MiR-92a inhibits proliferation and promotes apoptosis of OSCC cells through Wnt/ß-catenin signaling pathway.

OBJECTIVE: The aim of this study was to investigate the role of micro ribonucleic acid (miR)-92a in the pathogenesis of oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: The relative expression level of miR-92a in OSCC cell lines and normal oral epithelial keratinocyte cell lines was detected via quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Subsequently, the effects of miR-92a silencing on proliferation and apoptosis of OSCC cells were determined via cell counting kit-8 (CCK-8) assay, flow cytometry, and colony formation assay, respectively. Whether miR-92a could bind to the 3'-untranslated region (3'-UTR) of target mRNA was detected using Dual-Luciferase reporter gene assay. Furthermore, changes in Wnt/ß-catenin pathway-associated proteins were explored via Western blotting. RESULTS: The expression of miR-92a in OSCC cell lines was significantly higher than that of normal oral epithelial keratinocyte cell lines (p<0.05). Silencing of miR-92a significantly inhibited proliferation and promoted apoptosis of OSCC cells (p<0.05). Luciferase reporter gene assay confirmed that miR-92a could bind to the 3'-UTR of Kruppel-like factor 4 (KLF4) gene. After miR-92a silencing, the expressions of Wnt/ß-catenin pathway-associated proteins were remarkably down-regulated, including ß-catenin, c-Myc, and Wnt3a (p<0.05). CONCLUSIONS: Silencing of miR-92a inactivates the Wnt/ß-catenin signaling pathway by targeting KLF4, thereby inhibiting proliferation and promoting apoptosis of OSCC cells. Our findings suggest that miR-92a may be a potential therapeutic target for OSCC patients.

[Comparison of short-term and long-term efficacy between robot-assisted and thoracoscopy-laparoscopy-assisted radical esophageal cancer surgery].

Objective: To explore the safety and short-term and long-term efficacy of robot-assisted radical esophageal cancer surgery. Methods: A prospective randomized controlled trial was conducted. Patients who were preoperatively diagnosed as stage 0-IIIB esophageal squamous cell carcinoma and suitable for minimally invasive surgery in our hospital from January 1, 2014 to June 30, 2018 were prospectively enrolled. Those of age ≥75 years having received preoperative neoadjuvant therapy, contradicted to anesthesia or operation due to severe complications, with history of thoracotomy or laparotomy, with concurrent malignant tumors, without complete informations or refusing to participate in this study were excluded. Participants were randomly divided into the thoracoscopy-laparoscopy group and the robot group using a random number table in ratio of 1:1. Preoperative clinicopathological data, surgical data and postoperative outcomes were recorded. The patients were followed up mainly by telephone. Follow-up endpoint was recurrence of esophageal cancer and death. Kaplan-Meier method was used to estimate survival rate. The survival difference between the two groups was analyzed using the log-rank test. Results: According to above criteria, a total of 192 esophageal cancer patients were enrolled finally, including 144 males and 48 females with mean age of (61.9±8.6) years. The robot group had 94 cases, including 72 males and 22 females with mean age of (61.3±8.2) years, and the thoracoscopy-laparoscopy group had 98 cases, including 72 males and 26 females with mean age of (62.4±9.1) years. There were no significant differences in baseline data between the two groups (all P>0.05). Operation was abandoned in one case in each group due to extensive pleural cavity metastasis and one case in each group was converted to thoracotomy. The success rate of operation was 97.9% (92/94) in the robot group and 98.0% (96/98) in the thoracoscopy-laparoscopy group (χ(2)=0.002, P=0.996). The number of lymph nodes dissected in the robot group was significantly higher than that in the thoracoscopy-laparoscopy group (29.2±12.5 vs. 22.8±13.3, t=3.433, P=0.001), while there were no significant differences in operative time, intraoperative blood loss, R0 resection rate, postoperative 30-day mortality, postoperative hospital stay, ICU stay, time to withdrawal of chest drainage tube, ICU readmission, and postoperative morbidity of complications between the two groups (all P>0.05). The median follow-up time was 21 (3 to 57) months. During the follow-up, 3 cases and 4 cases were lost, and 2 cases and 3 cases died of other diseases in the robot group and in the thoracoscopy-laparoscopy group respectively. Recurrence occurred in 39 cases during follow-up, including 14 recurrences in the robotic group with 1- and 3-year recurrence-free survival rates of 92.4% and 87.6% respectively and the median recurrence time of 15 (9 to 42) months. There were 25 recurrences in the thoracoscopy-laparoscopy group with 1- and 3-year recurrence-free survival rates of 81.7% and 67.9% respectively and the median recurrence time of 9 (3 to 42) months. There was significant difference in recurrence-free survival between the two groups (χ(2)=4.193, P=0.041). Conclusions: The robotic surgical system has good oncology effect and surgical safety in the radical operation of esophageal cancer, which deserves further research and promotion.

A novel marine bacterium algicidal to the toxic dinoflagellate Alexandrium tamarense.

AIMS: This work is aiming at investigating algicidal characterization of a bacterium isolate DHQ25 against harmful alga Alexandrium tamarense. METHODS AND RESULTS: 16S rDNA sequence analysis showed that the most probable affiliation of DHQ25 belongs to the γ-proteobacteria subclass and the genus Vibrio. Bacterial isolate DHQ25 showed algicidal activity through an indirect attack. Xenic culture of A. tamarense was susceptible to the culture filtrate of DHQ25 by algicidal activity assay. Algicidal process demonstrated that the alga cell lysed and cellular substances released under the visual field of microscope. DHQ25 was a challenge controller of A. tamarense by the above characterizations of algicidal activity assay and algicidal process. CONCLUSION: Interactions between bacteria and harmful algal bloom (HAB) species proved to be an important factor regulating the population of these algae. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of a Vibrio sp. bacterium algicidal to the toxic dinoflagellate A. tamarense. The findings increase our knowledge of the role of bacteria in algal-bacterial interaction.

Analysis of community structure of a microbial consortium capable of degrading benzo(a)pyrene by DGGE.

A microbial consortium was obtained by enrichment culture of sea water samples collected from Botan oil port in Xiamen, China, using the persistent high concentration of a mixture of polycyclic aromatic hydrocarbons enrichment strategy. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial composition and community dynamic changes based on PCR amplification of 16S rRNA genes during batch culture enrichment. Using the spray-plate method, three bacteria, designated as BL01, BL02 and BL03, which corresponded to the dominant bands in the DGGE profiles, were isolated from the consortium. Sequence analysis showed that BL01, BL02 and BL03 were phylogenetically close to Ochrobactrum sp., Stenotrophomonas maltophilia and Pseudomonas fluorescens, respectively. The degradation of benzo(a)pyrene (BaP), a model high-molecular-weight polycyclic aromatic hydrocarbon (HMW PAH) compound was investigated using individual isolates, a mixture of the three isolates, and the microbial consortium (BL) originally isolated from the oil port sea water. Results showed that the order of degradative ability was BL>the mixture of the three isolates>individual isolates. BL degraded 44.07% of the 10 ppm BaP after 14 days incubation, which showed the highest capability for HMW PAH compound degradation.Our results revealed that this high selective pressure strategy was feasible and effective in enriching the HMW PAH-degraders from the original sea water samples.

Modified sublimation to isolate phenanthrene-degrading bacteria of the genera Sphingomonas and Burkholderia from Xiamen oil port.

Sublimation was developed by Alley and Brown (2000) in order to isolate bacterial strains that were capable of degrading water insoluble compounds. In this study, sublimation was modified by the use of nutritional agar plates, instead of mineral salt agar, to isolate phenanthrene-degrading bacteria from a mixed culture that had been enriched under the selective pressure of high phenanthrene content. Five strains were obtained with different morphology and degradation ability. Based on the 16S rDNA sequence, two of them were classified as species of the genus Sphingomonas; the others as species of the genus Burkholderia. Denaturing gradient gel electrophoresis (DGGE) was introduced to detect dynamic changes in the bacterial community during enrichment batch culture, and to determine any correlation between the five isolates and the phenanthrene-degrading consortium. The DGGE profile indicated that these five isolates corresponded to four dominant bands of the consortium. Compared to traditional means of isolation, we concluded that modified sublimation is effective and more convenient.

PAHs contamination and bacterial communities in mangrove surface sediments of the Jiulong River Estuary, China.

Sixteen sediment samples collected from eight transects in a mangrove swamp of the Jiulong River Estuary, Fujian, China were investigated for their content of polycyclic aromatic hydrocarbons (PAHs) and the biodegradation potential of the indigenous microorganisms. The bacterial community structures in the mangrove sediments and in enrichment cultures were also investigated. The results showed that the total PAHs concentration of mangrove sediments ranged from 280 to 1074 ng g(-1) dry weight, that the PAHs composition pattern in the mangrove sediments was dominated by high molecular weight PAH components (4-6 rings), and that Benzo[ghi]perylene and Indeno[1,2,3-cd]pyrene were the most dominant at different stations. Abundant PAH-degrading bacteria were found in all the stations, the values of phenanthrene-degrading bacteria ranged from 5.85 x 10(4) to 7.80 x 10(5) CFU g(-1) dry weight, fluoranthene-degrading bacteria ranged from 5.25 x 10(4) to 5.79 x 10(5) CFU g(-1) dry weight, pyrene-degrading bacteria ranged from 3.10 x 10(4) to 6.97 x 10(5) CFU g(-1) dry weight and the benzo(a)pyrene-degrading bacteria ranged from 5.25 x 10(4) to 7.26 x 10(5) CFU g(-1) dry weight. DGGE analysis of PCR-amplified 16S rDNA gene fragments confirmed that there was a remarkable shift in the composition of the bacterial community due to the addition of the different model PAH compound phenanthrene (three ring PAH), fluoranthene(four ring PAH), pyrene(four ring PAH) and benzo(a)pyrene(five ring PAH) during enrichment batch culture. Eleven strains were obtained with different morphology and different degradation ability. The presence of common bands for microbial species in the cultures and in the native mangrove sediment DNA indicated that these strains could be potential in situ PAH-degraders.

Organochlorine micropollutants in the Jiulong River Estuary and Western Xiamen Sea, China.

Organochlorine contaminants including 12 polychlorinated biphenyl (PCB) congeners and 18 insecticides were determined in water, pore water and sediments of the Jiulong River Estuary and Western Xiamen Sea, China. The results showed that the levels of the total PCBs ranged from non-detectable to 1500 ng l(-1) in water, from 209 to 3870 ng l(-1) in pore water, and from 2.78 to 14.8 ng g(-1) dry weight in sediments. Total organochlorine insecticide concentrations were from below the limit of detection to 2480 ng l(-1) in water, from 267 to 33400 ng l(-1) in pore water, and from 4.22 to 46.3 ng g(-1) dry weight in sediments. Concentrations of PCBs and insecticides in pore water were significantly higher than those in surface water, due to the high affinity of these hydrophobic compounds for sediment phase. The PCB congeners with the highest concentrations were CB153, CB180 and CB194, which together accounted for 68-87% of total PCBs in water, pore water and sediment. Among the hexachlorocyclohexane (HCH) compounds, beta-HCH was found to be a major isomer. Analysis of 1,1,1-trichloro-2,2-bis-chlorophenyl-ethane (DDT) and its metabolites showed that 1,1-dichloro-2[o-chlorophenyl]-2[p-chlorophenyl]-ethylene (DDE) was dominant in the group. In comparison to a 1998 study in the Western Xiamen Sea, levels of organochlorines were enhanced due probably to recent inputs and changes in sediments.

Microbial modulation in the biomass and toxin production of a red-tide causing alga.

The effect of S10, a strain of marine bacteria isolated from sediment in the Western Xiamen Sea, on the growth and paralytic shellfish poison (PSP) production in the alga Alexandrium tamarense (A. tamarense) was studied under controlled experimental conditions. The results of these experiments have shown that the growth of A. tamarense is obviously inhibited by S10 at high concentrations, however no evident effect on its growth was observed at low concentrations. Its PSP production was also inhibited by S10 at different concentrations, especially at low concentrations. The toxicity of this strain of A. tamarense is about (0.95-12.14) x 10(-6) MU/cell, a peak toxicity value of 12.14 x 10(-6) MU/cell appeared on the 14th day, after which levels decreased gradually. The alga grew well in conditions of pH 6-8 and salinities of 20-34 per thousand. The toxicity of the alga varied markedly at different pH and salinity levels. Toxicity decreased as pH increased, while it increased with salinity and reached a peak value at a salinity of 30 per thousand, after which it declined gradually. S10 at a concentration of 1.02 x 10(9) cells/ml inhibited growth and the PSP production of A. tamarense at different pH and salinity levels. S10 had the strongest inhibitory function on the growth of A. tamarense under conditions of pH 7 and a salinity of 34 per thousand. The best inhibitory effect on PSP production by A. tamarense was at pH 7, this inhibitory effect on PSP production did not relate to salinity. Interactions between marine bacteria and A. tamarense were also investigated using the flow cytometer technique (FCM) as well as direct microscope counting. S10 was identified as being a member of the genus Bacillus, the difference in 16S rDNA between S10 and Bacillus halmapalus was only 2%. The mechanism involved in the inhibition of growth and PSP production of A. tamarense by this strain of marine bacteria, and the prospect of using it and other marine bacteria in the bio-control of red-tides was discussed.

Aerobic and anaerobic metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium isolated from marine sediments.

This report describes the metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium (Marinobacter sp. strain CAB) isolated from marine sediments. Under aerobic and denitrifying conditions, this strain efficiently degraded this ubiquitous isoprenoid ketone. Several bacterial metabolites, 4,8,12-trimethyl-tridecan-1-ol, 4,8,12-trimethyltridecanal, 4,8,12-trimethyltridecanoic acid, Z-3,7-dimethylocten-2-oic acid, Z-3,7,11-trimethyldodecen-2-oic acid, and 6,10,14-trimethylpentadecan-2-ol, were formally identified, and different pathways were proposed to explain the formation of such isoprenoid compounds.

Prolonged pancreas preservation using a simplified UW solution containing polyethylene glycol.

The University of Wisconsin solution is considered the most effective universal flush and cold storage solution to date, and is now being widely applied clinically in organ transplantation. The results of this study show that Cardiosol, a modified cardioplegic solution containing 5% polyethylene glycol (PEG20M, MW 17,000 daltons), is significantly superior to UW (P less than 0.001) in the flush perfusion and hypothermic storage of pancreases for more than 36 hr prior to transplantation into streptozotocin-induced diabetic rats. When the pancreases were stored in Cardiosol, the 1-week survival rate was 7 of 10 (70%) after 24 hr of preservation; 7 of 12 (58%) after 36 hr; and 3 of 10 (30%) after 48 hr. In contrast, when the pancreases were stored in UW solution, the 1-week survival rate was 8 of 12 (67%) after 24 hr of preservation; after 36 hr, no animal survived (0 of 14). Intravenous glucose tolerance test K-values (decline in glucose concentration, percentage per minute) were normal in both groups receiving 24-hr-preserved pancreases, ranging from 2.60 to 4.16. Of interest, the peak insulin response 1 min following intravenous glucose was significantly higher (P less than 0.01) in the Cardiosol-preserved organs (303 +/- 29.8 microU/ml) (+/- SEM) than in the glands preserved in UW solution (112 +/- 47.9 microU/ml). We conclude that Cardiosol allows prolonged whole organ pancreas preservation in the rat transplant model.

Pancreas function index: an early monitor of allograft rejection.

The lack of reliable markers for the early diagnosis of allograft rejection is a major obstacle preventing improved results in clinical pancreas transplantation. Using a vascularized whole-pancreas transplant model in the rat, with exocrine ductal drainage into the lower urinary tract, we explored the possibility that a change in an index of pancreatic function (IPF), viz., urine volume, urine pH, and urine amylase (UA) in composite, may provide an earlier and more specific indicator of rejection than a decline in UA levels alone. Six Lewis-to-Lewis rat isograft and 12 ACI-to-Lewis allograft recipients were studied. Ten nontransplanted diabetic Lewis rats served as a control group. Euglycemia was restored in all the recipients of isografts, and was maintained for over a year. In the allograft group, rejection occurred on Days 7-9, with a mean graft survival time of 8.1 +/- 0.1 days. Peak UA levels and IPF during normal allograft function were 2422 +/- 353 U/ml and 100 +/- 14, respectively, whereas levels heralding rejection were 600 U/ml and 25 (P less than 0.05). The diagnosis of rejection based on the IPF resulted in significantly greater specificity and an earlier prediction time compared with UA alone (2.4 +/- 0.3 vs 1.6 +/- 0.2 days); the IPF permitted the successful prediction of rejection 3 or more days prior to hyperglycemia in 6 of 12 (50%) grafts, whereas only 1 of 12 (8%) rejection episodes was successfully predicted when the rejection criterion was based on UA. In conclusion, early diagnosis of rejection was achieved by a composite index of pancreatic function, improving the ability to predict pancreas-allograft rejection 24 to 48 hr prior to a fall in UA levels.

[An analysis of 26 cases of chronic myelomonocytic leukemia].

Chronic myelomonocytic leukemia (CMML) is a polymorphous malignant hematological stem cell disorder, characterized by abnormal hyperplasia of mature or immature cells of both monocytic and granulocytic series and with abnormal cellular morphology. It is an independent entity of chronic leukemia, as its prestage course is manifested by refractory anemia with monocytosis and the disease gradually evolves to CMML. In some cases, it finally becomes acute leukemia. In this study, the average white cell count of the patients was 29.3 x 10(9)/L.14 cases had leucocytosis, 7 leucopenia and 5 normal count. The absolute value of monocytes was 19 x 10(9)/L and the proportion of monocytes was 10-87%, with an average of 49%. In the leukopenic group with white cell count less than 4 x 10(9)/L, the absolute value of monocytes was less than 1 x 10(9)/L in 5 of the 7 cases. However, it was noticed that all the 5 cases had a proportion of monocytes greater than or equal to 10%. The authors would like to take this percentage as the diagnostic criteria for CMML, which is different from that adopted in FAB classification of 1982 as well as in the literatures. Statistics showed that P value of M/E, Mo/E, Mo/M were of apparent significance in the differentiation of CMML from normal controls and patients with other hematological diseases such as RA, RAEB, CML, CNL, M4 and M5.

Rapid purification of islets using magnetic microspheres coated with anti-acinar cell monoclonal antibodies.

A simple, rapid method of islet purification is important in large-scale human islet isolation. We have previously identified monoclonal antibodies specific for acinar cells, but not islets, and described an immunologic method of purification by selective lysis of the acinar cells. An attractive alternative to lysis of the acinar cell is depletion by a magnetic immunomicrosphere technique. We report in this study a rapid, reproducible method of rat islet purification utilizing magnetic microspheres coated with acinar-cell-specific monoclonal antibodies. Pancreatic digestion with collagenase followed by depletion of acinar cells with the magnetic immunomicrospheres (MIMS) yields large numbers of intact islets. We compared the islets thus obtained with hand-picked (HP) islets (control) for yield, purity, in vitro insulin secretory capacities, and in vivo functional viability. The islet yield with the MIMS method (n = 35) was 72.7% that obtained with the HP method (n = 6) (378 +/- 8 vs. 519 +/- 31 islets per pancreas). The purity of the MIMS-isolated islets was 84 +/- 1.9%, ranging from 75-95%. Static glucose stimulation showed excellent function (2-3-fold increase of insulin release over basal levels) with no statistical difference in insulin secretion between MIMS and HP islets. Under microscopic examination, both groups revealed a well-preserved structure with healthy endocrine cells. When 1321 +/- 59 MIMS islets were transplanted into streptozotocin-induced diabetic rats (n = 10), normoglycemia (less than 200 mg/dl) was restored in all recipients following transplantation, and 100% of them remained normoglycemic on day 120 postgrafting. In summary, a rapid, consistent, and simple method of isolating viable, purified rat islets is described. The broad interspecies crossreactivity of the McAb suggests that this technique may be generally useful for islet purification in large mammalia, including man.

Monitoring urinary amylase and pH in pancreas transplantation with urinary drainage in rats.

Whole pancreas isografts or allografts (ACI donors, RT1a) with bladder drainage of exocrine secretions were performed in Lewis rats (RR1(1] with streptozotocin-induced diabetes. Urinary amylase, pH, and volume and serum glucose were measured daily. They were analyzed alone, or in combination, to determine patterns in deviations from normal values, from isograft control values, or from a posttransplant baseline in relation to rejection (defined as reversion of plasma glucose of greater than 200 mg/dl) in nonimmunosuppressed recipients. Also studied were the sensitivity and specificity by which such deviations predicted rejection. Functioning grafts were associated with increased urinary amylase and pH compared with normal or diabetic controls; urinary volume was less than that of diabetic rats, but greater than that of normal rats. In nonimmunosuppressed allograft recipients (n = 9), rejection occurred at a mean (+/- SD) of 7.78 +/- 0.44 days. Serum glucose rose to above normal (greater than 134 mg/dl) 1 day before rejection in 3 animals (sensitivity 33%, false negative rate 66%; false positive rate in 9 isograft recipients, 44%). Urinary volume dropped below 3 ml at a mean of 3.17 +/- 0.98 days (range 2-5 days) before rejection in 6 animals (sensitivity 66%, false negative rate 33%; false positive rate 0%). Urinary pH fell below 7.25 at a mean of 3.13 +/- 1.81 days (range 1-5 days) before rejection in 8 rats (sensitivity of 89%, false negative rate 11%; false positive rate 29%). Urinary amylase dropped from a posttransplant peak at a mean of 3.56 +/- 1.42 days (range 1-6 days) before rejection in 9 animals (sensitivity 100%, false negative rate 0%; false positive rate 43%), and dropped below 1500 units per 24 hr at a mean of 2.00 +/- 1.32 days (range 1-5 days) before rejection in 8 animals (sensitivity 89%, false negative rate 11%; false positive rate 0%). A drop in urinary amylase combined with a drop in urinary volume or pH occurred at a mean of 3.22 +/- 1.48 days (range 1-5 days) before rejection in 9 rats (sensitivity 100%, false negative rate 0%; false positive rate 0%). In a separate group of 10 allograft recipients, immunosuppression with prednisone and cyclosporine was begun concomitant with, or within 2 days of, the drop in urinary amylase from the peak value; rejection did not occur in 3 animals and was delayed to a mean of 12.0 +/- 5.0 days posttransplant in 7 animals (P less than .05 compared with the nonimmunosuppressed group).(ABSTRACT TRUNCATED AT 400 WORDS)

Pretransplant cytotoxic conditioning produces effects consistent with clonal deletion mechanisms.

A rat cardiac allograft model (ACl to Lewis) was used to investigate the clonal deletion theory. Twelve groups of Lewis recipients received various combinations of donor-specific blood transfusions (DSTs), immediate post-DST immunosuppression with azathioprine/prednisone, and low-dose cyclosporine (1 mg/kg/day) posttransplant. DSTs and cyclosporine together gave modest prolongation of graft survival (from 6.0 to 17 days). DSTs plus immediate post-DST immunosuppression followed by low-dose cyclosporine prolonged graft survival to an average of 45 days. Third-party transfusions alone and in combination with immunosuppression did not significantly prolong allograft survival. Postoperative cyclosporine was required for the expression of this effect suggesting that clonal depression rather than clonal deletion had occurred. Combining DSTs with brief but intense preoperative immunosuppression may be a more effective method of pretransplant conditioning than DSTs alone.