Functional Characterization of CsSWEET5a, a Cucumber Hexose Transporter That Mediates the Hexose Supply for Pollen Development and Rescues Male Fertility in Arabidopsis.

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and ß-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed.

Effect of electroacupuncture treatment combined with rehabilitation care on serum sirt3 level and motor function in elderly patients with stroke hemiparesis.

OBJECTIVE: Acupuncture treatment helps to improve neurological and motor function in elderly patients with stroke hemiplegia. However, the exact mechanism by which electroacupuncture improves stroke hemiparesis is uncertain. The aim of this study was to determine the effect of electroacupuncture care on sirt3 levels in elderly patients with stroke hemiparesis. METHODS: One hundred and ten elderly patients with hemiplegia after first stroke were divided into an experimental group and a control group (n = 55 in each group). The control group was given conventional rehabilitation care by a rehabilitation therapist. In the experimental group, on the basis of conventional rehabilitation care, electroacupuncture was performed once a day for 28 days. RESULTS: Fugl-Meyer assessment (FMA) and barthel index (BI) scores were significantly higher, while neurologic deficit scale (NDS) and physiological state scores were significantly lower in both groups after 14 and 28 days of intervention compared to preintervention. The Generalized estimating equation (GEE) model also showed that the experimental group showed more favorable improvements in all outcomes at postintervention time points compared to the control group. After the intervention, serum sirt3 levels increased significantly in both groups compared to preintervention, and the increase was more pronounced in the experimental group. Consistently, the GEE model showed that serum sirt3 levels were significantly higher in the experimental group compared to the control group at postintervention time points. Correlation analysis revealed that serum sirt3 levels in the experimental group were negatively correlated with FMA and BI pre- and postintervention, while showing a significant positive correlation with NDS and physiological state scores. CONCLUSION: Electroacupuncture intervention led to significant improvements in motor function, activities of daily living and neurological function in elderly patients with stroke hemiplegia, which may be associate with increased serum sirt3 levels.

[Application of the interaction teaching mode integrated with virtual anatomy platform in teaching Meridian and Acupoints].

OBJECTIVE: To observe the effect of the interaction teaching mode integrated with Visible Body virtual anatomy platform in teaching Meridian and Acupoints. METHODS: A total of 60 students in the class of 2017 in the discipline of acupuncture-moxibustion and tuina, Xiangnan University were recruited and randomized into an observation group and a control group, 30 students in each one. In the control group, the traditional practical teaching mode was used. In the observation group, the interaction teaching mode integrated with virtual anatomy platform was adopted. The teaching duration was 10 class hours in both groups. After accomplishing the teaching schedule, the practical examination was conducted in the localization of commonly-used acupoints, very useful acupoints and the dangerous acupoints as well as acupuncture manipulation techniques. Moreover, the degree of satisfaction was investigated among the students in the two groups and the self-learning ability was evaluated in 3-month follow-up visit. RESULTS: In the observation group, the scores for the localization and acupuncture manipulation of commonly-used acupoints, very useful acupoints and the dangerous acupoints, as well as the degree of satisfaction of the 3 items, i.e. interesting, interaction and leaning-assistance were all higher than those in the control group (P<0.05). The degree of satisfaction in the acceptance and leaning-participation, as well as the scores of self-learning ability in 3-month follow-up visit were not different statistically between the two groups (P>0.05). CONCLUSION: The interaction teaching mode integrated with virtual anatomy platform improves the effect on teaching Meridian and Acupoints and achieves the high student satisfaction.

[Status of diagnosis and management of acute appendicitis in 2017: a national multi-center retrospective study].

OBJECTIVE: To analyze the current status of diagnosis and management of acute appendicitis (AA) in China. METHODS: Questionnaire survey was used to retrospectively collect data of hospitalized patients with AA from 43 medical centers nationwide in 2017 (Sort by number of cases provided: Jinling Hospital of Medical School of Nanjing University, The First Affiliated Hospital of Xinjiang Medical University, Lu'an People's Hospital, Tengzhou Central People's Hospital, Dalian Central Hospital, The Affiliated Hospital of Xuzhou Medical University, Dongying People's Hospital, Jinjiang Hospital of Traditional Chinese Medicine, Huangshan Shoukang Hospital, Xuyi People's Hospital, Nanjing Jiangbei People's Hospital, Lanzhou 940th Hospital of PLA, Heze Municipal Hospital, The First College of Clinical Medical Science of China Three Gorges University, Affiliated Jiujiang Hospital of Nanchang University, The Second People's Hospital of Hefei, Affiliated Central Hospital of Shandong Zaozhuang Mining Group, The Third People's Hospital of Kunshan City, Xuzhou First People's Hospital, The 81st Group Army Hospital of PLA, Linyi Central Hospital, The General Hospital of Huainan Eastern Hospital Group, The 908th Hospital of PLA, Liyang People's Hospital, The 901th Hospital of Joint Logistic Support Force, The Third Affiliated Hospital of Chongqing Medical University, The Fourth Hospital of Jilin University, Harbin Acheng District People's Hospital, The First Affiliated Hospital of Zhengzhou University, Nanjing Luhe People's Hospital, Taixing Municipal People's Hospital, Baotou Central Hospital, The Affiliated Hospital of Nantong University, Linyi People's Hospital, The 72st Group Army Hospital of PLA, Zaozhuang Municipal Hospital, People's Hospital of Dayu County, Taixing City Hospital of Traditional Chinese Medicine, Suzhou Municipal Hospital, Beijing Guang'anmen Hospital, Langxi County Hospital of Traditional Chinese Medicine, Nanyang Central Hospital, The Affiliated People's Hospital of Inner Mongolia Medical University).The diagnosis and management of AA were analyzed through unified summary. Different centers collected and summarized their data in 2017 and sent back the questionnaires for summary. RESULTS: A total of 8 766 AA patients were enrolled from 43 medical centers, including 4 711 males (53.7%) with median age of 39 years and 958 (10.9%) patients over 65 years old. Of 8 776 patients, 5 677 cases (64.6%) received one or more imaging examinations, and the other 3 099 (35.4%) did not receive any imaging examination. A total of 1 858 (21.2%) cases received medical treatment, mainly a combination of nitroimidazoles (1 107 cases, 59.8%) doublet regimen, followed by a single-agent regimen of non-nitroimidazoles (451 cases, 24.4%), a nitroimidazole-free doublet regimen (134 cases, 7.2%), a triple regimen of combined nitroimidazoles (116 cases, 6.3%), nitroimidazole alone (39 cases, 2.1%) and nitroimidazole-free triple regimen (3 cases, 0.2%). Of the 6 908 patients (78.8%) who underwent surgery, 4 319 (62.5%) underwent laparoscopic appendectomy and 2589 (37.5%) underwent open surgery. Ratio of laparotomy was higher in those patients under 16 years old (392 cases) or over 65 years old (258 cases) [15.1%(392/2 589) and 10.0%(258/2 589), respectively, compared with 8.5%(367/4 316) and 8.0%(347/4 316) in the same age group for laparoscopic surgery, χ²=91.415, P<0.001; χ²=15.915,P<0.001]. Patients with complicated appendicitis had higher ratio of undergoing open surgery as compared to those undergoing laparoscopic surgery [26.7%(692/2 589) vs. 15.6%(672/4 316), χ²=125.726, P<0.001].The cure rates of laparoscopic and open surgery were 100.0% and 99.8%(2 585/2 589) respectively without significant difference (P=0.206). Postoperative complication rates were 4.5%(121/2 589) and 4.7%(196/4 316) respectively, and the difference was not statistically significant (χ²=0.065, P=0.799). The incidence of surgical site infection was lower (0.6% vs. 1.7%, χ²=17.315, P<0.001), and hospital stay was shorter [6(4-7) days vs. 6(5-8) days, U=4 384 348.0, P<0.001] in the laparoscopic surgery group, while hospitalization cost was higher (median 12 527 yuan vs. 9 342 yuan, U=2 586 809.0, P<0.001). CONCLUSIONS: The diagnosis of acute appendicitis is still clinically based, supplemented by imaging examination. Appendectomy is still the most effective treatment at present. Laparoscopic appendectomy has become the main treatment strategy, but anti-infective drugs are also very effective.

Taishan Pinus massoniana pollen polysaccharide inhibits subgroup J avian leucosis virus infection by directly blocking virus infection and improving immunity.

Subgroup J avian leucosis virus (ALV-J) generally causes neoplastic diseases, immunosuppression and subsequently increases susceptibility to secondary infection in birds. The spread of ALV-J mainly depends on congenital infection and horizontal contact. Although ALV-J infection causes enormous losses yearly in the poultry industry worldwide, effective measures to control ALV-J remain lacking. In this study, we demonstrated that Taishan Pinus massoniana pollen polysaccharide (TPPPS), a natural polysaccharide extracted from Taishan Pinus massoniana pollen, can significantly inhibit ALV-J replication in vitro by blocking viral adsorption to host cells. Electron microscopy and blocking ELISA tests revealed that TPPPS possibly blocks viral adsorption to host cells by interacting with the glycoprotein 85 protein of ALV-J. Furthermore, we artificially established a congenitally ALV-J-infected chicken model to examine the anti-viral effects of TPPPS in vivo. TPPPS significantly inhibited viral shedding and viral loads in immune organs and largely eliminated the immunosuppression caused by congenital ALV-J infection. Additionally, pre-administration of TPPPS obviously reduced the size and delayed the occurrence of tumors induced by acute oncogenic ALV-J infection. This study revealed the prominent effects and feasible mechanisms of TPPPS in inhibiting ALV-J infection, thereby providing a novel prospect to control ALV-J spread.

Potentiation of Taishan Pinus massoniana pollen polysaccharide on the immune response and protection elicited by a highly pathogenic porcine reproductive and respiratory syndrome virus glycoprotein 5 subunit in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) heavily affects the global pork industry. Current available vaccine strategies have inherent drawbacks. In this work, the immune enhancement from Taishan Pinus massoniana pollen polysaccharide (TPPPS) and Freund's adjuvant on the efficacy of a PRRSV subunit vaccine were examined. Titers of specific anti-highly pathogenic PRRSV (HP-PRRSV) ELISA antibody and neutralizing antibody were significantly higher in pigs from the groups inoculated with medium- and high-dose TPPPS (mTPPPS, hTPPPS) adjuvant co-administered with a recombinant HP-PRRSV glycoprotein 5 subunit (GP5) than those from other groups (P < 0.05). Pigs inoculated with GP5 + Freund's adjuvant developed severely delayed humoral immune responses specific to GP5 within 28 days post-inoculation (dpi). The groups treated with mTPPPS and hTPPPS adjuvant exhibited the most potent immune enhancement effects on GP5 inoculation with cellular immunity developing, as shown by the level of T lymphocyte proliferation and the percentage of the CD3(+) T lymphocyte subpopulation. Although complete Freund's adjuvant elicited cell-mediated immune responses, the level of T lymphocyte proliferation in this group decreased quickly and no significant differences were observed compared with other adjuvant-alone groups at 56 dpi (P > 0.05). The ratio between CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocyte subpopulations indicated the inoculums of GP5 + mTPPPS and GP5 + hTPPPS induced consistently higher CD3(+)CD4(+) T lymphocyte subpopulations than other inoculums (P < 0.05). The immune responses caused by complete Freund's adjuvant were mainly mediated by CD3(+)CD8(+) T lymphocyte subpopulation (cytotoxic T lymphocytes) in the early stage of inoculation and had no significant difference compared with other adjuvant-alone groups after 28 dpi (P > 0.05). The low-dose TPPPS (lTPPPS) adjuvant also exhibited enhancement effects on humoral immune and T lymphocyte proliferation responses but these were significantly lower than the mTPPPS and hTPPPS doses (P < 0.05). Pigs challenged with HP-PRRSV from the GP5 + mTPPPS, GP5 + hTPPPS, and GP5 + Freund's adjuvant groups showed lower viremia, fewer clinical signs, and fewer pathological lung lesions compared with the groups of GP5-alone and GP5 + lTPPPS (P < 0.05). There were significant differences between the GP5-alone and GP5 + lTPPPS groups in detection indexes after viral challenge (P < 0.05). In conclusion, moderate doses of TPPPS as an adjuvant with GP5 show promise as a candidate for a HP-PRRSV subunit vaccine to efficiently prevent and control HP-PRRSV.

Arenobufagin intercalates with DNA leading to G2 cell cycle arrest via ATM/ATR pathway.

Arenobufagin, a representative bufadienolide, is the major active component in the traditional Chinese medicine Chan'su. It possesses significant antineoplastic activity in vitro. Although bufadienolide has been found to disrupt the cell cycle, the underlying mechanisms of this disruption are not defined. Here, we reported that arenobufagin blocked the transition from G2 to M phase of cell cycle through inhibiting the activation of CDK1-Cyclin B1 complex; The tumor suppressor p53 contributed to sustaining arrest at the G2 phase of the cell cycle in hepatocellular carcinoma (HCC) cells. Moreover, arenobufagin caused double-strand DNA breaks (DSBs) and triggered the DNA damage response (DDR), partly via the ATM/ATR-Chk1/Chk2-Cdc25C signaling pathway. Importantly, we used a synthetic biotinylated arenobufagin-conjugated chemical probe in live cells to show that arenobufagin accumulated mainly in the nucleus. The microscopic thermodynamic parameters measured using isothermal titration calorimetry (ITC) also demonstrated that arenobufagin directly bound to DNA in vitro. The hypochromicity in the UV-visible absorption spectrum, the significant changes in the circular dichroism (CD) spectrum of DNA, and the distinct quenching in the fluorescence intensity of the ethidium bromide (EB)-DNA system before and after arenobufagin treatment indicated that arenobufagin bound to DNA in vitro by intercalation. Molecular modeling suggested arenobufagin intercalated with DNA via hydrogen bonds between arenobufagin and GT base pairs. Collectively, these data provide novel insights into arenobufagin-induced cell cycle disruption that are valuable for the further discussion and investigation of the use of arenobufagin in clinical anticancer chemotherapy.

Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica.

A new quassinoid, bruceene A (1) along with seventeen known quassinoids (2-18) was isolated from the fruits of Brucea javanica. The structure of 1 was elucidated by extensive spectroscopic methods, and was further confirmed by single-crystal X-ray diffraction analysis. Isolation of similar quassinoids 1-3 as those in genus Ailanthus from genus Brucea, indicated the close chemotaxonomic relationship between these two genera, which further supported the phylogenetic study by DNA analysis. Compounds 5, 7, 10 and 12 with a 3-hydroxy-3-en-2-one moiety showed potent inhibitory activities against the MCF-7 and MDA-MB-231 cells with IC50 values in the ranges 0.063-0.182 µM and 0.081-0.238 µM, respectively; while glycosidation at 3-OH significantly decreased the cytotoxicity. It was also found that the most potent compound 7 induced apoptosis in MCF-7 cells via the intrinsic mitochondrial apoptotic pathway.

Hellebrigenin induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells through inhibition of Akt.

Hellebrigenin, one of bufadienolides belonging to cardioactive steroids, was found in skin secretions of toads and plants of Helleborus and Kalanchoe genera. In searching for natural constituents with anti-hepatoma activities, we found that hellebrigenin, isolated from traditional Chinese medicine Venenum Bufonis, potently reduced the viability and colony formation of human hepatocellular carcinoma cells HepG2, and went on to explore the underlying molecular mechanisms. Our results demonstrated that hellebrigenin triggered DNA damage through DNA double-stranded breaks and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser(1981)), p-Chk2 (Tyr(68)), p-CDK1 (Tyr(15)) and Cyclin B1, and down-regulation of p-CDC25C (Ser(216)). It was also found that hellebrigenin induced mitochondrial apoptosis, characterized by Bax translocation to mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c into cytosol and sequential activation of caspases and PARP. In addition, Akt expression and phosphorylation were inhibited by hellebrigenin, whereas Akt silencing with siRNA significantly blocked cell cycle arrest but enhanced apoptosis induced by hellebrigenin. Activation of Akt by human insulin-like growth factor I (hIGF-I) could obviously attenuate hellebrigenin-induced cell death. In summary, our study is the first to report the efficacy of hellebrigenin against HepG2 and elucidated its molecular mechanisms including DNA damage, mitochondria collapse, cell cycle arrest and apoptosis, which will contribute to the development of hellebrigenin into a chemotherapeutic agent in the treatment of liver cancer.

Effect of Sedum sarmentosum BUNGE extract on aristolochic acid-induced renal tubular epithelial cell injury.

Aristolochic acid (AA) is known as a potent mutagen that induces significant cytotoxic and mutagenic effects on renal tubular epithelial cells. Clinically, the persistent injury of AA results in the infiltration of inflammatory cells, epithelial-to-mesenchymal transition (EMT), and renal tubulointerstitial fibrosis. There are no truly effective pharmaceuticals. In this study, we investigated the potential role of the extract of Sedum sarmentosum Bunge (SSB), a traditional Chinese herbal medicine, on rat tubuloepithelial (NRK-52E) cells after AA injury in vitro. Evidence revealed that AA induced mitochondrial-pathway-mediated cellular apoptosis, accompanied by cell proliferation in a feedback mechanism. Treatment with SSB also induced cells to enter early apoptosis, but inhibited cell proliferation. In cultured NRK-52E cells, AA induced the imbalance of MMP-2/TIMP-2 and promoted EMT and ECM accumulation. SSB treatment significantly alleviated AA-induced NRK-52E cells fibrosis-like appearance, inhibited the induction of EMT, and deposition of ECM. SSB also decreased the activity of the NF-κB signaling pathway, resulting in down-regulated expression of NF-κB-controlled chemokines and pro-inflammatory cytokines, including MCP-1, MIF, and M-CSF, which may regulate the macrophage-mediated inflammatory reaction during renal fibrosis in vivo. Therefore, these findings suggest that SSB exerts protective effects against AA-induced tubular epithelial cells injury through suppressing the synthesis of inflammatory factors, EMT, and ECM production.

Comparative Cytogenetics Analysis of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians with Ct-1 DNA by Fluorescence In Situ Hybridization.

The chromosomes of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians were studied by FISH using C. farreri C(0)t-1 DNA probes. The results showed that C(0)t-1 DNA signals spread on all chromosomes in the three scallops, whereas signal density and intensity were different strikingly. Clustering brighter signals presented in the centromeric and telomeric regions of most C. farreri chromosomes, and in the centromeric or pericentromeric regions of several P. yessoensis chromosomes. Comparative analysis of the mapping indicated a relatively higher homology in the repetitive DNA sequences of the genome between C. farreri and P. yessoensis than that between C. farreri and A. irradians. In addition, FISH showed that the distribution of C(0)t-1 DNA clustering signals in C. farreri displayed completely similar signal bands between homologous chromosomes. Based on the C(0)t-1 DNA fluorescent bands, a more exact karyotype of C. farreri has been obtained. In this study, the comparative analysis based on C(0)t-1 DNA provides a new insight into the chromosomal reconstructions during the evolution process, and it is helpful for understanding an important source of genomic diversity, species relationships, and genome evolution.

[Pathogen identification of Pinellia ternata tuber disease and selection of fungicide].

The rotten tuber of Pinellia ternata was found as an important disease during the growing season in Tianshui production area. The isolated pathogens were tested following Koch's postulates and identified as Fusarium oxysporum. The suitable growth conditions for the F. oxysporum were 15-30 degrees C, pH 6-8, the optimal condition was 21.9 degrees C and pH 7.2. Some fungicides were demonstrated to be effective to inhibit the pathogen growth. 70% thiophanate-methyl and 58% metalaxyl MZ were most effective to inhibit the pathogen. The EC50 were 0.002 7, 0.066 2 g x L(-1), respectively.