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The conversion of D-fructose to 5-hydroxymethylfurfural (HMF) on a 1 mmol scale was achieved in good yield (68%) using NH(4)Cl as catalyst in isopropanol at 120°C. About 3% of 5-i-propoxymethylfurfural was formed. The reaction in ethanol at 100°C on a 10g scale gave a total yield of HMF and 5-ethoxymethylfurfural of 42%. No mineral acid such as H(2)SO(4) and HCl are required.

To investigate the effect of preoperative induction chemotherapy on treatment of recurrent liposarcoma. 21 patients with recurrent liposarcoma received the treatment of preoperative intra-arterial chemotherapy and surgical resection. Intra-arterial chemotherapy was given by subcutaneous implantable drug delivery system with infusion of cisplatin and doxorubicin followed by caffeine. After treatment, patients were followed up for 39 months. The liposarcoma changes in CT imaging were observed in 18 cases and there were 15 cases with medium or severe pathological changes caused by chemotherapy. At the end of the postoperative follow-up of 39 months, liposarcoma reoccurred locally in 2 cases; pulmonary metastasis occurred in 1 case and death in 3 cases. Preoperative intra-arterial chemotherapy is effective for highly malignant tumors such as recurrent liposarcoma and the judgment of prognosis is based on the postoperative pathological changes of such tumor.

In order to quantify energy saving effect of existing building envelope reconstruction, the paper makes the envelope reconstruction project of Jinan Lixia Office Building as a case, uses energy consumption simulation software DeST-C to make dynamic simulation analysis on several aspects such as natural room temperature, cold and hot load before and after reconstruction of experimental building in cold area. After reconstruction, accumulative hot load of building obtained by calculation decreases 33%, accumulative cold load decreases 15.67%, total load decreases 21.04% in the whole year, and the results show energy saving reconstruction effect of existing building envelope is remarkable.

To investigate the relativity of MGMT(O-6-methylguanine-DNA methyltransferase) gene methylation from patients with protein expression and osteosarcoma necrosis rate after chemotherapy. Fifty-one oteosarcoma tissues were collected, Methylation of MGMT gene promoter was detected by methylation-specific PCR method, and protein expression of MGMT was examined by immunohistochemistry procedure, the relationship between methylated MGMT gene expression and patients response to chemotherapy was analyzed. The positive ratio of methylation MGMT gene promoter in 51 patients was 23.5% (12 in 51). Negative percentage of protein expression of MGMT was 27.5% (14 in 51). It seemed that methylation of MGMT gene in osteosarcoma tissues had no evident relationship with the patient’s age, sexuality, and the size and type of neoplasms, etc. The necrosis rates of methylated MGMT of osteosarcoma (tumor grade from I to IV) were 0 (0/51), 3.9% (2/51), 5.9% (3/51), 13.7% (7/51), respectively. In contrast, the necrosis rates of unmethylated MGMT of osteosarcoma (tumor grade from I to IV) were 45.1% (23/51), 25.5% (13/51), 3.9% (2/51), 2.0% (1/51), respectively. It suggest that methylated and unmethylated MGMT gene of osteosarcoma have significant difference in protein expression. The unmethylated MGMT gene has higher positive protein expression (u = −4.92, P

This paper presents a Bayesian denoising method based on an anisotropic Markov Random Field (MRF) model in wavelet domain in order to improve the image denoising performance and reduce the computational complexity. The classical single-resolution image restoration method using MRFs and the maximum a posteriori (MAP) estimation is extended to the wavelet domain. To obtain the accurate MAP estimation, a novel anisotropic MRF model is proposed under this framework. As compared to the simple isotropic MRF model, this new model can capture the intrascale dependencies of wavelet coefficients significantly better. Simulation results demonstrate our proposed method has a good denoising performance while reducing the computational complexity.

The effect of the limb salvage of the treatment of Osteosarcoma Consolidated with Obvious Pathological Fractures is not very well, the purpose of this paper is to track the efficacy of limb salvage treatment when the patients accepted the artery intervention chemotherapy and enclosed 2 clinical case-reports. From January 2003 to September 2005, 2 clinical cases which one is a male, 29 years old, was confirmed osteosarcoma on the left distal femur, and the other is a female, 15 years old and has osteosarcoma on the right arm with obvious pathological fracture. After receiving arterial chemotherapy pump embedded, then started chemotherapy after the tumor biopsy, and the next process prosthesis replacement in limb salvage surgery after 5 times chemotherapy, follow on the next 5 times chemotherapy after the surgery. With an average follow-up 70 months, there are no postoperative infection and prosthesis loosening found, also didn’t detected tumor recurrence and metastasis, and the limb function recovered well. With the effective, neoadjuvant chemotherapy and comprehensive treatment, salvage treatment is not the contraindication of the patients with pathological fractures of combined primary osteosarcoma, and the treatment with long-term follow-up effectively.

In this paper, the stability of systems with interval time-varying delay is investigated. The time delay varies in an interval. By employing a new and tighter integral inequality and constructing an appropriate type of Lyapunov functional, the delay-dependent stability criteria are derived. Because neither any model transformation nor free weighting matrices are employed in the theoretical derivation, the developed stability criteria significantly improve and simplify the existing stability conditions.

To study the inhibitory effect of Hepatocyte growth factor (HGF) on the responsive hyperplasia of damaged astrocytes in vitro. We prepared damaged model of astrocytes to simulate the responsive hyperplasia of damaged astrocytes in vivo by culturing astrocytes in vitro; After the first day of Ad-HGF transfection, astrocytes were scratched, then after the first, the third, and the fifth day of scratch, we detect the expression amount of astrocytes specific glial fibrillary acidic protein (GFAP) and the ratio of S-phase cells with flow cytometry, both of which can reflect the proliferation status of damaged astrocytes; After HGF was added in scratched astrocytes, the activity of SPK and MAPK (P42/44) were detected by autoradiography and immunoblotting test; After adding different concentrations of HGF protein in astrocytes cultured in different serum concentrations and adding diverse concentrations of HGF protein, SPK and SPK inhibitor DMS in scratched astrocytes, we detect cell proliferation with 3H-TDR incorporation. The first day after Ad-HGF transfected astrocytes were scratched, the amount of GFAP secreted by astrocytes were decreased significantly (P < 0.05), and the cells in S phase were declined obviously. HGF has bidirectional regulation on SPK of scratched astrocytes: increases the SPK activity when HGF in low dose, while inhibits when in high dose. In addition, DMS can block the signal passage; HGF had no effects on MAPK (P42/44) of damaged astrocytes cells. In conclusion, after the transfection of Ad-HGF, it can inhibit the responsive hyperplasia of damaged astrocytes by the means of blocking SPK passage.

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which has been postulated to play a role in chromosomal organization in Archaea. Ssh10b is capable of significantly constraining negative DNA supercoils at elevated temperatures. In this study, the solution structure of the dimeric P62A mutant Ssh10b ([P62A]Ssh10b) was determined by multidimensional NMR spectroscopy. The backbone 15N dynamics, H/D exchange with and without the denaturant GdmSCN, and chemical and thermal denaturation experiments were performed to investigate the molecular basis of high thermostability of [P62A]Ssh10b. Data analysis has revealed an alpha/beta-hydrophobic core consisting of two alpha-helices and one beta-sheet which are stabilized by cooperative hydrophobic and hydrogen-bonding interactions. This stabilizing alpha/beta-hydrophobic core of [P62A]Ssh10b exhibiting highly restricted internal motions is composed of residues having highly protected amide protons which exchange with solvent mostly by means of a global unfolding process. The K40N mutation greatly destabilizes the mutant [P62A]Ssh10b because this mutation disturbs the packing of alpha-helix against the beta-sheet reducing the stability of the alpha/beta-hydrophobic core in the mutant protein. In comparison with homologous mesophilic and thermophilic proteins, it can be presumed that the stabilizing alpha/beta-hydrophobic core in the [P62A]Ssh10b structure greatly contributes to the high thermostability of the protein.

Applying the atom-bond electronegativity equalization method (ABEEM) to metallobiomolecules, the ABEEM parameters for transition metals (V, Cr, Mn, Fe, Co, Ni, Cu, and Zn) were calibrated through linear regression and least-squares optimization by choosing more than 300 training molecules. The quality of the ABEEM charge calculated in terms of the optimized electronegativity and hardness parameters for the training set is assessed by comparison with B3LYP/6-31G* charges. For a check, the ABEEM charges of some large metallobiomolecules have been calculated, and the obtained results correlate quite well to those calculated with the B3LYP/6-31G* method. The linear correlation coefficients R are all over 0.98. This shows that the ABEEM method can predict the charge distributions of large metallobiomolecules with high accuracy.

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future.

A yeast-like fungus, termed strain SD301, with the ability to produce a high concentration of squalene, was isolated from Shuidong Bay, China. The nucleotide sequence analysis of the internal transcribed spacer (ITS) region of SD301 indicated the strain belonged to Pseudozyma species. The highest biomass and squalene production of SD301 were obtained when glucose and yeast extracts were used as the carbon and nitrogen sources, respectively, with a C/N ratio of 3. The optimal pH and temperature were 6 and 25 °C, with 15 g L(-1) of supplemented sea salt. The maximum squalene productivity reached 0.039 g L(-1) h(-1) in batch fermentation, while the maximum squalene yield of 2.445 g L(-1) was obtained in fed-batch fermentation. According to our knowledge, this is the highest squalene yield produced thus far using fermentation technology, and the newly isolated strain Pseudozyma sp. SD301 is a promising candidate for commercial squalene production.

The mechanism, physiological relevance and evolutionary implication of selective RNA processing and stabilization (SRPS) remain elusive. Here we report the genome-wide maps of transcriptional start sites (TSs) and post-transcriptional processed sites (PSs) for Clostridium cellulolyticum. The PS-associated genes are preferably associated with subunits of heteromultimeric protein complexes, and the intergenic PSs (iPSs) are enriched in operons exhibiting highly skewed transcript-abundance landscape. Stem-loop structures associated with those iPSs located at 3′ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. In the cellulosome-encoding cip-cel operon, iPSs and stem-loops precisely regulate structure and abundance of the subunit-encoding transcripts processed from a primary polycistronic RNA, quantitatively specifying cellulosome stoichiometry. Moreover, cellulosome evolution is shaped by the number, position and biophysical nature of TSs, iPSs and stem-loops. Our findings unveil a genome-wide RNA-encoded strategy controlling in vivo stoichiometry of protein complexes., Selective RNA processing and stabilization (SRPS) can regulate bacterial operons, but the process is not well understood. Here, the authors show that the stoichiometry of cellulosome, a 12-subunit protein complex expressed from an operon in Gram-positive Clostridium cellullolyticum, is regulated by SRPS.

To observe the clinical effect of steel cable or greater trochanter reattachment (GTR) device combined with cemented hip hemiarthroplasty for unstable intertrochanteric fracture in elderlies. From July 2002 to June 2014, a total of 57 elderly patients with unstable intertrochanteric fracture, including 23 males and 34 females, were treated. Their ages ranged from 80 to 95 years, with the average of 83 years. According to Evans-Jensen classification, there were 18 type IIa cases, 13 type IIb cases and 26 type III cases. All patients received cemented bipolar femoral head replacement, using steel cable or GTR device to stabilize the unstable intertrochanteric fracture. All patients had successful operation procedure and were followed up for 36 months. Postoperative X-ray revealed satisfying postoperative position of artificial hip joint, without subsidence or loosening. Three cases with the use of steel cable alone to treat greater trochanter fracture suffered from rupture of steel cable. The patients using GTR device showed good reduction at the site of displaced greater trochanter fracture and a firm fixation. The clinical outcome measured with Harris hip score and Barthel Index at the time of final follow-up was significantly different between the groups. Hip hemiarthroplasty for elderly patients with unstable intertrochanteric fracture can meet the load bearing requirement at early stage and reduce postoperative complications prominently. Moreover, GTR devices can effectively solve the instability problem of posterior-lateral side of hip caused by displacement of greater trochanter in unstable intertrochanteric fracture.

Objective: The feasibility of intra-arterial chemotherapy for local recurrent extremity osteosarcoma with prosthesis replacement. Methods: 9 local recurrent limb osteosarcoma patients with prosthesis replacement who received treatment during the period from April 2010 to June 2013 at our hospital were recruited, including 3 patients with lung metastasis. All patients were treated with intra-arterial chemotherapy via implanted pump at affected limb. Results: The local recurrent neoplasms calcified and formed clear boundary with normal tissues after 3 to 5 cycles intra-arterial intervention chemotherapy, which indicates the osteosarcoma was controlled effectively to facilitate the osteosarcoma excision. No local recurrence happened during the follow-up visit varied from 12 to 38 months. The late result was better in 6 patients without lung metastasis than 3 patients with lung metastasis, 2 of who died and 1 currently survive without tumor. Conclusion: Intra-arterial intervention chemotherapy could effectively curb the progress of local recurrent osteosarcoma to facilitate the subsequent limb salvage treatment, and prolong the lifetime of patients with lung metastasis.

Ancient DNA was extracted from 13 skeletal remains from the burial groups of Khitan nobles, which were excavated in northeast China. The hypervariable segment I sequences (HVS I) of the mitochondrial DNA control region, in the 13 individuals, were used as genetic markers to determine the genetic relationships between the individuals and the genetic affinity to other interrelated populations by using the known database of mtDNA. Based on the phylogenetic analysis of these ancient DNA sequences, the genetic structures of two Khitan noble kindreds were obtained, including the Yel Yuzhi's kindred and the Xiao He's kindred. Furthermore, the relationships between the Khitan nobles and some modern interrelated populations were analyzed. On the basis of the result of the analysis, the gene flows of the ancient Khitans and their demographic expansion in history was deduced.

We report the three-dimensional structure of YggX from Salmonella enterica, determined by solution nuclear magnetic resonance (NMR) spectroscopy from protein labeled with carbon-13 and nitrogen-15 produced by Escherichia coli cells. The protein has a β1β2α1α2α3 fold that is unique to YggX and one of its homologs, a protein from Pseudomonas aeruginosa with 45% sequence identity whose X-ray structure [Protein Data Bank (PDB) 1T07] was determined by a structural genomics center. The NMR structure, which revealed that the C-terminal region of YggX is dynamically disordered, explains why electron density from the corresponding region was missing in the X-ray structure of the Pseudomonas protein. Because it has been hypothesized that YggX has a role in iron trafficking, we investigated the influence of Fe(II) on the 1H-15N NMR fingerprint region of nitrogen-15-labeled YggX. Several signals shifted or broadened upon the addition of excess Fe(II) under anoxic conditions, with His81 showing the largest effect. These results indicate that Fe(II) binds weakly to this protein at a region of the sequence conserved only in the subset of the YggX proteins from organisms similar to Salmonella. The finding that iron binds only weakly to YggX, and not to a highly conserved region of the structure, suggests that the role of this protein in iron homeostasis is more complex than previously thought. Proteins 2006. © 2005 Wiley-Liss, Inc.

Targetrons are mobile group II introns that can recognize their DNA target sites by base-pairing RNA-DNA interactions with the aid of site-specific binding reverse transcriptases. Targetron technology stands out from recently developed gene targeting methods because of the flexibility, feasibility, and efficiency, and is particularly suitable for the genetic engineering of difficult microorganisms, including cellulolytic bacteria that are considered promising candidates for biomass conversion via consolidated bioprocessing. Along with the development of the thermotargetron method for thermophiles, targetron technology becomes increasingly important for the metabolic engineering of industrial microorganisms aiming at biofuel/chemical production. To summarize the current progress of targetron technology and provide new insights on the use of the technology, this paper reviews the retrohoming mechanisms of both mesophilic and thermophilic targetron methods based on various group II introns, investigates the improvement of targetron tools for high target efficiency and specificity, and discusses the current applications in the metabolic engineering for bacterial producers. Although there are still intellectual property and technical restrictions in targetron applications, we propose that targetron technology will contribute to both biochemistry research and the metabolic engineering for industrial productions.

To assess the survival and prognostic significance of various demographic and radiographic parameters for conversion into total hip arthroplasty after treatment with a modified porous tantalum implant technology for early and intermediate stages of osteonecrosis of the femoral head (ONFH).This study included 45 patients (59 hips) with Steinberg Stage I-IV A ONFH undergoing progressively core decompression, impaction bone grafting of 5 mm-composite bone filling material and inserting of a porous tantalum implant. Weight-bearing was forbidden within the first 3 months after implants.A total of 57 hips (44 patients) were available during a mean follow-up period of 44.8 (11-62) months. Their mean age was 43 (21-70) years. The mean Harris hip score significantly improved from 59.93 ± 2.80 preoperative to 77.84 ± 2.95 at the last follow-up (P0.001). Overall, 11 hips (19.30%) were converted into total hip arthroplasty. The overall survival rate was 72.49% at 60 months postoperatively. The Cox proportional hazard model revealed that bone marrow edema was an independent prognostic factor related with a conversion into total hip arthroplasty.Higher survival rates may be obtained from modified tantalum implant technology for early and intermediate stages of ONFH. And prognosis was poor for patients of ONFH with bone marrow edema.

Background Clostridium cellulolyticum and other cellulolytic Clostridium strains are natural producers of lignocellulosic biofuels and chemicals via the consolidated bioprocessing (CBP) route, and systems metabolic engineering is indispensable to meet the cost-efficient demands of industry. Several genetic tools have been developed for Clostridium strains, and an efficient and stringent inducible genetic operation system is still required for the precise regulation of the target gene function. Results Here, we provide a stringent arabinose-inducible genetic operation (ARAi) system for C. cellulolyticum, including an effective gene expression platform with an oxygen-independent fluorescent reporter, a sensitive MazF-based counterselection genetic marker, and a precise gene knock-out method based on an inducible ClosTron system. A novel arabinose-inducible promoter derived from Clostridium acetobutylicum is employed in the ARAi system to control the expression of the target gene, and the gene expression can be up-regulated over 800-fold with highly induced stringency. The inducible ClosTron method of the ARAi system decreases the off-target frequency from 100% to 0, which shows the precise gene targeting in C. cellulolyticum. The inducible effect of the ARAi system is specific to a universal carbon source L-arabinose, implying that the system could be used widely for clostridial strains with various natural substrates. Conclusions The inducible genetic operation system ARAi developed in this study, containing both controllable gene expression and disruption tools, has the highest inducing activity and stringency in Clostridium by far. Thus, the ARAi system will greatly support the efficient metabolic engineering of C. cellulolyticum and other mesophilic Clostridium strains for lignocellulose bioconversion. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0214-2) contains supplementary material, which is available to authorized users.

Dockerin modules of the cellulosomal enzyme subunits play an important role in the assembly of the cellulosome by binding tenaciously to cohesin modules of the scaffoldin subunit. A previously reported NMR-derived solution structure of the type-I dockerin module from Ce148S of Clostridium thermocellum, which utilized two-dimensional homonuclear H-1-H-1 NOESY and three-dimensional N-15-edited NOESY distance restraints, displayed substantial conformational differences from subsequent structures of dockerin modules in complex with their cognate cohesin modules, raising the question whether the source of the observed differences resulted from cohesin-induced structural rearrangements. Here, we determined the solution structure of the Ce148S type-I dockerin based on N-15- and C-13-edited NOESY-derived distance restraints. The structure adopted a fold similar to X-ray crystal structures of dockerin modules in complex with their cohesin partners. A unique cis-peptide bond between Leu-65 and Pro-66 in the Ce148S type-I dockerin module was also identified in the present structure. Our structural analysis of the Ce148S type-I dockerin module indicates that it does not undergo appreciable cohesin-induced structural alterations but rather assumes an inherent calcium-dependent cohesin-primed conformation. (C) 2014 Elsevier Inc. All rights reserved.

Xinjiang is at the crossroads between East and West Eurasia, and it harbors a relatively complex genetic history. In order to better understand the population movements and interactions in this region, mitochondrial and Y chromosome analyses on 40 ancient human remains from the Tianshanbeilu site in eastern Xinjiang were performed. Twenty-nine samples were successfully assigned to specific mtDNA haplogroups, including the west Eurasian maternal lineages of U and W and the east Eurasian maternal lineages of A, C, D, F, G, Z, M7, and M10. In the male samples, two Y chromosome haplogroups, C* and N1 (xN1a, N1c), were successfully assigned. Our mitochondrial and Y-chromosomal DNA analyses combined with the archaeological studies revealed that the Di-qiang populations from the Hexi Corridor had migrated to eastern Xinjiang and admixed with the Eurasian steppe populations in the early Bronze Age.

Y chromosome haplogroup Q1a1 is found almost only in Han Chinese populations. However, it has not been found in ancient Han Chinese samples until now. Thus, the origin of haplogroup Q1a1 in Han Chinese is still obscure. This study attempts to provide answer to this question, and to uncover the origin and paternal genetic structure of the ancestors of the Han Chinese.Eighty-nine ancient human remains that were excavated from the presumed geographic source of the Han Chinese and dated to approximately 3,000 years ago were treated by the amelogenin gene polymerase chain reaction test, to determine their sex. Then, Y chromosome single nucleotide polymorphisms were subsequently analyzed from the samples detected as male.Samples from 27 individuals were successfully amplified. Their haplotypes could be attributed to haplogroups N, O*, O2a, O3a, and Q1a1. Analyses showed that the assigned haplogroup of each sample is correlated to the suspected social status and observed burial custom associated with the sample.The origins of the observed haplotypes and their distribution in present day Han Chinese and in the samples suggest that haplogroup Q1a1 was probably introduced into the Han Chinese population approximately 3,000 years ago.

The objective of this study was to explore the O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2′-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT–PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells’ ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

Clostridium includes a number of species, such as thermophilic Clostridium thermocellum and mesophilic Clostridium cellulolyticum, producing biofuels and chemicals from lignocellulose, while genetic engineering is necessary to improve wild-type strains to fulfill the requirement of industrialization. ClosTron system is widely used in the gene targeting of Clostridium because of its high efficiency and operability. However, the targetron plasmid present in cell hinders the successive gene disruption. To solve this problem, a pyrF-based screening system was developed and implemented in C. cellulolyticum strain H10 in this study for efficient targetron plasmid curing. The screening system was composed of a pyrF-deleted cell chassis (H10ΔpyrF) constructed via homologous recombination and a PyrF expression cassette located in a targetron plasmid containing an erythromycin resistance gene. With the screening system, the gene targeting could be achieved following a two-step procedure, including the first step of gene disruption through targetron transformation and erythromycin selection and the second step of plasmid curing by screening with 5-fluoroorotic acid. To test the developed screening system, successive inactivation of the major cellulosomal exocellulase Cel48F and the scaffoldin protein CipC was achieved in C. cellulolyticum, and the efficient plasmid curing was confirmed. With the assistance of the pyrF-based screening system, the targetron plasmid-cured colonies can be rapidly selected by one-plate screening instead of traditional days' unguaranteed screening, and the successive gene disruption becomes accomplishable with ClosTron system with improved stability and efficiency, which may promote the metabolic engineering of Clostridium species aiming at enhanced production of biofuels and chemicals.

Introduction The molecular links between shock-response and adaptation remain poorly understood, particularly for extremophiles. This has hindered rational engineering of solvent tolerance and correlated traits (e.g., productivity) in extremophiles. To untangle such molecular links, here we established a model that tracked the microevolution from shock to adaptation in thermophilic bacteria. Method Temporal dynamics of genomes and transcriptomes was tracked for Thermoanaerobacter sp. X514 which under increasing exogenous ethanol evolved from ethanol-sensitive wild-type (Strain X) to tolerance of 2%- (XI) and eventually 6%-ethanol (XII). Based on the reconstructed transcriptional network underlying stress tolerance, genetic engineering was employed to improve ethanol tolerance and production in Thermoanaerobacter. Results The spontaneous genome mutation rate (μg) of Thermoanaerobacter sp. X514, calculated at 0.045, suggested a higher mutation rate in thermophile than previously thought. Transcriptomic comparison revealed that shock-response and adaptation were distinct in nature, whereas the transcriptomes of XII resembled those of the extendedly shocked X. To respond to ethanol shock, X employed fructose-specific phosphotransferase system (PTS), Arginine Deiminase (ADI) pathway, alcohol dehydrogenase (Adh) and a distinct mechanism of V-type ATPase. As an adaptation to exogenous ethanol, XI mobilized resistance-nodulation-cell division (RND) efflux system and Adh, whereas XII, which produced higher ethanol than XI, employed ECF-type ϭ24, an alcohol catabolism operon and phase-specific heat-shock proteins (Hsps), modulated hexose/pentose-transport operon structure and reinforced membrane rigidity. Exploiting these findings, we further showed that ethanol productivity and tolerance can be improved simultaneously by overexpressing adh or ϭ24 in X. Conclusion Our work revealed thermophilic-bacteria specific features of adaptive evolution and demonstrated a rational strategy to engineer co-evolving industrial traits. As improvements of shock-response, stress tolerance and productivity have been crucial aims in industrial applications employing thermophiles, our findings should be valuable not just to the production of ethanol but also to a wide variety of biofuels and biochemicals.

nAbstract: OPEN ACCESS Background: Targetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a llribozymerr) and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity. Methodology/Principal Findings: Here, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67n100% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate. Conclusions/Significance: Thermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles. n

The indolocarbazole (ICZ) alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as a probe to isolate the 34.6-kb DNA region containing the spc gene cluster. Sequence analysis revealed genes for ICZ ring formation (spcO, D, P, C), sugar unit formation (spcA, B, E, K, J, I), glycosylation (spcN, G), methylation (spcMA, MB), as well as regulation (spcR). Their involvement in ICZ biosynthesis was confirmed by gene inactivation and heterologous expression in Streptomyces coelicolor M1152. This work represents the first cloning and characterization of an ICZ gene cluster isolated from a marine-derived actinomycete strain and would be helpful for thoroughly understanding the biosynthetic mechanism of ICZ glycosides.

BACKGROUND: Fucoidan, a sulfated polysaccharide extracted from brown algae, possesses potent antiinflammatory effects. AIMS: To examine the effect of fucoidan treatment on inflammation‐mediated dopaminergic neuronal damage and its potential mechanisms. METHODS: Microglial activation and injury of dopaminergic neurons were induced by intranigral injection of lipopolysaccharide (LPS), and the effects of fucoidan treatment on animal behavior, microglial activation and survival ratio of dopaminergic neurons were investigated. We further observed the efficacy of fucoidan on tumor necrosis factor‐alpha (TNF‐α) and the production of reactive oxygen species (ROS) in LPS‐activated primary microglia. RESULTS: Fucoidan significantly improved the behavioral manifestation, prevented the loss of dopaminergic neurons and inhibited the deleterious activation of microglia in the substantia nigra pars compacta of LPS‐treated rats. Further in vitro experiments indicated that the excessive production of TNF‐α and ROS in LPS‐induced primary microglia were significantly inhibited by fucoidan administration. CONCLUSION: This is the first study to demonstrate that fucoidan possesses neuroprotective effects on injured dopaminergic neurons in a LPS‐induced animal model of Parkinson's disease. The mechanisms underlying these effects may include its potent down‐regulation of intracellular ROS and subsequent proinflammatory cytokine release in LPS‐activated microglia.

a b s t r a c t To understand the mechanism of lipid accumulation and the corresponding metabolic changes of the microalga Nannochloropsis oceanica IMET1, the lipid content, fatty acid composition and metabolic profile were investigated via batch culture under nutrient deficiency and chemostatic culture under nitrate limitation. The results indicated that the triacylglycerol-neutral lipids were significantly accumulated through an acyl-CoA dependent pathway, while the polar lipids were partially converted to triacylglycerol through an acyl-CoA independent pathway. The fatty acid compositions of the polar lipids changed concurrently with the length of time of the nutrient deficiency, while the fatty acid compositions of the neutral lipids remained nearly consistent. The concentrations of several major osmolytes were significantly changed under chemostatic conditions with different nitrogen concentrations, which reflect the membrane property changes caused by the alteration of the polar lipid composition.

To investigate the clinical efficacy of intra-arterial chemotherapy by subcutaneous implantable delivery system (SIDS) in the treatment of distal femur neoplasm. From March 2002 to December 2009, 51 patients were treated with SIDS intra-femoral artery chemotherapy, followed by customized prosthetic reconstruction, including 45 patients of osteosarcoma and 6 malignant fibrous histiocytoma in distal femur. The average follow-up period was 64 months (ranging between 24 and 116 months) to track on the efficacy of chemotherapy, which shows that 96.1 % of patients got pain relief, 70.6 % of patients had significant radiological change, and 82.4 % of paitents with medium to severe pathological variation responded to the chemotherapy. Local recurrence happened in 4 cases, 3 cases are alive with disease, 4 cases died because of pulmonary metastases and other 40 patients are free of local recurrence or distant metastasis. SIDS intra-femur artery chemotherapy can improve clinical outcome of DFN, and provide effective method for treatment of DFN when combined with customized prosthetic reconstruction.

Cis-epoxysuccinic acid hydrolase (CESH) is an enzyme that catalyzes cis-epoxysuccinic acid to produce enantiomeric L(+)-tartaric acid. The production of tartaric acid by using CESH would be valuable in the chemical industry because of its high yield and selectivity, but the low stability of CESH hampers its application. To improve the stability of CESH, we fused five different carbohydrate-binding modules (CBMs) to CESH and immobilized the chimeric enzymes on cellulose. The effects of the fusion and immobilization on the activity, kinetics, and stability of CESH were compared. Activity measurements demonstrated that the fusion with CBMs and the immobilization on cellulose increased the pH and temperature adaptability of CESH. The chimeric enzymes showed significantly different enzyme kinetics parameters, among which the immobilized CBM30-CESH exhibited twofold catalytic efficiency compared with the native CESH. The half-life measurements indicated that the stability of the enzyme in its free form was slightly increased by the fusion with CBMs, whereas the immobilization on cellulose significantly increased the stability of the enzyme. The immobilized CBM30-CESH showed the longest half-life, which is more than five times the free native CESH half-life at 30 °C. Therefore, most CBMs can improve enzymatic properties, and CBM30 is the best fusion partner for CESH to improve both its enzymatic efficiency and its stability.

To summarize the experience in intraarterial neoadjuvant chemotherapy for extremity osteosarcoma. Between January 2002 and December 2007,111 patients with stage IIB extremity osteosarcoma received preoperative intraarterial therapy with subcutaneous implantation of chemotherapy pump as well as en bloc resection, and postoperative adjuvant chemotherapy. There were 63 males and 48 females with an average age of 18 (range, 14 ~ 39 years). The time from symptom onset to hospitalization varied from several days to 6 months. The induction chemotherapy regimen includes: epirubicin [50 ~ 70 mg/m(2) by 4-hour intraarterial infusion/day for 3 day] and cisplatin [100 ~ 120 mg/m(2) by 2-hour intraarterial infusion/day for 3 days] repetitively every 2 ~ 3 weeks. Among which 24 cases only received two cycles induction chemotherapy was set to nonstandard chemotherapy group and 87 cases received three to six cycles induction chemotherapy set to standard chemotherapy group. The number of preoperative chemotherapy-cycles of standard chemotherapy group depends on the clinical and radiographic evaluation of chemotherapy efficacy. Median follow-up time was 28(8 ~ 48) months. The rate of limb preservation surgery was 89.53% (77/86) in standard chemotherapy group,and was 37.5% (9/24) in nonstandard chemotherapy group. Kaplan-Meier survival analysis showed that the 3-year overall survival rate and disease free survival rate of all the 111 cases were 68.3% and 65.9% respectively. There were significant differences in overall survival rate (38.9%, 80.0%, P = 0.000), disease free survival rate (30.1%, 79.5%, P = 0.000), distant metastasis rate (66.67%, 16.09%, P = 0.0000) and local recurrence rate (58.33%, 13.79%, P = 0.0000) between nonstandard chemotherapy group and standard chemotherapy group. Standard intraarterial neo-adjuvant chemotherapy was more effective than nonstandard intraarterial induction chemotherapy to stage IIB extremity osteosarcoma.

To explore the relationship of motor deficits of the lower extremities with the imaging features of malignant spinal cord compression (MESCCs).From July 2006 through December 2008, 56 successive MESCC patients were treated at our department. All were evaluated by magnetic resonance imaging and computed tomography and were scored according to motor deficits Frankel grading on admission. Imaging assessment factors of main involved vertebrae were level of vertebral metastatic location, epidural space involvement, vertebral body involvement, lamina involvement, posterior protrusion of posterior wall, pedicle involvement, continuity of main involved vertebrae, fracture of anterior column, fracture of posterior wall, location in upper thoracic spine and/or cervicothoracic junction.Occurrence was the same between paralytic state of MESCCs and epidural space involvement of imaging features. Multiple regression equation showed that paralytic state had a linear regression relationship with imaging factors of lamina involvement (X1), posterior protrusion of posterior wall (X2), location in upper thoracic spine and/or cervicothoracic junction (X7) of main involved vertebrae. The optimal regression equation of paralytic state (Y) and imaging feature (X) was Y = -0.009 +0.639X, + 0.149X, +0.282X. Lamina involvement of main involved vertebrae has a greatest influence upon paralytic state of MESCC patients.Imaging factors of lamina involvement, posterior protrusion of posterior wall, location in upper thoracic spine and/or cervicothoracic junction of main involved vertebrae can predict the paralytic state of MESCC patients. MESCC with lamina involvement is more easily encroached on epidural space.

To summarize the experiences in intraarterial neoadjuvant chemotherapy for extremity osteosarcoma.Between January 2002 and December 2007, 111 patients with stage IIB extremity osteosarcoma received preoperative intraarterial chemotherapy after placing chemotherapy pump subcutaneously, en bloc resection and postoperative adjuvant chemotherapy. There were 63 males and 48 females with an average age of 18 years old (range: 14 - 39). The time from symptom onset to hospitalization varied from several days to 6 months. The induction chemotherapy regimen included epirubicin [50 - 70 mg/m(2) by 4-hour intraarterial infusion/day for 3 days] and epirubicin plus adriamycin [100 - 120 mg/m(2) by 2-hour intraarterial infusion/day for 3 days] repetitively every 2 - 3 weeks. Among which 24 cases only received 2 cycles of induction chemotherapy was assigned into the nonstandard chemotherapy group and 87 cases receiving 3 - 6 cycles of induction chemotherapy the standard chemotherapy group. The number of preoperative chemotherapeutical cycles of standard chemotherapy group depended on the clinical and radiographic evaluation of chemotherapy efficacy.The median follow-up time was 28 (8 - 48) months. The rate of extremity preservation surgery was 89.53% (77/86) in the standard chemotherapy group and 37.5% (9/24) in the nonstandard chemotherapy group. Kaplan-Meier survival analysis showed that the 3-year overall survival rate and disease-free survival rate of all 111 cases were 68.3% and 65.9% respectively. There were significant differences in overall survival rate (38.9%, 80.0%, P = 0.000), disease-free survival rate (30.1%, 79.5%, P = 0.000), distant metastatic rate (66.67%, 16.09%, P = 0.0000) and local recurrence rate (58.33%, 13.79%, P = 0.0000) between two groups.Standard intraarterial neo-adjuvant chemotherapy is more effective than nonstandard intraarterial induction chemotherapy to treat stage IIB extremity osteosarcoma.

1. Microglial activation plays an important role in the pathogenesis of neurodegenerative diseases by producing various pro-inflammatory cytokines. Microglia-derived nitric oxide (NO) is critical for the lipopolysaccharide (LPS)-induced selective loss of dopaminergic neurons. 2. Fucoidan is a sulphated polysaccharide extracted from brown seaweeds. It has a variety of biological actions, including anticoagulant, antiviral and anti-inflammatory effects. The aim of the present study was to investigate the effects of fucoidan on LPS-induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. 3. To investigate the effects of fucoidan on LPS-induced cellular activation in microglia, primary microglial cells were preincubated with fucoidan (31.25, 62.5 and 125 microg/mL) for 10 min, followed by stimulation with LPS (0.01 microg/mL). Then, cell shape and NO production were determined 24 h after LPS stimulation, whereas inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined at 6 and 18 h after LPS stimulation, respectively. To evaluate the inhibitory mechanisms involved, mitogen-activated protein kinase (MAPK) activation was also evaluated. 4. Lipopolysaccharide transformed cells into an amoeboid shape, whereas 62.5 microg/mL fucoidan inhibited this activation. Moreover, 125 microg/mL fucoidan significantly inhibited microglial NO production to 75% of that in LPS-treated group and also significantly diminished the expression of iNOS mRNA and protein by nearly 50%. Fucoidan (125 microg/mL) also suppressed phosphorylation of p38 and extracellular signal-regulated kinase (ERK) by approximately 50%, but not that of c-Jun N-terminal kinase. 5. The results provide the first evidence that fucoidan has a potent inhibitory effect against LPS-induced NO production by microglia. The results also suggest that this inhibitory action of fucoidan involves suppression of p38 and ERK phosphorylation.

Fucoidan is a natural polysaccharide extracted from brown seaweeds, with a wide variety of biological features, especially the anti-inflammatory and anti-oxidative effects. Studies indicated that the anti-inflammatory effect of fucoidan related to its capacity to interact with the selectin or scavenger receptor on the cell membrane. Fucoidan can also inhibit the synthesis and release of reactive oxygen species (ROS), as well as promote its clearance, showing the anti-oxidative activity.

Parkinson's disease is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of dopaminergic neurons in substantia nigra pars compacta, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Oxidative stress may contribute to MPTP- and Parkinson's disease-related neurodegeneration. Fucoidan is a sulfated polysaccharide extracted from brown seaweeds which possesses a wide variety of biological activities including potent antioxidative effects. Here we investigated the effect of fucoidan treatment on locomoter activities of animals, striatal dopamine and its metabolites and survival of nigral dopaminergic neurons in MPTP-induced animal model of Parkinsonism in C57/BL mice in vivo and on the neuronal damage induced by 1-methyl-4-phenylpyridinium (MPP(+)) in vitro, and to study the possible mechanisms. When administered prior to MPTP, fucoidan reduced behavioral deficits, increased striatal dopamine and its metabolites levels, reduced cell death, and led to a marked increase in tyrosine hydroxylase expression relative to mice treated with MPTP alone. Furthermore, we found that fucoidan inhibited MPTP-induced lipid peroxidation and reduction of antioxidant enzyme activity. In addition, pre-treatment with fucoidan significantly protected against MPP(+)-induced damage in MN9D cells. Taken together, these findings suggest that fucoidan has protective effect in MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative activity. (C) 2009 Elsevier B.V. All rights reserved.

In this paper, passivity and wave variables are first introduced, and then the reasons of communication time-varying delay are analyzed. For this time-varying delay a new-type time delay identification method based on the cross-correlation technology is proposed and this method can converge the time-varying delay parameter quickly. Finally this method is combined with wave integrals and a control scheme is proposed especially for time-varying delay. The effectiveness of the proposed method has been demonstrated through the simulation results.

The state observer and robust control for uncertain systems with time-varying state delay is studied. A sufficient condition is given for the existence of the state observer with delay and the observer-based robust controller for such uncertain system is also designed. The detail design procedures are presented by solving linear matrix inequalities (LMIs). At last, an illustrative numerical example is presented, which shows the effectiveness of the proposed method.

Assignment of individual compound identities within mixtures of thousands of metabolites in biological extracts is a major challenge for metabolomic technology. Mass spectrometry offers high sensitivity over a large dynamic range of abundances and molecular weights but is limited in its capacity to discriminate isobaric compounds. In this article, we have extended earlier studies using isotopic labeling for elemental composition elucidation (Rodgers, R. P.; Blumer, E. N.; Hendrickson, C. L.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2000, 11, 835-40) to limit the formulas consistent with any exact mass measurement by comparing observations of metabolites extracted from Arabidopsis thaliana plants grown with (I) (12)C and (14)N (natural abundance), (II) (12)C and (15)N, (III) (13)C and (14)N, or (IV) (13)C and (15)N. Unique elemental compositions were determined over a dramatically enhanced mass range by analyzing exact mass measurement data from the four extracts using two methods. In the first, metabolite masses were matched with a library of 11,000 compounds known to be present in living cells by using values calculated for each of the four isotopic conditions. In the second method, metabolite masses were searched against masses calculated for a constrained subset of possible atomic combinations in all four isotopic regimes. In both methods, the lists of elemental compositions from each labeling regime were compared to find common formulas with similar retention properties by HPLC in at least three of the four regimes. These results demonstrate that metabolic labeling can be used to provide additional constraints for higher confidence formula assignments over an extended mass range.

Robust stabilization of uncertain systems with time-varying state delay via output feedback is studied in this paper. The static output feedback controller is designed by solving a set of LMIs based on Lyapunov-Razumkhin approach. Then dynamic output feedback stabilization is transformed into the general static output feedback stabilization. In the controller design procedure there are not any requirements for the derivative of the delay, which allows fast time-varying delay.