Role of HAND2 gene and protein expression in endometrial carcinoma.

OBJECTIVES: To explore the relationship between HAND2 gene and protein expression and the development of endometrial carcinoma (EC). MATERIALS AND METHODS: The expression of HAND2 protein was detected by immunohistochemistry in 77 cases of EC paraffin block and their matched adjacent tissues. Western blot, real-time polymerase chain reaction (RT-PCR) were used to detect the expression of HAND2 protein and mRNA HAND2 expression in 34 cases of EC fresh tissue and paired adjacent tissues. RESULTS: The expression of HAND2 protein and the content of HAND2 mRNA in EC tissue were significantly lower than those in the non-tumorous tissue adjacent to EC. The positive expression rates of HAND2 protein in type I and type II EC were 19.67% and 50.00%, respectively. The expression of HAND2 protein in G1, G3, and G2 EC were 30.43%, 26.32%, 18.75%, respectively, with no statistically difference. The positive expression rates of HAND2 in the two groups of specimens with shallow and deep muscularis infiltrating were 40.63% and 15.56%, respectively. With the increase of EC FIGO stage, the positive expression rates of HAND2 protein decreased, and the difference was statistically significant (p <0.05). CONCLUSIONS: HAND2 mRNA and protein low expressed in EC tissues, which suggested the degree of endometrial malignancy. HAND2 may be helpful to the early diagnosis, treatment, and to evaluate the prognosis of endometrial cancer.

Overexpressed LEDGF is a novel biomarker of poor prognosis in patients with cervical cancer.

Lens epithelium-derived growth factor (LEDFG) can prevent cells apoptosis by activating stress proteins and anti-apoptotic protein, which are involved in the development of a variety of malignancies as some studies have shown. However, little is known about the role of LEDGF in cervical cancer. In this study, the authors collected 95 cases of the cervical cancer tissue samples and its matching tissue adjacent to carcinoma diagnosed by the Department of Pathology. mRNA expression of LEDFG in randomly selected 20 cervical can- cer tissues and 20 adjacent normal tissues was detected by quantitative real-time PCR (qRT-PCR). LEDFG protein expression in randomly selected 20 cervical cancer tissues and 20 adjacent normal tissues was detected by immunohistochemistry (IHC) and Western Blot (WB). All patients were followed up for about three years. The authors found that both mRNA and protein expression level of LEDFG was significantly higher in cancer tissues compared with normal controls (p < 0.05) and this overexpression was significantly correlated with the histologic grade, the immersion depth of interstitial, the invasion of vessel, and lymph node status of cervical cancer. Furthermore, the three-year survival rate of 34 patients with LEDGF positive expression having a survival rate of three years was 57.6%. The survival rate of three years with negative expression was 91.7%. The survival rate of patients with LEDGF positive ex- pression was significantly lower than those of the negative expression (p <0.01). In conclusion, the present results suggest that LEDFG expression is an independent prognostic biomarker for cervical cancer.

[Comparative study of proliferative and periodontal differentiation propensity of induced pluripotent stem cells at different passages].

OBJECTIVE: To compare the proliferative and periodontal specific differentiation abilities of induced pluripotent stem cells (iPSCs) at different passages, and to investigate whether long term culturing would have a negative influence on their proliferation and specific differentiation capacity, thus providing a theoretical basis for further in-depth research on periodontal regeneration and the possible clinical applications of iPSCs. METHODS: IPSCs derived from human gingival fibroblasts at passages 5, 10, 15 and 20 were recovered and cultured in vitro. Their morphology and proliferation rates were observed respectively. We further induced the iPSCs at different passages toward periodontal tissue under the treatment of growth/differentiation factor-5 (GDF-5) for 14 days through the EB routine, then compared the periodontal differentiation propensities between the different passages of iPSCs by detecting their calcified nodules formation by Alizarin red staining and assaying their relative periodontal tissue related marker expressions by qRT-PCR and immunofluorescence staining, including bone related markers: osteocalcin (OCN), bone sialoprotein (BSP); periodontal ligament related markers: periostin, vimentin; and cementum related markers: cementum attachment protein (CAP), cementum protein 1 (CEMP1). The untreated spontaneous differentiation groups were set as negative controls respectively. RESULTS: iPSCs at different passages all showed a high proliferative capacity when cultured in vitro and turned into a spindle-like shape similar to fibroblasts upon periodontal specific differentiation. All iPSCs formed typical calcified nodules upon GDF-5 induction by Alizarin red staining in comparison to their untreated controls. The relative calcium deposition at all passages had been significantly upgraded under the treatment of GDF-5 (P5: t=2.125, P=0.003; P10: t=2.246, P=0.021; P15: t=3.754, P=0.004; P20: t=3.933, P=0.002), but no significant difference in their calcium deposition were detected within passages 5, 10, 15 and 20 (periodontal differentiation: F=2.365, P=0.109; spontaneously differentiation: F=2.901, P=0.067). Periodontal tissue related marker expressions of iPSCs at all passages had also been significantly upgraded under the treatment of GDF-5 (P<0.05), but still, no significant difference in their expression levels of periodontal tissue related proteins were detected within passages (BSP: F=0.926 7, P=0.450; vimentin: F=0.917 1, P=0.455; CEMP1: F=2.129, P=0.1367). CONCLUSION: Our results preliminarily confirmed that long term culturing won't influence the proliferation capacity and periodontal specific differentiation propensity of iPSCs, as they can still proliferate and differentiate toward periodontal cells with high efficiency upon growth factor induction after continuous passaging. Therefore, iPSCs could be recognized as a promising cell source for future possible application in periodontal tissue regeneration.

Analysis of persistent changes to γ-aminobutyric acid receptor gene expression in Plutella xylostella subjected to sublethal amounts of spinosad.

A multi-generational approach was used to investigate the persistent effects of a sub-lethal dose of spinosad in Plutella xylostella. The susceptibility of various sub-populations of P. xylostella to spinosad and the effects of the insecticide on the gene expression of γ-aminobutyric acid receptor (GABAR) were determined. The results of a leaf dip bioassay showed that the sensitivity of P. xylostella to spinosad decreased across generations. The sub-strains had been previously selected based on a determined LC25 of spinosad. Considering that GABA-gated chloride channels are the primary targets of spinosad, the cDNA of P. xylostella was used to clone GABARα by using reverse transcription-polymerase chain reaction (RT-PCR). The mature peptide cDNA was 1477-bp long and contained a 1449-bp open reading frame encoding a protein of 483 amino acids. The resulting amino acid sequence was used to generate a neighbor-joining dendrogram, and homology search was conducted using NCBI BLAST. The protein had high similarity with the known GABAR sequence from P. xylostella. Subsequent semi-quantitative RT-PCR and real-time PCR analyses indicated that the GABAR transcript levels in the spinosad-resistant strain (RR, 145.82-fold) and in Sub1 strain (selected with LC25 spinosad for one generation) were the highest, followed by those in the spinosad-susceptible strain, the Sub10 strain (selected for ten generations), and the Sub5 strain (selected for five generations). This multi-generational study found significant correlations between spinosad susceptibility and GABAR gene expression, providing insights into the long-term effects of sub-lethal insecticide exposure and its potential to lead to the development of insecticide-resistant insect populations.

[Changes of microRNAs profiling in mesothelial cells exposed to multi-walled carbon nanotubes].

Objective: To explore the cytotoxicities of MWCNT to the mesothelial cells and screen the changes of microRNA profile after exposure to MWCNT. Methods: A LDH method was used to test the cytotoxicities of MWCNT to MeT-5A cell lines. And then the differentially expressed miRNAs between mesothelioma cells and normal mesothelial cells were selected from previous work of research group. Among the significant expression changed miRNAs, 5 were verified by RT-qPCR in mesothelioma cells. The same five ones were further tested in MeT-5A cells exposed to 10 µg/cm2 MWCNT for 8, 24, 48, 72 h by RT-qPCR. Target genes of 5 miRNAs were predicted using Targetscan and miRanda softwares. David6.7 was used to perform GO enrichment and KEGG pathway analysis of target genes. All the data were analyzed by one-way ANOVA and Dunnett-T test in SPSS17.0. Results: After 24 h exposure to MWCNT, cell proliferation was significantly suppressed at more than 20 µg/cm2 concentration. Among the differentially expressed miRNAs, 5 were chosen to further vestified, namely hsa-miR-155 (up-regulated) , hsa-miR-30 d-5p, hsa-miR-34c-5p, hsa-miR-28-5p and hsa-miR-324-5p (down-regulated) , which were consistent with the miRNA array results. The 5 miRNAs also had the same expression changes in MeT-5A cells after exposure to 10 µg/cm2 MWCNT for different time periods. The potential target genes of the 5 miRNAs may be AKAP13, CCND3, Twist and E-Cadherin, which mainly involved in TGF-ß signal pathway, small cell lung cancer, etc. Conclusion: MWCNT could induce to MeT-5A cells, and also cause miRNA expression changes. The differential changed miRNAs may involve in cancer related signal pathways.

Development of an atomic spectra research platform based on a 30-keV electron beam ion trap.

Electron Beam Ion Traps (EBITs) serve as efficient tools for producing and studying highly charged ions. In response to the diagnostic requirements of upcoming magnetic confinement fusion devices, a medium-energy atomic spectra research platform based on a compact EBIT is developed. This platform achieves a central magnetic field of up to 1.0 T, with electron beam currents reaching 20 mA and electron energies up to 30 keV, similar to the electron temperature on fusion reactors. The developed atomic spectra platform successfully provided spectral data for elements such as argon, xenon, iron, and tungsten. This platform stands as a valuable asset for advancing research in nuclear fusion, particularly concerning impurity spectroscopic diagnostics.

Development and test of the laser blow-off impurity injection system in experimental advanced superconducting tokamak.

The Laser Blow-Off (LBO) impurity injection system is a crucial tool for studying impurity transport and plasma behavior. Conducting proactive impurity transport research is challenging on experimental advanced superconducting tokamak (EAST) due to the uncontrollable generation of impurity sources; therefore, it is necessary to develop a laser blow-off impurity injection system for injecting controlled trace impurity particles. This study presents the design and test results of an LBO system for the EAST. The system aims to provide precise and repeatable control over the timing and quantity of impurity injection. The system primarily consists of a laser source, two mirrors, a moveable focusing lens, a target material, and a vacuum system. The movement of the focusing lens is achieved by a three-dimensional displacement system. The operation of the system is completed by a remote control system. With the accurate control system, the laser spot diameter is adjustable, allowing for modification of impurity injection quantity. The test results demonstrate that the system can rapidly detect external trigger signals and ensure precise timing for the impurity injection. Furthermore, this system can also quickly change the focal point of the laser spot, addressing the requirements for impurity injections during the experiments with less than 0.4 mm position error for laser spot focusing. Test results have shown that the aluminum film material can be peeled off by the LBO system when the laser energy exceeds 650 mJ and the smallest ablation spot is about 1 mm. This study is of significant importance for conducting plasma impurity transport research on the EAST.

Ethyl pyruvate significantly inhibits tumour necrosis factor-α, interleukin-1ß and high mobility group box 1 releasing and attenuates sodium taurocholate-induced severe acute pancreatitis associated with acute lung injury.

In this study, we examined the effect of ethyl pyruvate (EP) on pulmonary inflammation in rats with severe pancreatitis-associated acute lung injury (ALI). Severe acute pancreatitis (SAP) was induced in rats by the retrograde injection of 5% sodium taurocholate into the pancreatic duct. Rats were randomly divided into the following experimental groups: control group, SAP group and EP-treated group. The tissue specimens were harvested for morphological studies, Streptavidin-peroxidase immunohistochemistry examination. Pancreatic or lung tissue oedema was evaluated by tissue water content. Serum amylase and lung tissue malondialdehyde (MDA) and myeloperoxidase (MPO) were measured. Meanwhile, the nuclear factor-κB (NF-κB) activation, tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) levels and HMGB1 protein expression levels in the lung were studied. In the present study, we demonstrated that treatment with EP after SAP was associated with a reduction in the severity of SAP and lung injury. Treatment with EP significantly decreased the expression of TNF-α, IL-1ß, HMGB1 and ameliorated MDA concentration, MPO activity in the lung in SAP rats. Compared to SAP group, administration of EP prevented pancreatitis-induced increases in nuclear translocation of NF-κB in the lung. Similarly, treatment with EP significantly decreased the accumulation of neutrophils and markedly reduced the enhanced lung permeability. In conclusion, these results demonstrate that EP might play a therapeutic role in pulmonary inflammation in this SAP model.

[Independent and joint effect of chronotype and sleep duration on self-rated health in medical students].

Objective: To investigate the independent and joint effects of chronotype and sleep duration on self-rated health in medical students. Methods: A cross-sectional study was conducted in 1 526 medical students selected through proportional stratified cluster random sampling from a medical university in Zhejiang province. A questionnaire survey was conducted to collect the information about their basic demographic characteristics, chronotype, sleep duration, and other lifestyle factors such as midnight snack, sedentary behavior, physical activity, meal time, and self-rated health. The independent and joint effects of chronotype and sleep duration on self-rated health were assessed by logistic regression model after controlling for confounding variables. Results: The numbers of the students with evening chronotype, neutral chronotype, and morning chronotype were 664 (43.5%), 442 (29.0%), and 420 (27.5%), respectively. Among the medical students, 42.8% (653) had poor self-rated health. Compared with those with the morning chronotype, the adjusted ORs for those with neutral chronotype and evening chronotype were 1.69 (95%CI: 1.23-2.31) and 2.43 (95%CI: 1.81-3.26), respectively, trend test P<0.001. Compared with those with sleep duration of 8 h or above per night, the adjusted ORs for those with sleep duration of 7 and ≤6 h per night were 1.40 (95%CI: 1.07-1.84) and 2.38 (95%CI: 1.69-3.37), respectively, trend test P<0.001. In the joint effect, compared with those with the morning chronotype and sleep duration of 8 h or above per night, the adjusted OR for those with evening chronotype and sleep duration of ≤6 h per night was 6.53 (95%CI: 3.53-12.09). Conclusions: Both evening chronotype and insufficient sleep were associated with increased odds of poor self-rated health in medical students, and they had joint effects. Therefore, it is necessary to promote early to bed, early to rise and adequate sleep in medical student to maintain their health.

Development and preliminary test of a space-resolved vacuum-ultraviolet spectroscopy in EAST.

The impurity radiation from the divertor region of the EAST tokamak is dominantly in the wavelength range of vacuum ultraviolet (VUV) due to the elevated edge electron temperature. A space-resolved VUV spectroscopy is developed to measure impurity radiation in the divertor region. An eagle-type VUV spectrometer with a focal length of 1 m is adopted in this system, equipped with a spherical grating and a charged-coupled device (CCD) detector. The performance of the VUV spectrometer is preliminarily tested on a synchrotron radiation facility. The wavelength calibration is conducted near 65 nm. It is found that the wavelength range observed by the CCD detector is about 11.07 nm around the central wavelength of about 65 nm. With a linear dispersion of 0.0053 nm/pixel, it is possible to measure the ion temperature lower than 20 eV at the edge region by analyzing the Doppler broadening of a carbon line. These test results show that the performance of the VUV spectrometer is capable of measuring divertor radiation and analyzing the ion temperature of edge impurity ions.

Denitrosylation of nNOS induced by cerebral ischemia-reperfusion contributes to nitrosylation of CaMKII and its inhibition of autophosphorylation in hippocampal CA1.

OBJECTIVE: The aim of this study is to investigate the relation between CaMKII S-nitrosylation and its activation, as well as the underlying mechanism, after global cerebral ischemia-reperfusion. MATERIALS AND METHODS: The rat model of cerebral ischemia-reperfusion was established by four-vessel occlusion of 15 min and reperfusion of different times. nNOS inhibitor 7-nitroindazole (7-NI), exogenous nitric oxide donor GSNO (nitrosoglutathione), or N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 were administered before ischemia. The expressions of S-nitrosylation and phosphorylation of CaMKII and nNOS were detected by biotin switch assay, immunoblotting, and immunohistochemical staining after cerebral ischemia-reperfusion. The survival of hippocampal CA1 pyramidal cells after administration of the three drugs was examined by cresyl violet staining. RESULTS: Following cerebral ischemia-reperfusion, the S-nitrosylation of CaMKII was increased, accompanied by a decrease of phosphorylation, suggesting a decrease of activity (p<0.05). Meanwhile, the phosphorylation and S-nitrosylation of nNOS were notably decreased at the same time point (p<0.05). The administration of 7-NI, GSNO, and MK-801 increased the S-nitrosylation and phosphorylation of nNOS, leading to the attenuation of increased S-nitrosylation and decreased autophosphorylation of CaMKII after cerebral ischemia-reperfusion (p<0.05). Administration of MK-801, GSNO, and 7-NI significantly decreased the neuronal damage in rat hippocampal CA1 caused by cerebral ischemia-reperfusion (p<0.05). CONCLUSIONS: After cerebral ischemia-reperfusion, the decrease of autophosphorylation of CaMKII regulated by its S-nitrosylation may be due to the denitrosylation of nNOS and subsequent NO production. Increasing the phosphorylation of CaMKII by nNOS inhibitor, exogenous NO donor or NMDA receptor antagonist exerted neuroprotective effects against cerebral ischemia-reperfusion injury.

Effects of dietary rapeseed meal inclusion levels on growth performance, organ weight, and serum biochemical parameters in Cherry Valley ducks.

The aim of this study was to investigate the effects of the inclusion levels of different types of rapeseed meal (RSM) on performance, organ weight, and serum biochemical parameters in Cherry Valley ducks in the starter period and grower-finisher period. In Exp. 1, a total of 750 seven-day-old male ducklings were divided into 5 dietary treatments with 6 replicate pens of 25 birds per pen. The starter diets with the inclusion of 0, 5, 10, 15, or 20% of double-low RSM contained 0, 1.37, 2.15, 3.46, or 5.31 µmol glucosinolates (GLS)/g in the finished feed (from day 7 to 21). In Exp. 2, a total of 900 fifteen-day-old male ducklings were divided into 6 dietary treatments with 6 replicate pens of 25 birds per pen. The grower-finisher diets with the inclusion of 0, 5, 10, 15, 20, or 25% of Indian RSM contained 0, 7.67, 15.34, 24.66, 31.21, or 38.44 µmol GLS/g in the finished feed (from day 15 to 42). For ducklings in the starter period (Exp. 1), body weight gain and feed intake decreased linearly as the dietary double-low RSM inclusion level increased at day 7 to 14, while growth rate was not influenced by dietary double-low RSM inclusion levels at day 15 to 21 and day 7 to 21. For ducks in the grower-finisher period (Exp. 2), growth performance decreased linearly as the dietary RSM inclusion level increased from 5 to 20%. In addition, dietary RSM inclusion levels induced liver enlargement in ducklings at day 21 (5 to 20% double-low RSM with 1.37 to 5.31 µmol/g GLS) and thyroid enlargement accompanied by increased serum AST and ALP activities in ducks at day 42 (5 to 15% Indian RSM with 7.67 to 23.66 µmol/g GLS). Therefore, our results indicated that the upper limit of using RSM sources in feed formulation should consider the anti-nutritional factor of GLS content at different stages of duck growth.

Impact of adenovirus-mediated local expression of human tissue factor pathway inhibitor on vascular smooth muscular cell proliferation and apoptosis in the stent-implanted femoral artery of the rabbit.

This study examined the effect of gene transfer of local tissue factor pathway inhibitor (TFPI) on vascular smooth muscle cells (VSMCs) in stent-implanted arteries. Rabbit femoral arteries were balloon-injured, stent-implanted and infected with the replication-defective recombinant adenovirus-mediated TFPI gene (Ad-TFPI) or the beta-galactosidase gene (Ad-LacZ), or treated with saline solution. Expression of TFPI at the site of the stent was confirmed after 3 days using reverse transcription-polymerase chain reaction (RT-PCR). After 7 days, proliferating cells were visualized by immunostaining with antibodies to proliferating cell nuclear antigen (PCNA) and apoptotic cells were detected using the terminal deoxynucleotidyl mediated nick end labelling (TUNEL) technique. Cell proliferation was significantly decreased and apoptosis significantly increased in the media in the Ad-TFPI group compared with the other two groups. In conclusion, Ad-TFPI gene transfer can significantly suppress VSMC proliferation and induce its apoptosis in the media at the site of an implanted stent and may have potential for the treatment of instent re-stenosis.

Ischemic preconditioning negatively regulates plenty of SH3s-mixed lineage kinase 3-Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt.

Activation of Akt/protein kinase B has been recently reported to play an important role in ischemic tolerance. We here demonstrate that the decreased protein expression and phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) underlie the increased Akt-Ser-473 phosphorylation in the hippocampal CA1 subfield in ischemic preconditioning (IPC). Co-immunoprecipitation analysis reveals that Akt physically interacts with Rac1, a small Rho family GTPase required for mixed lineage kinase 3 (MLK3) autophosphorylation, and both this interaction and Rac1-Ser-71 phosphorylation induced by Akt are promoted in preconditioned rats. In addition, we show that Akt activation results in the disassembly of the plenty of SH3s (POSH)-MLK3-Rac1 signaling complex and down-regulation of the activation of MLK3/c-Jun N-terminal kinase (JNK) pathway. Akt activation results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c, and activation of caspase-3. The expression of Fas ligand is also decreased in the CA1 region. Akt activation protects against apoptotic neuronal death as shown in TUNEL staining following IPC. Intracerebral infusion of LY294002 before IPC reverses the increase in Akt phosphorylation and the decrease in JNK signaling activation, as well as the neuroprotective action of IPC. Our results suggest that activation of pro-apoptotic MLK3/JNK3 cascade can be suppressed through activating anti-apoptotic phosphoinositide 3-kinase/Akt pathway induced by a sublethal ischemic insult, which provides a functional link between Akt and the JNK family of stress-activated kinases in ischemic tolerance.

First measurement of the edge charge exchange recombination spectroscopy on EAST tokamak.

An edge toroidal charge exchange recombination spectroscopy (eCXRS) diagnostic, based on a heating neutral beam injection (NBI), has been deployed recently on the Experimental Advanced Superconducting Tokamak (EAST). The eCXRS, which aims to measure the plasma ion temperature and toroidal rotation velocity in the edge region simultaneously, is a complement to the exiting core CXRS (cCXRS). Two rows with 32 fiber channels each cover a radial range from ∼2.15 m to ∼2.32 m with a high spatial resolution of ∼5-7 mm. Charge exchange emission of Carbon VI CVI at 529.059 nm induced by the NBI is routinely observed, but can be tuned to any interested wavelength in the spectral range from 400 to 700 nm. Double-slit fiber bundles increase the number of channels, the fibers viewing the same radial position are binned on the CCD detector to improve the signal-to-noise ratio, enabling shorter exposure time down to 5 ms. One channel is connected to a neon lamp, which provides the real-time wavelength calibration on a shot-to-shot basis. In this paper, an overview of the eCXRS diagnostic on EAST is presented and the first results from the 2015 experimental campaign will be shown. Good agreements in ion temperature and toroidal rotation are obtained between the eCXRS and cCXRS systems.

Improved spatial calibration for the CXRS system on EAST.

A Charge eXchange Recombination Spectroscopy (CXRS) diagnostic system has been developed to measure profiles of ion temperature and rotation since 2014 on EAST. Several techniques have been developed to improve the spatial calibration of the CXRS diagnostic. The sightline location was obtained by measuring the coordinates of three points on each sightline using an articulated flexible coordinate measuring arm when the vessel was accessible. After vacuum pumping, the effect of pressure change in the vacuum vessel was evaluated by observing the movement of the light spot from back-illuminated sightlines on the first wall using the newly developed articulated inspection arm. In addition, the rotation of the periscope after vacuum pumping was derived by using the Doppler shift of neutral beam emission spectra without magnetic field. Combining these techniques, improved spatial calibration was implemented to provide a complete and accurate description of the EAST CXRS system. Due to the effects of the change of air pressure, a ∼0.4° periscope rotation, yielding a ∼20 mm movement of the major radius of observation positions to the lower field side, was derived. Results of Zeeman splitting of neutral beam emission spectra with magnetic field also showed good agreement with the calibration results.

amlC, another amylolytic gene maps close to the amlB locus in Streptomyces lividans TK24.

The region located upstream of the alpha-amylase gene (amlB) of Streptomyces lividans TK24 (Yin et al., 1997) contains a 2978-bp-long ORF divergent from amlB, and designated amlC. amlC Encodes a 993amino acid (aa) protein with a calculated molecular weight of 107.054kDa. On the basis of sequence similarity as well as enzymatic activity, AmlC is likely to belong to the 1, 4-alpha-D-glucan glucanohydrolase family. amlC is transcribed as a unique 3kb leaderless monocistronic mRNA. Primer extension experiments allowed the identification of promoter sequences that do not resemble the typical eubacterial promoter sequences. amlC was successfully disrupted and was mapped at approx. 700kb from a chromosomal end of S. lividans TK24, 100kb on the right of the amplifiable unit AUD1 (Volff et al., 1996). Nevertheless, amlC disruption seemed to be accompanied by extensive rearrangements of the 2500-kb DraI-II fragment of the chromosome.

Cloning and characterization of a new alpha-amylase gene from Streptomyces lividans TK24.

Streptomyces lividans TK24 possesses a very weak amylolytic activity, nevertheless Southern blot analysis carried out at high stringency revealed that this strain does contain a gene strongly related to the well expressed alpha-amylase gene (amlSL) of Streptomyces limosus. To clone this related gene, three genomic banks of S. lividans TK24 were constructed into the multicopy plasmid vector pIJ699 and transformed into the same strain. Two different genes were isolated. One (amlA) has been previously described, whereas the other (amlB) has never been described. Sub-cloning experiments localized amlB to a 3 kb BamHI-NotI fragment that was sequenced. Frame analysis on sequence data revealed the presence of a 1719 bp long open reading frame encoding a 573 amino acid protein of 61214 kDa. Northern blot analysis identified a unique 1.8 kb monocistronic transcript. Primer extension allowed the localization of the transcription start point 108 bp upstream of the translational start codon and demonstrated that the gene was transcribed from a unique typical eubacterial-like promoter. AmlB shares 74.7% amino acid identity with the alpha-amylase of S. limosus and only 27.2% with the amylolytic enzyme encoded by amlA.

Preparations for the motional Stark effect diagnostic on EAST.

Measurement and control of the current profile is essential for high performance and steady state operation of Experimental Advanced Superconducting Tokamak (EAST). For this purpose, a conventional Motional Stark Effect (MSE) diagnostics utilizing photoelastic modulators is proposed and investigated. The pilot experiment includes one channel to verify the feasibility of MSE, whose sightline intersects with Neutral Beam Injection at major radius of R = 2.12 m. A beam splitter is adopted for simultaneous measurements of Stark multiplets and their polarization directions. A simplified simulation code was also developed to explore the Stark splitting spectra. Finally, the filter is optimized based on the viewing geometry and neutral beam parameters.

TGF-ß1 suppression of microRNA-450b-5p expression: a novel mechanism for blocking myogenic differentiation of rhabdomyosarcoma.

Transforming growth factor beta 1 (TGF-ß1) is the most potent inhibitor of myogenic differentiation (MyoD) of rhabdomyosarcoma (RMS); however, the underlying mechanisms of this inhibition remain unclear. In this study, we identified novel TGF-ß1-related microRNAs (miRNAs); among these, miR-450b-5p is significantly regulated by TGF-ß1. We provide evidence that TGF-ß1 exerts it function by suppressing miR-450b-5p. Both in cultured cells and tumor implants, miR-450b-5p significantly arrested the growth of RMS and promoted its MyoD. Utilizing a bioinformatics approach, we identified miR-450b-5p target mRNAs. Among these candidates, only the expression of ecto-NOX disulfide-thiol exchanger 2 (ENOX2) and paired box 9 (PAX9) was augmented by miR-450b-5p knockdown examined by western blot; the engineered inhibition antagonized TGF-ß1-mediated differentiation inhibition. Furthermore, we found that the Smad3 and Smad4 pathways, but not Smad2, are the principal mediator of TGF-ß1 suppression of miR-450b-5p. Taken together, these results suggest that disrupting the TGF-ß1 suppression of miR-450b-5p, or knockdown of ENOX2 and PAX9, are effective approaches in inducing RMS MyoD.