[Preparation of interleukin-1ß-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice].

Objective: To prepare interleukin-1ß-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1ß-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1ß-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1ß-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1ß (IL-1ß) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1ß antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 µg/ml and 50.0 µg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

[A comparative study of transperitoneal transmesenteric approach versus paracolic sulci approach laparoscopic adrenal tumorectomy for treatment of primary hyperaldosteronism on left side].

Objective: To compare the therapeutic effect of transperitoneal transmesenteric approach versus paracolic sulci approach laparoscopic adrenal tumorectomy for treatment of left-sided primary hyperaldosteronism. Methods: From January 2017 to July 2019, the clinical data of 70 patients with left-sided primary hyperaldosteronism (PHA) who underwent surgery in the First Hospital of Lanzhou University and five other hospitals in Gansu Province were retrospectively analyzed. There are 43 male and 27 female patients. Among them,28 patients were performed transperitoneal transmesenteric approach laparoscopic adrenal tumorectomy and 42 patients were performed transperitoneal paracolic sulci approach laparoscopic adrenal tumorectomy. The general information and perioperative data of the two groups were compared. Results: All 70 cases of surgery were successfully completed. As compared with the paracolic sulci approach group, the operation time was significantly shorter in the transmesenteric approach group[(26.7±8.8)vs (38.9±7.1)min,P<0.001)], and the estimated blood loss was less in the transmesenteric approach group[45(30,50) vs 50(40,60)ml,P=0.042]. There was no statistically significant difference in the postoperative hospitalization days between the two groups[(4.4±1.0)vs(4.5±1.0)d, P=0.669)]. The electrolytes and aldosterone to renin ratio returned to a healthy level in the postoperative one month, and the blood pressure also returned to a healthy level in 53 (75.7%) patients. Conclusion: Transperitoneal transmesenteric approach laparoscopic adrenal tumorectomy is safe and feasible, with a short operation time and relatively less estimated blood loss.

[Minimally invasive lateral lumbar interbody fusion significantly improves the sagittal balance for adult degenerative scoliosis].

Objectives: To evaluate the effect of minimally invasive lateral lumbar interbody fusion (LLIF/OLIF) on the sagittal balance of adult degenerative scoliosis. Methods: From January 2014 to June 2017, a total of 23 patients with degenerative scoliosis underwent staged minimally invasive surgery in Shanghai Ruijin Hospital. All patients were implanted with LLIF or OLIF cage from the lateral approach first, and was followed by the posterior percutaneous pedicle screw fixation or pedicle screw fixation via Wiltse approach. If the sagittal deformity correction was not satisfactory after the first surgery, a posterior osteotomy can be performed during the second stage operation. A biplanar X-ray of the whole spine was taken with the EOS imaging system before and after surgery. The EOS software was used to measure and evaluate the patient's sagittal balance parameters including pelvic incidence (PI), pelvic tilt (PT), lumbar lordosis (LL), the sagittal vertical axis (SVA) and the coronal Cobb angle. The visual analogue scale (VAS) score for low back pain, the Oswestry Disability Index (ODI) score were evaluated before and after surgery. Paired t test or repeated measures ANOVA was used to compare the data before and after surgery. Results: There were 6 males and 17 females with a mean age of (72±4) years (62-79 years). Nine patients were treated with LLIF and 14 patients with OLIF. Sixteen cases were implanted with three cages, five with two cages and two with four cages. The mean follow-up period was 24.2 months (15-42 months). After the first operation, the Cobb angle of the patient was significantly improved (18°±7° vs 33°±8°, t=13.2, P<0.01). All the parameters for sagittal balance, including PI-LL (20°±8° vs 31°±8(o)), SVA ((5.3±2.0) cm vs (8.2±3.5) cm), PT (16°±6° vs 23°±4°) were all significantly improved as well (t=6.8, 4.5, 9.0, ALL P<0.01). At the last follow-up, the VAS score of low back pain (3.4±1.1 vs 6.3±1.0) and ODI scores (27.3%±3.0% vs 47.1%±5.9%) were also significantly improved (t=11.3, 17.8, both P<0.01). No major complications occurred in this group. Conclusions: Minimally invasive LLIF/OLIF can significantly improve the coronal and sagittal balance of adult degenerative scoliosis. Staged minimally invasive surgery can significantly alleviate pain and improve function in these patients.

Thermodynamic method for establishment of relationship between icephobicity/superhydrophobicity and microstructure-Based on computing for adhesion work.

Superhydrophobic surfaces (SHS) have potential in solving the icing of aircraft, high-voltage overhead transmission lines, and other power network devices exposed to the air. For this reason, we wish to establish the relationship between microstructure and the adhesion work by thermodynamic method, also for analysis of the relationship between the hydrophobicity and icephobicity (or anti-icing). Therefore, respectively considering Cassie-Baxter and Wenzel states, such relationship was theoretically established based on one/two-step surface model, enlightened by natural and artificial SHS. Among it, how to obtain the adhesion work of icing per unit ice-solid interface is the key to this study. Followed by it, hydrothermal experiment, chemical deposition, and etching methods were performed to verify our theoretical results. •How to model for the SHS based on the natural and artificial SHS;•Computation for adhesion work (waw) per unit area of a water droplet-SHS interface;•Computation for adhesion work (wai) per unit area of a frozen water droplet-SHS interface;•Computation for reduced adhesion work (wa2) after icing;•Hydrothermal experiment, chemical deposition and etching methods were used for validation of modeling.

Isolation and identification of pathogenic Aeromonas veronii from a dead Yangtze finless porpoise.

Diseases caused by Aeromonas veronii in freshwater fish have been widely reported, but other species such as aquatic mammals have probably been overlooked. Here, we identified one isolate of A. veronii from a Yangtze finless porpoise Neophocaena asiaeorientalis asiaeorientalis exhibiting disease and mortality, and subsequently confirmed its virulence in artificial infection of BALB/c mice. The bacterial isolate was identified as A. veronii based on physiological, biochemical, and phenotypic features, and homology of the 16S rRNA, cpn60, rpoB, dnaJ and gyrB genes. Our results expand the known host spectrum of A. veronii, which is of great importance for the etiology of porpoise, dolphin, and other cetacean diseases.

[Two-year clinical and radiological outcomes of minimally invasive surgery for severe adult degenerative scoliosis].

Objectives: To evaluate the efficacy of restoring the balance in the coronal and sagittal planes and to evaluate clinical outcomes in the treatment of severe adult degenerative scoliosis patients using staged lateral lumbar interbody fusion (LLIF) and percutaneous transpedicle screw fixation (pTSF). Methods: A retrospective analysis was made on 13 patients with severe adult degenerative scoliosis treated with staged surgery in Ruijin Hospital between May, 2014 and July, 2015. The operation data were collected, including operation time, estimated blood loss, and complications. Preoperative and postoperative standing anteroposterior and lateral lumbar radiographs were obtained by EOS image system in all patients for measurement of sagittal and coronal plane alignment. All patients were available for at least 24 months of following-up (18-32 months). Paired sample t test was used to compare the parameters of each group pre- and post-operation. Results: All of the 13 patients received LLIF with staged transpedicle screw fixation. Mean operation time was (231±48) minutes and (155±33) minutes for two-stage surgery. Mean blood loss was (253±80) ml during one-stage LLIF surgery and (326±99) ml during two-stage pTSF surgery. The interval between two surgical procedures was from 7 to 42 days (mean 14 days). In the coronal plane, the mean Cobb angle of curves was 43.1°±7.3° preoperatively, and it was 11.4°±4.0°after one-stage LLIF (t=10.16, P<0.001) and 5.5°±1.9° after two-stage pTSF (t=6.14, P<0.001). In the sagittal plane, lumbar lordosis (LL) changed after each procedure was as follows: LLIF from 16.8°±8.4° to 30.1°±6.8° (t=5.21, P<0.001) and PSF to 37.1°±4.0° (t=5.04, P<0.001). Sagittal vertical axis (SVA) was reduced from (10.5±2.4) cm to (5.3±2.1) cm after one-stage LLIF (t=8.52, P<0.001) and to (3.1±1.5) cm after two-stage pTSF (t=5.51, P<0.001). In the spino-pelvic parameter, pelvic incidence(PI)-LL mismatch decreased from 33.6°±9.6° to 17.8°±4.5° (t=5.31, P<0.001) after LLIF and to 9.9°±3.7° after pTSF (t=4.68, P<0.001). The visual analogue scale (VAS) of low back pain and leg pain and the Oswestry dability Index (ODI) improved significantly at the last follow-up (t=10.42, 8.94, 19.20, all P<0.01). Conclusions: LLIF significantly improves segmental and coronal plane alignment in patients with degenerative lumbar scoliosis, and these patients can be effectively corrected by staged minimally invasive surgery.

Alpha galactosidase A activity in Parkinson's disease.

Glucocerebrosidase (GCase, deficient in Gaucher disease) enzymatic activity measured in dried blood spots of Parkinson's Disease (PD) cases is within healthy range but reduced compared to controls. It is not known whether activities of additional lysosomal enzymes are reduced in dried blood spots in PD. To test whether reduction in lysosomal enzymatic activity in PD is specific to GCase, we measured GCase, acid sphingomyelinase (deficient in Niemann-Pick disease types A and B), alpha galactosidase A (deficient in Fabry), acid alpha-glucosidase (deficient in Pompe) and galactosylceramidase (deficient in Krabbe) enzymatic activities in dried blood spots of PD patients (n = 648) and controls (n = 317) recruited from Columbia University. Full sequencing of glucocerebrosidase (GBA) and the LRRK2 G2019S mutation was performed. Enzymatic activities were compared between PD cases and controls using t-test and regression models adjusted for age, gender, and GBA and LRRK2 G2019S mutation status. Alpha galactosidase A activity was lower in PD cases compared to controls both when only non-carriers were included (excluding all GBA and LRRK2 G2019S carriers and PD cases with age-at-onset below 40) [2.85 µmol/l/h versus 3.12 µmol/l/h, p = 0.018; after controlling for batch effect, p = 0.006 (468 PD cases and 296 controls)], and when including the entire cohort (2.89 µmol/l/h versus 3.10 µmol/l/h, p = 0.040; after controlling for batch effect, p = 0.011). Because the alpha galactosidase A gene is X-linked, we stratified the analyses by sex. Among women who were non-carriers of GBA and LRRK2 G2019S mutations (PD, n = 155; control, n = 194), alpha galactosidase A activity was lower in PD compared to controls (2.77 µmol/l/h versus 3.10 µmol/l/h, p = 0.044; after controlling for a batch effect, p = 0.001). The enzymatic activity of acid sphingomyelinase, acid alpha-glucosidase and galactosylceramidase was not significantly different between PD and controls. In non-carriers, most lysosomal enzyme activities were correlated, with the strongest association in GCase, acid alpha-glucosidase, and alpha galactosidase A (Pearson correlation coefficient between 0.382 and 0.532). In a regression model with all five enzymes among non-carriers (adjusted for sex and age), higher alpha galactosidase A activity was associated with lower odds of PD status (OR = 0.54; 95% CI:0.31-0.95; p = 0.032). When LRRK2 G2019S PD carriers (n = 37) were compared to non-carriers with PD, carriers had higher GCase, acid sphingomyelinase and alpha galactosidase A activity. We conclude that alpha galactosidase A may have a potential independent role in PD, in addition to GCase.

Berberine binds RXRα to suppress ß-catenin signaling in colon cancer cells.

Berberine, an isoquinoline alkaloid, is a traditional oriental medicine used to treat diarrhea and gastroenteritis. Recently, we reported that it could inhibit the growth of intestinal polyp in animals and in patients with the familial adenomatous polyposis by downregulating ß-catenin signaling. However, the intracellular target mediating the effects of berberine remains elusive. Here, we provide evidence that berberine inhibits ß-catenin function via directly binding to a unique region comprising residues Gln275, Arg316 and Arg371 in nuclear receptor retinoid X receptor alpha (RXRα), where berberine concomitantly binding to and synergistically activating RXRα with 9-cis-retinoic acid (9-cis-RA), a natural ligand binding to the classical ligand-binding pocket of RXRα. Berberine binding promotes RXRα interaction with nuclear ß-catenin, leading to c-Cbl mediated degradation of ß-catenin, and consequently inhibits the proliferation of colon cancer cells. Furthermore, berberine suppresses the growth of human colon carcinoma xenograft in nude mice in an RXRα-dependent manner. Together, our study not only identifies RXRα as a direct protein target for berberine but also dissects their binding mode and validates that berberine indeed suppresses ß-catenin signaling and cell growth in colon cancer via binding RXRα, which provide new strategies for the design of new RXRα-based antitumor agents and drug combinations.

Clinical significance and prognostic value of femoral lymph node metastasis in FIGO stage III vulvar carcinoma.

OBJECTIVES: To determine the clinical significance and prognostic value of femoral lymph node metastasis (FLNM) in patients with International Federation of Gynecology and Obstetrics (FIGO) stage III vulvar carcinoma. METHODS: The medical records of patients with vulvar carcinoma who underwent inguinofemoral lymphadenectomy between 1990 and 2013 were retrospectively reviewed. RESULTS: Of 66 patients with stage III vulvar carcinoma, 42 had superficial lymph node metastasis (SLNM) only and 24 had FLNM. Significantly higher rates of extracapsular invasion (P = 0.008), multiple nodal metastasis (P = 0.042), and advanced FIGO substage (P = 0.026) as well as a larger tumor diameter (≥4 cm, P = 0.023) and greater depth of invasion (≥5 mm, P = 0.020) were observed among patients with FLNM compared to those with SLNM only. After a median follow-up of 46 months (range, 6-172 months), 35 patients experienced relapse and 30 died from disease. The 5-year cancer-specific survival (CSS) rates were 70.1% and 30.8% for patients with SLNM only and FLNM, respectively (P = 0.001). In multivariate analysis, only FLNM was found to be an independent risk factor for reduced recurrence-free survival (RFS) and CSS among patients with stage III vulvar cancer (hazard ratio [HR] = 2.277, P = 0.037 for RFS; HR = 2.360, P = 0.042 for CSS). When the FLNM cases were considered together as stage IIIC, significant differences emerged in the RFS (P = 0.002) and CSS (P = 0.004) among the re-divided FIGO substages. CONCLUSIONS: FLNM represented an unfavorable status of node metastasis with a worse prognosis compared to that of SLNM alone, and this should be considered in a future FIGO staging system for vulvar cancer.

Extraction of proteins and preliminary characterization of physicochemical properties in Toona sinensis fruit.

We investigated the extraction of Toona sinensis fruit proteins and preliminarily characterized their physicochemical properties. The results showed that optimal extraction occurred under conditions of pH 10.5, a duration of 40 min, a liquid-to-solid ratio of 25:1, and a temperature of 40°C by an orthogonal design using T. sinensis fruit protein as the index and single factor. The total nitrogen content was 13.8 g/100 g and included 17 different amino acids. The glutamate level was highest at 35.37%, followed by arginine at 15.31%. The isoelectric point of T. sinensis fruit protein was between 6.8 and 10.0 with a typical absorption peak by infrared chromatography. Three protein bands were analyzed using SDS-polyacrylamide gel electrophoresis, with relative molecular weights of 55, 51, and 22 kDa. This study provides a theoretical basis for the comprehensive utilization of T. sinensis fruit by further investigating the biological activity of its proteins.

The effect of continuous perfusion of esmolol on cardiovascular risk in elderly patients undergoing noncardiac surgery.

We evaluated the effect of continuous perfusion of esmolol on cardiovascular risk during curative laparoscopic surgery for gastrointestinal cancer in elderly patients. Sixty patients with gastrointestinal cancer, aged from 60 to 80 years, were divided into an esmolol group (ES, n = 30) and a control group (NS, n = 30). ES patients were treated with esmolol at a dose of 0.3 mg/kg 3 min before tracheal intubation, and received continuous perfusion of esmolol at a dose of 50 µg/kg/min during operation. In NS, esmolol was replaced by saline. SBP/DBP, MAP, HR, SPO2, PETCO2, the depth of anesthesia and the value of RPP were recorded before anesthesia (T0), during intubation (T1), 10 min before pneumoperitoneum (T2), during pneumoperitoneum (T3), 30 min after incision (T4), at the end of surgery (T5), during extubation (T6) and 30 min after extubation (T7). The serum levels of cTnI, CK, CK-MB and LDH were measured before anesthesia, 6 and 30 h after surgery. HR, MAP, RPP at T1, T3, T6 in ES were obviously decreased compared with NS (P < 0.05). There were no significant differences between both groups at different time points with respects to the serum levels of cTnI, CK, CK-MB and LDH. No major adverse cardiocerebral event was observed in both groups in three postoperative months. The present study indicated that the application of esmolol during curative laparoscopic surgery for gastrointestinal cancer can effectively reduce the cardiovascular responses in intubation, operation and extubation, sustain hemodynamic stable, reduce myocardial oxygen consumption, and prevent perioperative adverse cardiovascular events, but had no significant myocardial protective effect.

Factors influencing suicidal ideation among Chinese patients with stomach cancer: qualitative study.

AIM: The aim of this qualitative descriptive study was to explore the influencing factors of suicidal ideation in Chinese patients diagnosed with stomach cancer. INTRODUCTION: More recently, links have been made between suicide and cancer. For inpatients with cancer, nurses are at the front line to cope with suicidal issues in clinical settings. METHOD: This study was part of a larger study that investigated psychological strains and related factors associated with suicidal ideation. Thirty-two patients with stomach cancer participated in a face-to-face interview. Thematic analysis was used to analyse the qualitative data. FINDINGS: Four themes were identified as protective factors against suicidal ideation: suicidal denial and fears of stigmatization; positive coping skills; family connectedness; and desire to live well and longer. DISCUSSION: Consistent with previous research, these four protective factors may encourage participants to think more positively about life and cancer. CONCLUSION: Our findings suggest four protective factors for targeted suicide prevention, which may play an important role in future suicide-prevention programmes for Chinese patients diagnosed with stomach cancer. IMPLICATIONS FOR NURSING: Nurses may help reduce patients' risk for suicidal ideation more effectively by discussing beliefs, family and meaning in life perspectives, as well as offering health education about positive coping skills. IMPLICATIONS FOR HEALTHY POLICY: Clinical nurses, hospitals and the government should work collaboratively with each other. It is suggested that Chinese national guidelines for working with the high-risk suicide populations should be considered within a future mental health working plan.

Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa.

Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops.

Correlation between Interleukin-17 gene polymorphism and gastric cancer susceptibility in Han Chinese population.

OBJECTIVE: The present study aims to investigate the correlation between Interleukin (IL)-17 gene polymorphism with gastric cancer susceptibility in Han Chinese population. PATIENTS AND METHODS: Between November 2013 and October 2014, 386 patients with gastric cancer who had undergone surgeries at our institution and 374 age- and sex-matched healthy controls were included in this study. Single nucleotide polymorphisms (SNPs) of IL-17 gene (rs2275913, rs3748067, rs4711998 and rs763780) in patients and health controls were studied by using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) and DNA sequencing technology. The correlation between IL-17 polymorphism and gastric cancer susceptibility was analyzed using logistic regression analysis. RESULTS: For IL17 rs2275913, no significant differences were observed in the frequencies of AA genotype and A allele between patients and controls (c2 = 0.870 p > 0.05; c2 = 0.814 p > 0.05). In IL17 rs3748067, the frequencies of TT genotype and T allele were significantly higher in patients than in controls (c2 = 12.82 p < 0.01; c2 = 12.805 p < 0.01). For IL17A rs4711998, no significant differences were observed in the frequencies of AA genotype and A allele between patients and controls (c2 = 2.636, p > 0.05; c2 = 1.462, p > 0.05). As for ILl7F rs763780, the frequencies of GG genotype and G allele in patients were significantly different from those in controls (c2 = 16.534, p < 0.01; c2 = 16.399, p < 0.01). CONCLUSIONS: Polymorphism of IL-17 rs3748067 and rs763780 is closely associated with gastric cancer development. Polymorphism of L-17 rs2275913 and rs4711998 may be correlated with the risk for gastric cancer.

Cloning and characterization of the nicotianamine synthase gene in Eruca vesicaria subsp sativa.

Nicotianamine (NA) is a ubiquitous metabolite in plants that bind heavy metals, is crucial for metal homeostasis, and is also an important metal chelator that facilitates long-distance metal transport and sequestration. NA synthesis is catalyzed by the enzyme nicotianamine synthase (NAS). Eruca vesicaria subsp sativa is highly tolerant to Ni, Pb, and Zn. In this study, a gene encoding EvNAS was cloned and characterized in E. vesicaria subsp sativa. The full-length EvNAS cDNA sequence contained a 111-bp 5'-untranslated region (UTR), a 155-bp 3'-UTR, and a 966-bp open reading frame encoding 322-amino acid residues. The EvNAS genomic sequence contained no introns, which is similar to previously reported NAS genes. The deduced translation of EvNAS contained a well-conserved NAS domain (1-279 amino acids) and an LIKI-CGEAEG box identical to some Brassica NAS and to the LIRL-box in most plant NAS, which is essential for DNA binding. Phylogenetic analysis indicated that EvNAS was most closely related to Brassica rapa NAS3 within the Cruciferae, followed by Thlaspi NAS1, Camelina NAS3, and Arabidopsis NAS3. A reverse transcription-polymerase chain reaction indicated that EvNAS expression was greatest in the leaves, followed by the flower buds and hypocotyls. EvNAS was moderately expressed in the roots.

Cloning and characterization of ADP-glucose pyrophosphorylase small subunit gene in Cyperus esculentus (yellow nutsedge).

ADP-glucose pyrophosphorylase (ADPGlcPPase) controls the first committed step of starch synthesis by catalyzing the biosynthesis of ADP-glucose from glucose-phosphate and ATP. It is a tetrameric protein consisting of two small and two large subunits. The small subunits have a catalytic function, while the large subunits regulate the enzyme activity. Cyperus esculentus (yellow nutsedge) is a perennial C4 plant grown from rhizomes and tubers. Previous studies on yellow nutsedge have mostly focused on the morphology and cultivation of tubers, their application in food, and biochemical analyses of the tubers. In this study, the gene encoding the ADPGlcPPase small subunit (CeAGPS) in yellow nutsedge was cloned and characterized. The full-length CeAGPS cDNA sequence contained an 81-bp 5'-untranslated region (UTR), a 188-bp 3'-UTR, and a 1539-bp open reading frame encoding 512-amino acid residues. The genomic sequence of CeAGPS comprises a nine exon-eight intron structure similar to the previously reported cotton and Arabidopsis thaliana AGPS genes. The deduced translation product of the CeAGPS gene contained a well-conserved catalytic domain and regulatory elements typical of plant AGPS. Reverse transcriptase polymerase chain reaction amplification of the target gene in various plant parts using gene-specific primers indicated that the expression of CeAGPS was most abundant in the tuber, and relatively lower in nutsedge roots.

The Friend leukaemia virus integration 1 (Fli-1) transcription factor affects lupus nephritis development by regulating inflammatory cell infiltration into the kidney.

The transcription factor Friend leukaemia virus integration 1 (Fli-1) is implicated in the pathogenesis of systemic lupus erythematosus in both human patients and murine models of lupus. Murphy Roths large (MRL)/lpr mice and New Zealand mixed (NZM)2410 mice, murine models of lupus, with decreased expression of Fli-1 had significantly prolonged survival and reduced nephritis. Lupus nephritis is a major cause of mortality and morbidity in patients, and inflammatory cell infiltration plays a key role in the development of the disease. To study how the expression of Fli-1 affects the infiltration of inflammatory cells into the kidneys, we generated congenic enhanced green fluorescent protein (GFP) transgenic MRL/lpr mice. A significantly increased number of GFP-expressing inflammatory cells infiltrated the kidneys of wild-type MRL/lpr mice compared to Fli-1 heterozygous (Fli-1(+/-)) MRL/lpr mice after injection of GFP(+) cells. Expression of inflammatory chemokine mRNA, including chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4 and CCL5, was significantly lower in the kidneys from Fli-1(+/-) MRL/lpr mice compared to wild-type littermates. Numbers of infiltrated cells into the kidneys correlate with expression levels of CCL2, CCL4 and CCL5, but not the titres of anti-dsDNA autoantibodies in these mice. Significantly increased inflammatory cells from wild-type MRL/lpr mice infiltrated into kidneys compared to the cells from Fli-1(+/-) MRL/lpr mice. The chemotaxis of inflammatory cells from Fli-1(+/-) MRL/lpr mice towards each chemokine was decreased significantly compared to inflammatory cells from wild-type MRL/lpr mice in the transwell migration assay in vitro. Our results indicate that Fli-1 affects lupus nephritis development by regulating the expression of chemokines in the kidney and the migration of inflammatory cells.

SHH-dependent knockout of HIF-1 alpha accelerates the degenerative process in mouse intervertebral disc.

Hypoxia-inducible factor-1alpha (HIF-1 alpha) has been reported to have an important role in the metabolism and synthesis of extracellular matrix of the nucleus pulposus cells (NPCs) and was assumed to be involved in the process of intervertebral disc degeneration. The objective of this study was to investigate the role of HIF-1alpha in disc degeneration in vivo using a conditional HIF-1alpha knockout (KO) mouse model. ShhCre transgenic mice were mated with HIF-1 alpha fl/fl mice to generate conditional HIF-1alpha KO mice (HIF-1alpha fl/fl-ShhCre+). Three mice of each genotype (Wide-type and HIF-1alpha KO) at the age of 3 days, 6, and 12 weeks were sacrificed after genotyping. Five lumbar disc samples were harvested from each mouse, with a total of 45 disc samples for each genotype. In situ hybridization and immunohistochemical analysis were used to check the efficacy of HIF-1alpha knockout. Histological grading of the disc degeneration was performed according to the classification system proposed by Boos et al. Picro-sirius red staining, Safranine O/fast green staining and immunohistochemical study were used to evaluate the expression of aggrecan, type-II collagen and vascular endothelial growth factor (VEGF). Histologic analysis revealed more NPC deaths and signs of degeneration in HIF-1alpha KO mice and the degeneration scores of HIF-1alpha KO mice were significantly higher than those of the Wide-type mice at the age of 6 weeks and 12 weeks. There were less expressions of aggrecan, type-II collagen and VEGF in the intervertebral discs of HIF1-alpha KO mice than in those of wild-type mice. Taken together, the results of our study indicated that HIF-1alpha is a pivotal contributor to NPC survival and the homeotasis of extracellular matrix through the HIF-1alpha/VEGF signaling pathway, and plays an important role in the development of disc degeneration.

Inhibition of ß-catenin signaling by nongenomic action of orphan nuclear receptor Nur77.

Dysregulation of ß-catenin turnover due to mutations of its regulatory proteins including adenomatous polyposis coli (APC) and p53 is implicated in the pathogenesis of cancer. Thus, intensive effort is being made to search for alternative approaches to reduce abnormally activated ß-catenin in cancer cells. Nur77, an orphan member of the nuclear receptor superfamily, has a role in the growth and apoptosis of cancer cells. Here, we reported that Nur77 could inhibit transcriptional activity of ß-catenin by inducing ß-catenin degradation via proteasomal degradation pathway that is glycogen synthase kinase 3ß and Siah-1 independent. Nur77 induction of ß-catenin degradation required both the N-terminal region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for ß-catenin binding. Nur77/ΔDBD, a Nur77 mutant lacking its DNA-binding domain, resided in the cytoplasm, interacted with ß-catenin, and induced ß-catenin degradation, demonstrating that Nur77-mediated ß-catenin degradation was independent of its DNA binding and transactivation, and might occur in the cytoplasm. In addition, we reported our identification of two digitalis-like compounds (DLCs), H-9 and ATE-i2-b4, which potently induced Nur77 expression and ß-catenin degradation in SW620 colon cancer cells expressing mutant APC protein in vitro and in animals. DLC-induced Nur77 protein was mainly found in the cytoplasm, and inhibition of Nur77 nuclear export by the CRM1-dependent nuclear export inhibitor leptomycin B or Jun N-terminal kinase inhibitor prevented the effect of DLC on inducing ß-catenin degradation. Together, our results demonstrate that ß-catenin can be degraded by cytoplasmic Nur77 through their interaction and identify H-9 and ATE-i2-b4 as potent activators of the Nur77-mediated pathway for ß-catenin degradation.