Effect of patchouli alcohol on macrophage mediated Helicobacter pylori digestion based on intracellular urease inhibition.

BACKGROUND: Helicobacter pylori infects almost half of the world population and is listed as a type I carcinoma factor since 1994. Pogostemon cablin (Blanco) Benth. (Labiatae) has been used to treat gastro-intestinal diseases for thousands of years in many east Asian countries, and the key ingredient, patchouli alcohol (PA), has been observed to exert anti-H. pylori and anti-urease activities. PURPOSE: We investigated the effect of PA on H. pylori urease and its subsequent influence on macrophage phagosome maturation and function. METHODS: In H. pylori experiment, the berthelot method and pH shock assay were adopted to evaluate the effect of PA on extracellular and intracellular H. pylori urease. And then, Q-PCR and Western blot were carried out to analyze the alterations in the expression of urease-related genes and proteins after PA treatment. In the H. pylori and macrophage cell (RAW264.7) co-culture experiment, the effects of PA on H. pylori-induced phagocytosis and intracellular killing of RAW264.7 were investigated using gentamycin protection assay, and the underlying mechanism was explored by immunofluorescence. RESULTS: PA at 25 and 50 µM inhibited intracellular H. pylori urease activity but not isolated urease by down-regulating the gene expression levels of ureB, ureE, ureI and nixA and reducing the protein expression level of UreB, thereby inhibiting the acid resistance of H. pylori. PA also recovered the function of macrophage bacterial digestion, and prior treatment with ammonium chloride inhibited the efficacy of PA. CONCLUSION: PA suppressed intracellular H. pylori urease function and maturation, which increased macrophage digestion ability.

Management and maintenance of the airway in cervical necrotising fasciitis: a retrospective analysis of 15 cases.

Cervical necrotising fasciitis is a progressive deep infection of the neck associated with high mortality, and skillful management of the airway is critical for operations under general anaesthesia. Tracheostomy under local anaesthesia has been considered the gold standard of airway management in patients with deep neck infections, but it may be difficult or impossible in advanced cases. We report here our experience over 6 years (January 2008 and December 2013) during which a total of 15 patients was diagnosed with cervical necrotising fasciitis. Of 6 patients, admitted between January 2008 and March 2010, 5 had routine tracheostomy under local anaesthesia, 1 had direct laryngoscopy intubation, and 9 who were admitted between Spring 2010 and December 2013 were treated with nasotracheal intubation. Postoperatively all patients were given moderate sedation and analgesia. Nasotracheal intubation was continued until the infection had been controlled. During intubation patency of the endotracheal tube was maintained by humidification with a continuous pump of 0.45% sodium chloride and suction. All 15 patients (10 men and 5 women, mean age 62 years, range 36-93) required an emergency drainage procedure under general anaesthesia. Fourteen of the 15 had evidence of compromise of the airway, but emergency intervention was not required. Since Spring 2010, 9 consecutive patients had required nasotracheal intubation, including 7 video laryngoscopies and 2 fibreoptic bronchoscopies. No other interventions were required. Patients were intubated postoperatively from 3 to 14 days, and there were no problems with the airway. Advanced techniques for control of the airway have a high rate of success in patients with necrotising fasciitis and could be an appropriate alternative to a traditional airway. Postoperative sedation and analgesia should be considered as routine management of pain and anxiety.

First Report of Gray Mold of Rhizoma paridis Caused by Botrytis cinerea in China.

Rhizoma paridis is a perennial, traditional Chinese medicinal herb. In May 2013, a disease was observed in an approximately 10 ha cultivated field in Enshi, Hubei Province, China. Approximately 80% of plants in the field were affected. Symptoms were visible on the basal leaves of affected plants. Chlorosis followed by necrosis started at the leaf tips and margins and gradually spread inward until the entire leaf was necrotic. Thick, gray mycelium and conidia were visible on both sides surface of leaves under wet, humid conditions. The leading edge of the chlorotic leaves was excised from 20 plant samples surface disinfested with 1% NaOCl solution for 1 min, rinsed in sterile water, air dried, and placed on potato dextrose agar (PDA). Plates were incubated at 22°C in the dark. Mycelia were initially hyaline and white, and became dark gray after 72 h. Mycelia were septate with dark branched conidiophores. Conidia were smooth, hyaline, ovoid, aseptate, and ranged from 8 to 14.5 × 7 to 8.5 µm. Numerous hard, small, irregular, and black sclerotia that were 1 to 3 × 2 to 5 mm were visible on PDA plates after 12 days. The fungus was identified as Botrytis cinerea on the basis of these characters (1). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 and ITS4 primer and sequenced (GenBank Accession No. KF265499). BLAST analysis of the PCR product showed 99% identity to Botryotinia fuckeliana (perfect stage of B. cinerea) (EF207415.1, EF207414.1). The pathogen was further identified to the species level as B. cinerea using gene sequences from glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) (2) (KJ638600, KJ638602, and KJ638601). Pathogenicity was tested by spraying the foliage of 40 two-year-old plants with a suspension of 106 conidia per ml of sterile distilled water. Each plant received 30 ml of the inoculum. Ten healthy potted plants were inoculated with sterilized water as control. All plants were covered with plastic bags for 5 days after inoculation to maintain high relative humidity and were placed in a growth chamber at 22°C. The first foliar lesions developed on leaves 7 days after inoculation and were similar to those observed in the field. No symptoms developed on the control plants. B. cinerea was consistently re-isolated from all artificially inoculated plants. The pathogenicity test was completed twice. To our knowledge, this is the first report of gray mold of R. paridis caused by B. cinerea in China. The root of R. paridis is the most commonly used Chinese herbal medicine to treat viper bites. In recent years, cultivation of this herb has increased in China because of its high value. Consequently, the economic importance of this disease is likely to increase with the greater prevalence of this host species. References: (1) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. Burgess Publishing Company, Minneapolis, MN, 1972. (2) M. Staats et al. Mol. Biol. Evol. 22:333, 2005.

First Report of Root Rot on Atractylodes macrocephala (Largehead Atractylodes Rhizome) Caused by Ceratobasidium sp. in China.

Atractylodes macrocephala is a perennial herbaceous plant (family Asteraceae) native to China. The biennial root, Largehead Atractylodes Rhizome (LAR), is the most commonly used Chinese herbal medicine to prevent early pregnancy loss due to miscarriage. From summer 2010 to spring 2012, symptoms of root rot were observed on LAR in Xianfeng county, Enshi city, Hubei Province, China. White mold on the root of LAR could be observed at an early growth stage in the field and the white mold spread over the entire plant after 10 days, which differs from root rot of LAR caused by Fusarium oxysporum and Rhizoctonia solani, neither of which are characterized as having mycelium spreading over the whole plant (4). Where root rot symptoms were present, rhizome yield was reduced by 15% on average, with up to 40% yield loss in some fields. Under humid conditions in mid-June, the disease in the field spread quickly and the rhizomes of LAR were completely rotted. After rainfall and increasing temperature from 16 to 35°C, white mycelium appeared and plants withered within a few weeks. In April 2011 and 2012, a fungus was consistently recovered from symptomatic rhizome samples after they were surface sterilized with 0.1% mercuric chloride solution and plated onto potato dextrose agar (PDA). Pale gray colonies with short aerial mycelia and brown sclerotia formed on PDA after 7 days incubation at 28°C. Binucleate cells were observed using light microscopy and the characteristics were matched with morphological characteristics of a Ceratobasidium sp (3). Genomic DNA of the culture was extracted, and the rDNA-internal transcribed spacer sequence (GenBank Accession No. JQ926741) showed 99% identity to Ceratobasidium sp (GenBank No. H269825.1). Mycelial plugs of the culture taken from PDA were inoculated onto 40 rhizomes of 1-year-old seedlings and plants were incubated with a 16-h photoperiod at 28°C and 90% relative humidity in an artificial climate chamber where they developed typical disease symptoms after 2 days. Ten rhizomes of 1-year-old seedlings and were treated with PDA plugs only. All seedlings inoculated with the pathogen were withered and the rhizomes were completely covered with gray mycelium 2 days after inoculation, which was similar to the symptoms observed in the field. After 7 days, the symptoms were more severe than those observed in the field, with seedlings rotted completely. The main stalk of all inoculated plants was covered with gray mycelia in 4 days, and the stalk became withered, which was similar to the symptoms observed in the field. No symptoms were observed on control seedlings and plants. Koch's postulates were fulfilled by successful reisolation of Ceratobasidium sp. from diseased seedlings. The pathogenicity tests were carried out twice. Ceratobasidium sp. has been reported to cause root rot of canola in Washington (2). It has also been observed on Rehmannia in China (1). To our knowledge, this is the first report of Ceratobasidium sp. causing root rot on LAR. References: (1) B. B. Chen et al. Chin. J. Chin. Material Medica (In Chinese) 9:1137, 2011. (2) K. L. Schroeder et al. Plant Dis. 96:591, 2012. (3) B. Sneh et al. Page 39 in: Identification of Rhizoctonia Species. The American Phytopathological Society, 1991. (4) S. X. Zang et al. J. Agric. Univ. Hebei (In Chinese) 28:73, 2005.