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At least 649 records · Page 36Linked to original sources
PubMed2004

Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation.

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.

Biomarkers↗
PubMed2003

A novel method for increasing the expression level of recombinant proteins.

Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.

Amino Acid Sequence↗
PubMed2002

Covert orienting of visuospatial attention in the early stages of aging.

Electrophysiological and behavioral responses were recorded in healthy young (19-23 years) and older (56-66 years) subjects during the execution of a visuospatial attention task. The objective was to test whether covert orienting of visuospatial attention (COVAT) is sensitive to the early stages of aging. All subjects responded faster to targets following valid than invalid cues. The amplitude of the P1 component of visual event-related potentials (ERP) was larger to targets following central valid cues at all SOAs. Subtle age-related changes were observed in P1 amplitude under peripheral cueing. Furthermore, older subjects presented longer reaction times (RTs) and lower P1 amplitudes regardless of the attention condition.

Adult↗
PubMed2006

Pedometer reliability, validity and daily activity targets among 10- to 15-year-old boys.

The aims of this study were to: (1) determine whether the number of pedometer counts recorded by adolescents differs according to the adiposity of the participant or location on the body; (2) assess the accuracy and reliability of pedometers during field activity; and (3) set adolescent pedometer-based physical activity targets. Seventy-eight 11- to 15-year-old Boy Scouts completed three types of activity: walking, fast walking and running. Each type was performed twice. Participants wore three pedometers and one activity monitor during all activities. Participants were divided into groups of normal weight (BMI < 85th percentile) and at risk of being overweight (BMI > or = 85th percentile). Intra-class correlations across the three activities indicated reliability (r = 0.51 - 0.92, P < 0.001). This conclusion was supported by narrow limits of agreement that were within a pre-set range that was practically meaningful. Multivariate analysis of covariance indicated adiposity group differences, but this difference was a function of the increased stature among the larger participants (P < 0.001). Ordinary least-squares regression models and multi-level regression models showed positive associations between the number of pedometer and activity monitor counts recorded by the three groups of participants during all activities (all P < 0.001). The mean number of counts recorded for all participants during the fast walk was 127 counts per minute. In conclusion, the pedometers provided an accurate assessment of adolescent physical activity, and a conservative estimate of 8000 pedometer counts in 60 min is equivalent to 60 min of moderate to vigorous physical activity.

Adolescent↗
PubMedDate not supplied

The role of antisense oligonucleotides in the wave of genomic information.

Technologies which efficiently dissect gene function and validate therapeutic targets are of great value in the post-sequencing era of the human genome project. The antisense oligonucleotide approach can directly use genomic sequence information, in a relatively time and cost effective manner, to define a gene's function and/or validate it as a potential therapeutic target. Antisense oligonucleotide inhibitors of gene expression may be applied to cellular assays (in vitro) or animal models of disease (in vivo). Information generated by this approach may then direct or supplement traditional drug discovery programs, or support development of the antisense oligonucleotide inhibitor, used to validate the target, as a drug.

Animals↗
PubMed2006

Targeted proteomics in biomarker validation: detection and quantification of proteins using a multi-dimensional peptide separation strategy.

Authentic biomarkers, distilling the essence of a complex, functionally significant process in a mammalian system into a precise, physicochemical measurement have been implicated as a tool of increasing importance for drug discovery and development. However, even in spite of recent technological advances, validating a new biomarker candidate, where generation of suitable antibodies is required, is still a long-lasting task. Methods to accelerate initial validation by MS approaches have been suggested, but all methods described so far are associated with serious drawbacks, finally leading to non-generic methods of detection and quantification. Moreover, when complex body fluids are used as samples, efficient debulking strategies are crucial to open a window of analytical sensitivity in the ng/mL range, where many diagnostically relevant analytes are present. Here we report the proof-of-principle of a multi-dimensional strategy for accelerated initial validation of biomarker candidates by MS, which promises to be generally applicable, sensitive and quantitative. The method presented employs a combination of electrophoretic and chromatographic steps on the peptide level, followed by MS quantification using isotopically labeled synthetic peptides as internal standards. Our proposed workflow includes up to four dimensions, finally resulting in a desired LOD sufficient to detect and quantify diagnostically relevant analytes from complex samples. Although the current state of the method only represents a starting point for further validation and development, it reveals great potential in biomarker validation.

Animals↗
PubMedDate not supplied

Identification and validation of candidate Myb target genes.

While a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene--c-myc--that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region.

Cell Line, Transformed↗
PubMed2000

Definition and validation of a reference target volume in early stage node-positive cervical carcinoma, based on lymphangiograms and CT-scans.

PURPOSE: To establish a reference planning target volume for postoperative radiotherapy in stage Ib and IIa N+ cervical carcinoma, based on 47 lymphangiograms and 15 CT-scans. METHODS: Radiation oncologists (n=17) from all radiotherapy institutes in The Netherlands were asked to define the clinical target volume (CTV) and planning target volume (PTV), and to delineate (on simulation films) the radiotherapy treatment portals following a radical hysterectomy with lymph node dissection for an early stage cervical carcinoma with positive iliac lymph nodes. A reference PTV was defined by using 47 normal lymphangiograms and CT-data of the pelvis from 15 patients who underwent surgery for cervical carcinoma. The simulation films were digitized and evaluated for adequacy in covering the PTV, previously individually determined by the radiation oncologists. Subsequently, the simulation films were also evaluated for adequacy in covering the reference PTV. RESULTS: Large variations were observed in the portals used and in treatment techniques. From the digitized films, it appeared that in 50% of the cases the defined PTV was not covered adequately. Furthermore, 71% of the treatment plans would not cover the lateral borders of the reference PTV sufficiently. CONCLUSIONS: There appears to be no consensus on the target volumes to be irradiated in postoperative radiotherapy of early stage cervical carcinoma. When a PTV defined on the basis of lymphangiograms and CT-data is taken as a reference, 71% of the treatment plans would not cover this PTV adequately. These findings indicate the need for a consensus in the design of standardized treatment volumes.

Carcinoma, Squamous Cell↗
PubMed1998

The discovery and validation of new drug targets in cancer.

Advances in our understanding of the signal transduction pathways involved in cellular growth control have provided several new strategies for cancer therapy. Recent advances now make it possible to develop selective inhibitors targeting genomic instability, the growth, survival, and invasion of the tumor, and its nourishment through the growth of new blood vessels.

Animals↗
PubMed2003

Inhibition of FLT3 in MLL. Validation of a therapeutic target identified by gene expression based classification.

We recently found that MLL-rearranged acute lymphoblastic leukemias (MLL) have a unique gene expression profile including high level expression of the receptor tyrosine kinase FLT3. We hypothesized that FLT3 might be a therapeutic target in MLL and found that 5 of 30 MLLs contain mutations in the activation loop of FLT3 that result in constitutive activation. Three are a newly described deletion of I836 and the others are D835 mutations. The recently described FLT3 inhibitor PKC412 proved cytotoxic to Ba/F3 cells dependent upon activated FLT3 containing either mutation. PKC412 is also differentially cytotoxic to leukemia cells with MLL translocations and FLT3 that is activated by either overexpression of the wild-type receptor or mutation. Finally, we developed a mouse model of MLL and used bioluminescent imaging to determine that PKC412 is active against MLL in vivo.

Animals↗
PubMed2005

Identification and validation of novel therapeutic targets for multiple myeloma.

In vitro and in vivo models have been developed that have allowed for delineation of mechanisms of multiple myeloma (MM) cell homing to bone marrow (BM); tumor cell adhesion to extracellular matrix proteins and BM stromal cells; and cytokine-mediated growth, survival, drug resistance, and migration within the BM milieu. Delineation of the signaling cascades mediating these sequelae has identified multiple novel therapeutic targets in the tumor cell and its BM microenvironment. Importantly, novel therapies targeting the tumor cell and the BM, as well as those targeting the tumor cell or BM alone, can overcome the growth, survival, conventional drug resistance, and migration of MM cells bound to BM using both in vitro and in vivo severe combined immunodeficiency mouse models of human MM. These studies have translated rapidly from the bench to the bedside in derived clinical trials, and have already led to the United States Food and Drug Administration approval of the novel proteasome inhibitor bortezomib for treatment of relapsed/refractory MM. Novel agents will need to be combined to enhance cytotoxicity, avoid development of drug resistance, and allow for use of lower doses in combination therapies. Genomics, proteomics, and cell signaling studies have helped to identify in vivo mechanisms of sensitivity versus resistance to novel therapies, as well as aiding in the rational application of combination therapies. These studies have therefore provided the framework for a new treatment paradigm targeting the MM cell in its BM milieu to overcome drug resistance and improve patient outcome in MM.

Animals↗
PubMed2005

Validation of the narcosis target lipid model for petroleum products: gasoline as a case study.

The narcosis target lipid model (NTLM) was used to predict the toxicity of water-accommodated fractions (WAFs) of six gasoline blending streams to algae (Pseudokirchnereilla subcapitata, formerly Selenastrum capricornutum), juvenile rainbow trout (Oncorhynchus mykiss), and water flea (Daphnia magna). Gasolines are comprised of hydrocarbons that on dissolution into the aqueous phase are expected to act via narcosis. Aquatic toxicity data were obtained using a lethal-loading test in which WAFs were prepared using different gasoline loadings. The compositions of the gasolines were determined by analysis of C3 to C13 hydrocarbons grouped in classes of n-alkanes, iso-alkanes, aromatics, cyclic alkanes, and olefins. A model was developed to compute the concentrations of hydrocarbon blocks in WAFs based on gasoline composition and loading. The model accounts for the volume change of the gasoline, which varies depending on loading and volatilization loss. The predicted aqueous composition of WAFs compared favorably to measurements, and the predicted aqueous concentrations of WAFs were used in the NTLM to predict the aquatic toxicity of the gasolines. For each gasoline loading and species, total toxic units (TUs) were computed with an assumption of additivity. The acute toxicity of gasolines was predicted to within a factor of two for algae and daphnids. Predicted TUs overestimated toxicity to trout because of experimental factors that were not considered in the model. This analysis demonstrates the importance of aliphatic hydrocarbon loss to headspace during WAF preparation and the contribution of both aromatic and aliphatic hydrocarbons test to the toxicity of gasolines in closed systems and loss of aliphatics to headspace during WAF preparation. Model calculations indicate that satisfactory toxicity predictions can be achieved by describing gasoline composition using a limited number of aromatic and aliphatic hydrocarbon blocks with different octanol-water partition coefficients.

Animals↗
PubMed2006

Novel approaches to antimalarial drug discovery.

Major advances in our understanding of malaria parasite biology have been made. Coupled with the completion of the malaria genome, this has presented exciting opportunities for target-based antimalarial drug discovery. However, the unraveling of more validated biological targets will not necessarily translate into the identification of new chemical entities that are effective against drug resistant parasites in the long term. As history has already shown, development of antiplasmodial agents aimed at a single parasite target or specialized process has failed to stem the tide of drug resistance. This review highlights recent starting points and/or approaches to antimalarial drug discovery with particular emphasis on innovative efforts, which are not necessarily based on the identification of new drug targets and attendant inhibitor design. Approaches covered include utilization of validated chemical scaffolds, bioprecursor and carrier prodrugs, double drug development and/or multi-therapeutic strategies, use of metallocenic scaffolds, the medicinal chemistry of antimalarial natural products and in silico drug design.

Animals↗
PubMed1993

Behavioral and computational models of spatial attention.

Pigeons pecked for food in a spatially cued choice reaction time (RT) task. A brief (50-ms) white light appeared on a left or right key and probabilistically predicted the location (on either the left or right key) of a subsequent target stimulus. The time between cue and target onset (stimulus onset asynchrony), the base rate of left cues, and the probability that the cue correctly predicted the target (cue validity) were experimentally varied. The mean RT to respond to the target key was faster on correctly cued trials (defining a validity effect), decreased for both valid and invalid trials as stimulus onset asynchrony increased (defining an alerting effect), showed a variety of base-rate effects, and did not depend on cue validity. It is shown with a computational-processing model that dynamic interactions of short-term and associative memory processes are sufficient to produce these attentionlike empirical phenomena.

Animals↗
PubMed2001

The urokinase plasminogen activator receptor (uPAR) as a target for the diagnosis and therapy of cancer.

The identification and characterization of validated molecular targets for cancer drug and diagnostic development is rapidly changing the way that promising new anti-cancer compounds are developed and evaluated. A significant body of in vitro and in vivo data has established the urokinase plasminogen activator (uPA) system as a promising target for cancer drug development. The uPA system has been demonstrated to have pleiotropic activities in the development of tumors, and in tumor progression and angiogenesis. There are multiple ways to target this system, the most straightforward being the development of small molecule active site inhibitors of the serine protease, uPA. However, compounds of this type have not entered into clinical trials, and issues related to selectivity and specificity of this class of inhibitors have yet to be satisfactorily resolved. Recent evidence suggests that in addition to uPA, its specific cell surface receptor (uPAR) may also be a suitable target for the design and development of cancer therapeutic and diagnostic agents. uPAR is central to several pathways implicated in tumor progression and angiogenesis. The binding of the uPA zymogen (scuPA) to uPAR appears to be a pre-requisite for efficient cell-surface activation of scuPA to the active two-chain form (tcuPA) by plasmin, and simple ligand occupancy of uPAR by scuPA initiates various signaling pathways leading to alterations in cell motility and adhesion. One therapeutic rationale that is currently being investigated is the simple displacement of scuPA or tcuPA from suPAR, which may effectively inhibit both the proteolytic and signal-transducing cascades. In addition, other approaches to the modulation of the activity of this system that may also be useful include blocking the interaction of uPAR with integrins and extracellular matrix proteins as well as strategies to down-regulate the expression of uPA and uPAR in target cells. This review will summarize these approaches, and also describe the targeting of uPAR for diagnosis and imaging.

Humans↗
PubMed2025

Integrating network pharmacology and experimental validation to uncover the synergistic effects of Huangqi ()-Ezhu () with 5-fluorouracil in colorectal cancer models.

OBJECTIVE: To evaluate the effects of Huangqi (Radix Astragali Mongolici)-Ezhu (Rhizoma Curcumae Phaeocaulis) (HQEZ) on colorectal cancer therapies and to elucidate the potential mechanisms of HQEZ, especially in combination with 5-Fluorouracil (5-FU). METHODS: The anti-tumor effects of HQEZ were evaluated in colorectal cancer models both in vivo and in vitro. The network pharmacological assay was used to investigate potential mechanisms of HQEZ. Potential target genes were selected by Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, protein-protein interaction network (PPI) and molecular docking. Within key targets, potential targets related to drug sensitivity, especially the sensitivity to 5-FU, were evaluated in HCT116 in vitro by immunofluorescence, quantitative real-time polymerase chain reaction (qPCR) and Western-blot. Then, changes in potential targets were assessed in tumors from tumor-bearing mice and the expression of these targets was also evaluated in colorectal cancer (COAD) patients from the Cancer Genome Atlas Program (TCGA) database. RESULTS: HQEZ significantly enhanced the anti-tumor activity of 5-FU in vivo and inhibit the growth of HCT116 in vitro. By network pharmacological analysis, key targets, such as protein kinase B (AKT1), epidermal growth factor receptor (EGFR), adenosine triphosphate (ATP) binding cassette subfamily B member 1 (ABCB1, also named multidrug resistance protein 1, MDR1), ATP binding cassette subfamily G member 2 (ABCG2), thymidylate synthetase (TYMS, also named TS), prostaglandin-endoperoxide synthase 2 (PTGS2), matrix metallopeptidase 2 (MMP2), MMP9, toll like receptor 4 (TLR4), TLR9 and dihydropyrimidine dehydrogenase (DPYD), were identified. Additionally, 4 potential core active ingredients (Folate, Curcumin, quercetin and kaempferol) were identified to be important for the treatment of colorectal cancer with HQEZ. In key targets, chemoresistance related targets were validated to be affected by HQEZ. Furthermore, 5-FU sensitivity related targets, including MDR1, TS, EGFR, ribonucleotide reductase catalytic subunit M1, Breast and Ovarian Cancer Susceptibility Protein 1 (BRCA1) and mutl homolog 1 were also significantly reduced by HQEZ both in vitro and in vivo. Finally, these validated key targets and 5-FU sensitivity related targets were demonstrated to be up-regulated in COAD patients based on TCGA database. CONCLUSION: HQEZ has synergistic effects on the anti-tumor activity of 5-FU in the treatment of colorectal cancer both in vivo and in vitro. The beneficial effect of HQEZ results from the inhibition of the drug sensitivity targets associated with 5-FU. The combination therapy of HQEZ with 5-FU or other chemotherapeutic drugs will also improve the anti-tumor efficacy of chemotherapy.

Humans↗