Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

PubMed Central

Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

2016-01-01

The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

Selection on Inversion Breakpoints Favors Proximity to Pairing Sensitive Sites in Drosophila melanogaster.

PubMed

Corbett-Detig, Russell B

2016-09-01

Chromosomal inversions are widespread among taxa, and have been implicated in a number of biological processes including adaptation, sex chromosome evolution, and segregation distortion. Consistent with selection favoring linkage between loci, it is well established that length is a selected trait of inversions. However, the factors that affect the distribution of inversion breakpoints remain poorly understood. "Sensitive sites" have been mapped on all euchromatic chromosome arms in Drosophila melanogaster, and may be a source of natural selection on inversion breakpoint positions. Briefly, sensitive sites are genomic regions wherein proximal structural rearrangements result in large reductions in local recombination rates in heterozygotes. Here, I show that breakpoints of common inversions are significantly more likely to lie within a cytological band containing a sensitive site than are breakpoints of rare inversions. Furthermore, common inversions for which neither breakpoint intersects a sensitive site are significantly longer than rare inversions, but common inversions whose breakpoints intersect a sensitive site show no evidence for increased length. I interpret these results to mean that selection favors inversions whose breakpoints disrupt synteny near to sensitive sites, possibly because these inversions suppress recombination in large genomic regions. To my knowledge this is the first evidence consistent with positive selection acting on inversion breakpoint positions. Copyright © 2016 by the Genetics Society of America.

Engineering synthetic TAL effectors with orthogonal target sites

PubMed Central

Garg, Abhishek; Lohmueller, Jason J.; Silver, Pamela A.; Armel, Thomas Z.

2012-01-01

The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs to accomplish near complete repression of target gene expression. PMID:22581776

Secure FAST: Security Enhancement in the NATO Time Sensitive Targeting Tool

DTIC Science & Technology

2010-11-01

designed to aid in the tracking and prosecuting of Time Sensitive Targets. The FAST tool provides user level authentication and authorisation in terms...level authentication and authorisation in terms of security. It uses operating system level security but does not provide application level security for...and collaboration tool, designed to aid in the tracking and prosecuting of Time Sensitive Targets. The FAST tool provides user level authentication and

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

PubMed Central

Alcántara-de la Cruz, Ricardo; Fernández-Moreno, Pablo T.; Ozuna, Carmen V.; Rojano-Delgado, Antonia M.; Cruz-Hipolito, Hugo E.; Domínguez-Valenzuela, José A.; Barro, Francisco; De Prado, Rafael

2016-01-01

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world. PMID:27752259

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.

2014-12-17

Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reactionmore » monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than

Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

PubMed

Rockah-Shmuel, Liat; Tawfik, Dan S

2012-12-01

DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

Selection on Inversion Breakpoints Favors Proximity to Pairing Sensitive Sites in Drosophila melanogaster

PubMed Central

Corbett-Detig, Russell B.

2016-01-01

Chromosomal inversions are widespread among taxa, and have been implicated in a number of biological processes including adaptation, sex chromosome evolution, and segregation distortion. Consistent with selection favoring linkage between loci, it is well established that length is a selected trait of inversions. However, the factors that affect the distribution of inversion breakpoints remain poorly understood. “Sensitive sites” have been mapped on all euchromatic chromosome arms in Drosophila melanogaster, and may be a source of natural selection on inversion breakpoint positions. Briefly, sensitive sites are genomic regions wherein proximal structural rearrangements result in large reductions in local recombination rates in heterozygotes. Here, I show that breakpoints of common inversions are significantly more likely to lie within a cytological band containing a sensitive site than are breakpoints of rare inversions. Furthermore, common inversions for which neither breakpoint intersects a sensitive site are significantly longer than rare inversions, but common inversions whose breakpoints intersect a sensitive site show no evidence for increased length. I interpret these results to mean that selection favors inversions whose breakpoints disrupt synteny near to sensitive sites, possibly because these inversions suppress recombination in large genomic regions. To my knowledge this is the first evidence consistent with positive selection acting on inversion breakpoint positions. PMID:27343234

LuciPHOr: Algorithm for Phosphorylation Site Localization with False Localization Rate Estimation Using Modified Target-Decoy Approach*

PubMed Central

Fermin, Damian; Walmsley, Scott J.; Gingras, Anne-Claude; Choi, Hyungwon; Nesvizhskii, Alexey I.

2013-01-01

The localization of phosphorylation sites in peptide sequences is a challenging problem in large-scale phosphoproteomics analysis. The intense neutral loss peaks and the coexistence of multiple serine/threonine and/or tyrosine residues are limiting factors for objectively scoring site patterns across thousands of peptides. Various computational approaches for phosphorylation site localization have been proposed, including Ascore, Mascot Delta score, and ProteinProspector, yet few address direct estimation of the false localization rate (FLR) in each experiment. Here we propose LuciPHOr, a modified target-decoy-based approach that uses mass accuracy and peak intensities for site localization scoring and FLR estimation. Accurate estimation of the FLR is a difficult task at the individual-site level because the degree of uncertainty in localization varies significantly across different peptides. LuciPHOr carries out simultaneous localization on all candidate sites in each peptide and estimates the FLR based on the target-decoy framework, where decoy phosphopeptides generated by placing artificial phosphorylation(s) on non-candidate residues compete with the non-decoy phosphopeptides. LuciPHOr also reports approximate site-level confidence scores for all candidate sites as a means to localize additional sites from multiphosphorylated peptides in which localization can be partially achieved. Unlike the existing tools, LuciPHOr is compatible with any search engine output processed through the Trans-Proteomic Pipeline. We evaluated the performance of LuciPHOr in terms of the sensitivity and accuracy of FLR estimates using two synthetic phosphopeptide libraries and a phosphoproteomic dataset generated from complex mouse brain samples. PMID:23918812

Computational Predictions Provide Insights into the Biology of TAL Effector Target Sites

PubMed Central

Grau, Jan; Wolf, Annett; Reschke, Maik; Bonas, Ulla; Posch, Stefan; Boch, Jens

2013-01-01

Transcription activator-like (TAL) effectors are injected into host plant cells by Xanthomonas bacteria to function as transcriptional activators for the benefit of the pathogen. The DNA binding domain of TAL effectors is composed of conserved amino acid repeat structures containing repeat-variable diresidues (RVDs) that determine DNA binding specificity. In this paper, we present TALgetter, a new approach for predicting TAL effector target sites based on a statistical model. In contrast to previous approaches, the parameters of TALgetter are estimated from training data computationally. We demonstrate that TALgetter successfully predicts known TAL effector target sites and often yields a greater number of predictions that are consistent with up-regulation in gene expression microarrays than an existing approach, Target Finder of the TALE-NT suite. We study the binding specificities estimated by TALgetter and approve that different RVDs are differently important for transcriptional activation. In subsequent studies, the predictions of TALgetter indicate a previously unreported positional preference of TAL effector target sites relative to the transcription start site. In addition, several TAL effectors are predicted to bind to the TATA-box, which might constitute one general mode of transcriptional activation by TAL effectors. Scrutinizing the predicted target sites of TALgetter, we propose several novel TAL effector virulence targets in rice and sweet orange. TAL-mediated induction of the candidates is supported by gene expression microarrays. Validity of these targets is also supported by functional analogy to known TAL effector targets, by an over-representation of TAL effector targets with similar function, or by a biological function related to pathogen infection. Hence, these predicted TAL effector virulence targets are promising candidates for studying the virulence function of TAL effectors. TALgetter is implemented as part of the open-source Java library

Characteristics of Food Industry Web Sites and "Advergames" Targeting Children

ERIC Educational Resources Information Center

Culp, Jennifer; Bell, Robert A.; Cassady, Diana

2010-01-01

Objective: To assess the content of food industry Web sites targeting children by describing strategies used to prolong their visits and foster brand loyalty; and to document health-promoting messages on these Web sites. Design: A content analysis was conducted of Web sites advertised on 2 children's networks, Cartoon Network and Nickelodeon. A…

Target-classification approach applied to active UXO sites

NASA Astrophysics Data System (ADS)

Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

2013-06-01

This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauch, Eva-Maria; Bernard, Steffen M.; La, David

Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein ismore » designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.« less

Long-Term Memories Bias Sensitivity and Target Selection in Complex Scenes

PubMed Central

Patai, Eva Zita; Doallo, Sonia; Nobre, Anna Christina

2014-01-01

In everyday situations we often rely on our memories to find what we are looking for in our cluttered environment. Recently, we developed a new experimental paradigm to investigate how long-term memory (LTM) can guide attention, and showed how the pre-exposure to a complex scene in which a target location had been learned facilitated the detection of the transient appearance of the target at the remembered location (Summerfield, Lepsien, Gitelman, Mesulam, & Nobre, 2006; Summerfield, Rao, Garside, & Nobre, 2011). The present study extends these findings by investigating whether and how LTM can enhance perceptual sensitivity to identify targets occurring within their complex scene context. Behavioral measures showed superior perceptual sensitivity (d′) for targets located in remembered spatial contexts. We used the N2pc event-related potential to test whether LTM modulated the process of selecting the target from its scene context. Surprisingly, in contrast to effects of visual spatial cues or implicit contextual cueing, LTM for target locations significantly attenuated the N2pc potential. We propose that the mechanism by which these explicitly available LTMs facilitate perceptual identification of targets may differ from mechanisms triggered by other types of top-down sources of information. PMID:23016670

Comparing phase-sensitive and phase-insensitive echolocation target images using a monaural audible sonar.

PubMed

Kuc, Roman

2018-04-01

This paper describes phase-sensitive and phase-insensitive processing of monaural echolocation waveforms to generate target maps. Composite waveforms containing both the emission and echoes are processed to estimate the target impulse response using an audible sonar. Phase-sensitive processing yields the composite signal envelope, while phase-insensitive processing that starts with the composite waveform power spectrum yields the envelope of the autocorrelation function. Analysis and experimental verification show that multiple echoes form an autocorrelation function that produces near-range phantom-reflector artifacts. These artifacts interfere with true target echoes when the first true echo occurs at a time that is less than the total duration of the target echoes. Initial comparison of phase-sensitive and phase-insensitive maps indicates that both display important target features, indicating that phase is not vital. A closer comparison illustrates the improved resolution of phase-sensitive processing, the near-range phantom-reflectors produced by phase-insensitive processing, and echo interference and multiple reflection artifacts that were independent of the processing.

Photoaffinity labeling in target- and binding-site identification

PubMed Central

Smith, Ewan; Collins, Ian

2015-01-01

Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

Parameter optimization, sensitivity, and uncertainty analysis of an ecosystem model at a forest flux tower site in the United States

USGS Publications Warehouse

Wu, Yiping; Liu, Shuguang; Huang, Zhihong; Yan, Wende

2014-01-01

Ecosystem models are useful tools for understanding ecological processes and for sustainable management of resources. In biogeochemical field, numerical models have been widely used for investigating carbon dynamics under global changes from site to regional and global scales. However, it is still challenging to optimize parameters and estimate parameterization uncertainty for complex process-based models such as the Erosion Deposition Carbon Model (EDCM), a modified version of CENTURY, that consider carbon, water, and nutrient cycles of ecosystems. This study was designed to conduct the parameter identifiability, optimization, sensitivity, and uncertainty analysis of EDCM using our developed EDCM-Auto, which incorporated a comprehensive R package—Flexible Modeling Framework (FME) and the Shuffled Complex Evolution (SCE) algorithm. Using a forest flux tower site as a case study, we implemented a comprehensive modeling analysis involving nine parameters and four target variables (carbon and water fluxes) with their corresponding measurements based on the eddy covariance technique. The local sensitivity analysis shows that the plant production-related parameters (e.g., PPDF1 and PRDX) are most sensitive to the model cost function. Both SCE and FME are comparable and performed well in deriving the optimal parameter set with satisfactory simulations of target variables. Global sensitivity and uncertainty analysis indicate that the parameter uncertainty and the resulting output uncertainty can be quantified, and that the magnitude of parameter-uncertainty effects depends on variables and seasons. This study also demonstrates that using the cutting-edge R functions such as FME can be feasible and attractive for conducting comprehensive parameter analysis for ecosystem modeling.

Target loads of atmospheric sulfur deposition for the protection and recovery of acid-sensitive streams in the Southern Blue Ridge Province.

PubMed

Sullivan, Timothy J; Cosby, Bernard J; Jackson, William A

2011-11-01

An important tool in the evaluation of acidification damage to aquatic and terrestrial ecosystems is the critical load (CL), which represents the steady-state level of acidic deposition below which ecological damage would not be expected to occur, according to current scientific understanding. A deposition load intended to be protective of a specified resource condition at a particular point in time is generally called a target load (TL). The CL or TL for protection of aquatic biota is generally based on maintaining surface water acid neutralizing capacity (ANC) at an acceptable level. This study included calibration and application of the watershed model MAGIC (Model of Acidification of Groundwater in Catchments) to estimate the target sulfur (S) deposition load for the protection of aquatic resources at several future points in time in 66 generally acid-sensitive watersheds in the southern Blue Ridge province of North Carolina and two adjoining states. Potential future change in nitrogen leaching is not considered. Estimated TLs for S deposition ranged from zero (ecological objective not attainable by the specified point in time) to values many times greater than current S deposition depending on the selected site, ANC endpoint, and evaluation year. For some sites, one or more of the selected target ANC critical levels (0, 20, 50, 100μeq/L) could not be achieved by the year 2100 even if S deposition was reduced to zero and maintained at that level throughout the simulation. Many of these highly sensitive streams were simulated by the model to have had preindustrial ANC below some of these target values. For other sites, the watershed soils contained sufficiently large buffering capacity that even very high sustained levels of atmospheric S deposition would not reduce stream ANC below common damage thresholds. Copyright © 2011 Elsevier Ltd. All rights reserved.

Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites.

PubMed

Betel, Doron; Koppal, Anjali; Agius, Phaedra; Sander, Chris; Leslie, Christina

2010-01-01

mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.

Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottea, J.A.; Payne, G.T.; Soderlund, D.M.

1990-08-01

Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate (({sup 3}H)BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of ({sup 3}H)BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited ({sup 3}H)BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealedmore » that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents.« less

Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vose, Sarah C.; Center for Children's Environmental Health Research, School of Public Health, University of California, Berkeley, CA 94720; Holland, Nina T.

2007-10-01

Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC{sub 50} valuesmore » of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.« less

Targeting Breast Cancer Recurrence via Hedgehog-mediated Sensitization of Breast Cancer Stem Cells

DTIC Science & Technology

2011-07-01

Hedgehog -mediated Sensitization of Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: David J. Robbins, Ph.D...June 2010 – 14 June 2011 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Breast Cancer Recurrence via Hedgehog -mediated Sensitization of...this award. Introduction The purpose of the research supported by this award is to determine if targeting the hedgehog signaling pathway in

Folate-containing reduction-sensitive lipid-polymer hybrid nanoparticles for targeted delivery of doxorubicin.

PubMed

Wu, Bo; Yu, Ping; Cui, Can; Wu, Ming; Zhang, Yang; Liu, Lei; Wang, Cai-Xia; Zhuo, Ren-Xi; Huang, Shi-Wen

2015-04-01

The development and evaluation of folate-targeted and reduction-triggered biodegradable nanoparticles are introduced to the research on targeted delivery of doxorubicin (DOX). This type of folate-targeted lipid-polymer hybrid nanoparticles (FLPNPs) is comprised of a poly(D,L-lactide-co-glycolide) (PLGA) core, a soybean lecithin monolayer, a monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16) reduction-sensitive shell, and a folic acid-targeted ligand. FLPNPs exhibited high size stability but fast disassembly in a simulated cancer cell reductive environment. The experiments on the release process in vitro revealed that as a reduction-sensitive drug delivery system, FLPNPs released DOX faster in the presence of 10 mM dithiothreitol (DTT). Results from flow cytometry, confocal image and in vitro cytotoxicity assays revealed that FLPNPs further enhanced cell uptake and generated higher cytotoxicity against human epidermoid carcinoma in the oral cavity than non-targeted redox-sensitive and targeted redox-insensitive controls. Furthermore, in vivo animal experiments demonstrated that systemic administration of DOX-loaded FLPNPs remarkably reduced tumor growth. Experiments on biodistribution of DOX-loaded FLPNPs showed that an increasing amount of DOX accumulated in the tumor. Therefore, FLPNPs formulations have proved to be a stable, controllable and targeted anticancer drug delivery system.

A Sensitive TLRH Targeted Imaging Technique for Ultrasonic Molecular Imaging

PubMed Central

Hu, Xiaowen; Zheng, Hairong; Kruse, Dustin E.; Sutcliffe, Patrick; Stephens, Douglas N.; Ferrara, Katherine W.

2010-01-01

The primary goals of ultrasound molecular imaging are the detection and imaging of ultrasound contrast agents (microbubbles), which are bound to specific vascular surface receptors. Imaging methods that can sensitively and selectively detect and distinguish bound microbubbles from freely circulating microbubbles (free microbubbles) and surrounding tissue are critically important for the practical application of ultrasound contrast molecular imaging. Microbubbles excited by low frequency acoustic pulses emit wide-band echoes with a bandwidth extending beyond 20 MHz; we refer to this technique as TLRH (transmission at a low frequency and reception at a high frequency). Using this wideband, transient echo, we have developed and implemented a targeted imaging technique incorporating a multi-frequency co-linear array and the Siemens Antares® imaging system. The multi-frequency co-linear array integrates a center 5.4 MHz array, used to receive echoes and produce radiation force, and two outer 1.5 MHz arrays used to transmit low frequency incident pulses. The targeted imaging technique makes use of an acoustic radiation force sub-sequence to enhance accumulation and a TLRH imaging sub-sequence to detect bound microbubbles. The radiofrequency (RF) data obtained from the TLRH imaging sub-sequence are processsed to separate echo signatures between tissue, free microbubbles, and bound microbubbles. By imaging biotin-coated microbubbles targeted to avidin-coated cellulose tubes, we demonstrate that the proposed method has a high contrast-to-tissue ratio (up to 34 dB) and a high sensitivity to bound microbubbles (with the ratio of echoes from bound microbubbles versus free microbubbles extending up to 23 dB). The effects of the imaging pulse acoustic pressure, the radiation force sub-sequence and the use of various slow-time filters on the targeted imaging quality are studied. The TLRH targeted imaging method is demonstrated in this study to provide sensitive and selective

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

2011-01-01

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion

PubMed Central

Kreibich, Saskia; Vonaesch, Pascale; Andritschke, Daniel; Rout, Samuel; Weidner, Kerstin; Sormaz, Milos; Songhet, Pascal; Horvath, Peter; Chabria, Mamta; Vogel, Viola; Spori, Doris M.; Jenny, Patrick; Hardt, Wolf-Dietrich

2012-01-01

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria. PMID:22911370

Initial basalt target site selection evaluation for the Mars penetrator drop test

NASA Technical Reports Server (NTRS)

Bunch, T. E.; Quaide, W. L.; Polkowski, G.

1976-01-01

Potential basalt target sites for an air drop penetrator test were described and the criteria involved in site selection were discussed. A summary of the background field geology and recommendations for optimum sites are also presented.

Mathematical description of drug-target interactions: application to biologics that bind to targets with two binding sites.

PubMed

Gibiansky, Leonid; Gibiansky, Ekaterina

2018-02-01

The emerging discipline of mathematical pharmacology occupies the space between advanced pharmacometrics and systems biology. A characteristic feature of the approach is application of advance mathematical methods to study the behavior of biological systems as described by mathematical (most often differential) equations. One of the early application of mathematical pharmacology (that was not called this name at the time) was formulation and investigation of the target-mediated drug disposition (TMDD) model and its approximations. The model was shown to be remarkably successful, not only in describing the observed data for drug-target interactions, but also in advancing the qualitative and quantitative understanding of those interactions and their role in pharmacokinetic and pharmacodynamic properties of biologics. The TMDD model in its original formulation describes the interaction of the drug that has one binding site with the target that also has only one binding site. Following the framework developed earlier for drugs with one-to-one binding, this work aims to describe a rigorous approach for working with similar systems and to apply it to drugs that bind to targets with two binding sites. The quasi-steady-state, quasi-equilibrium, irreversible binding, and Michaelis-Menten approximations of the model are also derived. These equations can be used, in particular, to predict concentrations of the partially bound target (RC). This could be clinically important if RC remains active and has slow internalization rate. In this case, introduction of the drug aimed to suppress target activity may lead to the opposite effect due to RC accumulation.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

PubMed Central

Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

2017-01-01

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

Sensitizing Black Adult and Youth Consumers to Targeted Food Marketing Tactics in Their Environments

PubMed Central

Isselmann DiSantis, Katherine; Kumanyika, Shiriki; Rohm Young, Deborah; Grier, Sonya A.; Lassiter, Vikki

2017-01-01

Food marketing environments of Black American consumers are heavily affected by ethnically-targeted marketing of sugar sweetened beverages, fast foods, and other products that may contribute to caloric overconsumption. This qualitative study assessed Black consumers’ responses to targeted marketing. Black adults (2 mixed gender groups; total n = 30) and youth (2 gender specific groups; total n = 35) from two U.S. communities participated before and after a sensitization procedure—a critical practice used to understand social justice concerns. Pre-sensitization focus groups elicited responses to scenarios about various targeted marketing tactics. Participants were then given an informational booklet about targeted marketing to Black Americans, and all returned for the second (post-sensitization) focus group one week later. Conventional qualitative content analysis of transcripts identified several salient themes: seeing the marketer’s perspective (“it’s about demand”; “consumers choose”), respect for community (“marketers are setting us up for failure”; “making wrong assumptions”), and food environments as a social justice issue (“no one is watching the door”; “I didn’t realize”). Effects of sensitization were reflected in participants’ stated reactions to the information in the booklet, and also in the relative occurrence of marketer-oriented themes and social justice-oriented themes, respectively, less and more after sensitization. PMID:29109377

Sensitizing Black Adult and Youth Consumers to Targeted Food Marketing Tactics in Their Environments.

PubMed

Isselmann DiSantis, Katherine; Kumanyika, Shiriki; Carter-Edwards, Lori; Rohm Young, Deborah; Grier, Sonya A; Lassiter, Vikki

2017-10-29

Food marketing environments of Black American consumers are heavily affected by ethnically-targeted marketing of sugar sweetened beverages, fast foods, and other products that may contribute to caloric overconsumption. This qualitative study assessed Black consumers' responses to targeted marketing. Black adults (2 mixed gender groups; total n = 30) and youth (2 gender specific groups; total n = 35) from two U.S. communities participated before and after a sensitization procedure-a critical practice used to understand social justice concerns. Pre-sensitization focus groups elicited responses to scenarios about various targeted marketing tactics. Participants were then given an informational booklet about targeted marketing to Black Americans, and all returned for the second (post-sensitization) focus group one week later. Conventional qualitative content analysis of transcripts identified several salient themes: seeing the marketer's perspective ("it's about demand"; "consumers choose"), respect for community ("marketers are setting us up for failure"; "making wrong assumptions"), and food environments as a social justice issue ("no one is watching the door"; "I didn't realize"). Effects of sensitization were reflected in participants' stated reactions to the information in the booklet, and also in the relative occurrence of marketer-oriented themes and social justice-oriented themes, respectively, less and more after sensitization.

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

PubMed Central

Shi, Tujin; Su, Dian; Liu, Tao; Tang, Keqi; Camp, David G.; Qian, Wei-Jun; Smith, Richard D.

2012-01-01

Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications (PTMs), as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed. PMID:22577010

Sites of ozone sensitivity in diverse maize inbred lines

USDA-ARS?s Scientific Manuscript database

Tropospheric ozone (O3) is an air pollutant that costs ~$14-26 billion in global crop losses and is projected to worsen in the future. Potential sites of O3 sensitivity in maize were tested by growing 200 inbred lines, including the nested association mapping population founder lines, under ambient...

Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo.

PubMed

Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

2017-01-01

In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro . Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo , we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy.

Interstitial telomeric sequences in human chromosomes cluster with common fragile sites, mutagen sensitive sites, viral integration sites, cancer breakpoints, proto-oncogenes and breakpoints involved in primate evolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adekunle, S.S.A.; Wyandt, H.; Mark, H.F.L.

1994-09-01

Recently we mapped the telomeric repeat sequences to 111 interstitial sites in the human genome and to sites of gaps and breaks induced by aphidicolin and sister chromatid exchange sites detected by BrdU. Many of these sites correspond to conserved fragile sites in man, gorilla and chimpazee, to sites of conserved sister chromatid exchange in the mammalian X chromosome, to mutagenic sensitive sites, mapped locations of proto-oncogenes, breakpoints implicated in primate evolution and to breakpoints indicated as the sole anomaly in neoplasia. This observation prompted us to investigate if the interstitial telomeric sites cluster with these sites. An extensive literaturemore » search was carried out to find all the available published sites mentioned above. For comparison, we also carried out a statistical analysis of the clustering of the sites of the telomeric repeats with the gene locations where only nucleotide mutations have been observed as the only chromosomal abnormality. Our results indicate that the telomeric repeats cluster most with fragile sites, mutagenic sensitive sites and breakpoints implicated in primate evolution and least with cancer breakpoints, mapped locations of proto-oncogenes and other genes with nucleotide mutations.« less

The impact of target site accessibility on the design of effective siRNAs.

PubMed

Tafer, Hakim; Ameres, Stefan L; Obernosterer, Gregor; Gebeshuber, Christoph A; Schroeder, Renée; Martinez, Javier; Hofacker, Ivo L

2008-05-01

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.

Protease-sensitive, polymer-caged liposomes: a method for making highly targeted liposomes using triggered release.

PubMed

Basel, Matthew T; Shrestha, Tej B; Troyer, Deryl L; Bossmann, Stefan H

2011-03-22

Liposomes have become useful and well-known drug delivery vehicles because of their ability to entrap drugs without chemically modifying them and to deliver them somewhat selectively to tumorous tissue via the enhanced permeation and retention (EPR) effect. Although useful, liposome preparations are still less than ideal because of imperfect specificity, slow release kinetics in the tumor, and leakiness prior to reaching the tumor site. Cancer-associated proteases (CAPs), which are differentially expressed in tumors, have also gained traction recently as a method for tumor targeting and drug delivery. By combining the EPR effect with CAPs sensitivity, a much more specific liposome can be produced. The method described here creates an improved liposome system that can target more specifically, with faster release kinetics and lower general leaking, by deliberately producing a very unstable liposome (loaded with hyperosmotic vehicle) that is subsequently stabilized by a cross-linked polymer shell containing consensus sequences for cancer-associated proteases (protease-triggered, caged liposomes). A cholesterol-anchored, graft copolymer, composed of a short peptide sequence for urokinase plasminogen activator (uPA) and poly(acrylic acid), was synthesized and incorporated into liposomes prepared at high osmolarities. Upon cross-linking of the polymers, the protease-triggered, caged liposomes showed significant resistance to osmotic swelling and leaking of contents. Protease-triggered, caged liposomes also showed significant and substantial differential release of contents in the presence of uPA, while bare liposomes showed no differential effect in the presence of uPA. Thus a protease-sensitive liposome system with fast release kinetics was developed that could be used for more specific targeting to tumors.

Recovery of perennial vegetation in military target sites in the eastern Mohave Desert, Arizona

USGS Publications Warehouse

Steiger, John W.; Webb, Robert H.

2000-01-01

The effect of the age of geomorphic surfaces on the recovery of desert vegetation in military target sites was studied in the Mohave and Cerbat Mountains of northwestern Arizona. The target sites were cleared of all vegetation during military exercises in 1942-1943 and have not been subsequently disturbed. The degree of recovery was measured by calculating percentage-similarity (PS) and correlation-coefficient indices on the basis of differences in cover, density, and volume of species growing in and out of each target site. PS values, ranging from 22.7 to 95.1 percent (100 percent = identical composition), indicate a wide range of recovery that is partially controlled by the edaphic properties of the geomorphic surfaces. Statistical analyses show a strong pattern that indicates a greater variability in the degree of recovery for sites on older surfaces than on younger surfaces and a weak pattern that indicates an inverse relation between the degree of recovery and geomorphic age. Comparisons of the different effects of target site construction on the edaphic characteristics of each target site provides an explanation for these patterns and suggests the soil properties critical to the recovery process. Statistically significant negative or positive response to disturbance for most species are independent of the age of the geomorphic surfaces; however, there is strong evidence for a shift in response for the common perennial species Acamptopappus sphaerocephalus, and to a lesser extent, Salazaria mexicana, Encelia farinosa, and Coldenia canescens, among different geomorphic surfaces.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

NASA Astrophysics Data System (ADS)

Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

2016-07-01

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Characteristics of food industry web sites and "advergames" targeting children.

PubMed

Culp, Jennifer; Bell, Robert A; Cassady, Diana

2010-01-01

To assess the content of food industry Web sites targeting children by describing strategies used to prolong their visits and foster brand loyalty; and to document health-promoting messages on these Web sites. A content analysis was conducted of Web sites advertised on 2 children's networks, Cartoon Network and Nickelodeon. A total of 290 Web pages and 247 unique games on 19 Internet sites were examined. Games, found on 81% of Web sites, were the most predominant promotion strategy used. All games had at least 1 brand identifier, with logos being most frequently used. On average Web sites contained 1 "healthful" message for every 45 exposures to brand identifiers. Food companies use Web sites to extend their television advertising to promote brand loyalty among children. These sites almost exclusively promoted food items high in sugar and fat. Health professionals need to monitor food industry marketing practices used in "new media." Published by Elsevier Inc.

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Su, Dian; Liu, Tao

2012-04-01

Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the pg/mL to low ng/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in the cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides or their posttranslational modifications (PTMs), as well as advances in MS instrumentation, whichmore » have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.« less

[Advances of tumor targeting peptides drug delivery system with pH-sensitive activities].

PubMed

Ma, Yin-yun; Li, Li; Huang, Hai-feng; Gou, San-hu; Ni, Jing-man

2016-05-01

The pH-sensitive peptides drug delivery systems, which target to acidic extracellular environment of tumor tissue, have many advantages in drug delivery. They exhibit a high specificity to tumor and low cytotoxicity, which significantly increase the efficacy of traditional anti-cancer drugs. In recent years the systems have received a great attention. The pH-sensitive peptides drug delivery systems can be divided into five types according to the difference in pH-responsive mechanism,type of peptides and carrier materials. This paper summarizes the recent progresses in the field with a focus on the five types of pH-sensitive peptides in drug delivery systems. This may provide a guideline to design and application of tumor targeting drugs.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

NASA Astrophysics Data System (ADS)

Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

2016-05-01

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike

Widespread occurrence of both metabolic and target-site herbicide resistance mechanisms in Lolium rigidum populations.

PubMed

Han, Heping; Yu, Qin; Owen, Mechelle J; Cawthray, Gregory R; Powles, Stephen B

2016-02-01

Lolium rigidum populations in Australia and globally have demonstrated rapid and widespread evolution of resistance to acetyl coenzyme A carboxylase (ACCase)-inhibiting and acetolactate synthase (ALS)-inhibiting herbicides. Thirty-three resistant L. rigidum populations, randomly collected from crop fields in a most recent resistance survey, were analysed for non-target-site diclofop metabolism and all known target-site ACCase gene resistance-endowing mutations. The HPLC profile of [(14) C]-diclofop-methyl in vivo metabolism revealed that 79% of these resistant L. rigidum populations showed enhanced capacity for diclofop acid metabolism (metabolic resistance). ACCase gene sequencing identified that 91% of the populations contain plants with ACCase resistance mutation(s). Importantly, 70% of the populations exhibit both non-target-site metabolic resistance and target-site ACCase mutations. This work demonstrates that metabolic herbicide resistance is commonly occurring in L. rigidum, and coevolution of both metabolic resistance and target-site resistance is an evolutionary reality. Metabolic herbicide resistance can potentially endow resistance to many herbicides and poses a threat to herbicide sustainability and thus crop production, calling for major research and management efforts. © 2015 Society of Chemical Industry.

Targeted Nanotechnology for Cancer Imaging

PubMed Central

Toy, Randall; Bauer, Lisa; Hoimes, Christopher; Ghaghada, Ketan B.; Karathanasis, Efstathios

2014-01-01

Targeted nanoparticle imaging agents provide many benefits and new opportunities to facilitate accurate diagnosis of cancer and significantly impact patient outcome. Due to the highly engineerable nature of nanotechnology, targeted nanoparticles exhibit significant advantages including increased contrast sensitivity, binding avidity and targeting specificity. Considering the various nanoparticle designs and their adjustable ability to target a specific site and generate detectable signals, nanoparticles can be optimally designed in terms of biophysical interactions (i.e., intravascular and interstitial transport) and biochemical interactions (i.e., targeting avidity towards cancer-related biomarkers) for site-specific detection of very distinct microenvironments. This review seeks to illustrate that the design of a nanoparticle dictates its in vivo journey and targeting of hard-to-reach cancer sites, facilitating early and accurate diagnosis and interrogation of the most aggressive forms of cancer. We will report various targeted nanoparticles for cancer imaging using X-ray computed tomography, ultrasound, magnetic resonance imaging, nuclear imaging and optical imaging. Finally, to realize the full potential of targeted nanotechnology for cancer imaging, we will describe the challenges and opportunities for the clinical translation and widespread adaptation of targeted nanoparticles imaging agents. PMID:25116445

Finding the target sites of RNA-binding proteins

PubMed Central

Li, Xiao; Kazan, Hilal; Lipshitz, Howard D; Morris, Quaid D

2014-01-01

RNA–protein interactions differ from DNA–protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. WIREs RNA 2014, 5:111–130. doi: 10.1002/wrna.1201 PMID:24217996

Target sites for the transposition of rat long interspersed repeated DNA elements (LINEs) are not random.

PubMed Central

Furano, A V; Somerville, C C; Tsichlis, P N; D'Ambrosio, E

1986-01-01

The long interspersed repeated DNA family of rats (LINE or L1Rn family) contains about 40,000 6.7-kilobase (kb) long members (1). LINE members may be currently mobile since their presence or absence causes allelic variation at three single copy loci (2, 3): insulin 1, Moloney leukemia virus integration 2 (Mlvi-2) (4), and immunoglobulin heavy chain (Igh). To characterize target sites for LINE insertion, we compared the DNA sequences of the unoccupied Mlvi-2 target site, its LINE-containing allele, and several other LINE-containing sites. Although not homologous overall, the target sites share three characteristics: First, depending on the site, they are from 68% to 86% (A+T) compared to 58% (A+T) for total rat DNA (5). Depending on the site, a 7- to 15-bp target site sequence becomes duplicated and flanks the inserted LINE member. The second is a version (0 or 1 mismatch) of the hexanucleotide, TACTCA, which is also present in the LINE member, in a highly conserved region located just before the A-rich right end of the LINE member. The third is a stretch of alternating purine/pyrimidine (PQ). The A-rich right ends of different LINE members vary in length and composition, and the sequence of a particularly long one suggests that it contains the A-rich target site from a previous transposition. PMID:3012480

RNase L targets distinct sites in influenza A virus RNAs.

PubMed

Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam; García-Sastre, Adolfo; Hesselberth, Jay R; Silverman, Robert H; Barton, David J

2015-03-01

Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Long-Circulating and pH-Sensitive Liposome Preparation Trapping a Radiotracer for Inflammation Site Detection.

PubMed

Mota, Luciene Das Graças; de Barros, André Luís Branco; Fuscaldi, Leonardo Lima; de Oliveira, Mônica Cristina; Cardoso, Valbert Nascimento

2015-06-01

Inflammatory and infectious diseases are one of the most common causes of mortality and morbidity. This paper aimed to prepare and to evaluate the ability of long-circulating and pH-sensitive liposomes, trapping a radiotracer, to identify inflamed focus. The physicochemical characterization of freeze-dried liposomes, using glucose as cryoprotectant, showed 80% of the vesicles with adequate mean diameter and good vesicle size homogeneity. Radiotracer encapsulation percentage in liposomes was 10.65%, of which 4.88% was adsorbed on the surface of the vesicles. Furthermore, liposomes presented positive zeta potential. Freeze-dried liposomes, stored for 180 days at 4 degrees C, did not show significant changes in the mean diameter, indicating good stability. Free radiotracer and radiolabeled liposomes were injected into inflammation focus-bearing rats, and ex-vivo biodistribution studies and scintigraphic images were performed. Results showed that radiopharmaceutical, free and encapsulated into liposomes, were able to identify the inflamed site. Target/non-target ratios, obtained by scintigraphic images, were greater than 1.5 at all investigated times. Data did not show significant differences between the free radiotracer and radiolabeled liposomes. Results suggest that this liposomal preparation could be employed as an alternative procedure for inflamed site detection by means of scintigraphic images. However, as the radiotracer is adsorbed onto the liposome surface by electrostatic forces, it is suggested that a neutral radiopharmaceutical be used to confirm the potential of this formulation as a scintigraphic probe for inflammation/infection detection.

PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing.

PubMed

Malina, Abba; Cameron, Christopher J F; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

2015-12-08

In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification.

Statistical Algorithms Accounting for Background Density in the Detection of UXO Target Areas at DoD Munitions Sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matzke, Brett D.; Wilson, John E.; Hathaway, J.

2008-02-12

Statistically defensible methods are presented for developing geophysical detector sampling plans and analyzing data for munitions response sites where unexploded ordnance (UXO) may exist. Detection methods for identifying areas of elevated anomaly density from background density are shown. Additionally, methods are described which aid in the choice of transect pattern and spacing to assure with degree of confidence that a target area (TA) of specific size, shape, and anomaly density will be identified using the detection methods. Methods for evaluating the sensitivity of designs to variation in certain parameters are also discussed. Methods presented have been incorporated into the Visualmore » Sample Plan (VSP) software (free at http://dqo.pnl.gov/vsp) and demonstrated at multiple sites in the United States. Application examples from actual transect designs and surveys from the previous two years are demonstrated.« less

PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing

PubMed Central

Malina, Abba; Cameron, Christopher J. F.; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

2015-01-01

In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification. PMID:26644285

Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma.

PubMed

Awasthi, Shivangi; Maity, Tapan; Oyler, Benjamin L; Qi, Yue; Zhang, Xu; Goodlett, David R; Guha, Udayan

2018-04-13

Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30-40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFR L858R mutation. However, in H1975 cells, which harbor the TKI-resistant EGFR L858R/T790M mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

PubMed Central

Pierce, M L; Ruffner, D E

1998-01-01

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

PubMed

Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

2014-01-01

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

PubMed Central

Azorsa, David O; Gonzales, Irma M; Basu, Gargi D; Choudhary, Ashish; Arora, Shilpi; Bisanz, Kristen M; Kiefer, Jeffrey A; Henderson, Meredith C; Trent, Jeffrey M; Von Hoff, Daniel D; Mousses, Spyro

2009-01-01

Background Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. Methods In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. Results Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. Conclusion These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine. PMID

An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

PubMed

Ma, Sanyuan; Liu, Yue; Liu, Yuanyuan; Chang, Jiasong; Zhang, Tong; Wang, Xiaogang; Shi, Run; Lu, Wei; Xia, Xiaojuan; Zhao, Ping; Xia, Qingyou

2017-04-01

Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

PubMed Central

Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B.

2009-01-01

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-π bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-α nAChR subunits. PMID:19321668

Free to be me: The relationship between the true self, rejection sensitivity, and use of online dating sites.

PubMed

Hance, Margaret A; Blackhart, Ginette; Dew, Megan

2018-01-01

Prior research (Blackhart et al., 2014) found that rejection-sensitive individuals are more likely to use online dating sites. The purpose of the current research was to explain the relationship between rejection sensitivity and online dating site usage. Study 1 examined whether true self mediated the relation between rejection sensitivity and online dating. Study 2 sought to replicate the findings of Study 1 and to examine whether self-disclosure moderated the relationship between true self and online dating in the mediation model. Results replicated those found by Blackhart et al. and also found that true self mediated the relationship between rejection sensitivity and online dating site usage. These findings suggest that rejection-sensitive individuals feel they can more easily represent their "true" selves in online environments, such as online dating sites, which partially explains why they are more likely to engage in online dating.

E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

PubMed

Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

2017-01-01

Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

Target-protecting dumbbell molecular probe against exonucleases digestion for sensitive detection of ATP and streptavidin.

PubMed

Chen, Jinyang; Liu, Yucheng; Ji, Xinghu; He, Zhike

2016-09-15

In this work, a versatile dumbbell molecular (DM) probe was designed and employed in the sensitively homogeneous bioassay. In the presence of target molecule, the DM probe was protected from the digestion of exonucleases. Subsequently, the protected DM probe specifically bound to the intercalation dye and resulted in obvious fluorescence signal which was used to determine the target molecule in return. This design allows specific and versatile detection of diverse targets with easy operation and no sophisticated fluorescence labeling. Integrating the idea of target-protecting DM probe with adenosine triphosphate (ATP) involved ligation reaction, the DM probe with 5'-end phosphorylation was successfully constructed for ATP detection, and the limitation of detection was found to be 4.8 pM. Thanks to its excellent selectivity and sensitivity, this sensing strategy was used to detect ATP spiked in human serum as well as cellular ATP. Moreover, the proposed strategy was also applied in the visual detection of ATP in droplet-based microfluidic platform with satisfactory results. Similarly, combining the principle of target-protecting DM probe with streptavidin (SA)-biotin interaction, the DM probe with 3'-end biotinylation was developed for selective and sensitive SA determination, which demonstrated the robustness and versatility of this design. Copyright © 2016 Elsevier B.V. All rights reserved.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

PubMed

Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

2018-06-29

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

PubMed

Menchon, Grégory; Prota, Andrea E; Lucena-Agell, Daniel; Bucher, Pascal; Jansen, Rolf; Irschik, Herbert; Müller, Rolf; Paterson, Ian; Díaz, J Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

2018-05-29

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Comparison of the Sensitivity of Surface Downward Longwave Radiation to Changes in Water Vapor at Two High Elevation Sites

NASA Technical Reports Server (NTRS)

Chen, Yonghua; Naud, Catherine M.; Rangwala, Imtiaz; Landry, Christopher C.; Miller, James R.

2014-01-01

Among the potential reasons for enhanced warming rates in many high elevation regions is the nonlinear relationship between surface downward longwave radiation (DLR) and specific humidity (q). In this study we use ground-based observations at two neighboring high elevation sites in Southwestern Colorado that have different local topography and are 1.3 kilometers apart horizontally and 348 meters vertically. We examine the spatial consistency of the sensitivities (partial derivatives) of DLR with respect to changes in q, and the sensitivities are obtained from the Jacobian matrix of a neural network analysis. Although the relationship between DLR and q is the same at both sites, the sensitivities are higher when q is smaller, which occurs more frequently at the higher elevation site. There is a distinct hourly distribution in the sensitivities at both sites especially for high sensitivity cases, although the range is greater at the lower elevation site. The hourly distribution of the sensitivities relates to that of q. Under clear skies during daytime, q is similar between the two sites, however under cloudy skies or at night, it is not. This means that the DLR-q sensitivities are similar at the two sites during daytime but not at night, and care must be exercised when using data from one site to infer the impact of water vapor feedbacks at another site, particularly at night. Our analysis suggests that care should be exercised when using the lapse rate adjustment to infill high frequency data in a complex topographical region, particularly when one of the stations is subject to cold air pooling as found here.

Herbicide Safeners Decrease Sensitivity to Herbicides Inhibiting Acetolactate-Synthase and Likely Activate Non-Target-Site-Based Resistance Pathways in the Major Grass Weed Lolium sp. (Rye-Grass)

PubMed Central

Duhoux, Arnaud; Pernin, Fanny; Desserre, Diane; Délye, Christophe

2017-01-01

Herbicides are currently pivotal to control weeds and sustain food security. Herbicides must efficiently kill weeds while being as harmless as possible for crops, even crops taxonomically close to weeds. To increase their selectivity toward crops, some herbicides are sprayed in association with safeners that are bioactive compounds exacerbating herbicide-degrading pathways reputedly specifically in crops. However, exacerbated herbicide metabolism is also a key mechanism underlying evolved non-target-site-based resistance to herbicides (NTSR) in weeds. This raised the issue of a possible role of safeners on NTSR evolution in weeds. We investigated a possible effect of the respective field rates of the two broadly used safeners cloquintocet-mexyl and mefenpyr-diethyl on the sensitivity of the troublesome global weed Lolium sp. (rye-grass) to the major herbicides inhibiting acetolactate-synthase (ALS) pyroxsulam and iodosulfuron + mesosulfuron, respectively. Three Lolium sp. populations were studied in three series of experiments. The first experiment series compared the frequencies of plants surviving application of each herbicide alone or in association with its safener. Safener co-application caused a net increase ranging from 5.0 to 46.5% in the frequency of plants surviving the field rate of their associated herbicide. In a second series of experiments, safener effect was assessed on individual plant sensitivity using vegetative propagation. A reduction in sensitivity to pyroxsulam and to iodosulfuron + mesosulfuron was observed for 44.4 and 11.1% of the plants in co-treatment with cloquintocet-mexyl and mefenpyr-diethyl, respectively. A third series of experiments investigated safener effect on the expression level of 19 Lolium sp. NTSR marker genes. Safeners showed an enhancing effect on the expression level of 10 genes. Overall, we demonstrated that cloquintocet-mexyl and mefenpyr-diethyl both reduced the sensitivity of Lolium sp. to their associated ALS

Herbicide Safeners Decrease Sensitivity to Herbicides Inhibiting Acetolactate-Synthase and Likely Activate Non-Target-Site-Based Resistance Pathways in the Major Grass Weed Lolium sp. (Rye-Grass).

PubMed

Duhoux, Arnaud; Pernin, Fanny; Desserre, Diane; Délye, Christophe

2017-01-01

Herbicides are currently pivotal to control weeds and sustain food security. Herbicides must efficiently kill weeds while being as harmless as possible for crops, even crops taxonomically close to weeds. To increase their selectivity toward crops, some herbicides are sprayed in association with safeners that are bioactive compounds exacerbating herbicide-degrading pathways reputedly specifically in crops. However, exacerbated herbicide metabolism is also a key mechanism underlying evolved non-target-site-based resistance to herbicides (NTSR) in weeds. This raised the issue of a possible role of safeners on NTSR evolution in weeds. We investigated a possible effect of the respective field rates of the two broadly used safeners cloquintocet-mexyl and mefenpyr-diethyl on the sensitivity of the troublesome global weed Lolium sp. (rye-grass) to the major herbicides inhibiting acetolactate-synthase (ALS) pyroxsulam and iodosulfuron + mesosulfuron, respectively. Three Lolium sp. populations were studied in three series of experiments. The first experiment series compared the frequencies of plants surviving application of each herbicide alone or in association with its safener. Safener co-application caused a net increase ranging from 5.0 to 46.5% in the frequency of plants surviving the field rate of their associated herbicide. In a second series of experiments, safener effect was assessed on individual plant sensitivity using vegetative propagation. A reduction in sensitivity to pyroxsulam and to iodosulfuron + mesosulfuron was observed for 44.4 and 11.1% of the plants in co-treatment with cloquintocet-mexyl and mefenpyr-diethyl, respectively. A third series of experiments investigated safener effect on the expression level of 19 Lolium sp. NTSR marker genes. Safeners showed an enhancing effect on the expression level of 10 genes. Overall, we demonstrated that cloquintocet-mexyl and mefenpyr-diethyl both reduced the sensitivity of Lolium sp. to their associated ALS

Novel and viable acetylcholinesterase target site for developing effective and environmentally safe insecticides.

PubMed

Pang, Yuan-Ping; Brimijoin, Stephen; Ragsdale, David W; Zhu, Kun Yan; Suranyi, Robert

2012-04-01

Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market.

Influence of quasi-specific sites on kinetics of target DNA search by a sequence-specific DNA-binding protein.

PubMed

Kemme, Catherine A; Esadze, Alexandre; Iwahara, Junji

2015-11-10

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such "quasi-specific" sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1's association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins.

Influence of Quasi-Specific Sites on Kinetics of Target DNA Search by a Sequence-Specific DNA-Binding Protein

PubMed Central

2015-01-01

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such “quasi-specific” sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1’s association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins. PMID:26502071

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster

PubMed Central

Remnant, Emily J.; Good, Robert T.; Schmidt, Joshua M.; Lumb, Christopher; Robin, Charles; Daborn, Phillip J.; Batterham, Philip

2013-01-01

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala301 to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala301 to Ser resistance mutation and Met360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser301 homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser301 change into an Ala301 background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene. PMID:23959864

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

PubMed

Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

2016-06-07

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane.

PubMed

James, Scott E; Greenberg, Philip D; Jensen, Michael C; Lin, Yukang; Wang, Jinjuan; Till, Brian G; Raubitschek, Andrew A; Forman, Stephen J; Press, Oliver W

2008-05-15

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

Renton's Quendall Terminals on List of EPA Superfund Sites Targeted for Immediate, Intense Attention

EPA Pesticide Factsheets

EPA released the list of Superfund sites that Administrator Pruitt has targeted for intense and immediate attention, including the Quendall Terminals Site, a former creosote facility on the shore of Lake Washington in Renton, Washington.

Herceptin conjugated PLGA-PHis-PEG pH sensitive nanoparticles for targeted and controlled drug delivery.

PubMed

Zhou, Zilan; Badkas, Apurva; Stevenson, Max; Lee, Joo-Youp; Leung, Yuet-Kin

2015-06-20

A dual functional nano-scaled drug carrier, comprising of a targeting ligand and pH sensitivity, has been made in order to increase the specificity and efficacy of the drug delivery system. The nanoparticles are made of a tri-block copolymer, poly(d,l lactide-co-glycolide) (PLGA)-b-poly(l-histidine) (PHis)-b-polyethylene glycol (PEG), via nano-precipitation. To provide the nanoparticle feature of endolysosomal escape and pH sensitivity, poly(l-histidine) was chosen as a proton sponge polymer. Herceptin, which specifically binds to HER2 antigen, was conjugated to the nanoparticles through click chemistry. The nanoparticles were characterized via dynamic light scattering (DLS) and transmission electron microscopy (TEM). Both methods showed the sizes of about 100nm with a uniform size distribution. The pH sensitivity was assessed by drug releases and size changes at different pH conditions. As pH decreased from 7.4 to 5.2, the drug release rate accelerated and the size significantly increased. During in vitro tests against human breast cancer cell lines, MCF-7 and SK-BR-3 showed significantly increased uptake for Herceptin-conjugated nanoparticles, as compared to non-targeted nanoparticles. Herceptin-conjugated pH-sensitive nanoparticles showed the highest therapeutic effect, and thus validated the efficacy of a combined approach of pH sensitivity and active targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced Hyperbranched Amplification with Zero Background Signal.

PubMed

Zhang, Yan; Wang, Xin-Yan; Zhang, Qianyi; Zhang, Chun-Yang

2017-11-21

DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH 2 -modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10 -6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

PubMed

Thapa, Bindu; Bahadur Kc, Remant; Uludağ, Hasan

2018-02-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells. © 2017 UICC.

Target distance-dependent variation of hearing sensitivity during echolocation in a false killer whale.

PubMed

Supin, Alexander Ya; Nachtigall, Paul E; Breese, Marlee

2010-06-01

Evidence of varying hearing sensitivity according to the target distance was obtained in a false killer whale Pseudorca crassidens during echolocation. Auditory evoked potentials (AEPs) triggered by echolocation clicks were recorded. The target distance varied from 1 to 6 m. The records contained AEPs to the self-heard emitted click and AEPs to the echoes. Mean level of echolocation clicks depended on distance (the longer the distance, the higher the click level), however, the effect of click level on AEP amplitude was eliminated by extracting AEPs to clicks of certain particular levels. The amplitude of the echo-provoked AEP was almost independent of distance, however, the amplitude of the AEP to the emitted click, did depend on distance within a range from 1 to 4 m: the longer the distance, the higher the amplitude. The latter result is interpreted as confirmational evidence that the animal is capable of varying hearing sensitivity according to target distance. The variation of hearing sensitivity may help to compensate for the echo attenuation with distance; as a secondary effect, this variation manifested itself in a variation of the amplitude of the AEP to emitted clicks.

Novel and Viable Acetylcholinesterase Target Site for Developing Effective and Environmentally Safe Insecticides

PubMed Central

Pang, Yuan-Ping; Brimijoin, Stephen; Ragsdale, David W; Zhu, Kun Yan; Suranyi, Robert

2012-01-01

Insect pests are responsible for human suffering and financial losses worldwide. New and environmentally safe insecticides are urgently needed to cope with these serious problems. Resistance to current insecticides has resulted in a resurgence of insect pests, and growing concerns about insecticide toxicity to humans discourage the use of insecticides for pest control. The small market for insecticides has hampered insecticide development; however, advances in genomics and structural genomics offer new opportunities to develop insecticides that are less dependent on the insecticide market. This review summarizes the literature data that support the hypothesis that an insect-specific cysteine residue located at the opening of the acetylcholinesterase active site is a promising target site for developing new insecticides with reduced off-target toxicity and low propensity for insect resistance. These data are used to discuss the differences between targeting the insect-specific cysteine residue and targeting the ubiquitous catalytic serine residue of acetylcholinesterase from the perspective of reducing off-target toxicity and insect resistance. Also discussed is the prospect of developing cysteine-targeting anticholinesterases as effective and environmentally safe insecticides for control of disease vectors, crop damage, and residential insect pests within the financial confines of the present insecticide market. PMID:22280344

Field-Effect Biosensors for On-Site Detection: Recent Advances and Promising Targets.

PubMed

Choi, Jaebin; Seong, Tae Wha; Jeun, Minhong; Lee, Kwan Hyi

2017-10-01

There is an explosive interest in the immediate and cost-effective analysis of field-collected biological samples, as many advanced biodetection tools are highly sensitive, yet immobile. On-site biosensors are portable and convenient sensors that provide detection results at the point of care. They are designed to secure precision in highly ionic and heterogeneous solutions with minimal hardware. Among various methods that are capable of such analysis, field-effect biosensors are promising candidates due to their unique sensitivity, manufacturing scalability, and integrability with computational circuitry. Recent developments in nanotechnological surface modification show promising results in sensing from blood, serum, and urine. This report gives a particular emphasis on the on-site efficacy of recently published field-effect biosensors, specifically, detection limits in physiological solutions, response times, and scalability. The survey of the properties and existing detection methods of four promising biotargets, exosomes, bacteria, viruses, and metabolites, aims at providing a roadmap for future field-effect and other on-site biosensors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultra-Sensitive Detection of Plasmodium falciparum by Amplification of Multi-Copy Subtelomeric Targets

PubMed Central

Hofmann, Natalie; Mwingira, Felista; Shekalaghe, Seif; Robinson, Leanne J.; Mueller, Ivo; Felger, Ingrid

2015-01-01

Background Planning and evaluating malaria control strategies relies on accurate definition of parasite prevalence in the population. A large proportion of asymptomatic parasite infections can only be identified by surveillance with molecular methods, yet these infections also contribute to onward transmission to mosquitoes. The sensitivity of molecular detection by PCR is limited by the abundance of the target sequence in a DNA sample; thus, detection becomes imperfect at low densities. We aimed to increase PCR diagnostic sensitivity by targeting multi-copy genomic sequences for reliable detection of low-density infections, and investigated the impact of these PCR assays on community prevalence data. Methods and Findings Two quantitative PCR (qPCR) assays were developed for ultra-sensitive detection of Plasmodium falciparum, targeting the high-copy telomere-associated repetitive element 2 (TARE-2, ∼250 copies/genome) and the var gene acidic terminal sequence (varATS, 59 copies/genome). Our assays reached a limit of detection of 0.03 to 0.15 parasites/μl blood and were 10× more sensitive than standard 18S rRNA qPCR. In a population cross-sectional study in Tanzania, 295/498 samples tested positive using ultra-sensitive assays. Light microscopy missed 169 infections (57%). 18S rRNA qPCR failed to identify 48 infections (16%), of which 40% carried gametocytes detected by pfs25 quantitative reverse-transcription PCR. To judge the suitability of the TARE-2 and varATS assays for high-throughput screens, their performance was tested on sample pools. Both ultra-sensitive assays correctly detected all pools containing one low-density P. falciparum–positive sample, which went undetected by 18S rRNA qPCR, among nine negatives. TARE-2 and varATS qPCRs improve estimates of prevalence rates, yet other infections might still remain undetected when absent in the limited blood volume sampled. Conclusions Measured malaria prevalence in communities is largely determined by the

Evaluation of Uncertainty and Sensitivity in Environmental Modeling at a Radioactive Waste Management Site

NASA Astrophysics Data System (ADS)

Stockton, T. B.; Black, P. K.; Catlett, K. M.; Tauxe, J. D.

2002-05-01

Environmental modeling is an essential component in the evaluation of regulatory compliance of radioactive waste management sites (RWMSs) at the Nevada Test Site in southern Nevada, USA. For those sites that are currently operating, further goals are to support integrated decision analysis for the development of acceptance criteria for future wastes, as well as site maintenance, closure, and monitoring. At these RWMSs, the principal pathways for release of contamination to the environment are upward towards the ground surface rather than downwards towards the deep water table. Biotic processes, such as burrow excavation and plant uptake and turnover, dominate this upward transport. A combined multi-pathway contaminant transport and risk assessment model was constructed using the GoldSim modeling platform. This platform facilitates probabilistic analysis of environmental systems, and is especially well suited for assessments involving radionuclide decay chains. The model employs probabilistic definitions of key parameters governing contaminant transport, with the goals of quantifying cumulative uncertainty in the estimation of performance measures and providing information necessary to perform sensitivity analyses. This modeling differs from previous radiological performance assessments (PAs) in that the modeling parameters are intended to be representative of the current knowledge, and the uncertainty in that knowledge, of parameter values rather than reflective of a conservative assessment approach. While a conservative PA may be sufficient to demonstrate regulatory compliance, a parametrically honest PA can also be used for more general site decision-making. In particular, a parametrically honest probabilistic modeling approach allows both uncertainty and sensitivity analyses to be explicitly coupled to the decision framework using a single set of model realizations. For example, sensitivity analysis provides a guide for analyzing the value of collecting more

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling.

PubMed

Su, Jiao; Zhang, Haijie; Jiang, Bingying; Zheng, Huzhi; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

2011-11-15

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

Highly sensitive and specific colorimetric detection of cancer cells via dual-aptamer target binding strategy.

PubMed

Wang, Kun; Fan, Daoqing; Liu, Yaqing; Wang, Erkang

2015-11-15

Simple, rapid, sensitive and specific detection of cancer cells is of great importance for early and accurate cancer diagnostics and therapy. By coupling nanotechnology and dual-aptamer target binding strategies, we developed a colorimetric assay for visually detecting cancer cells with high sensitivity and specificity. The nanotechnology including high catalytic activity of PtAuNP and magnetic separation & concentration plays a vital role on the signal amplification and improvement of detection sensitivity. The color change caused by small amount of target cancer cells (10 cells/mL) can be clearly distinguished by naked eyes. The dual-aptamer target binding strategy guarantees the detection specificity that large amount of non-cancer cells and different cancer cells (10(4) cells/mL) cannot cause obvious color change. A detection limit as low as 10 cells/mL with detection linear range from 10 to 10(5) cells/mL was reached according to the experimental detections in phosphate buffer solution as well as serum sample. The developed enzyme-free and cost effective colorimetric assay is simple and no need of instrument while still provides excellent sensitivity, specificity and repeatability, having potential application on point-of-care cancer diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Mitochondrial DNA Targets Increase Sensitivity of Malaria Detection Using Loop-Mediated Isothermal Amplification ▿

PubMed Central

Polley, Spencer D.; Mori, Yasuyoshi; Watson, Julie; Perkins, Mark D.; González, Iveth J.; Notomi, Tsugunori; Chiodini, Peter L.; Sutherland, Colin J.

2010-01-01

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per μl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per μl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays. PMID:20554824

NGR-modified pH-sensitive liposomes for controlled release and tumor target delivery of docetaxel.

PubMed

Gu, Zili; Chang, Minglu; Fan, Yang; Shi, Yanbin; Lin, Guimei

2017-12-01

As current tumor chemotherapy faces many challenges, it is important to develop drug delivery systems with increased tumor-targeting ability, enhanced therapeutic effects and reduced side effects. In this study, a pH-sensitive liposome was constructed containing CHEMS-anchored PEG2000 for extended circulation and NGR peptide as the targeting moiety. The NGR-modified docetaxel-loaded pH-sensitive extended-circulation liposomes (DTX/NGR-PLL) prepared possess suitable physiochemical properties, including particle size of approximately 200nm, drug encapsulation efficiency of approximately 70%, and pH-sensitive drug release properties. Experiments performed in vitro and in vivo on human fibrosarcoma cells (HT-1080) and human breast adenocarcinoma cells (MCF-7) verified the specific targeting ability and enhanced antitumor activity to HT-1080 cells. The results of intravenous administration demonstrated that NGR-modified liposomes can significantly and safely accumulate in tumor tissue in xenografted nude mice. In conclusion, the liposomes constructed hold promise as a safe and efficient drug delivery system for specific tumor treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion.

PubMed

Gladyshev, Eugene A; Arkhipova, Irina R

2009-12-15

Ribosomal DNA genes in many eukaryotes contain insertions of non-LTR retrotransposable elements belonging to the R2 clade. These elements persist in the host genomes by inserting site-specifically into multicopy target sites, thereby avoiding random disruption of single-copy host genes. Here we describe R9 retrotransposons from the R2 clade in the 28S RNA genes of bdelloid rotifers, small freshwater invertebrate animals best known for their long-term asexuality and for their ability to survive repeated cycles of desiccation and rehydration. While the structural organization of R9 elements is highly similar to that of other members of the R2 clade, they are characterized by two distinct features: site-specific insertion into a previously unreported target sequence within the 28S gene, and an unusually long target site duplication of 126 bp. We discuss the implications of these findings in the context of bdelloid genome organization and the mechanisms of target-primed reverse transcription.

Annualized earthquake loss estimates for California and their sensitivity to site amplification

USGS Publications Warehouse

Chen, Rui; Jaiswal, Kishor; Bausch, D; Seligson, H; Wills, C.J.

2016-01-01

Input datasets for annualized earthquake loss (AEL) estimation for California were updated recently by the scientific community, and include the National Seismic Hazard Model (NSHM), site‐response model, and estimates of shear‐wave velocity. Additionally, the Federal Emergency Management Agency’s loss estimation tool, Hazus, was updated to include the most recent census and economic exposure data. These enhancements necessitated a revisit to our previous AEL estimates and a study of the sensitivity of AEL estimates subjected to alternate inputs for site amplification. The NSHM ground motions for a uniform site condition are modified to account for the effect of local near‐surface geology. The site conditions are approximated in three ways: (1) by VS30 (time‐averaged shear‐wave velocity in the upper 30 m) value obtained from a geology‐ and topography‐based map consisting of 15 VS30 groups, (2) by site classes categorized according to National Earthquake Hazards Reduction Program (NEHRP) site classification, and (3) by a uniform NEHRP site class D. In case 1, ground motions are amplified using the Seyhan and Stewart (2014) semiempirical nonlinear amplification model. In cases 2 and 3, ground motions are amplified using the 2014 version of the NEHRP site amplification factors, which are also based on the Seyhan and Stewart model but are approximated to facilitate their use for building code applications. Estimated AELs are presented at multiple resolutions, starting with the state level assessment and followed by detailed assessments for counties, metropolitan statistical areas (MSAs), and cities. AEL estimate at the state level is ∼$3.7  billion, 70% of which is contributed from Los Angeles–Long Beach–Santa Ana, San Francisco–Oakland–Fremont, and Riverside–San Bernardino–Ontario MSAs. The statewide AEL estimate is insensitive to alternate assumptions of site amplification. However, we note significant differences in AEL estimates

Characterization of mechano-sensitive nano-containers for targeted vasodilation

NASA Astrophysics Data System (ADS)

Buscema, Marzia; Deyhle, Hans; Pfohl, Thomas; Hieber, Simone E.; Zumbuehl, Andreas; Müller, Bert

2016-04-01

Cardiovascular diseases are the worldwide number one cause of mortality. The blood flow in diseased human coronary arteries differs from the blood flow in the healthy vessels. This fact should be used for designing targeted localized delivery of vasodilators with a purely physical drug release trigger. Thus, we have proposed mechano-sensitive liposomes as mechano-sensitive containers. One has to tailor the liposome's properties, so that containers are stable under physiological conditions in health, but release their cargo near the constricted vessels at body temperature. In order to determine the shear stress threshold for release, both the morphology of the healthy and diseased human arteries and the mechanical property of the liposomes have to be known. We have shown that micro computed tomography (μCT) techniques allow visualizing the lumen of human coronary arteries and provide the basis for flow simulations to extract the wall shear stress of healthy and stenosed regions in human coronary arteries. The behavior of the mechano-sensitive liposomes is currently investigated by means of microfluidics and spatially resolved small-angle X-ray scattering. The liposomes are injected into micro-channels mimicking in vivo situation. The scattering signal from the liposomes reveals information about their size, shape, and wall thickness.

Instrumentation of Molecular Imaging on Site-Specific Targeting Fluorescent Peptide for Early Detection of Breast Cancer

NASA Astrophysics Data System (ADS)

Yu, Ping; Ma, Lixin

2012-02-01

In this work we developed two biomedical imaging techniques for early detection of breast cancer. Both image modalities provide molecular imaging capability to probe site-specific targeting dyes. The first technique, heterodyne CCD fluorescence mediated tomography, is a non-invasive biomedical imaging that uses fluorescent photons from the targeted dye on the tumor cells inside human breast tissue. The technique detects a large volume of tissue (20 cm) with a moderate resolution (1 mm) and provides the high sensitivity. The second technique, dual-band spectral-domain optical coherence tomography, is a high-resolution tissue imaging modality. It uses a low coherence interferometer to detect coherent photons hidden in the incoherent background. Due to the coherence detection, a high resolution (20 microns) is possible. We have finished prototype imaging systems for the development of both image modalities and performed imaging experiments on tumor tissues. The spectroscopic/tomographic images show contrasts of dense tumor tissues and tumor necrotic regions. In order to correlate the findings from our results, a diffusion-weighted magnetic resonance imaging (MRI) of the tumors was performed using a small animal 7-Telsa MRI and demonstrated excellent agreement.

Human Mars Ascent Vehicle Performance Sensitivities

NASA Technical Reports Server (NTRS)

Polsgrove, Tara P.; Thomas, Herbert D.

2016-01-01

Human Mars mission architecture studies have shown that the ascent vehicle mass drives performance requirements for the descent and in-space transportation elements. Understanding the sensitivity of Mars ascent vehicle (MAV) mass to various mission and vehicle design choices enables overall transportation system optimization. This paper presents the results of a variety of sensitivity trades affecting MAV performance including: landing site latitude, target orbit, initial thrust to weight ratio, staging options, specific impulse, propellant type and engine design.

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

PubMed Central

Tan, Benedict G.; Vijgenboom, Erik; Worrall, Jonathan A. R.

2014-01-01

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control. PMID:24121681

A mathematical model of single target site location by Brownian movement in subcellular compartments.

PubMed

Kuthan, Hartmut

2003-03-07

The location of distinct sites is mandatory for many cellular processes. In the subcompartments of the cell nucleus, only very small numbers of diffusing macromolecules and specific target sites of some types may be present. In this case, we are faced with the Brownian movement of individual macromolecules and their "random search" for single/few specific target sites, rather than bulk-averaged diffusion and multiple sites. In this article, I consider the location of a distant central target site, e.g. a globular protein, by individual macromolecules executing unbiased (i.e. drift-free) random walks in a spherical compartment. For this walk-and-capture model, the closed-form analytic solution of the first passage time probability density function (p.d.f.) has been obtained as well as the first and second moment. In the limit of a large ratio of the radii of the spherical diffusion space and central target, well-known relations for the variance and the first two moments for the exponential p.d.f. were found to hold with high accuracy. These calculations reinforce earlier numerical results and Monte Carlo simulations. A major implication derivable from the model is that non-directed random movement is an effective means for locating single sites in submicron-sized compartments, even when the diffusion coefficients are comparatively small and the diffusing species are present in one copy only. These theoretical conclusions are underscored numerically for effective diffusion constants ranging from 0.5 to 10.0 microm(2) s(-1), which have been reported for a couple of nuclear proteins in their physiological environment. Spherical compartments of submicron size are, for example, the Cajal bodies (size: 0.1-1.0 microm), which are present in 1-5 copies in the cell nucleus. Within a small Cajal body of radius 0.1 microm a single diffusing protein molecule (with D=0.5 microm(2) s(-1)) would encounter a medium-sized protein of radius 2.5 nm within 1 s with a probability near

Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites.

PubMed

Qudrat, Anam; Mosabbir, Abdullah Al; Truong, Kevin

2017-06-22

Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca 2+ -activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca 2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Centredale Manor Superfund Site in Rhode Island included on EPA List of Targeted for Immediate Attention

EPA Pesticide Factsheets

Today, the U.S. Environmental Protection Agency released the list of Superfund sites that Administrator Pruitt has targeted for immediate and intense attention. The Centredale Manor Restoration Project superfund site is one of the 21 sites on the list.

The allosteric site regulates the voltage sensitivity of muscarinic receptors.

PubMed

Hoppe, Anika; Marti-Solano, Maria; Drabek, Matthäus; Bünemann, Moritz; Kolb, Peter; Rinne, Andreas

2018-01-01

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M 1 -Rs and M 3 -Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G q protein cycle. In the presence of ACh, M 1 -R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M 3 -R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M 3 /M 1 -R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M 3 -Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects. Copyright © 2017 Elsevier Inc. All rights reserved.

Spy: a new group of eukaryotic DNA transposons without target site duplications.

PubMed

Han, Min-Jin; Xu, Hong-En; Zhang, Hua-Hao; Feschotte, Cédric; Zhang, Ze

2014-06-24

Class 2 or DNA transposons populate the genomes of most eukaryotes and like other mobile genetic elements have a profound impact on genome evolution. Most DNA transposons belong to the cut-and-paste types, which are relatively simple elements characterized by terminal-inverted repeats (TIRs) flanking a single gene encoding a transposase. All eukaryotic cut-and-paste transposons so far described are also characterized by target site duplications (TSDs) of host DNA generated upon chromosomal insertion. Here, we report a new group of evolutionarily related DNA transposons called Spy, which also include TIRs and DDE motif-containing transposase but surprisingly do not create TSDs upon insertion. Instead, Spy transposons appear to transpose precisely between 5'-AAA and TTT-3' host nucleotides, without duplication or modification of the AAATTT target sites. Spy transposons were identified in the genomes of diverse invertebrate species based on transposase homology searches and structure-based approaches. Phylogenetic analyses indicate that Spy transposases are distantly related to IS5, ISL2EU, and PIF/Harbinger transposases. However, Spy transposons are distinct from these and other DNA transposon superfamilies by their lack of TSD and their target site preference. Our findings expand the known diversity of DNA transposons and reveal a new group of eukaryotic DDE transposases with unusual catalytic properties. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI

PubMed Central

Shen, Betty; Heiter, Daniel F.; Chan, Siu-Hong; Wang, Hua; Xu, Shuang-Yong; Morgan, Richard D.; Wilson, Geoffrey G.; Stoddard, Barry L.

2010-01-01

The crystal structure of the rare-cutting HNH restriction endonuclease PacI in complex with its eight base pair target recognition sequence 5'-TTAATTAA-3' has been determined to 1.9 Å resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a ββα-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand-cleavage. PacI dramatically distorts its target sequence from Watson-Crick duplex DNA basepairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in non-canonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures. PMID:20541511

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

PubMed

Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

2013-04-10

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

Vascular targeting of a gold nanoparticle to breast cancer metastasis

PubMed Central

Peiris, Pubudu M.; Deb, Partha; Doolittle, Elizabeth; Doron, Gilad; Goldberg, Amy; Govender, Priya; Shah, Shruti; Rao, Swetha; Carbone, Sarah; Cotey, Thomas; Sylvestre, Meilyn; Singh, Sohaj; Schiemann, William P.; Lee, Zhenghong; Karathanasis, Efstathios

2015-01-01

The vast majority of breast cancer deaths are due to metastatic disease. While deep tissue targeting of nanoparticles is suitable for some primary tumors, vascular targeting may be a more attractive strategy for micrometastasis. This study combined a vascular targeting strategy with the enhanced targeting capabilities of a nanoparticle to evaluate the ability of a gold nanoparticle to specifically target the early spread of metastatic disease. As a ligand for the vascular targeting strategy, we utilized a peptide targeting alpha(v) beta(3) integrin, which is functionally linked to the development of micrometastases at a distal site. By employing a straightforward radiolabeling method to incorporate Technetium-99m into the gold nanoparticles, we used the high sensitivity of radionuclide imaging to monitor the longitudinal accumulation of the nanoparticles in metastatic sites. Animal and histological studies showed that vascular targeting of the nanoparticle facilitated highly accurate targeting of micrometastasis in the 4T1 mouse model of breast cancer metastasis using radionuclide imaging and a low dose of the nanoparticle. Due to the efficient targeting scheme, 14% of the injected AuNP deposited at metastatic sites in the lungs within 60 min after injection, indicating that the vascular bed of metastasis is a viable target site for nanoparticles. PMID:26036431

Analysis on establishing Chang'E-3 landing site as a reflectance calibration target

NASA Astrophysics Data System (ADS)

Liu, Bin; Fu, Xiaohui; Zeng, Xingguo; Yao, Meijuan; Zhang, Hongbo; Su, Yan; Zhao, Shu; Xue, Xiping; Li, Chunlai; Zou, Yongliao

2015-04-01

Recent lunar orbital observations suggested that the surface reflectance calculated based on the Apollo 16 standard area and Apollo 16 sample laboratory measurement is significantly different from its true value [1-3], one reason is the composition and maturity differences between the 62231 sampling site and the Apollo 16 standard site existed, the other reason is the physical properties of the returned lunar sample, such as porosity, have been changed during the sampling operations. So more new standard targets on the Moon, besides the widely used Apollo 16 area, are needed for imaging spectrometers on lunar missions to improve their reflectance calibration accuracies. The Chang'E-3 VIS/NIR Imaging Spectrometer (VNIS), which is just fixed at the front of the Yutu rover [4], equipped with a white spectralon panel as reflectance calibration standard, can perform in situ multispectral observations around the Chang'E-3 landing site without altering the physical and mineralogical natures of lunar soils. Therefore, it provides an opportunity to establish a new reliable standard target for in-flight reflectance calibration. The reflectance calibration target should be compositional homogeneous, the topography of which must be flat, and the reflectance should be identical with no nearby units of other different materials. As we have known, Chang'e-3 probe landed on the Mare Imbrium basin in the east part of Sinus Iridum, the landing site is relatively flat at a spatial coverage of ~660km2, and this region belongs to Eratosthenian low-Ti/high-Ti mare basalts [5-6]. According to much higher resolution topography data, elemental data and reflectance data of Chang'E-2 and Chang'E-3[7-8], we preliminary analyse the possibility on establishing Chang'E-3 landing site as a reflectance calibration target. Firstly, the overall terrain of the 4 km×4 km area around the landing site is flat, but there are still three bigger craters existed. Secondly, the composition on Chang'E-3

TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery.

PubMed

Vanwetswinkel, Sophie; Heetebrij, Robert J; van Duynhoven, John; Hollander, Johan G; Filippov, Dmitri V; Hajduk, Philip J; Siegal, Gregg

2005-02-01

We propose a ligand screening method, called TINS (target immobilized NMR screening), which reduces the amount of target required for the fragment-based approach to drug discovery. Binding is detected by comparing 1D NMR spectra of compound mixtures in the presence of a target immobilized on a solid support to a control sample. The method has been validated by the detection of a variety of ligands for protein and nucleic acid targets (K(D) from 60 to 5000 muM). The ligand binding capacity of a protein was undiminished after 2000 different compounds had been applied, indicating the potential to apply the assay for screening typical fragment libraries. TINS can be used in competition mode, allowing rapid characterization of the ligand binding site. TINS may allow screening of targets that are difficult to produce or that are insoluble, such as membrane proteins.

A multi-criteria targeting approach to neutral grassland conservation.

PubMed

Bayliss, Julian; Helyar, Alice; Lee, John T; Thompson, Stewart

2003-02-01

Resources for creating and managing rare habitats are limited, and a targeting approach aimed at identifying the most viable sites for habitat conservation is therefore desirable. This study developed a multi-criteria targeting approach to site conservation for two rare grassland types, based on a suite of biotic and abiotic factors managed within a Geographical Information System. A number of biotic and abiotic criteria were assessed to evaluate the biodiversity status of grassland sites. Biotic factors included species diversity, species richness and species rarity; and abiotic factors included patch area, position in the ecological unit and the influence of surrounding land use. Each criterion was given equal weighting and a final biodiversity value for each patch was calculated; the patch with the highest cumulative rank score was deemed the patch with the greatest biodiversity. Each site was then examined in relation to agricultural land under the existing management prescriptions of the Upper Thames Tributaries Environmentally Sensitive Area (UTTESA). Sites identified with high biodiversity potential, but currently not included under management prescriptions, were targeted for future inclusion in the ESA scheme. The targeting approach demonstrated how the national Lowland Meadows habitat action plan creation target of 500 ha could be achieved in the UTTESA. The fact that this target figure was so easily attained within this study area highlighted the possible underestimation of national habitat creation targets.

Immunological Reactivity Using Monoclonal and Polyclonal Antibodies of Autoimmune Thyroid Target Sites with Dietary Proteins

PubMed Central

Herbert, Martha

2017-01-01

Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods. Additionally, food interactions may play a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage. Our study was designed to investigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sites. We identified immune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones, thyroid receptors, enzymes, and transport proteins. We also measured immune reactivity of either target specific monoclonal or polyclonal antibodies for thyroid-stimulating hormone (TSH) receptor, 5′deiodinase, thyroid peroxidase, thyroglobulin, thyroxine-binding globulin, thyroxine, and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms. Dietary protein determinants included unmodified (raw) and modified (cooked and roasted) foods, herbs, spices, food gums, brewed beverages, and additives. There were no dietary protein immune reactions with TSH receptor, thyroid peroxidase, and thyroxine-binding globulin. However, specific antigen-antibody immune reactivity was identified with several purified food proteins with triiodothyronine, thyroxine, thyroglobulin, and 5′deiodinase. Laboratory analysis of immunological cross-reactivity between thyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potential immunoreactive role in autoimmune thyroid disease. PMID:28894619

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Searching target sites on DNA by proteins: Role of DNA dynamics under confinement

PubMed Central

Mondal, Anupam; Bhattacherjee, Arnab

2015-01-01

DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. PMID:26400158

CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.

PubMed

Fedoreyeva, L I; Vanyushin, B F

2011-06-01

Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyribooligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m(5)CAG, m(5)CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction--modification systems or some of their elements, at least, may exist.

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

PubMed Central

Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

2015-01-01

Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

Antigen sensitivity of CD22-specific chimeric T cell receptors is modulated by target epitope distance from the cell membrane

PubMed Central

James, Scott E.; Greenberg, Philip D.; Jensen, Michael C.; Lin, Yukang; Wang, Jinjuan; Till, Brian G.; Raubitschek, Andrew A.; Forman, Stephen J.; Press, Oliver W.

2008-01-01

We have targeted CD22 as a novel tumor-associated antigen for recognition by human CTL genetically modified to express chimeric T cell receptors (cTCR) recognizing this surface molecule. CD22-specifc cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower antigen sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of antigen engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope but constructed as a truncated CD22 molecule to approximate the length of a TCR:pMHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for antigen-induced triggering of effector functions has potential therapeutic applications, as such cells selectively lysed B cell lymphoma lines expressing high levels of CD22 but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength – and consequently antigen sensitivity – can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate antigen density. PMID:18453625

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

Vegetation and the importance of insecticide-treated target siting for control of Glossina fuscipes fuscipes.

PubMed

Esterhuizen, Johan; Njiru, Basilio; Vale, Glyn A; Lehane, Michael J; Torr, Stephen J

2011-09-01

Control of tsetse flies using insecticide-treated targets is often hampered by vegetation re-growth and encroachment which obscures a target and renders it less effective. Potentially this is of particular concern for the newly developed small targets (0.25 high × 0.5 m wide) which show promise for cost-efficient control of Palpalis group tsetse flies. Consequently the performance of a small target was investigated for Glossina fuscipes fuscipes in Kenya, when the target was obscured following the placement of vegetation to simulate various degrees of natural bush encroachment. Catches decreased significantly only when the target was obscured by more than 80%. Even if a small target is underneath a very low overhanging bush (0.5 m above ground), the numbers of G. f. fuscipes decreased by only about 30% compared to a target in the open. We show that the efficiency of the small targets, even in small (1 m diameter) clearings, is largely uncompromised by vegetation re-growth because G. f. fuscipes readily enter between and under vegetation. The essential characteristic is that there should be some openings between vegetation. This implies that for this important vector of HAT, and possibly other Palpalis group flies, a smaller initial clearance zone around targets can be made and longer interval between site maintenance visits is possible both of which will result in cost savings for large scale operations. We also investigated and discuss other site features e.g. large solid objects and position in relation to the water's edge in terms of the efficacy of the small targets.

Vascular Targeting of a Gold Nanoparticle to Breast Cancer Metastasis.

PubMed

Peiris, Pubudu M; Deb, Partha; Doolittle, Elizabeth; Doron, Gilad; Goldberg, Amy; Govender, Priya; Shah, Shruti; Rao, Swetha; Carbone, Sarah; Cotey, Thomas; Sylvestre, Meilyn; Singh, Sohaj; Schiemann, William P; Lee, Zhenghong; Karathanasis, Efstathios

2015-08-01

The vast majority of breast cancer deaths are due to metastatic disease. Although deep tissue targeting of nanoparticles is suitable for some primary tumors, vascular targeting may be a more attractive strategy for micrometastasis. This study combined a vascular targeting strategy with the enhanced targeting capabilities of a nanoparticle to evaluate the ability of a gold nanoparticle (AuNP) to specifically target the early spread of metastatic disease. As a ligand for the vascular targeting strategy, we utilized a peptide targeting alpha(v) beta(3) integrin, which is functionally linked to the development of micrometastases at a distal site. By employing a straightforward radiolabeling method to incorporate Technetium-99m into the AuNPs, we used the high sensitivity of radionuclide imaging to monitor the longitudinal accumulation of the nanoparticles in metastatic sites. Animal and histological studies showed that vascular targeting of the nanoparticle facilitated highly accurate targeting of micrometastasis in the 4T1 mouse model of breast cancer metastasis using radionuclide imaging and a low dose of the nanoparticle. Because of the efficient targeting scheme, 14% of the injected AuNP deposited at metastatic sites in the lungs within 60 min after injection, indicating that the vascular bed of metastasis is a viable target site for nanoparticles. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Sequences in Glycoprotein gp41, the CD4 Binding Site, and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

PubMed Central

O'Rourke, Sara M.; Schweighardt, Becky; Phung, Pham; Mesa, Kathryn A.; Vollrath, Aaron L.; Tatsuno, Gwen P.; To, Briana; Sinangil, Faruk; Limoli, Kay; Wrin, Terri

2012-01-01

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals. PMID:22933284

Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

PubMed Central

Geisler, Anja; Schön, Christian; Größl, Tobias; Pinkert, Sandra; Stein, Elisabeth A; Kurreck, Jens; Vetter, Roland; Fechner, Henry

2013-01-01

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs. PMID:23439498

Multifunctional pH-sensitive superparamagnetic iron-oxide nanocomposites for targeted drug delivery and MR imaging.

PubMed

Zhu, Lijuan; Wang, Dali; Wei, Xuan; Zhu, Xinyuan; Li, Jianqi; Tu, Chunlai; Su, Yue; Wu, Jieli; Zhu, Bangshang; Yan, Deyue

2013-08-10

A multifunctional pH-sensitive superparamagnetic iron-oxide (SPIO) nanocomposite system was developed for simultaneous tumor magnetic resonance imaging (MRI) and therapy. Small-size SPIO nanoparticles were chemically bonded with antitumor drug doxorubicin (DOX) and biocompatible poly(ethylene glycol) (PEG) through pH-sensitive acylhydrazone linkages, resulting in the formation of SPIO nanocomposites with magnetic targeting and pH-sensitive properties. These DOX-conjugated SPIO nanocomposites exhibited not only good stability in aqueous solution but also high saturation magnetizations. Under an acidic environment, the DOX was quickly released from the SPIO nanocomposites due to the cleavage of pH-sensitive acylhydrazone linkages. With the help of magnetic field, the DOX-conjugated SPIO nanocomposites showed high cellular uptake, indicating their magnetic targeting property. Comparing to free DOX, the DOX-conjugated SPIO nanocomposites showed better antitumor effect under magnetic field. At the same time, the relaxivity value of these SPIO nanocomposites was higher than 146s(-1)mM(-1) Fe, leading to ~4 times enhancement compared to that of free SPIO nanoparticles. As a negative contrast agent, these SPIO nanocomposites illustrated high resolution in MRI diagnosis of tumor-bearing mice. All of these results confirm that these pH-sensitive SPIO nanocomposites are promising hybrid materials for synergistic MRI diagnosis and tumor therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

pH-sensitive oncolytic adenovirus hybrid targeting acidic tumor microenvironment and angiogenesis

PubMed Central

Choi, Joung-Woo; Jung, Soo-Jung; Kasala, Dayananda; Hwang, June Kyu; Hu, Jun; Bae, You Han; Yun, Chae-Ok

2015-01-01

Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH 6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH 6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH 7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX. PMID:25575865

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells.

PubMed

Benakanakere, Indira; Johnson, Tyler; Sleightholm, Richard; Villeda, Virgilio; Arya, Monika; Bobba, Ravi; Freter, Carl; Huang, Chunfa

2014-01-01

Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment.

Sensitive and Specific Target Sequences Selected from Retrotransposons of Schistosoma japonicum for the Diagnosis of Schistosomiasis

PubMed Central

Xu, Jing; Zhu, Xing-Quan; Wang, Sheng-Yue; Xia, Chao-Ming

2012-01-01

Background Schistosomiasis japonica is a serious debilitating and sometimes fatal disease. Accurate diagnostic tests play a key role in patient management and control of the disease. However, currently available diagnostic methods are not ideal, and the detection of the parasite DNA in blood samples has turned out to be one of the most promising tools for the diagnosis of schistosomiasis. In our previous investigations, a 230-bp sequence from the highly repetitive retrotransposon SjR2 was identified and it showed high sensitivity and specificity for detecting Schistosoma japonicum DNA in the sera of rabbit model and patients. Recently, 29 retrotransposons were found in S. japonicum genome by our group. The present study highlighted the key factors for selecting a new perspective sensitive target DNA sequence for the diagnosis of schistosomiasis, which can serve as example for other parasitic pathogens. Methodology/Principal Findings In this study, we demonstrated that the key factors based on the bioinformatic analysis for selecting target sequence are the higher genome proportion, repetitive complete copies and partial copies, and active ESTs than the others in the chromosome genome. New primers based on 25 novel retrotransposons and SjR2 were designed and their sensitivity and specificity for detecting S. japonicum DNA were compared. The results showed that a new 303-bp sequence from non-long terminal repeat (LTR) retrotransposon (SjCHGCS19) had high sensitivity and specificity. The 303-bp target sequence was amplified from the sera of rabbit model at 3 d post-infection by nested-PCR and it became negative at 17 weeks post-treatment. Furthermore, the percentage sensitivity of the nested-PCR was 97.67% in 43 serum samples of S. japonicum-infected patients. Conclusions/Significance Our findings highlighted the key factors based on the bioinformatic analysis for selecting target sequence from S. japonicum genome, which provide basis for establishing powerful

Protecting Important Sites for Biodiversity Contributes to Meeting Global Conservation Targets

PubMed Central

Butchart, Stuart H. M.; Scharlemann, Jörn P. W.; Evans, Mike I.; Quader, Suhel; Aricò, Salvatore; Arinaitwe, Julius; Balman, Mark; Bennun, Leon A.; Bertzky, Bastian; Besançon, Charles; Boucher, Timothy M.; Brooks, Thomas M.; Burfield, Ian J.; Burgess, Neil D.; Chan, Simba; Clay, Rob P.; Crosby, Mike J.; Davidson, Nicholas C.; De Silva, Naamal; Devenish, Christian; Dutson, Guy C. L.; Fernández, David F. Día z; Fishpool, Lincoln D. C.; Fitzgerald, Claire; Foster, Matt; Heath, Melanie F.; Hockings, Marc; Hoffmann, Michael; Knox, David; Larsen, Frank W.; Lamoreux, John F.; Loucks, Colby; May, Ian; Millett, James; Molloy, Dominic; Morling, Paul; Parr, Mike; Ricketts, Taylor H.; Seddon, Nathalie; Skolnik, Benjamin; Stuart, Simon N.; Upgren, Amy; Woodley, Stephen

2012-01-01

Protected areas (PAs) are a cornerstone of conservation efforts and now cover nearly 13% of the world's land surface, with the world's governments committed to expand this to 17%. However, as biodiversity continues to decline, the effectiveness of PAs in reducing the extinction risk of species remains largely untested. We analyzed PA coverage and trends in species' extinction risk at globally significant sites for conserving birds (10,993 Important Bird Areas, IBAs) and highly threatened vertebrates and conifers (588 Alliance for Zero Extinction sites, AZEs) (referred to collectively hereafter as ‘important sites’). Species occurring in important sites with greater PA coverage experienced smaller increases in extinction risk over recent decades: the increase was half as large for bird species with>50% of the IBAs at which they occur completely covered by PAs, and a third lower for birds, mammals and amphibians restricted to protected AZEs (compared with unprotected or partially protected sites). Globally, half of the important sites for biodiversity conservation remain unprotected (49% of IBAs, 51% of AZEs). While PA coverage of important sites has increased over time, the proportion of PA area covering important sites, as opposed to less important land, has declined (by 0.45–1.14% annually since 1950 for IBAs and 0.79–1.49% annually for AZEs). Thus, while appropriately located PAs may slow the rate at which species are driven towards extinction, recent PA network expansion has under-represented important sites. We conclude that better targeted expansion of PA networks would help to improve biodiversity trends. PMID:22457717

Repopulation of calibrations with samples from the target site: effect of the size of the calibration.

NASA Astrophysics Data System (ADS)

Guerrero, C.; Zornoza, R.; Gómez, I.; Mataix-Solera, J.; Navarro-Pedreño, J.; Mataix-Beneyto, J.; García-Orenes, F.

2009-04-01

Near infrared (NIR) reflectance spectroscopy offers important advantages because is a non-destructive technique, the pre-treatments needed in samples are minimal, and the spectrum of the sample is obtained in less than 1 minute without the needs of chemical reagents. For these reasons, NIR is a fast and cost-effective method. Moreover, NIR allows the analysis of several constituents or parameters simultaneously from the same spectrum once it is obtained. For this, a needed steep is the development of soil spectral libraries (set of samples analysed and scanned) and calibrations (using multivariate techniques). The calibrations should contain the variability of the target site soils in which the calibration is to be used. Many times this premise is not easy to fulfil, especially in libraries recently developed. A classical way to solve this problem is through the repopulation of libraries and the subsequent recalibration of the models. In this work we studied the changes in the accuracy of the predictions as a consequence of the successive addition of samples to repopulation. In general, calibrations with high number of samples and high diversity are desired. But we hypothesized that calibrations with lower quantities of samples (lower size) will absorb more easily the spectral characteristics of the target site. Thus, we suspect that the size of the calibration (model) that will be repopulated could be important. For this reason we also studied this effect in the accuracy of predictions of the repopulated models. In this study we used those spectra of our library which contained data of soil Kjeldahl Nitrogen (NKj) content (near to 1500 samples). First, those spectra from the target site were removed from the spectral library. Then, different quantities of samples of the library were selected (representing the 5, 10, 25, 50, 75 and 100% of the total library). These samples were used to develop calibrations with different sizes (%) of samples. We used partial least

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2015-11-01

increased PhScN potency as a result of preventing endoproteolytic degradation. Finally, the in vivo lung extravasation and colonization data, as well as...successful colonization are late stages in breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony...Award Number: TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells

PubMed Central

2014-01-01

Background Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). Methods MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. Results We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Conclusion Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment. PMID:25401046

Outreach for Outreach: Targeting social media audiences to promote a NASA kids’ web site

NASA Astrophysics Data System (ADS)

Pham, C. C.

2009-12-01

The Space Place is a successful NASA web site that benefits upper elementary school students and educators by providing games, activities, and resources to stimulate interest in science, technology, engineering, and mathematics, as well as to inform the audience of NASA’s contributions. As online social networking grows to be a central component of modern communication, The Space Place has explored the benefits of integrating social networks with the web site to increase awareness of materials the web site offers. This study analyzes the capabilities of social networks, and specifically the demographics of Twitter and Facebook. It then compares these results with the content, audience, and perceived demographics of The Space Place web site. Based upon the demographic results, we identified a target constituency that would benefit from the integration of social networks into The Space Place web site. As a result of this study, a Twitter feed has been established that releases a daily tweet from The Space Place. In addition, a Facebook page has been created to showcase new content and prompt interaction among fans of The Space Place. Currently, plans are under way to populate the Space Place Facebook page. Each social network has been utilized in an effort to spark excitement about the content on The Space Place, as well as to attract followers to the main NASA Space Place web site. To pursue this idea further, a plan has been developed to promote NASA Space Place’s social media tools among the target audience.

Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

PubMed Central

Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

2013-01-01

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

Cost Effective, Ultra Sensitive Groundwater Monitoring for Site Remediation and Management: Standard Operating Procedures with QA/QC

DTIC Science & Technology

2015-05-01

in consultation with the site management . 4.0 DATA TYPES AND QUALITY CONTROL A sampling plan must account for the collection, handling, and...GUIDANCE DOCUMENT Cost-Effective, Ultra-Sensitive Groundwater Monitoring for Site Remediation and Management : Standard Operating Procedures...Groundwater Monitoring for Site Remediation and Management 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Halden, R.U., Roll, I.B. 5d

Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site Definition and Plasma Stability

DTIC Science & Technology

2015-11-01

systemic therapy to prevent breast cancer bone colony progression. Figure 6. Colocalization of Ac-PhscNGGK-Bio with DiI in lung– extravasated SUM149PT cells...breast cancer progression that are ultimately fatal. Hence, prevention of extravasation which leads to colony formation would increase life...1 Award Number: W81XWH-12-1-0097 TITLE: “Targeting Alpha5 Beta1 Integrin to Prevent Metastatic Breast Cancer Cell Invasion: PhScN Target Site

Hsmar1 Transposition Is Sensitive to the Topology of the Transposon Donor and the Target

PubMed Central

Claeys Bouuaert, Corentin; Chalmers, Ronald

2013-01-01

Hsmar1 is a member of the Tc1-mariner superfamily of DNA transposons. These elements mobilize within the genome of their host by a cut-and-paste mechanism. We have exploited the in vitro reaction provided by Hsmar1 to investigate the effect of DNA supercoiling on transposon integration. We found that the topology of both the transposon and the target affect integration. Relaxed transposons have an integration defect that can be partially restored in the presence of elevated levels of negatively supercoiled target DNA. Negatively supercoiled DNA is a better target than nicked or positively supercoiled DNA, suggesting that underwinding of the DNA helix promotes target interactions. Like other Tc1-mariner elements, Hsmar1 integrates into 5′-TA dinucleotides. The direct vicinity of the target TA provides little sequence specificity for target interactions. However, transposition within a plasmid substrate was not random and some TA dinucleotides were targeted preferentially. The distribution of intramolecular target sites was not affected by DNA topology. PMID:23341977

Rationally Designed Sensing Selectivity and Sensitivity of an Aerolysin Nanopore via Site-Directed Mutagenesis.

PubMed

Wang, Ya-Qian; Cao, Chan; Ying, Yi-Lun; Li, Shuang; Wang, Ming-Bo; Huang, Jin; Long, Yi-Tao

2018-04-27

Selectivity and sensitivity are two key parameters utilized to describe the performance of a sensor. In order to investigate selectivity and sensitivity of the aerolysin nanosensor, we manipulated its surface charge at different locations via single site-directed mutagenesis. To study the selectivity, we replaced the positively charged R220 at the entrance of the pore with negatively charged glutamic acid, resulting in barely no current blockages for sensing negatively charged oligonucleotides. For the sensitivity, we substituted the positively charged lumen-exposed amino acid K238 located at trans-ward third of the β-barrel stem with glutamic acid. This leads to a surprisingly longer duration time at +140 mV, which is about 20 times slower in translocation speed for Poly(dA) 4 compared to that of wild-type aerolysin, indicating the stronger pore-analyte interactions and enhanced sensitivity. Therefore, it is both feasible and understandable to rationally design confined biological nanosensors for single molecule detection with high selectivity and sensitivity.

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

PubMed Central

Miller, Megan B.; Yan, Yan; Machida, Kazuya; Kiraly, Drew D.; Levy, Aaron D.; Wu, Yi I.; Lam, TuKiet T.; Abbott, Thomas; Koleske, Anthony J.; Eipper, Betty A.; Mains, Richard E.

2017-01-01

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity. PMID:28418645

A Process-based, Climate-Sensitive Model to Derive Methane Emissions from Natural Wetlands: Application to 5 Wetland Sites, Sensitivity to Model Parameters and Climate

NASA Technical Reports Server (NTRS)

Walter, Bernadette P.; Heimann, Martin

1999-01-01

Methane emissions from natural wetlands constitutes the largest methane source at present and depends highly on the climate. In order to investigate the response of methane emissions from natural wetlands to climate variations, a 1-dimensional process-based climate-sensitive model to derive methane emissions from natural wetlands is developed. In the model the processes leading to methane emission are simulated within a 1-dimensional soil column and the three different transport mechanisms diffusion, plant-mediated transport and ebullition are modeled explicitly. The model forcing consists of daily values of soil temperature, water table and Net Primary Productivity, and at permafrost sites the thaw depth is included. The methane model is tested using observational data obtained at 5 wetland sites located in North America, Europe and Central America, representing a large variety of environmental conditions. It can be shown that in most cases seasonal variations in methane emissions can be explained by the combined effect of changes in soil temperature and the position of the water table. Our results also show that a process-based approach is needed, because there is no simple relationship between these controlling factors and methane emissions that applies to a variety of wetland sites. The sensitivity of the model to the choice of key model parameters is tested and further sensitivity tests are performed to demonstrate how methane emissions from wetlands respond to climate variations.

The ATP-binding site of type II topoisomerases as a target for antibacterial drugs.

PubMed

Maxwell, Anthony; Lawson, David M

2003-01-01

DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.

Occurrence Prospect of HDR and Target Site Selection Study in Southeastern of China

NASA Astrophysics Data System (ADS)

Lin, W.; Gan, H.

2017-12-01

Hot dry rock (HDR) geothermal resource is one of the most important clean energy in future. Site selection a HDR resource is a fundamental work to explore the HDR resources. This paper compiled all the HDR development projects domestic and abroad, and summarized the location of HDR geothermal geological index. After comparing the geological background of HDR in the southeast coastal area of China, Yangjiang Xinzhou in Guangdong province, Leizhou Peninsula area, Lingshui in Hainan province and Huangshadong in Guangzhou were selected from some key potential target area along the southeast coast of China. Deep geothermal field model of the study area is established based on the comprehensive analysis of the target area of deep geothermal geological background and deep thermal anomalies. This paper also compared the hot dry rock resources target locations, and proposed suggestions for the priority exploration target area and exploration scheme.

Mohawk Tannery Hazardous Waste Site in New Hampshire included on EPA List of Targeted for Immediate Attention

EPA Pesticide Factsheets

Today, the U.S. Environmental Protection Agency released the list of Superfund sites that Administrator Pruitt has targeted for immediate and intense attention. The former Mohawk Tannery facility (a.k.a. Granite State Leathers) is one of the 21 sites on th

HomoTarget: a new algorithm for prediction of microRNA targets in Homo sapiens.

PubMed

Ahmadi, Hamed; Ahmadi, Ali; Azimzadeh-Jamalkandi, Sadegh; Shoorehdeli, Mahdi Aliyari; Salehzadeh-Yazdi, Ali; Bidkhori, Gholamreza; Masoudi-Nejad, Ali

2013-02-01

MiRNAs play an essential role in the networks of gene regulation by inhibiting the translation of target mRNAs. Several computational approaches have been proposed for the prediction of miRNA target-genes. Reports reveal a large fraction of under-predicted or falsely predicted target genes. Thus, there is an imperative need to develop a computational method by which the target mRNAs of existing miRNAs can be correctly identified. In this study, combined pattern recognition neural network (PRNN) and principle component analysis (PCA) architecture has been proposed in order to model the complicated relationship between miRNAs and their target mRNAs in humans. The results of several types of intelligent classifiers and our proposed model were compared, showing that our algorithm outperformed them with higher sensitivity and specificity. Using the recent release of the mirBase database to find potential targets of miRNAs, this model incorporated twelve structural, thermodynamic and positional features of miRNA:mRNA binding sites to select target candidates. Copyright © 2012 Elsevier Inc. All rights reserved.

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

PubMed Central

Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

1992-01-01

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

PubMed

Häcker, Irina; Harrell Ii, Robert A; Eichner, Gerrit; Pilitt, Kristina L; O'Brochta, David A; Handler, Alfred M; Schetelig, Marc F

2017-03-07

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

Study of the pH-sensitive mechanism of tumor-targeting liposomes.

PubMed

Fan, Yang; Chen, Cong; Huang, Yiheng; Zhang, Fang; Lin, Guimei

2017-03-01

Currently, the phosphatidylethanolamine-based, pH-sensitive, liposome drug-delivery system has been widely developed for efficient, targeted cancer therapy. However, the mechanism of pH sensitivity was unclear; it is a main obstacle in controlling the preparation of pH-sensitive liposomes (PSLs).Therefore, our research is aimed at clarifying the pH-response mechanism of the various molecules that compose liposomes. We chose the small pH-sensitive molecules oleic acid (OA), linoleic acid (LA) and cholesteryl hemisuccinate (CHEMS) and the fundamental lipids cholesterol and phosphatidylethanolamine (PE) as test molecules. The PSLs were prepared using the thin-film hydration method and characterized in detail at various pH values (pH 5.0, 6.0 and 7.4), including particle size, ζ-potential, drug encapsulation efficiency and drug loading. The surface structure was observed by transmission electron microscopy (TEM), and the electrical conductivity of the liposome dispersion was also tested. The calorimetric analysis was conducted by Nano-differential scanning calorimetry (Nano-DSC). The in vitro drug release profile showed that PSLs exhibit good pH sensitivity. At neutral pH, the particle size was approximately 150nm, and it dramatically increased at pH 5.0. The ζ-potential increased as the pH decreased. The Nano-DSC results showed that cholesterol and CHEMS can both increase the stability and phase transfer temperature of PSLs. Conductivity increased to a maximum at pH 5.0 and was rather low at pH 7.4. In conclusion, results show that the three kinds of liposomes have pH responsive release characteristics in acidic pH. The OA-PSLs have a pH sensitive point of 5. Since CHEMS has a cholesterol-like structure, it can stabilizes the phospholipid bilayer under neutral conditions as shown in the Nano-DSC data, and because it has a special steroidal rigid structure, it exhibits better pH response characteristics under acidic conditions. Copyright © 2016 Elsevier B.V. All

pH-Sensitive Liposomes: Possible Clinical Implications

NASA Astrophysics Data System (ADS)

Yatvin, M. B.; Kreutz, W.; Horwitz, B. A.; Shinitzky, M.

1980-12-01

When pH-sensitive molecules are incorporated into liposomes, drugs can be specifically released from these vesicles by a change of pH in the ambient serum. Liposomes containing the pH-sensitive lipid palmitoyl homocysteine (PHC) were constructed so that the greatest pH differential (6.0 to 7.4) of drug release was obtained near physiological temperature. Such liposomes could be useful clinically if they enable drugs to be targeted to areas of the body in which pH is less than physiological, such as primary tumors and metastases or sites of inflammation and infection.

RGD-modified pH-sensitive liposomes for docetaxel tumor targeting.

PubMed

Chang, Minglu; Lu, Shanshan; Zhang, Fang; Zuo, Tiantian; Guan, Yuanyuan; Wei, Ting; Shao, Wei; Lin, Guimei

2015-05-01

Phosphatidylethanolamine-based pH-sensitive liposomes of various compositions have been described as efficient systems for delivery of therapeutic molecules into tumor cells. The aim of this work was to develop a drug delivery system based on pH-sensitive liposomes (PLPs) that were modified with arginine-glycine-aspartic acid (RGD) peptide to enhance the effectiveness of docetaxel treatment. Docetaxel/coumarin-6 loaded PLPs were prepared by the thin-film dispersion method and characterized in detail, including by particle size, polydispersity, zeta potential and drug encapsulation efficiency. In vitro studies using MCF-7, HepG2and A549 cells were employed to investigate cytotoxicity and cellular uptake of the drug solution or docetaxel/coumarin-6 loaded PLPs. The accumulation of 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled liposomes in vivo was studied through tumor section imaging of xenograft mouse models of MCF-7 24h after intravenous administration. The particle size of the non-coated or RGD modified PLPs ranged between 146 and 129nm. Drug release in vitro was modestly prolonged and had good pH sensitivity. In the in vitro study, RGD-coated PLPs showed higher cytotoxicity and cellular uptake relative to non-coated ones. The results of the in vivo study showed that RGD-coated PLPs had higher fluorescence, which suggested a more efficient accumulation than normal PLPs in tumors. In conclusion, these results confirmed RGD-modified PLPs as a potential drug delivery system to achieve controlled release and tumor targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Dual-targeted and pH-sensitive Doxorubicin Prodrug-Microbubble Complex with Ultrasound for Tumor Treatment

PubMed Central

Luo, Wanxian; Wen, Ge; Yang, Li; Tang, Jiao; Wang, Jianguo; Wang, Jihui; Zhang, Shiyu; Zhang, Li; Ma, Fei; Xiao, Liling; Wang, Ying; Li, Yingjia

2017-01-01

In this study, we investigated the potential of a dual-targeted pH-sensitive doxorubicin prodrug-microbubble complex (DPMC) in ultrasound (US)-assisted antitumor therapy. The doxorubicin prodrug (DP) consists of a succinylated-heparin carrier conjugated with doxorubicin (DOX) via hydrazone linkage and decorated with dual targeting ligands, folate and cRGD peptide. Combination of microbubble (MB) and DP, generated via avidin-biotin binding, promoted intracellular accumulation and improved therapeutic efficiency assisted by US cavitation and sonoporation. Aggregates of prepared DP were observed with an inhomogeneous size distribution (average diameters: 149.6±29.8 nm and 1036.2±38.8 nm, PDI: 1.0) while DPMC exhibited a uniform distribution (average diameter: 5.804±2.1 μm), facilitating its usage for drug delivery. Notably, upon US exposure, DPMC was disrupted and aggregated DP dispersed into homogeneous small-sized nanoparticles (average diameter: 128.6±42.3 nm, PDI: 0.21). DPMC could target to angiogenic endothelial cells in tumor region via αvβ3-mediated recognition and subsequently facilitate its specific binding to tumor cells mediated via recognition of folate receptor (FR) after US exposure. In vitro experiments showed higher tumor specificity and killing ability of DPMC with US than free DOX and DP for breast cancer MCF-7 cells. Furthermore, significant accumulation and specificity for tumor tissues of DPMC with US were detected using in vivo fluorescence and ultrasound molecular imaging, indicating its potential to integrate tumor imaging and therapy. In particular, through inducing apoptosis, inhibiting cell proliferation and antagonizing angiogenesis, DPMC with US produced higher tumor inhibition rates than DOX or DPMC without US in MCF-7 xenograft tumor-bearing mice while inducing no obvious body weight loss. Our strategy provides an effective platform for the delivery of large-sized or aggregated particles to tumor sites, thereby extending their

An alternate binding site for PPARγ ligands

PubMed Central

Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2014-01-01

PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer.

PubMed

Sprowl-Tanio, Stephanie; Habowski, Amber N; Pate, Kira T; McQuade, Miriam M; Wang, Kehui; Edwards, Robert A; Grun, Felix; Lyou, Yung; Waterman, Marian L

2016-01-01

There is increasing evidence that oncogenic Wnt signaling directs metabolic reprogramming of cancer cells to favor aerobic glycolysis or Warburg metabolism. In colon cancer, this reprogramming is due to direct regulation of pyruvate dehydrogenase kinase 1 ( PDK1 ) gene transcription. Additional metabolism genes are sensitive to Wnt signaling and exhibit correlative expression with PDK1. Whether these genes are also regulated at the transcriptional level, and therefore a part of a core metabolic gene program targeted by oncogenic WNT signaling, is not known. Here, we identify monocarboxylate transporter 1 (MCT-1; encoded by SLC16A1 ) as a direct target gene supporting Wnt-driven Warburg metabolism. We identify and validate Wnt response elements (WREs) in the proximal SLC16A1 promoter and show that they mediate sensitivity to Wnt inhibition via dominant-negative LEF-1 (dnLEF-1) expression and the small molecule Wnt inhibitor XAV939. We also show that WREs function in an independent and additive manner with c-Myc, the only other known oncogenic regulator of SLC16A1 transcription. MCT-1 can export lactate, the byproduct of Warburg metabolism, and it is the essential transporter of pyruvate as well as a glycolysis-targeting cancer drug, 3-bromopyruvate (3-BP). Using sulforhodamine B (SRB) assays to follow cell proliferation, we tested a panel of colon cancer cell lines for sensitivity to 3-BP. We observe that all cell lines are highly sensitive and that reduction of Wnt signaling by XAV939 treatment does not synergize with 3-BP, but instead is protective and promotes rapid recovery. We conclude that MCT-1 is part of a core Wnt signaling gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism.

Restoration of Corticosteroid Sensitivity in Chronic Obstructive Pulmonary Disease by Inhibition of Mammalian Target of Rapamycin.

PubMed

Mitani, Akihisa; Ito, Kazuhiro; Vuppusetty, Chaitanya; Barnes, Peter J; Mercado, Nicolas

2016-01-15

Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.

ALOMYbase, a resource to investigate non-target-site-based resistance to herbicides inhibiting acetolactate-synthase (ALS) in the major grass weed Alopecurus myosuroides (black-grass).

PubMed

Gardin, Jeanne Aude Christiane; Gouzy, Jérôme; Carrère, Sébastien; Délye, Christophe

2015-08-12

Herbicide resistance in agrestal weeds is a global problem threatening food security. Non-target-site resistance (NTSR) endowed by mechanisms neutralising the herbicide or compensating for its action is considered the most agronomically noxious type of resistance. Contrary to target-site resistance, NTSR mechanisms are far from being fully elucidated. A part of weed response to herbicide stress, NTSR is considered to be largely driven by gene regulation. Our purpose was to establish a transcriptome resource allowing investigation of the transcriptomic bases of NTSR in the major grass weed Alopecurus myosuroides L. (Poaceae) for which almost no genomic or transcriptomic data was available. RNA-Seq was performed from plants in one F2 population that were sensitive or expressing NTSR to herbicides inhibiting acetolactate-synthase. Cloned plants were sampled over seven time-points ranging from before until 73 h after herbicide application. Assembly of over 159M high-quality Illumina reads generated a transcriptomic resource (ALOMYbase) containing 65,558 potentially active contigs (N50 = 1240 nucleotides) predicted to encode 32,138 peptides with 74% GO annotation, of which 2017 were assigned to protein families presumably involved in NTSR. Comparison with the fully sequenced grass genomes indicated good coverage and correct representation of A. myosuroides transcriptome in ALOMYbase. The part of the herbicide transcriptomic response common to the resistant and the sensitive plants was consistent with the expected effects of acetolactate-synthase inhibition, with striking similarities observed with published Arabidopsis thaliana data. A. myosuroides plants with NTSR were first affected by herbicide action like sensitive plants, but ultimately overcame it. Analysis of differences in transcriptomic herbicide response between resistant and sensitive plants did not allow identification of processes directly explaining NTSR. Five contigs associated to NTSR in the F2

Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site

PubMed Central

Ding, Kejia; Wang, Annie; Boerneke, Mark A.; Dibrov, Sergey M.; Hermann, Thomas

2014-01-01

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex. PMID:24856063

Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.

PubMed

Kakimoto, Yuriko; Tashiro, Shinya; Kojima, Rieko; Morozumi, Yuki; Endo, Toshiya; Tamura, Yasushi

2018-04-18

Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

Targeting Bcl-2 stability to sensitize cells harboring oncogenic ras.

PubMed

Peng, Bo; Ganapathy, Suthakar; Shen, Ling; Huang, Junchi; Yi, Bo; Zhou, Xiaodong; Dai, Wei; Chen, Changyan

2015-09-08

The pro-survival factor Bcl-2 and its family members are critical determinants of the threshold of the susceptibility of cells to apoptosis. Studies are shown that cells harboring an oncogenic ras were extremely sensitive to the inhibition of protein kinase C (PKC) and Bcl-2 could antagonize this apoptotic process. However, it remains unrevealed how Bcl-2 is being regulated in this apoptotic process. In this study, we investigate the role of Bcl-2 stability in sensitizing the cells harboring oncogenic K-ras to apoptosis triggered by PKC inhibitor GO6976. We demonstrated that Bcl-2 in Swiss3T3 cells ectopically expressing or murine lung cancer LKR cells harboring K-ras rapidly underwent ubiquitin-dependent proteasome pathway after the treatment of GO6976, accompanied with induction of apoptosis. In this process, Bcl-2 formed the complex with Keap-1 and Cul3. The mutation of serine-17 and deletion of BH-2 or 4 was required for Bcl-2 ubiquitination and degradation, which elevate the signal threshold for the induction of apoptosis in the cells following PKC inhibition. Thus, Bcl-2 appears an attractive target for the induction of apoptosis by PKC inhibition in cancer cells expressing oncogenic K-ras.

Reconstructing the Qo Site of Plasmodium falciparum bc 1 Complex in the Yeast Enzyme

PubMed Central

Vallières, Cindy; Fisher, Nicholas; Meunier, Brigitte

2013-01-01

The bc 1 complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Qo pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Qo sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Qo mutants. We measured the bc 1 complex sensitivity to atovaquone, azoxystrobin, a Qo site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc 1 complex.The data obtained highlighted variations in the Qo site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Qo mutants could be useful and easy-to-use tools for the study of that class of antimalarials. PMID:23951230

Binding site and affinity prediction of general anesthetics to protein targets using docking.

PubMed

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

2012-05-01

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site

Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

PubMed Central

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G.

2012-01-01

Background The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explore whether a computational method, AutoDock, could serve as such a tool. Methods High-resolution crystal data of water soluble proteins (cytochrome C, apoferritin and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus, GLIC) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (https://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants are compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent co-crystallization data. Docking calculations for six general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known EC50 were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC50s and octanol/water partition coefficients for the six general anesthetics. Results All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (p=0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the

Sensitization of TRPA1 by Protein Kinase A

PubMed Central

Meents, Jannis E.; Fischer, Michael J. M.; McNaughton, Peter A.

2017-01-01

The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1. PMID:28076424

Site-Specific Targeting of Platelet-Rich Plasma via Superparamagnetic Nanoparticles

PubMed Central

Talaie, Tara; Pratt, Stephen J.P.; Vanegas, Camilo; Xu, Su; Henn, R. Frank; Yarowsky, Paul; Lovering, Richard M.

2015-01-01

Background: Muscle strains are one of the most common injuries treated by physicians. Standard conservative therapy for acute muscle strains usually involves short-term rest, ice, and nonsteroidal anti-inflammatory medications, but there is no clear consensus regarding treatments to accelerate recovery. Recently, clinical use of platelet-rich plasma (PRP) has gained momentum as an option for therapy and is appealing for many reasons, most notably because it provides growth factors in physiological proportions and it is autologous, safe, easily accessible, and potentially beneficial. Local delivery of PRP to injured muscles can hasten recovery of function. However, specific targeting of PRP to sites of tissue damage in vivo is a major challenge that can limit its efficacy. Hypothesis: Location of PRP delivery can be monitored and controlled in vivo with noninvasive tools. Study Design: Controlled laboratory study. Methods: Superparamagnetic iron oxide nanoparticles (SPIONs) can be visualized by both magnetic resonance imaging (MRI) (in vivo) and fluorescence microscopy (after tissue harvesting). PRP was labeled with SPIONs and administered by intramuscular injections of SPION-containing platelets. MRI was used to monitor the ability to manipulate and retain the location of PRP in vivo by placement of an external magnet. Platelets were isolated from whole blood and incubated with SPIONs. Following SPION incubation with PRP, a magnetic field was used to manipulate platelet location in culture dishes. In vivo, the tibialis anterior (TA) muscles of anesthetized Sprague-Dawley rats were injected with SPION-containing platelets, and MRI was used to track platelet position with and without a magnet worn over the TA muscles for 4 days. Results: The method used to isolate PRP yielded a high concentration (almost 4-fold increase) of platelets. In vitro experiments showed that the platelets successfully took up SPIONs and then rapidly responded to an applied magnetic field

Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli.

PubMed Central

Liu, J; Turnbough, C L

1994-01-01

In Escherichia coli, expression of the pyrC gene is regulated primarily by a translational control mechanism based on nucleotide-sensitive selection of transcriptional start sites at the pyrC promoter. When intracellular levels of CTP are high, pyrC transcripts are initiated predominantly with CTP at a site 7 bases downstream of the Pribnow box. These transcripts form a stable hairpin at their 5' ends that blocks ribosome binding. When the CTP level is low and the GTP level is high, conditions found in pyrimidine-limited cells, transcripts are initiated primarily with GTP at a site 9 bases downstream of the Pribnow box. These shorter transcripts are unable to form a hairpin at their 5' ends and are readily translated. In this study, we examined the effects of nucleotide sequence and position on the selection of transcriptional start sites at the pyrC promoter. We characterized promoter mutations that systematically alter the sequence at position 7 or 9 downstream of the Pribnow box or vary the spacing between the Pribnow box and wild-type transcriptional initiation region. The results reveal preferences for particular initiating nucleotides (ATP > or = GTP > UTP >> CTP) and for starting positions downstream of the Pribnow box (7 >> 6 and 8 > 9 > 10). The results indicate that optimal nucleotide-sensitive start site switching at the wild-type pyrC promoter is the result of competition between the preferred start site (position 7) that uses the poorest initiating nucleotide (CTP) and a weak start site (position 9) that uses a good initiating nucleotide (GTP). The sequence of the pyrC promoter also minimizes the synthesis of untranslatable transcripts and provides for maximum stability of the regulatory transcript hairpin. In addition, the results show that the effects of the mutations on pyrC expression and regulation are consistent with the current model for translational control. Possible effects of preferences for initiating nucleotides and start sites on the

Cooperation between a hierarchical set of recruitment sites targets the X chromosome for dosage compensation

PubMed Central

Albritton, Sarah Elizabeth; Kranz, Anna-Lena; Winterkorn, Lara Heermans; Street, Lena Annika; Ercan, Sevinc

2017-01-01

In many organisms, it remains unclear how X chromosomes are specified for dosage compensation, since DNA sequence motifs shown to be important for dosage compensation complex (DCC) recruitment are themselves not X-specific. Here, we addressed this problem in C. elegans. We found that the DCC recruiter, SDC-2, is required to maintain open chromatin at a small number of primary DCC recruitment sites, whose sequence and genomic context are X-specific. Along the X, primary recruitment sites are interspersed with secondary sites, whose function is X-dependent. A secondary site can ectopically recruit the DCC when additional recruitment sites are inserted either in tandem or at a distance (>30 kb). Deletion of a recruitment site on the X results in reduced DCC binding across several megabases surrounded by topologically associating domain (TAD) boundaries. Our work elucidates that hierarchy and long-distance cooperativity between gene-regulatory elements target a single chromosome for regulation. DOI: http://dx.doi.org/10.7554/eLife.23645.001 PMID:28562241

Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

PubMed

Lee, Ciaran M; Davis, Timothy H; Bao, Gang

2018-04-01

What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. © 2017 The Authors. Experimental Physiology © 2017 The Physiological Society.

SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

PubMed Central

Kim, Min Jung; Chia, Ian V.; Costantini, Frank

2008-01-01

Axin is a scaffold protein for the β-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-ΔC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.—Kim, M. J., Chia, I. V., Costantini, F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability. PMID:18632848

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

PubMed Central

Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

2012-01-01

Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation.

PubMed

Levy, Oren; Zhao, Weian; Mortensen, Luke J; Leblanc, Sarah; Tsang, Kyle; Fu, Moyu; Phillips, Joseph A; Sagar, Vinay; Anandakumaran, Priya; Ngai, Jessica; Cui, Cheryl H; Eimon, Peter; Angel, Matthew; Lin, Charles P; Yanik, Mehmet Fatih; Karp, Jeffrey M

2013-10-03

Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy to treat several diseases and are compelling to consider as vehicles for delivery of biological agents. However, MSCs appear to act through a seemingly limited "hit-and-run" mode to quickly exert their therapeutic impact, mediated by several mechanisms, including a potent immunomodulatory secretome. Furthermore, MSC immunomodulatory properties are highly variable and the secretome composition following infusion is uncertain. To determine whether a transiently controlled antiinflammatory MSC secretome could be achieved at target sites of inflammation, we harnessed mRNA transfection to generate MSCs that simultaneously express functional rolling machinery (P-selectin glycoprotein ligand-1 [PSGL-1] and Sialyl-Lewis(x) [SLeX]) to rapidly target inflamed tissues and that express the potent immunosuppressive cytokine interleukin-10 (IL-10), which is not inherently produced by MSCs. Indeed, triple-transfected PSGL-1/SLeX/IL-10 MSCs transiently increased levels of IL-10 in the inflamed ear and showed a superior antiinflammatory effect in vivo, significantly reducing local inflammation following systemic administration. This was dependent on rapid localization of MSCs to the inflamed site. Overall, this study demonstrates that despite the rapid clearance of MSCs in vivo, engineered MSCs can be harnessed via a "hit-and-run" action for the targeted delivery of potent immunomodulatory factors to treat distant sites of inflammation.

siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

PubMed

Vickers, Timothy A; Crooke, Stanley T

2012-07-01

While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

PubMed

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Fitness cost implications of phiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae)

USDA-ARS?s Scientific Manuscript database

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understandin...

A novel W1999S mutation and non-target site resistance impact on acetyl-CoA carboxylase inhibiting herbicides to varying degrees in a UK Lolium multiflorum population.

PubMed

Kaundun, Shiv Shankhar; Bailly, Geraldine C; Dale, Richard P; Hutchings, Sarah-Jane; McIndoe, Eddie

2013-01-01

Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance.

Targeted Delivery of LXR Agonist Using a Site-Specific Antibody-Drug Conjugate.

PubMed

Lim, Reyna K V; Yu, Shan; Cheng, Bo; Li, Sijia; Kim, Nam-Jung; Cao, Yu; Chi, Victor; Kim, Ji Young; Chatterjee, Arnab K; Schultz, Peter G; Tremblay, Matthew S; Kazane, Stephanie A

2015-11-18

Liver X receptor (LXR) agonists have been explored as potential treatments for atherosclerosis and other diseases based on their ability to induce reverse cholesterol transport and suppress inflammation. However, this therapeutic potential has been hindered by on-target adverse effects in the liver mediated by excessive lipogenesis. Herein, we report a novel site-specific antibody-drug conjugate (ADC) that selectively delivers a LXR agonist to monocytes/macrophages while sparing hepatocytes. The unnatural amino acid para-acetylphenylalanine (pAcF) was site-specifically incorporated into anti-CD11a IgG, which binds the α-chain component of the lymphocyte function-associated antigen 1 (LFA-1) expressed on nearly all monocytes and macrophages. An aminooxy-modified LXR agonist was conjugated to anti-CD11a IgG through a stable, cathepsin B cleavable oxime linkage to afford a chemically defined ADC. The anti-CD11a IgG-LXR agonist ADC induced LXR activation specifically in human THP-1 monocyte/macrophage cells in vitro (EC50-27 nM), but had no significant effect in hepatocytes, indicating that payload delivery is CD11a-mediated. Moreover, the ADC exhibited higher-fold activation compared to a conventional synthetic LXR agonist T0901317 (Tularik) (3-fold). This novel ADC represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.

Co-targeting the HER and IGF/insulin receptor axis in breast cancer, with triple targeting with endocrine therapy for hormone-sensitive disease.

PubMed

Chakraborty, Ashok; Hatzis, Christos; DiGiovanna, Michael P

2017-05-01

Interactions between HER2, estrogen receptor (ER), and insulin-like growth factor I receptor (IGF1R) are implicated in resistance to monotherapies targeting these receptors. We have previously shown in pre-clinical studies synergistic anti-tumor effects for co-targeting each pairwise combination of HER2, IGF1R, and ER. Strikingly, synergy for HER2/IGF1R targeting occurred not only in a HER2+ model, but also in a HER2-normal model. The purpose of the current study was therefore to determine the generalizability of synergistic anti-tumor effects of co-targeting HER2/IGF1R, the anti-tumor activity of triple-targeting HER2/IGF1R/ER in hormone-dependent cell lines, and the effect of using the multi-targeting drugs neratinib (pan-HER) and BMS-754807 (dual IGF1R/insulin receptor). Proliferation and apoptosis assays were performed in a large panel of cell lines representing varying receptor expression levels. Mechanistic effects were studied using phospho-protein immunoblotting. Analyses of drug interaction effects were performed using linear mixed-effects regression models. Enhanced anti-proliferative effects of HER/IGF-insulin co-targeting were seen in most, though not all, cell lines, including HER2-normal lines. For ER+ lines, triple targeting with inclusion of anti-estrogen generally resulted in the greatest anti-tumor effects. Double or triple targeting generally resulted in marked increases in apoptosis in the sensitive lines. Mechanistic studies demonstrated that the synergy between drugs was correlated with maximal inhibition of Akt and ERK pathway signaling. Dual HER/IGF-insulin targeting, and triple targeting with inclusion of anti-estrogen drugs, shows striking anti-tumor activity across breast cancer types, and drugs with broader receptor specificity may be more effective than single receptor selective drugs, particularly for ER- cells.

Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy.

PubMed

Li, Ying; Yu, Chuanfeng; Han, Huixia; Zhao, Caisheng; Zhang, Xiaoru

2016-07-15

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. Copyright © 2016 Elsevier B.V. All rights reserved.

Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella.

PubMed

Steinbach, Denise; Gutbrod, Oliver; Lümmen, Peter; Matthiesen, Svend; Schorn, Corinna; Nauen, Ralf

2015-08-01

Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [(3)H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4-S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where

Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies

DTIC Science & Technology

2016-10-01

2016 4 . TITLE AND SUBTITLE 5a. CONTRACT NUMBER Prostate Cancer Clinical Consortium Clinical Research Site: Targeted Therapies 5b. GRANT NUMBER 5c...new biomarker driven trials directly to patients W81XWH-14-2-0159 None listed 20 Table of Contents Page 1. Introduction…………………………………………………………. 4 2...Keywords……………………………………………………………. 4 3. Accomplishments ..……..…………………………………………... 4 4 . Impact…………………………...…………………………………… 8 5. Changes/Problems

MiR-214 inhibits cell migration, invasion and promotes the drug sensitivity in human cervical cancer by targeting FOXM1.

PubMed

Wang, Jian-Mei; Ju, Bao-Hui; Pan, Cai-Jun; Gu, Yan; Li, Meng-Qi; Sun, Li; Xu, Yan-Ying; Yin, Li-Rong

2017-01-01

MicroRNAs (miRNAs) play key roles in progression of cervical cancer. In the present study, we investigated the role of miR-214 in the process of migration, invasion and drug sensitivity to cisplatin in cervical cancer. We detected the differential expression of miR-214 in 19 cases cervical cancer tissues and normal tissues as well as 4 cervical cancer cells and one normal cervical cells by Real-time PCR. Then, wound healing assay, transwell invasion assay and MTT were used to detect the effects of migration, invasion and sensitivity to cisplatin of cervical cancer when miR-214 was overexpressed. Western blot, immunofluorescence and Flow Cytometry were used to detect the mechanism of migration, invasion and sensitivity to cisplatin. Next, bioinformatics analysis was used to find the target of miR-214. Through the luciferase reporter assay, Real-time PCR and western blot, we confirmed the binding relationship of miR-214 and FOXM1. In cervical cancer tissues, the expression of FOXM1 was detected by western blot and Immunohistochemistry. We also knocked down FOXM1 in cervical cancer cells, wound healing assay, transwell invasion assay and MTT were performed to detect the migration, invasion and sensitivity to cisplatin abilities of FOXM1. Western blot and Flow Cytometry were used to detect the mechanism of migration, invasion and sensitivity to cisplatin by FOXM1. Finally, we performed rescue expriments to confirm the function relationship between miR-214 and FOXM1. 1. Our results showed that miR-214 was frequently downregulated in tumor tissues and cancer cells especially in CIN III and cervical cancer stages. 2. Overexpression of miR-214 significantly inhibited migration and invasion of cervical cancer cells and prompted the sensitivity to cisplatin. 3. FOXM1 was identified as a target of miR-214 and down-regulated by miR-214. 4. Knocking down FOXM1 could inhibited migration and invasion of cervical cancer cells and prompted the sensitivity to cisplatin. 5. FOXM1 was

pH-sensitive inulin-based nanomicelles for intestinal site-specific and controlled release of celecoxib.

PubMed

Mandracchia, Delia; Trapani, Adriana; Perteghella, Sara; Sorrenti, Milena; Catenacci, Laura; Torre, Maria Luisa; Trapani, Giuseppe; Tripodo, Giuseppe

2018-02-01

Aiming at a site-specific drug release in the lower intestinal tract, this paper deals with the synthesis and physicochemical/biological characterization of pH-sensitive nanomicelles from an inulin (INU) amphiphilic derivative. To allow an intestinal site specific release of the payload, INU-Vitamin E (INVITE) bioconjugates were functionalized with succinic anhydride to provide the system with pH-sensitive groups preventing a premature release of the payload into the stomach. The obtained INVITESA micelles resulted nanosized, with a low critical aggregation concentration and the release studies showed a marked pH-dependent release. The drug loading stabilized the micelles against the acidic hydrolysis. From transport studies on Caco-2 cells, resulted that INVITESA nanomicelles cross the cellular monolayer but are actively re-transported in the secretory (basolateral-apical) direction when loaded in apical side. It suggests that the entrapped drug could not be absorbed before the release from the micelles, enabling so a local release of the active. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeting miR-381-NEFL axis sensitizes glioblastoma cells to temozolomide by regulating stemness factors and multidrug resistance factors.

PubMed

Wang, Zeyou; Yang, Jing; Xu, Gang; Wang, Wei; Liu, Changhong; Yang, Honghui; Yu, Zhibin; Lei, Qianqian; Xiao, Lan; Xiong, Jing; Zeng, Liang; Xiang, Juanjuan; Ma, Jian; Li, Guiyuan; Wu, Minghua

2015-02-20

MicroRNA-381 (miR-381) is a highly expressed onco-miRNA that is involved in malignant progression and has been suggested to be a good target for glioblastoma multiforme (GBM) therapy. In this study, we employed two-dimensional fluorescence differential gel electrophoresis (2-D DIGE) and MALDI-TOF/TOF-MS/MS to identify 27 differentially expressed proteins, including the significantly upregulated neurofilament light polypeptide (NEFL), in glioblastoma cells in which miR-381 expression was inhibited. We identified NEFL as a novel target molecule of miR-381 and a tumor suppressor gene. In human astrocytoma clinical specimens, NEFL was downregulated with increased levels of miR-381 expression. Either suppressing miR-381 or enforcing NEFL expression dramatically sensitized glioblastoma cells to temozolomide (TMZ), a promising chemotherapeutic agent for treating GBMs. The mechanism by which these cells were sensitized to TMZ was investigated by inhibiting various multidrug resistance factors (ABCG2, ABCC3, and ABCC5) and stemness factors (ALDH1, CD44, CKIT, KLF4, Nanog, Nestin, and SOX2). Our results further demonstrated that miR-381 overexpression reversed the viability of U251 cells exhibiting NEFL-mediated TMZ sensitivity. In addition, NEFL-siRNA also reversed the proliferation rate of U251 cells exhibiting locked nucleic acid (LNA)-anti-miR-381-mediated TMZ sensitivity. Overall, the miR-381-NEFL axis is important for TMZ resistance in GBM and may potentially serve as a novel therapeutic target for glioma.

Binding-Site Assessment by Virtual Fragment Screening

PubMed Central

Huang, Niu; Jacobson, Matthew P.

2010-01-01

The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules) would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock ∼11000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors. PMID:20404926

pH-sensitive and folic acid-targeted MPEG-PHIS/FA-PEG-VE mixed micelles for the delivery of PTX-VE and their antitumor activity.

PubMed

Di, Yan; Li, Ting; Zhu, Zhihong; Chen, Fen; Jia, Lianqun; Liu, Wenbing; Gai, Xiumei; Wang, Yingying; Pan, Weisan; Yang, Xinggang

2017-01-01

The aim of this study was to simultaneously introduce pH sensitivity and folic acid (FA) targeting into a micelle system to achieve quick drug release and to enhance its accumulation in tumor cells. Paclitaxel-(+)-α-tocopherol (PTX-VE)-loaded mixed micelles (PHIS/FA/PM) fabricated by poly(ethylene glycol) methyl ether-poly(histidine) (MPEG-PHIS) and folic acid-poly(ethylene glycol)-(+)-α-tocopherol (FA-PEG-VE) were characterized by dynamic light scattering and transmission electron microscopy (TEM). The mixed micelles had a spherical morphology with an average diameter of 137.0±6.70 nm and a zeta potential of -48.7±4.25 mV. The drug encapsulation and loading efficiencies were 91.06%±2.45% and 5.28%±0.30%, respectively. The pH sensitivity was confirmed by changes in particle size, critical micelle concentration, and transmittance as a function of pH. MTT assay showed that PHIS/FA/PM had higher cytotoxicity at pH 6.0 than at pH 7.4, and lower cytotoxicity in the presence of free FA. Confocal laser scanning microscope images demonstrated a time-dependent and FA-inhibited cellular uptake. In vivo imaging confirmed that the mixed micelles targeted accumulation at tumor sites and the tumor inhibition rate was 85.97%. The results proved that the mixed micelle system fabricated by MPEG-PHIS and FA-PEG-VE is a promising approach to improve antitumor efficacy.

pH-sensitive and folic acid-targeted MPEG-PHIS/FA-PEG-VE mixed micelles for the delivery of PTX-VE and their antitumor activity

PubMed Central

Di, Yan; Li, Ting; Zhu, Zhihong; Chen, Fen; Jia, Lianqun; Liu, Wenbing; Gai, Xiumei; Wang, Yingying; Pan, Weisan; Yang, Xinggang

2017-01-01

The aim of this study was to simultaneously introduce pH sensitivity and folic acid (FA) targeting into a micelle system to achieve quick drug release and to enhance its accumulation in tumor cells. Paclitaxel-(+)-α-tocopherol (PTX-VE)-loaded mixed micelles (PHIS/FA/PM) fabricated by poly(ethylene glycol) methyl ether-poly(histidine) (MPEG-PHIS) and folic acid-poly(ethylene glycol)-(+)-α-tocopherol (FA-PEG-VE) were characterized by dynamic light scattering and transmission electron microscopy (TEM). The mixed micelles had a spherical morphology with an average diameter of 137.0±6.70 nm and a zeta potential of −48.7±4.25 mV. The drug encapsulation and loading efficiencies were 91.06%±2.45% and 5.28%±0.30%, respectively. The pH sensitivity was confirmed by changes in particle size, critical micelle concentration, and transmittance as a function of pH. MTT assay showed that PHIS/FA/PM had higher cytotoxicity at pH 6.0 than at pH 7.4, and lower cytotoxicity in the presence of free FA. Confocal laser scanning microscope images demonstrated a time-dependent and FA-inhibited cellular uptake. In vivo imaging confirmed that the mixed micelles targeted accumulation at tumor sites and the tumor inhibition rate was 85.97%. The results proved that the mixed micelle system fabricated by MPEG-PHIS and FA-PEG-VE is a promising approach to improve antitumor efficacy. PMID:28860753

Tumor-targeted pH/redox dual-sensitive unimolecular nanoparticles for efficient siRNA delivery.

PubMed

Chen, Guojun; Wang, Yuyuan; Xie, Ruosen; Gong, Shaoqin

2017-08-10

A unique pH/redox dual-sensitive cationic unimolecular nanoparticle (NP) enabling excellent endosomal/lysosomal escape and efficient siRNA decomplexation inside the target cells was developed for tumor-targeted delivery of siRNA. siRNA was complexed into the cationic core of the unimolecular NP through electrostatic interactions. The cationic core used for complexing siRNA contained reducible disulfide bonds that underwent intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH) inside the cells, thereby facilitating the decomplexation of siRNA from the unimolecular NPs. The cationic polymers were conjugated onto the hyperbranched core (H40) via a pH-sensitive bond, which further facilitated the decomplexation of siRNA from the NPs. In vitro studies on the siRNA release behaviors showed that dual stimuli (pH=5.3, 10mM GSH) induced the quickest release of siRNA from the NPs. In addition, the imidazole groups attached to the cationic polymer segments enhanced the endosomal/lysosomal escape of NPs via the proton sponge effect. Intracellular tracking studies revealed that siRNA delivered by unimolecular NPs was efficiently released to the cytosol. Moreover, the GE11 peptide, an anti-EGFR peptide, enhanced the cellular uptake of NPs in MDA-MB-468, an EFGR-overexpressing triple negative breast cancer (TNBC) cell line. The GE11-conjugated, GFP-siRNA-complexed NPs exhibited excellent GFP gene silencing efficiency in GFP-MDA-MB-468 TNBC cells without any significant cytotoxicity. Therefore, these studies suggest that this smart unimolecular NP could be a promising nanoplatform for targeted siRNA delivery to EFGR-overexpressing cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeting Signal Transducers and Activators of Transcription-3 (Stat3) As a Novel Strategy In Sensitizing Breast Cancer To Egfr-Targeted Therapy

DTIC Science & Technology

2008-06-01

Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and...Novel Strategy In Sensitizing Breast Cancer To Egfr-Targeted Therapy 5b. GRANT NUMBER W81XWH-07-1-0390 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ...Hui-Wen Lo, Ph.D. 5d. PROJECT NUMBER 5e. TASK NUMBER Email: huiwen.lo@duke.edu 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME( S

49 CFR 325.77 - Computation of open site requirements-nonstandard sites.

Code of Federal Regulations, 2010 CFR

2010-10-01

... microphone target point is other than 50 feet (15.2 m), the test site must be an open site within a radius... microphone target point. (b) Plan view diagrams of nonstandard test sites are shown in Figures 3 and 4... (18.3 m) distance between the microphone location point and the microphone target point. (See § 325.79...

49 CFR 325.77 - Computation of open site requirements-nonstandard sites.

Code of Federal Regulations, 2011 CFR

2011-10-01

... microphone target point is other than 50 feet (15.2 m), the test site must be an open site within a radius... microphone target point. (b) Plan view diagrams of nonstandard test sites are shown in Figures 3 and 4... (18.3 m) distance between the microphone location point and the microphone target point. (See § 325.79...

TargetM6A: Identifying N6-Methyladenosine Sites From RNA Sequences via Position-Specific Nucleotide Propensities and a Support Vector Machine.

PubMed

Li, Guang-Qing; Liu, Zi; Shen, Hong-Bin; Yu, Dong-Jun

2016-10-01

As one of the most ubiquitous post-transcriptional modifications of RNA, N 6 -methyladenosine ( [Formula: see text]) plays an essential role in many vital biological processes. The identification of [Formula: see text] sites in RNAs is significantly important for both basic biomedical research and practical drug development. In this study, we designed a computational-based method, called TargetM6A, to rapidly and accurately target [Formula: see text] sites solely from the primary RNA sequences. Two new features, i.e., position-specific nucleotide/dinucleotide propensities (PSNP/PSDP), are introduced and combined with the traditional nucleotide composition (NC) feature to formulate RNA sequences. The extracted features are further optimized to obtain a much more compact and discriminative feature subset by applying an incremental feature selection (IFS) procedure. Based on the optimized feature subset, we trained TargetM6A on the training dataset with a support vector machine (SVM) as the prediction engine. We compared the proposed TargetM6A method with existing methods for predicting [Formula: see text] sites by performing stringent jackknife tests and independent validation tests on benchmark datasets. The experimental results show that the proposed TargetM6A method outperformed the existing methods for predicting [Formula: see text] sites and remarkably improved the prediction performances, with MCC = 0.526 and AUC = 0.818. We also provided a user-friendly web server for TargetM6A, which is publicly accessible for academic use at http://csbio.njust.edu.cn/bioinf/TargetM6A.

Visual cues of oviposition sites and spectral sensitivity of Cydia strobilella L.

PubMed

Jakobsson, Johan; Henze, Miriam J; Svensson, Glenn P; Lind, Olle; Anderbrant, Olle

2017-08-01

We investigated whether the spruce seed moth (Cydia strobilella L., Tortricidae: Grapholitini), an important pest in seed orchards of Norway spruce (Picea abies (L.) Karst.), can make use of the spectral properties of its host when searching for flowers to oviposit on. Spectral measurements showed that the flowers, and the cones they develop into, differ from a background of P. abies needles by a higher reflectance of long wavelengths. These differences increase as the flowers develop into mature cones. Electroretinograms (ERGs) in combination with spectral adaptation suggest that C. strobilella has at least three spectral types of photoreceptor; an abundant green-sensitive receptor with maximal sensitivity at wavelength λ max =526nm, a blue-sensitive receptor with λ max =436nm, and an ultraviolet-sensitive receptor with λ max =352nm. Based on our spectral measurements and the receptor properties inferred from the ERGs, we calculated that open flowers, which are suitable oviposition sites, provide detectable achromatic, but almost no chromatic contrasts to the background of needles. In field trials using traps of different spectral properties with or without a female sex pheromone lure, only pheromone-baited traps caught moths. Catches in baited traps were not correlated with the visual contrast of the traps against the background. Thus, visual contrast is probably not the primary cue for finding open host flowers, but it could potentially complement olfaction as a secondary cue, since traps with certain spectral properties caught significantly more moths than others. Copyright © 2017 Elsevier Ltd. All rights reserved.

Design criteria for a high energy Compton Camera and possible application to targeted cancer therapy

NASA Astrophysics Data System (ADS)

Conka Nurdan, T.; Nurdan, K.; Brill, A. B.; Walenta, A. H.

2015-07-01

The proposed research focuses on the design criteria for a Compton Camera with high spatial resolution and sensitivity, operating at high gamma energies and its possible application for molecular imaging. This application is mainly on the detection and visualization of the pharmacokinetics of tumor targeting substances specific for particular cancer sites. Expected high resolution (< 0.5 mm) permits monitoring the pharmacokinetics of labeled gene constructs in vivo in small animals with a human tumor xenograft which is one of the first steps in evaluating the potential utility of a candidate gene. The additional benefit of high sensitivity detection will be improved cancer treatment strategies in patients based on the use of specific molecules binding to cancer sites for early detection of tumors and identifying metastasis, monitoring drug delivery and radionuclide therapy for optimum cell killing at the tumor site. This new technology can provide high resolution, high sensitivity imaging of a wide range of gamma energies and will significantly extend the range of radiotracers that can be investigated and used clinically. The small and compact construction of the proposed camera system allows flexible application which will be particularly useful for monitoring residual tumor around the resection site during surgery. It is also envisaged as able to test the performance of new drug/gene-based therapies in vitro and in vivo for tumor targeting efficacy using automatic large scale screening methods.

Decaleside: a new class of natural insecticide targeting tarsal gustatory sites

NASA Astrophysics Data System (ADS)

Rajashekar, Yallappa; Rao, Lingamallu J. M.; Shivanandappa, Thimmappa

2012-10-01

Natural sources for novel insecticide molecules hold promise in view of their eco-friendly nature, selectivity, and mammalian safety. Recent progress in understanding the biology of insect olfaction and taste offers new strategies for developing selective pest control agents. We have isolated two natural insecticidal molecules from edible roots of Decalepis hamiltonii named Decalesides I and II, which are novel trisaccharides, highly toxic to household insect pests and stored-product insects. We have experimentally shown that insecticidal activity requires contact with tarsi on the legs but is not toxic orally. The insecticidal activity of molecules is lost by hydrolysis, and various sugars modify toxic response, showing that the insecticidal activity is via gustatory sites on the tarsi. Selective toxicity to insects by virtue of their gustatory site of action and the mammalian safety of the new insecticides is inherent in their chemical structure with 1-4 or 1-1 α linkage that is easily hydrolyzed by digestive enzymes of mammals. Decalesides represent a new chemical class of natural insecticides with a unique mode of action targeting tarsal chemosensory/gustatory system of insects.

Pseudotargeted MS Method for the Sensitive Analysis of Protein Phosphorylation in Protein Complexes.

PubMed

Lyu, Jiawen; Wang, Yan; Mao, Jiawei; Yao, Yating; Wang, Shujuan; Zheng, Yong; Ye, Mingliang

2018-05-15

In this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence. The development of this targeted approach is very easy as the same sample and the same LC-system were used for the discovery and the targeted analysis phases. No sample fractionation or enrichment was required for the discovery phase which allowed this method to analyze minute amount of sample. We applied this pseudotargeted MS method to quantitatively examine phosphopeptides in affinity purified endogenous Shc1 protein complexes at four temporal stages of EGF signaling and identified 82 phospho-sites. To our knowledge, this is the highest number of phospho-sites identified from the protein complexes. This pseudotargeted MS method is highly sensitive in the identification of low abundance phosphopeptides and could be a powerful tool to study phosphorylation-regulated assembly of protein complex.

Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

PubMed

Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

2016-04-01

An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. © The Author(s) 2016.

Highly Sensitive Detection of Target Biomolecules on Cell Surface Using Gold Nanoparticle Conjugated with Aptamer Probe

NASA Astrophysics Data System (ADS)

Kim, Hyonchol; Terazono, Hideyuki; Hayashi, Masahito; Takei, Hiroyuki; Yasuda, Kenji

2012-06-01

A method of gold nanoparticle (Au NP) labeling with backscattered electron (BE) imaging of field emission scanning electron microscopy (FE-SEM) was applied for specific detection of target biomolecules on a cell surface. A single-stranded DNA aptamer, which specifically binds to the target molecule on a human acute lymphoblastic leukemia cell, was conjugated with a 20 nm Au NP and used as a probe to label its target molecule on the cell. The Au NP probe was incubated with the cell, and the interaction was confirmed using BE imaging of FE-SEM through direct counting of the number of Au NPs attached on the target cell surface. Specific Au NP-aptamer probes were observed on a single cell surface and their spatial distributions including submicron-order localizations were also clearly visualized, whereas the nonspecific aptamer probes were not observed on it. The aptamer probe can be potentially dislodged from the cell surface with treatment of nucleases, indicating that Au NP-conjugated aptamer probes can be used as sensitive and reversible probes to label target biomolecules on cells.

Improving sensitivity and specificity of capturing and detecting targeted cancer cells with anti-biofouling polymer coated magnetic iron oxide nanoparticles.

PubMed

Lin, Run; Li, Yuancheng; MacDonald, Tobey; Wu, Hui; Provenzale, James; Peng, Xingui; Huang, Jing; Wang, Liya; Wang, Andrew Y; Yang, Jianyong; Mao, Hui

2017-02-01

Detecting circulating tumor cells (CTCs) with high sensitivity and specificity is critical to management of metastatic cancers. Although immuno-magnetic technology for in vitro detection of CTCs has shown promising potential for clinical applications, the biofouling effect, i.e., non-specific adhesion of biomolecules and non-cancerous cells in complex biological samples to the surface of a device/probe, can reduce the sensitivity and specificity of cell detection. Reported herein is the application of anti-biofouling polyethylene glycol-block-allyl glycidyl ether copolymer (PEG-b-AGE) coated iron oxide nanoparticles (IONPs) to improve the separation of targeted tumor cells from aqueous phase in an external magnetic field. PEG-b-AGE coated IONPs conjugated with transferrin (Tf) exhibited significant anti-biofouling properties against non-specific protein adsorption and off-target cell uptake, thus substantially enhancing the ability to target and separate transferrin receptor (TfR) over-expressed D556 medulloblastoma cells. Tf conjugated PEG-b-AGE coated IONPs exhibited a high capture rate of targeted tumor cells (D556 medulloblastoma cell) in cell media (58.7±6.4%) when separating 100 targeted tumor cells from 1×10 5 non-targeted cells and 41 targeted tumor cells from 100 D556 medulloblastoma cells spiked into 1mL blood. It is demonstrated that developed nanoparticle has higher efficiency in capturing targeted cells than widely used micron-sized particles (i.e., Dynabeads ® ). Copyright © 2016 Elsevier B.V. All rights reserved.

A Novel W1999S Mutation and Non-Target Site Resistance Impact on Acetyl-CoA Carboxylase Inhibiting Herbicides to Varying Degrees in a UK Lolium multiflorum Population

PubMed Central

Kaundun, Shiv Shankhar; Bailly, Geraldine C.; Dale, Richard P.; Hutchings, Sarah-Jane; McIndoe, Eddie

2013-01-01

Background Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). Methodology/Principal Findings The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. Conclusion/Significance This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance. PMID:23469130

Multiple Nucleosome Positioning Sites Regulate the CTCF-Mediated Insulator Function of the H19 Imprinting Control Region†

PubMed Central

Kanduri, Meena; Kanduri, Chandrasekhar; Mariano, Piero; Vostrov, Alexander A.; Quitschke, Wolfgang; Lobanenkov, Victor; Ohlsson, Rolf

2002-01-01

The 5′ region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites. PMID:11971967

An effective tumor-targeting strategy utilizing hypoxia-sensitive siRNA delivery system for improved anti-tumor outcome.

PubMed

Kang, Lin; Fan, Bo; Sun, Ping; Huang, Wei; Jin, Mingji; Wang, Qiming; Gao, Zhonggao

2016-10-15

Hypoxia is a feature of most solid tumors, targeting hypoxia is considered as the best validated yet not extensively exploited strategy in cancer therapy. Here, we reported a novel tumor-targeting strategy using a hypoxia-sensitive siRNA delivery system. In the study, 2-nitroimidazole (NI), a hydrophobic component that can be converted to hydrophilic 2-aminoimidazole (AI) through bioreduction under hypoxic conditions, was conjugated to the alkylated polyethyleneimine (bPEI1.8k-C6) to form amphiphilic bPEI1.8k-C6-NI polycations. bPEI1.8k-C6-NI could self-assemble into micelle-like aggregations in aqueous, which contributed to the improved stability of the bPEI1.8k-C6-NI/siRNA polyplexes, resulted in increased cellular uptake. After being transported into the hypoxic tumor cells, the selective nitro-to-amino reduction would cause structural change and elicit a relatively loose structure to facilitate the siRNA dissociation in the cytoplasm, for enhanced gene silencing efficiency ultimately. Therefore, the conflict between the extracellular stability and the intracellular siRNA release ability of the polyplexes was solved by introducing the hypoxia-responsive unit. Consequently, the survivin-targeted siRNA loaded polyplexes shown remarkable anti-tumor effect not only in hypoxic cells, but also in tumor spheroids and tumor-bearing mice, indicating that the hypoxia-sensitive siRNA delivery system had great potential for tumor-targeted therapy. Hypoxia is one of the most remarkable features of most solid tumors, and targeting hypoxia is considered as the best validated strategy in cancer therapy. However, in the past decades, there were few reports about using this strategy in the drug delivery system, especially in siRNA delivery system. Therefore, we constructed a hypoxia-sensitive siRNA delivery system utilizing a hypoxia-responsive unit, 2-nitroimidazole, by which the unavoidable conflict between improved extracellular stability and promoted intracellular si

MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1.

PubMed

Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin

2016-11-01

The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention.

Multi-targeted inhibition of tumor growth and lung metastasis by redox-sensitive shell crosslinked micelles loading disulfiram

NASA Astrophysics Data System (ADS)

Duan, Xiaopin; Xiao, Jisheng; Yin, Qi; Zhang, Zhiwen; Yu, Haijun; Mao, Shirui; Li, Yaping

2014-03-01

Metastasis, the main cause of cancer related deaths, remains the greatest challenge in cancer treatment. Disulfiram (DSF), which has multi-targeted anti-tumor activity, was encapsulated into redox-sensitive shell crosslinked micelles to achieve intracellular targeted delivery and finally inhibit tumor growth and metastasis. The crosslinked micelles demonstrated good stability in circulation and specifically released DSF under a reductive environment that mimicked the intracellular conditions of tumor cells. As a result, the DSF-loaded redox-sensitive shell crosslinked micelles (DCMs) dramatically inhibited cell proliferation, induced cell apoptosis and suppressed cell invasion, as well as impairing tube formation of HMEC-1 cells. In addition, the DCMs could accumulate in tumor tissue and stay there for a long time, thereby causing significant inhibition of 4T1 tumor growth and marked prevention in lung metastasis of 4T1 tumors. These results suggested that DCMs could be a promising delivery system in inhibiting the growth and metastasis of breast cancer.

Investigation of the LMJ ignition target sensitivity to the laser pulse shape with 2D integrated calculations

NASA Astrophysics Data System (ADS)

Cherfils, Catherine; Malinie, Guy; Boniface, Claude; Gauthier, Pascal; Laffite, Stephane; Loiseau, Pascal

2010-11-01

The A943 cryogenic target in a Rugby hohlraum is our current nominal design for ignition with 160 beams on the Laser MegaJoule (Laffite et al 2007, 49th Annual Meeting of the Division of Plasma Physics, Loiseau et al 2010, 40th Annual Anomalous Absorption Conference). In this study we redesign the laser pulse of the target under the form of a sum of six supergaussians, which is more amenable to a sensitivity study : four supergaussians are used to launch the four main shocks in the capsule, and two additional supergaussians are used first to remove the LEH windows and then to control the acceleration of the first shock, respectively. We use our 2D FCI2 code to compare the radiation hydro of the capsule, obtained with this new pulse, to what was previously obtained. We investigate the sensitivity of the yield on some parameters, which are the maximum powers and respective timings of the different components of the laser pulse.

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

PubMed

Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

2017-02-01

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1.

PubMed

Kulebyakin, Konstantin; Penkov, Dmitry; Blasi, Francesco; Akopyan, Zhanna; Tkachuk, Vsevolod

2016-12-02

Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic β-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to search new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability. Copyright © 2016 Elsevier Inc. All rights reserved.

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Acid-Sensitive Sheddable PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α siRNA in Chronic Inflammation Sites

PubMed Central

Aldayel, Abdulaziz M; Naguib, Youssef W; O'Mary, Hannah L; Li, Xu; Niu, Mengmeng; Ruwona, Tinashe B; Cui, Zhengrong

2016-01-01

There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot. PMID:27434685

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

PubMed Central

Abi-Ghanem, Josephine; Chusainow, Janet; Karimova, Madina; Spiegel, Christopher; Hofmann-Sieber, Helga; Hauber, Joachim; Buchholz, Frank; Pisabarro, M. Teresa

2013-01-01

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine. PMID:23275541

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

PubMed

Dong, L-F; Low, P; Dyason, J C; Wang, X-F; Prochazka, L; Witting, P K; Freeman, R; Swettenham, E; Valis, K; Liu, J; Zobalova, R; Turanek, J; Spitz, D R; Domann, F E; Scheffler, I E; Ralph, S J; Neuzil, J

2008-07-17

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

PubMed Central

Dong, Lan-Feng; Low, Pauline; Dyason, Jeffrey C.; Wang, Xiu-Fang; Prochazka, Lubomir; Witting, Paul K.; Freeman, Ruth; Swettenham, Emma; Valis, Karel; Liu, Ji; Zobalova, Renata; Turanek, Jaroslav; Spitz, Doug R.; Domann, Frederick E.; Scheffler, Immo E.; Ralph, Stephen J.; Neuzil, Jiri

2009-01-01

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ) binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and undergo apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy. PMID:18372923

Lung tumors with distinct p53 mutations respond similarly to p53 targeted therapy but exhibit genotype-specific statin sensitivity

PubMed Central

Turrell, Frances K.; Kerr, Emma M.; Gao, Meiling; Thorpe, Hannah; Doherty, Gary J.; Cridge, Jake; Shorthouse, David; Speed, Alyson; Samarajiwa, Shamith; Hall, Benjamin A.; Griffiths, Meryl; Martins, Carla P.

2017-01-01

Lung adenocarcinoma accounts for ∼40% of lung cancers, the leading cause of cancer-related death worldwide, and current therapies provide only limited survival benefit. Approximately half of lung adenocarcinomas harbor mutations in TP53 (p53), making these mutants appealing targets for lung cancer therapy. As mutant p53 remains untargetable, mutant p53-dependent phenotypes represent alternative targeting opportunities, but the prevalence and therapeutic relevance of such effects (gain of function and dominant-negative activity) in lung adenocarcinoma are unclear. Through transcriptional and functional analysis of murine KrasG12D-p53null, -p53R172H (conformational), and -p53R270H (contact) mutant lung tumors, we identified genotype-independent and genotype-dependent therapeutic sensitivities. Unexpectedly, we found that wild-type p53 exerts a dominant tumor-suppressive effect on mutant tumors, as all genotypes were similarly sensitive to its restoration in vivo. These data show that the potential of p53 targeted therapies is comparable across all p53-deficient genotypes and may explain the high incidence of p53 loss of heterozygosity in mutant tumors. In contrast, mutant p53 gain of function and their associated vulnerabilities can vary according to mutation type. Notably, we identified a p53R270H-specific sensitivity to simvastatin in lung tumors, and the transcriptional signature that underlies this sensitivity was also present in human lung tumors, indicating that this therapeutic approach may be clinically relevant. PMID:28790158

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.

PubMed

Boja, Emily S; Rodriguez, Henry

2012-04-01

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SETI target selection.

PubMed

Latham, D W; Soderblom, D R

1995-01-01

The NASA High Resolution Microwave Survey consists of two complementary elements: a Sky Survey of the entire sky to a moderate level of sensitivity; and a Targeted Search of nearby stars, one at a time, to a much deeper level of sensitivity. In this paper we propose strategies for target selection. We have two goals: to improve the chances of successful detection of signals from technical civilizations that inhabit planets around solar-type stars, and to minimize the chances of missing signals from unexpected sites. For the main Targeted Search survey of approximately 1000 nearby solar-type stars, we argue that the selection criteria should be heavily biased by what we know about the origin and evolution of life here on Earth. We propose that observations of stars with stellar companions orbiting near the habitable zone should be de-emphasized, because such companions would prevent the formation of habitable planets. We also propose that observations of stars younger than about three billion years should be de-emphasized in favor of older stars, because our own technical civilization took longer than three billion years to evolve here on Earth. To provide the information needed for the preparation of specific target lists, we have undertaken an inventory of a large sample of solar-type stars out to a distance of 60 pc, with the goal of characterizing the relevant astrophysical properties of these stars, especially their ages and companionship. To complement the main survey, we propose that a modest sample of the nearest stars should be observed without any selection biases whatsoever. Finally, we argue that efforts to identify stars with planetary systems should be expanded. If found, such systems should receive intensive scrutiny.

The transcription factor Prep1 controls hepatic insulin sensitivity and gluconeogenesis by targeting nuclear localization of FOXO1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kulebyakin, Konstantin; Penkov, Dmitry; IFOM – the FIRC Institute of Molecular Oncology, Via Adamello 16, Milan, 20139

Liver plays a key role in controlling body carbohydrate homeostasis by switching between accumulation and production of glucose and this way maintaining constant level of glucose in blood. Increased blood glucose level triggers release of insulin from pancreatic β-cells. Insulin represses hepatic glucose production and increases glucose accumulation. Insulin resistance is the main cause of type 2 diabetes and hyperglycemia. Currently thiazolidinediones (TZDs) targeting transcriptional factor PPARγ are used as insulin sensitizers for treating patients with type 2 diabetes. However, TZDs are reported to be associated with cardiovascular and liver problems and stimulate obesity. Thus, it is necessary to searchmore » new approaches to improve insulin sensitivity. A promising candidate is transcriptional factor Prep1, as it was shown earlier it could affect insulin sensitivity in variety of insulin-sensitive tissues. The aim of the present study was to evaluate a possible involvement of transcriptional factor Prep1 in control of hepatic glucose accumulation and production. We created mice with liver-specific Prep1 knockout and discovered that hepatocytes derived from these mice are much more sensitive to insulin, comparing to their WT littermates. Incubation of these cells with 100 nM insulin results in almost complete inhibition of gluconeogenesis, while in WT cells this repression is only partial. However, Prep1 doesn't affect gluconeogenesis in the absence of insulin. Also, we observed that nuclear content of gluconeogenic transcription factor FOXO1 was greatly reduced in Prep1 knockout hepatocytes. These findings suggest that Prep1 may control hepatic insulin sensitivity by targeting FOXO1 nuclear stability. - Highlights: • A novel model of liver-specific Prep1 knockout is established. • Ablation of Prep1 in hepatocytes increases insulin sensitivity. • Prep1 controls hepatic insulin sensitivity by regulating localization of FOXO1. • Prep1 regulates

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens.

PubMed

Morgens, David W; Wainberg, Michael; Boyle, Evan A; Ursu, Oana; Araya, Carlos L; Tsui, C Kimberly; Haney, Michael S; Hess, Gaelen T; Han, Kyuho; Jeng, Edwin E; Li, Amy; Snyder, Michael P; Greenleaf, William J; Kundaje, Anshul; Bassik, Michael C

2017-05-05

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens

PubMed Central

Morgens, David W.; Wainberg, Michael; Boyle, Evan A.; Ursu, Oana; Araya, Carlos L.; Tsui, C. Kimberly; Haney, Michael S.; Hess, Gaelen T.; Han, Kyuho; Jeng, Edwin E.; Li, Amy; Snyder, Michael P.; Greenleaf, William J.; Kundaje, Anshul; Bassik, Michael C.

2017-01-01

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens. PMID:28474669

Flexible, transparent and highly sensitive SERS substrates with cross-nanoporous structures for fast on-site detection.

PubMed

Wang, Yingcheng; Jin, Yuanhao; Xiao, Xiaoyang; Zhang, Tianfu; Yang, Haitao; Zhao, Yudan; Wang, Jiaping; Jiang, Kaili; Fan, Shoushan; Li, Qunqing

2018-05-30

A flexible and transparent film assembled from the cross-nanoporous structures of Au on PET (CNS of Au@PET) is developed as a versatile and effective SERS substrate for rapid, on-site trace analysis with high sensitivity. The fabrication of the CNS of Au can be achieved on a large scale at low cost by employing an etching process with super-aligned carbon nanotubes as a mask, followed by metal deposition. A strongly enhanced Raman signal with good uniformity can be obtained, which is attributed to the excitation of "hot spots" around the metal nanogaps and sharp edges. Using the CNS of Au@PET film as a SERS platform, real-time and on-site SERS detection of the food contaminant crystal violet (CV) is achieved, with a detection limit of CV solution on a tomato skin of 10-7 M. Owing to its ability to efficiently extract trace analytes, the resulting substrate also achieves detection of 4-ATP contaminants and thiram pesticides by swabbing the skin of an apple. A SERS detection signal for 4-ATP has a relative standard deviation of less than 10%, revealing the excellent reproducibility of the substrate. The flexible, transparent and highly sensitive substrates fabricated using this simple and cost-effective strategy are promising for practical application in rapid, on-site SERS-based detection.

The Acetylcholine Receptor and Its Ionic Channel as Targets for Drugs and Toxins

DTIC Science & Technology

1981-12-10

mlecular target that can have any number of different binding sites and is able to generate levels of energy barriers which &re a direct function of the...Albuquerque, E.X. XEatrachotoxi.-A 20- a -benzoate: A new radioactive ligand for voltage- sensitive sodium channels. Cell. Mol. Neurobio .. 1: 19-40

Assessment of Terra MODIS On-Orbit Polarization Sensitivity Using Pseudoinvariant Desert Sites

NASA Technical Reports Server (NTRS)

Wu, Aisheng; Geng, Xu; Wald, Andrew; Angal, Amit; Xiong, Xiaoxiong

2017-01-01

The Moderate Resolution Imaging Spectroradiometer (MODIS) is currently flying on NASA's Earth Observing System Terra and Aqua satellites, launched in 1999 and 2002, respectively. MODIS reflective solar bands in the visible wavelength range are known to be sensitive to polarized light based on prelaunch polarization sensitivity tests. After about five years of on-orbit operations, it was discovered that the polarization sensitivity at short wavelengths had shown a noticeable increase. In this paper, we examine the impact of polarization on measured top-of-atmosphere (TOA) reflectance based on MODIS Collection-6 L1B over pseudo invariant desert sites. The standard polarization correction equation is used in combination with simulated at-sensor radiances using the second simulation of a satellite signal in the Solar Spectrum, Vector Radiative Transfer Code (6SV). We ignore the polarization contribution from the surface and a ratio approach is used for both 6SV-derived in put parameters and observed TOA reflectance. Results indicate that significant gain corrections up to 25% are required near the end of scan for the 412 and 443 nm bands. The polarization correction reduces the seasonal fluctuations in reflectance trends and mirror side ratios from 30% and 12% to 10% and 5%, respectively, for the two bands. Comparison of the effectiveness of the polarization correction with the results from the NASA Ocean Biology Processing Group shows a good agreement in the corrected reflectance trending results and their seasonal fluctuations.

New support vector machine-based method for microRNA target prediction.

PubMed

Li, L; Gao, Q; Mao, X; Cao, Y

2014-06-09

MicroRNA (miRNA) plays important roles in cell differentiation, proliferation, growth, mobility, and apoptosis. An accurate list of precise target genes is necessary in order to fully understand the importance of miRNAs in animal development and disease. Several computational methods have been proposed for miRNA target-gene identification. However, these methods still have limitations with respect to their sensitivity and accuracy. Thus, we developed a new miRNA target-prediction method based on the support vector machine (SVM) model. The model supplies information of two binding sites (primary and secondary) for a radial basis function kernel as a similarity measure for SVM features. The information is categorized based on structural, thermodynamic, and sequence conservation. Using high-confidence datasets selected from public miRNA target databases, we obtained a human miRNA target SVM classifier model with high performance and provided an efficient tool for human miRNA target gene identification. Experiments have shown that our method is a reliable tool for miRNA target-gene prediction, and a successful application of an SVM classifier. Compared with other methods, the method proposed here improves the sensitivity and accuracy of miRNA prediction. Its performance can be further improved by providing more training examples.

L-baclofen-sensitive GABAB binding sites in the medial vestibular nucleus localized by immunocytochemistry

NASA Technical Reports Server (NTRS)

Holstein, G. R.; Martinelli, G. P.; Cohen, B.

1992-01-01

L-Baclofen-sensitive GABAB binding sites in the medial vestibular nucleus (MVN) were identified immunocytochemically and visualized ultrastructurally in L-baclofen-preinjected rats and monkeys, using a mouse monoclonal antibody with specificity for the p-chlorophenyl moiety of baclofen. Saline-preinjected animals showed no immunostain. In drug-injected animals, there was evidence for both pre- and postsynaptic GABAergic inhibition in MVN mediated by GABAB receptors. These neural elements could be utilized in control of velocity storage in the vestibulo-ocular reflex.

Evaluation of antitumor activity and cardiac toxicity of a bone-targeted ph-sensitive liposomal formulation in a bone metastasis tumor model in mice.

PubMed

Dos Santos Ferreira, Diego; Jesus de Oliveira Pinto, Bruno Luís; Kumar, Vidhya; Cardoso, Valbert Nascimento; Fernandes, Simone Odília; Souza, Cristina Maria; Cassali, Geovanni Dantas; Moore, Anna; Sosnovik, David E; Farrar, Christian T; Leite, Elaine Amaral; Alves, Ricardo José; de Oliveira, Mônica Cristina; Guimarães, Alexander Ramos; Caravan, Peter

2017-07-01

Chemotherapy for bone tumors is a major challenge because of the inability of therapeutics to penetrate dense bone mineral. We hypothesize that a nanostructured formulation with high affinity for bone could deliver drug to the tumor while minimizing off-target toxicity. Here, we evaluated the efficacy and toxicity of a novel bone-targeted, pH-sensitive liposomal formulation containing doxorubicin in an animal model of bone metastasis. Biodistribution studies with the liposome showed good uptake in tumor, but low accumulation of doxorubicin in the heart. Mice treated with the bone-targeted liposome formulation showed a 70% reduction in tumor volume, compared to 35% reduction for free doxorubicin at the same dose. Both cardiac toxicity and overall mortality were significantly lower for animals treated with the bone-targeted liposomes compared to free drug. Bone-targeted, pH-sensitive, doxorubicin containing liposomes represent a promising approach to selectively delivering doxorubicin to bone tumors while minimizing cardiac toxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Extrasynaptic Glycine Receptors of Rodent Dorsal Raphe Serotonergic Neurons: A Sensitive Target for Ethanol

PubMed Central

Maguire, Edward P; Mitchell, Elizabeth A; Greig, Scott J; Corteen, Nicole; Balfour, David J K; Swinny, Jerome D; Lambert, Jeremy J; Belelli, Delia

2014-01-01

Alcohol abuse is a significant medical and social problem. Several neurotransmitter systems are implicated in ethanol's actions, with certain receptors and ion channels emerging as putative targets. The dorsal raphe (DR) nucleus is associated with the behavioral actions of alcohol, but ethanol actions on these neurons are not well understood. Here, using immunohistochemistry and electrophysiology we characterize DR inhibitory transmission and its sensitivity to ethanol. DR neurons exhibit inhibitory ‘phasic' post-synaptic currents mediated primarily by synaptic GABAA receptors (GABAAR) and, to a lesser extent, by synaptic glycine receptors (GlyR). In addition to such phasic transmission mediated by the vesicular release of neurotransmitter, the activity of certain neurons may be governed by a ‘tonic' conductance resulting from ambient GABA activating extrasynaptic GABAARs. However, for DR neurons extrasynaptic GABAARs exert only a limited influence. By contrast, we report that unusually the GlyR antagonist strychnine reveals a large tonic conductance mediated by extrasynaptic GlyRs, which dominates DR inhibition. In agreement, for DR neurons strychnine increases their input resistance, induces membrane depolarization, and consequently augments their excitability. Importantly, this glycinergic conductance is greatly enhanced in a strychnine-sensitive fashion, by behaviorally relevant ethanol concentrations, by drugs used for the treatment of alcohol withdrawal, and by taurine, an ingredient of certain ‘energy drinks' often imbibed with ethanol. These findings identify extrasynaptic GlyRs as critical regulators of DR excitability and a novel molecular target for ethanol. PMID:24264816

pH-sensitive Au–BSA–DOX–FA nanocomposites for combined CT imaging and targeted drug delivery

PubMed Central

Huang, He; Yang, Da-Peng; Liu, Minghuan; Wang, Xiangsheng; Zhang, Zhiyong; Zhou, Guangdong; Liu, Wei; Cao, Yilin; Zhang, Wen Jie; Wang, Xiansong

2017-01-01

Albumin-based nanoparticles (NPs) as a drug delivery system have attracted much attention owing to their nontoxicity, non-immunogenicity, great stability and ability to bind to many therapeutic drugs. Herein, bovine serum albumin (BSA) was utilized as a template to prepare Au–BSA core/shell NPs. The outer layer BSA was subsequently conjugated with cis-aconityl doxorubicin (DOX) and folic acid (FA) to create Au–BSA–DOX–FA nanocomposites. A list of characterizations was undertaken to identify the successful conjugation of drug molecules and targeted agents. In vitro cytotoxicity using a cell counting kit-8 (CCK-8) assay indicated that Au–BSA NPs did not display obvious cytotoxicity to MGC-803 and GES-1 cells in the concentration range of 0–100 μg/mL, which can therefore be used as a safe drug delivery carrier. Furthermore, compared with free DOX, Au–BSA–DOX–FA nanocomposites exhibited a pH-sensitive drug release ability and superior antitumor activity in a drug concentration-dependent manner. In vivo computed tomography (CT) imaging experiments showed that Au–BSA–DOX–FA nanocomposites could be used as an efficient and durable CT contrast agent for targeted CT imaging of the folate receptor (FR) overexpressed in cancer tissues. In vivo antitumor experiments demonstrated that Au–BSA–DOX–FA nanocomposites have selective antitumor activity effects on FR-overexpressing tumors and no adverse effects on normal tissues and organs. In conclusion, the Au–BSA–DOX–FA nanocomposite exhibits selective targeting activity, X-ray attenuation activity and pH-sensitive drug release activity. Therefore, it can enhance CT imaging and improve the targeting therapeutic efficacy of FR-overexpressing gastric cancers. Our findings suggest that Au–BSA–DOX–FA nanocomposite is a novel drug delivery carrier and a promising candidate for cancer theranostic applications. PMID:28435261

Experimental Results of Site Calibration and Sensitivity Measurements in OTR for UWB Systems

NASA Astrophysics Data System (ADS)

Viswanadham, Chandana; Rao, P. Mallikrajuna

2017-06-01

System calibration and parameter accuracy measurement of electronic support measures (ESM) systems is a major activity, carried out by electronic warfare (EW) engineers. These activities are very critical and needs good understanding in the field of microwaves, antennas, wave propagation, digital and communication domains. EW systems are broad band, built with state-of-the art electronic hardware, installed on different varieties of military platforms to guard country's security from time to time. EW systems operate in wide frequency ranges, typically in the order of thousands of MHz, hence these are ultra wide band (UWB) systems. Few calibration activities are carried within the system and in the test sites, to meet the accuracies of final specifications. After calibration, parameters are measured for their accuracies either in feed mode by injecting the RF signals into the front end or in radiation mode by transmitting the RF signals on to system antenna. To carry out these activities in radiation mode, a calibrated open test range (OTR) is necessary in the frequency band of interest. Thus site calibration of OTR is necessary to be carried out before taking up system calibration and parameter measurements. This paper presents the experimental results of OTR site calibration and sensitivity measurements of UWB systems in radiation mode.

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

Lunar Orbit Insertion Targeting and Associated Outbound Mission Design for Lunar Sortie Missions

NASA Technical Reports Server (NTRS)

Condon, Gerald L.

2007-01-01

This report details the Lunar Orbit Insertion (LOI) arrival targeting and associated mission design philosophy for Lunar sortie missions with up to a 7-day surface stay and with global Lunar landing site access. It also documents the assumptions, methodology, and requirements validated by TDS-04-013, Integrated Transit Nominal and Abort Characterization and Sensitivity Study. This report examines the generation of the Lunar arrival parking orbit inclination and Longitude of the Ascending Node (LAN) targets supporting surface missions with global Lunar landing site access. These targets support the Constellation Program requirement for anytime abort (early return) by providing for a minimized worst-case wedge angle [and an associated minimum plane change delta-velocity (V) cost] between the Crew Exploration Vehicle (CEV) and the Lunar Surface Access Module (LSAM) for an LSAM launch anytime during the Lunar surface stay.

Targeting the Allosteric Site of Oncoprotein BCR-ABL as an Alternative Strategy for Effective Target Protein Degradation.

PubMed

Shimokawa, Kenichiro; Shibata, Norihito; Sameshima, Tomoya; Miyamoto, Naoki; Ujikawa, Osamu; Nara, Hiroshi; Ohoka, Nobumichi; Hattori, Takayuki; Cho, Nobuo; Naito, Mikihiko

2017-10-12

Protein degradation technology based on hybrid small molecules is an emerging drug modality that has significant potential in drug discovery and as a unique method of post-translational protein knockdown in the field of chemical biology. Here, we report the first example of a novel and potent protein degradation inducer that binds to an allosteric site of the oncogenic BCR-ABL protein. BCR-ABL allosteric ligands were incorporated into the SNIPER (Specific and Nongenetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers) platform, and a series of in vitro biological assays of binding affinity, target protein modulation, signal transduction, and growth inhibition were carried out. One of the designed compounds, 6 (SNIPER(ABL)-062), showed desirable binding affinities against ABL1, cIAP1/2, and XIAP and consequently caused potent BCR-ABL degradation.

Safe and efficient pH sensitive tumor targeting modified liposomes with minimal cytotoxicity.

PubMed

Wang, Lilin; Geng, Di; Su, Haijia

2014-11-01

Incorporating the pH-sensitivity of octylamine grafted poly aspartic acid (PASP) with the biocompatibility of liposomes, a novel pH sensitive drug delivery system, octylamine-graft-PASP (PASP-g-C8) modified liposomes (OPLPs), was obtained. Since hydrophobic chains have been grafted into PASP backbones, the octylamine chain could act as the "anchor" to implant onto liposomes. The structure of PASP-g-C8, involving long-chain and hydrophobic anchors can significantly enhance the stability of the drug carrier. The shortcoming of single PASP chain modified liposomes (PLPs), that cannot sustain a slow and controlled release especially in a physiological pH solution (resembling normal tissues of pH 7.4) is thus overcome. Drug release experiments were carried out and the result showed that OPLPs sustained a slow and steady release in comparison with PLPs in the physiological pH 7.4 environment. However, OPLPs can provide a fast release in subacid environment (pH 5.0 of resembled tumor tissues). The results of diameter analysis and zeta potential demonstrated that OPLPs presented a larger diameter and higher electronegativity. Furthermore, in the "chain-anchor" structure of PASP-g-C8, the degree of substitution (DS) of the "anchor" is a remarkable factor to alter the pH-sensitivity of OPLPs. The in vitro tumor inhibition and cell toxicity studies revealed that tumor cells treated with OPLPs survived only 35.0% after 48 h whereas normal cells survived 100% in the same condition. The pH sensitive OPLPs are promising tumor targeting drug delivery with high tumor inhibition and insignificant cytotoxicity. Copyright © 2014. Published by Elsevier B.V.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

PubMed

Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

2017-03-09

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

Eph A10-modified pH-sensitive liposomes loaded with novel triphenylphosphine-docetaxel conjugate possess hierarchical targetability and sufficient antitumor effect both in vitro and in vivo.

PubMed

Zhang, Jiulong; Yang, Chunrong; Pan, Shuang; Shi, Menghao; Li, Jie; Hu, Haiyang; Qiao, Mingxi; Chen, Dawei; Zhao, Xiuli

2018-11-01

Mitochondrial-targeting therapy was considered to be a promising approach for the efficient treatment of cancer while positive charge induced nonspecific cytotoxicity severely limits its application. To overcome this drawback, a novel mitochondria targeted conjugate triphenylphosphine-docetaxel (TD) has been synthesized successfully and incorporated it into liposomes (EPSLP/TD), which possessed excellent pH-sensitive characteristic, EphA 10 mediated active targetability as well as mitochondria-targeting capability. EPSLP/TD was characterized to have a small particle size, high-encapsulation efficiency and excellent pH-sensitive characteristic. Compared with DTX-loaded liposomes (EPSLP/DTX), EPSLP/TD possessed higher cytotoxicity against MCF-7 cell line. Mitochondrial-targeting assay demonstrated mitochondria-targeting moiety triphenylphosphine (TPP) could efficiently deliver DTX to mitochondria. Western immunoblotting assay indicated that EPSLP/TD could efficiently deliver antitumor drug to mitochondria and induce cell apoptosis via mitochondria-mediated apoptosis pathway. In vivo antitumor study demonstrated EPSLP/TD owed excellent in vivo antitumor activity. Histological assay demonstrated EPSLP/TD showed strongly apoptosis inducing effect, anti-proliferation effect and anti-angiogenesis effect. This work investigated the potential of hierarchical targeting pH-sensitive liposomes is a suitable carrier to activate mitochondria-mediated apoptosis pathway for cancer therapy.

Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

PubMed

Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

2014-12-05

As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

TargetMiner: microRNA target prediction with systematic identification of tissue-specific negative examples.

PubMed

Bandyopadhyay, Sanghamitra; Mitra, Ramkrishna

2009-10-15

Prediction of microRNA (miRNA) target mRNAs using machine learning approaches is an important area of research. However, most of the methods suffer from either high false positive or false negative rates. One reason for this is the marked deficiency of negative examples or miRNA non-target pairs. Systematic identification of non-target mRNAs is still not addressed properly, and therefore, current machine learning approaches are compelled to rely on artificially generated negative examples for training. In this article, we have identified approximately 300 tissue-specific negative examples using a novel approach that involves expression profiling of both miRNAs and mRNAs, miRNA-mRNA structural interactions and seed-site conservation. The newly generated negative examples are validated with pSILAC dataset, which elucidate the fact that the identified non-targets are indeed non-targets.These high-throughput tissue-specific negative examples and a set of experimentally verified positive examples are then used to build a system called TargetMiner, a support vector machine (SVM)-based classifier. In addition to assessing the prediction accuracy on cross-validation experiments, TargetMiner has been validated with a completely independent experimental test dataset. Our method outperforms 10 existing target prediction algorithms and provides a good balance between sensitivity and specificity that is not reflected in the existing methods. We achieve a significantly higher sensitivity and specificity of 69% and 67.8% based on a pool of 90 feature set and 76.5% and 66.1% using a set of 30 selected feature set on the completely independent test dataset. In order to establish the effectiveness of the systematically generated negative examples, the SVM is trained using a different set of negative data generated using the method in Yousef et al. A significantly higher false positive rate (70.6%) is observed when tested on the independent set, while all other factors are kept the

mTOR is a Promising Therapeutic Target Both in Cisplatin-Sensitive and Cisplatin-Resistant Clear Cell Carcinoma of the Ovary

PubMed Central

Mabuchi, Seiji; Kawase, Chiaki; Altomare, Deborah A.; Morishige, Kenichirou; Sawada, Kenjiro; Hayashi, Masami; Tsujimoto, Masahiko; Yamoto, Mareo; Klein-Szanto, Andres J.; Schilder, Russell J.; Ohmichi, Masahide; Testa, Joseph R.; Kimura, Tadashi

2009-01-01

Translational Relevance Clear cell carcinoma (CCC) of the ovary is a distinctive subtype of epithelial ovarian cancer associated with a poorer sensitivity to platinum-based chemotherapy and a worse prognosis than the more common serous adenocarcinoma (SAC). To improve survival, the development of new treatment strategies that target CCC more effectively is necessary. Our results show that mTOR is more frequently activated in CCCs than in SACs. Our data have relevance for the design of future clinical studies of first-line treatment for patients with CCC of the ovary. Moreover, the finding of increased expression of phospho-mTOR and greater sensitivity to RAD001 in cisplatin-resistant CCC cells than in cisplatin-sensitive cells suggests a novel treatment option for patients with recurrent disease after cisplatin-based first-line chemotherapy. Purpose mTOR (mammalian target of rapamycin) plays a central role in cell proliferation and is regarded as a promising target in cancer therapy including for ovarian cancer. This study aims to examine the role of mTOR as a therapeutic target in clear cell carcinoma (CCC) of the ovary which is regarded as aggressive, chemo-resistant histological subtype. Experimental Design Using tissue microarrays of 98 primary ovarian cancers (52 clear cell carcinomas and 46 serous adenocarcinomas), the expression of phospho-mTOR was assessed by immunohistochemistry. Then, the growth-inhibitory effect of mTOR inhibition by RAD001 (everolimus) was examined using 2 pairs of cisplatin-sensitive parental (RMG1 and KOC7C) and cisplatin-resistant human CCC cell lines (RMG1-CR and KOC7C-CR) both in vitro and in vivo. Results Immunohistochemical analysis demonstrated mTOR was more frequently activated in CCCs than in serous adenocarcinomas (86.6% vs 50%). Treatment with RAD001 markedly inhibited the growth of both RMG1 and KOC7C cells both in vitro and in vivo. Increased expression of phospho-mTOR was observed in cisplatin-resistant RMG1-CR and KOC7C

Targeting the cancer initiating cell: the Achilles' heel of cancer.

PubMed

McCubrey, James A; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Long, Jacquelyn M; Sattler, Jennifer A; Kempf, C Ruth; Laidler, Piotr; Steelman, Linda S

2011-01-01

We have isolated cell with the cancer initiating cell (CIC) phenotype from PC3 cells. The PC3/(CIC) cells are more resistant than the PC3/(BC) cells to chemotherapeutic drugs such as docetaxel which is used to treat prostate cancer. Thus these prostate CICs could lay dormant and persist even after chemotherapeutic drug treatment. Then when the chemotherapeutic drug is removed, they could potentially repopulate the original tumor site or metastize to a distant site. However, the prostate CICs were not significantly more resistant to drugs which target EGFR, NF-κB, Smo and the natural product genistein. Interesting the prostate CICs could be rendered more sensitive to docetaxel by inclusion of suboptimal doses of genistein, cyclopamine, and EGFR inhibitors. In contrast, addition of suboptimal amounts of genistein, cyclopamine, or EGFR inhibitors did not increase the sensitivity of the PC/(BC) cells to docetaxel. Similar results were observed when combination experiments were performed with cyclopamine and suboptimal doses of either genistein or docetaxel. The BC cells are usually more rapidly proliferating than the CICs. Thus the CICs are not as sensitive to docetaxel which targets replication. In contrast, the CICs could be rendered sensitive to docetaxel or cyclopamine by co-treatment with certain other drugs, including the natural product genistein which is present in the human diet of many people, especially Asians. Genistein is by itself only weakly toxic to prostate and other cancer cells. That is probably one of the big reasons that it can be used as a dietary supplement for prostate and breast cancers. It is clear from our studies that low doses of genistein can increase the sensitivity of prostate CICs to drugs such as docetaxel and cyclopamine, two drugs either used or under consideration for prostate cancer therapy.

Relative planetary radar sensitivities: Arecibo and Goldstone

NASA Technical Reports Server (NTRS)

Renzetti, N. A.; Thompson, T. W.; Slade, M. A.

1988-01-01

The increase of the Deep Space Network antennas from 64 meter to 70 meter diameter represents the first of several improvements that will be made over the next decade to enhance earth based radar sensitivity to solar system targets. The aperture increase at the Goldstone DSS-14 site, coupled with a proposed increase in transmitter power to 1000 kW, will improve the 3.5 cm radar by about one order of magnitude. Similarly, proposed Arecibo Observatory upgrades of a Gregorian feed structure and an increase of transmitter power to 1000 kW will increase the sensitivity of this radar about 20 fold. In addition, a Goldstone to Very Large Array bistatic observation with horizon to horizon tracking will have 3.5 times more sensitivity than will a Goldstone horizon to horizon monostatic observation. All of these improvements, which should be in place within the next decade, will enrich an already fertile field of planetary exploration.

Long-Gradient Separations Coupled with Selected Reaction Monitoring for Highly Sensitive, Large Scale Targeted Protein Quantification in a Single Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Fillmore, Thomas L.; Gao, Yuqian

2013-10-01

Long-gradient separations coupled to tandem MS were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional LC-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in LOQ for target proteins in human female serum. Based on at least one surrogate peptide per protein, an LOQ ofmore » 10 ng/mL was achieved for the two spiked proteins in non-depleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or sub-ng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and ELISA measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM offers at least 3 times higher multiplexing capacity than conventional LC-SRM due to ~3-fold increase in average peak widths for a 300-min gradient compared to a 45-min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.« less

Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

PubMed

Khan, M A; Hossain, M U; Rakib-Uz-Zaman, S M; Morshed, M N

2015-07-01

Ebola viruses (EBOVs) have been identified as an emerging threat in recent year as it causes severe haemorrhagic fever in human. Epitope-based vaccine design for EBOVs remains a top priority because a mere progress has been made in this regard. Another reason is the lack of antiviral drug and licensed vaccine although there is a severe outbreak in Central Africa. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites using various immunoinformatics and docking simulation tools. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell was checked for the selected protein. The peptide region spanning 9 amino acids from 42 to 50 and the sequence TLASIGTAF were found as the most potential B and T cell epitopes, respectively. This peptide could interact with 12 HLAs and showed high population coverage up to 80.99%. Using molecular docking, the epitope was further appraised for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post-therapeutic strategy, docking study of predicted 3D structure identified suitable therapeutic inhibitor against targeted protein. However, this computational epitope-based peptide vaccine designing and target site prediction against EBOVs open up a new horizon which may be the prospective way in Ebola viruses research; the results require validation by in vitro and in vivo experiments. © 2015 John Wiley & Sons Ltd.

TargetSpy: a supervised machine learning approach for microRNA target prediction.

PubMed

Sturm, Martin; Hackenberg, Michael; Langenberger, David; Frishman, Dmitrij

2010-05-28

Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences.In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well in human and drosophila

TargetSpy: a supervised machine learning approach for microRNA target prediction

PubMed Central

2010-01-01

Background Virtually all currently available microRNA target site prediction algorithms require the presence of a (conserved) seed match to the 5' end of the microRNA. Recently however, it has been shown that this requirement might be too stringent, leading to a substantial number of missed target sites. Results We developed TargetSpy, a novel computational approach for predicting target sites regardless of the presence of a seed match. It is based on machine learning and automatic feature selection using a wide spectrum of compositional, structural, and base pairing features covering current biological knowledge. Our model does not rely on evolutionary conservation, which allows the detection of species-specific interactions and makes TargetSpy suitable for analyzing unconserved genomic sequences. In order to allow for an unbiased comparison of TargetSpy to other methods, we classified all algorithms into three groups: I) no seed match requirement, II) seed match requirement, and III) conserved seed match requirement. TargetSpy predictions for classes II and III are generated by appropriate postfiltering. On a human dataset revealing fold-change in protein production for five selected microRNAs our method shows superior performance in all classes. In Drosophila melanogaster not only our class II and III predictions are on par with other algorithms, but notably the class I (no-seed) predictions are just marginally less accurate. We estimate that TargetSpy predicts between 26 and 112 functional target sites without a seed match per microRNA that are missed by all other currently available algorithms. Conclusion Only a few algorithms can predict target sites without demanding a seed match and TargetSpy demonstrates a substantial improvement in prediction accuracy in that class. Furthermore, when conservation and the presence of a seed match are required, the performance is comparable with state-of-the-art algorithms. TargetSpy was trained on mouse and performs well

Design of a sensitive aptasensor based on magnetic microbeads-assisted strand displacement amplification and target recycling.

PubMed

Li, Ying; Ji, Xiaoting; Song, Weiling; Guo, Yingshu

2013-04-03

A cross-circular amplification system for sensitive detection of adenosine triphosphate (ATP) in cancer cells was developed based on aptamer-target interaction, magnetic microbeads (MBs)-assisted strand displacement amplification and target recycling. Here we described a new recognition probe possessing two parts, the ATP aptamer and the extension part. The recognition probe was firstly immobilized on the surface of MBs and hybridized with its complementary sequence to form a duplex. When combined with ATP, the probe changed its conformation, revealing the extension part in single-strand form, which further served as a toehold for subsequent target recycling. The released complementary sequence of the probe acted as the catalyst of the MB-assisted strand displacement reaction. Incorporated with target recycling, a large amount of biotin-tagged MB complexes were formed to stimulate the generation of chemiluminescence (CL) signal in the presence of luminol and H2O2 by incorporating with streptavidin-HRP, reaching a detection limit of ATP as low as 6.1×10(-10)M. Moreover, sample assays of ATP in Ramos Burkitt's lymphoma B cells were performed, which confirmed the reliability and practicality of the protocol. Copyright © 2013 Elsevier B.V. All rights reserved.

Thermo-sensitively and magnetically ordered mesoporous carbon nanospheres for targeted controlled drug release and hyperthermia application.

PubMed

Chen, Lin; Zhang, Huan; Zheng, Jing; Yu, Shiping; Du, Jinglei; Yang, Yongzhen; Liu, Xuguang

2018-03-01

A multifunctional nanoplatform based on thermo-sensitively and magnetically ordered mesoporous carbon nanospheres (TMOMCNs) is developed for effective targeted controlled release of doxorubicin hydrochloride (DOX) and hyperthermia in this work. The morphology, specific surface area, porosity, thermo-stability, thermo-sensitivity, as well as magnetism properties of TMOMCNs were verified by high resolution transmission electron microscopy, field emission scanning electron microscopy, thermo-gravimetric analysis, X-ray diffraction, Brunauer-Emmeltt-Teller surface area analysis, dynamic light scattering and vibrating sample magnetometry measurement. The results indicate that TMOMCNs have an average diameter of ~146nm with a lower critical solution temperature at around 39.5°C. They are superparamagnetic with a magnetization of 10.15emu/g at 20kOe. They generate heat when inductive magnetic field is applied to them and have a normalized specific absorption rate of 30.23W/g at 230kHz and 290Oe, showing good potential for hyperthermia. The DOX loading and release results illustrate that the loading capacity is 135.10mg/g and release performance could be regulated by changing pH and temperature. The good targeting, DOX loading and release and hyperthermia properties of TMOMCNs offer new probabilities for high effectiveness and low toxicity of cancer chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Overcoming Multidrug Resistance through the GLUT1-Mediated and Enzyme-Triggered Mitochondrial Targeting Conjugate with Redox-Sensitive Paclitaxel Release.

PubMed

Ma, Pengkai; Chen, Jianhua; Bi, Xinning; Li, Zhihui; Gao, Xing; Li, Hongpin; Zhu, Hongyu; Huang, Yunfang; Qi, Jing; Zhang, Yujie

2018-04-18

Multidrug resistance (MDR) is thought to be the major obstacle leading to the failure of paclitaxel (PTX) chemotherapy. To solve this problem, a glucose transporter-mediated and matrix metalloproteinase 2 (MMP2)-triggered mitochondrion-targeting conjugate [glucose-polyethylene glycol (PEG)-peptide-triphenylphosponium-polyamidoamine (PAMAM)-PTX] composed of a PAMAM dendrimer and enzymatic detachable glucose-PEG was constructed for mitochondrial delivery of PTX. The conjugate was characterized by a 30 nm sphere particle, MMP2-sensitive PEG outer layer detachment from PAMAM, and glutathione (GSH)-sensitive PTX release. It showed higher cellular uptake both in glucose transporter 1 (GLUT1) overexpressing MCF-7/MDR monolayer cell (2D) and multicellular tumor spheroids (3D). The subcellular location study showed that it could specifically accumulate in the mitochondria. Moreover, it exhibited higher cytotoxicity against MCF-7/MDR cells, which significantly reverse the MDR of MCF-7/MDR cells. The MDR reverse might be caused by reducing the ATP content through destroying the mitochondrial membrane as well as by down-regulating P-gp expression. In vivo imaging and tissue distribution indicated more conjugate accumulated in the tumor of the tumor-bearing mice model. Consequently, the conjugate showed better tumor inhibition rate and lower body weight loss, which demonstrated that it possessed high efficiency and low toxicity. This study provides glucose-mediated GLUT targeting, MMP2-responsive PEG detachment, triphenylphosponium-mediated mitochondria targeting, and a GSH-sensitive intracellular drug release conjugate that has the potential to be exploited for overcoming MDR of PTX.

Photodynamic Modification Of Plasma Membrane Function In Erythrocytes Sensitized By Xanthenes: What Determines Potency?

NASA Astrophysics Data System (ADS)

Pooler, John P.

1988-02-01

Several xanthene sensitizers were compared as sensitizers of membrane function in erythrocytes and some of their physico-chemical properties were examined. Eosin derivatives that localize at different membrane sites were equally effective at sensitizing both ion leaks and inactivation of membrane cholinesterase, implying that a diffusible intermediate reacts with membrane targets. Assessments of membrane loading and calculations of diffusion distances for singlet oxygen indicate that amounts of membrane-located sensitizer are quantitatively much greater than amounts in the nearby reaction medium. Potency measurements and assessment of absorption spectra and singlet oxygen production in water-dioxane mixtures lead to the conclusion that differential sorption to membranes, photon capture in low polarity environments and conversion of excited states to singlet oxygen are they key determinants of sensitizing potency.

RNA interference as a method for target-site screening in the Western Corn Rootworm, Diabrotica virgifera virgifera

USDA-ARS?s Scientific Manuscript database

RNA interference (RNAi) is one of the most powerful and extraordinarily-specific means by which to silence genes. The ability of RNAi to silence genes makes it possible to ascertain function from genomic data, thereby making it an excellent choice for target-site screening. To test the efficacy of...

Glioma Dual-Targeting Nanohybrid Protein Toxin Constructed by Intein-Mediated Site-Specific Ligation for Multistage Booster Delivery

PubMed Central

Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Li, Dongdong; Xu, Fan; Wu, Aihua; Wang, Jinyu; Huang, Yongzhuo

2017-01-01

Malignant glioma is one of the most untreatable cancers because of the formidable blood-brain barrier (BBB), through which few therapeutics can penetrate and reach the tumors. Biologics have been booming in cancer therapy in the past two decades, but their application in brain tumor has long been ignored due to the impermeable nature of BBB against effective delivery of biologics. Indeed, it is a long unsolved problem for brain delivery of macromolecular drugs, which becomes the Holy Grail in medical and pharmaceutical sciences. Even assisting by targeting ligands, protein brain delivery still remains challenging because of the synthesis difficulties of ligand-modified proteins. Herein, we propose a rocket-like, multistage booster delivery system of a protein toxin, trichosanthin (TCS), for antiglioma treatment. TCS is a ribosome-inactivating protein with the potent activity against various solid tumors but lack of specific action and cell penetration ability. To overcome the challenge of its poor druggability and site-specific modification, intein-mediated ligation was applied, by which a gelatinase-cleavable peptide and cell-penetrating peptide (CPP)-fused recombinant TCS toxin can be site-specifically conjugated to lactoferrin (LF), thus constructing a BBB-penetrating, gelatinase-activatable cell-penetrating nanohybrid TCS toxin. This nanohybrid TCS system is featured by the multistage booster strategy for glioma dual-targeting delivery. First, LF can target to the BBB-overexpressing low-density lipoprotein receptor-related protein-1 (LRP-1), and assist with BBB penetration. Second, once reaching the tumor site, the gelatinase-cleavable peptide acts as a separator responsive to the glioma-associated matrix metalloproteinases (MMPs), thus releasing to the CPP-fused toxin. Third, CPP mediates intratumoral and intracellular penetration of TCS toxin, thereby enhancing its antitumor activity. The BBB penetration and MMP-2-activability of this delivery system were

Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Jun; Lei, Wan; Fu, Jian-Chun

2014-01-17

Highlight: •MiR-21 plays a significant role in 5-FU resistance. •This role might be attributed to targeting of hMSH2 as well as TP and DPD via miR-21 targeted hMSH2. •Indirectly targeted TP and DPD to influence 5-FU chemotherapy sensitivity. -- Abstract: 5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibilitymore » and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.« less

Site Targeted Press Coated Delivery of Methylprednisolone Using Eudragit RS 100 and Chitosan for Treatment of Colitis.

PubMed

Jagdale, Swati; Chandekar, Apoorva

2016-01-01

Inflammatory bowel disease (IBD) is one of the five most prevalent gastrointestinal disease burdens which commonly require lifetime care. Worldwide incidence rate of ulcerative colitis and Crohn's disease is about 16.8% and 13.4% respectively. Colitis is an inflammation of the colon. Colon targeted drug delivery will direct the drug to the colon. The drug will reach at the site of action and hence its side effects as well as dose can be reduced. Recent patent describes treatment of ulcerative colitis using anti CD3 antibodies, with nicotine and anti-depressant drugs, budesonide foam etc. Present study deals with optimization of site targeted methylprednisolone delivery for treatment of colitis. Chitosan and Eudragit RS 100 were used as coating polymers. Tablets were prepared by press coated technology. The core tablets contain drug, avicel as binder, croscarmellose sodium as super disintegrant and dicalcium phosphate as diluent. Drug excipient compatibility was carried out using FTIR, UV and DSC. Design of experiment was used to optimize the formulation. Tablets were evaluated for thickness, weight variation, hardness, swelling index, in-vitro drug release and release of drug in simulated media. Optimized batch (B2) contained chitosan 40% and eudragit RS 100 17.5%. B2 showed in-vitro drug release 85.65 ± 7.6% in 6.8 pH phosphate buffer and 96.7 ±9.1% in simulated media after 7.5 hours. In-vivo x-ray placebo study for formulation B2 had shown that the tablet reached to the ascending colon after 5 hours. This indicated a potential site targeted delivery of optimized batch B2.

Drought-induced legacy effects in wood growth across the Eastern and Midwestern U.S. are mediated by site climate, tree age, and drought sensitivity

NASA Astrophysics Data System (ADS)

Kannenberg, S.; Maxwell, J. T.; Pederson, N.; D'Orangeville, L.; Phillips, R.

2017-12-01

While it is widely known that drought reduces carbon (C) uptake in temperate forests, tree growth can also remain stagnant post-drought despite favorable climatic conditions. While such "legacy effects" are well established, the degree to which these effects depend on species identity or variability in site conditions is poorly quantified. We sought to uncover how site, species, climate, and tree age interact to affect the presence and magnitude of legacy effects in temperate trees following drought. To do this, we assembled dendrochronological records of 18 common species across 94 sites in Eastern and Midwestern U.S. forests and quantified drought-induced changes in wood growth in the year of the drought (hereafter "drought sensitivity") and the years after the drought (i.e., legacy effects). We predicted that species particularly prone to hydraulic damage (e.g., oaks) would have the least drought sensitivity yet experience larger legacy effects, and that this effect would be exacerbated at arid sites. Across all species and sites, wood growth was reduced by 14% in the year of the drought and by 7% post-drought. Surprisingly, legacy effects were smaller for oak species and larger across species known to be more drought sensitive (e.g. tulip poplar, maple, birch). As a result, we observed a positive relationship between a species' drought sensitivity and that species' legacy effect. These legacy effects were similar in size across a range of drought severities. Surprisingly, legacy effects were smaller in more arid sites - contrary to previous investigations in dryland ecosystems - perhaps indicating the role of adaptation in mediating a tree's recovery from drought. In addition, many species actually decreased the size of their legacy effects as they aged, despite no change in drought responses. Our results run contrary to our predictions, as species with the greatest drought sensitivity had the least ability to recover, and that younger mesic forests- not arid

Tumor-specific pH-responsive peptide-modified pH-sensitive liposomes containing doxorubicin for enhancing glioma targeting and anti-tumor activity.

PubMed

Zhao, Yang; Ren, Wei; Zhong, Ting; Zhang, Shuang; Huang, Dan; Guo, Yang; Yao, Xin; Wang, Chao; Zhang, Wei-Qiang; Zhang, Xuan; Zhang, Qiang

2016-01-28

The pH environment in gliomas is acidic. Therefore, in the present research, we selected our previously reported tumor-specific pH-responsive peptide H7K(R2)2 as a targeting ligand, which could respond to the acidic pH environment in gliomas, possessing CPP characteristics. The pH-sensitive liposomes were selected as carriers which could also respond to the acidic pH environment in gliomas triggering encapsulated drug release from these pH-sensitive liposomes. The H7K(R2)2-modified pH-sensitive liposomes containing doxorubicin (DOX-PSL-H7K(R2)2) were designed and prepared in order to evaluate their potential targeting of glioma tumor cells and their anti-tumor activity in mice with glioma tumor cells. DOX-PSL-H7K(R2)2 was prepared by the thin-film hydration method followed by remote loading using an ammonium sulfate gradient method. The in vitro release of DOX from pH-sensitive liposomes was tested and the in vitro targeting characteristics of H7K(R2)2-modified liposomes regarding C6 (rat C6 glioma cells) and U87-MG (human glioblastoma cells) were evaluated. The in vivo anti-tumor activity of DOX-PSL-H7K(R2)2 was also investigated in C6 tumor-bearing mice and in U87-MG orthotopic tumor-bearing nude mice. A specific targeting effect triggered by an acidic pH was observed in our in vitro experiments in C6 and U87-MG glioma cells. The pH-triggered DOX release from the pH-sensitive liposomes under acidic conditions was also confirmed in our in vitro experiment. Anti-tumor activity of DOX-PSL-H7K(R2)2 was found in C6 tumor-bearing mice and U87-MG orthotopic tumor-bearing nude mice in in vivo experiments. The antiangiogenic activity of DOX-PSL-H7K(R2)2 was confirmed in C6 tumor-bearing mice in the in vivo experiment. These H7K(R2)2-modified pH-sensitive liposomes containing anti-tumor drugs developed in this study are a promising delivery system involving the response stimuli at the acidic pH in the glioma tumor microenvironment and are suitable for anti-tumor therapy

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue.

PubMed

Bertsch, M; Mayburd, A L; Kassner, R J

2003-02-15

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.

MAPK Target Sites of Eyes Absent Are Not Required for Eye Development or Survival in Drosophila

PubMed Central

Jusiak, Barbara; Abulimiti, Abuduaini; Haelterman, Nele; Chen, Rui; Mardon, Graeme

2012-01-01

Eyes absent (Eya) is a highly conserved transcription cofactor and protein phosphatase that plays an essential role in eye development and survival in Drosophila. Ectopic eye induction assays using cDNA transgenes have suggested that mitogen activated protein kinase (MAPK) activates Eya by phosphorylating it on two consensus target sites, S402 and S407, and that this activation potentiates the ability of Eya to drive eye formation. However, this mechanism has never been tested in normal eye development. In the current study, we generated a series of genomic rescue transgenes to investigate how loss- and gain-of-function mutations at these two MAPK target sites within Eya affect Drosophila survival and normal eye formation: eya+GR, the wild-type control; eyaSAGR, which lacks phosphorylation at the two target residues; and eyaSDEGR, which contains phosphomimetic amino acids at the same two residues. Contrary to the previous studies in ectopic eye development, all eya genomic transgenes tested rescue both eye formation and survival equally effectively. We conclude that, in contrast to ectopic eye formation, MAPK-mediated phosphorylation of Eya on S402 and S407 does not play a role in normal development. This is the first study in Drosophila to evaluate the difference in outcomes between genomic rescue and ectopic cDNA-based overexpression of the same gene. These findings indicate similar genomic rescue strategies may prove useful for re-evaluating other long-standing Drosophila developmental models. PMID:23251383

Improving Sensitivity in Ultrasound Molecular Imaging by Tailoring Contrast Agent Size Distribution: In Vivo Studies

PubMed Central

Streeter, Jason E.; Gessner, Ryan; Miles, Iman; Dayton, Paul A.

2010-01-01

Molecular imaging with ultrasound relies on microbubble contrast agents (MCAs) selectively adhering to a ligand-specific target. Prior studies have shown that only small quantities of microbubbles are retained at their target sites, therefore, enhancing contrast sensitivity to low concentrations of microbubbles is essential to improve molecular imaging techniques. In order to assess the effect of MCA diameter on imaging sensitivity, perfusion and molecular imaging studies were performed with microbubbles of varying size distributions. To assess signal improvement and MCA circulation time as a function of size and concentration, blood perfusion was imaged in rat kidneys using nontargeted size-sorted MCAs with a Siemens Sequoia ultrasound system (Siemans, Mountain View, CA) in cadence pulse sequencing (CPS) mode. Molecular imaging sensitivity improvements were studied with size-sorted αvβ3-targeted bubbles in both fibrosarcoma and R3230 rat tumor models. In perfusion imaging studies, video intensity and contrast persistence was ≈8 times and ≈3 times greater respectively, for “sorted 3-micron” MCAs (diameter, 3.3 ± 1.95 μm) when compared to “unsorted” MCAs (diameter, 0.9 ± 0.45 μm) at low concentrations. In targeted experiments, application of sorted 3-micron MCAs resulted in a ≈20 times video intensity increase over unsorted populations. Tailoring size-distributions results in substantial imaging sensitivity improvement over unsorted populations, which is essential in maximizing sensitivity to small numbers of MCAs for molecular imaging. PMID:20236606

The cellulose synthase 3 (CesA3) gene of oomycetes: structure, phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides.

PubMed

Blum, Mathias; Gamper, Hannes A; Waldner, Maya; Sierotzki, Helge; Gisi, Ulrich

2012-04-01

Proper disease control is very important to minimize yield losses caused by oomycetes in many crops. Today, oomycete control is partially achieved by breeding for resistance, but mainly by application of single-site mode of action fungicides including the carboxylic acid amides (CAAs). Despite having mostly specific targets, fungicidal activity can differ even in species belonging to the same phylum but the underlying mechanisms are often poorly understood. In an attempt to elucidate the phylogenetic basis and underlying molecular mechanism of sensitivity and tolerance to CAAs, the cellulose synthase 3 (CesA3) gene was isolated and characterized, encoding the target site of this fungicide class. The CesA3 gene was present in all 25 species included in this study representing the orders Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales, and based on phylogenetic analyses, enabled good resolution of all the different taxonomic orders. Sensitivity assays using the CAA fungicide mandipropamid (MPD) demonstrated that only species belonging to the Peronosporales were inhibited by the fungicide. Molecular data provided evidence, that the observed difference in sensitivity to CAAs between Peronosporales and CAA tolerant species is most likely caused by an inherent amino acid configuration at position 1109 in CesA3 possibly affecting fungicide binding. The present study not only succeeded in linking CAA sensitivity of various oomycetes to the inherent CesA3 target site configuration, but could also relate it to the broader phylogenetic context. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

miR-25 modulates NSCLC cell radio-sensitivity through directly inhibiting BTG2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Zhiwei, E-mail: carlhe@126.com; Liu, Yi, E-mail: cassieliu@126.com; Xiao, Bing, E-mail: rockg714@aliyun.com

2015-02-13

A large proportion of the NSCLC patients were insensitive to radiotherapy, but the exact mechanism is still unclear. This study explored the role of miR-25 in regulating sensitivity of NSCLC cells to ionizing radiation (IR) and its downstream targets. Based on measurement in tumor samples from NSCLC patients, this study found that miR-25 expression is upregulated in both NSCLC and radio-resistant NSCLC patients compared the healthy and radio-sensitive controls. In addition, BTG expression was found negatively correlated with miR-25a expression in the both tissues and cells. By applying luciferase reporter assay, we verified two putative binding sites between miR-25 andmore » BTG2. Therefore, BTG2 is a directly target of miR-25 in NSCLC cancer. By applying loss-and-gain function analysis in NSCLC cell lines, we demonstrated that miR-25-BTG2 axis could directly regulated BTG2 expression and affect radiotherapy sensitivity of NSCLC cells. - Highlights: • miR-25 is upregulated, while BTG2 is downregulated in radioresistant NSCLC patients. • miR-25 modulates sensitivity to radiation induced apoptosis. • miR-25 directly targets BTG2 and suppresses its expression. • miR-25 modulates sensitivity to radiotherapy through inhibiting BTG2 expression.« less

Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

PubMed Central

Ashenberg, Orr; Padmakumar, Jai

2017-01-01

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors. PMID:28346537

EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets.

PubMed

Linardy, Evelyn M; Erskine, Simon M; Lima, Nicole E; Lonergan, Tina; Mokany, Elisa; Todd, Alison V

2016-01-15

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Microenvironmental Effect of 2'-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA.

PubMed

Imincan, Gülnur; Pei, Fen; Yu, Lijia; Jin, Hongwei; Zhang, Liangren; Yang, Xiaoda; Zhang, Lihe; Tang, XinJing

2016-04-19

2'-O-(1-Pyrenylmethyl)uridine modified oligoribonucleotides provide highly sensitive pyrene fluorescent probes for detecting specific nucleotide mutation of RNA targets. To develop more stable and cost-effective oligonucleotide probes, we investigated the local microenvironmental effects of nearby nucleobases on pyrene fluorescence in duplexes of RNAs and 2'-O-(1-pyrenylmethyl)uridine modified oligonucleotides. By incorporation of deoxyribonucleotides, ribonucleotides, 2'-MeO-nucleotides and 2'-F-nucleotides at both sides of 2'-O-(1-pyrenylmethyl)uridine (U(p)) in oligodeoxynucleotide probes, we synthesized a series of pyrene modified oligonucleotide probes. Their pyrene fluorescence emission spectra indicated that only two proximal nucleotides have a substantial effect on the pyrene fluorescence properties of these oligonucleotide probes hybridized with target RNA with an order of fluorescence sensitivity of 2'-F-nucleotides > 2'-MeO-nucleotides > ribonucleotides ≫ deoxyribonucleotides. While based on circular dichroism spectra, overall helix conformations (either A- or B-form) of the duplexes have marginal effects on the sensitivity of the probes. Instead, the local substitution reflected the propensity of the nucleotide sugar ring to adopt North type conformation and, accordingly, shifted their helix geometry toward a more A-type like conformation in local microenvironments. Thus, higher enhancement of pyrene fluorescence emission favored local A-type helix structures and more polar and hydrophobic environments (F > MeO > OH at 2' substitution) of duplex minor grooves of probes with the target RNA. Further dynamic simulation revealed that local microenvironmental effect of 2'-F-nucleotides or ribonucleotides was enough for pyrene moiety to move out of nucleobases to the minor groove of duplexes; in addition, 2'-F-nucleotide had less effect on π-stack of pyrene-modified uridine with upstream and downstream nucleobases. The present oligonucleotide probes

Novel therapeutic approaches for pulmonary arterial hypertension: Unique molecular targets to site-specific drug delivery.

PubMed

Vaidya, Bhuvaneshwar; Gupta, Vivek

2015-08-10

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by increased blood pressure in the small arterioles supplying blood to lungs for oxygenation. Advances in understanding of molecular and cellular biology techniques have led to the findings that PAH is indeed a cascade of diseases exploiting multi-faceted complex pathophysiology, with cellular proliferation and vascular remodeling being the key pathogenic events along with several cellular pathways involved. While current therapies for PAH do provide for amelioration of disease symptoms and acute survival benefits, their full therapeutic potential is hindered by patient incompliance and off-target side effects. To overcome the issues related with current therapy and to devise a more selective therapy, various novel pathways are being investigated for PAH treatment. In addition, inability to deliver anti-PAH drugs to the disease site i.e., distal pulmonary arterioles has been one of the major challenges in achieving improved patient outcomes and improved therapeutic efficacy. Several novel carriers have been explored to increase the selectivity of currently approved anti-PAH drugs and to act as suitable carriers for the delivery of investigational drugs. In the present review, we have discussed potential of various novel molecular pathways/targets including RhoA/Rho kinase, tyrosine kinase, endothelial progenitor cells, vasoactive intestinal peptide, and miRNA in PAH therapeutics. We have also discussed various techniques for site-specific drug delivery of anti-PAH therapeutics so as to improve the efficacy of approved and investigational drugs. This review will provide gainful insights into current advances in PAH therapeutics with an emphasis on site-specific drug payload delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

PubMed Central

Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

2014-01-01

Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

Local Sensitivity of Predicted CO 2 Injectivity and Plume Extent to Model Inputs for the FutureGen 2.0 site

DOE PAGES

Zhang, Z. Fred; White, Signe K.; Bonneville, Alain; ...

2014-12-31

Numerical simulations have been used for estimating CO2 injectivity, CO2 plume extent, pressure distribution, and Area of Review (AoR), and for the design of CO2 injection operations and monitoring network for the FutureGen project. The simulation results are affected by uncertainties associated with numerous input parameters, the conceptual model, initial and boundary conditions, and factors related to injection operations. Furthermore, the uncertainties in the simulation results also vary in space and time. The key need is to identify those uncertainties that critically impact the simulation results and quantify their impacts. We introduce an approach to determine the local sensitivity coefficientmore » (LSC), defined as the response of the output in percent, to rank the importance of model inputs on outputs. The uncertainty of an input with higher sensitivity has larger impacts on the output. The LSC is scalable by the error of an input parameter. The composite sensitivity of an output to a subset of inputs can be calculated by summing the individual LSC values. We propose a local sensitivity coefficient method and applied it to the FutureGen 2.0 Site in Morgan County, Illinois, USA, to investigate the sensitivity of input parameters and initial conditions. The conceptual model for the site consists of 31 layers, each of which has a unique set of input parameters. The sensitivity of 11 parameters for each layer and 7 inputs as initial conditions is then investigated. For CO2 injectivity and plume size, about half of the uncertainty is due to only 4 or 5 of the 348 inputs and 3/4 of the uncertainty is due to about 15 of the inputs. The initial conditions and the properties of the injection layer and its neighbour layers contribute to most of the sensitivity. Overall, the simulation outputs are very sensitive to only a small fraction of the inputs. However, the parameters that are important for controlling CO2 injectivity are not the same as those controlling

Onco-Regulon: an integrated database and software suite for site specific targeting of transcription factors of cancer genes

PubMed Central

Tomar, Navneet; Mishra, Akhilesh; Mrinal, Nirotpal; Jayaram, B.

2016-01-01

Transcription factors (TFs) bind at multiple sites in the genome and regulate expression of many genes. Regulating TF binding in a gene specific manner remains a formidable challenge in drug discovery because the same binding motif may be present at multiple locations in the genome. Here, we present Onco-Regulon (http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm), an integrated database of regulatory motifs of cancer genes clubbed with Unique Sequence-Predictor (USP) a software suite that identifies unique sequences for each of these regulatory DNA motifs at the specified position in the genome. USP works by extending a given DNA motif, in 5′→3′, 3′ →5′ or both directions by adding one nucleotide at each step, and calculates the frequency of each extended motif in the genome by Frequency Counter programme. This step is iterated till the frequency of the extended motif becomes unity in the genome. Thus, for each given motif, we get three possible unique sequences. Closest Sequence Finder program predicts off-target drug binding in the genome. Inclusion of DNA-Protein structural information further makes Onco-Regulon a highly informative repository for gene specific drug development. We believe that Onco-Regulon will help researchers to design drugs which will bind to an exclusive site in the genome with no off-target effects, theoretically. Database URL: http://www.scfbio-iitd.res.in/software/onco/NavSite/index.htm PMID:27515825

Biodistribution and pharmacokinetics of Mad2 siRNA-loaded EGFR-targeted chitosan nanoparticles in cisplatin sensitive and resistant lung cancer models.

PubMed

Nascimento, Ana Vanessa; Gattacceca, Florence; Singh, Amit; Bousbaa, Hassan; Ferreira, Domingos; Sarmento, Bruno; Amiji, Mansoor M

2016-04-01

The present study focuses on biodistribution profile and pharmacokinetic parameters of EGFR-targeted chitosan nanoparticles (TG CS nanoparticles) for siRNA/cisplatin combination therapy of lung cancer. Mad2 siRNA was encapsulated in EGFR targeted and nontargeted (NTG) CS nanoparticles by electrostatic interaction. The biodistribution of the nanoparticles was assessed qualitatively and quantitatively in cisplatin (DDP) sensitive and resistant lung cancer xenograft model. TG nanoparticles showed a consistent and preferential tumor targeting ability with rapid clearance from the plasma to infiltrate and sustain within the tumor up to 96 h. They exhibit a sixfold higher tumor targeting efficiency compared with the NTG nanoparticles. TG nanoparticles present as an attractive drug delivery platform for RNAi therapeutics against NSCLC.

Characterization of image heterogeneity using 2D Minkowski functionals increases the sensitivity of detection of a targeted MRI contrast agent.

PubMed

Canuto, Holly C; McLachlan, Charles; Kettunen, Mikko I; Velic, Marko; Krishnan, Anant S; Neves, Andre' A; de Backer, Maaike; Hu, D-E; Hobson, Michael P; Brindle, Kevin M

2009-05-01

A targeted Gd(3+)-based contrast agent has been developed that detects tumor cell death by binding to the phosphatidylserine (PS) exposed on the plasma membrane of dying cells. Although this agent has been used to detect tumor cell death in vivo, the differences in signal intensity between treated and untreated tumors was relatively small. As cell death is often spatially heterogeneous within tumors, we investigated whether an image analysis technique that parameterizes heterogeneity could be used to increase the sensitivity of detection of this targeted contrast agent. Two-dimensional (2D) Minkowski functionals (MFs) provided an automated and reliable method for parameterization of image heterogeneity, which does not require prior assumptions about the number of regions or features in the image, and were shown to increase the sensitivity of detection of the contrast agent as compared to simple signal intensity analysis. (c) 2009 Wiley-Liss, Inc.

A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.

PubMed

Clop, Alex; Marcq, Fabienne; Takeda, Haruko; Pirottin, Dimitri; Tordoir, Xavier; Bibé, Bernard; Bouix, Jacques; Caiment, Florian; Elsen, Jean-Michel; Eychenne, Francis; Larzul, Catherine; Laville, Elisabeth; Meish, Françoise; Milenkovic, Dragan; Tobin, James; Charlier, Carole; Georges, Michel

2006-07-01

Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov x Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3' UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

PubMed

Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

2018-01-02

Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

PubMed Central

Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

2009-01-01

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

Sensitive, site-specific, and stable vibrational probe of local protein environments: 4-azidomethyl-L-phenylalanine.

PubMed

Bazewicz, Christopher G; Liskov, Melanie T; Hines, Kevin J; Brewer, Scott H

2013-08-01

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-L-phenylalanine (pN₃CH₂Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN₃CH₂Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN₃Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN₃CH₂Phe to different protein environments to be measured. The photostability of pN₃CH₂Phe was also measured relative to the photoreactive UAA, pN₃Phe.

Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2-MPER region.

PubMed

Flyak, Andrew I; Kuzmina, Natalia; Murin, Charles D; Bryan, Christopher; Davidson, Edgar; Gilchuk, Pavlo; Gulka, Christopher P; Ilinykh, Philipp A; Shen, Xiaoli; Huang, Kai; Ramanathan, Palaniappan; Turner, Hannah; Fusco, Marnie L; Lampley, Rebecca; Kose, Nurgun; King, Hannah; Sapparapu, Gopal; Doranz, Benjamin J; Ksiazek, Thomas G; Wright, David W; Saphire, Erica Ollmann; Ward, Andrew B; Bukreyev, Alexander; Crowe, James E

2018-05-07

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMapp, which has been shown to be effective in nonhuman primate models of infection 1 and has been used under compassionate-treatment protocols in humans 2 . ZMapp is a mixture of three chimerized murine monoclonal antibodies (mAbs) 3-6 that target EBOV-specific epitopes on the surface glycoprotein 7,8 . However, ZMapp mAbs do not neutralize other species from the genus Ebolavirus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here, we describe three naturally occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane-proximal external region (MPER) of the glycoprotein. The identification of a conserved neutralizing antigenic site in the glycoprotein suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2-MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification.

PubMed

Feng, Kejun; Zhao, Jingjin; Wu, Zai-Sheng; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2011-03-15

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection. Copyright © 2010 Elsevier B.V. All rights reserved.

p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding.

PubMed

Itakura, Eisuke; Mizushima, Noboru

2011-01-10

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)-associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.

Recent Progress in the Design and Discovery of RXR Modulators Targeting Alternate Binding Sites of the Receptor.

PubMed

Su, Ying; Zeng, Zhiping; Chen, Ziwen; Xu, Dan; Zhang, Weidong; Zhang, Xiao-Kun

2017-01-01

Retinoid X receptors (RXRs) occupy a central position within the nuclear receptor superfamily. They not only function as important transcriptional factors but also exhibit diverse nongenomic biological activities. The pleiotropic actions of RXRs under both physiological and pathophysiological conditions confer RXRs important drug targets for the treatment of cancer, and metabolic and neurodegenerative diseases. RXR modulators have been studied for the purpose of developing both drug molecules and chemical tools for biological investigation of RXR. Development of RXR modulators has focused on small molecules targeting the canonical ligand-binding pocket. However, accumulating results have demonstrated that there are other binding mechanisms by which small molecules interact with RXR to act as RXR modulators. This review discusses the recent development in the design and discovery of RXR modulators with a focus on those targeting novel binding sites on RXR.

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants▿

PubMed Central

Ruckenstuhl, Christoph; Lang, Silvia; Poschenel, Andrea; Eidenberger, Armin; Baral, Pravas Kumar; Kohút, Peter; Hapala, Ivan; Gruber, Karl; Turnowsky, Friederike

2007-01-01

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines. PMID:17043127

Design and Synthesis of Irreversible Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and Interaction Sites.

PubMed

Wong, Michael H L; Bryan, Holly K; Copple, Ian M; Jenkins, Rosalind E; Chiu, Pak Him; Bibby, Jaclyn; Berry, Neil G; Kitteringham, Neil R; Goldring, Christopher E; O'Neill, Paul M; Park, B Kevin

2016-03-24

Semisynthetic triterpenoids such as bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate; CDDO-Me) (4) are potent inducers of antioxidant and anti-inflammatory signaling pathways, including those regulated by the transcription factor Nrf2. However, the reversible nature of the interaction between triterpenoids and thiols has hindered attempts to identify pharmacologically relevant targets and characterize the sites of interaction. Here, we report a shortened synthesis and SAR profiling of 4, enabling the design of analogues that react irreversibly with model thiols, as well as the model protein glutathione S-transferase P1, in vitro. We show that one of these analogues, CDDO-epoxide (13), is comparable to 4 in terms of cytotoxicity and potency toward Nrf2 in rat hepatoma cells and stably modifies specific cysteine residues (namely, Cys-257, -273, -288, -434, -489, and -613) within Keap1, the major repressor of Nrf2, both in vitro and in living cells. Supported by molecular modeling, these data demonstrate the value of 13 for identifying site(s) of interaction with pharmacologically relevant targets and informing the continuing development of triterpenoids as novel drug candidates.

Targeting Renal Cell Carcinoma with a HIF-2 antagonist

PubMed Central

Chen, Wenfang; Hill, Haley; Christie, Alana; Kim, Min Soo; Holloman, Eboni; Pavia-Jimenez, Andrea; Homayoun, Farrah; Ma, Yuanqing; Patel, Nirav; Yell, Paul; Hao, Guiyang; Yousuf, Qurratulain; Joyce, Allison; Pedrosa, Ivan; Geiger, Heather; Zhang, He; Chang, Jenny; Gardner, Kevin H.; Bruick, Richard K.; Reeves, Catherine; Hwang, Tae Hyun; Courtney, Kevin; Frenkel, Eugene; Sun, Xiankai; Zojwalla, Naseem; Wong, Tai; Rizzi, James P.; Wallace, Eli M.; Josey, John A.; Xie, Yang; Xie, Xian-Jin; Kapur, Payal; McKay, Renée M.; Brugarolas, James

2016-01-01

Clear cell Renal Cell Carcinoma (ccRCC) is characterized by VHL inactivation1,2. Because no other gene is mutated as frequently, and VHL mutations are truncal3, VHL inactivation is regarded as the governing event4. VHL loss activates HIF-2, and constitutive HIF-2 restores tumorigenesis in VHL-reconstituted ccRCC cells5. HIF-2 is implicated in angiogenesis and multiple other processes6–9, but angiogenesis is the main target of drugs like sunitinib10. HIF-2, a transcription factor, has been regarded as undruggable11. A structure-based design approach identified a selective HIF-2 antagonist (PT2399) that we evaluate using a tumorgraft (TG)/PDX platform12,13. PT2399 dissociated HIF-2 (an obligatory heterodimer [HIF-2α/HIF-1β])14 in human ccRCC suppressing tumorigenesis in 56% (10/18) lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumors, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant. Resistance occurred despite HIF-2 dissociation in tumors and evidence of Hif-2 inhibition in the mouse as determined by suppression of circulating erythropoietin, a HIF-2 target15 and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumors. Illustrating drug specificity, gene expression was largely unaffected by PT2399 in resistant tumors. Sensitive tumors exhibited a distinguishing gene expression signature, and generally higher HIF-2α levels. Prolonged PT2399 treatment led to resistance. We identified a binding site and second site suppressor mutation in HIF-2α and HIF-1β respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient with a sensitive TG had disease control for >11 months with the close analogue PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCC are, unexpectedly, HIF-2 independent, and set the stage for biomarker-driven clinical trials. PMID:27595394

The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

PubMed

Riga, Maria; Bajda, Sabina; Themistokleous, Christos; Papadaki, Stavrini; Palzewicz, Maria; Dermauw, Wannes; Vontas, John; Leeuwen, Thomas Van

2017-08-23

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.

PubMed

Gabriel, Gabriele V M; Viviani, Vadim R

2016-12-01

Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.

Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum.

PubMed

Dong, Hongyan; Yauk, Carole L; Rowan-Carroll, Andrea; You, Seo-Hee; Zoeller, R Thomas; Lambert, Iain; Wade, Michael G

2009-01-01

Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.

The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation.

PubMed

Rosenbaek, Lena L; Rizzo, Federica; Wu, Qi; Rojas-Vega, Lorena; Gamba, Gerardo; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

2017-10-11

The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na + and K + homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimulation. Thiazide-sensitive sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function.

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

PubMed

Ross, R L; McPherson, H R; Kettlewell, L; Shnyder, S D; Hurst, C D; Alder, O; Knowles, M A

2016-07-28

Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

Calculation of Absorbed Dose in Target Tissue and Equivalent Dose in Sensitive Tissues of Patients Treated by BNCT Using MCNP4C

NASA Astrophysics Data System (ADS)

Zamani, M.; Kasesaz, Y.; Khalafi, H.; Pooya, S. M. Hosseini

Boron Neutron Capture Therapy (BNCT) is used for treatment of many diseases, including brain tumors, in many medical centers. In this method, a target area (e.g., head of patient) is irradiated by some optimized and suitable neutron fields such as research nuclear reactors. Aiming at protection of healthy tissues which are located in the vicinity of irradiated tissue, and based on the ALARA principle, it is required to prevent unnecessary exposure of these vital organs. In this study, by using numerical simulation method (MCNP4C Code), the absorbed dose in target tissue and the equiavalent dose in different sensitive tissues of a patiant treated by BNCT, are calculated. For this purpose, we have used the parameters of MIRD Standard Phantom. Equiavelent dose in 11 sensitive organs, located in the vicinity of target, and total equivalent dose in whole body, have been calculated. The results show that the absorbed dose in tumor and normal tissue of brain equal to 30.35 Gy and 0.19 Gy, respectively. Also, total equivalent dose in 11 sensitive organs, other than tumor and normal tissue of brain, is equal to 14 mGy. The maximum equivalent doses in organs, other than brain and tumor, appear to the tissues of lungs and thyroid and are equal to 7.35 mSv and 3.00 mSv, respectively.

Signal-Switchable Electrochemiluminescence System Coupled with Target Recycling Amplification Strategy for Sensitive Mercury Ion and Mucin 1 Assay.

PubMed

Jiang, Xinya; Wang, Huijun; Wang, Haijun; Yuan, Ruo; Chai, Yaqin

2016-09-20

In the present work, we first found that mercury ion (Hg(2+)) has an efficient quenching effect on the electrochemiluminescence (ECL) of N-(aminobutyl)-N-(ethylisoluminol) (ABEI). Since we were inspired by this discovery, an aptamer-based ECL sensor was fabricated based on a Hg(2+) triggered signal switch coupled with an exonuclease I (Exo I)-stimulated target recycling amplification strategy for ultrasensitive determination of Hg(2+) and mucin 1 (MUC1). Concretely, the ECL intensity of ABEI-functionalized silver nanoparticles decorated graphene oxide nanocomposite (GO-AgNPs-ABEI) was initially enhanced by ferrocene labeled ssDNA (Fc-S1) (first signal switch "on" state) in the existence of H2O2. With the aid of aptamer, assistant ssDNA (S2) and full thymine (T) bases ssDNA (S3) modified Au nanoparticles (AuNPs-S2-S3) were immobilized on the sensing surface through the hybridization reaction. Then, via the strong and stable T-Hg(2+)-T interaction, an abundance of Hg(2+) was successfully captured on the AuNPs-S2-S3 and effectively inhibited the ECL reaction of ABEI (signal switch "off" state). Finally, the signal switch "on" state was executed by utilizing MUC1 as an aptamer-specific target to bind aptamer, leading to the large decrease of the captured Hg(2+). To further improve the sensitivity of the aptasensor, Exo I was implemented to digest the binded aptamer, which resulted in the release of MUC1 for achieving target recycling with strong detectable ECL signal even in a low level of MUC1. By integrating the quenching effect of Hg(2+) to reduce the background signal and target recycling for signal amplification, this proposed ECL aptasensor was successfully used to detect Hg(2+) and MUC1 sensitively with a wide linear response.

Sensitivity of the Modified Children's Yale-Brown Obsessive Compulsive Scale to Detect Change: Results from Two Multi-Site Trials

ERIC Educational Resources Information Center

Scahill, Lawrence; Sukhodolsky, Denis G.; Anderberg, Emily; Dimitropoulos, Anastasia; Dziura, James; Aman, Michael G.; McCracken, James; Tierney, Elaine; Hallett, Victoria; Katz, Karol; Vitiello, Benedetto; McDougle, Christopher

2016-01-01

Repetitive behavior is a core feature of autism spectrum disorder. We used 8-week data from two federally funded, multi-site, randomized trials with risperidone conducted by the Research Units on Pediatric Psychopharmacology Autism Network to evaluate the sensitivity of the Children's Yale-Brown Obsessive Compulsive Scale modified for autism…

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

PubMed Central

Mitchell, Sara N.; Rigden, Daniel J.; Dowd, Andrew J.; Lu, Fang; Wilding, Craig S.; Weetman, David; Dadzie, Samuel; Jenkins, Adam M.; Regna, Kimberly; Boko, Pelagie; Djogbenou, Luc; Muskavitch, Marc A. T.; Ranson, Hilary; Paine, Mark J. I.; Mayans, Olga; Donnelly, Martin J.

2014-01-01

The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae. PMID:24675797

A Sensitive Membrane-Targeted Biosensor for Monitoring Changes in Intracellular Chloride in Neuronal Processes

PubMed Central

Watts, Spencer D.; Suchland, Katherine L.; Amara, Susan G.; Ingram, Susan L.

2012-01-01

Background Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited. Methodology/Principal Findings In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30–40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k50 =  41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABAA agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons. Conclusions/Significance The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments. PMID:22506078

Prostate-Specific Membrane Antigen-Targeted Site-Directed Antibody-Conjugated Apoferritin Nanovehicle Favorably Influences In Vivo Side Effects of Doxorubicin.

PubMed

Dostalova, Simona; Polanska, Hana; Svobodova, Marketa; Balvan, Jan; Krystofova, Olga; Haddad, Yazan; Krizkova, Sona; Masarik, Michal; Eckschlager, Tomas; Stiborova, Marie; Heger, Zbynek; Adam, Vojtech

2018-06-11

Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.

Fluorescent CSC models evidence that targeted nanomedicines improve treatment sensitivity of breast and colon cancer stem cells.

PubMed

Gener, Petra; Gouveia, Luis Pleno; Sabat, Guillem Romero; de Sousa Rafael, Diana Fernandes; Fort, Núria Bergadà; Arranja, Alexandra; Fernández, Yolanda; Prieto, Rafael Miñana; Ortega, Joan Sayos; Arango, Diego; Abasolo, Ibane; Videira, Mafalda; Schwartz, Simo

2015-11-01

To be able to study the efficacy of targeted nanomedicines in marginal population of highly aggressive cancer stem cells (CSC), we have developed a novel in vitro fluorescent CSC model that allows us to visualize these cells in heterogeneous population and to monitor CSC biological performance after therapy. In this model tdTomato reporter gene is driven by CSC specific (ALDH1A1) promoter and contrary to other similar models, CSC differentiation and un-differentiation processes are not restrained and longitudinal studies are feasible. We used this model for preclinical validation of poly[(d,l-lactide-co-glycolide)-co-PEG] (PLGA-co-PEG) micelles loaded with paclitaxel. Further, active targeting against CD44 and EGFR receptors was validated in breast and colon cancer cell lines. Accordingly, specific active targeting toward surface receptors enhances the performance of nanomedicines and sensitizes CSC to paclitaxel based chemotherapy. Many current cancer therapies fail because of the failure to target cancer stem cells. This surviving population soon proliferates and differentiates into more cancer cells. In this interesting article, the authors designed an in vitro cancer stem cell model to study the effects of active targeting using antibody-labeled micelles containing chemotherapeutic agent. This new model should allow future testing of various drug/carrier platforms before the clinical phase. Copyright © 2015 Elsevier Inc. All rights reserved.

Transferrin receptor-targeted pH-sensitive micellar system for diminution of drug resistance and targetable delivery in multidrug-resistant breast cancer

PubMed Central

Gao, Wei; Ye, Guihua; Duan, Xiaochuan; Yang, Xiaoying; Yang, Victor C

2017-01-01

The emergence of drug resistance is partially associated with overproduction of transferrin receptor (TfR). To overcome multidrug resistance (MDR) and achieve tumor target delivery, we designed a novel biodegradable pH-sensitive micellar system modified with HAIYPRH, a TfR ligand (7pep). First, the polymers poly(l-histidine)-coupled polyethylene glycol-2000 (PHIS-PEG2000) and 7pep-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (7pep-DSPE-PEG2000) were synthesized, and the mixed micelles were prepared by blending of PHIS-PEG2000 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG2000) or 7pep-DSPE-PEG2000 (7-pep HD micelles). The micelles exhibited good size uniformity, high encapsulation efficiency, and a low critical micelle concentration. By changing the polymer ratio in the micellar formulation, the pH response range was specially tailored to pH ~6.0. When loaded with antitumor drug doxorubicin (DOX), the micelle showed an acid pH-triggering drug release profile. The cellular uptake and cytotoxicity study demonstrated that 7-pep HD micelles could significantly enhance the intracellular level and antitumor efficacy of DOX in multidrug-resistant cells (MCF-7/Adr), which attributed to the synergistic effect of poly(l-histidine)-triggered endolysosom escape and TfR-mediated endocytosis. Most importantly, the in vivo imaging study confirmed the target-ability of 7-pep HD micelles to MDR tumor. These findings indicated that 7-pep HD micelles would be a promising drug delivery system in the treatment of drug-resistant tumors. PMID:28223798

Generator-specific targets of mitochondrial reactive oxygen species.

PubMed

Bleier, Lea; Wittig, Ilka; Heide, Heinrich; Steger, Mirco; Brandt, Ulrich; Dröse, Stefan

2015-01-01

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H2O2) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4-Azidomethyl-L-Phenylalanine

PubMed Central

Bazewicz, Christopher G.; Liskov, Melanie T.; Hines, Kevin J.; Brewer, Scott H.

2013-01-01

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-Lphenylalanine (pN3CH2Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN3CH2Phe are compared to the vibrational reporters: 4-cyano-L-phenylalanine (pCNPhe) and 4-azido-L-phenylalanine (pN3Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN3CH2Phe to different protein environments to be measured. The photo-stability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe. PMID:23865850

Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature

PubMed Central

Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Tang, Yisi; Wang, Huiyuan; Xu, Qin; Li, Yaping; Li, Feng; Huang, Yongzhuo

2017-01-01

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation. PMID:27914267

A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

PubMed

Sun, Yueying; Sun, Yuanyuan; Tian, Weimin; Liu, Chenghui; Gao, Kejian; Li, Zhengping

2018-02-07

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.

Dual-pH Sensitive Charge-reversal Nanocomplex for Tumor-targeted Drug Delivery with Enhanced Anticancer Activity.

PubMed

Zhou, Qing; Hou, Yilin; Zhang, Li; Wang, Jianlin; Qiao, Youbei; Guo, Songyan; Fan, Li; Yang, Tiehong; Zhu, Lin; Wu, Hong

2017-01-01

Poly(β-L-malic acid) (PMLA), a natural aliphatic polyester, has been proven to be a promising carrier for anti-cancer drugs. In spite of excellent bio-compatibility, the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency. The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion. In this study, a dual pH-sensitive charge-reversal PMLA-based nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was developed for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release. The prepared nanocomplex showed a negative surface charge at the physiological pH, which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system, so as to prolong its circulation time. While at the tumor extracellular pH 6.8, the DMMA was hydrolyzed, leading to the charge reversal and exposure of the TAT on the polymeric micelles, thus enhancing the cellular internalization. Then, the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell. Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity, suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity.

Dual-pH Sensitive Charge-reversal Nanocomplex for Tumor-targeted Drug Delivery with Enhanced Anticancer Activity

PubMed Central

Zhou, Qing; Hou, Yilin; Zhang, Li; Wang, Jianlin; Qiao, Youbei; Guo, Songyan; Fan, Li; Yang, Tiehong; Zhu, Lin; Wu, Hong

2017-01-01

Poly(β-L-malic acid) (PMLA), a natural aliphatic polyester, has been proven to be a promising carrier for anti-cancer drugs. In spite of excellent bio-compatibility, the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency. The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion. In this study, a dual pH-sensitive charge-reversal PMLA-based nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was developed for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release. The prepared nanocomplex showed a negative surface charge at the physiological pH, which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system, so as to prolong its circulation time. While at the tumor extracellular pH 6.8, the DMMA was hydrolyzed, leading to the charge reversal and exposure of the TAT on the polymeric micelles, thus enhancing the cellular internalization. Then, the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell. Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity, suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity. PMID:28638469

Automated divertor target design by adjoint shape sensitivity analysis and a one-shot method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dekeyser, W., E-mail: Wouter.Dekeyser@kuleuven.be; Reiter, D.; Baelmans, M.

As magnetic confinement fusion progresses towards the development of first reactor-scale devices, computational tokamak divertor design is a topic of high priority. Presently, edge plasma codes are used in a forward approach, where magnetic field and divertor geometry are manually adjusted to meet design requirements. Due to the complex edge plasma flows and large number of design variables, this method is computationally very demanding. On the other hand, efficient optimization-based design strategies have been developed in computational aerodynamics and fluid mechanics. Such an optimization approach to divertor target shape design is elaborated in the present paper. A general formulation ofmore » the design problems is given, and conditions characterizing the optimal designs are formulated. Using a continuous adjoint framework, design sensitivities can be computed at a cost of only two edge plasma simulations, independent of the number of design variables. Furthermore, by using a one-shot method the entire optimization problem can be solved at an equivalent cost of only a few forward simulations. The methodology is applied to target shape design for uniform power load, in simplified edge plasma geometry.« less

Barriers to Liposomal Gene Delivery: from Application Site to the Target.

PubMed

Saffari, Mostafa; Moghimi, Hamid Reza; Dass, Crispin R

2016-01-01

Gene therapy is a therapeutic approach to deliver genetic material into cells to alter their function in entire organism. One promising form of gene delivery system (DDS) is liposomes. The success of liposome-mediated gene delivery is a multifactorial issue and well-designed liposomal systems might lead to optimized gene transfection particularly in vivo. Liposomal gene delivery systems face different barriers from their site of application to their target, which is inside the cells. These barriers include presystemic obstacles (epithelial barriers), systemic barriers in blood circulation and cellular barriers. Epithelial barriers differ depending on the route of administration. Systemic barriers include enzymatic degradation, binding and opsonisation. Both of these barriers can act as limiting hurdles that genetic material and their vector should overcome before reaching the cells. Finally liposomes should overcome cellular barriers that include cell entrance, endosomal escape and nuclear uptake. These barriers and their impact on liposomal gene delivery will be discussed in this review.

The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

PubMed

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D; Levin, Henry L

2009-03-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.

A Bayesian-Based Novel Methodology to Generate Reliable Site Response Mapping Sensitive to Data Uncertainties

NASA Astrophysics Data System (ADS)

Chakraborty, A.; Goto, H.

2017-12-01

The 2011 off the Pacific coast of Tohoku earthquake caused severe damage in many areas further inside the mainland because of site-amplification. Furukawa district in Miyagi Prefecture, Japan recorded significant spatial differences in ground motion even at sub-kilometer scales. The site responses in the damage zone far exceeded the levels in the hazard maps. A reason why the mismatch occurred is that mapping follow only the mean value at the measurement locations with no regard to the data uncertainties and thus are not always reliable. Our research objective is to develop a methodology to incorporate data uncertainties in mapping and propose a reliable map. The methodology is based on a hierarchical Bayesian modeling of normally-distributed site responses in space where the mean (μ), site-specific variance (σ2) and between-sites variance(s2) parameters are treated as unknowns with a prior distribution. The observation data is artificially created site responses with varying means and variances for 150 seismic events across 50 locations in one-dimensional space. Spatially auto-correlated random effects were added to the mean (μ) using a conditionally autoregressive (CAR) prior. The inferences on the unknown parameters are done using Markov Chain Monte Carlo methods from the posterior distribution. The goal is to find reliable estimates of μ sensitive to uncertainties. During initial trials, we observed that the tau (=1/s2) parameter of CAR prior controls the μ estimation. Using a constraint, s = 1/(k×σ), five spatial models with varying k-values were created. We define reliability to be measured by the model likelihood and propose the maximum likelihood model to be highly reliable. The model with maximum likelihood was selected using a 5-fold cross-validation technique. The results show that the maximum likelihood model (μ*) follows the site-specific mean at low uncertainties and converges to the model-mean at higher uncertainties (Fig.1). This result is

Intranuclear biophotonics by smart design of nuclear-targeting photo-/radio-sensitizers co-loaded upconversion nanoparticles.

PubMed

Fan, Wenpei; Shen, Bo; Bu, Wenbo; Zheng, Xiangpeng; He, Qianjun; Cui, Zhaowen; Ni, Dalong; Zhao, Kuaile; Zhang, Shengjian; Shi, Jianlin

2015-11-01

Biophotonic technology that uses light and ionizing radiation for positioned cancer therapy is a holy grail in the field of biomedicine because it can overcome the systemic toxicity and adverse side effects of conventional chemotherapy. However, the existing biophotonic techniques fail to achieve the satisfactory treatment efficacy, which remains a big challenge for clinical implementation. Herein, we develop a novel theranostic technique of "intranuclear biophotonics" by the smart design of a nuclear-targeting biophotonic system based on photo-/radio-sensitizers covalently co-loaded upconversion nanoparticles. These nuclear-targeting biophotonic agents can not only generate a great deal of multiple cytotoxic reactive oxygen species in the nucleus by making full use of NIR/X-ray irradiation, but also produce greatly enhanced intranuclear synergetic radio-/photodynamic therapeutic effects under the magnetic/luminescent bimodal imaging guidance, which may achieve the optimal efficacy in treating radio-resistant tumors. We anticipate that the highly effective intranuclear biophotonics will contribute significantly to the development of biophotonic techniques and open new perspectives for a variety of cancer theranostic applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ensemble-sensitivity Analysis Based Observation Targeting for Mesoscale Convection Forecasts and Factors Influencing Observation-Impact Prediction

NASA Astrophysics Data System (ADS)

Hill, A.; Weiss, C.; Ancell, B. C.

2017-12-01

The basic premise of observation targeting is that additional observations, when gathered and assimilated with a numerical weather prediction (NWP) model, will produce a more accurate forecast related to a specific phenomenon. Ensemble-sensitivity analysis (ESA; Ancell and Hakim 2007; Torn and Hakim 2008) is a tool capable of accurately estimating the proper location of targeted observations in areas that have initial model uncertainty and large error growth, as well as predicting the reduction of forecast variance due to the assimilated observation. ESA relates an ensemble of NWP model forecasts, specifically an ensemble of scalar forecast metrics, linearly to earlier model states. A thorough investigation is presented to determine how different factors of the forecast process are impacting our ability to successfully target new observations for mesoscale convection forecasts. Our primary goals for this work are to determine: (1) If targeted observations hold more positive impact over non-targeted (i.e. randomly chosen) observations; (2) If there are lead-time constraints to targeting for convection; (3) How inflation, localization, and the assimilation filter influence impact prediction and realized results; (4) If there exist differences between targeted observations at the surface versus aloft; and (5) how physics errors and nonlinearity may augment observation impacts.Ten cases of dryline-initiated convection between 2011 to 2013 are simulated within a simplified OSSE framework and presented here. Ensemble simulations are produced from a cycling system that utilizes the Weather Research and Forecasting (WRF) model v3.8.1 within the Data Assimilation Research Testbed (DART). A "truth" (nature) simulation is produced by supplying a 3-km WRF run with GFS analyses and integrating the model forward 90 hours, from the beginning of ensemble initialization through the end of the forecast. Target locations for surface and radiosonde observations are computed 6, 12, and

p62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

PubMed Central

Itakura, Eisuke

2011-01-01

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway. PMID:21220506

MicroRNA-101-3p suppresses cell proliferation, invasion and enhances chemotherapeutic sensitivity in salivary gland adenoid cystic carcinoma by targeting Pim-1

PubMed Central

Liu, Xiao-Yu; Liu, Zhi-Jian; He, Hong; Zhang, Chen; Wang, Yun-Long

2015-01-01

MicroRNAs (miRNAs) play critical roles in carcinogenesis and tumor progression. Recent research has revealed miR-101-3p as an important regulator in several cancers. Nevertheless, its function in salivary gland Adenoid cystic carcinoma (ACC), a relatively rare malignance with poor long-term survival rate arisen in head and neck region, remain unknown. In this study, down-regulated miR-101-3p expression was detected in ACC tissues and ACC cell lines with high potential for metastasis. Ectopic expression of miR-101-3p significantly repressed the invasion, proliferation, colony formation, and formation of nude mice xenografts and induced potent apoptosis in ACC cell lines. The provirus integration site for Moloney murine leukemia virus 1 (Pim-1) oncogene was subsequently confirmed as a direct target gene of miR-101-3p in ACC. Functional restoration assays revealed that miR-101-3p inhibits cell growth and invasion by directly decreasing Pim-1 expression. Protein levels of Survivin, Cyclin D1 and β-catenin were also down-regulated by miR-101-3p. miR-101-3p enhanced the sensitivity of cisplatin in ACC cell lines. Taken together, our results demonstrate that the novel miR-101-3p/Pim-1 axis provides excellent insights into the carcinogenesis and tumor progression of ACC and may be a promising therapeutic target for this type of cancer. PMID:26693056

Mitochondrial targets of photodynamic therapy and their contribution to cell death

NASA Astrophysics Data System (ADS)

Oleinick, Nancy L.; Usuda, Jitsuo; Xue, Liang-yan; Azizuddin, Kashif; Chiu, Song-mao; Lam, Minh C.; Morris, Rachel L.; Nieminen, Anna-Liisa

2002-06-01

In response to photodynamic therapy (PDT), many cells in culture or within experimental tumors are eliminated by apoptosis. PDT with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, causes prompt release of cytochrome c into the cytoplasm and activation of caspases-9 and -3, among other caspases, that are responsible for initiating cell degradation. Some cells appear resistant to apoptosis after PDT; however, if they have sustained sufficient damage, they will die by a necrotic process or through a different apoptotic pathway. In the case of PDT, the distinction between apoptosis and necrosis may be less important than the mechanism that triggers both processes, since critical lethal damage appears to occur during treatment and does not require the major steps in apoptosis to be expressed. We earlier showed, for example, that human breast cancer MCF-7 cells that lack caspase-3 are resistant to the induction of apoptosis by PDT, but are just as sensitive to the loss of clonogenicity as MCF-7 cells stably expressing transfected procaspase-3. Many photosensitizers that target mitochondria specifically attack the anti-apoptotic protein Bcl-2, generating a variety of crosslinked and cleaved photoproducts. Recent evidence suggests that the closely related protein Bcl-xL is also a target of Pc 4-PDT. Transient transfection of an expression vector encoding deletion mutants of Bcl-2 have identified the critical sensitive site in the protein that is required for photodamage. This region contains two alpha helices that form a secondary membrane anchorage site and are thought to be responsible for pore formation by Bcl-2. As specific protein targets are identified, we are becoming better able to model the critical events in PDT-induced cell death.

The novel cyst nematode effector protein 30D08 targets host nuclear functions to alter gene expression in feeding sites.

PubMed

Verma, Anju; Lee, Chris; Morriss, Stephanie; Odu, Fiona; Kenning, Charlotte; Rizzo, Nancy; Spollen, William G; Lin, Marriam; McRae, Amanda G; Givan, Scott A; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Baum, Thomas J; Mitchum, Melissa G

2018-05-04

Cyst nematodes deliver effector proteins into host cells to manipulate cellular processes and establish a metabolically hyperactive feeding site. The novel 30D08 effector protein is produced in the dorsal gland of parasitic juveniles, but its function has remained unknown. We demonstrate that expression of 30D08 contributes to nematode parasitism, the protein is packaged into secretory granules and it is targeted to the plant nucleus where it interacts with SMU2 (homolog of suppressor of mec-8 and unc-52 2), an auxiliary spliceosomal protein. We show that SMU2 is expressed in feeding sites and an smu2 mutant is less susceptible to nematode infection. In Arabidopsis expressing 30D08 under the SMU2 promoter, several genes were found to be alternatively spliced and the most abundant functional classes represented among differentially expressed genes were involved in RNA processing, transcription and binding, as well as in development, and hormone and secondary metabolism, representing key cellular processes known to be important for feeding site formation. In conclusion, we demonstrated that the 30D08 effector is secreted from the nematode and targeted to the plant nucleus where its interaction with a host auxiliary spliceosomal protein may alter the pre-mRNA splicing and expression of a subset of genes important for feeding site formation. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites

PubMed Central

Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

2015-01-01

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. DOI: http://dx.doi.org/10.7554/eLife.11553.001 PMID:26609810

Detection of canonical A-to-G editing events at 3' UTRs and microRNA target sites in human lungs using next-generation sequencing.

PubMed

Soundararajan, Ramani; Stearns, Timothy M; Griswold, Anthony L; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

2015-11-03

RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3' untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3' UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states.

Target-site resistance to neonicotinoids.

PubMed

Crossthwaite, Andrew J; Rendine, Stefano; Stenta, Marco; Slater, Russell

2014-10-01

Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.

Linking Oviposition Site Choice to Offspring Fitness in Aedes aegypti: Consequences for Targeted Larval Control of Dengue Vectors

PubMed Central

Wong, Jacklyn; Morrison, Amy C.; Stoddard, Steven T.; Astete, Helvio; Chu, Yui Yin; Baseer, Imaan; Scott, Thomas W.

2012-01-01

Background Current Aedes aegypti larval control methods are often insufficient for preventing dengue epidemics. To improve control efficiency and cost-effectiveness, some advocate eliminating or treating only highly productive containers. The population-level outcome of this strategy, however, will depend on details of Ae. aegypti oviposition behavior. Methodology/Principal Findings We simultaneously monitored female oviposition and juvenile development in 80 experimental containers located across 20 houses in Iquitos, Peru, to test the hypothesis that Ae. aegypti oviposit preferentially in sites with the greatest potential for maximizing offspring fitness. Females consistently laid more eggs in large vs. small containers (β = 9.18, p<0.001), and in unmanaged vs. manually filled containers (β = 5.33, p<0.001). Using microsatellites to track the development of immature Ae. aegypti, we found a negative correlation between oviposition preference and pupation probability (β = −3.37, p<0.001). Body size of emerging adults was also negatively associated with the preferred oviposition site characteristics of large size (females: β = −0.19, p<0.001; males: β = −0.11, p = 0.002) and non-management (females: β = −0.17, p<0.001; males: β = −0.11, p<0.001). Inside a semi-field enclosure, we simulated a container elimination campaign targeting the most productive oviposition sites. Compared to the two post-intervention trials, egg batches were more clumped during the first pre-intervention trial (β = −0.17, P<0.001), but not the second (β = 0.01, p = 0.900). Overall, when preferred containers were unavailable, the probability that any given container received eggs increased (β = 1.36, p<0.001). Conclusions/Significance Ae. aegypti oviposition site choice can contribute to population regulation by limiting the production and size of adults. Targeted larval control strategies may unintentionally lead to

pH- and ion-sensitive polymers for drug delivery

PubMed Central

Yoshida, Takayuki; Lai, Tsz Chung; Kwon, Glen S; Sako, Kazuhiro

2013-01-01

Introduction Drug delivery systems (DDSs) are important for effective, safe, and convenient administration of drugs. pH- and ion-responsive polymers have been widely employed in DDS for site-specific drug release due to their abilities to exploit specific pH- or ion-gradients in the human body. Areas covered Having pH-sensitivity, cationic polymers can mask the taste of drugs and release drugs in the stomach by responding to gastric low pH. Anionic polymers responsive to intestinal high pH are used for preventing gastric degradation of drug, colon drug delivery and achieving high bioavailability of weak basic drugs. Tumor-targeted DDSs have been developed based on polymers with imidazole groups or poly(β-amino ester) responsive to tumoral low pH. Polymers with pH-sensitive chemical linkages, such as hydrazone, acetal, ortho ester and vinyl ester, pH-sensitive cell-penetrating peptides and cationic polymers undergoing pH-dependent protonation have been studied to utilize the pH gradient along the endocytic pathway for intracellular drug delivery. As ion-sensitive polymers, ion-exchange resins are frequently used for taste-masking, counterion-responsive drug release and sustained drug release. Polymers responding to ions in the saliva and gastrointestinal fluids are also used for controlled drug release in oral drug formulations. Expert opinion Stimuli-responsive DDSs are important for achieving site-specific and controlled drug release; however, intraindividual, interindividual and intercellular variations of pH should be considered when designing DDSs or drug products. Combination of polymers and other components, and deeper understanding of human physiology are important for development of pH- and ion-sensitive polymeric DDS products for patients. PMID:23930949

Thiamethoxam acts as a target-site synergist of spinosad in resistant strains of Frankliniella occidentalis.

PubMed

Guillén, Juan; Bielza, Pablo

2013-02-01

Previous studies have suggested that the resistance mechanism towards spinosad in Frankliniella occidentalis (Pergande) is an altered target site. Like the neonicotinoids, the spinosyns act on nicotinic acetylcholine receptors (nAChRs) in insects, but at a distinct site. The changes in nAChRs related to spinosad resistance in thrips might involve interaction with neonicotinoids. In this study, the efficacy of spinosad and neonicotinoids, alone and in combination, was evaluated in susceptible and spinosad-resistant thrips strains. The neonicotinoids tested were imidacloprid, thiacloprid, acetamiprid, thiamethoxam and clothianidin. No cross-resistance was shown between spinosad and any of the neonicotinoids. However, an increased toxicity was observed when a mixture of spinosad with thiamethoxam or clothianidin was tested. No synergism was found in the susceptible strains. The more spinosad-resistant the thrips strain, the stronger was the synergism. Data suggest that spinosad and thiamethoxam may interact at the nAChRs in spinosad-resistant thrips, facilitating enhanced insecticidal action. Copyright © 2012 Society of Chemical Industry.

Theranostic pH-sensitive nanoparticles for highly efficient targeted delivery of doxorubicin for breast tumor treatment.

PubMed

Pan, Changqie; Liu, Yuqing; Zhou, Minyu; Wang, Wensheng; Shi, Min; Xing, Malcolm; Liao, Wangjun

2018-01-01

A multifunctional theranostic nanoplatform integrated with environmental responses has been developed rapidly over the past few years as a novel treatment strategy for several solid tumors. We synthesized pH-sensitive poly(β-thiopropionate) nanoparticles with a supermagnetic core and folic acid (FA) conjugation (FA-doxorubicin-iron oxide nanoparticles [FA-DOX@ IONPs]) to deliver an antineoplastic drug, DOX, for the treatment of folate receptor (FR)-overexpressed breast cancer. In addition to an imaging function, the nanoparticles can release their payloads in response to an environment of pH 5, such as the acidic environment found in tumors. After chemical ( 1 H nuclear magnetic resonance) and physical (morphology and super-magnetic) characterization, FA-DOX@IONPs were shown to demonstrate pH-dependent drug release profiles. Western blotting analysis revealed the expression of FRs in three breast cancer cell lines, MCF-7, BT549, and MD-MBA-231. The cell counting kit-8 assay and transmission electron microscopy showed that FA-DOX@IONPs had the strongest cytotoxicity against breast cancer cells, compared with free DOX and non-FR targeted nanoparticles (DOX@IONPs), and caused cellular apoptosis. The FA-DOX@IONP-mediated cellular uptake and intracellular internalization were clarified by fluorescence microscopy. FA-DOX@IONPs plus magnetic field treatment suppressed in vivo tumor growth in mice to a greater extent than either treatment alone; furthermore, the nanoparticles exerted no toxicity against healthy organs. Magnetic resonance imaging was successfully applied to monitor the nanoparticle accumulation. Our results suggest that theranostic pH-sensitive nanoparticles with dual targeting could enhance the available therapies for cancer.

Deletion of Tsc2 in Nociceptors Reduces Target Innervation, Ion Channel Expression, and Sensitivity to Heat

PubMed Central

Carlin, Dan; Golden, Judith P.; Monk, Kelly R.

2018-01-01

Abstract The mechanistic target of rapamycin complex 1 (mTORC1) is known to regulate cellular growth pathways, and its genetic activation is sufficient to enhance regenerative axon growth following injury to the central or peripheral nervous systems. However, excess mTORC1 activation may promote innervation defects, and mTORC1 activity mediates injury-induced hypersensitivity, reducing enthusiasm for the pathway as a therapeutic target. While mTORC1 activity is required for full expression of some pain modalities, the effects of pathway activation on nociceptor phenotypes and sensory behaviors are currently unknown. To address this, we genetically activated mTORC1 in mouse peripheral sensory neurons by conditional deletion of its negative regulator Tuberous Sclerosis Complex 2 (Tsc2). Consistent with the well-known role of mTORC1 in regulating cell size, soma size and axon diameter of C-nociceptors were increased in Tsc2-deleted mice. Glabrous skin and spinal cord innervation by C-fiber neurons were also disrupted. Transcriptional profiling of nociceptors enriched by fluorescence-associated cell sorting (FACS) revealed downregulation of multiple classes of ion channels as well as reduced expression of markers for peptidergic nociceptors in Tsc2-deleted mice. In addition to these changes in innervation and gene expression, Tsc2-deleted mice exhibited reduced noxious heat sensitivity and decreased injury-induced cold hypersensitivity, but normal baseline sensitivity to cold and mechanical stimuli. Together, these data show that excess mTORC1 activity in sensory neurons produces changes in gene expression, neuron morphology and sensory behavior. PMID:29766046

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

DTIC Science & Technology

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

MicroRNA-9 functions as a tumor suppressor and enhances radio-sensitivity in radio-resistant A549 cells by targeting neuropilin 1.

PubMed

Xiong, Kai; Shao, Li Hong; Zhang, Hai Qin; Jin, Linlin; Wei, Wei; Dong, Zhuo; Zhu, Yue Quan; Wu, Ning; Jin, Shun Zi; Xue, Li Xiang

2018-03-01

Radiotherapy is commonly used to treat lung cancer but may not kill all cancer cells, which may be attributed to the radiotherapy resistance that often occurs in non-small cell lung cancer (NSCLC). At present, the molecular mechanism of radio-resistance remains unclear. Neuropilin 1 (NRP1), a co-receptor for vascular endothelial growth factor (VEGF), was demonstrated to be associated with radio-resistance of NSCLC cells via the VEGF-phosphoinositide 3-kinase-nuclear factor-κB pathway in our previous study. It was hypothesized that certain microRNAs (miRs) may serve crucial functions in radio-sensitivity by regulating NRP1. Bioinformatics predicted that NRP1 was a potential target of miR-9, and this was validated by luciferase reporter assays. Functionally, miR-9-transfected A549 cells exhibited a decreased proliferation rate, increased apoptosis rate and attenuated migratory and invasive abilities. Additionally, a high expression of miR-9 also significantly enhanced the radio-sensitivity of A549 cells in vitro and in vivo . These data improve understanding of the mechanisms of cell radio-resistance, and suggest that miR-9 may be a molecular target for the prediction of radio-sensitivity in NSCLC.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

PubMed

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

The challenge of modelling nitrogen management at the field scale: simulation and sensitivity analysis of N2O fluxes across nine experimental sites using DailyDayCent

NASA Astrophysics Data System (ADS)

Fitton, N.; Datta, A.; Hastings, A.; Kuhnert, M.; Topp, C. F. E.; Cloy, J. M.; Rees, R. M.; Cardenas, L. M.; Williams, J. R.; Smith, K.; Chadwick, D.; Smith, P.

2014-09-01

The United Kingdom currently reports nitrous oxide emissions from agriculture using the IPCC default Tier 1 methodology. However Tier 1 estimates have a large degree of uncertainty as they do not account for spatial variations in emissions. Therefore biogeochemical models such as DailyDayCent (DDC) are increasingly being used to provide a spatially disaggregated assessment of annual emissions. Prior to use, an assessment of the ability of the model to predict annual emissions should be undertaken, coupled with an analysis of how model inputs influence model outputs, and whether the modelled estimates are more robust that those derived from the Tier 1 methodology. The aims of the study were (a) to evaluate if the DailyDayCent model can accurately estimate annual N2O emissions across nine different experimental sites, (b) to examine its sensitivity to different soil and climate inputs across a number of experimental sites and (c) to examine the influence of uncertainty in the measured inputs on modelled N2O emissions. DailyDayCent performed well across the range of cropland and grassland sites, particularly for fertilized fields indicating that it is robust for UK conditions. The sensitivity of the model varied across the sites and also between fertilizer/manure treatments. Overall our results showed that there was a stronger correlation between the sensitivity of N2O emissions to changes in soil pH and clay content than the remaining input parameters used in this study. The lower the initial site values for soil pH and clay content, the more sensitive DDC was to changes from their initial value. When we compared modelled estimates with Tier 1 estimates for each site, we found that DailyDayCent provided a more accurate representation of the rate of annual emissions.

Rhodium(II) proximity-labeling identifies a novel target site on STAT3 for inhibitors with potent anti-leukemia activity

PubMed Central

Minus, Matthew B.; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M.; Long, Xin; Krueger, Michael; Stevens, Alexandra; Kolosov, Mikhail I.; Sison, Edward Allen R.; Ball, Zachary T.

2015-01-01

Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. PMID:26480340

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala.

PubMed

Rizzo, Nidia; Gramajo, Rodrigo; Escobar, Maria Cabrera; Arana, Byron; Kroeger, Axel; Manrique-Saide, Pablo; Petzold, Max

2012-10-30

In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. The study was conducted as a cluster randomized community trial using "reduction of the vector population" as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical rains occurred in the area and

Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala

PubMed Central

2012-01-01

Background In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs) alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. Methods The study was conducted as a cluster randomized community trial using “reduction of the vector population” as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods) and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices) at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material) and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.). The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. Results At baseline (during the dry season) a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae). After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed) in 970 study households, tropical

Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance.

PubMed

Moretti, Marcelo L; Van Horn, Christopher R; Robertson, Renae; Segobye, Kabelo; Weller, Stephen C; Young, Bryan G; Johnson, William G; Douglas Sammons, R; Wang, Dafu; Ge, Xia; d' Avignon, André; Gaines, Todd A; Westra, Philip; Green, Amanda C; Jeffery, Taylor; Lespérance, Mackenzie A; Tardif, François J; Sikkema, Peter H; Christopher Hall, J; McLean, Michael D; Lawton, Mark B; Schulz, Burkhard

2018-05-01

The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Protein arginine (N)-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins.

PubMed

Verbiest, Vincent; Montaudon, Danièle; Tautu, Michel T; Moukarzel, Joyce; Portail, Jean-Pierre; Markovits, Judith; Robert, Jacques; Ichas, François; Pourquier, Philippe

2008-04-30

PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.

The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification.

PubMed

Sanosyan, Armen; Fayd'herbe de Maudave, Alexis; Bollore, Karine; Zimmermann, Valérie; Foulongne, Vincent; Van de Perre, Philippe; Tuaillon, Edouard

2017-01-01

Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples. Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples. BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays. Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load.

The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification

PubMed Central

Fayd’herbe de Maudave, Alexis; Bollore, Karine; Zimmermann, Valérie; Foulongne, Vincent; Van de Perre, Philippe; Tuaillon, Edouard

2017-01-01

Background Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples. Methods Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples. Results BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays. Conclusions Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

Structural basis for microRNA targeting

DOE PAGES

Schirle, Nicole T.; Sheu-Gruttadauria, Jessica; MacRae, Ian J.

2014-10-31

MicroRNAs (miRNAs) control expression of thousands of genes in plants and animals. miRNAs function by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. In this paper, we determined crystal structures of human Argonaute-2 (Ago2) bound to a defined guide RNA with and without target RNAs representing miRNA recognition sites. These structures suggest a stepwise mechanism, in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing. Pairing to nt 2 to 5 promotes conformational changes that expose nt 2 to 8 and 13 to 16 for further target recognition. Interactions withmore » the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner, whereas an adenosine binding-pocket opposite guide nt 1 further facilitates target recognition. Spurious slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion. Finally, these results explain the conserved nucleotide-pairing patterns in animal miRNA target sites first observed over two decades ago.« less

The binding sites of inhibitory monoclonal antibodies on acetylcholinesterase. Identification of a novel regulatory site at the putative "back door".

PubMed

Simon, S; Le Goff, A; Frobert, Y; Grassi, J; Massoulié, J

1999-09-24

We investigated the target sites of three inhibitory monoclonal antibodies on Electrophorus acetylcholinesterase (AChE). Previous studies showed that Elec-403 and Elec-410 are directed to overlapping but distinct epitopes in the peripheral site, at the entrance of the catalytic gorge, whereas Elec-408 binds to a different region. Using Electrophorus/rat AChE chimeras, we identified surface residues that differed between sensitive and insensitive AChEs: the replacement of a single Electrophorus residue by its rat homolog was able to abolish binding and inhibition, for each antibody. Reciprocally, binding and inhibition by Elec-403 and by Elec-410 could be conferred to rat AChE by the reverse mutation. Elec-410 appears to bind to one side of the active gorge, whereas Elec-403 covers its opening, explaining why the AChE-Elec-410 complex reacts faster than the AChE-Elec-403 or AChE-fasciculin complexes with two active site inhibitors, m-(N,N, N-trimethyltammonio)trifluoro-acetophenone and echothiophate. Elec-408 binds to the region of the putative "back door," distant from the peripheral site, and does not interfere with the access of inhibitors to the active site. The binding of an antibody to this novel regulatory site may inhibit the enzyme by blocking the back door or by inducing a conformational distortion within the active site.

Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification.

PubMed

Zhu, Linling; Zhang, Junying; Wang, Fengyang; Wang, Ya; Lu, Linlin; Feng, Chongchong; Xu, Zhiai; Zhang, Wen

2016-04-15

Amyloid-beta (Aβ) oligomers are highly toxic species in the process of Aβ aggregation and are regarded as potent therapeutic targets and diagnostic markers for Alzheimer's disease (AD). Herein, a label-free molecular beacon (MB) system integrated with enzyme-free amplification strategy was developed for simple and highly selective assay of Aβ oligomers. The MB system was constructed with abasic site (AP site)-containing stem-loop DNA and a fluorescent ligand 2-amino-5,6,7-trimethyl-1,8-naphyridine (ATMND), of which the fluorescence was quenched upon binding to the AP site in DNA stem. Enzyme-free amplification was realized by target-triggered continuous opening of two delicately designed MBs (MB1 and MB2). Target DNA hybridization with MB1 and then MB2 resulted in the release of two ATMND molecules in one binding event. Subsequent target recycling could greatly amplify the detection sensitivity due to the greatly enhanced turn-on emission of ATMND fluorescence. Combining with Aβ oligomers aptamers, the strategy was applied to analyze Aβ oligomers and the results showed that it could quantify Aβ oligomers with high selectivity and monitor the Aβ aggregation process. This novel method may be conducive to improve the diagnosis and pathogenic study of Alzheimer's disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Targeting of EGFR, VEGFR2, and Akt by Engineered Dual Drug Encapsulated Mesoporous Silica-Gold Nanoclusters Sensitizes Tamoxifen-Resistant Breast Cancer.

PubMed

Kumar, B N Prashanth; Puvvada, Nagaprasad; Rajput, Shashi; Sarkar, Siddik; Mahto, Madhusudan Kr; Yallapu, Murali M; Pathak, Amita; Emdad, Luni; Das, Swadesh K; Reis, Rui L; Kundu, S C; Fisher, Paul B; Mandal, Mahitosh

2018-05-30

Tamoxifen administration enhanced overall disease-free survival and diminished mortality rates in cancer patients. However, patients with breast cancer often fail to respond for tamoxifen therapy due to the development of a drug-resistant phenotype. Functional analysis and molecular studies suggest that protein mutation and dysregulation of survival signaling molecules such as epidermal growth factor receptor, vascular endothelial growth factor receptor 2, and Akt contribute to tamoxifen resistance. Various strategies, including combinatorial therapies, show chemosensitize tamoxifen-resistant cancers. Based on chemotoxicity issues, researchers are actively investigating alternative therapeutic strategies. In the current study, we fabricate a mesoporous silica gold cluster nanodrug delivery system that displays exceptional tumor-targeting capability, thus promoting accretion of drug indices at the tumor site. We employ dual drugs, ZD6474, and epigallocatechin gallate (EGCG) that inhibit EGFR2, VEGFR2, and Akt signaling pathways since changes in these signaling pathways confer tamoxifen resistance in MCF 7 and T-47D cells. Mesoporous silica gold cluster nanodrug delivery of ZD6474 and EGCG sensitize tamoxifen-resistant cells to apoptosis. Western and immune-histochemical analyses confirmed the apoptotic inducing properties of the nanoformulation. Overall, results with these silica gold nanoclusters suggest that they may be a potent nanoformulation against chemoresistant cancers.

Folate-receptor-targeted NIR-sensitive polydopamine nanoparticles for chemo-photothermal cancer therapy

NASA Astrophysics Data System (ADS)

Li, Hao; Jin, Zhen; Cho, Sunghoon; Jeon, Mi Jeong; Du Nguyen, Van; Park, Jong-Oh; Park, Sukho

2017-10-01

We propose the use of folate-receptor-targeted, near-infrared-sensitive polydopamine nanoparticles (NPs) for chemo-photothermal cancer therapy as an enhanced type of drug-delivery system which can be synthesized by in situ polymerization and conjugation with folic acid. The NPs consist of a Fe3O4/Au core, coated polydopamine, conjugated folic acid, and loaded anti-cancer drug (doxorubicin). The proposed multifunctional NPs show many advantages for therapeutic applications such as good biocompatibility and easy bioconjugation. The polydopamine coating of the NPs show a higher photothermal effect and thus more effective cancer killing compared to Fe3O4/Au nanoparticles at the same intensity as near-infrared laser irradiation. In addition, the conjugation of folic acid was shown to enhance cancer cellular uptake efficiency via the folate receptor and thus improve chemotherapeutic efficiency. Through in vitro cancer cell treatment testing, the proposed multifunctional NPs showed advanced photothermal and chemotherapeutic performance. Based on these enhanced anti-cancer properties, we expect that the proposed multifunctional NPs can be used as a drug-delivery system in cancer therapy.

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection▿ †

PubMed Central

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D.; Levin, Henry L.

2009-01-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. PMID:19109383

Chemical structure determines target organ carcinogenesis in rats

PubMed Central

Carrasquer, C. A.; Malik, N.; States, G.; Qamar, S.; Cunningham, S.L.; Cunningham, A.R.

2012-01-01

SAR models were developed for 12 rat tumour sites using data derived from the Carcinogenic Potency Database. Essentially, the models fall into two categories: Target Site Carcinogen – Non-Carcinogen (TSC-NC) and Target Site Carcinogen – Non-Target Site Carcinogen (TSC-NTSC). The TSC-NC models were composed of active chemicals that were carcinogenic to a specific target site and inactive ones that were whole animal non-carcinogens. On the other hand, the TSC-NTSC models used an inactive category also composed of carcinogens but to any/all other sites but the target site. Leave one out validations produced an overall average concordance value for all 12 models of 0.77 for the TSC-NC models and 0.73 for the TSC-NTSC models. Overall, these findings suggest that while the TSC-NC models are able to distinguish between carcinogens and non-carcinogens, the TSC-NTSC models are identifying structural attributes that associate carcinogens to specific tumour sites. Since the TSC-NTSC models are composed of active and inactive compounds that are genotoxic and non-genotoxic carcinogens, the TSC-NTSC models may be capable of deciphering non-genotoxic mechanisms of carcinogenesis. Together, models of this type may also prove useful in anticancer drug development since they essentially contain chemicals moieties that target specific tumour site. PMID:23066888

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine*

PubMed Central

Syeda, Ruhma; Santos, Jose S.; Montal, Mauricio

2016-01-01

KCNQ (voltage-gated K+ channel family 7 (Kv7)) channels control cellular excitability and underlie the K+ current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K+ current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels. PMID:26627826

Testing of stack-unit/aquifer sensitivity analysis using contaminant plume distribution in the subsurface of Savannah River Site, South Carolina, USA

USGS Publications Warehouse

Rine, J.M.; Shafer, J.M.; Covington, E.; Berg, R.C.

2006-01-01

Published information on the correlation and field-testing of the technique of stack-unit/aquifer sensitivity mapping with documented subsurface contaminant plumes is rare. The inherent characteristic of stack-unit mapping, which makes it a superior technique to other analyses that amalgamate data, is the ability to deconstruct the sensitivity analysis on a unit-by-unit basis. An aquifer sensitivity map, delineating the relative sensitivity of the Crouch Branch aquifer of the Administrative/Manufacturing Area (A/M) at the Savannah River Site (SRS) in South Carolina, USA, incorporates six hydrostratigraphic units, surface soil units, and relevant hydrologic data. When this sensitivity map is compared with the distribution of the contaminant tetrachloroethylene (PCE), PCE is present within the Crouch Branch aquifer within an area classified as highly sensitive, even though the PCE was primarily released on the ground surface within areas classified with low aquifer sensitivity. This phenomenon is explained through analysis of the aquifer sensitivity map, the groundwater potentiometric surface maps, and the plume distributions within the area on a unit-by- unit basis. The results of this correlation show how the paths of the PCE plume are influenced by both the geology and the groundwater flow. ?? Springer-Verlag 2006.

Testing the capability of ORCHIDEE land surface model to simulate Arctic ecosystems: Sensitivity analysis and site-level model calibration

NASA Astrophysics Data System (ADS)

Dantec-Nédélec, S.; Ottlé, C.; Wang, T.; Guglielmo, F.; Maignan, F.; Delbart, N.; Valdayskikh, V.; Radchenko, T.; Nekrasova, O.; Zakharov, V.; Jouzel, J.

2017-06-01

The ORCHIDEE land surface model has recently been updated to improve the representation of high-latitude environments. The model now includes improved soil thermodynamics and the representation of permafrost physical processes (soil thawing and freezing), as well as a new snow model to improve the representation of the seasonal evolution of the snow pack and the resulting insulation effects. The model was evaluated against data from the experimental sites of the WSibIso-Megagrant project (www.wsibiso.ru). ORCHIDEE was applied in stand-alone mode, on two experimental sites located in the Yamal Peninsula in the northwestern part of Siberia. These sites are representative of circumpolar-Arctic tundra environments and differ by their respective fractions of shrub/tree cover and soil type. After performing a global sensitivity analysis to identify those parameters that have most influence on the simulation of energy and water transfers, the model was calibrated at local scale and evaluated against in situ measurements (vertical profiles of soil temperature and moisture, as well as active layer thickness) acquired during summer 2012. The results show how sensitivity analysis can identify the dominant processes and thereby reduce the parameter space for the calibration process. We also discuss the model performance at simulating the soil temperature and water content (i.e., energy and water transfers in the soil-vegetation-atmosphere continuum) and the contribution of the vertical discretization of the hydrothermal properties. This work clearly shows, at least at the two sites used for validation, that the new ORCHIDEE vertical discretization can represent the water and heat transfers through complex cryogenic Arctic soils—soils which present multiple horizons sometimes with peat inclusions. The improved model allows us to prescribe the vertical heterogeneity of the soil hydrothermal properties.

Detection of canonical A-to-G editing events at 3′ UTRs and microRNA target sites in human lungs using next-generation sequencing

PubMed Central

Soundararajan, Ramani; Stearns, Timothy M.; Griswold, Anthony J.; Mehta, Arpit; Czachor, Alexander; Fukumoto, Jutaro; Lockey, Richard F.; King, Benjamin L.; Kolliputi, Narasaiah

2015-01-01

RNA editing is a post-transcriptional modification of RNA. The majority of these changes result from adenosine deaminase acting on RNA (ADARs) catalyzing the conversion of adenosine residues to inosine in double-stranded RNAs (dsRNAs). Massively parallel sequencing has enabled the identification of RNA editing sites in human transcriptomes. In this study, we sequenced DNA and RNA from human lungs and identified RNA editing sites with high confidence via a computational pipeline utilizing stringent analysis thresholds. We identified a total of 3,447 editing sites that overlapped in three human lung samples, and with 50% of these sites having canonical A-to-G base changes. Approximately 27% of the edited sites overlapped with Alu repeats, and showed A-to-G clustering (>3 clusters in 100 bp). The majority of edited sites mapped to either 3′ untranslated regions (UTRs) or introns close to splice sites; whereas, only few sites were in exons resulting in non-synonymous amino acid changes. Interestingly, we identified 652 A-to-G editing events in the 3′ UTR of 205 target genes that mapped to 932 potential miRNA target binding sites. Several of these miRNA edited sites were validated in silico. Additionally, we validated several A-to-G edited sites by Sanger sequencing. Altogether, our study suggests a role for RNA editing in miRNA-mediated gene regulation and splicing in human lungs. In this study, we have generated a RNA editome of human lung tissue that can be compared with other RNA editomes across different lung tissues to delineate a role for RNA editing in normal and diseased states. PMID:26486088

Targeting renal cell carcinoma with a HIF-2 antagonist.

PubMed

Chen, Wenfang; Hill, Haley; Christie, Alana; Kim, Min Soo; Holloman, Eboni; Pavia-Jimenez, Andrea; Homayoun, Farrah; Ma, Yuanqing; Patel, Nirav; Yell, Paul; Hao, Guiyang; Yousuf, Qurratulain; Joyce, Allison; Pedrosa, Ivan; Geiger, Heather; Zhang, He; Chang, Jenny; Gardner, Kevin H; Bruick, Richard K; Reeves, Catherine; Hwang, Tae Hyun; Courtney, Kevin; Frenkel, Eugene; Sun, Xiankai; Zojwalla, Naseem; Wong, Tai; Rizzi, James P; Wallace, Eli M; Josey, John A; Xie, Yang; Xie, Xian-Jin; Kapur, Payal; McKay, Renée M; Brugarolas, James

2016-11-03

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when

The relationship between the target effective site concentration of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients

PubMed Central

Fan, Xiaochong; Ma, Minyu; Li, Zhisong; Gong, Shengkai; Zhang, Wei; Wen, Yuanyuan

2015-01-01

Objective: To study the relationship between the target effective site concentration (Ce) of rocuronium and the degree of recovery from neuromuscular blockade in elderly patients. Methods: 50 elderly patients (ASA grade II) scheduled for selective surgical procedure under general anaesthesia were randomly divided into two groups, A and B, with 25 cases in each group. The Ce of rocuronium for intubation was 3 μg·ml-1 in both groups, and the Ce during operation were 0.8 and 1.0 μg·ml-1 in group A and B, respectively. When target controlled infusion of rocuronium was stopped, without the administration of reversal agents for neuromuscular blockade, the relationship between Ce and the first twitch height (T1) was studied by regression analysis. Results: There was a significant linear relationship between Ce and T1, and there was no statistical difference in regression coefficient and interception between group A and B (P>0.05). Conclusion: The degree of recovery from neuromuscular blockade could be judged by the target effective site concentration of rocuronium at the time of reversal from neuromuscular blockade in the elderly patients. PMID:26629159

Target-site mutations conferring resistance to glyphosate in feathertop Rhodes grass (Chloris virgata) populations in Australia.

PubMed

Ngo, The D; Krishnan, Mahima; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

2018-05-01

Chloris virgata is a warm-season, C 4 , annual grass weed affecting field crops in northern Australia that has become an emerging weed in southern Australia. Four populations with suspected resistance to glyphosate were collected in South Australia, Queensland and New South Wales, Australia, and compared with one susceptible (S) population to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the rate of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in four populations of C. virgata (V12, V14.2, V14.16 and V15). GR plants were 2-9.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. GR and S plants did not differ in glyphosate absorption and translocation. Target-site EPSPS mutations corresponding to Pro-106-Leu (V14.2) and Pro-106-Ser (V15, V14.16 and V12) substitutions were found in GR populations. The population with Pro-106-Leu substitution was 2.9-4.9-fold more resistant than the three other populations with Pro-106-Ser substitution. This report confirms glyphosate resistance in C. virgata and shows that target-site EPSPS mutations confer resistance to glyphosate in this species. The evolution of glyphosate resistance in C. virgata highlights the need to identify alternative control tactics. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Photoelectrochemical DNA Biosensor Based on Dual-Signal Amplification Strategy Integrating Inorganic-Organic Nanocomposites Sensitization with λ-Exonuclease-Assisted Target Recycling.

PubMed

Shi, Xiao-Mei; Fan, Gao-Chao; Shen, Qingming; Zhu, Jun-Jie

2016-12-28

Sensitive and accurate analysis of DNA is crucial to better understanding of DNA functions and early diagnosis of fatal disease. Herein, an enhanced photoelectrochemical (PEC) DNA biosensor was proposed based on dual-signal amplification via coupling inorganic-organic nanocomposites sensitization with λ-exonuclease (λ-Exo)-assisted target recycling. The short DNA sequence about chronic myelogenous leukemia (CML, type b3a2) was selected as target DNA (tDNA). ZnO nanoplates were deposited with CdS nanocrystals to form ZnO/CdS hetero-nanostructure, and it was used as PEC substrate for immobilizing hairpin DNA (hDNA). CdTe quantum dots (QDs) covalently linked with meso-tetra(4-carboxyphenyl)porphine (TCPP) to form CdTe/TCPP inorganic-organic nanocomposites, which were utilized as sensitization agents labeling at the terminal of probe DNA (pDNA). When the hDNA-modified sensing electrode was incubated with tDNA and λ-Exo, hDNA hybridized with tDNA, and meanwhile it could be recognized and cleaved by λ-Exo, resulting in the release of tDNA. The rest of nonhybridized hDNA would continuously hybridize with the released tDNA, cleave by λ-Exo, and set free the tDNA again. After λ-Exo-assisted tDNA recycling, more amounts of short DNA (sDNA) fragments coming from digestion of hDNA produced on the electrode and hybridized with CdTe/TCPP-labeled pDNA (pDNA-CdTe/TCPP conjugates). In this case, the sensitization of CdTe/TCPP inorganic-organic nanocomposites occurred, which evidently extend the absorption range and strengthened the absorption intensity of light energy, and accordingly the photocurrent signal significantly promoted. Through introducing the dual-signal amplification tactics, the developed PEC assay allowed a low calculated detection limit of 25.6 aM with a wide detection scope from 0.1 fM to 5 pM for sensitive and selective determination of tDNA.

Targeting hunter distribution based on host resource selection and kill sites to manage disease risk.

PubMed

Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K

2013-10-01

Endemic and emerging diseases are rarely uniform in their spatial distribution or prevalence among cohorts of wildlife. Spatial models that quantify risk-driven differences in resource selection and hunter mortality of animals at fine spatial scales can assist disease management by identifying high-risk areas and individuals. We used resource selection functions (RSFs) and selection ratios (SRs) to quantify sex- and age-specific resource selection patterns of collared (n = 67) and hunter-killed (n = 796) nonmigratory elk (Cervus canadensis manitobensis) during the hunting season between 2002 and 2012, in southwestern Manitoba, Canada. Distance to protected area was the most important covariate influencing resource selection and hunter-kill sites of elk (AICw = 1.00). Collared adult males (which are most likely to be infected with bovine tuberculosis (Mycobacterium bovis) and chronic wasting disease) rarely selected for sites outside of parks during the hunting season in contrast to adult females and juvenile males. The RSFs showed selection by adult females and juvenile males to be negatively associated with landscape-level forest cover, high road density, and water cover, whereas hunter-kill sites of these cohorts were positively associated with landscape-level forest cover and increasing distance to streams and negatively associated with high road density. Local-level forest was positively associated with collared animal locations and hunter-kill sites; however, selection was stronger for collared juvenile males and hunter-killed adult females. In instances where disease infects a metapopulation and eradication is infeasible, a principle goal of management is to limit the spread of disease among infected animals. We map high-risk areas that are regularly used by potentially infectious hosts but currently underrepresented in the distribution of kill sites. We present a novel application of widely available data to target hunter distribution based on host resource

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

PubMed

Minus, Matthew B; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M; Long, Xin; Krueger, Michael J; Stevens, Alexandra; Kolosov, Mikhail I; Tweardy, David J; Sison, Edward Allan R; Redell, Michele S; Ball, Zachary T

2015-10-26

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

PubMed Central

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Confirming therapeutic target of protopine using immobilized β2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

PubMed

Liu, Guangxin; Wang, Pei; Li, Chan; Wang, Jing; Sun, Zhenyu; Zhao, Xinfeng; Zheng, Xiaohui

2017-07-01

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β 2 -adrenoceptor (β 2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β 2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β 2 -AR by the 2 methods were (1.00 ± 0.06) × 10 5 M -1 and (1.52 ± 0.14) × 10 4 M -1 . The numbers of binding sites were (1.23 ± 0.07) × 10 -7 M and (9.09 ± 0.06) × 10 -7 M, respectively. These results indicated that β 2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser 169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis. Copyright © 2017 John Wiley & Sons, Ltd.

Redox sensitive Pyk2 as a target for therapeutics in breast cancer.

PubMed

Felty, Quentin

2011-01-01

Breast cancer progression is dependent on the formation of new blood vessels that not only help the tumor by supplying additional nutrients, but also allow cancer cells to spread from the breast to distant sites in the body. Several studies suggest a positive correlation between new vessel formation and estrogens. Estrogenic environmental chemicals such as PCBs have been shown to increase the expression of factors known to promote vessel formation in breast tumors. These studies highlight a growing concern that women exposed to estrogenic environmental compounds may be more susceptible to either aggressive metastatic tumors or a high recurrence of breast cancer. Our concept offers a fundamental new understanding of the way the environment contributes to breast cancer progression. This review will be focused on a highly novel Pyk2 signaling complex as a target for therapy of estrogen dependent breast tumor angiogenesis. A better understanding of the role of Pyk2 signaling in estrogen dependent tumor vascularization may lead to the development of a new therapy against aggressive breast cancer using small molecule inhibitors of Pyk2.

Novel GABA receptor pesticide targets.

PubMed

Casida, John E; Durkin, Kathleen A

2015-06-01

The γ-aminobutyric acid (GABA) receptor has four distinct but overlapping and coupled targets of pesticide action importantly associated with little or no cross-resistance. The target sites are differentiated by binding assays with specific radioligands, resistant strains, site-directed mutagenesis and molecular modeling. Three of the targets are for non-competitive antagonists (NCAs) or channel blockers of widely varied chemotypes. The target of the first generation (20th century) NCAs differs between the larger or elongated compounds (NCA-IA) including many important insecticides of the past (cyclodienes and polychlorocycloalkanes) or present (fiproles) and the smaller or compact compounds (NCA-IB) highly toxic to mammals and known as cage convulsants, rodenticides or chemical threat agents. The target of greatest current interest is designated NCA-II for the second generation (21st century) of NCAs consisting for now of isoxazolines and meta-diamides. This new and uniquely different NCA-II site apparently differs enough between insects and mammals to confer selective toxicity. The fourth target is the avermectin site (AVE) for allosteric modulators of the chloride channel. NCA pesticides vary in molecular surface area and solvent accessible volume relative to avermectin with NCA-IBs at 20-22%, NCA-IAs at 40-45% and NCA-IIs at 57-60%. The same type of relationship relative to ligand-docked length is 27-43% for NCA-IBs, 63-71% for NCA-IAs and 85-105% for NCA-IIs. The four targets are compared by molecular modeling for the Drosophila melanogaster GABA-R. The principal sites of interaction are proposed to be: pore V1' and A2' for NCA-IB compounds; pore A2', L6' and T9' for NCA-IA compounds; pore T9' to S15' in proximity to M1/M3 subunit interface (or alternatively an interstitial site) for NCA-II compounds; and M1/M3, M2 interfaces for AVE. Understanding the relationships of these four binding sites is important in resistance management and in the discovery and use

Topography of eye-position sensitivity of saccades evoked electrically from the cat's superior colliculus.

PubMed

McIlwain, J T

1990-03-01

Saccades evoked electrically from the deep layers of the superior colliculus have been examined in the alert cat with its head fixed. Amplitudes of the vertical and horizontal components varied linearly with the starting position of the eye. The slopes of the linear-regression lines provided an estimate of the sensitivity of these components to initial eye position. In observations on 29 sites in nine cats, the vertical and horizontal components of saccades evoked from a given site were rarely influenced to the same degree by initial eye position. For most sites, the horizontal component was more sensitive than the vertical component. Sensitivities of vertical and horizontal components were lowest near the representations of the horizontal and vertical meridians, respectively, of the collicular retinotopic map, but otherwise exhibited no systematic retinotopic dependence. Estimates of component amplitudes for saccades evoked from the center of the oculomotor range also diverged significantly from those predicted from the retinotopic map. The results of this and previous studies indicate that electrical stimulation of the cat's superior colliculus cannot yield a unique oculomotor map or one that is in register everywhere with the sensory retinotopic map. Several features of these observations suggest that electrical stimulation of the colliculus produces faulty activation of a saccadic control system that computes target position with respect to the head and that small and large saccades are controlled differently.

Prediction of TF target sites based on atomistic models of protein-DNA complexes

PubMed Central

Angarica, Vladimir Espinosa; Pérez, Abel González; Vasconcelos, Ana T; Collado-Vides, Julio; Contreras-Moreira, Bruno

2008-01-01

Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs) plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition. PMID:18922190

The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription

PubMed Central

Jennings, Barbara H.

2014-01-01

Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in “active” chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation

Testing CMAQ chemistry sensitivities in base case and emissions control runs at SEARCH and SOS99 surface sites in the southeastern US

NASA Astrophysics Data System (ADS)

Arnold, J. R.; Dennis, Robin L.

CMAQ was run to simulate urban and regional tropospheric conditions in the southeastern US over 14 days in July 1999 at 32, 8 and 2 km grid spacings. Runs were made with either of two older mechanisms, Carbon Bond IV (CB4) and the Regional Acid Deposition Model, version 2 (RADM2), and with the more recent and complete California Statewide Air Pollution Research Center, version 1999 mechanism (SAPRC99) in a sensitivity matrix with a full emissions base case and separate 50% control scenarios for emissions of nitrogen oxides (NO X) and volatile organic compounds (VOC). Results from the base case were compared to observations at the Southeastern Aerosol Research and Characterization Study (SEARCH) site at Jefferson Street in Atlanta, GA (JST) and the Southern Oxidant Study (SOS) Cornelia Fort Airpark (CFA) site downwind of Nashville, TN. In the base case, SAPRC99 predicted more ozone (O 3) than CB4 or RADM2 almost every hour and especially for afternoon maxima at both JST and CFA. Performance of the 8 km models at JST was better than that of the 32 km ones for all chemistries, reducing the 1 h peak bias by as much as 30 percentage points; at CFA only the RADM2 8 km model improved. The 2 km solutions did not show improved performance over the 8 km ones at either site, with normalized 1 h bias in the peak O 3 ranging from 21% at CFA to 43% at JST. In the emissions control cases, SAPRC99 was generally more responsive than CB4 and RADM2 to NO X and VOC controls, excepting hours at JST with predicted increased O 3 from NO X control. Differential sensitivity to chemical mechanism varied by more than ±10% for NO X control at JST and CFA, and in a similar range for VOC control at JST. VOC control at the more strongly NO X- limited urban CFA site produced a differential sensitivity response of <5%. However, even when differential sensitivities in control cases were small, neither their sign nor their magnitude could be reliably determined from model performance in the full

Assessment of nevirapine bioavailability from targeted sites in the human gastrointestinal tract.

PubMed

Macha, Sreeraj; Yong, Chan-Loi; MacGregor, Thomas R; Castles, Mark; Quinson, Anne-Marie; Rouyrre, Nicolas; Wilding, Ian

2009-12-01

This study investigated absorption of nevirapine (NVP) from targeted sites of the gastrointestinal tract using remotely activated capsules and gamma scintigraphy. A total of 24 participants were randomized to receive 50 mg NVP orally as a suspension or via remotely activated capsules for release into the ascending colon. The 24 participants were then rerandomized into parallel groups of n = 8 for drug release into the ileum, jejunum, or descending colon. The mean gastric emptying time of capsules ranged from 0.88 to 3.35 hours. The small intestinal and colon transit time ranged from 4.08 to 7.76 hours and 17.6 to 21.2 hours, respectively, and capsule recovery time ranged from 27.6 to 34.4 hours. The relative bioavailability ratio of NVP in the jejunum was 1.06 (90% confidence interval [CI]: 1.00-1.12) compared to suspension. In the ileum, ascending colon, and descending colon, bioavailability decreased to 0.89 (0.80-0.99), 0.82 (0.71-0.95), and 0.58 (0.22-1.53), respectively. The absorption rate decreased by approximately 10-fold from the jejunum (3.83 h(-1)) to the descending colon (0.338 h(-1)), and t(max) increased from 2.42 hours (jejunum) to 16.3 hours (descending colon). Overall, NVP is absorbed from all 4 sites of the gastrointestinal tract, and the rate of absorption decreased from the jejunum to the descending colon. Relative bioavailability of NVP was in the order of jejunum > ileum > ascending colon > descending colon.

The therapeutic potential of allosteric ligands for free fatty acid sensitive GPCRs.

PubMed

Hudson, Brian D; Ulven, Trond; Milligan, Graeme

2013-01-01

G protein coupled receptors (GPCRs) are the most historically successful therapeutic targets. Despite this success there are many important aspects of GPCR pharmacology and function that have yet to be exploited to their full therapeutic potential. One in particular that has been gaining attention in recent times is that of GPCR ligands that bind to allosteric sites on the receptor distinct from the orthosteric site of the endogenous ligand. As therapeutics, allosteric ligands possess many theoretical advantages over their orthosteric counterparts, including more complex modes of action, improved safety, more physiologically appropriate responses, better target selectivity, and reduced likelihood of desensitisation and tachyphylaxis. Despite these advantages, the development of allosteric ligands is often difficult from a medicinal chemistry standpoint due to the more complex challenge of identifying allosteric leads and their often flat or confusing SAR. The present review will consider the advantages and challenges associated with allosteric GPCR ligands, and examine how the particular properties of these ligands may be exploited to uncover the therapeutic potential for free fatty acid sensitive GPCRs.

A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).

PubMed

Unwin, Richard D; Griffiths, John R; Whetton, Anthony D

2009-01-01

The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

Molecular dynamics simulations and statistical coupling analysis of GPI12 in L. major: functional co-evolution and conservedness reveals potential drug-target sites.

PubMed

Singh, Shailza; Mandlik, Vineetha; Shinde, Sonali

2015-03-01

GPI12 represents an important enzyme in the GPI biosynthetic pathway of several parasites like 'Leishmania'. GPI activity is generally regulated through either the hindrance in GPI complex assembly formation or the modulation of the lipophosphoglycan (LPG) flux to either reduce or enhance the pathogenicity in an organism. Of the various GPI molecules known, GPI12 is an important enzyme in the GPI biosynthetic pathway which can be exploited as a target due to the substrate specificity difference in parasites and humans. In the present study, the functional importance of the co-evolving residues of the GPI12 protein of Leishmania has been highlighted using the GPI proteins belonging to the GlcNAC-deacetylase family. Exploring the active site of the GPI12 protein and designing inhibitors against the functional residues provide ways and means to change the efficiency of deacetylation activity of the enzyme. The activity of de-N-acetylase is low in the absence of metal ions like zinc. Hence we designed eight small molecules in order to modulate the activity of GPI12. Compound 8 was found to be an appropriate choice to target the agonist (GPI12) active site thereby targeting the residues which were essential in the Zn binding and chelation activity. Inhibition of these sites offered a strong constraint to block the protein activity and in turn GPI biosynthesis.

MT119, a new planar-structured compound, targets the colchicine site of tubulin arresting mitosis and inhibiting tumor cell proliferation.

PubMed

Zhang, Zhixiang; Meng, Tao; Yang, Na; Wang, Wei; Xiong, Bing; Chen, Yi; Ma, Lanping; Shen, Jingkang; Miao, Ze-Hong; Ding, Jian

2011-07-01

Microtubule-targeted drugs are now indispensable for the therapy of various cancer types worldwide. In this article, we report MT119 [6-[2-(4-methoxyphenyl) -ethyl]-9-[(pyridine-3-ylmethyl)amino]pyrido[2',1':2,3]imida-zo[4,5-c]isoquinolin-5(6H)-one] as a new microtubule-targeted agent. MT119 inhibited tubulin polymerization significantly both in tumor cells and in cell-free systems, which was followed by the disruption of mitotic spindle assembly. Surface plasmon resonance-based analyses showed that MT119 bound to purified tubulin directly, with the K(D) value of 10.6 μM. The binding of MT119 in turn caused tubulin conformational changes as evidenced by the quenched tryptophan fluorescence, the reduction of the bis-ANS reactivity and the decreased DTNB-sulfhydryl reaction rate. Competitive binding assays further revealed that MT119 bound to tubulin at its colchicine site. Consequently, by inhibiting tubulin polymerization, MT119 arrested different tumor cells at mitotic phase, which contributed to its potent antitumor activity in vitro. MT119 was also similarly cytotoxic to vincristine-, adriamycin- or mitoxantrone-resistant cancer cells and to their corresponding parental cells. Together, these data indicate that MT119 represents a new class of colchicine-site-targeted inhibitors against tubulin polymerization, which might be a promising starting point for future cancer therapeutics. Copyright © 2010 UICC.

Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3 Single Amino Acid near the Active Site Influences Inhibitor Sensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Bum Soo; Allali-Hassani, Abdellah; Tempel, Wolfram

2010-07-06

Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoK{alpha} ({alpha}1 and {alpha}2) and ChoK{beta} isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoK{alpha}1 and ChoK{beta} isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoK{alpha}1 occupied the choline-bindingmore » pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoK{beta} was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoK{alpha} isoforms ({alpha}1 and {alpha}2) compared with ChoK{beta}. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoK{alpha}2 (equivalent to Leu-419 of ChoK{alpha}1), or the corresponding residue Phe-352 of ChoK{beta}, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.« less

Targeting smooth emergence: the effect site concentration of remifentanil for preventing cough during emergence during propofol-remifentanil anaesthesia for thyroid surgery.

PubMed

Lee, B; Lee, J-R; Na, S

2009-06-01

The administration of short-acting opioids can be a reliable and safe method to prevent coughing during emergence from anaesthesia but the proper dose or effect site concentration of remifentanil for this purpose has not been reported. We therefore investigated the effect site concentration (Ce) of remifentanil for preventing cough during emergence from anaesthesia with propofol-remifentanil target-controlled infusion. Twenty-three ASA I-II grade female patients, aged 23-66 yr undergoing elective thyroidectomy were enrolled in this study. EC(50) and EC(95) of remifentanil for preventing cough were determined using Dixon's up-and-down method and probit analysis. Propofol effect site concentration at extubation, mean arterial pressure, and heart rate (HR) were compared in patients with smooth emergence and without smooth emergence. Three out of 11 patients with remifentanil Ce of 1.5 ng ml(-1) and all seven patients with Ce of 2.0 ng ml(-1) did not cough during emergence; the EC(50) of remifentanil that suppressed coughing was 1.46 ng ml(-1) by Dixon's up-and-down method, and EC(95) was 2.14 ng ml(-1) by probit analysis. Effect site concentration of propofol at awakening was similar in patients with a smooth emergence and those without smooth emergence, but HR and arterial pressure were higher in those who coughed during emergence. Clinically significant hypoventilation was not seen in any patient. We found that the EC(95) of effect site concentration of remifentanil to suppress coughing at emergence from anaesthesia was 2.14 ng ml(-1). Maintaining an established Ce of remifentanil is a reliable method of abolishing cough and thereby targeting smooth emergence from anaesthesia.

Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

PubMed Central

Korrapati, Anil Babu; Swaminathan, Gokul; Singh, Aarti; Khanna, Navin; Swaminathan, Sathyamangalam

2012-01-01

Background Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy. Methodology/Principal Findings We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. Conclusion/Significance The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection. PMID:22848770

Radioligand Recognition of Insecticide Targets.

PubMed

Casida, John E

2018-04-04

Insecticide radioligands allow the direct recognition and analysis of the targets and mechanisms of toxic action critical to effective and safe pest control. These radioligands are either the insecticides themselves or analogs that bind at the same or coupled sites. Preferred radioligands and their targets, often in both insects and mammals, are trioxabicyclooctanes for the γ-aminobutyric acid (GABA) receptor, avermectin for the glutamate receptor, imidacloprid for the nicotinic receptor, ryanodine and chlorantraniliprole for the ryanodine receptor, and rotenone or pyridaben for NADH + ubiquinone oxidoreductase. Pyrethroids and other Na + channel modulator insecticides are generally poor radioligands due to lipophilicity and high nonspecific binding. For target site validation, the structure-activity relationships competing with the radioligand in the binding assays should be the same as that for insecticidal activity or toxicity except for rapidly detoxified or proinsecticide analogs. Once the radioligand assay is validated for relevance, it will often help define target site modifications on selection of resistant pest strains, selectivity between insects and mammals, and interaction with antidotes and other chemicals at modulator sites. Binding assays also serve for receptor isolation and photoaffinity labeling to characterize the interactions involved.

NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogbomo, Henry; Hahn, Anke; Geiler, Janina

2006-01-06

The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe,more » and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.« less

Genetic variation in IL-16 miRNA target site and time to prostate cancer diagnosis in African American men

PubMed Central

Hughes, Lucinda; Ruth, Karen; Rebbeck, Timothy R.; Giri, Veda N.

2013-01-01

Background Men with a family history of prostate cancer and African American men are at high risk for prostate cancer and in need of personalized risk estimates to inform screening decisions. This study evaluated genetic variants in genes encoding microRNA (miRNA) binding sites for informing of time to prostate cancer diagnosis among ethnically-diverse, high-risk men undergoing prostate cancer screening. Methods The Prostate Cancer Risk Assessment Program (PRAP) is a longitudinal screening program for high-risk men. Eligibility includes men ages 35-69 with a family history of prostate cancer or African descent. Participants with ≥ 1 follow-up visit were included in the analyses (n=477). Genetic variants in regions encoding miRNA binding sites in four target genes (ALOX15, IL-16, IL-18, and RAF1) previously implicated in prostate cancer development were evaluated. Genotyping methods included Taqman® SNP Genotyping Assay (Applied Biosystems) or pyrosequencing. Cox models were used to assess time to prostate cancer diagnosis by risk genotype. Results Among 256 African Americans with ≥ one follow-up visit, the TT genotype at rs1131445 in IL-16 was significantly associated with earlier time to prostate cancer diagnosis vs. the CC/CT genotypes (p=0.013), with a suggestive association after correction for false-discovery (p=0.065). Hazard ratio after controlling for age and PSA for TT vs. CC/CT among African Americans was 3.0 (95% CI 1.26-7.12). No association to time to diagnosis was detected among Caucasians by IL-16 genotype. No association to time to prostate cancer diagnosis was found for the other miRNA target genotypes. Conclusions Genetic variation in IL-16 encoding miRNA target site may be informative of time to prostate cancer diagnosis among African American men enrolled in prostate cancer risk assessment, which may inform individualized prostate cancer screening strategies in the future. PMID:24061634

Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.

PubMed Central

Dubois, B W; Cherian, S F; Evers, A S

1993-01-01

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein. PMID:8341659

PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing.

PubMed

Rastogi, Achal; Murik, Omer; Bowler, Chris; Tirichine, Leila

2016-07-01

With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.

A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper).

PubMed

Liu, Zewen; Williamson, Martin S; Lansdell, Stuart J; Denholm, Ian; Han, Zhaojun; Millar, Neil S

2005-06-14

Neonicotinoids, such as imidacloprid, are nicotinic acetylcholine receptor (nAChR) agonists with potent insecticidal activity. Since its introduction in the early 1990s, imidacloprid has become one of the most extensively used insecticides for both crop protection and animal health applications. As with other classes of insecticides, resistance to neonicotinoids is a significant threat and has been identified in several pest species, including the brown planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. In this study, radioligand binding experiments have been conducted with whole-body membranes prepared from imidacloprid-susceptible and imidacloprid-resistant strains of N. lugens. The results reveal a much higher level of [3H]imidacloprid-specific binding to the susceptible strain than to the resistant strain (16.7 +/- 1.0 and 0.34 +/- 0.21 fmol/mg of protein, respectively). With the aim of understanding the molecular basis of imidacloprid resistance, five nAChR subunits (Nlalpha1-Nlalpha4 and Nlbeta1) have been cloned from N. lugens.A comparison of nAChR subunit genes from imidacloprid-sensitive and imidacloprid-resistant populations has identified a single point mutation at a conserved position (Y151S) in two nAChR subunits, Nlalpha1 and Nlalpha3. A strong correlation between the frequency of the Y151S point mutation and the level of resistance to imidacloprid has been demonstrated by allele-specific PCR. By expression of hybrid nAChRs containing N. lugens alpha and rat beta2 subunits, evidence was obtained that demonstrates that mutation Y151S is responsible for a substantial reduction in specific [3H]imidacloprid binding. This study provides direct evidence for the occurrence of target-site resistance to a neonicotinoid insecticide.

Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

PubMed Central

Alquezar-Planas, David E.; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N.; Lenz, Dorina; Helgen, Kristofer M.; Roca, Alfred L.; Hartman, Stefanie

2016-01-01

Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

Unusual Properties of Regulatory DNA from the Drosophila Engrailed Gene: Three ``pairing-Sensitive'' Sites within a 1.6-Kb Region

PubMed Central

Kassis, J. A.

1994-01-01

We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called ``pairing-sensitive sites'' (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter. PMID:8005412

Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.

PubMed

Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria S; Allende, Miguel L; Calcaterra, Nora B

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.

Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

PubMed Central

Mouguelar, Valeria S.; Allende, Miguel L.; Calcaterra, Nora B.

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates. PMID:23667590

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine.

PubMed

Syeda, Ruhma; Santos, Jose S; Montal, Mauricio

2016-02-05

KCNQ (voltage-gated K(+) channel family 7 (Kv7)) channels control cellular excitability and underlie the K(+) current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K(+) current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of specific metabolic pathways as druggable targets regulating the sensitivity to cyanide poisoning.

PubMed

Sips, Patrick Y; Shi, Xu; Musso, Gabriel; Nath, Anjali K; Zhao, Yanbin; Nielson, Jason; Morningstar, Jordan; Kelly, Amy E; Mikell, Brittney; Buys, Eva; Bebarta, Vikhyat; Rutter, Jared; Davisson, V Jo; Mahon, Sari; Brenner, Matthew; Boss, Gerry R; Peterson, Randall T; Gerszten, Robert E; MacRae, Calum A

2018-01-01

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning.

Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction.

PubMed

Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl; Hejazi, Mohammad Saeid

2014-01-01

Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA), raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting "A site" of 16S rRNA) to riboswitches via docking method. There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8) hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with "16S rRNA A site". Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA "A site" suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

An electrophysiological investigation of reinforcement effects in attention deficit/hyperactivity disorder: Dissociating cue sensitivity from down-stream effects on target engagement and performance.

PubMed

Chronaki, Georgia; Soltesz, Fruzsina; Benikos, Nicholas; Sonuga-Barke, Edmund J S

2017-12-01

Neural hypo-sensitivity to cues predicting positive reinforcement has been observed in ADHD using the Monetary Incentive Delay (MID) task. Here we report the first study using an electrophysiological analogue of this task to distinguish between (i) cue related anticipation of reinforcement and downstream effects on (ii) target engagement and (iii) performance in a clinical sample of adolescents with ADHD and controls. Thirty-one controls and 32 adolescents with ADHD aged 10-16 years performed the electrophysiological (e)-MID task - in which preparatory cues signal whether a response to an upcoming target will be reinforced or not - under three conditions; positive reinforcement, negative reinforcement (response cost) and no consequence (neutral). We extracted values for both cue-related potentials known to be, both, associated with response preparation and modulated by reinforcement (Cue P3 and Cue CNV) and target-related potentials (target P3) and compared these between ADHD and controls. ADHD and controls did not differ on cue-related components on neutral trials. Against expectation, adolescents with ADHD displayed Cue P3 and Cue CNV reinforcement-related enhancement (versus neutral trials) compared to controls. ADHD individuals displayed smaller target P3 amplitudes and slower and more variable performance - but effects were not modulated by reinforcement contingencies. When age, IQ and conduct problems were controlled effects were marginally significant but the pattern of results did not change. ADHD was associated with hypersensitivity to positive (and marginally negative) reinforcement reflected on components often thought to be associated with response preparation - however these did not translate into improved attention to targets. In the case of ADHD, upregulated CNV may be a specific marker of hyper-arousal rather than an enhancement of anticipatory attention to upcoming targets. Future studies should examine the effects of age, IQ and conduct problems

Parameter Estimation and Sensitivity Analysis of an Urban Surface Energy Balance Parameterization at a Tropical Suburban Site

NASA Astrophysics Data System (ADS)

Harshan, S.; Roth, M.; Velasco, E.

2014-12-01

Forecasting of the urban weather and climate is of great importance as our cities become more populated and considering the combined effects of global warming and local land use changes which make urban inhabitants more vulnerable to e.g. heat waves and flash floods. In meso/global scale models, urban parameterization schemes are used to represent the urban effects. However, these schemes require a large set of input parameters related to urban morphological and thermal properties. Obtaining all these parameters through direct measurements are usually not feasible. A number of studies have reported on parameter estimation and sensitivity analysis to adjust and determine the most influential parameters for land surface schemes in non-urban areas. Similar work for urban areas is scarce, in particular studies on urban parameterization schemes in tropical cities have so far not been reported. In order to address above issues, the town energy balance (TEB) urban parameterization scheme (part of the SURFEX land surface modeling system) was subjected to a sensitivity and optimization/parameter estimation experiment at a suburban site in, tropical Singapore. The sensitivity analysis was carried out as a screening test to identify the most sensitive or influential parameters. Thereafter, an optimization/parameter estimation experiment was performed to calibrate the input parameter. The sensitivity experiment was based on the "improved Sobol's global variance decomposition method" . The analysis showed that parameters related to road, roof and soil moisture have significant influence on the performance of the model. The optimization/parameter estimation experiment was performed using the AMALGM (a multi-algorithm genetically adaptive multi-objective method) evolutionary algorithm. The experiment showed a remarkable improvement compared to the simulations using the default parameter set. The calibrated parameters from this optimization experiment can be used for further model

MicroRNA-100 regulates pancreatic cancer cells growth and sensitivity to chemotherapy through targeting FGFR3.

PubMed

Li, Zhipeng; Li, Xu; Yu, Chao; Wang, Min; Peng, Feng; Xiao, Jie; Tian, Rui; Jiang, Jianxin; Sun, Chengyi

2014-12-01

We intended to investigate the role of microRNA 100 (miR-100) in regulating pancreatic cancer cells' growth in vitro and tumor development in vivo. QTR-PCR was used to examine the expression of miR-100 in pancreatic cancer cell lines and tumor cells from human patients. Lentivirual vector containing miR-100 mimics (lv-miR-100) was used to overexpress miR-100 in MIA PaCa-2 and FCPAC-1 cells. The effects of overexpressing miR-100 on pancreatic cancer cell proliferation and chemosensitivity to cisplatin were examined by cell proliferation essay in vitro. MIA PaCa-2 cells with endogenously overexpressed miR-100 were transplanted into null mice to examine tumor growth in vivo. The predicted target of miR-100, fibroblast growth factor receptor 3 (FGFR3), was downregulated by siRNA to examine its effect on pancreatic cancer cells. We found miR-100 was markedly underexpressed in both pancreatic cancer cell lines and tumor cells from patients. In cancer cells, transfection of lv-miR-100 was able to upregulate endogenous expression of miR-100, inhibited cancer cell proliferation, and increased sensitivities to cisplatin. Overexpressing miR-100 led to significant inhibition on tumor formation in vivo. Luciferase essay showed FGFR3 was direct target of miR-100. FGFR3 was significantly downregulated by overexpressing miR-100 in pancreatic cancer cells and knocking down FGFR3 by siRNA exerted similar effect as miR-100. Our study demonstrated that miR-100 played an important role in pancreatic cancer development, possibly through targeting FGFR3. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer.

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

PubMed Central

Cain-Hom, Carol; Splinter, Erik; van Min, Max; Simonis, Marieke; van de Heijning, Monique; Martinez, Maria; Asghari, Vida

2017-01-01

Abstract Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events. PMID:28053125

Newly Engineered Magnetic Erythrocytes for Sustained and Targeted Delivery of Anti-Cancer Therapeutic Compounds

PubMed Central

Taranta, Monia; Naldi, Ilaria

2011-01-01

Cytotoxic chemotherapy of cancer is limited by serious, sometimes life-threatening, side effects that arise from toxicities to sensitive normal cells because the therapies are not selective for malignant cells. So how can they be selectively improved? Alternative pharmaceutical formulations of anti-cancer agents have been investigated in order to improve conventional chemotherapy treatment. These formulations are associated with problems like severe toxic side effects on healthy organs, drug resistance and limited access of the drug to the tumor sites suggested the need to focus on site-specific controlled drug delivery systems. In response to these concerns, we have developed a new drug delivery system based on magnetic erythrocytes engineered with a viral spike fusion protein. This new erythrocyte-based drug delivery system has the potential for magnetic-controlled site-specific localization and highly efficient fusion capability with the targeted cells. Here we show that the erythro-magneto-HA virosomes drug delivery system is able to attach and fuse with the target cells and to efficiently release therapeutic compounds inside the cells. The efficacy of the anti-cancer drug employed is increased and the dose required is 10 time less than that needed with conventional therapy. PMID:21373641

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

PubMed

Denys, A; Allain, F; Foxwell, B; Spik, G

1997-08-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

Retroviral integration: Site matters

PubMed Central

Demeulemeester, Jonas; De Rijck, Jan

2015-01-01

Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co‐opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine‐grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success. PMID:26293289

Effect of co-administration of probiotics with polysaccharide based colon targeted delivery systems to optimize site specific drug release.

PubMed

Prudhviraj, G; Vaidya, Yogyata; Singh, Sachin Kumar; Yadav, Ankit Kumar; Kaur, Puneet; Gulati, Monica; Gowthamarajan, K

2015-11-01

Significant clinical success of colon targeted dosage forms has been limited by their inappropriate release profile at the target site. Their failure to release the drug completely in the colon may be attributed to changes in the colonic milieu because of pathological state, drug effect and psychological stress accompanying the diseased state or, a combination of these. Alteration in normal colonic pH and bacterial picture leads to incomplete release of drug from the designed delivery system. We report the effectiveness of a targeted delivery system wherein the constant replenishment of the colonic microbiota is achieved by concomitant administration of probiotics along with the polysaccharide based drug delivery system. Guar gum coated spheroids of sulfasalazine were prepared. In the dissolution studies, these spheroids showed markedly higher release in the simulated colonic fluid. In vivo experiments conducted in rats clearly demonstrated the therapeutic advantage of co-administration of probiotics with guar gum coated spheroids. Our results suggest that concomitant use of probiotics along with the polysaccharide based delivery systems can be a simple strategy to achieve satisfactory colon targeting of drugs. Copyright © 2015 Elsevier B.V. All rights reserved.

Radiation Sensitization in Cancer Therapy.

ERIC Educational Resources Information Center

Greenstock, Clive L.

1981-01-01

Discusses various aspects of radiation damage to biological material, including free radical mechanisms, radiation sensitization and protection, tumor hypoxia, mechanism of hypoxic cell radiosensitization, redox model for radiation modification, sensitizer probes of cellular radiation targets, pulse radiolysis studies of free radical kinetics,…

Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae.

PubMed

Wilmes, Anja; Hanna, Reem; Heathcott, Rosemary W; Northcote, Peter T; Atkinson, Paul H; Bellows, David S; Miller, John H

2012-04-15

Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC(50) of 35μM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC(50)=19μM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray 'hits', peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking. Copyright © 2012 Elsevier B.V. All rights reserved.

An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

PubMed Central

Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. Steven; Qian, Wei-Jun

2010-01-01

Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell line by applying anti-phosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that the majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems. PMID:19534553

A sensitive electrochemical aptasensor for multiplex antibiotics detection based on high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted cascade target recycling.

PubMed

Yan, Zhongdan; Gan, Ning; Li, Tianhua; Cao, Yuting; Chen, Yinji

2016-04-15

A multiplex electrochemical aptasensor was developed for simultaneous detection of two antibiotics such as chloramphenicol (CAP) and oxytetracycline (OTC), and high-capacity magnetic hollow porous nanotracers coupling exonuclease-assisted target recycling was used to improve sensitivity. The cascade amplification process consists of the exonuclease-assisted target recycling amplification and metal ions encoded magnetic hollow porous nanoparticles (MHPs) to produce voltammetry signals. Upon the specific recognition of aptamers to targets (CAP and OTC), exonuclease I (Exo I) selectively digested the aptamers which were bound with CAP and OTC, then the released CAP and OTC participated new cycling to produce more single DNA, which can act as trigger strands to hybrid with nanotracers to generate further signal amplification. MHPs were used as carriers to load more amounts of metal ions and coupling with Exo I assisted cascade target recycling can amplify the signal for about 12 folds compared with silica based nanotracers. Owing to the dual signal amplification, the linear range between signals and the concentrations of CAP and OTC were obtained in the range of 0.0005-50 ng mL(-1). The detection limits of CAP and OTC were 0.15 and 0.10 ng mL(-1) (S/N=3) which is more than 2 orders lower than commercial enzyme-linked immunosorbent immunoassay (ELISA) method, respectively. The proposed method was successfully applied to simultaneously detection of CAP and OTC in milk samples. Besides, this aptasensor can be applied to other antibiotics detection by changing the corresponding aptamer. The whole scheme is facile, selective and sensitive enough for antibiotics screening in food safety. Copyright © 2015 Elsevier B.V. All rights reserved.

Voltage-sensitive styryl dyes as singlet oxygen targets on the surface of bilayer lipid membrane.

PubMed

Sokolov, V S; Gavrilchik, A N; Kulagina, A O; Meshkov, I N; Pohl, P; Gorbunova, Yu G

2016-08-01

Photosensitizers are widely used as photodynamic therapeutic agents killing cancer cells by photooxidation of their components. Development of new effective photosensitive molecules requires profound knowledge of possible targets for reactive oxygen species, especially for its singlet form. Here we studied photooxidation of voltage-sensitive styryl dyes (di-4-ANEPPS, di-8-ANEPPS, RH-421 and RH-237) by singlet oxygen on the surface of bilayer lipid membranes commonly used as cell membrane models. Oxidation was induced by irradiation of a photosensitizer (aluminum phthalocyanine tetrasulfonate) and monitored by the change of dipole potential on the surface of the membrane. We studied the drop of the dipole potential both in the case when the dye molecules were adsorbed on the same side of the lipid bilayer as the photosensitizer (cis-configuration) and in the case when they were adsorbed on the opposite side (trans-configuration). Based on a simple model, we determined the rate of oxidation of the dyes from the kinetics of change of the potential during and after irradiation. This rate is proportional to steady-state concentration of singlet oxygen in the membrane under irradiation. Comparison of the oxidation rates of various dyes reveals that compounds of ANEPPS series are more sensitive to singlet oxygen than RH type dyes, indicating that naphthalene group is primarily responsible for their oxidation. Copyright © 2016 Elsevier B.V. All rights reserved.

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

PubMed

Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

Field-sensitivity To Rheological Parameters

NASA Astrophysics Data System (ADS)

Freund, Jonathan; Ewoldt, Randy

2017-11-01

We ask this question: where in a flow is a quantity of interest Q quantitatively sensitive to the model parameters θ-> describing the rheology of the fluid? This field sensitivity is computed via the numerical solution of the adjoint flow equations, as developed to expose the target sensitivity δQ / δθ-> (x) via the constraint of satisfying the flow equations. Our primary example is a sphere settling in Carbopol, for which we have experimental data. For this Carreau-model configuration, we simultaneously calculate how much a local change in the fluid intrinsic time-scale λ, limit-viscosities ηo and η∞, and exponent n would affect the drag D. Such field sensitivities can show where different fluid physics in the model (time scales, elastic versus viscous components, etc.) are important for the target observable and generally guide model refinement based on predictive goals. In this case, the computational cost of solving the local sensitivity problem is negligible relative to the flow. The Carreau-fluid/sphere example is illustrative; the utility of field sensitivity is in the design and analysis of less intuitive flows, for which we provide some additional examples.

Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

PubMed

Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

2017-08-15

MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

Solid Waste Landfill Site Selection in the Sense of Environment Sensitive Sustainable Urbanization: Izmir, Turkey Case

NASA Astrophysics Data System (ADS)

TÜdeş, Şule; Kumlu, Kadriye Burcu Yavuz

2017-10-01

Each stage of the planning process should be based on the natural resource protection, in the sense of environmental sensitive and sustainable urban planning. Values, which are vital for the continuity of the life in the Earth, as soil, water, forest etc. should be protected from the undesired effects of the pollution and the other effects caused by the high urbanization levels. In this context, GIS-MCDM based solid waste landfill site selection is applied for Izmir, Turkey, where is a significant attraction place for tourism. As Multi criteria Decision Making (MCDM) technique, Analytical Hierarchy Process (AHP) is used. In this study, geological, tectonically and hydrological data, as well as agricultural land use, slope, distance to the settlement areas and the highways are used as inputs for AHP analysis. In the analysis stage, those inputs are rated and weighted. The weighted criteria are evaluated via GIS, by using weighted overlay tool. Therefore, an upper-scale analysis is conducted and a map, which shows the alternative places for the solid waste landfill sites, considering the environmental protection and evaluated in the context of environmental and urban criteria, are obtained.

A new highly sensitive and specific real-time PCR assay targeting the malate dehydrogenase gene of Kingella kingae and application to 201 pediatric clinical specimens.

PubMed

Houmami, Nawal El; Durand, Guillaume André; Bzdrenga, Janek; Darmon, Anne; Minodier, Philippe; Seligmann, Hervé; Raoult, Didier; Fournier, Pierre-Edouard

2018-06-06

Kingella kingae is a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium by culture and broad-range 16S rRNA gene polymerase chain reaction (PCR) assays from clinical specimens have proven unsatisfactory and were gradually let out for the benefit of specific real-time PCR tests targeting the groEL gene and RTX locus of K. kingae by the late 2000s. However, recent studies showed that real-time PCR (RT-PCR) assays targeting the Kingella sp. RTX locus that are currently available for the diagnosis of K. kingae infection lack of specificity because they could not distinguish between K. kingae and the recently described K. negevensis species. Furthermore, in silico analysis of the groEL gene from a large collection of 45 K. kingae strains showed that primers and probes from K. kingae groEL -based RT-PCR assays display a few mismatches with K. kingae groEL variations that may result in a decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative to groEL - and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, a K. kingae -specific RT-PCR assay targeting the malate dehydrogenase ( mdh ) gene was developed for predicting no mismatch against 18 variants of the K. kingae mdh gene from 20 distinct sequences types of K. kingae This novel K. kingae -specific RT-PCR assay demonstrated a high specificity and sensitivity and was successfully used to diagnose K. kingae infections and carriage in 104 clinical specimens from children aged between 7 months and 7 years old. Copyright © 2018 American Society for Microbiology.

Site-targeted complement inhibition by a complement receptor 2-conjugated inhibitor (mTT30) ameliorates post-injury neuropathology in mouse brains.

PubMed

Rich, Megan C; Keene, Chesleigh N; Neher, Miriam D; Johnson, Krista; Yu, Zhao-Xue; Ganivet, Antoine; Holers, V Michael; Stahel, Philip F

2016-03-23

Intracerebral complement activation after severe traumatic brain injury (TBI) leads to a cascade of neuroinflammatory pathological sequelae that propagate host-mediated secondary brain injury and adverse outcomes. There are currently no specific pharmacological agents on the market to prevent or mitigate the development of secondary cerebral insults after TBI. A novel chimeric CR2-fH compound (mTT30) provides targeted inhibition of the alternative complement pathway at the site of tissue injury. This experimental study was designed to test the neuroprotective effects of mTT30 in a mouse model of closed head injury. The administration of 500 μg mTT30 i.v. at 1 h, 4 h and 24 h after head injury attenuated complement C3 deposition in injured brains, reduced the extent of neuronal cell death, and decreased post-injury microglial activation, compared to vehicle-injected placebo controls. These data imply that site-targeted alternative pathway complement inhibition may represent a new promising therapeutic avenue for the future management of severe TBI. Copyright © 2016. Published by Elsevier Ireland Ltd.

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-07-01

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

Direction, site and the muzzle target distance of bullet in the head and neck at close range as an indication of suicide or homicide.

PubMed

Suwanjutha, T

1988-05-01

Direction, site and muzzle target distance can indicate suicide or homicide. This conclusion can be drawn from autopsies of 57 cases of suicide and 68 cases of homicide by handgun fired at close range to the head and neck together with going to the crimescene in some cases. This study was carried out in Bangkok during the period from January 1983 to January 1986. In order to determine whether it was suicide or homicide, the path of the bullet, the site, the muzzle target distance must be considered. The angle of the bullet would be either elevated (from below upward), horizontal or an angle of depression (from above downward). For suicide, the direction of the bullet should be at an angle of elevation in the majority of cases. The position of the handgun in relation to the head in suicide was most often in tight contact and near contact. For homicide, the direction of the bullet should be horizontal in most cases. The bullet was at close range in the majority of the cases. There are 8 common sites for suicide and homicide and 10 different sites in the case of homicide which are at neck, left cheek, left aural region, lip, left occipital area orbit, chin, left eyebrow, submental and nose.

Myosin‑II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis