[Effects and mechanism of human umbilical vein endothelial cells-derived exosomes on wound healing in diabetic rabbits].

Objective: The investigate the effects and mechanism of exosomes derived from human umbilical vein endothelial cells (HUVECs) on wound healing in diabetes rabbits. Methods: The experimental research methods were used. The primary vascular endothelial cells (VECs) and human skin fibroblasts (HSFs) were extracted from skin tissue around ulcer by surgical excision of two patients with diabetic ulcer (the male aged 49 years and the female aged 58 years) admitted to Xiangya Third Hospital of Central South University in June 2019. The cells were successfully identified through morphological observation and flow cytometry. The HUVEC exosomes were extracted by ultracentrifugation and identified successfully by morphological observation, particle size detection, and Western blotting detection. Twenty female 3-month-old New Zealand rabbits were taken to create one type 2 diabetic full-thickness skin defect wound respectively on both sides of the back. The wounds were divided into exosomes group and phosphate buffer solution (PBS) group and treated accordingly, with 20 wounds in each group, the time of complete tissue coverage of wound was recorded. On PID 14, hematoxylin-eosin staining or Masson staining was performed to observe angiogenesis or collagen fiber hyperplasia (n=20). The VECs and HSFs were co-cultured with HUVEC exosomes for 24 h to observe the uptake of HUVEC exosomes by the two kinds of cells. The VECs and HSFs were divided to exosome group treated with HUVEC exosomes and PBS group treated with PBS to detect the cell proliferation on 4 d of culture with cell count kit 8, to detect and calculate the cell migration rate at 24 and 48 h after scratch by scratch test, to detect the cell migration number at 24 h of culture with Transwell test, and to detect the mRNA expressions of nuclear factor-erythroid 2-related factor 2 (NRF2) and transcription activating factor 3 (ATF3) by real time fluorescence quantitative reverse transcription polymerase chain reaction. Besides, the number of vascular branches and vascular length were observed in the tube forming experiment after 12 h of culture of VECs (n=3). The VECs and HSFs were taken and divided into PBS group and exosome group treated as before, and NRF2 interference group, ATF3 interference group, and no-load interference group with corresponding gene interference. The proliferation and migration of the two kinds of cells, and angiogenesis of VECs were detected as before (n=3). Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and least significant difference test. Results: The time of complete tissue coverage of wound in exosome group was (17.9±1.9) d, which was significantly shorter than (25.2±2.3) d in PBS group (t=4.54, P<0.05). On PID14, the vascular density of wound in PBS group was significantly lower than that in exosome group (t=10.12, P<0.01), and the collagen fiber hyperplasia was less than that in exosome group. After 24 h of culture, HUVEC exosomes were successfully absorbed by VECs and HSFs. The proliferative activity of HSFs and VECs in exosome group was significantly higher than that in PBS group after 4 d of culture (with t values of 54.73 and 7.05, respectively, P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs (with t values of 3.42 and 11.87, respectively, P<0.05 or P<0.01) and VECs (with t values of 21.42 and 5.49, respectively, P<0.05 or P<0.01) in exosome group were significantly higher than those in PBS group. After 24 h of culture, the migration numbers of VECs and HSFs in exosome group were significantly higher than those in PBS group (with t values of 12.31 and 16.78, respectively, P<0.01). After 12 h of culture, the mRNA expressions of NRF2 in HSFs and VECs in exosome group were significantly higher than those in PBS group (with t values of 7.52 and 5.78, respectively, P<0.05 or P<0.01), and the mRNA expressions of ATF3 were significantly lower than those in PBS group (with t values of 13.44 and 8.99, respectively, P<0.01). After 12 h of culture, the number of vascular branches of VECs in exosome group was significantly more than that in PBS group (t=17.60, P<0.01), and the vascular length was significantly longer than that in PBS group (t=77.30, P<0.01). After 4 d of culture, the proliferation activity of HSFs and VECs in NRF2 interference group was significantly lower than that in PBS group and exosome group (P<0.05 or P<0.01); the proliferation activity of HSFs and VECs in ATF3 interference group was significantly higher than that in PBS group (P<0.05 or P<0.01) and significantly lower than that in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in ATF3 interference group were significantly higher than those in PBS group (P<0.05 or P<0.01) and significantly lower than those in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in NRF2 interference group were significantly lower than those in PBS group and exosome group (P<0.05 or P<0.01). After 24 h of culture, the migration numbers of VECs and HSFs in ATF3 interference group were significantly more than those in PBS group (P<0.05) and significantly less than those in exosome group (P<0.05 or P<0.01); the migration numbers of VECs and HSFs in NRF2 interference group were significantly less than those in PBS group and exosome group (P<0.01). After 12 h of culture, the vascular length and number of branches of VECs in NRF2 interference group were significantly decreased compared with those in PBS group and exosome group (P<0.01); the vascular length and number of branches of VECs in ATF3 interference group were significantly increased compared with those in PBS group (P<0.01) and were significantly decreased compared with those in exosome group (P<0.01). Conclusions: HUVEC exosomes can promote the wound healing of diabetic rabbits by promoting the proliferation and migration of VECs and HSFs, and NRF2 and ATF3 are obviously affected by exosomes in this process, which are the possible targets of exosome action.

[Advances in nutritional support therapy for stroke prevention and treatment].

As a serious disease of death and disability, stroke constitutes a serious threat to human health. Because of stroke patients often have high-risk factors of malnutrition such as dysphagia and autonomic eating disorder, the hospitalization time, mortality and disability rate of stroke patients increases. Nutritional therapy can effectively improve the malnutrition of patients, which are of great significance for the treatment and rehabilitation of stroke and the prevention of its complications. Nutrients are important components of nutrition therapy, and different ways of nutrition therapy directly affect the effect of treatment. This article summarizes effects of nutrients and different nutritional treatments on stroke prevention, morbidity and treatment, and provides a theoretical basis and new thinking for further reducing the incidence rate of stroke, improving the quality of life in patients and reducing the financial burden of society and family.

[The relationship of resilience with cognitive function in patients with first-episode schizophrenia].

Objective: To investigate the correlation between resilience and cognitive function in patients with schizophrenia. Methods: Fifty-nine patients with first-episode schizophrenia and 86 healthy controls were enrolled. Patients with schizophrenia were enrolled from the psychiatric outpatient and inpatient Department of the Third Affiliated Hospital of Sun Yat-sen University from September 2017 to January 2020, while healthy controls were recruited through advertising. The levels of resilience and cognitive function were compared between the two groups.Meanwhile, the partial correlation analysis of resilience and cognitive function of the two groups was performed. Results: There was no statistically significant difference in gender, marriage and age between the two groups (all P>0.05), and there were 39 males and 20 females with an average age of (23.8±7.4) years in the schizophrenia group, while 47 males and 39 females with an average age of (22.9±4.7) years in the healthy control group. However, there was a significant difference inyears of education between the two groups (P<0.05). The total score of resilience [(56.9±16.7) vs(68.0±14.4)] and scores ofthree factorsin patients with schizophrenia were significantly lower than that in healthy controls(all P<0.05). The total score of MATRICS Consensus Cognitive Battery (MCCB)[(23±12) vs (42±11)] and each subscale score in patients with schizophrenia were significantly lower than that in healthy controls(all P<0.05). Partial correlation analysis showed that the total score of resilience and tenacity were correlated with symbol coding of schizophrenia(partial correlation coefficients were 0.286, 0.289, respectively, both P<0.05). The total score of resilience and the scores of tenacity, strength and optimism were all correlated with emotion management ability of schizophrenia(partial correlation coefficients were 0.334, 0.271, 0.382, 0.308, respectively, all P<0.05). In the healthy controls, the total score of resilience, tenacity and optimism were correlated with symbol coding(partial correlation coefficients were 0.268, 0.225, 0.291, respectively, all P<0.05). Strength and optimism were correlated with Hopkins verbal learning test (HVLT)(partial correlation coefficients were 0.268, 0.225, respectively, both P<0.05). Strength was correlated with spatial span, continuous performance test(partial correlation coefficients were 0.244, 0.217, respectively, bothP<0.05). The total scores of resilience and tenacity, strength and optimism were correlated with emotional management ability(partial correlation coefficients were 0.306, 0.230, 0.286, 0.289, respectively, all P<0.05), while the total scores of resilience, strength and optimism were correlated with the total score of MCCB(partial correlation coefficients were 0.291, 0.359, 0.287, respectively, all P<0.05). Conclusion: The current study suggests that resilience and cognitive function of patients with first-episode schizophrenia areimpaired significantly. Resilience in patients with schizophrenia isrelated to partial neurocognitive function and emotion management ability in social cognitive function.

[Correlation of the peripheral serum complement protein levels and cognitive function in first-episode drug-naive patients with schizophrenia].

Objective: To explore the role of peripheral serum complement protein in the pathogenesis of cognitive impairment by analyzing the correlation between peripheral serum levels of complement protein and cognitive function in first-episode drug-naive patients with schizophrenia. Methods: A total of 66 first-episode drug-naive schizophrenics (schizophrenia group) from the Third Affiliated Hospital of Sun Yat-sen University and 88 healthy volunteers (control group) were enrolled. Peripheral serum levels of complements (C3, C4 and CH50) were separately examined by liposome immunoassay and turbidimetric inhibition immunoassay. The MATRICS Consensus Cognitive Battery (MCCB) was used to assess the cognitive function. Results: Peripheral serum levels of C4, but not C3 and CH50, were significantly lower in patients with schizophrenia [0.20(0.16, 0.25) g/L] than those in the control group [0.23 (0.19, 0.27) g/L] (P<0.05). Moreover the peripheral serum levels of C3, C4 and CH50 were positively correlated with MCCB verbal fluency (r=0.258, r=0.283 and r=0.330, all P<0.05), and the peripheral serum levels of CH50 were negatively correlated with attention and alertness (r=-0.257, P<0.05). Conclusion: The decrease of peripheral serum complement C4 protein levels may be involved in the mechamism of cognitive impairment in schizophrenia.

PAX3 silencing suppresses gastric cancer proliferation and angiogenesis via MET/PI3K signaling.

PAX3 is the key factor in cell signal transduction pathway and may be involved in the regulation of cancer cell proliferation, differentiation and migration. The aim of the study was to investigate the effects and mechanism of PAX3 silencing on the gastric cancer. Specific PAX3 silencing was performed both in vitro and in vivo using small-interfering RNAs (siRNAs). The proliferation, apoptosis and angiogenesis of gastric cancer cells were assessed using MTT assay, flow cytometry and in vitro tube formation assay. Mice with gastric xenografts, which expressed either si-PAX3 or non-coding siRNA (si-NC), were developed and the effects of PAX3 silencing on tumor progression were evaluated. PCNA is a proliferating cell nuclear antigen and can be used as an index for evaluating cell proliferation status. Immunocytochemistry assay was used to quantify PAX3 and PCNA expression. After 4 weeks of tumor inoculation, tumor tissues were weighed. Tumor tissue morphology and apoptosis were evaluated using HE staining and TUNEL assay. In order to investigate the effect of silencing PAX3 on cell apoptosis, angiogenesis and MET/PI3K pathway, quantitative real-time PCR (qRT-PCR) or western blot were used to detect the expression levels of caspase-3, VEGF, MET, p-MET, PI3K and p-PI3K. After PAX3 silencing, PAX3 expression was significantly decreased in two gastric cancer cell lines, MKN-28 and SGC-7901 (p<0.05 vs Control). PAX3 silencing reduced cell proliferation, induced cell apoptosis and inhibited tube formation. PAX3 and PCNA expression were also significantly decreased. In mice, silencing PAX3 significantly inhibited tumor growth and decreased microvessel density in tumor. PAX3 silencing also decreased cell density in tumors, which concurred with increased apoptosis and PAX3 expression. PAX3 silencing upregulated the expression of caspase-3, downregulated the expression of VEGF, phosphorylation of PI3K and MET. Our data showed that these anti-tumor effects of PAX3 silencing might be attributed to its role in inducing cell apoptosis and inhibiting angiogenesis.

[Determination of samarium oxide and lanthanum oxide in the air of workplace by inductively coupled plasmamass spectrometry].

Objective: To establish a method for the determination ofsamarium oxide and lanthanum oxide by inductively coupled plasmamass spectrometryin the air of workplace. Methods: Samarium, lanthanum and their compounds in the air of workplace were collected through microporous filter. The samples were digested by nitricacid and perhydrol (V/V=4∶1) and detected by inductively coupled plasmamass spectrometry. Results: The linear range ofsamarium oxide and lanthanum oxide was 0-50.00 µg/L, Sm(2)O(3): y=0.0119x, r=0.9999; La(2)O(3): y=0.0617x, r=0.9998. The detection limits were less than 0.1 µg/L, and the minimum detection concentration were less than 1.52×10(-5) mg/m(3). The sampling efficiency were 100%, the recovery rates were 95.70%-102.01%, and the precision were 0.78%-1.58%. Conclusion: The indicators established in this study are conformed with the requirements of Chinese Occupational Standars of GBZ/T 210.4-2008, "The Guidelines for the Development of Occupational Hygiene StandarsMehods Part 4: Determination of Chemical Substances in the Air of Workplace".

[Analysis of the impact of job characteristics and organizational support for workplace violence].

Objective: To analyze the effect of job characteristics and organizational support for workplace violence, explore the influence path and the theoretical model, and provide a theoretical basis for reducing workplace violence. Methods: Stratified random sampling was used to select 813 medical staff, conductors and bus drivers in Chongqing with a self-made questionnaire to investigate job characteristics, organization attitude toward workplace violence, workplace violence, fear of violence, workplace violence, etc from February to October, 2014. Amos 21.0 was used to analyze the path and to establish a theoretical model of workplace violence. Results: The odds ratio of work characteristics and organizational attitude to workplace violence were 6.033 and 0.669, respectively, and the path coefficients were 0.41 and-0.14, respectively (P<0.05). The Fitting indexes of the model: Chi-square (χ(2)) =67.835, The ratio of the chi-square to the degree of freedom (χ(2)/df) =5.112, Good-of-fit index (GFI) =0.970, Adjusted good-of-fit index (AGFI) =0.945, Normed fit index (NFI) =0.923, Root mean square error of approximation (RMSEA) =0.071, Fit criterion (Fmin) =0.092, so the model fit well with the data. Conclusion: The job characteristic is a risk factor for workplace violence while organizational attitude is a protective factor for workplace violence, so changing the job characteristics and improving the enthusiasm of the organization to deal with workplace violence are conducive to reduce workplace violence and increase loyalty to the unit.

Occurrence and Genetic Diversity of Grapevine berry inner necrosis virus from Grapevines in China.

To investigate the prevalence and genetic diversity of Grapevine berry inner necrosis virus (GINV) in China, 195 grapevine samples from 15 Chinese provinces and regions were tested using reverse-transcription polymerase chain reaction. The samples included symptomatic and asymptomatic cultivars, with 35.9% (70 of 195) of samples testing positive for GINV. Seventeen samples had obvious ring spot symptoms, and 94.1% (16 of 17) tested positive for GINV, suggesting that GINV may be highly associated with the ring spot symptom. The genetic diversity of GINV isolates was analyzed based on the partial nucleotide and amino acid sequences of the coat protein (CP) and movement protein (MP) genes. Phylogenetic analyses of the MP and CP gene sequences divided the GINV isolates into three groups. The majority of the Chinese isolates were in groups 1 and 2, and only one Chinese isolate, along with a previously reported Japanese isolate, was in group 3. This is the first report on the genetic diversity of GINV isolates and their prevalence and distribution in China.

[Effects of statin intervention on mild coronary plaque progression assessed by serial coronary CT angiography].

OBJECTIVE: To assess the effects of statin treatment on mild coronary plaque progression by serial coronary CT angiography. METHODS: A total of 120 consecutive patients (74 men, ages(58.9±8.1)years) with mild (≤50%luminal narrowing and lesion length<20 mm) non-calcified plaque detected by coronary CT angiography during September 2012 and December 2013 were prospectively enrolled in this study.Subjects were divided into three groups: no statin (n=36), statin lowering LDL-C <50% (n=43), and statin lowering LDL-C ≥50%(n=41). Serial scans were performed after a median interval of 705 (interquartile range: 467, 803) days.Total plaque volume, percent plaque volume for both baseline and follow-up were measured.Baseline and follow-up data were compared. RESULTS: Compared with baseline, total plaque volume in no statin group showed increasing trend by the end of follow-up ((97.3±57.8) mm(3) vs. (82.2±57.7) mm(3,) P=0.075). However, no significant change was observed as for total plaque volume ((78.5±45.2) mm(3) vs.(77.6±50.5) mm(3), P=0.910) in the statin lowering LDL-C <50% group.Total plaque volume was significantly reduced by the end of follow-up ((61.5 ± 46.1) mm(3) vs.(77.7±48.1) mm(3), P=0.024) in the statin lowering LDL-C ≥50% group.Percent plaque volume in no statin group was significantly increased by the end of follow-up ((51.9±16.5)% vs.(45.9±12.8)%, P=0.036). However, no significant change was observed as for percent plaque volume ((49.1±13.7)% vs.(47.5±14.9)%, P=0.554) in the statin lowering LDL-C <50% group. Percent plaque volume was significantly reduced by the end of follow-up ((39.1±17.1)% vs.(48.2±15.0)%, P=0.003) in the statin lowering LDL-C ≥50% group. Multivariable linear regression analysis showed that both higher baseline total plaque volume(ß=-0.50, P<0.001) and statin lowering LDL-C ≥50%(ß=-0.32, P=0.001) were independent determinants of plaque regression. CONCLUSION: This study suggests that LDL-C reduction ≥50% post statin treatment can retard plaque progression, and even induce regression of mild non-calcified coronary plaque, patients with greater baseline coronary plaque volume are more likely to benefit from statin therapy.

Evaluation methods on the nutritional status of stroke patients.

OBJECTIVE: This study was designed to assess the effect of particular tools on the nutritional status of patients with stroke risk factors; to analyze these risk factors; to construct an assessment table; and to enable nurses to conduct fast and accurate assessment of the nutritional status of patients with stroke. PATIENTS AND METHODS: Various nutritional assessment tools were employed to assess the nutritional status of stroke patients [(Nutritional Risk Screening 2002, NRS2002); (mini nutritional assessment, MNA), (subjective global assessment SGA), (malnutrition universal screening, MUST); (body composition, BCA)]. The leading disease-related factors of cerebral apoplexy were observed in patients with malnutrition. And a statistical analysis was conducted. RESULTS: The significant risk factors of cerebral apoplexy in malnourished patients older than 70 years were swallowing dysfunctions, disturbance of consciousness and reliance or half-reliance on feeding practices. The significant risk factors of malnutrition in patients with cerebral apoplexy were the decline in upper limb muscle strength, decline in the performance of various activities, loss of appetite and gastrointestinal symptoms. CONCLUSIONS: Disorders that affect the nutritional status of stroke patients can be used as evaluation tools, as described in the evaluation table. The clinical relevance of this study includes the following: to enable the clinical nursing staff to easily assess the patient's nutritional status in a timely manner; to improve compliance with nutritional evaluation; to provide clinical nutrition support to patients with stroke; and to provide a scientific basis for the improvement of the clinical outcomes of patients with cerebral apoplexy.

Gene diagnosis of micrometastases in regional lymph nodes of patients with stage I non-small cell lung cancer: impact on staging and prognosis.

PURPOSE: The long-term survival of patients with completely resected stage I non-small cell lung cancer (NSCLC) is not optimal because of undetected lymph node micrometastasis at the time of surgery. The aim of this study is to evaluate the role of survivin and livin mRNA expression in histopathologically negative lymph nodes of stage I NSCLC patients as markers of micrometastasis. METHODS: Clinical data and tissue samples of primary tumor and lymph nodes were collected from 44 patients with stage I NSCLC. Reverse-transcriptase-PCR (RT-PCR) was used to detect survivin and livin mRNA expression in these tumor and lymph node samples. RESULTS: Survivin mRNA was detected in all tumors, and livin mRNA was detectable in 39 of the 44 primary tumors. The cut-off values of survivin and livin mRNA levels for diagnosing micrometastasis in lymph nodes were set up according to the expression of survivin and livin mRNA in control lymph nodes. Fifteen (34.1 %) of 44 stage I NSCCL patients had micrometastasis in lymph nodes by survivin and/or livin mRNA positive expression. Survival analysis showed higher rate of cancer recurrences and tumor-related death in patients with lymph node micrometastasis (P < 0.001 and P = 0.001, respectively). Tumor-free survival and overall survival were significantly worse in patients with lymph node micrometastasis compared with those without such micrometastasis (P = 0.007 and P = 0.01, respectively). CONCLUSION: RT-RCR assay for survivin and livin mRNA can be considered as useful diagnostic tool for the detection of lymph node micrometastasis for stage I NSCLC patients.

First Report of Syringa oblata and S. reticulata Leafroll Disease in China.

Syringa oblata is an important ornamental tree widely grown in China. In September of 2008, S. oblata plants exhibiting symptoms of leafroll and yellowing were found in a garden on the Northwest A&F University campus. Samples were collected from this site. Total DNA was extracted from 0.5 g of phloem tissue from leaf midribs and stems of each sample. DNA samples were analyzed with a nested PCR assay using phytoplasma 16S rDNA universal primers R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (1), which amplified a 1,452- and 1,246-bp product, respectively. We tested all 30 lilac samples, 20 of which had symptoms of leafroll and yellowing. These produced the expected 1,452- and 1,246-bp PCR products In contrast, the remaining 10 samples from symptomless trees yielded no PCR products. We also surveyed another lilac variety (Syringa reticulata), which is widely grown on the campus, and tested 50 samples with the above method. Again, 1.4- and 1.2-kb PCR products were amplified from all 30 trees displaying leafroll and yellowing symptoms, but not from the other 20 samples from symptomless trees. A comparative analysis of sequences derived from the two hosts showed that the phytoplasmas infecting them were most similar (>99%) to paulownia witches'-broom (PaWB) phytoplasma (GenBank Accession No. EF199937). Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with endonucleases AluI and MseI indicated that all symptomatic plants were infected by the phytoplasmas belonging to aster yellow group (16SrI) subgroup D (16SrI-D) PaWB phytoplasma (2). 16S rDNA sequence comparisons and RFLP analysis of the cloned 16S rDNA from S. oblata (GenBank Accession No. FJ445224) and S. reticulate (GenBank Accession No. FJ445225) indicated that the phytoplasmas infecting them were nearly identical (99.8% identity). To our knowledge, this is the first report of the presence of the phytoplasma associated with a leafroll disease of S. oblata and S. reticulata in China. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

First Report of Elm Yellows Phytoplasma Infecting Clover in China.

In the summer of 2008, phyllody and enlarged petioles resembling symptoms of phytoplasma infection were observed on clover (Trifolium repens) plants in lawns on the campus of Northwest A&F University. Typical phytoplasma-like bacteria were observed in the phloem cells when ultra-thin sections from leaf midrib tissues were examined with transmission electron microscopy. Nested PCR assays were used to verify the association of phytoplasma with the disease. Total DNA was extracted from the phloem of leaf midribs from 20 symptomatic plants and six symptomless plants using the modified CTAB method (1). Using the phytoplasma universal primer pair R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (4), PCR products of 1.4 and 1.2 kb were amplified, respectively, from symptomatic plants only. Jujube witches'-broom (JWB) and paulownia witches'-broom (PaWB) phytoplasma DNA samples served as controls and were used to study group relationships. After sequencing of the 16S rDNA fragment (GenBank Accession No. FJ436792), a BLAST search determined that the clover phytoplasma shared closest homology (99.6%) with JWB (GenBank Accession No. FJ154846) phytoplasmas compared with lesser identity (90.4%) with PaWB (GenBank Accession No. EF199937). Subsequent restriction fragment length polymorphism analysis of the PCR-amplified 1.2-kb 16S rDNA R16(1)F1/R1 fragment indicated that the phytoplasma associated with the disease belongs to subgroup 16SrV-B of the elm yellows phytoplasma group. Clover phyllody phytoplasma were previously reported to infect clover in Canada (GenBank Accession No. L33762) (3) and Italy (GenBank Accession No. X77482) (2). The phytoplasma reported here shared 86.7 and 90.0% identity with the clover phyllody phytoplasma above, respectively, much lower than that with Elm yellows phytoplasma group. To our knowledge, this is the first report of Elm yellows phytoplasma infecting clover in China. References:(1) E. Angelini et al. Vitis 40:79, 2001. (2) G. Firrao et al. Eur. J. Plant Pathol. 102:817, 1996. (3) N. A. Harrison et al. Plant Pathol. 52:147, 2003. (4) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

A Phytoplasma Associated with an Outbreak of an Unusual Disease of Chrysanthemum in China in 2008.

In August of 2008, a disease of chrysanthemum (Dendranthema morifolium (Ramat.) Tzvel) caused losses of 70 to 80% in one of the largest chrysanthemum gardens in Yangling, Shanxi Province, China. Chrysanthemum plants in nearby areas also were affected to various degrees. Symptoms included flattened stems, shortening of internodes, yellowing of leaf margins, root death, and dwarfing of plants. Affected plants eventually collapsed. On the basis of these symptoms, a phytoplasma was suspected. Total nucleic acids were extracted from 0.5 g of phloem tissue from stems of eight symptomatic and eight asymptomatic plants by the cetyltrimethylammoniumbromide (CTAB) method (1). To amplify phytoplasma DNA, primer pairs R16mF2/R16mR1, followed by R16F2n/R16R1 (2), were used in a nested PCR. A final amplicon product (1.2 kb) was obtained from all symptomatic plants but not from asymptomatic ones. Restriction fragment length polymorphism (RFLP) analyses of R16F2n/R16R1 amplicons with MseI, AluI, HhaI, HaeIII, KpnI, RsaI, and HpaII endonucleases indicated that all symptomatic plants, but none of the asymptomatic plants, contained a phytoplasma strain of group 16SrI, subgroup B (3). A search of rDNA sequences in GenBank revealed a similarity (>99%) to aster yellow phytoplasma, 16SrI group, thereby confirming strain identity based on RFLP analysis. These results indicate the disease of chrysanthemum is associated with a phytoplasma related to the aster yellow phytoplasma group. Sequences were deposited in GenBank (Accession No. FJ543467). A vector of this phytoplasma in chrysanthemum has not been identified. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 48:1153, 1998.

Stable Agrobacterium-mediated genetic transformation of London plane tree (Platanus acerifolia Willd.).

London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene beta-glucuronidase (GUS), we have followed the transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD(600) of 0.8-1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer. Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established. By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines, the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome.

Biochemical properties of the P42 protein encoded by RNA segment 6 of influenza C virus.

P42, encoded by a colinear transcript of Influenza C virus RNA segment 6 (M gene), is an integral membrane protein which is cleaved by signal peptidase to generate M1' and CM2 composed of N-terminal 259 amino acids and C-terminal 115 amino acids, respectively. Herein, the biochemical features of P42 were investigated. N-glycosylated form of P42, designated P44, forms disulphide-linked dimers and tetramers. P44 is transported to the Golgi apparatus, but not to the trans-Golgi, since P44 is completely sensitive to endoglycosidase H. P44 and P42 are unstable irrespective of N-glycosylation or oligomerization. 26S proteasome inhibitor, lactacystin prevented the degradation of P42 as well as M1', but not that of P44 efficiently, suggesting that P44 is degraded by another protease besides the 26S proteasome.

Role of individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of influenza C virus hemagglutinin-esterase protein.

The hemagglutinin-esterase (HE) glycoprotein of influenza C virus is composed of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain (R). The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Here, we investigated the role of the individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of the HE protein by eliminating each of the glycosylation sites by site-specific mutagenesis. Comparison of electrophoretic mobility between the wild-type and the mutant proteins showed that while seven of the glycosylation sites are used, one (position 131) is not. Analysis of reactivity of the mutants with anti-HE monoclonal antibodies demonstrated that glycosylation at position 144 is essential for the formation of conformation-dependent epitopes. It was also evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144), is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant HEs lacking one of these three sites failed to undergo the trimer assembly. Removal of an oligosaccharide chain at position 144 or 189 resulted in a decrease in the esterase activity. By contrast, two mutants lacking an oligosaccharide chain at position 26 or 603, which were defective not only in cell surface expression but in trimerization, possessed full-enzyme activity, suggesting that the HE monomers present within the cell have acetylesterase activity. Fusion activity of cells expressing each of mutant HEs was found to be comparable with the ability of the protein to be transported to the cell surface, suggesting that there is no specific oligosaccharide chain that plays a critical role in promoting membrane fusion.