High prevalence of Schistosoma japonicum by perfusion in naturally exposed water buffalo in a region of the Philippines endemic for human schistosomiasis.

In the past decade, ecological surveys emphasized rats and dogs as the most significant animal reservoirs for Schistosoma japonicum (S.j) in the Philippines. However, recent studies demonstrated 51-91% prevalence of schistosomiasis among water buffalo using qPCR in the Sj endemic regions in the Philippines. In order to resolve the inconsistency of reported surveys regarding Sj endemicity among carabao, a domestic water buffalo that is the most important draught animal, we introduced 42 schistosome negative water buffalo to Macanip, Jaro municipality, Leyte, the Philippines, a subsistence rice-farming village that has been the focus of schistosomiasis japonica studies of our group for the past 20 years. We conducted perfusion to the remaining 34 buffalo that survived 10 months of nature exposure and Typhoon Haiyan. Thirty-three water buffalo were found to be positive with at least 1 pair of worms from the mesenteric vein. The infection rate is 97%, with the worm burden of 94 (95% confidence interval, 49-138 worms) worms. To our knowledge, this is the first report about S. japonicum worm burden in naturally infected water buffalo in the Philippines. The fact that with less than one-year of exposure, in this human schistosomiasis endemic area, only 1 out of 34 water buffalo was uninfected is striking. Urgent attention is needed for a cost-effective technique for monitoring Sj infection in animals and humans. Meanwhile, intervention implementation, including water buffalo treatment and vaccination, should be taken into consideration.

[Study of intratympanic methylprednisolone injections in diabetics with a sudden sensorineural hearing loss].

OBJECTIVE: To evaluate the effects of intratympanic methylprednisolone injection by microcatheter in diabetics with a sudden hearing loss. METHODS: From July 2005 to November 2009, 113 diabetics with a sudden hearing loss within an onset of 10 days at our department were recruited. But they received no previous intervention and were assigned to treatment and control groups. Treatment group were made by microcatheter connected with an insulin bump. Microcatheter was placed in a round window niche and methylprednisolone (62.5 mg/ml) infused at a rate of 10 microl/h for 14 days. Then the microcatheter was extracted. Simultaneously vasodilation, neurotrophic, thrombolysis and insulin hypoglycemia were administered in all patients. Pure tone test was conducted at Days 10 and 20 after intervention. RESULTS: The outcome was as follows: cure (n = 6), efficacy (n = 19), effect (n = 12) and no effect (n = 11) respectively. The overall effective rate of 77.08% in the treatment group was superior to that in the control group. And there was statistical difference (P < 0.05). Pure tone average (PTA) of two groups showed no statistical difference. After 10 days, the PTA values were (66 ± 21) versus (76 ± 14) dB in the treatment and control groups respectively. At Day 20, the values were (50 ± 16) and (59 ± 12) dB respectively. The improvement of pure tone threshold at Days 10 and 20 had significant statistical difference (P < 0.05). Neither group had hypoglycemia or diabetic complications during treatment. And the prognosis had no obvious correlation with the severity of diabetes. CONCLUSION: The therapy of intratympanic methylprednisolone injection by microcatheter connected with micropump is both effective and feasible in diabetics with a sudden hearing loss.

[Evaluation on the immuno-protective efficacy of the recombinant antigen SjPGAM-SjEnol against Schistosoma japonicum in mice].

OBJECTIVE: To construct and express the recombinant plasmid pET32a-SjPGAM-SjEnol and evaluate its immuno-protective efficacy against the infection of Schistosoma japonicum in mice. METHODS: The peptides of SjPGAM and SjEnol containing the multivalent epitopes with higher binding capacity of human MHC II and mouse H2-dII but low homology with the host were analyzed and screened through bioinformatics. The corresponding nucleotide sequence of selected epitopes was spliced and the recombinant plasmid pET32a-SjPGAM-SjEnol was constructed and expressed in Escherichia coli BL21 cells. The antigenicity of the recombinant protein was detected by Western blotting and the protective effect induced with the recombinant was evaluated in mice. 55 BALB/c mice were randomly divided into 5 groups each with 11. Mice from groups A, B and C were injected with a mixture of recombinant protein (27 microg) pET32a-SjPGAM-SjEnol (A), pETL28a-SjPGAM (B) and pET28a-SjEnol (C) respectively together with 206 adjuvant, mice from groups D and E received adjuvant or PBS only, all injected for three times at two-week intervals. Mice were then challenged with 40 +/- 2 cercariae of S. japonicum at two weeks after the last vaccination, and sacrificed for perfusion by 6 weeks post infection. Adult worms were collected, the number of eggs in a gram of liver tissue was counted, and the rates of worm reduction and egg reduction were calculated. Serum samples were collected before vaccination, every one week after each inoculation and before sacrifice, and specific IgG was detected by ELISA. RESULTS: The sequences encoding the 96-147 aa of SjPGAM and 233-312 aa of SjEonl were chosen for constructing the recombinant plasmid, a cDNA fragment with the length of 447 bp was amplified by PCR. The recombinant plasmid was expressed in E. coli with a molecular weight of Mr 33,000. Western blotting revealed that the fusion protein was recognized by the rabbit serum specific to SjSWAP, and showed an adequate antigenicity. Vaccination experiment showed that when compared with those of the blank control, the worm reduction rate in group A was 39.7%, significantly higher than that of groups B (18.5%) and C (14.7%) (P < 0.05). The liver egg reduction rate in group A was 64.9%, also higher than that of groups B (47.5%, P < 0.05) and C (30.5%, P < 0.01). ELISA showed that the serum specific IgG in group A (2.372 +/- 0.268) was much higher than that of groups D (0.490 +/- 0.138) (P < 0.01 and E (0.220 +/- 0.088) (P < 0.01). CONCLUSION: The recombinant plasmid pET32a-SjPGAM-SjEnol has been constructed, and recombinant protein pET32a-SjPGAM-SjEnol induces higher immune-protection against S. japonicum than that of SjPGAM and SjEonl.

[Effects of increased planting density with reduced nitrogen fertilizer application on greenhouse gas emission in double-season rice fields under water saving and simple cultivation mode]. / èŠ‚æ°´è½»ç®€æ ½åŸ¹æ¨¡å¼ä¸‹å¢žå¯†å‡æ°®å¯¹åŒå­£ç¨»ç”°æ¸©å®¤æ°”ä½“æŽ’æ”¾çš„å½±å“.

To clarify the effects of increased density and reduced nitrogen on greenhouse gas emission in double-season paddy fields under the water-saving and simple cultivation mode, we used Luliangyou 996 (early rice) and Fengyuanyou 299 (late rice) as materials to collect greenhouse gas by closed static box method, monitored the dynamics of CH4 and N2O emissions from different combinations of increased density and reduced nitrogen for early and late rice, and explored changes in cumulative CH4 and N2O emissions, global warming potential (GWP) and greenhouse gas intensity (GHGI) as affected by different combinations of increased density and reduced nitrogen from double-season rice fields. The results showed that the cumulative emissions of CH4 and N2O from different combinations were significantly different. Compared with the control (CK), cumulative CH4 emission, GWP and GHGI in the two seasons decreased by 50.8%, 37.3%, and 42.9% for the combination of increased density and reduced nitrogen IR2(the amount of nitrogen applied of early rice was 86.4 kg·hm-2, the density was 360000 holes·hm-2; the amount of nitrogen applied of late rice was 108 kg·hm-2, the density was 320000 holes·hm-2), respectively. IR2 of early rice had the lowest N2O cumulative emission, being 33.7% lower than CK. IR1(the amount of nitrogen applied of early rice was 103.2 kg·hm-2, the density was 320000 holes·hm-2; the amount of nitrogen applied of late rice was 129 kg·hm-2, the density was 280000 holes·hm-2) of late rice had the lowest N2O cumulative emission, being decreased by 94.9%. IR2 had the lowest annual total GWP and GHGI of double-season paddy fields. Compared with other treatments of increased density and reduced nitrogen, the IR2 treatment, where nitrogen fertilizer in both early and late rice was reduced by 28.0%, the density of early rice was increased by 28.6%, and the density of late rice was increased by 33.3%, was an effective and safe option for simultaneously ensuring high yield and reducing greenhouse gas emissions.

[Immune protection of tegument protein rSj29 against Schistosoma japonicum in mice].

OBJECTIVE: To clone, express and characterize a tegument protein gene of Schistosoma japonicum (Sj29) , and investigate the immune protection of the recombinant protein against S. japonicum in mice. METHODS: The gene coding for Sj29 protein was amplified by PCR, and the sequence was analyzed by bioinformatics tools. Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced the recombinant with IPTG. The recombinant protein (rSj29) was purified by His-binding-resin affinity chromatography and characterized by Western blotting. Three groups each with 10 BALB/c mice were immunized subcutaneously three times (two weeks interval) respectively with 100 microl recombinant rSj29 (0.1 mg/ml) , adjuvant or PBS. At the 15th day after the final inoculation, each mouse was challenged by 40 +/- 2 cercariae of S. japonicum. At the 53rd day after infection, the mice were sacrificed to obtain the number of adult worms, number of eggs in liver and feces. Serum samples were collected at pre-immunization and certain time after immunization, and were analyzed for IgG by ELISA. The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique. mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR. RESULTS: A 576 bp Sj29 gene fragment was obtained. The recombinant protein rSj29 with Mr 22,900 was expressed in the form of inclusion body. The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice. The number of adult worms (15.4 +/- 5.9), number of hepatic eggs (40,143.3 +/- 2,995.9) and number of fecal eggs (3,803.9 +/- 110.9) in recombinant protein group were significantly higher than those of PBS control group (20 +/- 3.4, 49,318.1 +/- 6,648.3, 5,238.1 +/- 303.5, respectively) (P < 0.05) . There was a high level of specific IgG against rSj29 (maximum dilution 1:32000) in recombinant protein group. Immunohistochemical analysis showed the Sj29 protein expressed on the surface of different stages of S. japonicum. mRNA level of Sj29 was the highest at the 32nd day post-infection. CONCLUSION: The recombinant protein rSj29 induces certain degree of protective immunity in mice.

[Effects of winter cropping mode on soil organic carbon and carbon management index of double rice paddy]. / 双季稻田冬季种植模式对土壤有机碳和碳库管理指数的影响.

To explore the effects of winter crops recycling into paddy field on soil carbon pool, we cultivated rape, Chinese milk vetch, ryegrass and potato in winter season after harvesting double-season rice, with fallow as control (CK). The contents of soil organic carbon (SOC) and labile organic carbon (LOC) in the harvest of early and late rice were measured, and the stable carbon, activity of carbon pool, carbon pool index, carbon pool activity index and carbon pool management index (CMI) were calculated. Results showed that winter cover crops returning increased SOC content by 1%-8% and 3%-18% after harvest of early rice and late rice, respectively in comparison with CK. Cultivating rape, Chinese milk vetch and ryegrass all increased the LOC content, with enhancement of 16.2%-84.2% and 24.4%-28.1% after harvest of early rice and late rice, respectively. The CMI of all winter crop treatments presented a growth ratio of 1.4%-41.8% compared to CK. In conclusion, the cultivation of winter crop and recycling to field promoted soil carbon fixation and soil quality, among which the ryegrass and Chinese milk vetch showed better comprehensive effect.

[Differences and Relationship Between Rhizosphere Characteristics and Methane Emissions of Double-cropping Rice Variety].

Six field varieties of early rice and late rice were selected as test materials for field experiments to explore the difference in CH4 emissions among different rice varieties, and Static Obscura-Gas Chromatography was used to determine the CH4 gas. The results demonstrated a significant difference in the CH4 emissions flux between early and late rice varieties. The average yield of total fertility CH4 emissions was highest in Xiangzaoxian 24 and lowest in Zhuliangyou 819, with a difference of 34.6%. Of the late rice varieties, Tyou 15 was the highest and the Ziyou 299 was the lowest, with a difference of 33.9%. Differences in CH4 emissions and the greenhouse effect of unit yields between different double cropping rice varieties differed significantly. The cumulative CH4 emissions from early rice varieties ranged from 198.3-303.44 kg·hm-2, and the lowest emissions were from Zhuliangyou 819. The greenhouse effect per yield ranged from 0.67 to 1.40 kg·kg-1, and Luliangyou 996 had the lowest emission value. The late-season rice varieties exhibited significantly higher cumulative CH4 emissions compared to early rice, ranging from 291.93 to 388.28 kg·hm-2, and Ziyou 299 had the lowest emission value. The greenhouse effect of per yields rice varieties, while the late rice varieties were contrary to early rice. Reducing carbon and nitrogen concentrations in the rhizosphere and increasing Eh values could reduce CH4 emissions.

Microdissected thin anterolateral thigh perforator flaps with multiple perforators: A series of case reports.

INTRODUCTION: The study aimed to explore the effect of microdissected thin anterolateral thigh (MTALT) perforator flap with multiple perforators on patients with complex defects on the hand, elbow, heel, or knee. METHODS: From March 2012 to February 2013, 5 patients with complex defects on the hand, elbow, heel, or knee were included. During the flap preparation, 2 to 3 perforators penetrating the fascia of the anterolateral femoral area were initially detected, and the deep fascia was incised. The superficial fascia layer of the flap and the deep adipose were then dissected, and removed after verifying the distribution of the blood vessels using an operating microscope. The whole flap was then elevated, and transposed to the recipient areas for microsurgical reparation. RESULTS: Two cases of post-burn scar contracture and 3 cases of traumatic tissue defects were successfully reconstructed with these multiple-perforator MTALT flaps. No complication was reported, and secondary operative procedure was not needed in any patient in the follow-up. CONCLUSION: MTALT perforator flap with multiple perforators is safe and reliable for patients with complex defects on the hand, elbow, heel, or knee.

[Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

OBJECTIVE: To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. METHODS: Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. RESULT: Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. CONCLUSION: The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

Vaccination with recombinant paramyosin in Montanide ISA206 protects against Schistosoma japonicum infection in water buffalo.

BACKGROUND: Schistosomiasis japonica is a zoonosis and presents significant public health problems in China and the Philippines. Vaccines targeting domestic animals constitute attractive control measures. METHODS: We conducted three vaccine trials to evaluate the protective efficacy of recombinant full-length paramyosin (rSj97) in water buffalo. Animals were immunized with 3 doses of rSj97 adjuvanted with ISA206 at 250µg/dose or 500µg/dose at 4wk intervals before challenge with 1000 Schistosoma japonicum cercariae. The primary outcome was worm burden assessed by portal perfusion 8-10weeks post challenge. Safety measures included weight, temperature, body condition score, hemogram and routine assays for hepatic and renal function. RESULTS: The three-dose regimen was well tolerated in all three trials. In the first trial, vaccinated buffalo had 51.5% lower worm burden post challenge compared to controls. In the second trial, buffalo immunized with 500µg/dose of rSj97 had 57.8% lower worm burden compared to controls (p=0.026). A similar but not significant reduction (60.9%) was observed with animals administered with 250ug rSj97/dose. In the third trial, buffalo immunized with a 500µg/dose of rSj97 had 57.8% lower worm burden compared to controls (p=0.014). CONCLUSIONS: These findings indicated that rSj97 is a safe and promising vaccine candidate for schistosomiasis japonica in water buffalo.

Nested-PCR assay for detection of Schistosoma japonicum infection in domestic animals.

BACKGROUND: Schistosomiasis japonica is a common zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. The prevalence and infectivity of this disease in domestic animals in China have significantly decreased and, for this reason, diagnostics with a higher sensitivity have become increasingly necessary. It was reported that polymerase chain reaction (PCR)-based methods could be used to detect schistosome infection in humans and animals and presented a high sensitivity and specificity. The present study aimed to develop a PCR-based method for detection of Schistosoma japonicum infection in domestic animals. METHODS: A specific nested-PCR assay was developed to detect S. japonicum infection in domestic animals via amplification of a 231-bp DNA fragment of retrotransposon SjR2. The developed assay was first used in sera and dry blood filter paper (DBFP) from goats and buffaloes at different time points of infection. Then, 78 DBFPs from 39 artificially-infected bovines at 14 and 28 days post-infection and 42 DBFPs from schistosome-negative bovines from the city of Huangshan in the Anhui province were used to evaluate the diagnostic validity. Furthermore, this assay was used to detect S. japonicum infection in domestic animals in Dongzhi and Wangjiang counties. RESULTS: The expected PCR product was detected in eggs and adult worms of S. japonicum and blood samples from S. japonicum-infected goats and water buffaloes, but not from Fasciola and Haemonchus contortus worms. The nested-PCR assay could detect the target S. japonicum DNA in DBFPs from goats and buffaloes after day 3 post-infection. The sensitivity in buffaloes at 14 and 28 days post-infection was 92.30% (36/39) and 100% (39/39), respectively. The specificity was 97.60% (41/42). The positivity rates in Dongzhi and Wangjiang counties were 6.00% and 8.00% in bovines and 22.00% and 16.67% in goats, respectively. The positivity rates in goats in both counties were higher than those in bovines with a significant difference in Dongzhi County but not in Wangjiang County (P < 0.05 and P = 0.23, respectively). CONCLUSIONS: Our results suggest that the developed nested-PCR assay may be used for the diagnosis of S. japonicum infection in domestic animals, and the control of S. japonicum infection in goats should be paid more attention.

Chemokine ligand 20 enhances progression of hepatocellular carcinoma via epithelial-mesenchymal transition.

AIM: To identify the mechanisms of chemokine ligand 20 (CCL20)-induced hepatocellular carcinoma (HCC) metastasis and evaluate it as a prognostic marker. METHODS: Expression of CCL20 was evaluated by immunohistochemistry in HCC tissues from 62 patients who underwent curative resection. The relationship between CCL20 expression and clinicopathologic features was analyzed. Univariate and multivariate analyses were performed to evaluate its predictive value for recurrence and survival of HCC patients. The expression levels of epithelial-mesenchymal transition (EMT)-and signaling pathway-related proteins were evaluated by Western blotting and immunocytochemistry. The effects of CCL20 on HCC cell proliferation and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenoltetrazolium bromide (MTT) and Transwell assays. RESULTS: CCL20 immunoreactivity was detected in all 62 patient specimens. CCL20 expression was associated with preoperative alpha-fetoprotein level (P = 0.043), tumor size (P = 0.000), tumor number (P = 0.008), vascular invasion (P = 0.014), and tumor differentiation (P = 0.007). Patients with high CCL20 expression had poorer recurrence-free and overall survivals compared to those with low CCL20 expression (both P < 0.001). CCL20 induced EMT-like changes in HCC cells and increased their proliferation and migration ability (P < 0.05). Western blotting and immunofluorescence staining showed that CCL20 induced an EMT-like phenotype in HCC cells, and increased expression of phosphorylated AKT, ß-catenin and vimentin, and decreased E-cadherin expression (P < 0.05). The correlation analysis revealed that high CCL20 expression in HCC tissue specimens was negatively correlated with E-cadherin expression (13.33%, 4/30), and positively correlated with vimentin (90.0%, 27/30), ß-catenin (96.67%, 29/30) and p-AKT (76.67%, 23/30) expression. CONCLUSION: CCL20 expression is associated with HCC recurrence and patient survival and promotes HCC cell proliferation and migration by inducing EMT-like changes via PI3K/AKT and Wnt/ß-catenin pathways.

[Effects of Water and Nitrogenous Fertilizer Coupling on CH4 and N2O Emission from Double-Season Rice Paddy Field].

To provide support for the efficient use of water and fertilizer technology to double-season rice cultivation, water and fertilizer coupling mode was applied in this research, including two irrigation methods and four N levels. The irrigation methods were flood irrigation and intermittent irrigation, while four N levels were high-N, middle-N, low-N and none-N. Field experiment was conducted to study the effect of water and fertilizer coupling mode on CH4 and N2O emission. The results showed that the accumulated CH4 emissions were significantly reduced by intermittent irrigation, in comparison with flood irrigation, the reduction in early rice season were from 13. 18 kg.hm-2 to 87. 90 kg.hm-2, and were from 74. 48 kg.hm-2 to 131. 07 kg.hm-2 in late rice season, with a rate of 24. 4% -67. 4% and 42. 5% -65. 5% respectively; whereas the accumulated N20 emissions were increased, the increment were from 0. 03 kg.hm-2 to 0. 24 kg.hm-2 in early rice season and from 0. 35 kg.hm-2 to 1. 53 kg.hm-2 in late rice season when compared flood irrigation, increased by 6.2% -18. 3% and 40.2% - 80.9% respectively. On the whole, intermittent irrigation reduces the warming potential of greenhouse gases (GWP), which were decreased by 18. 8% to 58. 6% in early rice season and by 34. 4% to 60. 1% in late rice season, and the reduction of total GWP were from 2 388 to 4 151 kg. hm-2 (CO2 eq), with a rate of 41% -54% . Through correlation analysis it found that CH4 emissions from soil were significantly related with soil solution Eh and solution CH4 concentration. In comparison with the flood irrigation, the application of intermittent irrigation in double-season rice cultivation was conducive to CH4 reduction, though the increase came in N2O, but the GWPs were significantly reduced. Comprehensively, intermittent irrigation matching with middle-N is more benefit to double-season rice cultivation.

Linc00675 is a novel marker of short survival and recurrence in patients with pancreatic ductal adenocarcinoma.

AIM: To detect linc00675 expression in pancreatic ductal adenocarcinoma (PDAC), to analyze the relationship between the expression level of linc00675 and the clinical pathological characteristics, to explore the biological functions of linc00675, and to determine whether linc00675 has independent prognostic value in PDAC. METHODS: We studied linc00675 expression among eight histologically confirmed PDAC tissue samples and four chronic pancreatitis tissue samples through microarray screening. RT-qPCR was conducted to further investigate linc00675 expression in PDAC cell lines as well as archived tissues from a large cohort of PDAC patients. The correlations between the level of lnc00675 and clinicopathological characteristics and survival in patients with pancreatic cancer were evaluated using Correlation analysis. Univariate and multivariate analyses were conducted to predict whether lnc00675 expression is an independent prognostic and recurrence factor in patients with pancreatic cancer. After downregulating the expression of linc00675 through siRNA, MTT assay, flow cytometry, transwell assay and Western blot were used to explore the biological function of linc00675 in proliferation, invasion, and cell cycle progression of pancreatic cancer cells. The relative molecular expression levels of epithelial-mesenchymal transition were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: The expression of Linc00675 in PDAC tissue samples was shown to be 672 times that in chronic pancreatitis tissue samples by microarray screening (P = 3.69 × 10(-5)). This finding was confirmed in tumor tissues from 90 patients with PDAC compared with adjacent normal tissue samples by quantitative RT-PCR. We found that linc00675 overexpression positively correlated with lymph node metastasis (P = 0.005), perineural invasion (P = 0.006), and poor survival (P < 0.001). Univariate and multivariate analyses showed that linc00675 expression served as an independent predictor of overall survival (P = 0.009). Additionally, receiver operating characteristic curve analysis showed that high linc00675 might serve as a predictor of tumor progression within 6 mo to a year after surgery. In vitro functional analysis demonstrated that knockdown of linc00675 attenuated pancreatic cancer cell proliferation and invasion as well as induced S phase arrest. Suppression of linc00675 in pancreatic cancer cells resulted can reverse the progress of epithelial-mesenchymal transition. CONCLUSION: Linc00675 may function as an oncogene during PDAC development, and its expression is an independent predictor of unfavorable prognosis in patients with PDAC.

Low RASSF6 expression in pancreatic ductal adenocarcinoma is associated with poor survival.

AIM: To analyze RASSF6 expression in pancreatic ductal adenocarcinoma (PDAC) and to determine whether RASSF6 has an independent prognostic value in PDAC. METHODS: We studied RASSF6 expression in 96 histologically confirmed PDAC samples and 20 chronic pancreatitis specimens using immunohistochemistry and real-time quantitative reverse transcription-PCR. PDAC issues were then classified as RASSF6 strongly positive, weakly positive or negative. RASSF6 mRNA and protein expression in PDAC samples with strong positive staining was further evaluated using real-time PCR and Western blot analysis. Lastly, correlations between RASSF6 staining and patients' clinicopathological variables and outcomes were assessed. RESULTS: RASSF6 was negatively expressed in 51 (53.1%) PDAC samples, weakly positively expressed in 29 (30.2%) and strongly positively expressed in 16 (16.7%), while its expression was much higher in para-tumor tissues and chronic pancreatitis tissues. Positive relationships between RASSF6 expression and T-stage (P = 0.047) and perineural invasion (P = 0.026) were observed. The median survival time of strongly and weakly positive and negative RASSF6 staining groups was 33 mo, 15 mo and 11 mo, respectively. Cox multivariate analysis indicated that RASSF6 was an independent prognostic indicator of overall survival in patients with PDAC. A survival curve analysis revealed that increased RASSF6 expression was correlated with better overall survival (P = 0.009). CONCLUSION: RASSF6 expression is an independent biomarker of an unfavorable prognosis in patients with PDAC.

[Preliminary study on pro-apoptotic gene BAD of Schistosoma japonicum].

OBJECTIVE: To understand the characteristics of pro-apoptotic gene SjBAD of Schistosoma japonicum, such as its biology, immunology, and transcriptional expression, and evaluate its potential of the recombinant protein as a vaccine candidate for schistosomiasis. METHODS: SjBAD was amplified by PCR and subeloned into a pET-28a(+) vector, and the recombinant plasmid was transformed into competent E. coli BL21 for producing recombinant protein. The expressions of SjBAD in different development stages of schistosomula and 42-day male and female worms were determined by real-time PCR. The immunogenicity of the recombinant protein was analyzed by Western blotting and ELISA. The potential of this protein as a vaccine candidate molecule was assessed by testing the worm reduction rate and liver egg reduction rate in the BALB/c mice immunized by the recombinant antigen SjBAD. RESULTS: SjBAD was successfully cloned, the recombinant plasmid pET-28a(+)-SjBAD was successfully expressed in E. coli, and the molecular weight of the recombinant protein was around 22 kDa. Western-blotting showed that the recombinant protein had good immunogenicity. The recombinant protein could induce high level of specific IgG antibodies in the BALB/c mice. SjBAD was expressed in all tested 7-, 14-, 21-, 28-, 35- and 42-day worms, and was highly expressed in 14-day schistosomula, while the expression level in 42-day male worms was higher than that in 42-day female worms. Two in- dependent animal trials showed that 30.82% and 27.87% worm reduction rates, as well as 42.52% and 45.84% liver eggs reduction rates were obtained in the rSjBAD vaccinated group compared with those of the blank control group (both P < 0.05). CONCLUSIONS: The proapoptotic gene SjBAD is successfully cloned and expressed. The gene is expressed in different development stages of S. japonicum. The rSjBAD vaccinated BALB/c mice can obtain a partial protective immunity against S. japonicum infection.

[Cloning, expressing and functional analysis of SjMF4, a novel Schistosoma japonicum gene].

Based on the phenomenon of the natural anti-schistosomiasis in Microtus fortis, the sera from normal Microtus fortis were employed to immunoscreen the cDNA library of adult Schistosoma japonicum (Chinese strain), two positive clones were obtained, RACE technique was further applied to amplify one of the clones, and a cDNA fragment with an ORF was identified. Sequencing revealed that it was a novel gene of Schistosoma japonicum, and it was named SjMF4 (Schistosoma japonicum Microtus fortis 4). Then the structure and functional motifs of SjMF4 were analysed. The gene was subcloned into pET-28a(+) vector; the recombinant protein showed good antigenicity in Western blotting. The gene was further subcloned into eukaryotic expression vector pcDNA3 to construct the DNA vaccine containing SjMF4. Immune experiments in mice showed significant protection that the recombinant plasmid did induce 28.64%+/-3.82% worm reduction and 21.73%+/-3.98% egg reduction than controls against the Schistosoma japonicum cercaria challenge.

[Characterization of CH4, N2O emission and selection of rice cultivars in double cropping rice fields].

Methane and nitrous oxide emission fluxes of rice cultivars in the double cropping rice fields were measured by static chamber-gas chromatography in field experiments. The results showed a single-peak pattern in seasonal variations of methane emission and a double-peak pattern in variations of nitrous oxide emission for the early season rice, and a single-peak pattern in variations of both methane and nitrous oxide emission for the late season rice. There was significant difference among the rice cultivars in the average emission fluxes of methane and nitrous oxide during the whole growing season. The ranges for the methane and nitrous oxide average emission fluxes were 0.58 mg x (m2 x h)(-1) and 5.89 microg x (m2 x h)(-1) for the early-season rice cultivars, and 4.06 mg x (m2 x h)(-1) and 5.70 microg (m2 x h)(-1) for the late-season rice, respectively. The ranges of GWP (global warming potential) contributed by the amount of greenhouse gases emission and GWP per unit yield of rice cultivars were 2.92 kg x hm(-2) and 0.097 kg x kg(-1) for the early-season rice cultivars, and 2 256 kg x hm(-2) and 0.28 kg x kg(-1) for the late-season rice cultivars, respectively. The GWP and GWP per unit yield of rice types were sorted in a descending order: traditional rice > the super hybrid rice > the hybrid rice. For the early season rice, the methane and nitrous oxide emission fluxes of the blank region without rice were 27.1%-31.8% and 33.6%-88.3% of those measured for the region with rice planted, respectively, whereas the corresponding percentages for the late season rice were 23.8%-28.8% and 38.6%-45.3%, respectively. Based on these results, Luliangyou No. 819, Jinyou No. 402 and Xiangzaoxian No. 24 were selected as suitable early-season rice cultivars, whereas Yueyou No. 9113 and Xiangwanxian No. 12 should be chosen for late-season rice planting.

[Cloning, expression and immune function analysis of a gene encoding inositol monophosphate in Schistosoma japonicum].

OBJECTIVE: To clone and express a full-length cDNA encoding inositol monophosphate of Schistosoma japonicum (SjIM), and to access its immunoprotection in BALB/c mice for schistosomisis. METHODS: A full-length cDNA encoding the S. japonicum inositol monophosphate was isolated from 42 d schistosomes cDNAs. The expression profiles in different developmental stages were detected by real-time quantitative RT-PCR. The open reading frame (ORF) was subcloned into a pET28a(+) vector and transformed into BL21 and the recombinant protein was induced by IPTG. The immune characters of the purified recombinant protein were analyzed by Western blotting and immunoprotection in BALB/c mice. RESULTS: Bioinformatics analysis indicated that SjIM had an ORF of 834 base pairs that encoded 278 amino acids. Real-time quantitative RT-PCR analysis revealed that SjIM was upregulated in 35-day-old schistosomes, while the expression level in females was higher than that in male worms in 42nd day. Western blotting showed that the recombinant SjIM was immunogenic. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjIM could induce 48.76% and 41.29% reductions in the numbers of worms and eggs in the liver, respectively. CONCLUSIONS: The gene of SjIM is obtained from schistosomes cDNAs and the recombinant SjIM protein is induced successfully in E. coli. These aforementioned results demonstrate that the recombinant SjIM cand induce partial protection against schistosomiasis in BALB/c mice.

[Identification and preliminary analysis of a novel full-length cDNA encoding retinoid X receptor 2 from Schistosoma japonicum].

OBJECTIVE: To clone and preliminarily analyze the full-length cDNA encoding retinoid X receptor 2 (RXR2) from Schistosoma japonicum. METHODS: The rapid amplification cDNA ends (RACE)was applied to get a full-length cDNA encoding retinoid X receptor 2 from S. japonicum (SjRXR2). The transcription of SjRXR2 was detected by real-time PCR. By bioinformatical technology, the gene structure was analyzed and the antibody epitope was predicted. The polyclonal antibodies were raised in mice immunized with the synthesis peptide. Western blot was applied to detect its expression in the worm. RESULTS: The full-length cDNA of SjRXR2 was 5 960 bp and contained an open reading frame encoding a 1 435 amino acid which had a predicted molecular weight 159 kDa. Bioinformatical analysis indicated that SjRXR2 had a highly conserved DNA binding domain (DBD) and a moderate conserved ligand binding domain (LBD). The relative mRNA (s) of SjRXR2 with higher expressions at Day 21 and 42 were evaluated in five different S. japonicum developmental stages. The Western blot analysis showed that polyclonal antibodies were able to specifically recognize the protein with molecular around 150 kDa from the extract of S. japonicum. CONCLUSION: A full-length cDNA encoding retinoid X receptor 2 (RXR2) from S. japonicum is obtained which provides preliminary information for further investigation of SjRXR2 functions in S. japonicum.