Proteomic analysis reveals the mechanisms of Mycena dendrobii promoting transplantation survival and growth of tissue culture seedlings of Dendrobium officinale.

AIMS: Dendrobium officinale is an important traditional Chinese medicinal herb. Its seedlings generally show low survival and growth when transferred from in vitro tissue culture to a greenhouse or field environment. In this study, the effect of Mycena dendrobii on the survival and growth of D. officinale tissue culture seedlings and the mechanisms involved was explored. METHODS AND RESULTS: Mycena dendrobii were applied underneath the roots of D. officinale tissue culture seedlings. The seedling survival and growth were analysed. The root proteins induced by M. dendrobii were identified using two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF-MS). Mycena dendrobii treatment significantly enhanced survival and growth of D. officinale seedlings. Forty-one proteins induced by M. dendrobii were identified. Among them, 10 were involved in defence and stress response, two were involved in the formation of root or mycorrhizae, and three were related to the biosynthesis of bioactive constituents. CONCLUSIONS: These results suggest that enhancing stress tolerance and promoting new root formation induced by M. dendrobii may improve the survival and growth of D. officinale tissue culture seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a foundation for future use of M. dendrobii in the large-scale cultivation of Dendrobiums.

First Report of Leaf Spot Caused by Nigrospora oryzae on Dendrobium candidum in China.

Dendrobium (Dendrobium candidum Wall. ex Lindl.) is a perennial herb in the Orchidaceae family. It has been used as traditional medicinal plant in China, Malaysia, Laos, and Thailand (2). Fungal disease is one of the most important factors affecting the development of Dendrobium production. During summer 2012, chocolate brown spots were observed on leaves of 2-year-old Dendrobium seedlings in a greenhouse in Hangzhou, Zhejiang Province, China, situated at 30.26°N and 120.19°E. Approximately 80% of the plants in each greenhouse were symptomatic. Diseased leaves exhibited irregular, chocolate brown, and necrotic lesions with a chlorotic halo, reaching 0.8 to 3.2 cm in diameter. Affected leaves began to senesce and withered in autumn, and all leaves of diseased plants fell off in the following spring. Symptomatic leaf tissues were cut into small pieces (4 to 5 mm long), surface-sterilized (immersed in 75% ethanol for 30 s, and then 1% sodium hypochlorite for 60 s), rinsed three times in sterilized distilled water, and then cultured on potato dextrose agar (PDA) amended with 30 mg/liter of kanamycin sulfate (dissolved in ddH2O). Petri plates were incubated in darkness at 25 ± 0.5°C, and a grey mycelium with a white border developed after 4 days. Fast-growing white mycelia were isolated from symptomatic leaf samples, and the mycelia became gray-brown with the onset of sporulation after 5 days. Conidia were unicellular, black, elliptical, and 11.4 to 14.3 µm (average 13.1 µm) in diameter. Based on these morphological and pathogenic characteristics, the isolates were tentatively identified as Nigrospora oryzae (1). Genomic DNA was extracted from a representative isolate F12-F, and a ~600-bp fragment was amplified and sequenced using the primers ITS1 and ITS4 (4). BLAST analysis showed that F12-F ITS sequence (Accession No. KF516962) had 99% similarity with the ITS sequence of an N. oryzae isolate (JQ863242.1). Healthy Dendrobium seedlings (4 months old) were used in pathogenicity tests under greenhouse conditions. Leaves were inoculated with mycelial plugs (5 mm in diameter) from a 5-day-old culture of strain F12-F, and sterile PDA plugs served as controls. Seedlings were covered with plastic bags for 5 days and maintained at 25 ± 0.5°C and 80 ± 5% relative humidity. Eight seedlings were used in each experiment, which was repeated three times. After 5 days, typical chocolate brown spots and black lesions were observed on inoculated leaves, whereas no symptoms developed on controls, which fulfilled Koch's postulates. This shows that N. oryzae can cause leaf spot of D. candidum. N. oryzae is a known pathogen for several hosts but has not been previously reported on any species of Dendrobium in China (3). To our knowledge, on the basis of literature, this is the first report of leaf spot of D. candidum caused by N. oryzae in China. References: (1) H. J. Hudson. Trans. Br. Mycol. Soc. 46:355, 1963. (2) Q. Jin et al. PLoS One. 8(4):e62352, 2013. (3) P. Sharma et al. J. Phytopathol. 161:439, 2013. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Genetic linkage map of EST-SSR and SRAP markers in the endangered Chinese endemic herb Dendrobium (Orchidaceae).

Dendrobium officinale is an endangered orchid from southeast Asia that is known for its medicinal properties in traditional Chinese medicine. We constructed an integrated genetic linkage map of an F(1) population derived from an interspecific cross between D. officinale and D. aduncum (both, 2n = 38), using expressed sequence tag-simple sequence repeats (EST-SSR) and sequence-related amplified polymorphism (SRAP). A total of 349 polymorphic loci, including 261 SRAP loci and 88 EST-SSR loci, were identified for genetic linkage analysis. The software JoinMap 4.0 was used to construct the genetic maps. A total of 157 loci were arranged into 27 major linkage groups, each containing a minimum of four markers, and a further 23 markers were distributed to five triplets and four doublets, the frame map covered a total distance of 1580.4 cM, with a mean of 11.89 cM between adjacent markers. This primary map of the D. officinale and D. aduncum hybrid provides a basis for genetic studies and should facilitate future studies of medical traits mapping and marker-assisted selection in Dendrobium species breeding programs.

Cellular neural network analysis for two-dimensional bioheat transfer equation.

The cellular neural network (CNN) method is applied to solve the Pennes bioheat transfer equation, and its feasibility is demonstrated. Numerical solutions were obtained for a cellular neural network for a two-dimensional steady-state temperature field obtained from focused and unfocused ultrasound heat sources. Transient-state temperature fields were also studied and compared with experimental results obtained elsewhere. The cellular neural networks' key features of asynchronous parallel processing, continuous-time dynamics and local interaction enable real-time temperature field estimation for clinical hyperthermia.

Antibiotic treatment of orbital cellulitis: an analysis of pathogenic bacteria and bacterial susceptibility.

The proper choice of effective antibiotics is a mainstay for the treatment of orbital cellulitis. The lack of native data regarding the microorganism causing the infection and its antibiotic sensitivity prompted us to conduct this study. We retrospectively collected 29 cases of orbital cellulitis admitted to Chung-Ho Memorial Hospital of Kaohsiung Medical College from January 1994 to September 1998. The effectiveness of antibiotics with bacterial susceptibility was analyzed. Of the 29 cases, fifteen were male and fourteen female. The patients ranged in age from 7 months to 79 years (mean, 37.6 years). Sinusitis (9 cases, 31.0%) is the most common etiology. Fourteen cases received both medical and surgical treatments. Eighteen cases had purulent discharge from the infection areas sent for culture isolation of the microorganism. The culture positive rate was 50% (9 in 18 cases). The Staphylococcus aureus (5 cases) was the most common pathogen. The bacterial susceptibility test showed drug resistance of 100% for penicillin G (seven out of seven cases; 7/7), 100% for ampicillin (10/10), and 0% for amikacin (0/3) and vancomycin (0/7). Penicillin and ampicillin are not effective for those isolated bacteria. Oxacillin and gentamicin, frequently used in first line treatment, might encounter drug resistance in some cases. Amikacin and vancomycin, without any resistance in bacterial susceptibility tests, could be used in vision-threatening, critical, and intractable cases.

Fungal corneal ulcers of onion harvesters in southern Taiwan.

Fungal corneal ulcers related to agriculture has been reported throughout the world, especially in tropical areas. Most of them were sporadic and had histories of ocular trauma or use of topical corticosteroids and topical antibiotics. Five onion harvesters had fungal corneal ulcers during the same harvest period in Southern Taiwan. The authors think that this is the first report of a group occurrence relating to agricultural workers. Although all of the patients improved after medical and surgical management, their vision was greatly decreased. It is suggested that the tropical climate, the harvest procedure, the characteristic monsoon, and lack of eye protection were involved. Therefore, the importance of the eye protection, hygiene education, and improving medical care to reduce the occurrence of fungal corneal ulcer in agriculture workers must be emphasised.

Calcium-induced changes on crystallins in organ-cultured porcine lens.

In this study, intact porcine lenses were cultured in vitro for 7 days supplemented with commercial balanced salt solution (BSS) which is usually used as an irrigation solution during intraocular surgery, and the lenses were maintained under various culture conditions, e.g. temperature and CO2 concentration. The intact porcine lenses after 7 days culture were analyzed with optical density scanner, gel permeation chromatography on TSK HM-55 column and SDS-PAGE (polyacrylamide gel electrophoresis). It was found that lenses exhibited the least opacity when lenses were cultured with Ca(+2)-free BSS buffer, CO2-free incubator and maintained at a temperature of 25 degrees C. After the lenses were cultured with Ca(+2)-free BSS or BSS medium, the composition of crystallins in lenses was separated with TSK HM-55 column. It was indicated that the percentage of high molecular weight (HMW) protein and (alpha-crystallin increased, and gamma-crystallin decreased in lenses incubated with BSS medium compared with lenses incubated with Ca(+2)-free BSS medium. Following an increase in the concentration of calcium in the medium from 4.3 mM, 20 mM, 50 mM, 100 mM to 200 mM, the opacity of the lens was measured with a densitometer. The changed percentage of various crystallins was similar to lenses with BSS media that increased in HMW protein and alpha-crystallin, decreasing in gamma-crystallin. In the case of lens protein pattern, the crystallin washed from TSK HM-55 gel was separated with SDS-PAGE (polyacrylamide gel electrophoresis). It was indicated that some of proteins disappeared when lenses were incubated with various concentrations of calcium. The vanished pH proteins were 20.5 kDa at 50 mM calcium, 20.5 kDa and 21 kDa at 100 mM, 20.5 kDa, 21 kDa, 22 kDa and 23 kDa at 200 mM which were compared with the protein bands in the presence of 20 mM calcium in BSS medium. This study indicates that the commercial balanced salt solution (BSS) which is usually used as an irrigating solution during intraocular operations may increase the risk for lens opacity because of the calcium contained in the solution.

[Clinical study on effect of kang' aibao oral liquid in treating 103 malignant tumor patients].

OBJECTIVE: To observe the clinical efficacy of Kangaibao oral liquid(KABOL), a Chinese herbal preparation in treating malignant turmor of middle or advanced stage. METHODS: A comparative study was done by observing the effect of 103 patients treated with KABOL, and another 90 patients treated with chemotherapy in the same period were taken as a control group for comparison. RESULTS: The immunologic function, short-term effective rate (complete relieved rate and partial relieved rate), survival quality, 1-, 2- and 3-year survival rate, median suvival time, and the CEA declining rate of the KABOL group were significantly higher than those of the control group. CONCLUSIONS: KABOL has the effects of inhibiting tumor growth, prolonging the survival time and improving the quality of survival of tumor patient.

Genetic transformation of Lycium barbarum L. via A. tumefaciens.

A system for transformation and regeneration of Lycium barbarum L., an important Chinese medical plant, has been established. Young stem segments from Lycium barbarum L. were infected with Agrobacterium tumefaciens C58cl(pGV3850::neo1103), and the transformed calli selected from the callus induction medium containing 50 micrograms/ml kanamycin could regenerate buds on differentiation medium containing 25 micrograms/ml kanamycin. 30% of the regenerated buds were normal in morphology. The normal buds could develop into whole plantlets after they were transferred to the rooting medium to induce roots. Nopaline detection, NPT-II enzyme activity assay and Southern blotting hybridization indicated that the foreign genes had been integrated into the genome of Lycium barbarum L. and expressed in the plant. In the processes of experiments, it was found that (i) after the pre-processes, the explants which formed callus quickly were easy to transform; (ii) the rate of normal regenerated plants from transgenic calli was higher than that from the untransgenic ones.

The inhibitory effect of Chinese tea and its polyphenols on in vitro and in vivo N-nitrosation.

The objective of this study is to evaluate the possible role of Chinese tea as a natural inhibitor of N-nitrosation, and to compare the relative inhibitory potency of various kinds of Chinese tea in vitro and in vivo. Studies on the inhibitory effect of 145 samples of Chinese tea on the formation of N-nitrosomorpholine (NMOR) showed that the inhibitory potency of the 7 types of Chinese tea differed greatly, the average blocking rate of green tea (89.04%, n = 60), crush, tear, and curl (CTC) black tea (86.99%, n = 13), brick tea (85.40%, n = 7), jasmine tea (84.99%, n = 21), Oolong tea (82.37%, n = 9), sun-dried tea (61.99%, n = 13) and Chinese Gongfu black tea (54.98%, n = 22), were positively correlated with their polyphenols contents. The inhibitory effect of green tea and black tea on endogenous N-nitrosation was also confirmed in 3 human volunteer experiments. Drinking tea after meal had a greater effect than drinking it before meal. It is concluded that Chinese tea can exert a relatively strong inhibitory potency for N-Nitroso compounds (NOC) formation both in vitro and in humans, and the active constituents may be related to their polyphenols contents, especially the tea catechin derivatives. The results also demonstrated that the amounts of NMOR formed in vitro depended on the molecular structure of tea catechin derivatives and their molar ratios to nitrite.

Comprehensive assessment of long-term postoperative continence in pediatric imperforate anus. Clinical, manometric and contrast studies.

109 patients with imperforate anus were followed up postoperatively for 5 to 28 years. Postoperative continence was evaluated by anorectal manometry, barium enema and clinical symptoms. A comprehensive assessment system, combining the clinical and objective systems was designed which can reflect postoperative anal continence function completely and objectively. It is useful for determining the causes of incontinence and its treatment.

[Determination of hydroxygenkwanin and genkwanin in flos Genkwa before and after processing by a TLC-scanner].

Thin-layer chromatography and a dual wavelength TLC-scanner (Shimadzu CS-910) were used for the determination of hydroxygenkwanin and genkwanin in flos Genkwa before and after processing with vinegar. The result shows that the amounts of hydroxygenkwanin and genkwanin in processed products were increased. The amounts of the said two constituents were different in various products.

Studies on the nature of prolactin-like immunoreactivity in bromelin-treated cervical mucus.

Prolactin has been reported to be present in cervical mucus at concentrations higher than those found in blood. Our initial findings appeared to confirm this and the material fulfilled criteria of validity generally applied when an immunoassay is employed on a new biological matrix, i.e. parallelism and chromatographic identity. Further experiments demonstrated that prolactin concentrations in cervical mucus were less than 40 mU/L and the prolactin-like immunoreactivity originally detected was due to the action of the enzyme bromelin which was used to liquefy the mucus. Bromelin has a similar molecular weight to prolactin and appeared to digest prolactin tracer and reduce its ability to bind antiserum in a manner paralleling the effect of adding pituitary prolactin.