Functional Identification of Regulatory Elements within the Promoter Region of Platelet-Derived Growth Factor 2

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2,the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure.In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor

The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.

Damage identification of beam-like structures with contiguous and distributed damage

Structural health monitoring of existing infrastructure is currently an active field of research, where elaborate experimental programs and advanced analytical methods are used in identifying the current state of health of critical structures. Change of static deflection as the indicator of damage is the simplest tool in a structural health monitoring scenario of bridges that is least exploited in damage identification strategies. In this paper, some simple and elegant equations based on loss of symmetry due to damage are derived and presented for identification of damage in a bridge girder modeled as a simply supported beam using changes in static deflections and dynamic parameters. A single contiguous and distributed damage, typical of reinforced or prestressed concrete structures, is assumed for the structure. The methodology is extended for a base-line-free as well as base-line-inclusive measurement. Measurement strategy involves application of loads only at two symmetric points one at a time and deflection measurements at those symmetric points as well as at the midspan of the beam. A laboratory-based experiment is used to validate the approach. Copyright (c) 2012 John Wiley & Sons, Ltd.

A novel index for identification of weak nodes for reactive compensation to improve voltage stability

In this study, a new reactive power loss index (RPLI) is proposed for identification of weak buses in the system. This index is further used for determining the optimal locations for placement of reactive compensation devices in the power system for additional voltage support. The new index is computed from the reactive power support and loss allocation algorithm using Y-bus method for the system under intact condition and as well as critical/severe network contingencies cases. Fuzzy logic approach is used to select the important and critical/severe line contingencies from the contingency list. The inherent characteristics of the reactive power in system operation is properly addressed while determining the reactive power loss allocation to load buses. The proposed index is tested on sample 10-bus equivalent system and 72-bus practical equivalent system of Indian southern region power grid. The validation of the weak buses identification from the proposed index with that from other existing methods in the literature is carried out to demonstrate the effectiveness of the proposed index. Simulation results show that the identification of weak buses in the system from the new RPLI is completely non-iterative, thus requires minimal computational efforts as compared with other existing methods in the literature.

Ultrasonic Guided Wave Sensor Network Optimization for Monitoring Rivet Line

Guided waves using piezo-electric wafer active sensors (PWAS) is one of the useful techniques of damage detection. Sensor network optimization with minimal network hardware footprint and maximal area of coverage remains a challenging problem. PWAS sensors are placed at discrete locations in order to inspect damages in plates and the idea has the potential to be extended to assembled structures. Various actuator-sensor configurations are possible within the network in order to identify and locate damages. In this paper we present a correlation based approach to monitor cracks emanating from rivet line using a simulated guided wave signal whose sensor is operating in pulse echo mode. Discussions regarding the identification of phase change due to reflections from the crack are also discussed in this paper.

Identification of type and degree of railway ballast fouling using ground coupled GPR antennas

GPR is widely used for ballast fouling identification, however, there are no robust guidelines to find the degree and type of fouling quantitatively. In this study, GPR studies were carried out on model and actual railway tracks using three ground coupled antennas and considering three fouling materials. Three ground coupled antennas viz., 100 MHz, 500 MHz and 800 MHz antennas were used for the initial survey and it was found that the 800 MHz ground coupled antenna is an optimum one to get quality results. Three major fouling materials viz., screened/broken ballast, coal and iron ore were used to construct prototype model sections, which were 1/2 of the actual Indian broad-gauge railway track. A separate model section has been created for each degree and type of fouling and GPR surveys were carried out. GPR study shows that increasing the fouling content results in a decrease in the Electromagnetic Wave (EMW) velocity and an increase in the dielectric constant. EMW velocity of ballast fouled with screened ballast was found to be more than coal fouled ballast and iron ore fouled ballast at any degree of fouling and EMW velocity of iron ore fouled ballast was found to be less than coal and screen ballast fouled ballast. Dielectric constant of iron ore fouled ballast was found to be higher than coal and screen ballast fouled ballast for all degrees of fouling. Average slope of the trend line of screen ballast fouled section is low (25.6 degrees), coal fouled ballast is medium (27.8 degrees) and iron ore fouled ballast is high (47.6 degrees). (C) 2016 Elsevier B.V. All rights reserved.

3D line drawing from point clouds using chromatic stereo and shading

1 PDF document (8 pp., English).-- Contributed to: VSMM'08: 14th International Conference on Virtual Systems and Multimedia (Limassol, Cyprus, Oct 20-25, 2008)

Identification of treatments targeting PMLexpressing breast cancers

The main aim of this project is to find a suitable ATO concentration to combine with already approved chemotherapeutic agents to find that synergistic effect in triple negative breast cancer MDA-MB 231 cell line, as a new strategy to treat the disease.

Larval development and identification of the genus Triglops (Scorpaeniformes: Cottidae)

Prior to Pietsch’s (1993) revision of the genus Triglops, identification of their larvae was difficult; six species co-occur in the eastern North Pacific Ocean and Bering Sea and three co-occur in the western North Atlantic Ocean. We examined larvae from collections of the Alaska Fisheries Science Center and Atlantic Reference Centre and used updated meristic data, pigment patterns, and morphological characters to identify larvae of Triglops forficatus, T. macellus, T. murrayi, T. nybelini, T. pingeli, and T. scepticus; larvae of T. metopias, T. dorothy, T. jordani, and T. xenostethus have yet to be identified and are thus not included in this paper. Larval Triglops are characterized by a high myomere count (42–54), heavy dorsolateral pigmentation on the gut, and a pointed snout. Among species co-occurring in the eastern North Pacific Ocean, T. forficatus, T. macellus, and T. pingeli larvae are distinguished from each other by meristic counts and presence or absence of a series of postanal ventral melanophores. Triglops scepticus is differentiated from other eastern North Pacific Ocean larvae by having 0–3 postanal ventral melanophores, a large eye, and a large body depth. Among species co-occurring in the western North Atlantic Ocean, T. murrayi and T. pingeli larvae are distinguished from each other by meristic counts (vertebrae, dorsal-fin rays, and anal-fin rays once formed), number of postanal ventral melanophores, and first appearance and size of head spines. Triglops nybelini is distinguished from T. murrayi and T. pingeli by a large eye, pigment on the lateral line and dorsal midline in flexion larvae, and a greater number of dorsal-fin rays and pectoral-fin rays once formed.

Identification and characterization of a novel envelope protein in Rana grylio virus

Viral envelope proteins have been proposed to play significant roles in virus infection and assembly. In this study, an envelope protein gene, 53R, was cloned and characterized from Rana grylio virus (RGV), a member of the family Iridoviridae. Database searches found its homologues in all sequenced iricloviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blot detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found an association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R-GFP fusion protein in pEGFP-N3/53R-transfected cells. Furthermore, detergent extraction and Western blot detection confirmed that RGV 53R was associated with virion membrane. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that it may play an important role in virus assembly. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.

Rapid identification of saponins in plant extracts by electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/tandem mass spectrometry

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and liquid chromatography coupled with on-line mass spectrometry (LC/MS/MS) were applied to characterize saponins in crude extracts from Panax ginseng. The MSn data of the [M - H](-) ions of saponins can provide structural information on the sugar sequences of the saccharide chains and on the sapogins of saponins. By ESI-MSn, non-isomeric saponins and isomeric saponins with different aglycones can be determined rapidly in plant extracts. LC/MS/MS is a good complementary analytical tool for determination of isomeric saponins. These approaches constitute powerful analytical tools far rapid screening and structural assignment of saponins in plant extracts. Copyright (C) 2000 John Wiley & Sons, Ltd.

Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides

Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157 bp, including 1512 bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57 kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of Porphyra lines (Rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique AFLP,fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germplasm identification-AFLP) was designed for identification of the 27 Porphyra lines. In addition, 21 specific AFLP markers from 15 Porphyra lines were identified; 6 AFLP markers from 4 Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of the Porphyra lines.

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.