[Effect of exosome derived from ovarian cancer cell on the differentiation of fibroblast].

Objective: To study the effects of exosome secreted by ovarian cancer (OC) cell on the differentiation and metastasis of normal fibroblasts (NFs). Methods: NFs were collected from patients who underwent hysteromyoma resection in the Affiliated Hospital of Qingdao University from May to December 2019. Exosome was extracted from the culture supernatant of SKOV3 cells by using ultra-high-speed centrifugation. The NFs were co-cultured with condition medium (CM), exosome of SKOV3 (SKOV3-exo) and control medium. The expression levels of fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) were detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. The metastatic ability of NFs was detected by Transwell array. Results: Under the transmission electron microscope, the extracellular vesicles extracted from the culture supernatant of SKOV3 were 30-100 nm in diameter with cup holder-like bilayer membrane structure, and the protein expression levels of TSG101 and HSP27 in exosomes (1.00±0.05 and 1.12±0.13) were higher than those of ovarian cancer SKOV3 cells (0.22±0.21 and 0.36±0.14, respectively, P<0.05). PKH67 fluorescently labeled exosomes could be taken up by NFs. The expression levels of α-SMA and FAP mRNA in CM group(2.91±0.15 and 3.21±0.33)and SKOV3-exo group (3.50±0.21 and 4.63±0.24, respectively) were higher than that in blank group (1.00±0.06 and 1.00±0.13, P<0.05). The protein expression levels of α-SMA and FAP in CM group and SKOV3-exo group (0.89±0.11 and 1.25±0.09, 0.81±0.09 and 1.20±0.12) were higher than those in the blank group (0.12±0.31 and 0.11±0.19, respectively, P<0.05). The migrated numbers of cells in the CM group and SKOV3-exo group [(215.01±14.80) and (389.72±19.43), respectively] were higher than that in the blank group [(113.73±4.70), P<0.05]. Conclusion: The exosome secreted by SKOV3 cells can be taken up by NFs, which makes it to differentiate into cancer associated fibroblasts (CAFs) and significantly enhances its metastatic ability, indicating that OC cells may promote the transformation of normal ovarian mesenchymal fibroblasts to CAFs through exosome pathways, and then promote the development of ovarian cancer.

[Relationship between myocardial microRNA-30a expression and myocardial fibrosis in rats post myocardial infarction].

OBJECTIVE: To explore the potential role and mechanism of microRNA(miR)-30a in myocardial fibrosis after myocardial infarction (MI). METHODS: Rats were randomly divided into 1 week MI group (n=11), 2 weeks MI group (n=13) and 4 weeks MI group (n=15) by applying random number table after left anterior descending coronary artery ligation. Rats in Sham group were examined at respective time points (n=16). Heart function was monitored by echocardiography. Myocardial collagen volume fraction (CVF) was determined on Masson stained sections. Myocardial expression of collagen Ⅰ and Ⅲ was determined by immunohistochemistry. The myocardial mRNA level of miR-30a, TGF-ß1 and CTGF were detected by real time-quantitative PCR analysis. The myocardial protein levels of TGF-ß1 and CTGF were measured by Western blot analysis. RESULTS: The LVEDD ((8.37±0.58) mm) and LVESD ((6.12±0.82) mm) in 4 weeks MI group were significantly higher than those in Sham group ((6.08±0.57) mm, (4.17±0.60) mm), all P<0.01. The FS ((27.0±3.9) %) and LVEF ((51.0±6.3) %) in 4 weeks MI group were significantly lower than those in Sham group ((47.0±2.1) %, (82.0±2.3)%), all P<0.01. The level of myocardial CVF in 1 week MI group, 2 weeks MI group and 4 weeks MI group were significantly higher than in Sham group (all P<0.01) in a time-dependent manner. The level of myocardial collagen Ⅰ and Ⅲ was increased gradually from 1 week to 4 weeks post MI compared with Sham group (all P<0.01). The collagen Ⅰ/Ⅲ ratio was similar between 1 week MI group and Sham group (P=0.58), however, which was significantly higher in 2 weeks MI group and 4 weeks MI group compared with Sham group (all P<0.01), and the ratio was significantly higher in 4 weeks MI group than 2 weeks MI group (P<0.01). The level of miR-30a was significantly and gradually reduced in all MI groups compared with Sham group (all P<0.01). The mRNA and protein levels of TGF-ß1 and CTGF were significantly and gradually increased after MI compared with Sham group (all P<0.001). CONCLUSIONS: Our results indicate that overexpression of miR-30a after MI might be a potential strategy for suppressing myocardial fibrosis by modulating the mRNA and protein levels of TGF-ß1 and CTGF.

A three-phase epidemiological study of short and long sleepers in a middle-aged Chinese population: prevalence and characteristics.

Epidemiological studies of short and long sleepers have not been conducted previously. We collected socioeconomic, psychological, and polysomnographic characteristics of 6501 parents (3252 men and 3249 women) of 4036 primary school children in Guangzhou city. The study data were collected in three phases. The overall prevalence of short (5 h or less) and long (10 h or more) sleep duration was 0.52 and 0.64%, respectively. Long sleepers had higher Eysenck Personality Questionnaire neuroticism scores [odds ratio (OR)=1.224, 95% confidence interval (CI)=1.047-1.409] and lower education levels (OR=0.740, 95%CI=0.631-0.849) than short sleepers. In the polysomnographic assessment, short, long, and normal sleepers (7-8 h) shared similar durations of Stage 3 sleep (short=25.7±10.7, long=20.3±7.9, and normal=28.0±12.8 min, F=1.402, P=0.181). In daytime multiple sleep latency tests, short sleepers (10/19, 52.6%) were more prone to have a short sleep latency (≤ 8 min) than long sleepers (2/23, 8.7%). In addition to different sleep durations, neuroticism might also contribute to differences between short and long sleepers in social achievements. Stage 3 sleep might be essential for humans. The short sleep latency (≤ 8 min) of short sleepers in multiple sleep latency tests should be interpreted cautiously, since it was of the same severity as required for a diagnosis of narcolepsy or idiopathic hypersomnia.

A three-phase epidemiological study of short and long sleepers in a middle-aged Chinese population: prevalence and characteristics

Epidemiological studies of short and long sleepers have not been conducted previously. We collected socioeconomic, psychological, and polysomnographic characteristics of 6501 parents (3252 men and 3249 women) of 4036 primary school children in Guangzhou city. The study data were collected in three phases. The overall prevalence of short (5 h or less) and long (10 h or more) sleep duration was 0.52 and 0.64%, respectively. Long sleepers had higher Eysenck Personality Questionnaire neuroticism scores [odds ratio (OR)=1.224, 95% confidence interval (CI)=1.047-1.409] and lower education levels (OR=0.740, 95%CI=0.631-0.849) than short sleepers. In the polysomnographic assessment, short, long, and normal sleepers (7-8 h) shared similar durations of Stage 3 sleep (short=25.7±10.7, long=20.3±7.9, and normal=28.0±12.8 min, F=1.402, P=0.181). In daytime multiple sleep latency tests, short sleepers (10/19, 52.6%) were more prone to have a short sleep latency (≤8 min) than long sleepers (2/23, 8.7%). In addition to different sleep durations, neuroticism might also contribute to differences between short and long sleepers in social achievements. Stage 3 sleep might be essential for humans. The short sleep latency (≤8 min) of short sleepers in multiple sleep latency tests should be interpreted cautiously, since it was of the same severity as required for a diagnosis of narcolepsy or idiopathic hypersomnia.

First Report of Bacterial Blight of Guar Caused by Xanthomonas axonopodis pv. cyamopsidis in China.

Bacterial blight was observed on field-grown guar (Cyamopsis tetragonoloba L.) for the first time in China. The disease outbreak occurred in the Xinjiang Uyghur Autonomous Region after several weeks of unusually heavy rains during late summer 2013. The disease incidence was generally 40 to 50%, although values as high as 80% were observed in several fields. Initial field symptoms included water-soaked spots on leaves, pods, petioles, and stems. During later stages of infection, the color of the spots became dark. We also observed large, angular, necrotic lesions at leaf tips, black streaks on petioles and stems, split stems, defoliation, wilting or top withering, vascular necrosis, and dieback. Samples of diseased leaves, stems, petioles, pods, and seeds were surface sterilized, ground, and then plated onto King's B medium. Plates were incubated at 28°C for 72 h. Fifteen bacterial strains with yellow-pigmented, opaque, and round colonies were isolated. These strains were aerobic, gram-negative rods with a single, polar flagellum. They were positive for H2S, esculin, oxidase, tobacco hypersensitivity, indole production from tryptophan, nitrate reduction to nitrite, and the utilization of glucose, mannose, trehalose, galactose, and starch. The maximum salt tolerance of the strains was 2 to 3%. Pathogenicity tests using eight strains were conducted in July 2013. A bacterial culture was suspended in sterile water with a final concentration of 108 CFU/ml. Eight 4-week-old guar plants were inoculated by (i) spraying the bacterial suspension on the leaves until runoff, or (ii) puncturing the stems with a needle that had been dipped into the bacterial suspension. Sterile water was used as a negative control. Plants were kept in a mist room with 100% relative humidity for 24 h. Stem and leaf symptoms similar to those of the original plants were observed on the inoculated guar plants within 10 days of inoculation. No symptoms developed on the negative control plants. Yellow bacterial colonies re-isolated from inoculated plant tissues were morphologically identical to the original. 16S rDNA was amplified using universal primers (Pa 5'-AGTTTGATCCTGGCTCAG-3' and Ph 5'-TACCTTGTTACGACTTCGTCCCA-3') and sequenced. A BLAST search of the NCBI GenBank database indicated that the 16S rDNA sequences of three strains (accession nos. KF563926, KF563927, and KF563928) had 99.9% identity to Xanthomonas axonopodis strain XV938 (AF123091). Under greenhouse conditions, bacterial strains wilted asparagus bean and pea but rarely infected bean, kidney bean, faba bean, mung bean, soybean, red bean, pea, garbanzo bean, and peanut. Based on morphology, pathogenicity tests, 16S rDNA sequencing, and host plant specificity, the pathogen was confirmed as X. axonopodis pv. cyamopsidis (synonym: X. campestris pv. cyamopsidis [Patel et al., 1953]). To our knowledge, this is the first report of bacterial blight of guar caused by X. axonopodis pv. cyamopsidis in China. Guar has recently been introduced in Xinjiang Province. Our findings indicate that bacterial blight may pose a threat to the economic sustainability of guar production in the region. References: (1) I. A. Milyutina et a1. FEMS Microbiol. Lett. 239:17, 2004. (2) I. M. G. Almeida et al. Summa Phytopathol. 18:255, 1992. (3) J. D. Mihail et al. Plant Dis. 69:811, 1985.

First Report of Powdery Mildew Caused by Podosphaera fusca on Herba eupatorii in China.

Herba eupatorii, one of the most important Chinese medicinal herbs, belongs to the Asteraceae family. In June 2012, a previously unknown disease, tentatively identified as powdery mildew, was observed on H. eupatorii growing in Shangqiu, in eastern Henan Province, China. Symptoms began as white mycelium partially covering upper leaf surfaces; as the disease progressed, it spread to cover entire leaf surfaces. The infected leaves became yellow and necrotic at advanced stages of infection. Specimens consisting of infected leaves were maintained at the Plant-Microbe Interaction Laboratory at Shangqiu Normal University. Microscopic observations of the morphology of the fungus revealed oval primary conidia measuring 18 to 27 × 15 to 22 µm. A long unbranched germ tube that germinated laterally from the ends of conidia was observed in some samples. Conidiophores were cylindrical, simple unbranched, and composed of a basal cell with a swollen base and three to six barrel-shaped conidia formed in chains, measuring 112 to 180 × 9 to 12 µm. Mycelial appressoria were nipple-shaped. Chasmothecia were not observed in the collected samples. To verify the identity of the fungus, the internal transcribed spacer (ITS) rDNA was amplified with ITS1 and ITS4 primers (3) and sequenced. The sequences were deposited as GenBank Accession No. JX546297. Comparison with sequences in the GenBank database revealed that the ITS sequence was 100% homologous with the sequence of Podosphaera fusca on Calendula officinalis (AB525914) (2) and Syneilesis palmata (AB040349) (1). The ITS sequence analysis verified that the causal agent was P. fusca, which is reported to be a cosmopolitan powdery mildew fungus, parasitic on numerous plant species in the Asteraceae family. Koch's postulates were completed by inoculating healthy H. eupatorii plants with a conidial suspension (prepared in distilled water) of 105 conidia/ml collected from infected plants. Five plants were sprayed until the suspension ran off the leaves, while five additional plants were sprayed with distilled water as a control. Plants were maintained in a climate cell under the following conditions: day, 24°C, 16 h; night, 20°C, 8 h; 85% humidity. After 10 days, inoculated plants developed symptoms similar to those observed in the field, whereas control plants remained healthy. Further examination showed that the inoculated plants were infected by P. fusca. To our knowledge, this is the first report of P. fusca affecting H. eupatorii in China. Because there are no fungicides labeled for use on this plant, the appearance of powdery mildew caused by P. fusca could result in substantial production loss of H. eupatorii. References: (1) T. Hirata et al. Can. J. Bot. 78:1521, 2000. (2) S. Takamatsu et al. Persoonia 24:38, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Boll Rot of Cotton Caused by Pantoea agglomerans in China.

Since the summer of 2006, bacterial boll rot of cotton has been observed on fruits of 'Xinluzao 31' (Xinluzao 6 × Acala) in Xinjiang Province. It resulted in as much as 20% yield loss in several fields. Symptoms do not appear on the outer carpel. In the infected cotton bolls, fibers do not mature completely and seed tissue exhibits brown necrotic coloration. Lint and seeds from 24 surface-disinfested cotton bolls were triturated and plated onto King's medium B (KB). Plates were incubated at 28°C for 48 h. Forty eight strains with yellow pigmentation on KB were characterized. All were nonfluorescent on KB, gram negative, facultatively anaerobic, unable to produce indole from tryptophan, able to reduce nitrate to nitrite, and produce acid from glucose, cellobiose, lactose, melibiose, and melonate. In addition, 16S rDNA in seven strains was amplified with universal primers (1). The PCR products were cloned into pGEM-T easy vector and sequenced. A BLAST search of the seven sequences against the GenBank nucleotide library indicated 100% identity with the 16S rDNA sequence of Enterobacter agglomerans strain A80. Then an additional primer pair, pagF and pagR (3), was used for more specific amplification of Pantoea agglomerans 16S rDNA, which resulted in single highly specific fragments of approximately 1 kb. On the basis of morphological, physiological, biochemical characteristics, and 16S rRNA gene sequence analysis, we identified the bacterium to be P. agglomerans. To confirm pathogenicity, cell suspensions (1 × 108 CFU/ml) of eight representative strains were used to inoculate cotton at peak bolling stage in the field. Cell suspensions, or water as the control, were applied to stigma scars, wall sutures, and scratch wounds on bracts, calyxes, and bolls. Alternatively, a needle was used to puncture through a drop of suspension placed on the boll wall suture and bracts. At least 20 bolls or flowers were inoculated with each bacterial strain per inoculation method. Infection occurred only when bacterial injections breached the endocarp of the boll either through the carpel wall or a suture between carpel sections. Disease symptoms developed 1 week postinoculation. The inoculated organism was reisolated from the diseased tissues. P. agglomerans is generally regarded to be a soil saprophyte or leaf epiphyte, but strains can opportunistically infect plants triggering gall formations or human wounds causing septic arthritis. The disease symptoms and pathogen characteristics observed in this study are identical to those reported in the United States (2). To our knowledge, this is the first report of P. agglomerans causing boll rot of cotton in China. References: (1) S. Manulisi and I. Barash. Mol. Plant Pathol. 4:307, 2003. (2) E. G. Medrano et al. J. Appl. Microbiol. 103:436, 2007. (3) S. Vorwerk et al. Agric. For. Entomol. 9:57, 2007.

First Report of Acidovorax avenae subsp. citrulli Infecting Edible Seed Watermelon (Citrullus lanatus var. lanatus) in China.

Edible seed watermelon, Citrullus lanatus var. lanatus, is a type of watermelon from which only the seeds are consumed. Between 2003 and 2005, a bacterial disease was discovered on this type of watermelon in several regions of Xinjiang Province in western China, with a diseased area of 40,000 ha. The average disease incidence on fruit and seedlings was approximately 30 and 20%, respectively. Symptoms were most noticeable on fruit. Lesions on the fruit rind were first noticeable as small pinpoint water-soaked areas. As the lesions rapidly enlarged, black, starshaped cracks formed in the rind, and the rind along with the flesh around the spots hardened. If no secondary invaders or saprophytes entered through the cracks, the inside flesh often disintegrated into dry stiff cavities. The fruit with necrotic spots became markedly malformed. The pathogen also infected seedlings. First symptoms on the seedling appeared as dark, water-soaked lesions on the underside and edge of the cotyledons. As the cotyledon expanded, the lesions become necrotic and often extended along the length of the midrib and eventually merged into large, black, withered areas. To determine the causal agent of the disease, 54 bacterial strains were isolated from cotyledons, fruits, or seeds of edible seed watermelon. By spray inoculation, these strains were inoculated onto edible seed watermelon cv. Xinzigua1 at the two-true-leaf stage with bacterial concentrations at 107 CFU/ml. All strains were strongly virulent to cv. Xinzigua1, which is planted in large areas in Xinjiang, by forming many water-soaked spots on cotyledons and true leaves. There were no symptoms in the control that was sprayed with sterile deionized water. Through biological and biochemical characteristics, including Gram reaction and tests for catalase, oxidase, and oxygen requirement, all strains were identified as Acidovorax avenae subsp. citrulli. To verify pathogen identification, specific polymerase chain reaction was also carried out. One set of 16S primers, WFB1/WFB2 for A. avenae subsp. citrulli, was used (1). A single unique band of approximately 360 bp was amplified for all strains tested. To our knowledge, this is the first report of A. avenae subsp. citrulli infecting edible seed watermelon. Reference: (1) R. R. Walcott and R. D. Gitaitis. Plant Dis.84:470, 2000.

[The association of Graves' disease with serum soluble interleukin 2 receptor].

The serum soluble interleukin 2 receptor (sIL2R) was measured in 38 first visited patients with Graves' disease and 29 normal controls. The serum sIL2R in 17 patients with Graves' disease was determined after treatment with antithyroid drugs (propylthiouracil) for a short period (1.2 +/- 0.5 months). The serum sIL2R was measured by sandwich enzyme linked immunosorbent assay. The sIL2R was significantly higher in patients before (3.04 +/- 0.19 U/ml) and after treatment (2.56 +/- 0.41 U/ml) than in normal controls (2.20 +/- 0.27 U/ml, P < 0.01). The mean value of serum sIL2R in 17 patients after treatment (2.56 +/- 0.41 U/ml) was substantially decreased as compared with that before treatment (2.99 +/- 0.14 U/ml, P < 0.01). The serum level of sIL2R in pretreatment patients was correlated significantly with T3(r = 0.5032, P < 0.05), but was not obviously related to T4 or rT3. These findings suggest that the human lymphocytes in patients with Graves' disease were activated in vivo and that sIL2R may be an useful immunological indicator of disease activity.

The treatment of chronic hematogenous osteomyelitis.

Eighty-five patients with a total of 103 foci of chronic hematogenous osteomyelitis were treated in the period from 1965-1982. Only patients who had been followed for two or more years of treatment were included in the series for evaluation. All foci were treated surgically with thorough debridement. According to the management of the wounds, patients were divided into three groups: wound healing by secondary intention in cases where skin closure was impossible; primary closure of wound with or without pedicle muscle transfer in cases of a small debrided cavity or in cases where a nearby skeletal muscle is available; and closed irrigation and suction drainage of the wound cavity. After a long-term follow-up period, satisfactory results to varying degrees were obtained in each group. Closed intermittent irrigation and suction drainage with high concentrations of antibiotic solutions gave the best results. In instances of failure, the causes may be due to inadequate removal of infected sclerotic bone and sequestra, obstruction of drainage tubes, resistance to antibiotics, or inadequate systemic antibiotic treatment. The use of myocutaneous flap transference to close the postoperative wound of chronic osteomyelitis was introduced, and preliminary results are encouraging.