[Effects of gelatin methacrylate anhydride hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the treatment of full-thickness skin defect wounds in mice].

Objective: To investigate the effects of gelatin methacrylate anhydride (GelMA) hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) in the treatment of full-thickness skin defect wounds in mice. Methods: This study was an experimental study. hUCMSCs-sEVs were extracted by ultracentrifugation, their morphology was observed through transmission electron microscope, and the expression of CD9, CD63, tumor susceptibility gene 101 (TSG101), and calnexin was detected by Western blotting. The human umbilical vein endothelial cells (HUVECs), the 3rd and 4th passages of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs) were all divided into blank control group (routinely cultured) and hUCMSC-sEV group (cultured with the cell supernatant containing hUCMSCs-sEVs). The cell scratch test was performed and the cell migration rates at 6, 12, and 24 h after scratching were calculated, the cell Transwell assay was performed and the number of migration cells at 12 h after culture was calculated, and the proportion of proliferating cells was detected by 5-acetylidene-2'-deoxyuridine and Hoechst staining at 24 h after culture, with sample numbers being all 3. The simple GelMA hydrogel and the GelMA hydrogel loaded with hUCMSCs-sEVs (hereinafter referred to as hUCMSC-sEV/GelMA hydrogel) were prepared. Then the micromorphology of 2 kinds of hydrogels was observed under scanning electron microscope, the distribution of hUCMSCs-sEVs was observed by laser scanning confocal microscope, and the cumulative release rates of hUCMSCs-sEVs at 0 (immediately), 2, 4, 6, 8, 10, and 12 d after soaking hUCMSC-sEV/GelMA hydrogel in phosphate buffer solution (PBS) were measured and calculated by protein colorimetric quantification (n=3). Twenty-four 6-week-old male C57BL/6J mice were divided into PBS group, hUCMSC-sEV alone group, GelMA hydrogel alone group, and hUCMSC-sEV/GelMA hydrogel group according to the random number table, with 6 mice in each group, and after the full-thickness skin defect wounds on the back of mice in each group were produced, the wounds were performed with PBS injection, hUCMSC-sEV suspenson injection, simple GelMA coverage, and hUCMSC-sEV/GelMA hydrogel coverage, respectively. Wound healing was observed on post injury day (PID) 0 (immediately), 4, 8, and 12, and the wound healing rates on PID 4, 8, and 12 were calculated, and the wound tissue was collected on PID 12 for hematoxylin-eosin staining to observe the structure of new tissue, with sample numbers being both 6. Results: The extracted hUCMSCs-sEVs showed a cup-shaped structure and expressed CD9, CD63, and TSG101, but barely expressed calnexin. At 6, 12, and 24 h after scratching, the migration rates of HEKs (with t values of 25.94, 20.98, and 20.04, respectively), HDFs (with t values of 3.18, 5.68, and 4.28, respectively), and HUVECs (with t values of 4.32, 19.33, and 4.00, respectively) in hUCMSC-sEV group were significantly higher than those in blank control group (P<0.05). At 12 h after culture, the numbers of migrated HEKs, HDFs, and HUVECs in hUCMSC-sEV group were 550±23, 235±9, and 856±35, respectively, which were significantly higher than 188±14, 97±6, and 370±32 in blank control group (with t values of 22.95, 23.13, and 17.84, respectively, P<0.05). At 24 h after culture, the proportions of proliferating cells of HEKs, HDFs, and HUVECs in hUCMSC-sEV group were significantly higher than those in blank control group (with t values of 22.00, 13.82, and 32.32, respectively, P<0.05). The inside of simple GelMA hydrogel showed a loose and porous sponge-like structure, and hUCMSCs-sEVs was not observed in it. The hUCMSC-sEV/GelMA hydrogel had the same sponge-like structure, and hUCMSCs-sEVs were uniformly distributed in clumps. The cumulative release rate curve of hUCMSCs-sEVs from hUCMSC-sEV/GelMA hydrogel tended to plateau at 2 d after soaking, and the cumulative release rate of hUCMSCs-sEVs was (59.2±1.8)% at 12 d after soaking. From PID 0 to 12, the wound areas of mice in the 4 groups gradually decreased. On PID 4, 8, and 12, the wound healing rates of mice in hUCMSC-sEV/GelMA hydrogel group were significantly higher than those in the other 3 groups (P<0.05); the wound healing rates of mice in GelMA hydrogel alone group and hUCMSC-sEV alone group were significantly higher than those in PBS group (P<0.05). On PID 8 and 12, the wound healing rates of mice in hUCMSC-sEV alone group were significantly higher than those in GelMA hydrogel alone group (P<0.05). On PID 12, the wounds of mice in hUCMSC-sEV/GelMA hydrogel group showed the best wound epithelization, loose and orderly arrangement of dermal collagen, and the least number of inflammatory cells, while the dense arrangement of dermal collagen and varying degrees of inflammatory cell infiltration were observed in the wounds of mice in the other 3 groups. Conclusions: hUCMSCs-sEVs can promote the migration and proliferation of HEKs, HDFs, and HUVECs which are related to skin wound healing, and slowly release in GelMA hydrogel. The hUCMSC-sEV/GelMA hydrogel as a wound dressing can significantly improve the healing speed of full-thickness skin defect wounds in mice.

[A multicenter clinical analysis of short-term efficacy of laparoscopic radical resection of hilar cholangiocarcinoma].

Objective: To investigate the feasibility and safety of laparoscopic radical resection of hilar cholangiocarcinoma at multiple centers in China. Methods: Between December 2015 and August 2019, the clinical data of 143 patients who underwent LRHC in Affiliated Hospital of North Sichuan Medical College, Second Hospital of Hebei Medical University, Affiliated Hospital of Xuzhou Medical University, Affiliated Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Hunan Provincial People's Hospital, the First Hospital Affiliated to Army Medical University, Sir Run Run Shaw Hospital Affiliated to Medical College of Zhejiang University, West China Hospital of Sichuan University, Nanfang Hospital of Southern Medical University and the First Affiliated Hospital of Chongqing Medical University were collected prospectively. There were 92 males and 51 females with age of (64±11) years (range: 53 to 72 years). Bismuth type: type I, 38 cases (26.6%), type Ⅱ, 19 cases (13.3%), type Ⅲa, 15 cases (10.5%), type Ⅲb, 28 cases (19.6%) and type Ⅳ, 43 cases (30.0%). The patients within the first 10 operation cases in each operation time (the first 10 patients in each operation team) were divided into group A (77 cases), and the patients after 10 cases in each operation time were classified as group B (66 cases); the cases with more than 10 cases in the center were further divided into group A(1) (116 cases), and the center with less than 10 cases was set as group A(2) (27 cases). T test or Wilcoxon test was used to compare the measurement data between groups, and the chi square test or Fisher exact probability method was used to compare the counting data between groups. Kaplan Meier curve was used for survival analysis. Results: All patients successfully completed laparoscopic procedure. The mean operation time was (421.3±153.4) minutes (range: 159 to 770 minutes), and the intraoperative blood loss was 100 to 1 500 ml (median was 300 ml) .Recent post-operative complications contained bile leakage, abdominal bleeding, abdominal infection, gastrointestinal bleeding, and delay gastric emptying, pulmonary infection, liver failure, et al.The post-operative hospital stay was (15.9±9.2) days. The operation time in group B was relatively reduced ( (429.5±190.7)minutes vs. (492.3±173.1)minutes, t=2.063, P=0.041) and the blood loss (465 ml vs. 200 ml) was also reduced (Z=2.021, P=0.043) than that in group B. The incidence of postoperative biliary fistula and lung infection in patients in group A was significantly higher than that in group B (χ(2)=4.341, 0.007; P=0.037, 0.047) .Compared with group A(2), the operation time in group A(1) was relatively reduced( (416.3±176.5)minutes vs. (498.1±190.4)minutes, t=2.136, P=0.034) , the incidence of bile leakage and abdominal cavity infection in group A(1) was lower than that in group A(2) (χ(2)=7.537, 3.162; P=0.006, 0.046) . Kaplan Meier survival curve showed that the difference of short-term survival time between group A and group B was statistically significant (P<0.05) . Conclusions: The completion of laparoscopic hilar cholangiocarcinoma radical surgery is based on improved surgical skills, and proficiency in standardized operation procedures.It is feasible for laparoscopic radical resection of hilar cholangiocarcinoma to well experienced surgeon with cases be strictly screened, but it is not recommended for widespread promotion at this exploratory stage.

[Application of intestinal stent in prevention of anastomotic leakage after rectal cancer operation].

Objective: To observe preventive effect of intestinal stent against anastomotic leakage after rectal cancer operation. Methods: A retrospective cohort study was carried out. Clinical data of 107 patients with low rectal cancer undergoing laparoscopic radical resection from January 2015 to August 2019 were retrospectively analyzed. Intestinal stent was placed intraoperatively in 48 cases and was not placed in 59 cases. Postoperative Wexner score for anal function and incidence of anastomotic leakage were compared between patients with and without intstinal stent. Results: There was no significant differences in age, distance between tumor and the anal verge, operative time and postoperative Wexner score for anal function between the two groups (all P>0.05). After a month of follow-up, the incidence of anastomotic leakage was 16.9% (10/59) in the non-stent group, while no anastomotic leakage was found in the stent group (P=0.002). Conclusion: Placement of intestinal stent can effectively prevent anastomotic leakage after low rectal cancer surgery.

[Influence factors and differences of posterior corneal elevation measured by Pentacam system combined with Corvis ST].

Objective: To investigate the influence factors and differences of abnormal posterior corneal elevation by Pentacam system and Corvis ST. Methods: This retrospective case series study included 227 eyes of 144 patients (90 males, 139 eyes; 54 females, 88 eyes) from December 2017 to October 2018 who were going to receive corneal refractive surgery at the Corneal Refraction Department of Qingdao Eye Hospital. The general data of the patients including gender, age, refractive parameters, optimal correction of spherical and cylindrical diopters were collected. All patients underwent Pentacam system and Corvis ST measurement. According to the back difference (BD) of Pentacam parameters, BD<12 µm was set as the control group (59 patients, 118 eyes) and BD≥12 µm as the high BD group (85 patients, 109 eyes). In the high BD group, BD≤16 µm was set as the suspicious group (44 patients, 53 eyes), while BD>16 µm was set as the abnormal group (41 patients, 56 eyes). Seven parameters of Pentacam and 15 parameters of Corvis ST were selected. The Pentacam parameters included BD, anterior surface keratometry (ASK), posterior surface keratometry (PSK), anterior surface astigmatism (AAstig), posterior surface astigmatism (PAstig), central corneal thickness (CCT), and corneal diameter (W-W). The parameters of Corvis ST included the first applanation time (AT(1)), the first applanation length (AL(1)), the first applanation velocity (AV(1)), the second applanation time (AT(2)), the second applanation length (AL(2)), the second applanation velocity (AV(2)), highest concavity time (HCT), highest concavity peak distance (HC-PD), highest concavity deformation amplitude (HC-DA), highest concavity radius (HC-R), the ratio of deformation amplitude (DA ratio), Integr. Radius, corneal thickness thinnest/pachymetric progression (ARTh), SPA1 (resultant pressure divided by deflection amplitude at the first applanation), and the Corvis Biomechanical Index (CBI). The comparison between the groups was analyzed with Independent sample t test, Kruskal-Wallis H test, and Bonferroni test. Spearman rank correlation analysis was used to explore the correlation factors of BD, and the main factors affecting BD were found through multiple linear regression. Results: There were no statistically significant differences between the control group and the high BD group in age, spherical diopters, and cylindrical diopters (t=-3.311, -1.808, -2.359; P=0.071, 0.072, 0.121, respectively). In Pentacam parameters, ASK, PSK, PAstig, and W-W showed significant differences among groups (Z=18.492, 31.547, 10.773, 70.167; P<0.05). AAstig and CCT showed no statistical difference between groups (P>0.05). Compared with the control group [42.80 (41.98, 44.00)], ASK increased in the abnormal group [43.40 (42.20, 44.40)] significantly (t=-4.292; P<0.05). PSK of the suspicious group [-6.50 (-6.60, -6.35)] and the abnormal group [-6.50 (-6.70, -6.33)] increased significantly compared with the control group [-6.30 (-6.50, -6.20)] (t=4.492, 4.618; P<0.05). Compared with the control group [0.40 (0.30, 0.50)], PAstig of the suspicious group [0.40 (0.30, 0.40)] and the abnormal group [0.40 (0.30, 0.40)] increased significantly (t=2.796, 2.515; P=0.016, 0.036). Compared with the control group [11.50 (11.40, 11.80)], W-W of the suspicious group [11.40 (11.00, 11.60)] and the abnormal group [11.10 (10.90, 11.30)] decreased, and W-W of the abnormal group also decreased significantly compared with the suspicious group (t=3.235, 8.353, 4.282; P<0.05). The correlation analysis between BD and Pentacam parameters of patients in each group showed that BD was negatively correlated with W-W (r=-0.614, -0.304, -0.396, -0.661, P<0.05) in the control group, the suspicious group, the abnormal group, and all patients, while BD had a low correlation with other parameters or no significant correlation. The correlation analysis of BD and Corvis ST parameters in patients showed that only in the suspicious group, BD was positively correlated with AV(1), HCT, and HC-DA (r=0.332, 0.361, 0.382, P<0.05), while no significant correlation was found between BD and other Corvis ST parameters in each group. In order to further explore the main factors affecting BD, Pentacam parameters and Corvis ST parameters were selected as independent variables with BD as the dependent variable to establish a multivariate linear regression analysis model. There was no collinearity between variables W-W, ASK, PSK, HC-PD, SPA1, and CCT (tolerance<0.100). The equation test result was F=37.221, P<0.001, adjusted r(2)=0.504, and the fitting was good. Conclusions: Among the Pentacam parameters, W-W, ASK, and PSK are the main factors affecting the change of BD. HC-PD and SPA1 in the Corvis ST parameters may also have some influence on BD. The Pentacam system combined with Corvis ST is a very useful differential diagnosis system for patients with abnormal BD. (Chin J Ophthalmol, 2020, 56:110-117).

[The role of neutrophil in the pathogenesis of chronic rhinosinusitis].

SummaryChronic nasal-sinusitis is a chronic inflammatory disease characterized by persistent inflammation in the nasal and nasal mucosa. The pathogenesis of CRS is extremely complex and there is currently a lack of effective therapy. The reason for inaccurate diagnosis and invalid treatment of CRS is its sophisticated and unclear mechanism. The pathogenesis of CRS from Asian populations is neutrophil infiltration mediated by Th1/Th17 mixture. Consequently, exploring the function of neutrophil in the pathogenesis of CRS plays an important role in clinical diagnosis and treatment for CRS patients in China.

[The possible failing reasons of balloon catheter dilation procedure in the management of chronic rhinosinusitis].

Objective:To investigate the effectiveness of balloon catheter dilation (BCD) in the treatment of chronic rhinosinusitis, and to analyse the possible factors which lead to BCD failure, as well as to provide basic reference for BCD clinical usage.Method:Forty-six sinuses of 32 patients with chronic rhinosinusitis were underwent "Balloon-only" BCD or "FESS assisted" BCD at our institution between September 2014 and December 2016. By recording details of the operation of all the subjects in this study and following up the clinical symptoms, nasal endoscopy, computed tomography of the sinuses, and postoperative complications six months after operation, to evaluate the difficulty, safety, effectiveness and especially, the failing reasons of BCD.Result:BCD was approached in 46 sinuses (19 maxillary sinus, 22 frontal and 5 sphenoid), and succeeded in 13 maxillary sinuses, 19 frontal sinuses, and 4 sphenoid sinuses. Of the 13 maxillary sinuses, there were 9 sinuses underwent "Balloon-only" procedure, other 4 cases underwent "FESS assisted" procedure. There were 3 cases of frontal sinus failed, because of the frontal recess anatomical complexity and the twisted drainage. Of the 5 sphenoid sinuses, 4 cases succeeded, including fungal sphenoiditis cases, in which the mould was completely cleared through the dilated ostia, and 1 case failed. All the patients were followed up for 1, 3 and 6 months of patient's quality of life, nasal endoscope, computed tomography of the sinuses. The results showed that the SNOT-20 scores of the quality of life in significant relief of symptoms, nasal mucosa status improved significantly compared with the preoperative, dilated ostium remains open, no obvious scar formation, no severe operative complications.Conclusion:Balloon catheter dilation in the treatment of chronic rhinosinusitis is safe and effective. But the operation indications is limited, and many factors influence the success rate of BCD, so, preoperatively gaining the information of nasal cavity and anatomical structure around ostium according to patients' nasal endoscopy and sinus CT is critical to success of BCD.

[Study on the change of optical zone after femtosecond laser assisted laser in situ keratomileusis].

Objective: To explore the change of optical zone after femtosecond laser assisted laser in sitn keratomileusis(FS-LASIK) so as to provide the reference for measurement and design of clinical optical zone. Methods: This retrospective case series study covers 41 eyes of 24 patients (7 males and 17 females, aged from 18 to 42 years old) with myopia and myopic astigmatism who have received FS-LASIK surgery at Corneal Refractive Department of Qingdao Eye Hospital and completed over 6 months of clinical follow-up. Pentacam system (with the application of 6 corneal topographic map modes including: the pure axial curvature topographic map, the pure tangential curvature topographic map, the axial curvature difference topographic map, the tangential curvature difference topographic map, the postoperative front elevation map and the corneal thickness difference topographic map), combined with transparent concentric software (a system independently developed by Qingdao Eye Hospital) was used to measure the optical zone at 1, 3 and 6 months postoperatively, the optical zone diameters measurement results among different follow-up times in group were analyzed with the repeated measures analysis of variance, and the actual measured values and the theoretical design values of the optical zone were analyzed with independent-samples t-testing. Spearman correlation coefficient (r(s)) have been applied to evaluate the relationship between postoperative optical zone measurement values and the potential influencing factors. Results: The optical zone diameters measured by pure axial curvature topographic map at 1, 3 and 6 months after FS-LASIK showed (6.55±0.50)mm, (6.50±0.53)mm and (6.48±0.53)mm respectively. The differences between values are of no statistical significance (F=1.60, P=0.21), the optical zone diameter measured by pure tangential curvature topographic map at 1, 3 and 6 months after FS-LASIK showed (5.44±0.46)mm, (5.46±0.52)mm and (5.44±0.50)mm respectively, the differences between values are of no statistical significance (F=0.17, P=0.85). The optical zone diameters measured by postoperative front elevation map at 1, 3 and 6 months after FS-LASIK showed (5.06±0.28)mm, (5.12±0.32)mm and (5.17±0.28)mm respectively. The differences between the values of 3 and 6 months postoperatively are of no statistical significance (F=6.14, P=0.15), the optical zone diameters measured by axial curvature difference topographic map at 1, 3 and 6 months after FS-LASIK showed (6.51±0.37)mm, (6.45±0.41)mm and (6.41±0.40)mm respectively, and the differences between the values of 3 and 6 months postoperatively are of no statistical significance (F=7.25, P=0.05). The optical zone diameters measured by tangential curvature difference topographic map at 1, 3 and 6 months after FS-LASIK showed (5.21±0.23)mm, (5.16±0.19)mm and (5.17±0.20) mm respectively, and the differences between the values of 1 and 3 months postoperatively are of statistical significance (F=1.75, P=0.04). The optical zone diameters measured by corneal thickness difference topographic map at 1, 3 and 6 months after FS-LASIK showed (6.53±0.40)mm, (6.39±0.43)mm and (6.41±0.47)mm respectively, and the differences between the values of 1 and 3 months postoperatively are of statistical significance (F=1.67, P=0.032). The actual measured optical zone values from the 6 different modes of Pentacam system are less than the theoretical design values (7.75 mm), and the differences were statistical significance (t=-15.42, -29.39, -59.27, -21.47, -81.69, -18.22, P<0.01). Conclusions: The optical zone measurement values tend to be stable at 3 months after FS-LASIK. The actual measured values from all the 6 different modes of Pentacam system were less than the theoretical design values. The results from pure tangential curvature topographic map, the tangential curvature difference topographic map and the postoperative front elevation map showed greater variation with clear border, which was beneficial for eccentric research. The results from pure axial curvature topographic map, the axial curvature difference topographic map and the corneal thickness difference topographic map were close to the theoretically designed values. Furthermore, the axial curvature difference topographic map showed clearer border and less variation thus maybe more favorable for measuring optical zone in clinical application.(Chin J Ophthalmol, 2018, 54: 39-47).

[Tumor-secreted vascular endothelial growth factor A increases the pulmonary metastasis from nasopharyngeal carcinoma].

Objective: Vascular endothelial growth factor A (VEGFA) was investigated as the key protein which might promote the specific metastasis progress of nasopharyngeal carcinoma. Methods: Sixteen specimens of pulmonary metastasis carcinoma and counterparts in primary nasopharyngeal carcinoma tissue were collected from patients. The expression of VEGFA through immunohistochemistry was investigated.VEGFA was knocked down by siRNA in two cell lines of nasopharyngeal carcinoma (CNE-1 and 5-8F), MTT and Transwell test were used to explore the role of VEGFA in praxiology. Then shRNA was used to cultivate the stable CNE-1 cell line with down-regulated-expression of VEGFA. The nude mice models were built through tail vein injection of specific nasopharyngeal carcinoma cells, and lungs were collected to perform further metastasis analysis. Results: Previous genetic studies showed that VEGFA had higher expression in metastasis tissue, and the result was validated in the present study using immunohistochemistry. The percentage of positive cells was 84.8% in pulmonary metastasis group, 51.5% in primary tissue group (t=8.639, P<0.05), average optical density was 0.154 in pulmonary metastasis group, 0.061 in primary tissue group (t=18.791, P<0.05). Low expression of VEGFA inhibited cell viability of optical density value of CNE-1 in siRNA gourp was 0.715, 0.902 in control group (t=7.274, P<0.05); 5-8F in siRNA group was 0.715, 0.935 in control group (t=7.751, P<0.05). Number counting suppressed migration of CNE-1 in siRNA group was 52 per high-power lens, 124 per high-power lens in control group (t=29.380, P<0.05), 5-8F in siRNA group was 65 per high-power lens, 155 per high-power lens in control group (t=18.181, P<0.05). Number counting invasion of CNE-1 in siRNA gourp was 38 per high-power lens, 86 per high-power lens in control group (t=27.665, P<0.05), 5-8F in siRNA group was 52 per high-power lens, 116 per high-power lens in control group (t=40.972, P<0.05) in vitro. Furthermore, knock-down of VEGFA in nasopharyngeal carcinoma reduced the pulmonary metastasis in vivo. Number counting of tumor volumes in shRNA group was 2.4, and 11.0 in control group (t=6.143, P<0.05); average optical density of immunohistochemistry in shRNA group was 0.033, and 0.176 in control group (t=15.734, P<0.05). Conclusions: Results above reveal the overexpression of VEGFA in nasopharyngeal carcinoma can facilitate the pulmonary metastasis. Targeting VEGFA may provide a new biomarker of clinical study.

Contrast Functions of αA- and αB-Crystallins in Cancer Development.

α-Crystallins, initially identified as the structural proteins of the ocular lens, belong to the small heat shock protein family. They play significant roles in maintaining the lens transparency and preventing protein aggregation. α-Crystallins exist in two isoforms: αA and αB, and they display differential tissue distribution. Their mutations are implicated in several human diseases including cardiac myopathies, neurodegenerative diseases, cataracts and various types of cancers. Increased αB expression was detected in retinoblastoma, breast cancer, glioblastoma, prostate and renal cell carcinomas, indicating its role in promoting tumor growth. A complex picture emerges for αA. Although earlier studies suggest that αA may promote cancer development, recent studies from our laboratory demonstrate that αA can act as a tumor suppressor inhibiting cell transformation and retarding cell migration through modulating MAP kinase activity. In this review, we summarize the recent progress about the functions of αA and αB in cancer development.

Sumoylation Pathway as Potential Therapeutic Targets in Cancer.

Sumoylation is a covalent protein posttranslational modification that conjugates the small ubiquitin-like peptide SUMO to substrate. Sumoylation is critically implicated in multiple biological processes, including cell proliferation, differentiation, senescence and apoptosis, etc. Therefore, it is not surprising that dysregulation of sumoylation has been implicated in tumorigenesis and different types of cancer were found to be addicted to functional sumoylation pathway. The potential role for sumoylation as a therapeutic target in caner is emerging. In this review, we summarize current knowledge regarding the involvement of sumoylation in genome stability and DNA damage response. We will further discuss the therapeutic potential of sumoylation as synthetic lethal partner and as a key signaling pathway in cancer stem cells.

[Detection and screen of pulmonary metastasis-related signature genes in nasopharyngeal carcinoma].

Objective: To establish an animal model of pulmonary metastasis from nasopharyngeal carcinoma (NPC) and to investigate differential genes associated with pulmonary metastasis. Methods: CNE cell line was used to construct the stable metastasis CNE/Luc cell line which steadily expresses the fluorescent enzyme genes. The CNE/Luc cells were injected into immunodeficiency mice through tail vein, and with the in vivo imaging technology, the mice with pulmonary metastasis were filtered out. The pulmonary metastasis cells, were separated and injected into the tail vein of other nude mice to obtain the tissue-specificity metastasis cells confirmed by fluorescent imaging system. Based on the gene chip, the differential genes associated with pulmonary metastasis for NPC were found. Results: The gene expression profiles of nasopharyngeal carcinoma cell line CNE/Luc and their lung metastasis-associated subpopulation CNE/Luc-2 were constructed by gene chip technology. Ten metastasis-related genes were screened by software analysis, namely as TP53, PIK3CA, MET, KRAS, VEGFA, EDNRB, GSK3B, FOXO3, SOD2, and BIRC3. Conclusions: Some genes including TP53, PIK3CA, MET, KRAS, VEGFA, EDNRB, GSK3B, FOXO3, SOD2, and BIRC3 are indicated to have important roles in the lung metastasis of nasopharyngeal carcinoma.

[Influences of exendin-4 on the secretion function of islet beta cells from rats in the early stage of severe scald].

Objective: To observe the secretion function changes of islet beta cells isolated from rats in the early stage of severe scald, and to explore the influence of them. Methods: Thirty-six Wistar rats were divided into sham injury (SI) group, sham injury+ exendin-4 (SIE) group, scald (S) group, and scald+ exendin-4 (SE) group according to the random number table, with 9 rats in each group. Rats in groups S and SE were inflicted with 50% total body surface area full-thickness scald by a 12-s immersion of back and a 6-s immersion of abdomen in 94 ℃ hot water. Rats in groups SI and SIE were sham injured through immersion of back and abdomen in 37 ℃ warm water. Rats in groups S and SE were subcutaneously injected with exendin-4 (4 µg/kg) twice a day post injury, while rats in groups SI and SIE were subcutaneously injected with sterile water in the same volume. At post injury hour (PIH) 72, the following detections were performed. Eight rats of each group were respectively selected to measure level of fasting blood glucose with cutting-tail method, and to detect plasma level of glucagon-like peptide 1 (GLP-1) and serum level of insulin by enzyme-linked immunosorbent assay (ELISA). The insulin resistant index (HOMA-IR) was calculated. Six rats of each group were respectively selected for islet isolation. The isolated rat islets were stimulated with RPMI 1640 medium containing 2.8 or 16.7 mmol/L glucose, respectively. Insulin content in supernatant was detected by ELISA, and insulin secretion index was calculated with 6 samples in each group. The isolated islets from 3 rats of each group were selected for the observation of the super-structure of islet beta cells under transmission electron microscope. The number of docked granules in per 10 µm membrane of islet beta cells and the ratio of insulin vesicles to the total insulin granules were calculated with 3 samples in each group. Data were processed with one-way analysis of variance and LSD test. Results: (1) Compared with that in group S, levels of fasting blood glucose of rats in group SI, SIE, and SE were significantly decreased (P<0.05 or P<0.01). (2) Compared with those in group SI, plasma level of GLP-1 of rats in group SIE was significantly increased (P<0.05), while serum level of insulin and HOMA-IR of rats did not change obviously (with P values above 0.05). Plasma levels of GLP-1 of rats in groups S and SE were significantly decreased (with P values below 0.01), while serum levels of insulin and HOMA-IR were obviously increased (with P values below 0.01). Compared with those in group SIE, plasma levels of GLP-1 of rats in groups S and SE were significantly decreased (with P values below 0.01), while serum levels of insulin and HOMA-IR were significantly increased (with P values below 0.01). Compared with those in group S, plasma level of GLP-1 and serum level of insulin of rats in group SE were significantly increased (with P values below 0.01), while HOMA-IR was significantly decreased (P<0.05). (3) There was no statistically significant difference in the insulin secretion content of rats in the 4 groups when stimulated with 2.8 mmol/L glucose (P>0.05). Under stimulation of 16.7 mmol/L glucose, compared with that in group SI, the insulin secretion content of rats in groups SIE and SE were significantly increased (P<0.05 or P<0.01), while in group S it was significantly decreased (P<0.05). Compared with that in group SE, the insulin secretion content of rats in group S was significantly decreased (P<0.01) . Compared with that in group S, the insulin secretion content of rats in group SE was significantly increased (P<0.01). Compared with that in group SI (2.25±0.20), the insulin secretion index of rats in group SE (2.68±0.24) was significantly increased (P<0.05). Compared with that in group SIE (2.47±0.18), the insulin secretion index of rats in group S (2.11±0.28) was significantly decreased (P<0.05). Compared with that in group S, the insulin secretion index of rats in group SE was significantly increased (P<0.01). (4) Compared with those in group SI, the number of docked granules per 10 µm membrane of islet beta cells in group SE was significantly increased (P<0.05), while the ratio of insulin vesicles of rat islet beta cells in group S was significantly increased (P<0.01). Compared with those in group SE, the number of docked granules per 10 µm membrane of islet beta cells in group S was significantly decreased (P<0.01), while the ratio of insulin vesicles of rat islet beta cells was significantly increased (P<0.05). Compared with those in group S, the number of docked granules per 10 µm membrane of islet beta cells in group SE was significantly increased (P<0.01), while the ratio of insulin vesicles of rat islet beta cells was significantly decreased (P<0.05). Conclusions: In the early stage of severe scald in rats, level of GLP-1 is decreased and the insulin secretion function of islet beta cells is injured. Long-lasting GLP-1 analogous exendin-4 can improve the secretion function of isolated islet beta cells from severely scalded rats.

Expression and prognostic influence of NF-κB and EGFR in esophageal cancer.

The goal of this study was to investigate the expression profiles of nuclear factor-kappa B (NF-κB) and epidermal growth factor receptor (EGFR) in esophageal cancer and to determine their association with tumor prognosis. This study included 40 esophageal cancer patients [22 men and 18 women; average age = 62.7 ± 3.9 years; tumor-node-metastasis (TNM) staging: 12 patients with stage I, 13 patients with stage II, and 15 patients with stage III disease]. Tumor tissues and tumor-adjacent tissue specimens were collected during radical resections at our hospital. Immunohistochemical staining was used to examine these tissues for NF-κB and EGFR expression. Follow-up of all patients included gathering information such as the 3-year survival rate. We found that NF-κB and EGFR expression was significantly higher in tumor tissues compared to tumor-adjacent normal tissues. Expression was not related to gender or age, but was positively associated with the degree of tumor infiltration. NF-κB and EGFR expression levels gradually increased with higher TNM stage, but this difference was not significant. Follow-up results showed that patients with higher NF-κB and EGFR levels had a lower survival rate and unfavorable prognosis. In conclusion, we found that NF-κB and EGFR expression was significantly elevated during the occurrence and development of esophageal carcinoma, and expression of these factors appears to be correlated with cancer progression. Higher expression of both genes is associated with an unfavorable prognosis.

The tumor suppressor, p53 regulates the γA-crystallin gene during mouse lens development.

The tumor suppressor, p53 regulates a large number of target genes to control cell proliferation and apoptosis. In addition, it is also implicated in the regulation of cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates several genes including c-Maf and Prox1, two important transcription factors for lens differentiation, and αA and ßA3/A1, the lens differentiation markers. In the present study, we present evidence to show that the γA-crystallin gene distal promoter and the first intron also contain p53 binding sites and are capable of mediating p53 control during mouse lens development. First, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from human lens epithelial cells (HLE) directly binds to the p53 binding sites present in the γA-crystallin gene. Second, the exogenous wild type p53 induces the dose-dependent expression of the luciferase reporter gene driven by the basic promoter containing the γA-crystallin gene p53 binding site. In contrast, the exogenous dominant negative mutant p53 causes a dose-dependent inhibition of the same promoter. Third, ChIP assays revealed that p53 binds to the γA-crystallin gene promoter in vivo. Finally, in the p53 knockout mouse lenses, the expression level of the γAcrystallin gene was found attenuated in comparison with that in the wild type mouse lenses. Together, our results reveal that p53 regulates γA-crystallin gene expression during mouse lens development. Thus, p53 directly regulates all 3 types of crystallin genes to control lens differentiation.

Effect of glutathione and Y27632 on the viability of cryopreserved porcine adipose-derived stem cells.

BACKGROUND: Recently it has been reported that reduced glutathione (GSH) and/or Rho-associated kinase (ROCK) inhibitor supplemented in cryopreservation solution could improve the viability of cells. OBJECTIVE: To identify the cryopreservation efficiency of GSH and ROCK inhibitor on porcine ADSCs. MATERIALS AND METHODS: Porcine ADSCs were separated and cultured. Cells at the 4th passage were suspended in cryopreservation solution supplemented with Y-27632 and GSH or both, and then frozen and thawed. The viability of cryopreserved ADSCs was compared using a MTT assay. RESULTS: The addition of GSH and Y-27632 to cryopreservation solution (dimethyl sulfoxide) and post-thaw culture medium significantly improves the post-thaw viability of ADSCs. CONCLUSION: GSH and Y-27632 are able to increase the survival of ADSCs, and they have an additive effect, as compared to GSH or Y27632 alone.

First Report of Forsythia Shoot Blight Caused by Phytophthora nicotianae in Connecticut.

Blighting of Forsythia × intermedia 'Showoff' was first observed affecting several hundred plants in a commercial nursery in Connecticut in September 2012. Symptoms included wilting, leaf and stem blight, and dieback progressing to plant death. A Phytophthora sp. was isolated from symptomatic tissues on half-strength potato dextrose agar (½PDA). Colonies were white and cottony on ½PDA, reaching 9 mm in 15 days at 25°C, but colorless and inconspicuous on pimaricin, ampicillin, rifampicin, pentachloronitrobenzene agar (PARP) with sparse and limited aerial mycelium, reaching 60 mm in 15 days at 25°C. The characteristics of the pathogen were observed and measured from a 3-month-old colony on ½PDA. Sporangia were abundant, various in shape, ovoid, ellipsoid to pyriform or limoniform, occasionally gourd shaped or irregular; (17.9) 27.2 to 41.4 (47.3) × (12.6) 19.1 to 30.5 (36.7) µm (n = 30), length/breadth ratio 1.4 ± 0.2, papillate and noncaducous. Papillae measured 2.9 ± 0.8 × 3.4 ± 0.8 µm (n = 10). Chlamydospores were present, 23.4 ± 3.1 × 22 ± 3.3 µm (n = 10). Oogonia and oospores were not observed. Arachnoid mycelia were present. These morphological characteristics are consistent with Phytophthora nicotianae Breda de Haan (1). The identity of the pathogen was confirmed as P. nicotianae by BLAST analysis of ITS, Cox II, and beta tubulin gene sequences (99% match for the three sequences, E value = 0). Pathogenicity tests were conducted four times on healthy liners of Forsythia × intermedia 'Showoff' grown in 10-cm-diameter pots. Leaves and stems were wounded by pricking with a sterile needle and six plants were inoculated with 0.25 cm2 plugs of the pathogen growing on ½PDA placed on three leaves and in three stem nodes and covered with Parafilm. Controls consisted of an equal number of plants wounded and inoculated with ½PDA alone. All plants were placed inside high humidity chambers for 24 h and then transferred to a greenhouse for up to 1 month. Typical symptoms developed within 1 week of inoculation and the pathogen was re-isolated from diseased tissue. Disease incidence was nearly 100% of inoculated leaves and stems and not observed in control plants without the pathogen. Three replicate 6-week-old broadleaf tobacco 'C9' plants were each inoculated with tobacco or forsythia isolates of P. nicotianae or sterile media alone, by wounding stems and placing colonized 0.25 cm2 ½PDA plugs into wounds and covering with Parafilm. After 1 week, stems were split and the length of internal necrosis in the stem measured. Disease resulted from inoculation with both the tobacco and forsythia isolates and stem necrosis averaged 43 and 23 mm for tobacco or forsythia isolates, respectively. No necrosis was observed in the pathogen-free controls. P. nicotianae has been reported from the basal stem and roots of F. viridissima in Italy (2) and from shoots of Forsythia × intermedia in Virginia (3). To our knowledge, this is the first report of P. nicotianae causing shoot blight on Forsythia in the northeastern United States. References: (1) J. van. Breda de Haan. Mededeelingenuit's Lands Plantentuin Batavia. 15:57, 1896. (2) S. O. Cacciola et al. Plant Dis. 78:525, 1994. (3) C. X. Hong et al. Plant Dis. 89:430, 2005.

First Report of Potato Virus H on Solanum muricatum in China.

Solanum muricatum, commonly known as pepino, pepino dulce, or tree melon, is a perennial shrub well known for its attractive, sweet, flavorful fruits and is frequently cultivated as an annual. It has gained increasing popularity in China and is grown as a cash crop in many provinces. S. muricatum belongs to the family Solanaceae and is closely related to tomato, eggplant, and potato. In 2012, during a study of serological relationships between PVH and PVM on potatoes, potato virus H (PVH) was detected serendipitously in symptomless pepino plantlets in Beijing, grown from tissue culture stocks. PVH is a recently discovered carlavirus reported from potato plants from Huhhot, Inner Mongolia Autonomous Region. Since then, it was found on potatoes in Yunnan, Hebei, Liaoning, Heilongjiang, and Xinjiang provinces. PVH induces mild symptoms with a slight leaf curl in systemic leaves, but most often it is almost symptomless or latent on potatoes (2). To confirm the presence of PVH on S. muricatum, surveys were conducted in 2012 and 2013 in Gansu, Yunnan, and Guangxi provinces and Beijing. Fruits and leaves were collected randomly from pepino plants displaying no obvious symptoms. For PVH detection, a combination of RT-PCR, genome sequencing and serological assays were used. RNAs extracted from fruits and leaves were amplified using RT-PCR with primer pairs PVHCPF and PVHCPR (2), and extracted samples were probed by Western blotting with the specific polyclonal antiserum against PVH (2). Among the 50 plants randomly collected, fruits and leaves of nine plants tested positive for PVH. Subsequently, an RT-PCR product of the expected size (2.6 kb) encompassing the triple-gene block, the capsid protein gene, and the cysteine-rich protein gene, was amplified with a specific primer pair (PVHB1F 5'-TGATGGAATTTACAAAAAC-3' and PVHUR 5'-CTTATGCGCATCTATCAATC-3'), and then cloned into pMD19-T (TaKaRa, Dalian, China) and sequenced (PVH-Pepino with GenBank Accession No. KF546312). Further sequence comparison showed that PVH-Pepino shared 91 to 98% nucleotide sequence identity in the genes mentioned above with those of the reported potato isolates PVH-Ho and PVH-YN (HM584819 and JQ904630, respectively). PVH-Pepino shared deduced amino acid identity of 98 to 99% in CP gene with PVH-Ho and PVH-YN, respectively, but only shared 57 to 67% amino acid identities with other reported carlaviruses (1,2). Thus, latent infection of PVH on S. muricatum was confirmed. To our knowledge, this is the first report of S. muricatum as a natural host of PVH. Our results suggest that PVH, as a new member of the genus Carlavirus, has a wider host range than originally expected. Potatoes and pepinos are crops widely grown in China. The fact that no symptoms were expressed by PVH in pepino plants (symptomless carrier) and only mild symptoms expressed by PVH in diseased potatoes makes detection and remediation of this disease more difficult. Although this finding does not show that PVH is economically important to pepino, this cannot be excluded in the presence of other viruses (2). References: (1) A. King et al. Page 881 in: Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, London, 2011. (2) Y. Y. Li et al. PLOS ONE 8(6):e69255, 2013.

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.).

Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

p53 directly regulates αA- and ßA3/A1-crystallin genes to modulate lens differentiation.

It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the αA and ßA3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.

PP-1α and PP-1γ display antagonism and differential roles in tumorigenicity of lung cancer cells.

Protein serine/threonine phosphatases are important cellular signaling molecules and play major roles in regulating many different functions including cell proliferation, senescence, programmed cell death, and oncogenic cell transformation. Among different serine/threonine phosphatases, PP-1 and PP-2A contribute to more than 90% phosphatase activities in eukaryotes. While the functions of PP-2A in cell transformation and tumorigenesis have been well established, the role of PP-1 in carcinogenesis remains to be further explored. Moreover, PP-1 exists in different isoforms, whether these isoforms have differential functions in tumorigenesis remains to be examined. In the present study, we demonstrated that in lung cancer 1299 cells, PP1α and PP- 1 & γ exist in an antagonizing balance. In the parent H1299 cells, PP-1γ is dominant, about 4-fold higher than that of PP-1α. Overexpression of PP-1α significantly down-regulates PP-1γ at both mRNA and protein levels. In contrast, knockdown of PP-1α leads to upregulation of PP-1γ. Moreover, overexpression of PP-1α significantly attenuates the ability of the H1299 cells in promoting tumorigenicity as tested in immuno-deficient nude mice. This attenuation is derived from the halted cell cycle progression, which is largely attributed by the changed RB-E2F activity. Together, our results demonstrate that PP-1α and PP-1γ not only antagonize each other in lung cancer cells, but also display differential functions in tumorigenicity.