[Mechanism of intestinal injury induced by WNT2B high-expressed fibroblasts in Crohn's disease].

Objective: To explore the mechanism of intestinal tissue damage induced by macrophages activated by WNT2B high-expressed fibroblasts. Methods: This study involved biological information analysis, pathological tissue research and cell experimental research. The biological information of the colon tissue from the children with inflammatory bowel disease in previous study was analyzed again with single-cell sequencing. The pathological tissues were collected by colonoscopy from 10 children with Crohn's disease treated in the Department of Gastroenterology of Guangzhou Women and Children's Medical Center from July 2022 to September 2022. According to the findings of colonoscopy, tissues with obvious inflammation or ulceration were classified as the inflammatory group, while tissues with slight inflammation and no ulceration were classified as the non-inflammatory group. HE staining was performed to observe the pathological changes of the colon tissues. Macrophage infiltration and CXCL12 expression were detected by immunofluorescence. In terms of cell experiments, fibroblasts transfected with WNT2B plasmid or empty plasmid were co-cultured with salinomycin treated or non-treated macrophages, respectively; the expression of proteins through Wnt classical pathway were detected by western blotting. Macrophages treated with SKL2001 were used as the experimental group, and those with phosphate buffer as the control group. The expression and secretion of CXCL12 in macrophages were detected by quantitative Real-time PCR and enzyme-linked immunosorbent assay (ELISA). T-test or rank sum test were used for the comparison between groups. Results: Single-cell sequencing analysis suggested that macrophages were the main cells in inflammatory bowel disease colon tissue, and there was interaction between WNT2B high-expressed fibroblasts and macrophages. HE staining of the 10 patients ((9.3±3.8) years old, 7 males and 3 females) showed that the pathological score of colon tissue in the inflammatory group was higher than that in the non-inflammatory group (4 (3, 4) vs. 2 (1, 2) points, Z=3.05, P=0.002). Tissue immunofluorescence indicated that the number of infiltrating macrophages in the inflammatory group was significantly higher than that in the non-inflammatory group under high power field of view (72.8±10.4 vs.8.4±3.5, t=25.10, P<0.001), as well as the number of cells expressing CXCL12 (14.0±3.5 vs. 4.7±1.9, t=14.68, P<0.001). In cell experiments, western blotting suggested an elevated level of glycogen synthase kinase-3ß phosphorylation in macrophages co-cultured with fibroblast transfected with WNT2B plasmid, and salinmycin could reverse this change. Real-time PCR suggested that the transcription level of CXCL12 in the experimental group was higher than that in the control group (6.42±0.04 vs. 1.00±0.03, t=183.00, P<0.001), as well as the expression and secretion of CXCL12 by ELISA ((465±34) vs. (77±9) ng/L, t=13.21, P=0.006). Conclusion: WNT2B high-expressed fibroblasts can secrete WNT2B protein and activate the Wnt classical signaling pathway thus enhancing the expression and secretion of CXCL12 in macrophages, inducing the development of intestinal inflammation of Crohn's disease.

Acute Toxicity and Hazardous Concentrations of Zinc to Native Freshwater Organisms Under Different pH Values in China.

Zinc bioavailability to aquatic organisms varies greatly under different pH values. In the present study, five native species in China and four common international test species were selected to investigate the influence of changing pH on acute zinc toxicity. The results showed that the higher trophic levels exhibited increasing sensitivity to zinc as pH decreased. However, when the pH value was between 8 and 11, the acute toxicity of zinc was relatively constant. In addition, by using a species-sensitivity distribution (SSD) method, the short-term hazardous concentrations of zinc at different pH values (based on the 5th percentiles of the pH-specific SSDs) were determined to be 17.26 µg/L (pH 4), 48.31 µg/L (pH 5), 80.34 µg/L (pH 6) and 230.6 µg/L (pH 7), respectively. The present study provides useful information for deriving water quality criteria and assessing the risks of metals in the near future.

[The role of poloxamer 188 for cord blood mononuclear cells into megakaryocytes cultivation and induction in three-dimensional WAVE Bioreactor].

Objective: To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC). Methods: The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14. Results: In the two-dimensional (2D) culture, CD41(+), CD41(+)/CD61(+), CD61(+) megakaryocytic numbers increased significantly after adding P188 (all P<0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group (P<0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P>0.05). Conclusion: 3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.

Genetic and epigenetic alterations induced by different levels of rye genome integration in wheat recipient.

The narrow genetic variation present in common wheat (Triticum aestivum) varieties has greatly restricted the improvement of crop yield in modern breeding systems. Alien addition lines have proven to be an effective means to broaden the genetic diversity of common wheat. Wheat-rye addition lines, which are the direct bridge materials for wheat improvement, have been wildly used to produce new wheat cultivars carrying alien rye germplasm. In this study, we investigated the genetic and epigenetic alterations in two sets of wheat-rye disomic addition lines (1R-7R) and the corresponding triticales. We used expressed sequence tag-simple sequence repeat, amplified fragment length polymorphism, and methylation-sensitive amplification polymorphism analyses to analyze the effects of the introduction of alien chromosomes (either the entire genome or sub-genome) to wheat genetic background. We found obvious and diversiform variations in the genomic primary structure, as well as alterations in the extent and pattern of the genomic DNA methylation of the recipient. Meanwhile, these results also showed that introduction of different rye chromosomes could induce different genetic and epigenetic alterations in its recipient, and the genetic background of the parents is an important factor for genomic and epigenetic variation induced by alien chromosome addition.

Double-loop locking cross-stitch suture and suspension fixation for medial collateral ligament origin reconstruction.

The purpose of this study was to summarize the clinical application and results of the double-loop locking cross-stitch suture and suspension fixation method for medial collateral ligament origin reconstruction. Thirty-six patients (21 males, 15 females) with an average age of 40 years (range = 17-58 years), who underwent treatment for acute fracture of the medial collateral ligament at our hospital from February 2008 to May 2009, were included in this study. All patients presented unilateral injuries (17 right-sided, 19 left-sided) and underwent repair with the double-loop locking cross-stitch suture and suspension fixation method. All incisions in this group of patients healed by first intention. Thirty-two patients were followed up for 6-20 months (average = 12 months). There were no reports of wound infection, ligament re-fracture or other complications in the follow-up period. Based on the Lysholm knee-scoring scale, the patients received a 100% excellent and good rating (20 patients - excellent score, 12 patients - good score) postoperatively. The advantages of the double-loop locking cross-stitch suture and suspension fixation method are a smaller incision, reliable fixation, and early restoration of knee joint stability. It is, therefore, an effective and low-risk method for the reconstruction of medial collateral ligament origin.

Cross-leg repair of large soft-tissue defects in distal sites of the feet by distally based neuro-fasciocutaneous flaps with perforating vessels.

The objective of this study was to introduce a method for repairing large soft-tissue defects on the foot. Distally based neuro-fasciocutaneous flaps with perforating vessels were designed along the saphenous and sural neurovascular axes. The cutaneous perforating branches of the major arteries of the lower extremities were used as pedicles, which provided a rotation arc for the cross-leg flap to cover the large-sized soft-tissue defects on the foot. We transferred 6 neurocutaneous vascular axial flaps, including 4 saphenous neurocutaneous axial flaps (ranging from 25 x 13 to 17 x 9 cm in area) with posterior tibial perforators as the pedicle, and 2 sural neurocutaneous axial flaps (ranging from 29 x 12 to 18 x 7 cm in area) supplied by the perforating branches of the peroneal vessels. These 6 cases of neuro-fasciocutaneous flaps survived with satisfactory cosmetic appearances and functional results on follow-up at 8 to 17 months post-surgery. Placing a distally based neuro-fasciocutaneous cross-leg flap with perforating vessels is an effective method for repairing large-sized soft-tissue defects on the foot.

First Report of Streptomyces galilaeus Associated with Common Scab in China.

During a survey of potato scab pathogens in China from 2003 to 2012, a new pathogen was found in Shanxi and Neimenggu provinces. The incidence was approximately 20% of all recovered strains. The lesions caused by the pathogen were slightly raised and similar to those caused by Streptomyces scabies (3). Lesions were excised (approximately 10 mm3) from 40 infected tubers, surface-disinfested with 0.3% NaOCl for 30 s, rinsed in sterile water three times, cut into 5 mm3, then sliced into 1-mm pieces, and plated on water agar amended with ampicillin (50 µg/ml). Plates were incubated at 28°C in the dark for 4 days. The spores of Streptomyces sp. strains growing from the tuber pieces were collected from single bacterial colonies and cultured on oatmeal agar. To fulfill Koch's postulates, one strain, CPS-2, was grown at 28°C for 10 days and the spores were washed from the plates as inoculum. One hundred milliliters of inoculum (1 × 105 CFU/ml) was mixed with autoclaved soil and vermiculite (1:1) in each pot (15 cm in diameter). Cut tubers were planted in the pots (potato cv. Favorita, one plant per pot, five replicates) and grown under greenhouse conditions (22 ± 5°C). Typical common scab symptoms consisting of small, brown, raised lesions developed on potato tubers 12 weeks after planting. The same strain was re-isolated from the lesions of the new scabby tubers. Non-inoculated plants, treated as described above, but without strain CPS-2, remained healthy. The CPS-2 strain was identified based on morphological and physiological characterization and 16S rDNA sequence. On yeast-malt extract agar, the test strain produced grayish-white aerial hypha, reddish brown substrate mycelium and pigments, and loose spiral spore chains. Spores were smooth and were 0.8 to 0.9 × 1.1 to 1.2 µm in size (diameter and length). The ability of the strain to use single sources of carbon and nitrogen was verified according to the International Streptomyces project (4). The strain grew in media supplemented with L-arabinose, D-fructose, D-glucose, rhamnose, raffinose, meso-inositol, sucrose, and D-xylose, but not D-mannitol. It used L-hydroxyproline, L-methionine, and L-histidine, and produced melanin on tyrosine and peptone yeast extract agar. The strain did not grow at a pH less than 5.0 and was sensitive to streptomycin (20 µg/ml), phenol (0.1%), and crystal violet (0.5 µg/mL), but not to penicillin (10 IU/ml). The strain also produced hydrogen sulfide. The biological characteristics of strain CPS-2 were in accord with Streptomyces galilaeus. CPS-2 produced thaxtomin A in oatmeal liquid medium and the txt AB gene fragment was successfully amplified using specific primers (2). The 16S rDNA sequence of CPS-2 was amplified by PCR with primers 16S1-F: 5'-CATTCACGGAGAGTTTGATCC-3' and 16S1-R: 5'-AGAAAGGAGGTGATCCAGCC-3' (1) and sequenced. A BLAST search of the 16S rDNA sequence for CPS-2 was conducted using the NCBI GenBank database, resulting in 99.8% similarity to S. galilaeus (NR_040857). The 16S rDNA sequence for CPS-2 (1,388 bp) was deposited in GenBank (AY621378). To our knowledge, this is the first report of S. galilaeus causing common scab of potato in China. References: (1) R. A. Bukhalid et al. Appl. Environ. Microbiol. 68:738, 2002. (2) R. Flores-González et al. Plant Pathol. 57:162, 2008. (3) D. H. Lambert and R. Loria. Int. J. Syst. Bacteriol. 39:387, 1989. (4) E. B. Shirling and D. Gottlieb. Int. J. Syst. Bacteriol. 16:313, 1966.

First Report of Alternaria alternata Causing Blight on Zanthoxylum piperitum in China.

Since 2009, brown leaf spot and panicle blight of Zanthoxylum piperitum (L.) DC. ("liujin," commonly known as Japanese pepper and Japanese pricklyash) has been observed on 40% of the plants in the test field of Foresty Academy of Science in Hebei Province of China. When symptoms formed on leaves, a thick yellow spot appeared, which then turned brown. When on the spikes, brown lesions were observed initially on the grain, which then spread down to fruit stem, and finally the whole spike wilted and dried up. Yield and quality losses were considerable. A fungus was isolated consistently from the diseased tissues using potato dextrose agar (PDA) (1). Three representative isolates were chosen for further characterization. All the isolates grew at 28°C on PDA and potato carrot agar (PCA) medium. Fungal colonies were initially white, then became olivaceous with some white mycelium on the top of the colony, and turned brown with age. When observed with the microscope, crineous septate hypha appeared, and conidiophore peduncles were upright or slightly curved, with a few branches, 33.0 to 75.0 µm long and 4.0 to 5.5 µm wide. Conidia were crineous short clubs or near oval in shape, 22.5 to 40.0 µm long and 8.0 to 13.5 µm wide, with a short conical beak, and had one to four longitudinal cross walls. On PCA, condia had three to seven transepta and one to five longisepta, and were produced in a branched, long chain with more than five conidia. The pathogen was identified based on morphological characteristics as Alternaria alternata (Fr.:Fr.) Keissl. (3). DNA was extracted from mycelium and PCR was performed on the internal transcribed spacer (ITS) region with primers ITS1 and ITS4. A 570-bp fragment was amplified and sequenced (GenBank Accession No. JQ973810). BLASTn analysis revealed there was 100% sequence identity with A. alternata strains (GU566303 and GQ121322). To further identify the fungus, A. alternata species-specific primers AAF2/AAR3 (2) were used to generate an amplicon which was then sequenced (JX308287). Sequence comparison showed there was 100% sequence identity with A. alternata (JQ927300 and JQ907485). Pathogenicity tests were performed by spraying with a cultured suspension (106 spores/ml) of approximately 100 µl onto healthy leaves in 15-cm-diameter glass dishes containing sterilized filter paper soaked with sterilized water at room temperature. Control plants were inoculated with sterile distilled water. Ten days after inoculation, symptoms were observed in all inoculated leaves and appeared to be identical to those observed in the field. No symptoms were noted on the control leaves. Identical results were also obtained when spikes were inoculated. The fungi reisolated from symptomatic plants were A. alternata. To our knowledge, this is the first report of A. alternata causing leaf spots and panicle blight of Z. piperitum in China. References: (1) O. D. Dhingra and J. B. Sinclair. Basic Plant Pathology Methods. CRC Press, Boca Raton, FL, 1995. (2) P. Konstantinova. et al. Mycol. Res. 106:23, 2002. (3) T. Y. Zhang. China fungi records (Alternaria) (Volume 16) (in Chinese). Beijing: Science Press, 2003.

First Report of Peanut Pod Rot Caused by Neocosmospora vasinfecta in Northern China.

From 2006 to 2010, peanut (Arachis hypogaea) pod rot became more prevalent in northern China, especially in the Sha River drainage area. The incidence of pod rot ranged from 30 to 100%. Typical symptoms were black rot of the pods, but no obvious morphological abnormality of the aboveground parts of infected plants was observed. Brown or black spots appeared on many pods when initially infected and then all peanut pods became black and rotten. The same fungus was isolated from 54 surface-disinfested lesions (85.2% of all lesions) on potato dextrose agar (PDA) media. One isolate, designated as HBXLb, was chosen for further characterization. In culture, both anamorph and teleomorph were present. Mycelia of the fungus grew quickly (colonies were 3.2 cm in diameter in 3 days) and became white and floccose on PDA at 28°C. The hyaline, elongated-to-cylindrical conidia aggregated on the slimy heads of conidiogenous cells that developed on undifferentiated hyphae after incubation for 3 to 4 days. Conidial sizes varied from 5 to 10 × 1.5 to 3 µm (n = 50) and were mostly single celled. Some conidia appeared slightly curved. The morphology was consistent with Acremonium spp. Numerous ascomata (perithecia) formed within 10 to 14 days when incubated at 28°C under light and dark conditions. Perithecia were orange-brown, strawberry shaped (300 to 400 µm in diameter), and ostiolate on the top. Cylindrical asci, with an average size of 90 to 110 × 7.5 to 9 µm, were present inside the ascomata with each containing eight ascospores in a row. The ascospores were brownish, spherical to ellipsoidal, and 10 to 15 × 8 to 12 µm. The cultural and morphological characteristics of isolate HBXLb matched the description of N. vasinfecta (2). The internal transcribed spacer (ITS) region of rDNA was amplified by the primer pairs ITS4/ITS5. A 525-bp amplicon (ITS4-5.8s-ITS5) was obtained and sequenced (GenBank Accession No. HM461901). The ITS sequence was a 100% match to N. vasinfecta strain N-JXLN01 (GenBank Accession No. GU213063) by BLASTn in GenBank. Pathogenicity tests were conducted on detached pods of peanut cultivar Jihua 4. Forty surface-disinfested peanut pods were soaked in a conidial suspension (105 conidia per ml) for 2 min and 40 pods were soaked in sterile water as a control. Then all peanut pods were maintained in moist petri dishes under darkness for 14 days at 28°C. Brown or black spots appeared on all pods inoculated with the fungus within 10 days, while the controls remained healthy. Symptoms were similar to those originally observed in the field, and N. vasinfecta could be reisolated from all infected pods. This fungus previously has been reported as the pathogen of foot rot of peanut in South Africa (1), Taiwan (4), and Australia (3). To our knowledge, this is the first report of peanut pod rot caused by N. vasinfecta in China. References: (1) S. W. Baard et al. Phytophylactica 17:49, 1985. (2) O. A. Cornely et al. Emerg. Infect. Dis. 7:149, 2001. (3) M. F. Fuhlbohm et al. Australas. Plant Dis. Notes 2:3, 2007. (4) J. W. Huang et al. Plant Pathol. Bull. 1:203, 1992.

First Report of Neocosmospora striata Causing Peanut Pod Rot in China.

In 2008, an outbreak of pod rot of peanut (Arachis hypogaea L.) occurred on most of the peanut cultivars in the Old Yellow River drainage area, the largest peanut-growing region in China. Disease incidence reached as high as 90% in some fields, causing severe yield losses. The black rot of pods and blackened, nonrotting taproots is similar to symptoms of peanut black rot caused by Cylindrocladium parasiticum, but the reddish orange perithecia of C. parasiticum were not found on the taproots close to the surface of the soil. The foliage of affected plants was generally asymptomatic, but some plants turned greener. This pod rot disease was further investigated in 2008 and 2010. Twenty-three Fusarium-like isolates were obtained from symptomatic, surface-disinfested pods with a frequency of 82%. These isolates were fast growing, with flat, thin, and grayish white colonies when cultured on potato dextrose agar (PDA) at 28°C for 3 to 4 days. The hyaline, elongated to cylindrical conidia, aggregated in slimy heads on conidiogenous cells developed from undifferentiated hyphae when observed with the light microscope. The size of conidia (single celled or one septum) varied from 3 to 9 µm long and 1.5 to 3.5 µm wide on the basis of the measurement of 50 spores. Some conidia appeared slightly curved. Ascomata formed within 10 to 14 days, with a punctate appearance on the colony. The cerebriform ascomata were dark brown, pyriform, ostiolate, glabrous, 120 to 170 × 90 to 130 µm, and with necks 30 to 50 µm long. Asci measured 60 to 90 × 6 to 10 µm, were cylindrical to cylindric-clavate, thin walled, and had an apical ring. Ascospore arrangement was obliquely uniseriate or partially biseriate, very pale yellow to hyaline, ellipsoidal, and measured 8 to 12 × 4.5 to 6 µm. Some spores had a median transverse straight or curved septum and were slightly constricted at the septum, with 6 to 10 thin, transverse, hyaline flanges. Morphological characteristics of the isolates with ascomata dark brown and ascospores with 6 to 10 transverse hyaline flanges matched the description for Neocosmospora striata (1). The internal transcribed spacer (ITS) region of rDNA was amplified from extracted template DNA with primer pairs ITS4/ITS5 and sequenced. A 591-bp amplicon (GenBank Accession No. HM461900) had 99% sequence identity with Fusarium solani (HQ607968 and HQ608009) and N. vasinfecta (GU213063), which indicated that these fungi belong to the genus Neocosmospora or Fusarium, although there is no direct sequence evidence that they are N. striata. N. striata has only been previously reported in Japan (2). This species is unique because of the dark brown ascomata and there is no comparable species (1). Koch's postulates were completed by surface-disinfesting 80 peanut pods of cv. Jihua 9813 and soaking them in conidial suspensions (105 conidia/ml) for 2 min. Another 80 other pods soaked in sterile water served as controls. All peanuts were incubated in moist petri dishes under darkness at 28°C. Symptoms similar to those originally observed in the field formed within 10 days on all inoculated peanut pods and not the controls. N. striata was reisolated from all affected peanut pods. To our knowledge, this is first report of N. striata causing peanut pod rot in China and the first description of the anamorph of the fungus. References: (1) P. F. Cannon et al. Trans. Br. Mycol. Soc. 82:673, 1984. (2) S. Udagawa et al. Trans. Mycol. Soc. Jpn. 16:340, 1975.

Seedling and Slow Rusting Resistance to Leaf Rust in Chinese Wheat Cultivars.

Identification of resistance genes is important for developing leaf rust resistant wheat (Triticum aestivum) cultivars. A total of 102 Chinese winter wheat cultivars and advanced lines were inoculated with 24 pathotypes of Puccinia triticina for postulation of leaf rust resistance genes effective at the seedling stage. These genotypes were also planted in the field for characterization of slow rusting responses to leaf rust in the 2006-07 and 2007-08 cropping seasons. Fourteen leaf rust resistance genes-Lr1, Lr2a, Lr3bg, Lr3ka, Lr14a, Lr16, Lr17a, Lr18, Lr20, Lr23, Lr24, Lr26, Lr34, and LrZH84-either singly or in combinations, were postulated in 65 genotypes, whereas known resistance genes were not identified in the other 37 accessions. Resistance gene Lr26 was present in 44 accessions. Genes Lr14a and Lr34 were each detected in seven entries. Lr1 and Lr3ka were each found in six cultivars, and five lines possessed Lr16. Lr17a and Lr18 were each identified in four lines. Three cultivars were postulated to possess Lr3bg. Genes Lr20, Lr24, and LrZH84 were each present in two cultivars. Each of the genes Lr2a and Lr23 may exist in one line. Fourteen genotypes showed slow leaf rusting resistance in two cropping seasons.

Visfatin stimulates production of monocyte chemotactic protein-1 and interleukin-6 in human vein umbilical endothelial cells.

Monocyte chemotactic protein-1 and interleukin-6 are important inflammatory cytokines, which have close relationships with atherosclerosis. Visfatin is a novel adipokine involved in regulation of inflammatory cytokines, however, associations of visfatin with cytokines (MCP-1, IL-6) in human umbilical vein endothelial cells are unclear. The aim of this study was to determine whether visfatin has effects on the expression of MCP-1 and IL-6 in human umbilical vein endothelial cells. Enzyme-linked immunosorbent assay were used for measuring MCP-1 and IL-6 production in human umbilical vein endothelial cells. Real-time quantitative reverse-transcription polymerase chain reaction was used for determining MCP-1 and IL-6 mRNA expression. For the pathway determination following inhibitors were used: wortmannin [phosphatiylinositol 3-kinase (PI3K)], SB203580 [p38 mitogen-activated protein kinase (MAPK)], PD98059 [extracellular signal-regulated kinase (ERK) 1/2)], JNK inhibitor II [c-Jun NH 2-terminal kinase (JNK)]. We demonstrated that visfatin could obviously upregulate secretion of MCP-1and IL-6 in a dose- and time-dependent manner in human umbilical vein endothelial cells. Visfatin-induced effects were diminished by SB203580, wortmannin, and PD98059. In summary, these results suggest that visfatin-induced MCP-1 and IL-6 production involve p38 MAPK, PI3K, and ERK 1/2 pathways in human umbilical vein endothelial cells as determined by inhibition with specific inhibitors.

Molecular mapping of leaf rust resistance gene LrZH84 in Chinese wheat line Zhou 8425B.

Leaf rust, caused by Puccinia triticina, is one of the most widespread diseases in common wheat (Triticum aestivum L.) worldwide. With the objective of identifying and mapping new genes for resistance to leaf rust, F(1), F(2) plants and F(3) lines from a cross between resistant line Zhou 8425B and susceptible line Chinese Spring were inoculated with Chinese P. triticina races THTT and MBHP in the greenhouse. A total of 793 pairs of SSR primers were used to test the parents and resistant and susceptible bulks. Seven polymorphic chromosome 1B markers were used for genotyping the F(2) and F(3) populations. Zhou 8425B carried a single dominant resistance gene, temporarily designated LrZH84, linked to SSR markers gwm582 and barc8 with genetic distances of 3.9 and 5.2 cM, respectively. The Xbarc8 allele co-segregated with Lr26 in the F(3) population. The Xgwm582 allele associated with LrZH84 was identified as a leaf rust resistance gene and shown to be present in the Predgornaia 2 parent of Zhou 8425B. The seedling reaction pattern of LrZH84 was different from those of lines with Lr26, Lr33, Lr44 and Lr46, all of which are located in chromosome 1B. It was concluded that LrZH84 is likely to be a new leaf rust resistance gene.

Recent epidemiological changes in meningococcal disease may be due to the displacement of serogroup A by serogroup C in Hefei City, China.

OBJECTIVES: To determine the change in epidemiological characteristics of meningococcal disease in the Hefei City area and to provide valuable information for developing timely and appropriate public health interventions. METHODS: Meningococcal disease was identified according to the National Disease Surveillance System. Data were collected using standardised questionnaires. Serological and bacteria culture testing was performed on cerebrospinal fluid and serum specimens from suspected cases for evidence of Neisseria meningitidis infection. RESULTS: From July 2003 to June 2007, meningococcal disease was confirmed in 386 cases in the total population. This worked out at an annual incidence of 1.19-2.86 per 100,000 population, which was significantly higher than that in 2002-3 (0.27 cases per 100,000 population). The increase in incidence was accompanied by a shift in age distribution, more cases being reported in adolescents and young adults: the median age increased to 15 years (range 2 months to 78 years). When assessed by age group, middle-school students (aged 12-17) had the highest incidence (6.57 per 100 000 population) and the highest proportion (31.4%). The N meningitidis serogroup was identified in 135 (35.0%) of the cases of meningococcal disease; all were serogroup C. No cases due to serogroup A or other strains were found during the study period. The mean case-fatality rate was 7.3%, with a peak of 16.9% in children younger than 6. Since winter 2003, a vaccination campaign has been partially implemented, but the effectiveness has been limited. CONCLUSIONS: The incidence of meningococcal disease has substantially increased in Hefei City, which may be due to the replacement of serogroup A by serogroup C. A shift in age distribution of cases to adolescents and young adults was found.

First Report of Potato Scab Caused by Streptomyces turgidiscabies in China.

Potato scab, caused by several plant pathogenic Streptomyces species, is known to occur in potato-planting areas worldwide. Symptoms of disease on potato tubers are shallow, raised, or pitted corky lesions (2). In 1998, Streptomyces turgidiscabies was reported as a new potato scab pathogen from Hokkaido, Japan (3). Potato scab has been observed in many potato-cultivation areas in China and incidence of the disease was approximately 6 to 10% in some fields in 2006 (in our survey). To investigate the casual agent of scab disease, isolations were made from scabby potato tubers collected from different areas using oatmeal agar. Identification of an isolate from Shaanxi Province was based on morphological and physiological characterization followed by 16S rRNA confirmation. Characteristics were gray, aerial hypha, rectiflexuous spore chains, a smooth spore surface, and spores that were 0.5 to 0.7 × 1.0 to 1.2 µm. The strain did not produce melanin on tyrosine-peptone-yeast extract agar media, did not produce diffusible pigments, used all the International Streptomyces Project (ISP) sugars (4) as single carbon sources, used l-hydroxyproline, l-tyrosine, and l-histidine as single nitrogen sources but not l-methionine, grew at pH 4.5, was susceptible to streptomycin (20 µg ml-1), phenol (0.1%), and penicillin (10 IU ml-1) but not to crystal violet (0.5 µg ml-1), and produced H2S. The identification was confirmed by comparison of its 16S rRNA sequence with the GenBank database using the BLAST program. The 16S rRNA sequence was amplified by PCR with primers S1: 5'-CATTCACGGAGAGTTTGATCC-3' and S2: 5'-AGAAAGGAGGTGATCCAGCC-3' and sequenced. BLASTn analysis of the sequence obtained showed the highest similarity (99.9%) with S. turgidiscabies type strain ATCC 700248 (GenBank Accession No. AB026221). The sequence was submitted to GenBank (Accession No. AM889495). Pathogenicity of the strain was tested in the greenhouse on potato tubers of cv. Favorita grown in pots (one plant per pot, three replicates). One hundred milliliters of inoculum (1 × 105 CFU ml-1) of the strain was mixed with sterile soil and vermiculite (1:1) in each pot. Potato plants were grown at 25°C and the soil was allowed to dry between waterings. The immature potato tubers were used to evaluate scab symptoms 10 weeks after planting. All tubers inoculated with the pathogen developed typical common scab symptoms consisting of erumpent, brown, corky lesions, which is different from the symptoms caused by S. reticuliscabiei (1). The noninoculated control tubers did not show scab symptoms. S. turgidiscabies was reisolated from lesions of diseased immature tubers. The pathogenicity test indicates that S. turgidiscabies caused scab disease on potato tubers. To our knowledge, this is the first report of S. turgidiscabies causing potato scab disease in China. References: (1) K. Bouchek-Mechiche et al. Int. J. Syst. Evol. Microbiol. 56:2771, 2006. (2) R. Loria et al. Plant Dis. 81:836, 1997. (3) K. Miyajima et al. Int. J. Syst. Bacteriol. 48:495, 1998. (4) E. B. Shirling and D. Gottlieb. Int. J. Syst. Bacteriol. 16:313, 1966.

First Report of Cercospora concors Causing Cercospora Leaf Blotch of Potato in Inner Mongolia, North China.

Cercospora leaf blotch disease of potato (Solanum tuberosum L.) caused by Cercospora concors (Casp.) Sacc (synonym Mycovellosiella concors (Casp.) Deighton) occurs worldwide but mainly has been reported in the cool and temperate climates of Europe, Asia, North America, and eastern Africa. Cercospora leaf blotch is usually a minor disease and may go unnoticed since it commonly occurs simultaneously with other potato leaf diseases such as late blight (caused by Phytophthora infestans) and early blight (caused by Alternaria solani) (2). Symptoms of Cercospora leaf blotch first appear on lower leaves as small, yellowish green, irregular blotches and later may appear on middle and upper leaves. As the leaves expand, the blotches enlarge and become purplish brown or black. Conidiophores and conidia form on the underside of the lesions, giving the lesions a mildewed appearance similar to late blight. Necrotic lesions are distinguished from those caused by the early blight pathogen A. solani by the lack of concentric rings (1). In more severe epidemics of Cercospora leaf blotch, potato leaves may be killed, stem lesions become dark and entire plants die, but no resulting yield loss from the disease has been documented. Potato tubers are not infected. From August to September of 2005, yellow-brown lesions appeared on the upper side of potato leaves (cv. Zihuabai, certified virus free) and gray mildew developed on the underside of leaves in potato field trials conducted in Jining County, 41°N, 113°E of Inner Mongolia, North China. The infections were observed mostly on lower and middle leaves of plants; 20 to 30% of plants were infected. In the laboratory, the mildew was scraped with a sterile scalpel and examined microscopically. The conidiophores were irregular in width, grayish, and highly branched. The conidia were numerous, light to dark, straight or slightly bent, cylindrical or obclavate, with conspicuous scars, and zero to six septa. The mature spores were from 16 to 59 µm long and 4 to 6 µm wide. The teleomorph of the fungus was not found. On the basis of the morphological characters, the causal agent was identified as C. concors. C. concors has been previously identified from potato leaves in the Engshi District of Hubei Province, China (3), but to our knowledge, this is the first report of the fungus causing Cercospora leaf blotch of potato in Inner Mongolia, North China. References: (1) G. D. Franc and B. I. Christ. Page 22 in: Compendium of Potato Diseases. 2nd ed. W. R. Stevenson et al., eds. American Phytopathological Society, St. Paul, MN, 2001. (2) E. R. French. Page 19 in: Compendium of Potato Diseases. 2nd ed. W. R. Stevenson et al., eds. American Phytopathological Society, St. Paul, MN, 2001. (3) S. M. Tian et al. China Potato J. 1:13, 1997.

Gelatinolytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 in rat brain after implantation of 9L rat glioma cells.

The matrix metalloproteinases (MMPs) have come to be highlighted by their close relation to the cell invasion of gliomas. The inhibitors of MMPs have undergone extensive development because of its effectiveness against tumor invasion and angiogenesis. Therefore, a suitable animal model is necessary for searching new MMPs inhibitors against gliomas. In this study, we established an experimental model by implanting 9L glioma cells stereotactically into Fisher344 (F344) rat's brain, and the expression and enzymatic activity of MMP-2 and MMP-9 in 9L glioma cells and in tumor tissue was determined by means of reverse transcription polymerase chain reaction (RT-PCR), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) zymography, in situ film zymography and immunostaining. The results of RT-PCR showed that the mRNA level of MMP-2 in 9L glioma cells was higher than that of MMP-9, and the mRNA expression of MMP-9 was increased along with the growth of malignant gliomas. SDS-PAGE zymography revealed that the expression of MMP-2 and MMP-9 were significantly increased in tumor tissues, and the MMP-9 wasn't detected in normal tissue. The positive stain of MMP-2 and MMP-9 was enhanced with the growth of malignant gliomas, especially for MMP-9. The expression of active gelatinase was found in tumor tissue. In conclusion, the expression of active MMP-2 and MMP-9 was increased in 9L/F344 rat brain during the growth of malignant gliomas at different time intervals, which indicate that 9L/F344 animal model may be a prospective animal model to test new MMPs inhibitors.

Predicting secretory protein signal sequence cleavage sites by fusing the marks of global alignments.

A newly synthesized secretory protein in cells bears a special sequence, called signal peptide or sequence, which plays the role of "address tag" in guiding the protein to wherever it is needed. Such a unique function of signal sequences has stimulated novel strategies for drug design or reprogramming cells for gene therapy. To realize these new ideas and plans, however, it is important to develop an automated method for fast and accurately identifying the signal sequences or their cleavage sites. In this paper, a new method is developed for predicting the signal sequence of a query secretory protein by fusing the results from a series of global alignments through a voting system. The very high success rates thus obtained suggest that the novel approach is very promising, and that the new method may become a useful vehicle in identifying signal sequence, or at least serve as a complementary tool to the existing algorithms of this field.

Site-directed mutagenesis of gentisate 1,2-dioxygenases from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2.

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is the first enzyme in gentisate pathway that catalyses the ring fission of gentisate to form maleylpyruvate. Phylogenetic tree of amino acid sequences from 11 GDOs demonstrates that the GDOs from different genus share identities between 12.1% and 64.8%. According to the alignment result, four highly conserved histidine residues in GDO from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2 were chosen to be substituted with aspartate residues. Enzyme analysis indicated that substitution of any of these four histidine residues had resulted in the complete loss of its catalytic activity.

Arg169 is essential for catalytic activity of 3-hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1.

3-Hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 is an enzyme that utilizes 3-hydroxybenzoate (3-HBA) as substrate yielding gentisate. Site-directed mutagenesis was carried out to define which residues may be involved in catalytic reaction. Substitution of arginine to glutamate at position 169 of the enzyme resulted in the complete loss of catalytic activity. This indicated Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis.