular N-H⋯O and O-H⋯O hydrogen bonds link the mol-ecules into a two-dimensional array parallel to the ab plane.

Elimination of the mycotoxin citrinin production in the industrial important strain Monascus purpureus SM001.

The application of the high-producing pigments industrial strain Monascus purpureus SM001 has been greatly limited by the synchronous production of mycotoxin citrinin. Here we have tried both traditional mutagenesis and metabolic engineering methods to eliminate the production of citrinin. Traditional chemical and physical mutagens were applied to induce mutagenesis, and a bio-screening method based on the antibacterial activity of citrinin against Bacillus subtilis was designed to select mutants. Among the resulting four citrinin-free mutants, only mutant MU2411 can maintain the similar pigments yield. A binary vector system was constructed and successfully disrupted the polyketide synthase gene pksCT in M. purpureus SM001 through the Agrobacterium tumefaciens-mediated transformation. The resulting citrinin-free DeltapksCT mutants maintained the same level of pigments yield. The established Monascus genetic system was comprehensively evaluated and showed high efficiency and specificity, which provides us a potential approach to manipulate and improve industrial Monascus strains.