Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

A toll receptor from Chinese shrimp Fenneropenaeus chinensis is responsive to Vibrio anguillarum infection

Toll-like receptors (TLRs) are an evolutionarily ancient family of pattern recognition receptors (PRRs), playing a crucial role in innate immune responses. Here we present a Toll homolog from Chinese shrimp Fenneropenaeus chinensis, designated FcToll. The full-length cDNA of FcToll is 4115 bp including a poly A-tail of 16 bp, encoding a putative protein of 931 amino acids. The predicted protein consists of an extracellular domain with a potential signal peptide, 16 leucine-rich repeats (LRR), two LRR-C-terminal (LRR-CT) motifs, and two LRR-N-terminal (LRR-NT) motifs, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic Toll/Interteukin-IR (TIR) domain of 139 residues. Genomic structure of FcToll gene contains five exons and four introns. Phylogenetic analysis revealed that it belongs to insect-type invertebrate Toll family. Transcripts of FcToll gene were constitutively expressed in various tissues, with predominant level in lymphoid organ. Real-time PCR assays demonstrated that expression patterns of FcToll were distinctly modulated after bacterial or viral stimulation, with significant enhancement after 5 h post-Vibrio anguillorum challenge but markedly reduced levels immediately after white spot syndrome virus (WSSV) exposure. These results suggest that FcToll might be involved in innate host defense, especially against the pathogen V. anguillarum. (c) 2008 Elsevier Ltd. All rights reserved.

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C-18 column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2 x 10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N = 3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain. (C) 2008 Elsevier B.V. All rights reserved.

可集成的制造执行系统

制造执行系统作为企业上层事务处理和低层设备控制系统之间的中间桥梁 ,在生产计划的优化调度、生产过程的改进等方面起着越来越重要的作用 ,并得到工业界广泛的认可和应用。本文总结了制造执行系统的发展历程和存在的问题 ,简要介绍了制造执行系统的功能、信息交互关系 ,以及信息技术对制造执行系统的冲击 ,进而引入工作流管理技术、智能Agent和面向对象技术 ,提出了可集成制造执行系统的体系结构、运行机制和开发方法 ,并对该系统的市场进行了分析与预测。

基于8098单片机的气压伺服及数据采集系统

本文介绍了一种以8098单片机为核心器件的气压伺服及数据来集系统，包括硬件设计，软件编程及测控原理。

AE指数的可靠性研究

Auroral electrojet index (AE) are usually used to quantitatively describe the activity of the geomagnetic field in the polar region. AE is a means to identify the level of a substorm as well. The auroral electrojet indices (AU, AL, and AE) have served well for more than four decades as measures of magnetospheric substorm activity. However, as substorm studies have progressed considerably during the past several years, the accuracy of the present electrojet indices have become an important issue. Thus it is fortunate for us to reexamine and evaluate the accuracy of the present electrojet indices and improve them if necessary. For a better use of the present indices and for future improvement we examine the limitations of the auroral electrojet indices as an accurate quantitative measure of the auroral electrojets and magnetospheric substorms. Some of the limitations arise from the data availability and also from the present simplified scheme in deriving them, but some of them originate in the definition themselves. In the present paper, we analyze the characteristics of aurora, ionospheric current system and the AE index. It is noted that for the pseudo-breakup events the variations of AE ( > 500 nT) clearly show the sudden increase and slow decrease phases. However, aurora does not expand to poleward or equatorward, and the ionospheric currents presents the features of the magnetic convection. We mainly focus on investigating what time the AE could be used to identify the subtorm.

Technika role play oraz gry fabularne na lektoracie języka angielskiego a autonomizacja studentów

Publikacja zawiera wybór 14 artykułów zaprezentowanych w trakcie sympozjum nt. „Autonomizacja studentów a efektywność dydaktyki na poziomie zaawansowanym”, które odbyło się w dn. 4-5 lutego 2002 roku w Uniwersytecie im. Adama Mickiewicza w Poznaniu na zakończenie projektu badawczego pod tym samym tytułem. Autorów łączy innowacyjność podejścia, ukierunkowana na autonomizację studentów. Artykuły obejmują następujące grupy zagadnień: opis stosowanych procedur dydaktycznych, badania i obserwacje efektów tych innowacji, a także wybrane ujęcia teoretyczne. Zbiór stanowi reprezentatywny przegląd aktualnej refleksji i stanu badań empirycznych w szeroko pojętym ujęciu autonomizacyjnym.

Base pairing interaction between 5′- and 3′-UTRs controls icaR mRNA translation in Staphylococcus aureus

UPNa. Instituto de Agrobiotecnología. Laboratorio de Biofilms Microbianos.

Performance Analysis of a WWW Server

This paper addresses the problem of analyzing performance of WWW servers. The web has experienced a phenomenal growth and has become the most popular Internet application. As a consequence of its large popularity, the Internet has suffered from various performance problems, such as network congestion and overloaded servers. These days, it is not uncommon to find servers refusing connections because they are overloaded. Performance has always been a key issue in the design and operation of on-line systems. With regard to Internet, performance is also critical, because users want fast and easy access to all objects (i.e., documents, pictures, audio, and video) available on the net. Thus, it is important to understand WWW performance issues. This paper focuses on the performance analysis of a Web server. Using a synthetic benchmark (WebStone), we analyze three different Web server software running on top of a Windows NT platform and performing some typical WWW tasks.

Simultaneous two-wavelength transmission quantitative phase microscopy with a color camera.

We present a quantitative phase microscopy method that uses a Bayer mosaic color camera to simultaneously acquire off-axis interferograms in transmission mode at two distinct wavelengths. Wrapped phase information is processed using a two-wavelength algorithm to extend the range of the optical path delay measurements that can be detected using a single temporal acquisition. We experimentally demonstrate this technique by acquiring the phase profiles of optically clear microstructures without 2pi ambiguities. In addition, the phase noise contribution arising from spectral channel crosstalk on the color camera is quantified.

Identification of microRNAs expressed in two mosquito vectors, Aedes albopictus and Culex quinquefasciatus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes. RESULTS: We identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection. CONCLUSIONS: Aedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

Variation in use of surveillance colonoscopy among colorectal cancer survivors in the United States.

BACKGROUND: Clinical practice guidelines recommend colonoscopies at regular intervals for colorectal cancer (CRC) survivors. Using data from a large, multi-regional, population-based cohort, we describe the rate of surveillance colonoscopy and its association with geographic, sociodemographic, clinical, and health services characteristics. METHODS: We studied CRC survivors enrolled in the Cancer Care Outcomes Research and Surveillance (CanCORS) study. Eligible survivors were diagnosed between 2003 and 2005, had curative surgery for CRC, and were alive without recurrences 14 months after surgery with curative intent. Data came from patient interviews and medical record abstraction. We used a multivariate logit model to identify predictors of colonoscopy use. RESULTS: Despite guidelines recommending surveillance, only 49% of the 1423 eligible survivors received a colonoscopy within 14 months after surgery. We observed large regional differences (38% to 57%) across regions. Survivors who received screening colonoscopy were more likely to: have colon cancer than rectal cancer (OR = 1.41, 95% CI: 1.05-1.90); have visited a primary care physician (OR = 1.44, 95% CI: 1.14-1.82); and received adjuvant chemotherapy (OR = 1.75, 95% CI: 1.27-2.41). Compared to survivors with no comorbidities, survivors with moderate or severe comorbidities were less likely to receive surveillance colonoscopy (OR = 0.69, 95% CI: 0.49-0.98 and OR = 0.44, 95% CI: 0.29-0.66, respectively). CONCLUSIONS: Despite guidelines, more than half of CRC survivors did not receive surveillance colonoscopy within 14 months of surgery, with substantial variation by site of care. The association of primary care visits and adjuvant chemotherapy use suggests that access to care following surgery affects cancer surveillance.

Immunization with cocktail of HIV-derived peptides in montanide ISA-51 is immunogenic, but causes sterile abscesses and unacceptable reactogenicity.

BACKGROUND: A peptide vaccine was produced containing B and T cell epitopes from the V3 and C4 Envelope domains of 4 subtype B HIV-1 isolates (MN, RF, CanO, & Ev91). The peptide mixture was formulated as an emulsion in incomplete Freund's adjuvant (IFA). METHODS: Low-risk, healthy adult subjects were enrolled in a randomized, placebo-controlled dose-escalation study, and selected using criteria specifying that 50% in each study group would be HLA-B7+. Immunizations were scheduled at 0, 1, and 6 months using a total peptide dose of 1 or 4 mg. Adaptive immune responses in16 vaccine recipients and two placebo recipients after the 2nd immunization were evaluated using neutralization assays of sera, as well as ELISpot and ICS assays of cryopreserved PBMCs to assess CD4 and CD8 T-cell responses. In addition, (51)Cr release assays were performed on fresh PBMCs following 14-day stimulation with individual vaccine peptide antigens. RESULTS: 24 subjects were enrolled; 18 completed 2 injections. The study was prematurely terminated because 4 vaccinees developed prolonged pain and sterile abscess formation at the injection site-2 after dose 1, and 2 after dose 2. Two other subjects experienced severe systemic reactions consisting of headache, chills, nausea, and myalgia. Both reactions occurred after the second 4 mg dose. The immunogenicity assessments showed that 6/8 vaccinees at each dose level had detectable MN-specific neutralizing (NT) activity, and 2/7 HLA-B7+ vaccinees had classical CD8 CTL activity detected. However, using both ELISpot and ICS, 8/16 vaccinees (5/7 HLA-B7+) and 0/2 controls had detectable vaccine-specific CD8 T-cell responses. Subjects with moderate or severe systemic or local reactions tended to have more frequent T cell responses and higher antibody responses than those with mild or no reactions. CONCLUSIONS: The severity of local responses related to the formulation of these four peptides in IFA is clinically unacceptable for continued development. Both HIV-specific antibody and T cell responses were induced and the magnitude of response correlated with the severity of local and systemic reactions. If potent adjuvants are necessary for subunit vaccines to induce broad and durable immune responses, careful, incremental clinical evaluation is warranted to minimize the risk of adverse events. TRIAL REGISTRATION: ClinicalTrials.gov NCT00000886.

Secuestro de carbono y nitrógeno y emisiones de óxido nitroso del suelo bajo diferentes sistemas de labranza y secuencias de cultivo en la región semiárida pampeana argentina

El objetivo de esta tesis fue estudiar la influencia de diferentes secuencias de cultivos soja - maíz (sj - mz) y soja - soja (sj - sj), y sistemas de labranza, siembra directa con cultivo de cobertura (SDcc) y labranza reducida (LR); sobre los stocks de carbono orgánico total (COT) y nitrógeno total (Nt) y las emisiones de óxido nitroso (N2O) del suelo en condiciones de campo. El experimento está situado en Manfredi, Córdoba, Argentina. El clima de la región es semiárido y el suelo del experimento es un Haplustol éntico. Luego de 13 años se realizó un muestreo de suelo hasta 100 cm de profundidad y se analizó densidad aparente y composición química del suelo. Las emisiones de N2O se midieron durante un año a campo. Los stocks de COT y Nt comparado con LR y los tratamientos bajo sj-sj los menores stocks, tendencia que se mantuvo hasta los 100 cm. La siembra directa promueve la acumulación de COT y Nt y el efecto es mayor cuanto mayor es la inclusión de gramíneas en la secuencia. Respecto de las emisiones de N2O en general variaron considerablemente con el tiempo y las tasas más altas se asocian acon períodos de altos contenidos de agua y nitratos en el suelo. La aplicación de fertilizante nitrogenado en maíz aumentó las emisiones de N2O del suelo. Las emisiones anuales de N - N2O medidas fueron inferiores a las calculadas en base a Intergovernmental Panel on Climate Change (IPCC), esta metodología sobreestimó las emisioens de N2O, para las condiciones estudiadas.