WorldWideScience

Sample records for targeted glycoproteomic identification

  1. Microbial glycoproteomics

    DEFF Research Database (Denmark)

    Halim, Adnan; Anonsen, Jan Haug

    2017-01-01

    Mass spectrometry-based "-omics" technologies are important tools for global and detailed mapping of post-translational modifications. Protein glycosylation is an abundant and important post translational modification widespread throughout all domains of life. Characterization of glycoproteins...... and research in this area is rapidly accelerating. Here, we review recent developments in glycoproteomic technologies with a special focus on microbial protein glycosylation....

  2. pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.

    Science.gov (United States)

    Liu, Ming-Qi; Zeng, Wen-Feng; Fang, Pan; Cao, Wei-Qian; Liu, Chao; Yan, Guo-Quan; Zhang, Yang; Peng, Chao; Wu, Jian-Qiang; Zhang, Xiao-Jin; Tu, Hui-Jun; Chi, Hao; Sun, Rui-Xiang; Cao, Yong; Dong, Meng-Qiu; Jiang, Bi-Yun; Huang, Jiang-Ming; Shen, Hua-Li; Wong, Catherine C L; He, Si-Min; Yang, Peng-Yuan

    2017-09-05

    The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15 N/ 13 C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

  3. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

    DEFF Research Database (Denmark)

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick

    2015-01-01

    developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied...... the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines....... However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery...

  4. Mining the O-glycoproteome using zinc-finger nuclease-glycoengineered SimpleCell lines

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Vakhrushev, Sergey Y; Vester-Christensen, Malene B

    2011-01-01

    Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN target...

  5. N-linked (N-) glycoproteomics of urinary exosomes. [Corrected].

    Science.gov (United States)

    Saraswat, Mayank; Joenväära, Sakari; Musante, Luca; Peltoniemi, Hannu; Holthofer, Harry; Renkonen, Risto

    2015-02-01

    Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes. © 2015 by The

  6. Glycoproteomic analysis of serum from patients with gastric precancerous lesions

    DEFF Research Database (Denmark)

    Gomes, Catarina; Almeida, Andreia; Ferreira, José Alexandre

    2013-01-01

    Gastric cancer is preceded by a carcinogenesis pathway that includes gastritis caused by Helicobacter pylori infection, chronic atrophic gastritis that may progress to intestinal metaplasia (IM), dysplasia, and ultimately gastric carcinoma of the more common intestinal subtype. The identification...... of glycosylation changes in circulating serum proteins in patients with precursor lesions of gastric cancer is of high interest and represents a source of putative new biomarkers for early diagnosis and intervention. This study applies a glycoproteomic approach to identify altered glycoproteins expressing...... the simple mucin-type carbohydrate antigens T and STn in the serum of patients with gastritis, IM (complete and incomplete subtypes), and control healthy individuals. The immunohistochemistry analysis of the gastric mucosa of these patients showed expression of T and STn antigens in gastric lesions, with STn...

  7. Advances in mass spectrometry driven O-glycoproteomics

    DEFF Research Database (Denmark)

    Levery, Steven B; Steentoft, Catharina; Halim, Adnan

    2015-01-01

    is becoming available as well. SCOPE OF REVIEW: Here we discuss the challenges of analysis of O-glycans and new O-glycoproteomics strategies focusing on O-GalNAc and O-Man glycoproteomes. MAJOR CONCLUSIONS: A variety of strategies are now available for proteome-wide analysis of O-glycosylation sites enabling...

  8. Camouflage, detection and identification of moving targets.

    Science.gov (United States)

    Hall, Joanna R; Cuthill, Innes C; Baddeley, Roland; Shohet, Adam J; Scott-Samuel, Nicholas E

    2013-05-07

    Nearly all research on camouflage has investigated its effectiveness for concealing stationary objects. However, animals have to move, and patterns that only work when the subject is static will heavily constrain behaviour. We investigated the effects of different camouflages on the three stages of predation-detection, identification and capture-in a computer-based task with humans. An initial experiment tested seven camouflage strategies on static stimuli. In line with previous literature, background-matching and disruptive patterns were found to be most successful. Experiment 2 showed that if stimuli move, an isolated moving object on a stationary background cannot avoid detection or capture regardless of the type of camouflage. Experiment 3 used an identification task and showed that while camouflage is unable to slow detection or capture, camouflaged targets are harder to identify than uncamouflaged targets when similar background objects are present. The specific details of the camouflage patterns have little impact on this effect. If one has to move, camouflage cannot impede detection; but if one is surrounded by similar targets (e.g. other animals in a herd, or moving background distractors), then camouflage can slow identification. Despite previous assumptions, motion does not entirely 'break' camouflage.

  9. Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

    Science.gov (United States)

    Sun, Bingyun; Hood, Leroy

    2014-06-06

    The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

  10. Nanorobot architecture for medical target identification

    International Nuclear Information System (INIS)

    Cavalcanti, Adriano; Shirinzadeh, Bijan; Freita, Robert A Jr; Hogg, Tad

    2008-01-01

    This work has an innovative approach for the development of nanorobots with sensors for medicine. The nanorobots operate in a virtual environment comparing random, thermal and chemical control techniques. The nanorobot architecture model has nanobioelectronics as the basis for manufacturing integrated system devices with embedded nanobiosensors and actuators, which facilitates its application for medical target identification and drug delivery. The nanorobot interaction with the described workspace shows how time actuation is improved based on sensor capabilities. Therefore, our work addresses the control and the architecture design for developing practical molecular machines. Advances in nanotechnology are enabling manufacturing nanosensors and actuators through nanobioelectronics and biologically inspired devices. Analysis of integrated system modeling is one important aspect for supporting nanotechnology in the fast development towards one of the most challenging new fields of science: molecular machines. The use of 3D simulation can provide interactive tools for addressing nanorobot choices on sensing, hardware architecture design, manufacturing approaches, and control methodology investigation

  11. Nanorobot architecture for medical target identification

    Energy Technology Data Exchange (ETDEWEB)

    Cavalcanti, Adriano [CAN Center for Automation in Nanobiotech, Melbourne VIC 3168 (Australia); Shirinzadeh, Bijan [Robotics and Mechatronics Research Laboratory, Department of Mechanical Engineering, Monash University, Clayton, Melbourne VIC 3800 (Australia); Freita, Robert A Jr [Institute for Molecular Manufacturing, Pilot Hill, CA 95664 (United States); Hogg, Tad [Hewlett-Packard Laboratories, Palo Alto, CA 94304 (United States)

    2008-01-09

    This work has an innovative approach for the development of nanorobots with sensors for medicine. The nanorobots operate in a virtual environment comparing random, thermal and chemical control techniques. The nanorobot architecture model has nanobioelectronics as the basis for manufacturing integrated system devices with embedded nanobiosensors and actuators, which facilitates its application for medical target identification and drug delivery. The nanorobot interaction with the described workspace shows how time actuation is improved based on sensor capabilities. Therefore, our work addresses the control and the architecture design for developing practical molecular machines. Advances in nanotechnology are enabling manufacturing nanosensors and actuators through nanobioelectronics and biologically inspired devices. Analysis of integrated system modeling is one important aspect for supporting nanotechnology in the fast development towards one of the most challenging new fields of science: molecular machines. The use of 3D simulation can provide interactive tools for addressing nanorobot choices on sensing, hardware architecture design, manufacturing approaches, and control methodology investigation.

  12. Glycoproteomic profile in wine: a 'sweet' molecular renaissance

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Antonacci, Donato; Larsen, Martin Røssel

    2010-01-01

    strategy in combination with tandem mass spectrometry was performed in order to analyze the glycoproteome of white wine. A total of 28 glycoproteins and 44 glycosylation sites were identified. The identified glycoproteins were from grape and yeast origin. In particular, several glycoproteins derived from...... grape, like invertase and pathogenesis-related (PR) proteins, and from the yeast, were found after the vinification process. Bioinformatic analysis revealed sequence similarity between the identified grape glycoproteins and known plant allergens. This study is an important step forward in order...

  13. Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells

    DEFF Research Database (Denmark)

    Vakhrushev, Sergey Y; Steentoft, Catharina; Vester-Christensen, Malene B

    2013-01-01

    Characterizing protein GalNAc-type O-glycosylation has long been a major challenge, and as a result, our understanding of this glycoproteome is particularly poor. Recently, we presented a novel strategy for high throughput identification of O-GalNAc glycosites using zinc finger nuclease gene......-engineered "SimpleCell" lines producing homogeneous truncated O-glycosylation. Total lysates of cells were trypsinized and subjected to lectin affinity chromatography enrichment, followed by identification of GalNAc O-glycopeptides by nLC-MS/MS, with electron transfer dissociation employed to specify sites of O...... of O-GalNAc attached to tyrosine. Furthermore, we uncovered ≈ 220 O-glycosites wherein the peptides were clearly identified, but the glycosites could not be unambiguously assigned to specific positions. The improved strategy should greatly facilitate high throughput characterization of the human GalNAc...

  14. Drug target identification in protozoan parasites.

    Science.gov (United States)

    Müller, Joachim; Hemphill, Andrew

    2016-08-01

    Despite the fact that diseases caused by protozoan parasites represent serious challenges for public health, animal production and welfare, only a limited panel of drugs has been marketed for clinical applications. Herein, the authors investigate two strategies, namely whole organism screening and target-based drug design. The present pharmacopoeia has resulted from whole organism screening, and the mode of action and targets of selected drugs are discussed. However, the more recent extensive genome sequencing efforts and the development of dry and wet lab genomics and proteomics that allow high-throughput screening of interactions between micromolecules and recombinant proteins has resulted in target-based drug design as the predominant focus in anti-parasitic drug development. Selected examples of target-based drug design studies are presented, and calcium-dependent protein kinases, important drug targets in apicomplexan parasites, are discussed in more detail. Despite the enormous efforts in target-based drug development, this approach has not yet generated market-ready antiprotozoal drugs. However, whole-organism screening approaches, comprising of both in vitro and in vivo investigations, should not be disregarded. The repurposing of already approved and marketed drugs could be a suitable strategy to avoid fastidious approval procedures, especially in the case of neglected or veterinary parasitoses.

  15. OPTICAL correlation identification technology applied in underwater laser imaging target identification

    Science.gov (United States)

    Yao, Guang-tao; Zhang, Xiao-hui; Ge, Wei-long

    2012-01-01

    The underwater laser imaging detection is an effective method of detecting short distance target underwater as an important complement of sonar detection. With the development of underwater laser imaging technology and underwater vehicle technology, the underwater automatic target identification has gotten more and more attention, and is a research difficulty in the area of underwater optical imaging information processing. Today, underwater automatic target identification based on optical imaging is usually realized with the method of digital circuit software programming. The algorithm realization and control of this method is very flexible. However, the optical imaging information is 2D image even 3D image, the amount of imaging processing information is abundant, so the electronic hardware with pure digital algorithm will need long identification time and is hard to meet the demands of real-time identification. If adopt computer parallel processing, the identification speed can be improved, but it will increase complexity, size and power consumption. This paper attempts to apply optical correlation identification technology to realize underwater automatic target identification. The optics correlation identification technology utilizes the Fourier transform characteristic of Fourier lens which can accomplish Fourier transform of image information in the level of nanosecond, and optical space interconnection calculation has the features of parallel, high speed, large capacity and high resolution, combines the flexibility of calculation and control of digital circuit method to realize optoelectronic hybrid identification mode. We reduce theoretical formulation of correlation identification and analyze the principle of optical correlation identification, and write MATLAB simulation program. We adopt single frame image obtained in underwater range gating laser imaging to identify, and through identifying and locating the different positions of target, we can improve

  16. Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics

    DEFF Research Database (Denmark)

    Mysling, Simon; Palmisano, Giuseppe; Højrup, Peter

    2010-01-01

    Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE...

  17. Recent Advances in Target Characterization and Identification by Photoaffinity Probes

    Directory of Open Access Journals (Sweden)

    Sang J. Chung

    2013-08-01

    Full Text Available Target identification of biologically active molecules such as natural products, synthetic small molecules, peptides, and oligonucleotides mainly relies on affinity chromatography, activity-based probes, or photoaffinity labeling (PAL. Amongst them, activity-based probes and PAL have offered great advantages in target identification technology due to their ability to form covalent bonds with the corresponding targets. Activity-based probe technology mainly relies on the chemical reactivity of the target proteins, thereby limiting the majority of the biological targets to enzymes or proteins which display reactive residues at the probe-binding site. In general, the probes should bear a reactive moiety such as an epoxide, a Michael acceptor, or a reactive alkyl halide in their structures. On the other hand, photoaffinity probes (PAPs are composed of a target-specific ligand and a photoactivatable functional group. When bound to the corresponding target proteins and activated with wavelength-specific light, PAPs generate highly reactive chemical species that covalently cross-link proximal amino acid residues. This process is better known as PAL and is widely employed to identify cellular targets of biologically active molecules. This review highlights recent advances in target identification by PAL, with a focus on the structure and chemistry of the photoaffinity probes developed in the recent decade, coupled to the target proteins identified using these probes.

  18. [Thalidomide teratogenicity and its direct target identification].

    Science.gov (United States)

    Ito, Takumi; Ando, Hideki; Handa, Hiroshi

    2015-01-01

    Half a century ago, thalidomide was developed as a sedative drug and was wildly used over 40 countries. However the drug has serious birth defects such as amelia and phocomelia. Now thalidomide is regarded as a clinically effective drug and used for the treatment of multiple myeloma under strict controls. The direct target of thalidomide had been a long-standing question. We identified cereblon as a primary direct target protein for thalidomide teratogenicity using new affinity bead technology in 2010. In this review, we introduce an overview of thalidomide teratogenicity, a story about how we identified cereblon, and recent advances in cereblon studies.

  19. Identification of synaptic targets of Drosophila pumilio.

    Directory of Open Access Journals (Sweden)

    Gengxin Chen

    2008-02-01

    Full Text Available Drosophila Pumilio (Pum protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3'UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3'UTR of discs large (dlg1, the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB, which is an anatomical site of memory storage.

  20. An integrated sample pretreatment platform for quantitative N-glycoproteome analysis with combination of on-line glycopeptide enrichment, deglycosylation and dimethyl labeling

    Energy Technology Data Exchange (ETDEWEB)

    Weng, Yejing; Qu, Yanyan; Jiang, Hao; Wu, Qi [National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); University of the Chinese Academy of Sciences, Beijing 100039 (China); Zhang, Lihua, E-mail: lihuazhang@dicp.ac.cn [National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Yuan, Huiming [National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); Zhou, Yuan [National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China); University of the Chinese Academy of Sciences, Beijing 100039 (China); Zhang, Xiaodan; Zhang, Yukui [National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 (China)

    2014-06-23

    Highlights: • An integrated platform for quantitative N-glycoproteome analysis was established. • On-line enrichment, deglycosylation and labeling could be achieved within 160 min. • A N{sub 2}-assisted interface was applied to improve the compatibility of the platform. • The platform exhibited improved quantification accuracy, precision and throughput. - Abstract: Relative quantification of N-glycoproteomes shows great promise for the discovery of candidate biomarkers and therapeutic targets. The traditional protocol for quantitative analysis of glycoproteomes is usually off-line performed, and suffers from long sample preparation time, and the risk of sample loss or contamination due to manual manipulation. In this study, a novel integrated sample preparation platform for quantitative N-glycoproteome analysis was established, with combination of online N-glycopeptide capture by a HILIC column, sample buffer exchange by a N{sub 2}-assisted HILIC–RPLC interface, deglycosylation by a hydrophilic PNGase F immobilized enzymatic reactor (hIMER) and solid dimethyl labeling on a C18 precolumn. To evaluate the performance of such a platform, two equal aliquots of immunoglobulin G (IgG) digests were sequentially pretreated, followed by MALDI-TOF MS analysis. The signal intensity ratio of heavy/light (H/L) labeled deglycosylated peptides with the equal aliquots was 1.00 (RSD = 6.2%, n = 3), much better than those obtained by the offline protocol, with H/L ratio as 0.76 (RSD = 11.6%, n = 3). Additionally, the total on-line sample preparation time was greatly shortened to 160 min, much faster than that of offline approach (24 h). Furthermore, such an integrated pretreatment platform was successfully applied to analyze the two kinds of hepatocarcinoma ascites syngeneic cell lines with high (Hca-F) and low (Hca-P) lymph node metastasis rates. For H/L labeled Hca-P lysates with the equal aliquots, 99.6% of log 2 ratios (H/L) of quantified glycopeptides ranged from −1

  1. Snapshot spectral and polarimetric imaging; target identification with multispectral video

    Science.gov (United States)

    Bartlett, Brent D.; Rodriguez, Mikel D.

    2013-05-01

    As the number of pixels continue to grow in consumer and scientific imaging devices, it has become feasible to collect the incident light field. In this paper, an imaging device developed around light field imaging is used to collect multispectral and polarimetric imagery in a snapshot fashion. The sensor is described and a video data set is shown highlighting the advantage of snapshot spectral imaging. Several novel computer vision approaches are applied to the video cubes to perform scene characterization and target identification. It is shown how the addition of spectral and polarimetric data to the video stream allows for multi-target identification and tracking not possible with traditional RGB video collection.

  2. Detection and identification of human targets in radar data

    Science.gov (United States)

    Gürbüz, Sevgi Z.; Melvin, William L.; Williams, Douglas B.

    2007-04-01

    Radar offers unique advantages over other sensors, such as visual or seismic sensors, for human target detection. Many situations, especially military applications, prevent the placement of video cameras or implantment seismic sensors in the area being observed, because of security or other threats. However, radar can operate far away from potential targets, and functions during daytime as well as nighttime, in virtually all weather conditions. In this paper, we examine the problem of human target detection and identification using single-channel, airborne, synthetic aperture radar (SAR). Human targets are differentiated from other detected slow-moving targets by analyzing the spectrogram of each potential target. Human spectrograms are unique, and can be used not just to identify targets as human, but also to determine features about the human target being observed, such as size, gender, action, and speed. A 12-point human model, together with kinematic equations of motion for each body part, is used to calculate the expected target return and spectrogram. A MATLAB simulation environment is developed including ground clutter, human and non-human targets for the testing of spectrogram-based detection and identification algorithms. Simulations show that spectrograms have some ability to detect and identify human targets in low noise. An example gender discrimination system correctly detected 83.97% of males and 91.11% of females. The problems and limitations of spectrogram-based methods in high clutter environments are discussed. The SNR loss inherent to spectrogram-based methods is quantified. An alternate detection and identification method that will be used as a basis for future work is proposed.

  3. Environmental Aspects, Objectives and Targets Identification Process

    International Nuclear Information System (INIS)

    Green, R.

    2002-01-01

    The purpose of this report is to document the environmental aspects and associated environmental impacts of the Bechtel SAIC Company (BSC) scope of work, evaluate the significance of those environmental aspects based on established criteria, and establish environmental objectives and targets for specific environmental aspects. This report is intended to be used by environmental staff in the evaluation of BSC work packages during the annual risk-based planning process. This report shall be fully reviewed and revised annually during the annual work planning process to reflect changes in BSC operations, facilities, and scope of work. Planned BSC work will be evaluated to determine if the work is covered by a previously defined activity, product or service (see Table 2); if work activities require redefinition or addition of a new activity; and if the significant evaluation for each environment aspect is still valid based on scope of planned work. New workscope initiated during the fiscal year through the Baseline Change Proposal process (i.e., not as part of the annual work plan) also will be reviewed for new environmental aspects and determination of whether the new workscope would change the significance rating of any environmental aspect. If a new environmental aspect is identified in a new work activity, product, or service but the aspect is not determined to be significant (see Section 4), then this report can be changed through an interim change notice (ICN). This report can be changed five times through an ICN before a full revision is required. However, if new workscope causes an environmental aspect to be graded as significant using the evaluation process in Section 4, this report shall be revised though a full review and revision

  4. Microarray-Based Identification of Transcription Factor Target Genes

    NARCIS (Netherlands)

    Gorte, M.; Horstman, A.; Page, R.B.; Heidstra, R.; Stromberg, A.; Boutilier, K.A.

    2011-01-01

    Microarray analysis is widely used to identify transcriptional changes associated with genetic perturbation or signaling events. Here we describe its application in the identification of plant transcription factor target genes with emphasis on the design of suitable DNA constructs for controlling TF

  5. Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry.

    Science.gov (United States)

    Zhang, Yong; Xie, Xinfang; Zhao, Xinyuan; Tian, Fang; Lv, Jicheng; Ying, Wantao; Qian, Xiaohong

    2018-01-06

    Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took

  6. Hadron identification in a fixed target experiment at the SSC

    International Nuclear Information System (INIS)

    Nelson, K.S.; Cox, B.

    1993-01-01

    This article presents the design criteria and expected performance of a hadron identification system in a fixed target experiment at the SSC. The proposed SFT spectrometer will be used as a model for the discussion. Two primary uses of hadron identification is a B physics experiment are flavor tagging and the rejection of background due to particle reflections in the reconstruction of exclusive decay modes. In the first case it is shown that use of kaons can increase substantially the number of events which can be tagged. In the latter case, decays in which particles are mis-identified can form a background to a desired decay mode

  7. Target identification using Zernike moments and neural networks

    Science.gov (United States)

    Azimi-Sadjadi, Mahmood R.; Jamshidi, Arta A.; Nevis, Andrew J.

    2001-10-01

    The development of an underwater target identification algorithm capable of identifying various types of underwater targets, such as mines, under different environmental conditions pose many technical problems. Some of the contributing factors are: targets have diverse sizes, shapes and reflectivity properties. Target emplacement environment is variable; targets may be proud or partially buried. Environmental properties vary significantly from one location to another. Bottom features such as sand, rocks, corals, and vegetation can conceal a target whether it is partially buried or proud. Competing clutter with responses that closely resemble those of the targets may lead to false positives. All the problems mentioned above contribute to overly difficult and challenging conditions that could lead to unreliable algorithm performance with existing methods. In this paper, we developed and tested a shape-dependent feature extraction scheme that provides features invariant to rotation, size scaling and translation; properties that are extremely useful for any target classification problem. The developed schemes were tested on an electro-optical imagery data set collected under different environmental conditions with variable background, range and target types. The electro-optic data set was collected using a Laser Line Scan (LLS) sensor by the Coastal Systems Station (CSS), located in Panama City, Florida. The performance of the developed scheme and its robustness to distortion, rotation, scaling and translation was also studied.

  8. Integrated proteomic and N-glycoproteomic analyses of doxorubicin sensitive and resistant ovarian cancer cells reveal glycoprotein alteration in protein abundance and glycosylation

    Science.gov (United States)

    Hou, Junjie; Zhang, Chengqian; Xue, Peng; Wang, Jifeng; Chen, Xiulan; Guo, Xiaojing; Yang, Fuquan

    2017-01-01

    Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and

  9. Screening Technologies for Target Identification in Pancreatic Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Michl, Patrick, E-mail: michlp@med.uni-marburg.de; Ripka, Stefanie; Gress, Thomas; Buchholz, Malte [Department of Gastroenterology and Endocrinology, University Hospital, Philipps-University Marburg, Baldinger Strasse, D-35043 Marburg (Germany)

    2010-12-29

    Pancreatic cancer exhibits an extraordinarily high level of resistance to almost any kind of systemic therapy evaluated in clinical trials so far. Therefore, the identification of novel therapeutic targets is urgently required. High-throughput screens have emerged as an important tool to identify putative targets for diagnosis and therapy in an unbiased manner. More than a decade ago, microarray technology was introduced to identify differentially expressed genes in pancreatic cancer as compared to normal pancreas, chronic pancreatitis and other cancer types located in close proximity to the pancreas. In addition, proteomic screens have facilitated the identification of differentially secreted proteins in body fluids of pancreatic cancer patients, serving as possible biomarkers. Recently, RNA interference-based loss-of-function screens have been used to identify functionally relevant genes, whose knock-down has impact on pancreatic cancer cell viability, thereby representing potential new targets for therapeutic intervention. This review summarizes recent results of transcriptional, proteomic and functional screens in pancreatic cancer and discusses potentials and limitations of the respective technologies as well as their impact on future therapeutic developments.

  10. Screening Technologies for Target Identification in Pancreatic Cancer

    International Nuclear Information System (INIS)

    Michl, Patrick; Ripka, Stefanie; Gress, Thomas; Buchholz, Malte

    2010-01-01

    Pancreatic cancer exhibits an extraordinarily high level of resistance to almost any kind of systemic therapy evaluated in clinical trials so far. Therefore, the identification of novel therapeutic targets is urgently required. High-throughput screens have emerged as an important tool to identify putative targets for diagnosis and therapy in an unbiased manner. More than a decade ago, microarray technology was introduced to identify differentially expressed genes in pancreatic cancer as compared to normal pancreas, chronic pancreatitis and other cancer types located in close proximity to the pancreas. In addition, proteomic screens have facilitated the identification of differentially secreted proteins in body fluids of pancreatic cancer patients, serving as possible biomarkers. Recently, RNA interference-based loss-of-function screens have been used to identify functionally relevant genes, whose knock-down has impact on pancreatic cancer cell viability, thereby representing potential new targets for therapeutic intervention. This review summarizes recent results of transcriptional, proteomic and functional screens in pancreatic cancer and discusses potentials and limitations of the respective technologies as well as their impact on future therapeutic developments

  11. Atlas-based identification of targets for functional radiosurgery

    International Nuclear Information System (INIS)

    Stancanello, Joseph; Romanelli, Pantaleo; Modugno, Nicola; Cerveri, Pietro; Ferrigno, Giancarlo; Uggeri, Fulvio; Cantore, Giampaolo

    2006-01-01

    Functional disorders of the brain, such as Parkinson's disease, dystonia, epilepsy, and neuropathic pain, may exhibit poor response to medical therapy. In such cases, surgical intervention may become necessary. Modern surgical approaches to such disorders include radio-frequency lesioning and deep brain stimulation (DBS). The subthalamic nucleus (STN) is one of the most useful stereotactic targets available: STN DBS is known to induce substantial improvement in patients with end-stage Parkinson's disease. Other targets include the Globus Pallidus pars interna (GPi) for dystonia and Parkinson's disease, and the centromedian nucleus of the thalamus (CMN) for neuropathic pain. Radiosurgery is an attractive noninvasive alternative to treat some functional brain disorders. The main technical limitation to radiosurgery is that the target can be selected only on the basis of magnetic resonance anatomy without electrophysiological confirmation. The aim of this work is to provide a method for the correct atlas-based identification of the target to be used in functional neurosurgery treatment planning. The coordinates of STN, CMN, and GPi were identified in the Talairach and Tournoux atlas and transformed to the corresponding regions of the Montreal Neurological Institute (MNI) electronic atlas. Binary masks describing the target nuclei were created. The MNI electronic atlas was deformed onto the patient magnetic resonance imaging-T1 scan by applying an affine transformation followed by a local nonrigid registration. The first transformation was based on normalized cross correlation and the second on optimization of a two-part objective function consisting of similarity criteria and weighted regularization. The obtained deformation field was then applied to the target masks. The minimum distance between the surface of an implanted electrode and the surface of the deformed mask was calculated. The validation of the method consisted of comparing the electrode-mask distance to

  12. Glycomics and glycoproteomics focused on aging and age-related diseases--Glycans as a potential biomarker for physiological alterations.

    Science.gov (United States)

    Miura, Yuri; Endo, Tamao

    2016-08-01

    Since glycosylation depends on glycosyltransferases, glycosidases, and sugar nucleotide donors, it is susceptible to the changes associated with physiological and pathological conditions. Therefore, alterations in glycan structures may be good targets and biomarkers for monitoring health conditions. Since human aging and longevity are affected by genetic and environmental factors such as diseases, lifestyle, and social factors, a scale that reflects various environmental factors is required in the study of human aging and longevity. We herein focus on glycosylation changes elucidated by glycomic and glycoproteomic studies on aging, longevity, and age-related diseases including cognitive impairment, diabetes mellitus, and frailty. We also consider the potential of glycan structures as biomarkers and/or targets for monitoring physiological and pathophysiological changes. Glycan structures are altered in age-related diseases. These glycans and glycoproteins may be involved in the pathophysiology of these diseases and, thus, be useful diagnostic markers. Age-dependent changes in N-glycans have been reported previously in cohort studies, and characteristic N-glycans in extreme longevity have been proposed. These findings may lead to a deeper understanding of the mechanisms underlying aging as well as the factors influencing longevity. Alterations in glycosylation may be good targets and biomarkers for monitoring health conditions, and be applicable to studies on age-related diseases and healthy aging. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Genome-wide identification of direct HBx genomic targets

    KAUST Repository

    Guerrieri, Francesca

    2017-02-17

    Background The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV. Results ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication. Conclusions Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

  14. A comparative glycoproteome study of developing endosperm in the hexose-deficient miniature1 (mn1 seed mutant and its wild type Mn1 in maize

    Directory of Open Access Journals (Sweden)

    Cecilia eSilva-Sanchez

    2014-02-01

    Full Text Available In maize developing seeds, transfer cells are prominently located at the basal endosperm transfer layer (BETL. As the first filial cell layer, BETL is a gateway to sugars, nutrients and water from mother plant; and anchor of numerous functions such as sucrose turnover, auxin and cytokinin biosynthesis/accumulation, energy metabolism, defense response, and signaling between maternal and filial generations. Previous studies showed that basal developing endosperms of miniature1 (mn1 mutant seeds lacking the Mn1-encoded cell wall invertase II, are also deficient for hexose. Given the role of glucose as one of the key sugars in protein glycosylation and proper protein folding; we performed a comparative large scale glycoproteome profiling of total proteins of these two genotypes (mn1 mutant vs Mn1 wild type using 2D gel electrophoresis and glycosylation/total protein staining, followed by image analysis. Protein identification was done by LC-MS/MS. A total of 413 spots were detected; from which, 113 spots matched between the two genotypes. Of these, 45 showed > 20% decrease/increase in glycosylation level and were selected for protein identification. A large number of identified proteins showed decreased glycosylation levels in mn1 developing endosperms as compared to the Mn1. Functional classification of proteins, showed mainly of post-translational modification, protein turnover, chaperone activities, carbohydrate and amino acid biosynthesis / transport, and cell wall biosynthesis. These proteins and activities were related to endoplasmic reticulum (ER stress and unfolded protein response (UPR as a result of the low glycolsylation levels of the mutant proteins. Overall, these results provide for the first time a global glycoproteome profile of maize BETL-enriched basal endosperm to better understand their role in seed development in maize.

  15. Probing the O-glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery

    DEFF Research Database (Denmark)

    Vieira Campos, Diana Alexandra; Freitas, Daniela; Gomes, Joana

    2015-01-01

    biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "SimpleCell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SC lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses...... at least partially truncated O-glycans. Overall we identified 499 O-glycoproteins and 1,236 O-glycosites in gastric cancer SCs, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer...... with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to demonstrate that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set...

  16. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Vakhrushev, Sergey; Joshi, Hiren Jitendra

    2013-01-01

    Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O-glycosylation is hig......Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O......-glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc-transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O...

  17. Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

    DEFF Research Database (Denmark)

    Vester-Christensen, Malene B; Halim, Adnan; Joshi, Hiren Jitendra

    2013-01-01

    The metazoan O-mannose (O-Man) glycoproteome is largely unknown. It has been shown that up to 30% of brain O-glycans are of the O-Man type, but essentially only alpha-dystroglycan (α-DG) of the dystrophin-glycoprotein complex is well characterized as an O-Man glycoprotein. Defects in O-Man...... glycosylation underlie congenital muscular dystrophies and considerable efforts have been devoted to explore this O-glycoproteome without much success. Here, we used our SimpleCell strategy using nuclease-mediated gene editing of a human cell line (MDA-MB-231) to reduce the structural heterogeneity of O-Man...... glycans and to probe the O-Man glycoproteome. In this breast cancer cell line we found that O-Man glycosylation is primarily found on cadherins and plexins on β-strands in extracellular cadherin and Ig-like, plexin and transcription factor domains. The positions and evolutionary conservation of O-Man...

  18. Identification of apoptosis-related PLZF target genes

    International Nuclear Information System (INIS)

    Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes; Campillo, Jose Antonio; Parrado, Antonio

    2007-01-01

    The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localization is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression

  19. Identification of acoustic targets off Angola using General ...

    African Journals Online (AJOL)

    % of the animal protein in the diet of the coastal population. The primary means of estimating stock abundance is through acoustic surveys, a method that is sensitive to identification of echo traces. This paper describes commonly encountered ...

  20. Utilizing random Forest QSAR models with optimized parameters for target identification and its application to target-fishing server.

    Science.gov (United States)

    Lee, Kyoungyeul; Lee, Minho; Kim, Dongsup

    2017-12-28

    The identification of target molecules is important for understanding the mechanism of "target deconvolution" in phenotypic screening and "polypharmacology" of drugs. Because conventional methods of identifying targets require time and cost, in-silico target identification has been considered an alternative solution. One of the well-known in-silico methods of identifying targets involves structure activity relationships (SARs). SARs have advantages such as low computational cost and high feasibility; however, the data dependency in the SAR approach causes imbalance of active data and ambiguity of inactive data throughout targets. We developed a ligand-based virtual screening model comprising 1121 target SAR models built using a random forest algorithm. The performance of each target model was tested by employing the ROC curve and the mean score using an internal five-fold cross validation. Moreover, recall rates for top-k targets were calculated to assess the performance of target ranking. A benchmark model using an optimized sampling method and parameters was examined via external validation set. The result shows recall rates of 67.6% and 73.9% for top-11 (1% of the total targets) and top-33, respectively. We provide a website for users to search the top-k targets for query ligands available publicly at http://rfqsar.kaist.ac.kr . The target models that we built can be used for both predicting the activity of ligands toward each target and ranking candidate targets for a query ligand using a unified scoring scheme. The scores are additionally fitted to the probability so that users can estimate how likely a ligand-target interaction is active. The user interface of our web site is user friendly and intuitive, offering useful information and cross references.

  1. A Comprehensive, Open-source Platform for Mass Spectrometry-based Glycoproteomics Data Analysis.

    Science.gov (United States)

    Liu, Gang; Cheng, Kai; Lo, Chi Y; Li, Jun; Qu, Jun; Neelamegham, Sriram

    2017-11-01

    Glycosylation is among the most abundant and diverse protein post-translational modifications (PTMs) identified to date. The structural analysis of this PTM is challenging because of the diverse monosaccharides which are not conserved among organisms, the branched nature of glycans, their isomeric structures, and heterogeneity in the glycan distribution at a given site. Glycoproteomics experiments have adopted the traditional high-throughput LC-MS n proteomics workflow to analyze site-specific glycosylation. However, comprehensive computational platforms for data analyses are scarce. To address this limitation, we present a comprehensive, open-source, modular software for glycoproteomics data analysis called GlycoPAT (GlycoProteomics Analysis Toolbox; freely available from www.VirtualGlycome.org/glycopat). The program includes three major advances: (1) "SmallGlyPep," a minimal linear representation of glycopeptides for MS n data analysis. This format allows facile serial fragmentation of both the peptide backbone and PTM at one or more locations. (2) A novel scoring scheme based on calculation of the "Ensemble Score (ES)," a measure that scores and rank-orders MS/MS spectrum for N- and O-linked glycopeptides using cross-correlation and probability based analyses. (3) A false discovery rate (FDR) calculation scheme where decoy glycopeptides are created by simultaneously scrambling the amino acid sequence and by introducing artificial monosaccharides by perturbing the original sugar mass. Parallel computing facilities and user-friendly GUIs (Graphical User Interfaces) are also provided. GlycoPAT is used to catalogue site-specific glycosylation on simple glycoproteins, standard protein mixtures and human plasma cryoprecipitate samples in three common MS/MS fragmentation modes: CID, HCD and ETD. It is also used to identify 960 unique glycopeptides in cell lysates from prostate cancer cells. The results show that the simultaneous consideration of peptide and glycan

  2. A new disaster victim identification management strategy targeting "near identification-threshold" cases: Experiences from the Boxing Day tsunami.

    Science.gov (United States)

    Wright, Kirsty; Mundorff, Amy; Chaseling, Janet; Forrest, Alexander; Maguire, Christopher; Crane, Denis I

    2015-05-01

    The international disaster victim identification (DVI) response to the Boxing Day tsunami, led by the Royal Thai Police in Phuket, Thailand, was one of the largest and most complex in DVI history. Referred to as the Thai Tsunami Victim Identification operation, the group comprised a multi-national, multi-agency, and multi-disciplinary team. The traditional DVI approach proved successful in identifying a large number of victims quickly. However, the team struggled to identify certain victims due to incomplete or poor quality ante-mortem and post-mortem data. In response to these challenges, a new 'near-threshold' DVI management strategy was implemented to target presumptive identifications and improve operational efficiency. The strategy was implemented by the DNA Team, therefore DNA kinship matches that just failed to reach the reporting threshold of 99.9% were prioritized, however the same approach could be taken by targeting, for example, cases with partial fingerprint matches. The presumptive DNA identifications were progressively filtered through the Investigation, Dental and Fingerprint Teams to add additional information necessary to either strengthen or conclusively exclude the identification. Over a five-month period 111 victims from ten countries were identified using this targeted approach. The new identifications comprised 87 adults, 24 children and included 97 Thai locals. New data from the Fingerprint Team established nearly 60% of the total near-threshold identifications and the combined DNA/Physical method was responsible for over 30%. Implementing the new strategy, targeting near-threshold cases, had positive management implications. The process initiated additional ante-mortem information collections, and established a much-needed, distinct "end-point" for unresolved cases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Effect of Various Environmental Stressors on Target Detection, Identification, and Marksmanship

    National Research Council Canada - National Science Library

    Tikuisis, Peter; Keefe, Allan A

    2007-01-01

    .... Using a small arms trainer (SAT), the detection, identification, and engagement of targets were tested under a variety of environmentally stressful conditions including heat and cold exposure, noise, fatiguing exercise, and sleep...

  4. Target Detection, Identification, and Marksmanship Under Various Types of Physiological Strain

    National Research Council Canada - National Science Library

    Tikuisis, Peter

    2006-01-01

    .... Using a small arms trainer (SAT), target detection, identification, and engagement were tested under a variety of conditions including heat and cold exposure, fatiguing exercise, and sleep deprivation, with caffeine intervention...

  5. Target identification of natural and traditional medicines with quantitative chemical proteomics approaches.

    Science.gov (United States)

    Wang, Jigang; Gao, Liqian; Lee, Yew Mun; Kalesh, Karunakaran A; Ong, Yong Siang; Lim, Jaehong; Jee, Joo-Eun; Sun, Hongyan; Lee, Su Seong; Hua, Zi-Chun; Lin, Qingsong

    2016-06-01

    Natural and traditional medicines, being a great source of drugs and drug leads, have regained wide interests due to the limited success of high-throughput screening of compound libraries in the past few decades and the recent technology advancement. Many drugs/bioactive compounds exert their functions through interaction with their protein targets, with more and more drugs showing their ability to target multiple proteins, thus target identification has an important role in drug discovery and biomedical research fields. Identifying drug targets not only furthers the understanding of the mechanism of action (MOA) of a drug but also reveals its potential therapeutic applications and adverse side effects. Chemical proteomics makes use of affinity chromatography approaches coupled with mass spectrometry to systematically identify small molecule-protein interactions. Although traditional affinity-based chemical proteomics approaches have made great progress in the identification of cellular targets and elucidation of MOAs of many bioactive molecules, nonspecific binding remains a major issue which may reduce the accuracy of target identification and may hamper the drug development process. Recently, quantitative proteomics approaches, namely, metabolic labeling, chemical labeling, or label-free approaches, have been implemented in target identification to overcome such limitations. In this review, we will summarize and discuss the recent advances in the application of various quantitative chemical proteomics approaches for the identification of targets of natural and traditional medicines. Copyright © 2016. Published by Elsevier Inc.

  6. Study on the knowledge base system for the identification of typical target

    International Nuclear Information System (INIS)

    Qin Kai; Zhao Yingjun

    2008-01-01

    Based on the research on target knowledge base, target database, texture analysis, shape analysis, this paper proposed a new knowledge based method for typical target identification from remote sensing image. By extracting the texture characters and shape characters, joining with spatial analysis in GIS, reasoning according to the prior knowledge in the knowledge base, this method can identify and ex- tract typical target from remote sensing images. (authors)

  7. Deconstruction of O-glycosylation-GalNAc-T isoforms direct distinct subsets of the O-glycoproteome

    DEFF Research Database (Denmark)

    Schjoldager, Katrine T; Joshi, Hiren J; Kong, Yun

    2015-01-01

    GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc......-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and GalNAc-T2) in liver cells and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas...... distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and GalNAc-T2. The non-redundant O-glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc-T2 isoform, these support a role in lipid...

  8. Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

    2013-01-01

    Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome...... with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated...... in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells....

  9. Genome-wide identification of KANADI1 target genes.

    Directory of Open Access Journals (Sweden)

    Paz Merelo

    Full Text Available Plant organ development and polarity establishment is mediated by the action of several transcription factors. Among these, the KANADI (KAN subclade of the GARP protein family plays important roles in polarity-associated processes during embryo, shoot and root patterning. In this study, we have identified a set of potential direct target genes of KAN1 through a combination of chromatin immunoprecipitation/DNA sequencing (ChIP-Seq and genome-wide transcriptional profiling using tiling arrays. Target genes are over-represented for genes involved in the regulation of organ development as well as in the response to auxin. KAN1 affects directly the expression of several genes previously shown to be important in the establishment of polarity during lateral organ and vascular tissue development. We also show that KAN1 controls through its target genes auxin effects on organ development at different levels: transport and its regulation, and signaling. In addition, KAN1 regulates genes involved in the response to abscisic acid, jasmonic acid, brassinosteroids, ethylene, cytokinins and gibberellins. The role of KAN1 in organ polarity is antagonized by HD-ZIPIII transcription factors, including REVOLUTA (REV. A comparison of their target genes reveals that the REV/KAN1 module acts in organ patterning through opposite regulation of shared targets. Evidence of mutual repression between closely related family members is also shown.

  10. Drug target identification using side-effect similarity

    DEFF Research Database (Denmark)

    Campillos, Monica; Kuhn, Michael; Gavin, Anne-Claude

    2008-01-01

    Targets for drugs have so far been predicted on the basis of molecular or cellular features, for example, by exploiting similarity in chemical structure or in activity across cell lines. We used phenotypic side-effect similarities to infer whether two drugs share a target. Applied to 746 marketed...... drugs, a network of 1018 side effect-driven drug-drug relations became apparent, 261 of which are formed by chemically dissimilar drugs from different therapeutic indications. We experimentally tested 20 of these unexpected drug-drug relations and validated 13 implied drug-target relations by in vitro...... binding assays, of which 11 reveal inhibition constants equal to less than 10 micromolar. Nine of these were tested and confirmed in cell assays, documenting the feasibility of using phenotypic information to infer molecular interactions and hinting at new uses of marketed drugs....

  11. Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Neubert, Patrick; Halim, Adnan; Zauser, Martin

    2016-01-01

    -transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized proteinO-mannosyltransferases. We find thatO-mannosylation is not only a prominent modification of cell wall...

  12. Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

    Science.gov (United States)

    Stutz, Charles C.; Zhang, Xiaobin; Shusta, Eric V.

    2018-01-01

    The blood-brain barrier (BBB) represents a large obstacle for the treatment of central nervous system diseases. Targeting endogenous nutrient transporters that transcytose the BBB is one promising approach to selectively and noninvasively deliver a drug payload to the brain. The main limitations of the currently employed transcytosing receptors are their ubiquitous expression in the peripheral vasculature and the inherent low levels of transcytosis mediated by such systems. In this review, approaches designed to increase the repertoire of transcytosing receptors which can be targeted for the purpose of drug delivery are discussed. In particular, combinatorial protein libraries can be screened on BBB cells in vitro or in vivo to isolate targeting peptides or antibodies that can trigger transcytosis. Once these targeting reagents are discovered, the cognate BBB transcytosis system can be identified using techniques such as expression cloning or immunoprecipitation coupled with mass spectrometry. Continued technological advances in BBB genomics and proteomics, membrane protein manipulation, and in vitro BBB technology promise to further advance the capability to identify and optimize peptides and antibodies capable of mediating drug transport across the BBB. PMID:23789958

  13. Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

    Directory of Open Access Journals (Sweden)

    Murphy Dennis J

    2007-07-01

    Full Text Available Abstract Background Mycobacterium tuberculosis, the causative agent of tuberculosis (TB, infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections. Methods The availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality. Results Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB, enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability

  14. Neural Network Target Identification System for False Alarm Reduction

    Science.gov (United States)

    Ye, David; Edens, Weston; Lu, Thomas T.; Chao, Tien-Hsin

    2009-01-01

    A multi-stage automated target recognition (ATR) system has been designed to perform computer vision tasks with adequate proficiency in mimicking human vision. The system is able to detect, identify, and track targets of interest. Potential regions of interest (ROIs) are first identified by the detection stage using an Optimum Trade-off Maximum Average Correlation Height (OT-MACH) filter combined with a wavelet transform. False positives are then eliminated by the verification stage using feature extraction methods in conjunction with neural networks. Feature extraction transforms the ROIs using filtering and binning algorithms to create feature vectors. A feed forward back propagation neural network (NN) is then trained to classify each feature vector and remove false positives. This paper discusses the test of the system performance and parameter optimizations process which adapts the system to various targets and datasets. The test results show that the system was successful in substantially reducing the false positive rate when tested on a sonar image dataset.

  15. PDTD: a web-accessible protein database for drug target identification

    Directory of Open Access Journals (Sweden)

    Gao Zhenting

    2008-02-01

    Full Text Available Abstract Background Target identification is important for modern drug discovery. With the advances in the development of molecular docking, potential binding proteins may be discovered by docking a small molecule to a repository of proteins with three-dimensional (3D structures. To complete this task, a reverse docking program and a drug target database with 3D structures are necessary. To this end, we have developed a web server tool, TarFisDock (Target Fishing Docking http://www.dddc.ac.cn/tarfisdock, which has been used widely by others. Recently, we have constructed a protein target database, Potential Drug Target Database (PDTD, and have integrated PDTD with TarFisDock. This combination aims to assist target identification and validation. Description PDTD is a web-accessible protein database for in silico target identification. It currently contains >1100 protein entries with 3D structures presented in the Protein Data Bank. The data are extracted from the literatures and several online databases such as TTD, DrugBank and Thomson Pharma. The database covers diverse information of >830 known or potential drug targets, including protein and active sites structures in both PDB and mol2 formats, related diseases, biological functions as well as associated regulating (signaling pathways. Each target is categorized by both nosology and biochemical function. PDTD supports keyword search function, such as PDB ID, target name, and disease name. Data set generated by PDTD can be viewed with the plug-in of molecular visualization tools and also can be downloaded freely. Remarkably, PDTD is specially designed for target identification. In conjunction with TarFisDock, PDTD can be used to identify binding proteins for small molecules. The results can be downloaded in the form of mol2 file with the binding pose of the probe compound and a list of potential binding targets according to their ranking scores. Conclusion PDTD serves as a comprehensive and

  16. Gene silencing in Tribolium castaneum as a tool for the targeted identification of candidate RNAi targets in crop pests.

    Science.gov (United States)

    Knorr, Eileen; Fishilevich, Elane; Tenbusch, Linda; Frey, Meghan L F; Rangasamy, Murugesan; Billion, Andre; Worden, Sarah E; Gandra, Premchand; Arora, Kanika; Lo, Wendy; Schulenberg, Greg; Valverde-Garcia, Pablo; Vilcinskas, Andreas; Narva, Kenneth E

    2018-02-01

    RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T 0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.

  17. Target identification for stereotactic thalamotomy using diffusion tractography.

    Directory of Open Access Journals (Sweden)

    Zsigmond Tamás Kincses

    Full Text Available BACKGROUND: Stereotactic targets for thalamotomy are usually derived from population-based coordinates. Individual anatomy is used only to scale the coordinates based on the location of some internal guide points. While on conventional MR imaging the thalamic nuclei are indistinguishable, recently it has become possible to identify individual thalamic nuclei using different connectivity profiles, as defined by MR diffusion tractography. METHODOLOGY AND PRINCIPAL FINDINGS: Here we investigated the inter-individual variation of the location of target nuclei for thalamotomy: the putative ventralis oralis posterior (Vop and the ventral intermedius (Vim nucleus as defined by probabilistic tractography. We showed that the mean inter-individual distance of the peak Vop location is 7.33 mm and 7.42 mm for Vim. The mean overlap between individual Vop nuclei was 40.2% and it was 31.8% for Vim nuclei. As a proof of concept, we also present a patient who underwent Vop thalamotomy for untreatable tremor caused by traumatic brain injury and another patient who underwent Vim thalamotomy for essential tremor. The probabilistic tractography indicated that the successful tremor control was achieved with lesions in the Vop and Vim respectively. CONCLUSIONS: Our data call attention to the need for a better appreciation of the individual anatomy when planning stereotactic functional neurosurgery.

  18. PET imaging of T cells: Target identification and feasibility assessment.

    Science.gov (United States)

    Auberson, Yves P; Briard, Emmanuelle; Rudolph, Bettina; Kaupmann, Klemen; Smith, Paul; Oberhauser, Berndt

    2018-06-01

    Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are however no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: PKC , Lck, ZAP70 and Itk. Ultimately, we focused on Itk (interleukin-2-inducible T cell kinase) and identified a tool molecule with properties suitable for in vivo imaging of T cells, (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)-phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydro-cyclopropa[f]-indazole-3-carboxamide (23). While not having the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Identification of novel androgen receptor target genes in prostate cancer

    Directory of Open Access Journals (Sweden)

    Gerald William L

    2007-06-01

    Full Text Available Abstract Background The androgen receptor (AR plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa. However, little is known about AR target genes that mediate the receptor's roles in disease progression. Results Using Chromatin Immunoprecipitation (ChIP Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant and LNCaP (androgen-dependent PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells – D-dopachrome tautomerase (DDT, Protein kinase C delta (PRKCD, Glutathione S- transferase theta 2 (GSTT2, Transient receptor potential cation channel subfamily V member 3 (TRPV3, and Pyrroline-5-carboxylate reductase 1 (PYCR1 – most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT, was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. Conclusion AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are

  20. Integrative Bioinformatics Approaches for Identification of Drug Targets in Hypertension.

    Science.gov (United States)

    Hemerich, Daiane; van Setten, Jessica; Tragante, Vinicius; Asselbergs, Folkert W

    2018-01-01

    High blood pressure or hypertension is an established risk factor for a myriad of cardiovascular diseases. Genome-wide association studies have successfully found over nine hundred loci that contribute to blood pressure. However, the mechanisms through which these loci contribute to disease are still relatively undetermined as less than 10% of hypertension-associated variants are located in coding regions. Phenotypic cell-type specificity analyses and expression quantitative trait loci show predominant vascular and cardiac tissue involvement for blood pressure-associated variants. Maps of chromosomal conformation and expression quantitative trait loci (eQTL) in critical tissues identified 2,424 genes interacting with blood pressure-associated loci, of which 517 are druggable. Integrating genome, regulome and transcriptome information in relevant cell-types could help to functionally annotate blood pressure associated loci and identify drug targets.

  1. Vitamin D Pathway Status and the Identification of Target Genes in the Mouse Mammary Gland

    Science.gov (United States)

    2014-11-01

    breast cancer stem cells with oncolytic herpes simplex virus. Cancer Gene Therapy 2012;19(10):707-14. June 21, 2012 – Poster Presentation – Presented...AD_________________ Award Number: W81XWH-11-1-0152 TITLE: Vitamin D Pathway Status and the Identification of Target Genes in the Mouse Mammary... Identification of Target Genes in the 5b. GRANT NUMBER W81XWH-11-1-0152 Mouse Mammary Gland 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT

  2. Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

    Science.gov (United States)

    Cao, Xueyan; Song, Dahe; Yang, Mei; Yang, Ning; Ye, Qing; Tao, Dongbing; Liu, Biao; Wu, Rina; Yue, Xiqing

    2017-11-29

    Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

  3. Platforms for the identification of GPCR targets, and of orthosteric and allosteric modulators.

    Science.gov (United States)

    Franco, Rafael; Canela, Enric I; Casado, Vicent; Ferre, Sergi

    2010-04-01

    The review provides a summary of old and new approaches for GPCR target identification and for the screening of molecules acting on GPCR targets. The new findings in the field are presented as well as an opinion about how these developments may help GPCR drug discovery. Importance in the field: GPCRs have been the most useful family of proteins in terms of targets for drug discovery. The expectations for GPCR target identification and discovery of new drugs acting on 'old' or 'new' GPCR targets are very high. Given the fact that the pace at which new 'GPCR drugs' appear in the market is decreasing and since the new developments in the field are not being translated into drug discovery there is a need to review the field from a critical perspective. To overcome the limitation of the old approaches used in GPCR target identification and drugs discovery new approaches are required. In particular successful approaches in GPCR drug discovery should take into account that the real GPCR targets for a given disease are not GPCR monomers but GPCR heteromers. The reader will gain an overview of the strategies currently used and their pros and cons. The reader will also understand that new strategies may help in accelerating the access of GPCR into the market, and also notice that successful strategies should take advantage of the new findings in the field of GPCRs.

  4. CSF N-glycoproteomics for early diagnosis in Alzheimer's disease.

    Science.gov (United States)

    Palmigiano, Angelo; Barone, Rita; Sturiale, Luisa; Sanfilippo, Cristina; Bua, Rosaria Ornella; Romeo, Donata Agata; Messina, Angela; Capuana, Maria Luisa; Maci, Tiziana; Le Pira, Francesco; Zappia, Mario; Garozzo, Domenico

    2016-01-10

    This work aims at exploring the human CSF (Cerebrospinal fluid) N-glycome by MALDI MS techniques, in order to assess specific glycosylation pattern(s) in patients with Alzheimer's disease (n:24) and in subjects with mild cognitive impairment (MCI) (n:11), these last as potential AD patients at a pre-dementia stage. For comparison, 21 healthy controls were studied. We identified a group of AD and MCI subjects (about 40-50% of the studied sample) showing significant alteration of CSF N-glycome profiling, consisting of a decrease in the overall sialylation degree and an increase in species bearing bisecting GlcNAc. Noteworthy, all the MCI patients that converted to AD within the clinical follow-up, had an abnormal CSF glycosylation profile. Based on the studied cohort, CSF glycosylation changes may occur before an AD clinical onset. Previous studies specifically focused on the key role of glycosyltransferase GnT-III on AD-pathogenesis, addressing the patho-mechanism to specific sugar modification of BACE-1 glycoprotein with bisecting GlcNAc. Our patients addressed protein N-glycosylation changes at an early phase of the whole biomolecular misregulation on AD, pointing to CSF N-glycome analyses as promising tool to enhance early detection of AD and also suggesting alternative therapeutics target molecules, such as specific glyco-enzymes. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2014-01-01

    Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein...... reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" (12C) and "heavy" (13C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems....... extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide...

  6. Chemical deamidation: a common pitfall in large-scale N-linked glycoproteomic mass spectrometry-based analyses

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Melo-Braga, Marcella Nunes; Engholm-Keller, Kasper

    2012-01-01

    for false positives. The confusion arises since the protein N-glycosidase F (PNGase F) reaction used to separate N-glycans from formerly glycosylated peptides catalyses the cleavage and deamidates the asparagine residue. This is typically viewed as beneficial since it acts to highlight the modification site......-linked consensus sites based on common N-linked glycoproteomics strategies without proper control experiments. Beside showing the spontaneous deamidation we provide alternative methods for validation that should be used in such experiments....

  7. C-mii: a tool for plant miRNA and target identification.

    Science.gov (United States)

    Numnark, Somrak; Mhuantong, Wuttichai; Ingsriswang, Supawadee; Wichadakul, Duangdao

    2012-01-01

    MicroRNAs (miRNAs) have been known to play an important role in several biological processes in both animals and plants. Although several tools for miRNA and target identification are available, the number of tools tailored towards plants is limited, and those that are available have specific functionality, lack graphical user interfaces, and restrict the number of input sequences. Large-scale computational identifications of miRNAs and/or targets of several plants have been also reported. Their methods, however, are only described as flow diagrams, which require programming skills and the understanding of input and output of the connected programs to reproduce. To overcome these limitations and programming complexities, we proposed C-mii as a ready-made software package for both plant miRNA and target identification. C-mii was designed and implemented based on established computational steps and criteria derived from previous literature with the following distinguishing features. First, software is easy to install with all-in-one programs and packaged databases. Second, it comes with graphical user interfaces (GUIs) for ease of use. Users can identify plant miRNAs and targets via step-by-step execution, explore the detailed results from each step, filter the results according to proposed constraints in plant miRNA and target biogenesis, and export sequences and structures of interest. Third, it supplies bird's eye views of the identification results with infographics and grouping information. Fourth, in terms of functionality, it extends the standard computational steps of miRNA target identification with miRNA-target folding and GO annotation. Fifth, it provides helper functions for the update of pre-installed databases and automatic recovery. Finally, it supports multi-project and multi-thread management. C-mii constitutes the first complete software package with graphical user interfaces enabling computational identification of both plant miRNA genes and mi

  8. A conceptual framework for the identification of candidate drugs and drug targets in acute promyelocytic leukemia

    DEFF Research Database (Denmark)

    Marstrand, T T; Borup, R; Willer, A

    2010-01-01

    regulation, and (ii) the identification of candidate drugs and drug targets for therapeutic interventions. Significantly, our study provides a conceptual framework that can be applied to any subtype of AML and cancer in general to uncover novel information from published microarray data sets at low cost...

  9. Isolation, Characterization and Identification of Environmental Bacterial Isolates with Screening for Antagonism Against Three Bacterial Targets

    Science.gov (United States)

    2017-04-01

    ISOLATES WITH SCREENING FOR ANTAGONISM AGAINST THREE BACTERIAL TARGETS 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Identification of environmental isolates followed the flowchart from “Bergey’s Manual of Determinative Bacteriology” (Holt et al. 1994), which

  10. Identification of distant drug off-targets by direct superposition of binding pocket surfaces.

    Science.gov (United States)

    Schumann, Marcel; Armen, Roger S

    2013-01-01

    Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target ("distant off-targets"). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target ("distant off-target").

  11. Metabolic modeling to identify engineering targets for Komagataella phaffii: The effect of biomass composition on gene target identification.

    Science.gov (United States)

    Cankorur-Cetinkaya, Ayca; Dikicioglu, Duygu; Oliver, Stephen G

    2017-11-01

    Genome-scale metabolic models are valuable tools for the design of novel strains of industrial microorganisms, such as Komagataella phaffii (syn. Pichia pastoris). However, as is the case for many industrial microbes, there is no executable metabolic model for K. phaffiii that confirms to current standards by providing the metabolite and reactions IDs, to facilitate model extension and reuse, and gene-reaction associations to enable identification of targets for genetic manipulation. In order to remedy this deficiency, we decided to reconstruct the genome-scale metabolic model of K. phaffii by reconciling the extant models and performing extensive manual curation in order to construct an executable model (Kp.1.0) that conforms to current standards. We then used this model to study the effect of biomass composition on the predictive success of the model. Twelve different biomass compositions obtained from published empirical data obtained under a range of growth conditions were employed in this investigation. We found that the success of Kp1.0 in predicting both gene essentiality and growth characteristics was relatively unaffected by biomass composition. However, we found that biomass composition had a profound effect on the distribution of the fluxes involved in lipid, DNA, and steroid biosynthetic processes, cellular alcohol metabolic process, and oxidation-reduction process. Furthermore, we investigated the effect of biomass composition on the identification of suitable target genes for strain development. The analyses revealed that around 40% of the predictions of the effect of gene overexpression or deletion changed depending on the representation of biomass composition in the model. Considering the robustness of the in silico flux distributions to the changing biomass representations enables better interpretation of experimental results, reduces the risk of wrong target identification, and so both speeds and improves the process of directed strain development

  12. PharmMapper 2017 update: a web server for potential drug target identification with a comprehensive target pharmacophore database.

    Science.gov (United States)

    Wang, Xia; Shen, Yihang; Wang, Shiwei; Li, Shiliang; Zhang, Weilin; Liu, Xiaofeng; Lai, Luhua; Pei, Jianfeng; Li, Honglin

    2017-07-03

    The PharmMapper online tool is a web server for potential drug target identification by reversed pharmacophore matching the query compound against an in-house pharmacophore model database. The original version of PharmMapper includes more than 7000 target pharmacophores derived from complex crystal structures with corresponding protein target annotations. In this article, we present a new version of the PharmMapper web server, of which the backend pharmacophore database is six times larger than the earlier one, with a total of 23 236 proteins covering 16 159 druggable pharmacophore models and 51 431 ligandable pharmacophore models. The expanded target data cover 450 indications and 4800 molecular functions compared to 110 indications and 349 molecular functions in our last update. In addition, the new web server is united with the statistically meaningful ranking of the identified drug targets, which is achieved through the use of standard scores. It also features an improved user interface. The proposed web server is freely available at http://lilab.ecust.edu.cn/pharmmapper/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. A novel algorithm for finding optimal driver nodes to target control complex networks and its applications for drug targets identification.

    Science.gov (United States)

    Guo, Wei-Feng; Zhang, Shao-Wu; Shi, Qian-Qian; Zhang, Cheng-Ming; Zeng, Tao; Chen, Luonan

    2018-01-19

    The advances in target control of complex networks not only can offer new insights into the general control dynamics of complex systems, but also be useful for the practical application in systems biology, such as discovering new therapeutic targets for disease intervention. In many cases, e.g. drug target identification in biological networks, we usually require a target control on a subset of nodes (i.e., disease-associated genes) with minimum cost, and we further expect that more driver nodes consistent with a certain well-selected network nodes (i.e., prior-known drug-target genes). Therefore, motivated by this fact, we pose and address a new and practical problem called as target control problem with objectives-guided optimization (TCO): how could we control the interested variables (or targets) of a system with the optional driver nodes by minimizing the total quantity of drivers and meantime maximizing the quantity of constrained nodes among those drivers. Here, we design an efficient algorithm (TCOA) to find the optional driver nodes for controlling targets in complex networks. We apply our TCOA to several real-world networks, and the results support that our TCOA can identify more precise driver nodes than the existing control-fucus approaches. Furthermore, we have applied TCOA to two bimolecular expert-curate networks. Source code for our TCOA is freely available from http://sysbio.sibcb.ac.cn/cb/chenlab/software.htm or https://github.com/WilfongGuo/guoweifeng . In the previous theoretical research for the full control, there exists an observation and conclusion that the driver nodes tend to be low-degree nodes. However, for target control the biological networks, we find interestingly that the driver nodes tend to be high-degree nodes, which is more consistent with the biological experimental observations. Furthermore, our results supply the novel insights into how we can efficiently target control a complex system, and especially many evidences on the

  14. On-orbit real-time robust cooperative target identification in complex background

    Directory of Open Access Journals (Sweden)

    Wen Zhuoman

    2015-10-01

    Full Text Available Cooperative target identification is the prerequisite for the relative position and orientation measurement between the space robot arm and the to-be-arrested object. We propose an on-orbit real-time robust algorithm for cooperative target identification in complex background using the features of circle and lines. It first extracts only the interested edges in the target image using an adaptive threshold and refines them to about single-pixel-width with improved non-maximum suppression. Adapting a novel tracking approach, edge segments changing smoothly in tangential directions are obtained. With a small amount of calculation, large numbers of invalid edges are removed. From the few remained edges, valid circular arcs are extracted and reassembled to obtain circles according to a reliable criterion. Finally, the target is identified if there are certain numbers of straight lines whose relative positions with the circle match the known target pattern. Experiments demonstrate that the proposed algorithm accurately identifies the cooperative target within the range of 0.3–1.5 m under complex background at the speed of 8 frames per second, regardless of lighting condition and target attitude. The proposed algorithm is very suitable for real-time visual measurement of space robot arm because of its robustness and small memory requirement.

  15. High-Speed Target Identification System Based on the Plume’s Spectral Distribution

    Directory of Open Access Journals (Sweden)

    Wenjie Lang

    2015-01-01

    Full Text Available In order to recognize the target of high speed quickly and accurately, an identification system was designed based on analysis of the distribution characteristics of the plume spectrum. In the system, the target was aligned with visible light tracking module, and the spectral analysis of the target’s plume radiation was achieved by interference module. The distinguishing factor recognition algorithm was designed on basis of ratio of multifeature band peaks and valley mean values. Effective recognition of the high speed moving target could be achieved after partition of the active region and the influence of target motion on spectral acquisition was analyzed. In the experiment the small rocket combustion was used as the target. The spectral detection experiment was conducted at different speeds 2.0 km away from the detection system. Experimental results showed that spectral distribution had significant spectral offset in the same sampling period for the target with different speeds, but the spectral distribution was basically consistent. Through calculation of the inclusion relationship between distinguishing factor and distinction interval of the peak value and the valley value at the corresponding wave-bands, effective identification of target could be achieved.

  16. Identification of control targets in Boolean molecular network models via computational algebra.

    Science.gov (United States)

    Murrugarra, David; Veliz-Cuba, Alan; Aguilar, Boris; Laubenbacher, Reinhard

    2016-09-23

    Many problems in biomedicine and other areas of the life sciences can be characterized as control problems, with the goal of finding strategies to change a disease or otherwise undesirable state of a biological system into another, more desirable, state through an intervention, such as a drug or other therapeutic treatment. The identification of such strategies is typically based on a mathematical model of the process to be altered through targeted control inputs. This paper focuses on processes at the molecular level that determine the state of an individual cell, involving signaling or gene regulation. The mathematical model type considered is that of Boolean networks. The potential control targets can be represented by a set of nodes and edges that can be manipulated to produce a desired effect on the system. This paper presents a method for the identification of potential intervention targets in Boolean molecular network models using algebraic techniques. The approach exploits an algebraic representation of Boolean networks to encode the control candidates in the network wiring diagram as the solutions of a system of polynomials equations, and then uses computational algebra techniques to find such controllers. The control methods in this paper are validated through the identification of combinatorial interventions in the signaling pathways of previously reported control targets in two well studied systems, a p53-mdm2 network and a blood T cell lymphocyte granular leukemia survival signaling network. Supplementary data is available online and our code in Macaulay2 and Matlab are available via http://www.ms.uky.edu/~dmu228/ControlAlg . This paper presents a novel method for the identification of intervention targets in Boolean network models. The results in this paper show that the proposed methods are useful and efficient for moderately large networks.

  17. Identification and validation nucleolin as a target of curcumol in nasopharyngeal carcinoma cells.

    Science.gov (United States)

    Wang, Juan; Wu, Jiacai; Li, Xumei; Liu, Haowei; Qin, Jianli; Bai, Zhun; Chi, Bixia; Chen, Xu

    2018-06-30

    Identification of the specific protein target(s) of a drug is a critical step in unraveling its mechanisms of action (MOA) in many natural products. Curcumol, isolated from well known Chinese medicinal plant Curcuma zedoary, has been shown to possess multiple biological activities. It can inhibit nasopharyngeal carcinoma (NPC) proliferation and induce apoptosis, but its target protein(s) in NPC cells remains unclear. In this study, we employed a mass spectrometry-based chemical proteomics approach reveal the possible protein targets of curcumol in NPC cells. Cellular thermal shift assay (CETSA), molecular docking and cell-based assay was used to validate the binding interactions. Chemical proteomics capturing uncovered that NCL is a target of curcumol in NPC cells, Molecular docking showed that curcumol bound to NCL with an -7.8 kcal/mol binding free energy. Cell function analysis found that curcumol's treatment leads to a degradation of NCL in NPC cells, and it showed slight effects on NP69 cells. In conclusion, our results providing evidences that NCL is a target protein of curcumol. We revealed that the anti-cancer effects of curcumol in NPC cells are mediated, at least in part, by NCL inhibition. Many natural products showed high bioactivity, while their mechanisms of action (MOA) are very poor or completely missed. Understanding the MOA of natural drugs can thoroughly exploit their therapeutic potential and minimize their adverse side effects. Identification of the specific protein target(s) of a drug is a critical step in unraveling its MOA. Compound-centric chemical proteomics is a classic chemical proteomics approach which integrates chemical synthesis with cell biology and mass spectrometry (MS) to identify protein targets of natural products determine the drug mechanism of action, describe its toxicity, and figure out the possible cause of off-target. It is an affinity-based chemical proteomics method to identify small molecule-protein interactions

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

    Science.gov (United States)

    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

  19. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells

    Science.gov (United States)

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging. PMID:28636640

  20. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells.

    Science.gov (United States)

    Lake, Michael P; Bouchard, Louis-S

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.

  1. Computational Identification of MicroRNAs and Their Targets from Finger Millet (Eleusine coracana).

    Science.gov (United States)

    Usha, S; Jyothi, M N; Suchithra, B; Dixit, Rekha; Rai, D V; Nagesh Babu, R

    2017-03-01

    MicroRNAs are endogenous small RNAs regulating intrinsic normal growth and development of plant. Discovering miRNAs, their targets and further inferring their functions had become routine process to comprehend the normal biological processes of miRNAs and their roles in plant development. In this study, we used homology-based analysis with available expressed sequence tag of finger millet (Eleusine coracana) to predict conserved miRNAs. Three potent miRNAs targeting 88 genes were identified. The newly identified miRNAs were found to be homologous with miR166 and miR1310. The targets recognized were transcription factors and enzymes, and GO analysis showed these miRNAs played varied roles in gene regulation. The identification of miRNAs and their targets is anticipated to hasten the pace of key epigenetic regulators in plant development.

  2. RISC RNA sequencing for context-specific identification of in vivo microRNA targets.

    Science.gov (United States)

    Matkovich, Scot J; Van Booven, Derek J; Eschenbacher, William H; Dorn, Gerald W

    2011-01-07

    MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo. To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs). We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1645 mRNAs consistently targeted to mouse cardiac RISCs. We used this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing "seed" sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts. RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context and is applicable to any tissue and any disease state.

  3. From targeting to implementation: The role of identification of fuel poor households

    International Nuclear Information System (INIS)

    Dubois, Ute

    2012-01-01

    This paper proposes to evaluate the efficiency of fuel poverty policies by looking at them as a three-step process. These steps are policy targeting, identification of households and implementation of measures. Each of these steps are costly and can face feasibility problems. This is particularly true in the case of fuel poverty policies, because of the multidimensional aspect of fuel poverty. Therefore, public policies in that field are necessarily imperfect, and this should be taken into account in the analysis of policies. This three-step framework is applied to the French fuel poverty policy, which has been developed in 2010. As its practical modalities of implementation are still under discussion, we mainly discuss the choices made in terms of targeting and highlight the role of identification of fuel poor households as a key factor for the success of that policy. The general idea behind this discussion is that the efficiency of such policies has to be viewed in a comprehensive way, which includes the three policy steps. - Highlights: ► This paper analyses fuel poverty policy as a three-step process. ► The steps are targeting, identification and implementation. ► Feasibility and implementation problems should be considered in policy formulation. ► Errors of inclusion and exclusion limit the effectiveness of fuel poverty policy. ► We apply this framework to the recent French fuel poverty policy.

  4. Efficient and robust identification of cortical targets in concurrent TMS-fMRI experiments

    Science.gov (United States)

    Yau, Jeffrey M.; Hua, Jun; Liao, Diana A.; Desmond, John E.

    2014-01-01

    Transcranial magnetic stimulation (TMS) can be delivered during fMRI scans to evoke BOLD responses in distributed brain networks. While concurrent TMS-fMRI offers a potentially powerful tool for non-invasively investigating functional human neuroanatomy, the technique is currently limited by the lack of methods to rapidly and precisely localize targeted brain regions – a reliable procedure is necessary for validly relating stimulation targets to BOLD activation patterns, especially for cortical targets outside of motor and visual regions. Here we describe a convenient and practical method for visualizing coil position (in the scanner) and identifying the cortical location of TMS targets without requiring any calibration or any particular coil-mounting device. We quantified the precision and reliability of the target position estimates by testing the marker processing procedure on data from 9 scan sessions: Rigorous testing of the localization procedure revealed minimal variability in coil and target position estimates. We validated the marker processing procedure in concurrent TMS-fMRI experiments characterizing motor network connectivity. Together, these results indicate that our efficient method accurately and reliably identifies TMS targets in the MR scanner, which can be useful during scan sessions for optimizing coil placement and also for post-scan outlier identification. Notably, this method can be used generally to identify the position and orientation of MR-compatible hardware placed near the head in the MR scanner. PMID:23507384

  5. Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries

    Directory of Open Access Journals (Sweden)

    Marco Cavallari

    2017-07-01

    Full Text Available Unbiased and simultaneous identification of a specific antibody and its target antigen has been difficult without prior knowledge of at least one interaction partner. Immunization with complex mixtures of antigens such as whole organisms and tissue extracts including tumoral ones evokes a highly diverse immune response. During such a response, antibodies are generated against a variety of epitopes in the mixture. Here, we propose a surface display design that is suited to simultaneously identify camelid single domain antibodies and their targets. Immune libraries of single-domain antigen recognition fragments from camelid heavy chain-only antibodies (VHH were attached to the peptidoglycan of Gram-positive Staphylococcus aureus employing its endogenous housekeeping sortase enzyme. The sortase transpeptidation reaction covalently attached the VHH to the bacterial peptidoglycan. The reversible nature of the reaction allowed the recovery of the VHH from the bacterial surface and the use of the VHH in downstream applications. These staphylococcal surface display libraries were used to rapidly identify VHH as well as their targets by immunoprecipitation (IP. Our novel bacterial surface display platform was stable under harsh screening conditions, allowed fast target identification, and readily permitted the recovery of the displayed VHH for downstream analysis.

  6. Identification of Cell Surface Targets through Meta-analysis of Microarray Data

    Directory of Open Access Journals (Sweden)

    Henry Haeberle

    2012-07-01

    Full Text Available High-resolution image guidance for resection of residual tumor cells would enable more precise and complete excision for more effective treatment of cancers, such as medulloblastoma, the most common pediatric brain cancer. Numerous studies have shown that brain tumor patient outcomes correlate with the precision of resection. To enable guided resection with molecular specificity and cellular resolution, molecular probes that effectively delineate brain tumor boundaries are essential. Therefore, we developed a bioinformatics approach to analyze micro-array datasets for the identification of transcripts that encode candidate cell surface biomarkers that are highly enriched in medulloblastoma. The results identified 380 genes with greater than a two-fold increase in the expression in the medulloblastoma compared with that in the normal cerebellum. To enrich for targets with accessibility for extracellular molecular probes, we further refined this list by filtering it with gene ontology to identify genes with protein localization on, or within, the plasma membrane. To validate this meta-analysis, the top 10 candidates were evaluated with immunohistochemistry. We identified two targets, fibrillin 2 and EphA3, which specifically stain medulloblastoma. These results demonstrate a novel bioinformatics approach that successfully identified cell surface and extracellular candidate markers enriched in medulloblastoma versus adjacent cerebellum. These two proteins are high-value targets for the development of tumor-specific probes in medulloblastoma. This bioinformatics method has broad utility for the identification of accessible molecular targets in a variety of cancers and will enable probe development for guided resection.

  7. Structure identification by Mass Spectrometry Non-Targeted Analysis using the US EPA’s CompTox Chemistry Dashboard

    Science.gov (United States)

    Identification of unknowns in mass spectrometry based non-targeted analyses (NTA) requires the integration of complementary pieces of data to arrive at a confident, consensus structure. Researchers use chemical reference databases, spectral matching, fragment prediction tools, r...

  8. Baby or bathwater? Referrals of "non-cases" in a targeted early identification intervention for psychosis.

    Science.gov (United States)

    Jordan, Gerald; Kinkaid, Miriam; Iyer, Srividya N; Joober, Ridha; Goldberg, Karen; Malla, Ashok; Shah, Jai L

    2018-03-14

    To explore the unintended impact of a targeted case identification (TCI) campaign for first episode psychosis (FEP) on people not experiencing FEP ("non-cases") with respect to referral patterns and reasons for being a non-case. Sources of referral, reasons for being a non-case, and subsequent referral destinations of non-cases were examined before and after a TCI. Following the TCI, a greater proportion of non-cases lived outside the study catchment area. A smaller proportion was referred by the parent hospital's emergency room or had a substance-induced psychosis. TCIs for FEP may have unintended effects, with implications for early case identification and early intervention services.

  9. Muddled mechanisms: recent progress towards antimalarial target identification [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Rachel L. Edwards

    2016-10-01

    Full Text Available In the past decade, malaria rates have plummeted as a result of aggressive infection control measures and the adoption of artemisinin-based combination therapies (ACTs. However, a potential crisis looms ahead. Treatment failures to standard antimalarial regimens have been reported in Southeast Asia, and devastating consequences are expected if resistance spreads to the African continent. To prevent a potential public health emergency, the antimalarial arsenal must contain therapeutics with novel mechanisms of action (MOA. An impressive number of high-throughput screening (HTS campaigns have since been launched, identifying thousands of compounds with activity against one of the causative agents of malaria, Plasmodium falciparum. Now begins the difficult task of target identification, for which studies are often tedious, labor intensive, and difficult to interpret. In this review, we highlight approaches that have been instrumental in tackling the challenges of target assignment and elucidation of the MOA for hit compounds. Studies that apply these innovative techniques to antimalarial target identification are described, as well as the impact of the data in the field.

  10. Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

    Directory of Open Access Journals (Sweden)

    Moizza Mansoor

    2008-01-01

    Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

  11. Systematic identification of combinatorial drivers and targets in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Adel Tabchy

    Full Text Available There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

  12. Systematic identification of combinatorial drivers and targets in cancer cell lines.

    Science.gov (United States)

    Tabchy, Adel; Eltonsy, Nevine; Housman, David E; Mills, Gordon B

    2013-01-01

    There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

  13. Chemical Genomics and Emerging DNA Technologies in the Identification of Drug Mechanisms and Drug Targets

    DEFF Research Database (Denmark)

    Olsen, Louise Cathrine Braun; Færgeman, Nils J.

    2012-01-01

    and validate therapeutic targets and to discover drug candidates for rapidly and effectively generating new interventions for human diseases. The recent emergence of genomic technologies and their application on genetically tractable model organisms like Drosophila melanogaster,Caenorhabditis elegans...... critical roles in the genomic age of biological research and drug discovery. In the present review we discuss how simple biological model organisms can be used as screening platforms in combination with emerging genomic technologies to advance the identification of potential drugs and their molecular...

  14. In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Silvia Schiarea

    Full Text Available The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s, and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation were analyzed by high-resolution accurate mass liquid chromatography-multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man2(Xyl (Fuc GlcNAc2, Man3(Xyl (Fuc GlcNAc2, GlcNAcMan3(Xyl (Fuc GlcNAc2 and GlcNAc 2Man3(Xyl (Fuc GlcNAc2. These carry both core β1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants, but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit.

  15. Computational identification of 18 micrornas and their targets in three species of rose

    International Nuclear Information System (INIS)

    Baloch, I.A.; Barozai, M.Y.K.; Achakzai, A.K.K.

    2015-01-01

    MicroRNAs (miRNAs) are non-protein coding, small endogenous RNAs. Their length ranges from 18-26 nucleotides (nt). The miRNAs convergence property becomes a rational approach for the hunt of novel miRNAs in other organisms by homology search. As presently very little miRNAs are reported for rose species, so this study deals with the identification of miRNAs in different species of rose. Consequently 18 miRNA belonging to 17 miRNA families were identified in 3 species of rose (Rosa hybrid, Rosa chinensis and Rosa virginiana). All of the identified miRNA families (miR156, 160, 164, 166, 398, 482, 831, 837, 838, 841, 847, 3436, 3627, 6135, 6285, 6287 and 6288) are being reported for the first time in rose. Precursors of the identified miRNAs form stable minimum free energy (MFE) stem-loop structures and the mature miRNAs are found in the stem portions of their corresponding precursors. 11 putative targets of the miRNAs have also been identified. The identified targets are various proteins including transcription factors. Identification of 18 miRNAs will be supportive to explore the gene regulation phenomenon in various species of roses and it will be a good contribution for understanding the post transcriptional gene regulation in various stages of the life cycles of roses. (author)

  16. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Michael P Lake

    Full Text Available Transmission electron microscopy (TEM can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.

  17. Drug Target Identification and Elucidation of Natural Inhibitors for : An Study

    Directory of Open Access Journals (Sweden)

    Surya Narayan Rath

    2016-12-01

    Full Text Available Environmental microbes like Bordetella petrii has been established as a causative agent for various infectious diseases in human. Again, development of drug resistance in B. petrii challenged to combat against the infection. Identification of potential drug target and proposing a novel lead compound against the pathogen has a great aid and value. In this study, bioinformatics tools and technology have been applied to suggest a potential drug target by screening the proteome information of B. petrii DSM 12804 (accession No. PRJNA28135 from genome database of National Centre for Biotechnology information. In this regards, the inhibitory effect of nine natural compounds like ajoene (Allium sativum, allicin (A. sativum, cinnamaldehyde (Cinnamomum cassia, curcumin (Curcuma longa, gallotannin (active component of green tea and red wine, isoorientin (Anthopterus wardii, isovitexin (A. wardii, neral (Melissa officinalis, and vitexin (A. wardii have been acknowledged with anti-bacterial properties and hence tested against identified drug target of B. petrii by implicating computational approach. The in silico studies revealed the hypothesis that lpxD could be a potential drug target and with recommendation of a strong inhibitory effect of selected natural compounds against infection caused due to B. petrii, would be further validated through in vitro experiments.

  18. Identification of miRNAs and their target genes in developing soybean seeds by deep sequencing

    Directory of Open Access Journals (Sweden)

    Chen Shou-Yi

    2011-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs regulate gene expression by mediating gene silencing at transcriptional and post-transcriptional levels in higher plants. miRNAs and related target genes have been widely studied in model plants such as Arabidopsis and rice; however, the number of identified miRNAs in soybean (Glycine max is limited, and global identification of the related miRNA targets has not been reported in previous research. Results In our study, a small RNA library and a degradome library were constructed from developing soybean seeds for deep sequencing. We identified 26 new miRNAs in soybean by bioinformatic analysis and further confirmed their expression by stem-loop RT-PCR. The miRNA star sequences of 38 known miRNAs and 8 new miRNAs were also discovered, providing additional evidence for the existence of miRNAs. Through degradome sequencing, 145 and 25 genes were identified as targets of annotated miRNAs and new miRNAs, respectively. GO analysis indicated that many of the identified miRNA targets may function in soybean seed development. Additionally, a soybean homolog of Arabidopsis SUPPRESSOR OF GENE SLIENCING 3 (AtSGS3 was detected as a target of the newly identified miRNA Soy_25, suggesting the presence of feedback control of miRNA biogenesis. Conclusions We have identified large numbers of miRNAs and their related target genes through deep sequencing of a small RNA library and a degradome library. Our study provides more information about the regulatory network of miRNAs in soybean and advances our understanding of miRNA functions during seed development.

  19. Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

    Science.gov (United States)

    Nongonierma, Alice B; FitzGerald, Richard J

    2018-06-01

    Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

  20. Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing.

    Science.gov (United States)

    Hanson, E; Ingold, S; Haas, C; Ballantyne, J

    2018-05-01

    The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable 'source level' information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and

  1. Targeted sequencing of clade-specific markers from skin microbiomes for forensic human identification.

    Science.gov (United States)

    Schmedes, Sarah E; Woerner, August E; Novroski, Nicole M M; Wendt, Frank R; King, Jonathan L; Stephens, Kathryn M; Budowle, Bruce

    2018-01-01

    The human skin microbiome is comprised of diverse communities of bacterial, eukaryotic, and viral taxa and contributes millions of additional genes to the repertoire of human genes, affecting human metabolism and immune response. Numerous genetic and environmental factors influence the microbiome composition and as such contribute to individual-specific microbial signatures which may be exploited for forensic applications. Previous studies have demonstrated the potential to associate skin microbial profiles collected from touched items to their individual owner, mainly using unsupervised methods from samples collected over short time intervals. Those studies utilize either targeted 16S rRNA or shotgun metagenomic sequencing to characterize skin microbiomes; however, these approaches have limited species and strain resolution and susceptibility to stochastic effects, respectively. Clade-specific markers from the skin microbiome, using supervised learning, can predict individual identity using skin microbiomes from their respective donors with high accuracy. In this study the hidSkinPlex is presented, a novel targeted sequencing method using skin microbiome markers developed for human identification. The hidSkinPlex (comprised of 286 bacterial (and phage) family-, genus-, species-, and subspecies-level markers), initially was evaluated on three bacterial control samples represented in the panel (i.e., Propionibacterium acnes, Propionibacterium granulosum, and Rothia dentocariosa) to assess the performance of the multiplex. The hidSkinPlex was further evaluated for prediction purposes. The hidSkinPlex markers were used to attribute skin microbiomes collected from eight individuals from three body sites (i.e., foot (Fb), hand (Hp) and manubrium (Mb)) to their host donor. Supervised learning, specifically regularized multinomial logistic regression and 1-nearest-neighbor classification were used to classify skin microbiomes to their hosts with up to 92% (Fb), 96% (Mb

  2. Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation.

    Directory of Open Access Journals (Sweden)

    Erica Larschan

    Full Text Available Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to "chromatin entry sites," which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.

  3. Network Understanding of Herb Medicine via Rapid Identification of Ingredient-Target Interactions

    Science.gov (United States)

    Zhang, Hai-Ping; Pan, Jian-Bo; Zhang, Chi; Ji, Nan; Wang, Hao; Ji, Zhi-Liang

    2014-01-01

    Today, herb medicines have become the major source for discovery of novel agents in countermining diseases. However, many of them are largely under-explored in pharmacology due to the limitation of current experimental approaches. Therefore, we proposed a computational framework in this study for network understanding of herb pharmacology via rapid identification of putative ingredient-target interactions in human structural proteome level. A marketing anti-cancer herb medicine in China, Yadanzi (Brucea javanica), was chosen for mechanistic study. Total 7,119 ingredient-target interactions were identified for thirteen Yadanzi active ingredients. Among them, about 29.5% were estimated to have better binding affinity than their corresponding marketing drug-target interactions. Further Bioinformatics analyses suggest that simultaneous manipulation of multiple proteins in the MAPK signaling pathway and the phosphorylation process of anti-apoptosis may largely answer for Yadanzi against non-small cell lung cancers. In summary, our strategy provides an efficient however economic solution for systematic understanding of herbs' power.

  4. Mycoplasma non-coding RNA: identification of small RNAs and targets

    Directory of Open Access Journals (Sweden)

    Franciele Maboni Siqueira

    2016-10-01

    Full Text Available Abstract Background Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system. Results A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity. Conclusion This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms.

  5. Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

    2013-03-22

    Highlights: ► We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ► We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ► SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ► SS-related genes were selected from database by in silico analyses. ► 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly

  6. Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis

    Directory of Open Access Journals (Sweden)

    Jolly Emmitt R

    2005-11-01

    Full Text Available Abstract Background A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. Results Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. Conclusion We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

  7. Hazard identification and risk assessment for biologics targeting the immune system.

    Science.gov (United States)

    Weir, Andrea B

    2008-01-01

    Biologic pharmaceuticals include a variety of products, such as monoclonal antibodies, fusion proteins and cytokines. Products in those classes include immunomodulatory biologics, which are intended to enhance or diminish the activity of the immune system. Immunomodulatory biologics have been approved by the U.S. FDA for a variety of indications, including cancer and inflammatory conditions. Prior to gaining approval for marketing, sponsoring companies for all types of products must demonstrate a product's safety in toxicology studies conducted in animals and show safety and efficacy in clinical trials conducted in patients. The overall goal of toxicology studies, which applies to immunomodulatory and other product types, is to identify the hazards that products pose to humans. Because biologics are generally highly selective for specific targets (receptors/epitopes), conducting toxicology studies in animal models with the target is essential. Such animals are referred to as pharmacologically relevant. Endpoints routinely included in toxicology studies, such as hematology, organ weight and histopathology, can be used to assess the effect of a product on the structure of the immune system. Additionally, specialized endpoints, such as immunophenotyping and immune function tests, can be used to define effects of immunomodulatory products on the immune system. Following hazard identification, risks posed to patients are assessed and managed. Risks can be managed through clinical trial design and risk communication, a practice that applies to immunomodulatory and other product types. Examples of risk management in clinical trial design include establishing a safe starting dose, defining the appropriate patient population and establishing appropriate patient monitoring. Risk communication starts during clinical trials and continues after product approval. A combination of hazard identification, risk assessment and risk management allows for drug development to proceed

  8. Transcriptome-wide identification of Rauvolfia serpentina microRNAs and prediction of their potential targets.

    Science.gov (United States)

    Prakash, Pravin; Rajakani, Raja; Gupta, Vikrant

    2016-04-01

    MicroRNAs (miRNAs) are small non-coding RNAs of ∼ 19-24 nucleotides (nt) in length and considered as potent regulators of gene expression at transcriptional and post-transcriptional levels. Here we report the identification and characterization of 15 conserved miRNAs belonging to 13 families from Rauvolfia serpentina through in silico analysis of available nucleotide dataset. The identified mature R. serpentina miRNAs (rse-miRNAs) ranged between 20 and 22nt in length, and the average minimal folding free energy index (MFEI) value of rse-miRNA precursor sequences was found to be -0.815 kcal/mol. Using the identified rse-miRNAs as query, their potential targets were predicted in R. serpentina and other plant species. Gene Ontology (GO) annotation showed that predicted targets of rse-miRNAs include transcription factors as well as genes involved in diverse biological processes such as primary and secondary metabolism, stress response, disease resistance, growth, and development. Few rse-miRNAs were predicted to target genes of pharmaceutically important secondary metabolic pathways such as alkaloids and anthocyanin biosynthesis. Phylogenetic analysis showed the evolutionary relationship of rse-miRNAs and their precursor sequences to homologous pre-miRNA sequences from other plant species. The findings under present study besides giving first hand information about R. serpentina miRNAs and their targets, also contributes towards the better understanding of miRNA-mediated gene regulatory processes in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Pharmacologic Management of Duchenne Muscular Dystrophy: Target Identification and Preclinical Trials

    Science.gov (United States)

    Kornegay, Joe N.; Spurney, Christopher F.; Nghiem, Peter P.; Brinkmeyer-Langford, Candice L.; Hoffman, Eric P.; Nagaraju, Kanneboyina

    2014-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked human disorder in which absence of the protein dystrophin causes degeneration of skeletal and cardiac muscle. For the sake of treatment development, over and above definitive genetic and cell-based therapies, there is considerable interest in drugs that target downstream disease mechanisms. Drug candidates have typically been chosen based on the nature of pathologic lesions and presumed underlying mechanisms and then tested in animal models. Mammalian dystrophinopathies have been characterized in mice (mdx mouse) and dogs (golden retriever muscular dystrophy [GRMD]). Despite promising results in the mdx mouse, some therapies have not shown efficacy in DMD. Although the GRMD model offers a higher hurdle for translation, dogs have primarily been used to test genetic and cellular therapies where there is greater risk. Failed translation of animal studies to DMD raises questions about the propriety of methods and models used to identify drug targets and test efficacy of pharmacologic intervention. The mdx mouse and GRMD dog are genetically homologous to DMD but not necessarily analogous. Subcellular species differences are undoubtedly magnified at the whole-body level in clinical trials. This problem is compounded by disparate cultures in clinical trials and preclinical studies, pointing to a need for greater rigor and transparency in animal experiments. Molecular assays such as mRNA arrays and genome-wide association studies allow identification of genetic drug targets more closely tied to disease pathogenesis. Genes in which polymorphisms have been directly linked to DMD disease progression, as with osteopontin, are particularly attractive targets. PMID:24936034

  10. Comparative genomics identification of a novel set of temporally regulated hedgehog target genes in the retina.

    Science.gov (United States)

    McNeill, Brian; Perez-Iratxeta, Carol; Mazerolle, Chantal; Furimsky, Marosh; Mishina, Yuji; Andrade-Navarro, Miguel A; Wallace, Valerie A

    2012-03-01

    The hedgehog (Hh) signaling pathway is involved in numerous developmental and adult processes with many links to cancer. In vertebrates, the activity of the Hh pathway is mediated primarily through three Gli transcription factors (Gli1, 2 and 3) that can serve as transcriptional activators or repressors. The identification of Gli target genes is essential for the understanding of the Hh-mediated processes. We used a comparative genomics approach using the mouse and human genomes to identify 390 genes that contained conserved Gli binding sites. RT-qPCR validation of 46 target genes in E14.5 and P0.5 retinal explants revealed that Hh pathway activation resulted in the modulation of 30 of these targets, 25 of which demonstrated a temporal regulation. Further validation revealed that the expression of Bok, FoxA1, Sox8 and Wnt7a was dependent upon Sonic Hh (Shh) signaling in the retina and their regulation is under positive and negative controls by Gli2 and Gli3, respectively. We also show using chromatin immunoprecipitation that Gli2 binds to the Sox8 promoter, suggesting that Sox8 is an Hh-dependent direct target of Gli2. Finally, we demonstrate that the Hh pathway also modulates the expression of Sox9 and Sox10, which together with Sox8 make up the SoxE group. Previously, it has been shown that Hh and SoxE group genes promote Müller glial cell development in the retina. Our data are consistent with the possibility for a role of SoxE group genes downstream of Hh signaling on Müller cell development. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  11. An integrative in-silico approach for therapeutic target identification in the human pathogen Corynebacterium diphtheriae.

    Directory of Open Access Journals (Sweden)

    Syed Babar Jamal

    Full Text Available Corynebacterium diphtheriae (Cd is a Gram-positive human pathogen responsible for diphtheria infection and once regarded for high mortalities worldwide. The fatality gradually decreased with improved living standards and further alleviated when many immunization programs were introduced. However, numerous drug-resistant strains emerged recently that consequently decreased the efficacy of current therapeutics and vaccines, thereby obliging the scientific community to start investigating new therapeutic targets in pathogenic microorganisms. In this study, our contributions include the prediction of modelome of 13 C. diphtheriae strains, using the MHOLline workflow. A set of 463 conserved proteins were identified by combining the results of pangenomics based core-genome and core-modelome analyses. Further, using subtractive proteomics and modelomics approaches for target identification, a set of 23 proteins was selected as essential for the bacteria. Considering human as a host, eight of these proteins (glpX, nusB, rpsH, hisE, smpB, bioB, DIP1084, and DIP0983 were considered as essential and non-host homologs, and have been subjected to virtual screening using four different compound libraries (extracted from the ZINC database, plant-derived natural compounds and Di-terpenoid Iso-steviol derivatives. The proposed ligand molecules showed favorable interactions, lowered energy values and high complementarity with the predicted targets. Our proposed approach expedites the selection of C. diphtheriae putative proteins for broad-spectrum development of novel drugs and vaccines, owing to the fact that some of these targets have already been identified and validated in other organisms.

  12. Identification and characterization of angiogenesis targets through proteomic profiling of endothelial cells in human cancer tissues.

    Directory of Open Access Journals (Sweden)

    Mehdi Mesri

    Full Text Available Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.

  13. Sleep and eyewitness memory: Fewer false identifications after sleep when the target is absent from the lineup.

    Science.gov (United States)

    Stepan, Michelle E; Dehnke, Taylor M; Fenn, Kimberly M

    2017-01-01

    Inaccurate eyewitness identifications are the leading cause of known false convictions in the United States. Moreover, improving eyewitness memory is difficult and often unsuccessful. Sleep consistently strengthens and protects memory from interference, particularly when a recall test is used. However, the effect of sleep on recognition memory is more equivocal. Eyewitness identification tests are often recognition based, thus leaving open the question of how sleep affects recognition performance in an eyewitness context. In the current study, we investigated the effect of sleep on eyewitness memory. Participants watched a video of a mock-crime and attempted to identify the perpetrator from a simultaneous lineup after a 12-hour retention interval that either spanned a waking day or night of sleep. In Experiment 1, we used a target-present lineup and, in Experiment 2, we used a target-absent lineup in order to investigate correct and false identifications, respectively. Sleep reduced false identifications in the target-absent lineup (Experiment 2) but had no effect on correct identifications in the target-present lineup (Experiment 1). These results are discussed with respect to memory strength and decision making strategies.

  14. Sleep and eyewitness memory: Fewer false identifications after sleep when the target is absent from the lineup

    Science.gov (United States)

    Dehnke, Taylor M.; Fenn, Kimberly M.

    2017-01-01

    Inaccurate eyewitness identifications are the leading cause of known false convictions in the United States. Moreover, improving eyewitness memory is difficult and often unsuccessful. Sleep consistently strengthens and protects memory from interference, particularly when a recall test is used. However, the effect of sleep on recognition memory is more equivocal. Eyewitness identification tests are often recognition based, thus leaving open the question of how sleep affects recognition performance in an eyewitness context. In the current study, we investigated the effect of sleep on eyewitness memory. Participants watched a video of a mock-crime and attempted to identify the perpetrator from a simultaneous lineup after a 12-hour retention interval that either spanned a waking day or night of sleep. In Experiment 1, we used a target-present lineup and, in Experiment 2, we used a target-absent lineup in order to investigate correct and false identifications, respectively. Sleep reduced false identifications in the target-absent lineup (Experiment 2) but had no effect on correct identifications in the target-present lineup (Experiment 1). These results are discussed with respect to memory strength and decision making strategies. PMID:28877169

  15. Sleep and eyewitness memory: Fewer false identifications after sleep when the target is absent from the lineup.

    Directory of Open Access Journals (Sweden)

    Michelle E Stepan

    Full Text Available Inaccurate eyewitness identifications are the leading cause of known false convictions in the United States. Moreover, improving eyewitness memory is difficult and often unsuccessful. Sleep consistently strengthens and protects memory from interference, particularly when a recall test is used. However, the effect of sleep on recognition memory is more equivocal. Eyewitness identification tests are often recognition based, thus leaving open the question of how sleep affects recognition performance in an eyewitness context. In the current study, we investigated the effect of sleep on eyewitness memory. Participants watched a video of a mock-crime and attempted to identify the perpetrator from a simultaneous lineup after a 12-hour retention interval that either spanned a waking day or night of sleep. In Experiment 1, we used a target-present lineup and, in Experiment 2, we used a target-absent lineup in order to investigate correct and false identifications, respectively. Sleep reduced false identifications in the target-absent lineup (Experiment 2 but had no effect on correct identifications in the target-present lineup (Experiment 1. These results are discussed with respect to memory strength and decision making strategies.

  16. Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL.

    Science.gov (United States)

    Wen, Qiang; Goldenson, Benjamin; Silver, Serena J; Schenone, Monica; Dancik, Vlado; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy A; An, W Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S; Moore, Christopher B; Bliss-Moreau, Meghan; Verplank, Lynn; Tolliday, Nicola J; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S; Smith, Franklin O; Woods, William G; Taub, Jeffrey; Scherer, Christina A; Bradner, James E; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E; Gould, Robert J; Clemons, Paul A; Carr, Steven A; Root, David E; Schreiber, Stuart L; Stern, Andrew M; Crispino, John D

    2012-08-03

    The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Identification of Five Novel Variants in Chinese Oculocutaneous Albinism by Targeted Next-Generation Sequencing.

    Science.gov (United States)

    Qiu, Biyuan; Ma, Tao; Peng, Chunyan; Zheng, Xiaoqin; Yang, Jiyun

    2018-04-01

    The diagnosis of oculocutaneous albinism (OCA) is established using clinical signs and symptoms. OCA is, however, a highly genetically heterogeneous disease with mutations identified in at least nineteen unique genes, many of which produce overlapping phenotypic traits. Thus, differentiating genetic OCA subtypes for diagnoses and genetic counseling is challenging, based on clinical presentation alone, and would benefit from a comprehensive molecular diagnostic. To develop and validate a more comprehensive, targeted, next-generation-sequencing-based diagnostic for the identification of OCA-causing variants. The genomic DNA samples from 28 OCA probands were analyzed by targeted next-generation sequencing (NGS), and the candidate variants were confirmed through Sanger sequencing. We observed mutations in the TYR, OCA2, and SLC45A2 genes in 25/28 (89%) patients with OCA. We identified 38 pathogenic variants among these three genes, including 5 novel variants: c.1970G>T (p.Gly657Val), c.1669A>C (p.Thr557Pro), c.2339-2A>C, and c.1349C>G (p.Thr450Arg) in OCA2; c.459_470delTTTTGCTGCCGA (p.Ala155_Phe158del) in SLC45A2. Our findings expand the mutational spectrum of OCA in the Chinese population, and the assay we developed should be broadly useful as a molecular diagnostic, and as an aid for genetic counseling for OCA patients.

  18. Direct Quantification of Methane Emissions Across the Supply Chain: Identification of Mitigation Targets

    Science.gov (United States)

    Darzi, M.; Johnson, D.; Heltzel, R.; Clark, N.

    2017-12-01

    Researchers at West Virginia University's Center for Alternative Fuels, Engines, and Emissions have recently participated in a variety of studies targeted at direction quantification of methane emissions from across the natural gas supply chain. These studies included assessing methane emissions from heavy-duty vehicles and their fuel stations, active unconventional well sites - during both development and production, natural gas compression and storage facilities, natural gas engines - both large and small, two- and four-stroke, and low-throughput equipment associated with coal bed methane wells. Engine emissions were sampled using conventional instruments such as Fourier transform infrared spectrometers and heated flame ionization detection analyzers. However, to accurately quantify a wide range of other sources beyond the tailpipe (both leaks and losses), a full flow sampling system was developed, which included an integrated cavity-enhanced absorption spectrometer. Through these direct quantification efforts and analysis major sources of methane emissions were identified. Technological solutions and best practices exist or could be developed to reduce methane emissions by focusing on the "lowest-hanging fruit." For example, engine crankcases from across the supply chain should employ vent mitigation systems to reduce methane and other emissions. An overview of the direct quantification system and various campaign measurements results will be presented along with the identification of other targets for additional mitigation.

  19. Detection and Identification of Multiple Stationary Human Targets Via Bio-Radar Based on the Cross-Correlation Method

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2016-10-01

    Full Text Available Ultra-wideband (UWB radar has been widely used for detecting human physiological signals (respiration, movement, etc. in the fields of rescue, security, and medicine owing to its high penetrability and range resolution. In these applications, especially in rescue after disaster (earthquake, collapse, mine accident, etc., the presence, number, and location of the trapped victims to be detected and rescued are the key issues of concern. Ample research has been done on the first issue, whereas the identification and localization of multi-targets remains a challenge. False positive and negative identification results are two common problems associated with the detection of multiple stationary human targets. This is mainly because the energy of the signal reflected from the target close to the receiving antenna is considerably stronger than those of the targets at further range, often leading to missing or false recognition if the identification method is based on the energy of the respiratory signal. Therefore, a novel method based on cross-correlation is proposed in this paper that is based on the relativity and periodicity of the signals, rather than on the energy. The validity of this method is confirmed through experiments using different scenarios; the results indicate a discernible improvement in the detection precision and identification of the multiple stationary targets.

  20. Detection and Identification of Multiple Stationary Human Targets Via Bio-Radar Based on the Cross-Correlation Method.

    Science.gov (United States)

    Zhang, Yang; Chen, Fuming; Xue, Huijun; Li, Zhao; An, Qiang; Wang, Jianqi; Zhang, Yang

    2016-10-27

    Ultra-wideband (UWB) radar has been widely used for detecting human physiological signals (respiration, movement, etc.) in the fields of rescue, security, and medicine owing to its high penetrability and range resolution. In these applications, especially in rescue after disaster (earthquake, collapse, mine accident, etc.), the presence, number, and location of the trapped victims to be detected and rescued are the key issues of concern. Ample research has been done on the first issue, whereas the identification and localization of multi-targets remains a challenge. False positive and negative identification results are two common problems associated with the detection of multiple stationary human targets. This is mainly because the energy of the signal reflected from the target close to the receiving antenna is considerably stronger than those of the targets at further range, often leading to missing or false recognition if the identification method is based on the energy of the respiratory signal. Therefore, a novel method based on cross-correlation is proposed in this paper that is based on the relativity and periodicity of the signals, rather than on the energy. The validity of this method is confirmed through experiments using different scenarios; the results indicate a discernible improvement in the detection precision and identification of the multiple stationary targets.

  1. The search and identification of the new immunodiagnostic targets of bacillus anthracis spore

    International Nuclear Information System (INIS)

    Biketov, S.; Dunaytsev, I.; Baranova, E.; Marinin, L.; Dyatlov, I.

    2009-01-01

    Spores of Bacillus anthracis have been used as bio warfare agent to bio terrorize purposes. As efficiency of anti-epidemic measures included urgent prevention and treatment is determined by terms within which the bio agent is identified. Direct and rapid spore detection by antibodies based detection system is very attractive alternative to current PCR-based assays or routine phenotyping which are the most accurate but are also complex, time-consumption and expensive. The main difficulty with respect to such kind of anthrax spores detection is a cross-reaction with spores of closely related bacteria. For development of species-specific antibodies to anthrax spores recombinant scFvs or hybridoma technique were used. In both case surface spore antigens contained species-specific epitopes are need. Among exosporium proteins only ExsF(BxpB), ExsK and SoaA are specific to B.cereus group. On the surface of B. anthracis spores, a unique tetrasaccharides containing an novel monosaccharide - anthrose, was discovered. It was shown that anthrose can be serving as species-specific target for B. anthracis spores detection. We have revealed that EA1 isolated from spore of Russians strain STI-1 contain carbohydrate which formed species-specific epitopes and determine immunogenicity of this antigen. Antibodies to this antigen specifically recognized the surface target of B. anthracis spores and do not reacted with others Bacillus spore. Based on these antibodies we developed the test-systems in different formats for rapid direct detection and identification of B. anthracis spores. The results of trial these test-systems with using more than 50 different Bacillus strains were indicated that carbohydrate of EA1 isolated from spore is effective immunodiagnostic target for anthrax spores bio detection.(author)

  2. Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

    Directory of Open Access Journals (Sweden)

    Anna Frenzel

    Full Text Available Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C. Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

  3. Comparative genomics allowed the identification of drug targets against human fungal pathogens

    Directory of Open Access Journals (Sweden)

    Martins Natalia F

    2011-01-01

    Full Text Available Abstract Background The prevalence of invasive fungal infections (IFIs has increased steadily worldwide in the last few decades. Particularly, there has been a global rise in the number of infections among immunosuppressed people. These patients present severe clinical forms of the infections, which are commonly fatal, and they are more susceptible to opportunistic fungal infections than non-immunocompromised people. IFIs have historically been associated with high morbidity and mortality, partly because of the limitations of available antifungal therapies, including side effects, toxicities, drug interactions and antifungal resistance. Thus, the search for alternative therapies and/or the development of more specific drugs is a challenge that needs to be met. Genomics has created new ways of examining genes, which open new strategies for drug development and control of human diseases. Results In silico analyses and manual mining selected initially 57 potential drug targets, based on 55 genes experimentally confirmed as essential for Candida albicans or Aspergillus fumigatus and other 2 genes (kre2 and erg6 relevant for fungal survival within the host. Orthologs for those 57 potential targets were also identified in eight human fungal pathogens (C. albicans, A. fumigatus, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Paracoccidioides lutzii, Coccidioides immitis, Cryptococcus neoformans and Histoplasma capsulatum. Of those, 10 genes were present in all pathogenic fungi analyzed and absent in the human genome. We focused on four candidates: trr1 that encodes for thioredoxin reductase, rim8 that encodes for a protein involved in the proteolytic activation of a transcriptional factor in response to alkaline pH, kre2 that encodes for α-1,2-mannosyltransferase and erg6 that encodes for Δ(24-sterol C-methyltransferase. Conclusions Our data show that the comparative genomics analysis of eight fungal pathogens enabled the identification of

  4. Identification of fungal DNA barcode targets and PCR primers based on Pfam protein families and taxonomic hierarchy

    NARCIS (Netherlands)

    Lewis, C.T.; Bilkhu, S.; Robert, V.; Eberhardt, U.; Szoke, S.; Seifert, K.A.; Lévesque, C.A.

    2011-01-01

    Abstract: DNA barcoding is the application of DNA sequences of standardized genetic markers for the identification of eukaryotic organisms. We attempted to identify alternative candidate barcode gene targets for the fungal biota from available fungal genomes using a taxonomy-aware processing

  5. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  6. A distributed computational search strategy for the identification of diagnostics targets: application to finding aptamer targets for methicillin-resistant staphylococci.

    Science.gov (United States)

    Flanagan, Keith; Cockell, Simon; Harwood, Colin; Hallinan, Jennifer; Nakjang, Sirintra; Lawry, Beth; Wipat, Anil

    2014-06-30

    The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

  7. A distributed computational search strategy for the identification of diagnostics targets: Application to finding aptamer targets for methicillin-resistant staphylococci

    Directory of Open Access Journals (Sweden)

    Flanagan Keith

    2014-06-01

    Full Text Available The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

  8. Identification of direct regulatory targets of the transcription factor Sox10 based on function and conservation

    Directory of Open Access Journals (Sweden)

    Lee Sanghyuk

    2008-09-01

    Full Text Available Abstract Background Sox10, a member of the Sry-related HMG-Box gene family, is a critical transcription factor for several important cell lineages, most notably the neural crest stem cells and the derivative peripheral glial cells and melanocytes. Thus far, only a handful of direct target genes are known for this transcription factor limiting our understanding of the biological network it governs. Results We describe identification of multiple direct regulatory target genes of Sox10 through a procedure based on function and conservation. By combining RNA interference technique and DNA microarray technology, we have identified a set of genes that show significant down-regulation upon introduction of Sox10 specific siRNA into Schwannoma cells. Subsequent comparative genomics analyses led to potential binding sites for Sox10 protein conserved across several mammalian species within the genomic region proximal to these genes. Multiple sites belonging to 4 different genes (proteolipid protein, Sox10, extracellular superoxide dismutase, and pleiotrophin were shown to directly interact with Sox10 by chromatin immunoprecipitation assay. We further confirmed the direct regulation through the identified cis-element for one of the genes, extracellular superoxide dismutase, using electrophoretic mobility shift assay and reporter assay. Conclusion In sum, the process of combining differential expression profiling and comparative genomics successfully led to further defining the role of Sox10, a critical transcription factor for the development of peripheral glia. Our strategy utilizing relatively accessible techniques and tools should be applicable to studying the function of other transcription factors.

  9. eap Gene as novel target for specific identification of Staphylococcus aureus.

    Science.gov (United States)

    Hussain, Muzaffar; von Eiff, Christof; Sinha, Bhanu; Joost, Insa; Herrmann, Mathias; Peters, Georg; Becker, Karsten

    2008-02-01

    The cell surface-associated extracellular adherence protein (Eap) mediates adherence of Staphylococcus aureus to host extracellular matrix components and inhibits inflammation, wound healing, and angiogenesis. A well-characterized collection of S. aureus and non-S. aureus staphylococcal isolates (n = 813) was tested for the presence of the Eap-encoding gene (eap) by PCR to investigate the use of the eap gene as a specific diagnostic tool for identification of S. aureus. Whereas all 597 S. aureus isolates were eap positive, this gene was not detectable in 216 non-S. aureus staphylococcal isolates comprising 47 different species and subspecies of coagulase-negative staphylococci and non-S. aureus coagulase-positive or coagulase-variable staphylococci. Furthermore, non-S. aureus isolates did not express Eap homologs, as verified on the transcriptional and protein levels. Based on these data, the sensitivity and specificity of the newly developed PCR targeting the eap gene were both 100%. Thus, the unique occurrence of Eap in S. aureus offers a promising tool particularly suitable for molecular diagnostics of this pathogen.

  10. Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

    Science.gov (United States)

    Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

    2014-05-01

    Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

  11. Identification of Multiple Cryptococcal Fungicidal Drug Targets by Combined Gene Dosing and Drug Affinity Responsive Target Stability Screening

    Directory of Open Access Journals (Sweden)

    Yoon-Dong Park

    2016-08-01

    Full Text Available Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development.

  12. Combined effects of expectations and visual uncertainty upon detection and identification of a target in the fog.

    Science.gov (United States)

    Quétard, Boris; Quinton, Jean-Charles; Colomb, Michèle; Pezzulo, Giovanni; Barca, Laura; Izaute, Marie; Appadoo, Owen Kevin; Mermillod, Martial

    2015-09-01

    Detecting a pedestrian while driving in the fog is one situation where the prior expectation about the target presence is integrated with the noisy visual input. We focus on how these sources of information influence the oculomotor behavior and are integrated within an underlying decision-making process. The participants had to judge whether high-/low-density fog scenes displayed on a computer screen contained a pedestrian or a deer by executing a mouse movement toward the response button (mouse-tracking). A variable road sign was added on the scene to manipulate expectations about target identity. We then analyzed the timing and amplitude of the deviation of mouse trajectories toward the incorrect response and, using an eye tracker, the detection time (before fixating the target) and the identification time (fixations on the target). Results revealed that expectation of the correct target results in earlier decisions with less deviation toward the alternative response, this effect being partially explained by the facilitation of target identification.

  13. Systems integration of biodefense omics data for analysis of pathogen-host interactions and identification of potential targets.

    Directory of Open Access Journals (Sweden)

    Peter B McGarvey

    2009-09-01

    Full Text Available The NIAID (National Institute for Allergy and Infectious Diseases Biodefense Proteomics program aims to identify targets for potential vaccines, therapeutics, and diagnostics for agents of concern in bioterrorism, including bacterial, parasitic, and viral pathogens. The program includes seven Proteomics Research Centers, generating diverse types of pathogen-host data, including mass spectrometry, microarray transcriptional profiles, protein interactions, protein structures and biological reagents. The Biodefense Resource Center (www.proteomicsresource.org has developed a bioinformatics framework, employing a protein-centric approach to integrate and support mining and analysis of the large and heterogeneous data. Underlying this approach is a data warehouse with comprehensive protein + gene identifier and name mappings and annotations extracted from over 100 molecular databases. Value-added annotations are provided for key proteins from experimental findings using controlled vocabulary. The availability of pathogen and host omics data in an integrated framework allows global analysis of the data and comparisons across different experiments and organisms, as illustrated in several case studies presented here. (1 The identification of a hypothetical protein with differential gene and protein expressions in two host systems (mouse macrophage and human HeLa cells infected by different bacterial (Bacillus anthracis and Salmonella typhimurium and viral (orthopox pathogens suggesting that this protein can be prioritized for additional analysis and functional characterization. (2 The analysis of a vaccinia-human protein interaction network supplemented with protein accumulation levels led to the identification of human Keratin, type II cytoskeletal 4 protein as a potential therapeutic target. (3 Comparison of complete genomes from pathogenic variants coupled with experimental information on complete proteomes allowed the identification and

  14. Genome-wide identification of microRNA targets in the neglected disease pathogens of the genus Echinococcus.

    Science.gov (United States)

    Macchiaroli, Natalia; Maldonado, Lucas L; Zarowiecki, Magdalena; Cucher, Marcela; Gismondi, María Inés; Kamenetzky, Laura; Rosenzvit, Mara Cecilia

    2017-06-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in biological processes such as development. MiRNAs silence target mRNAs by binding to complementary sequences in the 3'untranslated regions (3'UTRs). The parasitic helminths of the genus Echinococcus are the causative agents of echinococcosis, a zoonotic neglected disease. In previous work, we performed a comprehensive identification and characterization of Echinococcus miRNAs. However, current knowledge about their targets is limited. Since target prediction algorithms rely on complementarity between 3'UTRs and miRNA sequences, a major limitation is the lack of accurate sequence information of 3'UTR for most species including parasitic helminths. We performed RNA-seq and developed a pipeline that integrates the transcriptomic data with available genomic data of this parasite in order to identify 3'UTRs of Echinococcus canadensis. The high confidence set of 3'UTRs obtained allowed the prediction of miRNA targets in Echinococcus through a bioinformatic approach. We performed for the first time a comparative analysis of miRNA targets in Echinococcus and Taenia. We found that many evolutionarily conserved target sites in Echinococcus and Taenia may be functional and under selective pressure. Signaling pathways such as MAPK and Wnt were among the most represented pathways indicating miRNA roles in parasite growth and development. Genome-wide identification and characterization of miRNA target genes in Echinococcus provide valuable information to guide experimental studies in order to understand miRNA functions in the parasites biology. miRNAs involved in essential functions, especially those being absent in the host or showing sequence divergence with respect to host orthologs, might be considered as novel therapeutic targets for echinococcosis control. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Highly Efficient Release of Glycopeptides from Hydrazide Beads by Hydroxylamine Assisted PNGase F Deglycosylation for N-Glycoproteome Analysis.

    Science.gov (United States)

    Huang, Junfeng; Wan, Hao; Yao, Yating; Li, Jinan; Cheng, Kai; Mao, Jiawei; Chen, Jin; Wang, Yan; Qin, Hongqiang; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

    2015-10-20

    Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.

  16. Putative drug and vaccine target protein identification using comparative genomic analysis of KEGG annotated metabolic pathways of Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Damte, Dereje; Suh, Joo-Won; Lee, Seung-Jin; Yohannes, Sileshi Belew; Hossain, Md Akil; Park, Seung-Chun

    2013-07-01

    In the present study, a computational comparative and subtractive genomic/proteomic analysis aimed at the identification of putative therapeutic target and vaccine candidate proteins from Kyoto Encyclopedia of Genes and Genomes (KEGG) annotated metabolic pathways of Mycoplasma hyopneumoniae was performed for drug design and vaccine production pipelines against M.hyopneumoniae. The employed comparative genomic and metabolic pathway analysis with a predefined computational systemic workflow extracted a total of 41 annotated metabolic pathways from KEGG among which five were unique to M. hyopneumoniae. A total of 234 proteins were identified to be involved in these metabolic pathways. Although 125 non homologous and predicted essential proteins were found from the total that could serve as potential drug targets and vaccine candidates, additional prioritizing parameters characterize 21 proteins as vaccine candidate while druggability of each of the identified proteins evaluated by the DrugBank database prioritized 42 proteins suitable for drug targets. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Combined N-glycome and N-glycoproteome analysis of the Lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes

    DEFF Research Database (Denmark)

    Dam, Svend Secher; Thaysen-Andersen, Morten; Stenkjær, Eva

    2013-01-01

    Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial...... interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed...

  18. Identification of highly effective target genes for RNAi-mediated control of emerald ash borer, Agrilus planipennis.

    Science.gov (United States)

    Rodrigues, Thais B; Duan, Jian J; Palli, Subba R; Rieske, Lynne K

    2018-03-22

    Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), caused up to 90% mortality of larvae and adults. In EAB eggs, larvae, and adults, the hsp is expressed at higher levels when compared to that of shi. Ingestion of dsHSP and dsSHI caused mortality in both neonate larvae and adults. Administration of a mixture of both dsRNAs worked better than either dsRNA by itself. In contrast, injection of EAB.dsHSP and EAB.dsSHI did not cause mortality in T. castaneum. Thus, the two genes identified cause high mortality in the EAB with no apparent phenotype effects in a non-target organism, the red flour beetle, and could be used in RNAi-mediated control of this invasive pest.

  19. 20180318 - Structure identification by Mass Spectrometry Non-Targeted Analysis using the US EPA’s CompTox Chemistry Dashboard (ACS Spring)

    Science.gov (United States)

    Identification of unknowns in mass spectrometry based non-targeted analyses (NTA) requires the integration of complementary pieces of data to arrive at a confident, consensus structure. Researchers use chemical reference databases, spectral matching, fragment prediction tools, r...

  20. Leveraging 3D chemical similarity, target and phenotypic data in the identification of drug-protein and drug-adverse effect associations.

    Science.gov (United States)

    Vilar, Santiago; Hripcsak, George

    2016-01-01

    Drug-target identification is crucial to discover novel applications for existing drugs and provide more insights about mechanisms of biological actions, such as adverse drug effects (ADEs). Computational methods along with the integration of current big data sources provide a useful framework for drug-target and drug-adverse effect discovery. In this article, we propose a method based on the integration of 3D chemical similarity, target and adverse effect data to generate a drug-target-adverse effect predictor along with a simple leveraging system to improve identification of drug-targets and drug-adverse effects. In the first step, we generated a system for multiple drug-target identification based on the application of 3D drug similarity into a large target dataset extracted from the ChEMBL. Next, we developed a target-adverse effect predictor combining targets from ChEMBL with phenotypic information provided by SIDER data source. Both modules were linked to generate a final predictor that establishes hypothesis about new drug-target-adverse effect candidates. Additionally, we showed that leveraging drug-target candidates with phenotypic data is very useful to improve the identification of drug-targets. The integration of phenotypic data into drug-target candidates yielded up to twofold precision improvement. In the opposite direction, leveraging drug-phenotype candidates with target data also yielded a significant enhancement in the performance. The modeling described in the current study is simple and efficient and has applications at large scale in drug repurposing and drug safety through the identification of mechanism of action of biological effects.

  1. Automatic identification of bird targets with radar via patterns produced by wing flapping

    NARCIS (Netherlands)

    Zaugg, S.; Saporta, G.; van Loon, E.; Schmaljohann, H.; Liechti, F.

    2008-01-01

    Bird identification with radar is important for bird migration research, environmental impact assessments (e.g. wind farms), aircraft security and radar meteorology. In a study on bird migration, radar signals from birds, insects and ground clutter were recorded. Signals from birds show a typical

  2. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    International Nuclear Information System (INIS)

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian; Ansari, Nariman; Esner, Milan; Bickle, Marc; Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H.; Parczyk, Karsten; Prechtl, Stefan; Steigemann, Patrick

    2014-01-01

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  3. 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions

    Energy Technology Data Exchange (ETDEWEB)

    Wenzel, Carsten; Riefke, Björn; Gründemann, Stephan; Krebs, Alice; Christian, Sven; Prinz, Florian; Osterland, Marc; Golfier, Sven; Räse, Sebastian [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Ansari, Nariman [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Esner, Milan; Bickle, Marc [Max Planck Institute of Molecular Cell Biology and Genetics, High-Throughput Technology Development Studio (TDS), Dresden (Germany); Pampaloni, Francesco; Mattheyer, Christian; Stelzer, Ernst H. [Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt (Germany); Parczyk, Karsten; Prechtl, Stefan [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany); Steigemann, Patrick, E-mail: Patrick.Steigemann@bayer.com [Bayer Pharma AG, Global Drug Discovery, Muellerstrasse 178, 13353 Berlin (Germany)

    2014-04-15

    Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions. - Highlights: • Establishment of a novel method for 3D cell culture based high-content screening. • First reported high

  4. Identification of Cell Surface Proteins as Potential Immunotherapy Targets in 12 Pediatric Cancers

    Energy Technology Data Exchange (ETDEWEB)

    Orentas, Rimas J. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Yang, James J. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States); Wen, Xinyu; Wei, Jun S. [Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States); Mackall, Crystal L. [Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD (United States); Khan, Javed, E-mail: rimas.orentas@nih.gov [Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Gaithersburg, MD (United States)

    2012-12-17

    Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.

  5. CRISPR Approaches to Small Molecule Target Identification. | Office of Cancer Genomics

    Science.gov (United States)

    A long-standing challenge in drug development is the identification of the mechanisms of action of small molecules with therapeutic potential. A number of methods have been developed to address this challenge, each with inherent strengths and limitations. We here provide a brief review of these methods with a focus on chemical-genetic methods that are based on systematically profiling the effects of genetic perturbations on drug sensitivity.

  6. Identification of drug targets by chemogenomic and metabolomic profiling in yeast

    KAUST Repository

    Wu, Manhong; Zheng, Ming; Zhang, Weiruo; Suresh, Sundari; Schlecht, Ulrich; Fitch, William L.; Aronova, Sofia; Baumann, Stephan; Davis, Ronald; St.Onge, Robert; Dill, David L.; Peltz, Gary

    2012-01-01

    OBJECTIVE: To advance our understanding of disease biology, the characterization of the molecular target for clinically proven or new drugs is very important. Because of its simplicity and the availability of strains with individual deletions in all

  7. Multiresolution, Multi-Scale Target Identification and Tracking using the Anisotropic Diffusion Pyramid

    National Research Council Canada - National Science Library

    Acton, Scott

    1998-01-01

    ...: the anisotropic diffusion pyramid and the morphological pyramid. Coarse-to-fine target searches are implemented within the image pyramids, providing a lOOX improvement in computational expense over standard correlation-based approaches...

  8. Approaches to the identification of new nonhormonal targets for male contraception.

    Science.gov (United States)

    Kopf, Gregory S

    2008-10-01

    Fertility control is a global health issue with major personal and societal impact. Although, currently, there are several different options for contraception, the technologies behind these are antiquated, and the options for male-based contraception (i.e., withdrawal, condom and vasectomy) are inadequate. The genomic, proteomic and bioinformatic revolutions have provided new tools and new targets for contraceptive development, and the results of such approaches have identified gene products that play critical roles in male reproduction, thus expanding the array of potential targets for novel and innovative male-based contraceptives. This article will review the types of targets being considered in the development of nonhormonal male contraceptives and the technologies used to identify and validate these targets.

  9. Two-dimensional hidden semantic information model for target saliency detection and eyetracking identification

    Science.gov (United States)

    Wan, Weibing; Yuan, Lingfeng; Zhao, Qunfei; Fang, Tao

    2018-01-01

    Saliency detection has been applied to the target acquisition case. This paper proposes a two-dimensional hidden Markov model (2D-HMM) that exploits the hidden semantic information of an image to detect its salient regions. A spatial pyramid histogram of oriented gradient descriptors is used to extract features. After encoding the image by a learned dictionary, the 2D-Viterbi algorithm is applied to infer the saliency map. This model can predict fixation of the targets and further creates robust and effective depictions of the targets' change in posture and viewpoint. To validate the model with a human visual search mechanism, two eyetrack experiments are employed to train our model directly from eye movement data. The results show that our model achieves better performance than visual attention. Moreover, it indicates the plausibility of utilizing visual track data to identify targets.

  10. Identification of novel small-molecule Ulex europaeus I mimetics for targeted drug delivery.

    Science.gov (United States)

    Hamashin, Christa; Spindler, Lisa; Russell, Shannon; Schink, Amy; Lambkin, Imelda; O'Mahony, Daniel; Houghten, Richard; Pinilla, Clemencia

    2003-11-17

    Lectin mimetics have been identified that may have potential application towards targeted drug delivery. Synthetic multivalent polygalloyl constructs effectively competed with Ulex europaeus agglutinin I (UEA1) for binding to intestinal Caco-2 cell membranes.

  11. Typicality aids search for an unspecified target, but only in identification and not in attentional guidance.

    Science.gov (United States)

    Castelhano, Monica S; Pollatsek, Alexander; Cave, Kyle R

    2008-08-01

    Participants searched for a picture of an object, and the object was either a typical or an atypical category member. The object was cued by either the picture or its basic-level category name. Of greatest interest was whether it would be easier to search for typical objects than to search for atypical objects. The answer was"yes," but only in a qualified sense: There was a large typicality effect on response time only for name cues, and almost none of the effect was found in the time to locate (i.e., first fixate) the target. Instead, typicality influenced verification time-the time to respond to the target once it was fixated. Typicality is thus apparently irrelevant when the target is well specified by a picture cue; even when the target is underspecified (as with a name cue), it does not aid attentional guidance, but only facilitates categorization.

  12. Effect of Various Environmental Stressors on Target Detection, Identification, and Marksmanship

    Science.gov (United States)

    2007-03-01

    diverses conditions environnementales difficiles, dont l’exposition à la chaleur et au froid, le bruit, l’exercice épuisant et la privation de sommeil...urban scenario used for marksmanship testing. The arrow indicates a walking target and the centrepiece depicting a poster of a soldier was a permanent... poster of a soldier was a permanent display that was used to hide targets. The heating and cooling protocols caused significant separations in core

  13. Identification of drug targets by chemogenomic and metabolomic profiling in yeast

    KAUST Repository

    Wu, Manhong

    2012-12-01

    OBJECTIVE: To advance our understanding of disease biology, the characterization of the molecular target for clinically proven or new drugs is very important. Because of its simplicity and the availability of strains with individual deletions in all of its genes, chemogenomic profiling in yeast has been used to identify drug targets. As measurement of drug-induced changes in cellular metabolites can yield considerable information about the effects of a drug, we investigated whether combining chemogenomic and metabolomic profiling in yeast could improve the characterization of drug targets. BASIC METHODS: We used chemogenomic and metabolomic profiling in yeast to characterize the target for five drugs acting on two biologically important pathways. A novel computational method that uses a curated metabolic network was also developed, and it was used to identify the genes that are likely to be responsible for the metabolomic differences found. RESULTS AND CONCLUSION: The combination of metabolomic and chemogenomic profiling, along with data analyses carried out using a novel computational method, could robustly identify the enzymes targeted by five drugs. Moreover, this novel computational method has the potential to identify genes that are causative of metabolomic differences or drug targets. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

  14. Statistical Use of Argonaute Expression and RISC Assembly in microRNA Target Identification

    Science.gov (United States)

    Stanhope, Stephen A.; Sengupta, Srikumar; den Boon, Johan; Ahlquist, Paul; Newton, Michael A.

    2009-01-01

    MicroRNAs (miRNAs) posttranscriptionally regulate targeted messenger RNAs (mRNAs) by inducing cleavage or otherwise repressing their translation. We address the problem of detecting m/miRNA targeting relationships in homo sapiens from microarray data by developing statistical models that are motivated by the biological mechanisms used by miRNAs. The focus of our modeling is the construction, activity, and mediation of RNA-induced silencing complexes (RISCs) competent for targeted mRNA cleavage. We demonstrate that regression models accommodating RISC abundance and controlling for other mediating factors fit the expression profiles of known target pairs substantially better than models based on m/miRNA expressions alone, and lead to verifications of computational target pair predictions that are more sensitive than those based on marginal expression levels. Because our models are fully independent of exogenous results from sequence-based computational methods, they are appropriate for use as either a primary or secondary source of information regarding m/miRNA target pair relationships, especially in conjunction with high-throughput expression studies. PMID:19779550

  15. Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

    Science.gov (United States)

    Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

    2017-03-21

    This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon

  16. Large-scale identification of microRNA targets in murine Dgcr8-deficient embryonic stem cell lines.

    Directory of Open Access Journals (Sweden)

    Matthew P A Davis

    Full Text Available Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3' UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.

  17. Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

    International Nuclear Information System (INIS)

    Quintana, Anita M; Liu, Fan; O'Rourke, John P; Ness, Scott A

    2011-01-01

    The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells

  18. Identification of attractive drug targets in neglected-disease pathogens using an in silico approach.

    Directory of Open Access Journals (Sweden)

    Gregory J Crowther

    Full Text Available BACKGROUND: The increased sequencing of pathogen genomes and the subsequent availability of genome-scale functional datasets are expected to guide the experimental work necessary for target-based drug discovery. However, a major bottleneck in this has been the difficulty of capturing and integrating relevant information in an easily accessible format for identifying and prioritizing potential targets. The open-access resource TDRtargets.org facilitates drug target prioritization for major tropical disease pathogens such as the mycobacteria Mycobacterium leprae and Mycobacterium tuberculosis; the kinetoplastid protozoans Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi; the apicomplexan protozoans Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii; and the helminths Brugia malayi and Schistosoma mansoni. METHODOLOGY/PRINCIPAL FINDINGS: Here we present strategies to prioritize pathogen proteins based on whether their properties meet criteria considered desirable in a drug target. These criteria are based upon both sequence-derived information (e.g., molecular mass and functional data on expression, essentiality, phenotypes, metabolic pathways, assayability, and druggability. This approach also highlights the fact that data for many relevant criteria are lacking in less-studied pathogens (e.g., helminths, and we demonstrate how this can be partially overcome by mapping data from homologous genes in well-studied organisms. We also show how individual users can easily upload external datasets and integrate them with existing data in TDRtargets.org to generate highly customized ranked lists of potential targets. CONCLUSIONS/SIGNIFICANCE: Using the datasets and the tools available in TDRtargets.org, we have generated illustrative lists of potential drug targets in seven tropical disease pathogens. While these lists are broadly consistent with the research community's current interest in certain specific proteins, and suggest

  19. Identification of potential target genes of ROR-alpha in THP1 and HUVEC cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Gulec, Cagri, E-mail: cagri.gulec@gmail.com; Coban, Neslihan, E-mail: neslic@istanbul.edu.tr; Ozsait-Selcuk, Bilge, E-mail: ozsaitb@istanbul.edu.tr; Sirma-Ekmekci, Sema, E-mail: semasirma@gmail.com; Yildirim, Ozlem, E-mail: ozlm-yildirim@hotmail.com; Erginel-Unaltuna, Nihan, E-mail: nihanerginel@yahoo.com

    2017-04-01

    ROR-alpha is a nuclear receptor, activity of which can be modulated by natural or synthetic ligands. Due to its possible involvement in, and potential therapeutic target for atherosclerosis, we aimed to identify ROR-alpha target genes in monocytic and endothelial cell lines. We performed chromatin immunoprecipitation (ChIP) followed by tiling array (ChIP-on-chip) for ROR-alpha in monocytic cell line THP1 and endothelial cell line HUVEC. Following bioinformatic analysis of the array data, we tested four candidate genes in terms of dependence of their expression level on ligand-mediated ROR-alpha activity, and two of them in terms of promoter occupancy by ROR-alpha. Bioinformatic analyses of ChIP-on-chip data suggested that ROR-alpha binds to genomic regions near the transcription start site (TSS) of more than 3000 genes in THP1 and HUVEC. Potential ROR-alpha target genes in both cell types seem to be involved mainly in membrane receptor activity, signal transduction and ion transport. While SPP1 and IKBKA were shown to be direct target genes of ROR-alpha in THP1 monocytes, inflammation related gene HMOX1 and heat shock protein gene HSPA8 were shown to be potential target genes of ROR-alpha. Our results suggest that ROR-alpha may regulate signaling receptor activity, and transmembrane transport activity through its potential target genes. ROR-alpha seems also to play role in cellular sensitivity to environmental substances like arsenite and chloroprene. Although, the expression analyses have shown that synthetic ROR-alpha ligands can modulate some of potential ROR-alpha target genes, functional significance of ligand-dependent modulation of gene expression needs to be confirmed with further analyses.

  20. Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells

    International Nuclear Information System (INIS)

    Ailan, He; Shuanglin, Xiang; Xiangwen, Xiao; Daolong, Ren; Lu, Gan; Xiaofeng, Ding; Xi, Qiao; Xingwang, Hu; Rushi, Liu; Jian, Zhang

    2009-01-01

    Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis. To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453. 20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them. This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future

  1. Computational identification of conserved microRNAs and their targets from expression sequence tags of blueberry (Vaccinium corybosum).

    Science.gov (United States)

    Li, Xuyan; Hou, Yanming; Zhang, Li; Zhang, Wenhao; Quan, Chen; Cui, Yuhai; Bian, Shaomin

    2014-01-01

    MicroRNAs (miRNAs) are a class of endogenous, approximately 21nt in length, non-coding RNA, which mediate the expression of target genes primarily at post-transcriptional levels. miRNAs play critical roles in almost all plant cellular and metabolic processes. Although numerous miRNAs have been identified in the plant kingdom, the miRNAs in blueberry, which is an economically important small fruit crop, still remain totally unknown. In this study, we reported a computational identification of miRNAs and their targets in blueberry. By conducting an EST-based comparative genomics approach, 9 potential vco-miRNAs were discovered from 22,402 blueberry ESTs according to a series of filtering criteria, designated as vco-miR156-5p, vco-miR156-3p, vco-miR1436, vco-miR1522, vco-miR4495, vco-miR5120, vco-miR5658, vco-miR5783, and vco-miR5986. Based on sequence complementarity between miRNA and its target transcript, 34 target ESTs from blueberry and 70 targets from other species were identified for the vco-miRNAs. The targets were found to be involved in transcription, RNA splicing and binding, DNA duplication, signal transduction, transport and trafficking, stress response, as well as synthesis and metabolic process. These findings will greatly contribute to future research in regard to functions and regulatory mechanisms of blueberry miRNAs.

  2. The single lineup paradigm: A new way to manipulate target presence in eyewitness identification experiments.

    Science.gov (United States)

    Oriet, Chris; Fitzgerald, Ryan J

    2018-02-01

    The suspect in eyewitness lineups may be guilty or innocent. These possibilities are traditionally simulated in eyewitness identification studies using a dual-lineup paradigm: All witnesses observe the same perpetrator and then receive one of two lineups. In this paradigm, the suspect's guilt is manipulated by including the perpetrator in one lineup and an innocent suspect in the other. The lineup is then filled with people matched to either the suspect (resulting in different fillers in perpetrator-present and perpetrator-absent lineups) or to the perpetrator (resulting in the same fillers in each lineup). An inescapable feature of the dual-lineup paradigm is that the perpetrator-present and perpetrator-absent lineups differ not only in the suspect's guilt, but also in their composition. Here, we describe a single-lineup paradigm: Subjects observe one of two perpetrators and then all subjects receive the same lineup containing one of the perpetrators. This alternative paradigm allows manipulation of the suspect's guilt without changing the lineup's composition. In three experiments, we applied the single-lineup paradigm to explore suspect-filler similarity and consistently found that increasing similarity reduced perpetrator identifications but did little to prevent innocent suspect misidentifications. Conversely, when fillers were matched to the perpetrator using a dual-lineup paradigm, increasing similarity reduced identification of perpetrators and innocent suspects. This finding suggests that the effect of filler similarity may depend on the person to whom the fillers are matched. We suggest that the single-lineup paradigm is a more ecologically valid and better controlled approach to creating suspect-matched lineups in laboratory investigations of eyewitness memory than existing procedures. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

  3. Identification of Rv0222 from RD4 as a novel serodiagnostic target for tuberculosis

    DEFF Research Database (Denmark)

    Rosenkrands, Ida; Aagaard, Claus; Weldingh, Karin

    2008-01-01

    tuberculosis patients and healthy controls led to identification of Rv0222 as the most promising serodiagnostic antigen. Recognition of the Rv0222 was compared with the 38 kDa protein and a fusion protein of the RD1 proteins ESAT-6 and CFP10 in a serum panel from pulmonary tuberculosis (TB) patients from......Forty-seven Mycobacterium tuberculosis genes from the 'regions of difference' RD2-7, RD9-13 and RD15 were cloned and expressed, and the purified recombinant proteins were screened for their serodiagnostic potential. Evaluation of six selected proteins in serum samples from Danish resident...

  4. Metabolic network analysis-based identification of antimicrobial drug targets in category A bioterrorism agents.

    Directory of Open Access Journals (Sweden)

    Yong-Yeol Ahn

    Full Text Available The 2001 anthrax mail attacks in the United States demonstrated the potential threat of bioterrorism, hence driving the need to develop sophisticated treatment and diagnostic protocols to counter biological warfare. Here, by performing flux balance analyses on the fully-annotated metabolic networks of multiple, whole genome-sequenced bacterial strains, we have identified a large number of metabolic enzymes as potential drug targets for each of the three Category A-designated bioterrorism agents including Bacillus anthracis, Francisella tularensis and Yersinia pestis. Nine metabolic enzymes- belonging to the coenzyme A, folate, phosphatidyl-ethanolamine and nucleic acid pathways common to all strains across the three distinct genera were identified as targets. Antimicrobial agents against some of these enzymes are available. Thus, a combination of cross species-specific antibiotics and common antimicrobials against shared targets may represent a useful combinatorial therapeutic approach against all Category A bioterrorism agents.

  5. Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.

    Science.gov (United States)

    Bosse, Kristopher R; Raman, Pichai; Zhu, Zhongyu; Lane, Maria; Martinez, Daniel; Heitzeneder, Sabine; Rathi, Komal S; Kendsersky, Nathan M; Randall, Michael; Donovan, Laura; Morrissy, Sorana; Sussman, Robyn T; Zhelev, Doncho V; Feng, Yang; Wang, Yanping; Hwang, Jennifer; Lopez, Gonzalo; Harenza, Jo Lynne; Wei, Jun S; Pawel, Bruce; Bhatti, Tricia; Santi, Mariarita; Ganguly, Arupa; Khan, Javed; Marra, Marco A; Taylor, Michael D; Dimitrov, Dimiter S; Mackall, Crystal L; Maris, John M

    2017-09-11

    We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Identification of the Schistosoma mansoni Molecular Target for the Antimalarial Drug Artemether

    KAUST Repository

    Lepore, Rosalba

    2011-11-28

    Plasmodium falciparum and Schistosoma mansonii are the parasites responsible for most of the malaria and schistosomiasis cases in the world. Notwithstanding their many differences, the two agents have striking similarities in that they both are blood feeders and are targets of an overlapping set of drugs, including the well-known artemether molecule. Here we explore the possibility of using the known information about the mode of action of artemether in Plasmodium to identify the molecular target of the drug in Schistosoma and provide evidence that artemether binds to SmSERCA, a putative Ca2+-ATPase of Schistosoma. We also predict the putative binding mode of the molecule for both its Plasmodium and Schistosoma targets. Our analysis of the mode of binding of artemether to Ca2+-ATPases also provides an explanation for the apparent paradox that, although the molecule has no side effect in humans, it has been shown to possess antitumoral activity. © 2011 American Chemical Society.

  7. Identification of target organs of copper nanoparticles with ICP-MS technique

    International Nuclear Information System (INIS)

    Zhen Chen; Huan Meng; Yun Wang; Chengcheng Zhang; Yuliang Zhao

    2007-01-01

    Nanosized copper particles are widely used in fields of lubricants, polymers/plastic, metallic coating and ink. Recently, we found that copper particles in different sizes can lead to different toxicological effects. To clarify the target organs of copper particles of different sizes, the inductively coupled plasma mass spectroscopy (ICP-MS) was employed to evaluate the distribution of copper in different organs of mice after a single dose oral exposure. The results suggest that the main target organs for copper nanoparticles are kidney, liver and blood. Liver is the main damaged organ. (author)

  8. Identification of Sarcosine as a Target Molecule for the Canine Olfactory Detection of Prostate Carcinoma.

    Science.gov (United States)

    Pacik, Dalibor; Plevova, Mariana; Urbanova, Lucie; Lackova, Zuzana; Strmiska, Vladislav; Necas, Alois; Heger, Zbynek; Adam, Vojtech

    2018-03-21

    The hypothesis that dogs can detect malignant tumours through the identification of specific molecules is nearly 30 years old. To date, several reports have described the successful detection of distinct types of cancer. However, is still a lack of data regarding the specific molecules that can be recognized by a dog's olfactory apparatus. Hence, we performed a study with artificially prepared, well-characterized urinary specimens that were enriched with sarcosine, a widely reported urinary biomarker for prostate cancer (PCa). For the purposes of the study, a German shepherd dog was utilized for analyses of 60 positive and 120 negative samples. Our study provides the first evidence that a sniffer dog specially trained for the olfactory detection of PCa can recognize sarcosine in artificial urine with a performance [sensitivity of 90%, specificity of 95%, and precision of 90% for the highest amount of sarcosine (10 µmol/L)] that is comparable to the identification of PCa-diagnosed subjects (sensitivity of 93.5% and specificity of 91.6%). This study casts light on the unrevealed phenomenon of PCa olfactory detection and opens the door for further studies with canine olfactory detection and cancer diagnostics.

  9. Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

    Science.gov (United States)

    Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Ingrassia, Valeria; Valenti, Vincenza; Giammanco, Antonina; Fayer, Francesca; Misiano, Gabriella; Cocorullo, Gianfranco; Scrimali, Chiara; Palesano, Ornella; Altieri, Grazia I; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

    Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  10. Identification of new possible targets for leukemia treatment by kinase activity profiling

    NARCIS (Netherlands)

    Ter Elst, Arja; Diks, Sander H.; Kampen, Kim R.; Hoogerbrugge, Peter M.; Ruijtenbeek, Rob; Boender, Piet J.; Sikkema, Arend H.; Scherpen, Frank J. G.; Kamps, Willem A.; Peppelenbosch, Maikel P.; de Bont, Eveline S. J. M.

    To date, the biology of acute leukemia has been unclear, and defining new therapeutic targets without prior knowledge remains complicated. The use of high-throughput techniques would enable us to learn more about the biology of the disease, and make it possible to directly assess a broader range of

  11. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Science.gov (United States)

    Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

    2011-01-01

    Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  12. Identification of target cells by immunohistochemical detection of covalently rearranged estradiol in rehydrated paraffin sections.

    Science.gov (United States)

    Jungblut, P W; Sierralta, W D

    1998-04-01

    Estradiol is released from the binding niche of the receptor and covalently arrested in the molecular vicinity by the Mannich reaction during target fixation in acetic acid/formaldehyde. The exposed steroid is freely accessible for appropriate antibodies. It can be visualized in sections by the second antibody/enzyme technique in high resolution and without enhancements.

  13. Genome-wide identification of structural variants in genes encoding drug targets

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Dahmcke, Christina Mackeprang

    2012-01-01

    The objective of the present study was to identify structural variants of drug target-encoding genes on a genome-wide scale. We also aimed at identifying drugs that are potentially amenable for individualization of treatments based on knowledge about structural variation in the genes encoding...

  14. Doing worse, but feeling happy : Social comparison and identification in response to upward and downward targets

    NARCIS (Netherlands)

    Groothof, H.; Siero, F.W.; Buunk, Abraham (Bram)

    2007-01-01

    We investigated people's responses to exposure to downward and upward targets. In Study 1, among 197 participants, it was predicted and found that such exposure led to a contrast effect on self-evaluation, and to an assimilation effect on affect. In Study 2, among 148 participants, it was predicted

  15. Identification of clinical target areas in the brainstem of prion‐infected mice

    Science.gov (United States)

    Mirabile, Ilaria; Jat, Parmjit S.; Brandner, Sebastian

    2015-01-01

    Aims While prion infection ultimately involves the entire brain, it has long been thought that the abrupt clinical onset and rapid neurological decline in laboratory rodents relates to involvement of specific critical neuroanatomical target areas. The severity and type of clinical signs, together with the rapid progression, suggest the brainstem as a candidate location for such critical areas. In this study we aimed to correlate prion pathology with clinical phenotype in order to identify clinical target areas. Method We conducted a comprehensive survey of brainstem pathology in mice infected with two distinct prion strains, which produce different patterns of pathology, in mice overexpressing prion protein (with accelerated clinical onset) and in mice in which neuronal expression was reduced by gene targeting (which greatly delays clinical onset). Results We identified specific brainstem areas that are affected by prion pathology during the progression of the disease. In the early phase of disease the locus coeruleus, the nucleus of the solitary tract, and the pre‐Bötzinger complex were affected by prion protein deposition. This was followed by involvement of the motor and autonomic centres of the brainstem. Conclusions Neurodegeneration in the locus coeruleus, the nucleus of the solitary tract and the pre‐Bötzinger complex predominated and corresponded to the manifestation of the clinical phenotype. Because of their fundamental role in controlling autonomic function and the overlap with clinical signs in sporadic Creutzfeldt–Jakob disease, we suggest that these nuclei represent key clinical target areas in prion diseases. PMID:25311251

  16. Identification and in vitro characterization of phage-displayed VHHs targeting VEGF

    DEFF Research Database (Denmark)

    Farajpour, Zahra; Rahbarizadeh, Fatemeh; Kazemi, Bahram

    2014-01-01

    Vascular endothelial growth factor (VEGF) is a potential target for cancer treatment because of its role in angiogenesis and its overexpression in most human cancers. Currently, anti-VEGF antibodies have been shown to be promising tools for therapeutic applications. However, large size, poor tumo...

  17. Identification and characterization of DNAzymes targeting DNA methyltransferase I for suppressing bladder cancer proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xiangbo; Zhang, Lu; Ding, Nianhua; Yang, Xinghui; Zhang, Jin; He, Jiang; Li, Zhi; Sun, Lun-Quan, E-mail: lunquansun@csu.edu.cn

    2015-05-29

    Epigenetic inactivation of genes plays a critical role in many important human diseases, especially in cancer. A core mechanism for epigenetic inactivation of the genes is methylation of CpG islands in genome DNA, which is catalyzed by DNA methyltransferases (DNMTs). The inhibition of DNMTs may lead to demethylation and expression of the silenced tumor suppressor genes. Although DNMT inhibitors are currently being developed as potential anticancer agents, only limited success is achieved due to substantial toxicity. Here, we utilized a multiplex selection system to generate efficient RNA-cleaving DNAzymes targeting DNMT1. The lead molecule from the selection was shown to possess efficient kinetic profiles and high efficiency in inhibiting the enzyme activity. Transfection of the DNAzyme caused significant down-regulation of DNMT1 expression and reactivation of p16 gene, resulting in reduced cell proliferation of bladder cancers. This study provides an alternative for targeting DNMTs for potential cancer therapy. - Highlights: • Identified DNMT1-targeted DNAzymes by multiplex selection system. • Biochemically characterized a lead DNAzyme with high kinetic efficiency. • Validated DNMT1-targeted DNAzyme in its enzymatic and cellular activities.

  18. Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

    Directory of Open Access Journals (Sweden)

    O'Rourke John P

    2011-01-01

    Full Text Available Abstract Background The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. Methods We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. Results By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Conclusions Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer

  19. Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.

    Science.gov (United States)

    Conwell, Sara E; White, Anne E; Harper, J Wade; Knipe, David M

    2015-01-01

    The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions. During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To

  20. Comprehensive Identification of SUMO2/3 Targets and Their Dynamics during Mitosis

    DEFF Research Database (Denmark)

    Schou, Julie; Kelstrup, Christian D; Hayward, Daniel G

    2014-01-01

    During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been...... identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins...... from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein...

  1. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Directory of Open Access Journals (Sweden)

    Yu-Hsuan Tu

    Full Text Available Lactoferricin B (LfcinB is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP assays. Sixteen proteins were identified, and an E. coli interaction database (EcID analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  2. Dana-Farber Cancer Institute: Identification of Therapeutic Targets Across Cancer Types | Office of Cancer Genomics

    Science.gov (United States)

    The Dana Farber Cancer Institute CTD2 Center focuses on the use of high-throughput genetic and bioinformatic approaches to identify and credential oncogenes and co-dependencies in cancers. This Center aims to provide the cancer research community with information that will facilitate the prioritization of targets based on both genomic and functional evidence, inform the most appropriate genetic context for downstream mechanistic and validation studies, and enable the translation of this information into therapeutics and diagnostics.

  3. Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea

    OpenAIRE

    Lapp, T.; Zaher, S. S.; Haas, C. T.; Becker, D. L.; Thrasivoulou, C.; Chain, B. M.; Larkin, D. F. P.; Noursadeghi, M.

    2015-01-01

    Purpose: We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment. Methods: Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned me...

  4. Proteomic Identification of Non-Gal Antibody Targets After Pig-to-Primate Cardiac Xenotransplantation

    Science.gov (United States)

    Byrne, Guerard W.; Stalboerger, Paul G.; Davila, Eduardo; Heppelmann, Carrie J.; Gazi, Mozammel H.; McGregor, Hugh C. J.; LaBreche, Peter T.; Davies, William R.; Rao, Vinay P.; Oi, Keiji; Tazelaar, Henry D.; Logan, John S.; McGregor, Christopher G. A.

    2008-01-01

    Background Experience with non-antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non-Gal endothelial cell (EC) antigens. Methods To gauge the diversity of the non-Gal antibody response we used antibody derived from CD46 transgenic heterotopic cardiac xenografts performed without T-cell immunosuppression, Group A (n = 4) and Gal knockout (GT-KO) heart transplants under tacrolimus and sirolimus immunosuppression, Group B (n = 8). Non-Gal antibody was measured by flow cytometry and by Western blots using GT-KO EC membrane antigens. A nanoLC/MS/MS analysis of proteins recovered from 2D gels was used to identify target antigens. Results Group A recipients exhibited a mixed cellular and humoral rejection. Group B recipients mainly exhibited classical DXR. Western blot analysis showed a non-Gal antibody response induced by GT+ and GT-KO hearts to an overlapping set of pig aortic EC membrane antigens. Proteomic analysis identified 14 potential target antigens but failed to define several immunodominant targets. Conclusions These experiments indicate that the non-Gal antibody response is directed to a number of stress response and inflammation related pig EC antigens and a few undefined targets. Further analysis of these antibody specificities using alternative methods is required to more fully define the repertoire of non-Gal antibody responses. PMID:18957049

  5. Identification of hookworm DAF-16/FOXO response elements and direct gene targets.

    Directory of Open Access Journals (Sweden)

    Xin Gao

    2010-08-01

    Full Text Available The infective stage of the parasitic nematode hookworm is developmentally arrested in the environment and needs to infect a specific host to complete its life cycle. The canine hookworm (Ancylostoma caninum is an excellent model for investigating human hookworm infections. The transcription factor of A. caninum, Ac-DAF-16, which has a characteristic fork head or "winged helix" DNA binding domain (DBD, has been implicated in the resumption of hookworm development in the host. However, the precise roles of Ac-DAF-16 in hookworm parasitism and its downstream targets are unknown. In the present study, we combined molecular techniques and bioinformatics to identify a group of Ac-DAF-16 binding sites and target genes.The DNA binding domain of Ac-DAF-16 was used to select genomic fragments by in vitro genomic selection. Twenty four bound genomic fragments were analyzed for the presence of the DAF-16 family binding element (DBE and possible alternative Ac-DAF-16 bind motifs. The 22 genes linked to these genomic fragments were identified using bioinformatics tools and defined as candidate direct gene targets of Ac-DAF-16. Their developmental stage-specific expression patterns were examined. Also, a new putative DAF-16 binding element was identified.Our results show that Ac-DAF-16 is involved in diverse biological processes throughout hookworm development. Further investigation of these target genes will provide insights into the molecular basis by which Ac-DAF-16 regulates its downstream gene network in hookworm infection.

  6. Identification of Androgen Receptor and Beta-Catenin Target Genes in Prostate and Prostate Cancer

    Science.gov (United States)

    2013-10-01

    Anderson A, Yang GY, Arbeit JM, and Auborn KJ. Indole- 3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice...signaling is prevalent in many cancers, most notably colorectal cancer; however their role in prostate cancer is becoming increasingly appreciated...condition 2 gives no background bands. F) Schematic of human PSA promoter and or β-catenin binding sites (TBE). G-H) Target specific PCR for PSA (G) or

  7. Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting.

    Directory of Open Access Journals (Sweden)

    Aman P Mann

    Full Text Available Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA against E-selectin (ESTA-1 by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (K(D = 47 nM while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition of sLe(x positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1 that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery.

  8. Targeted Nanodiamonds for Identification of Subcellular Protein Assemblies in Mammalian Cells

    OpenAIRE

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conj...

  9. Crowd Sourcing a New Paradigm for Interactome Driven Drug Target Identification in Mycobacterium tuberculosis

    Science.gov (United States)

    Rohira, Harsha; Bhat, Ashwini G.; Passi, Anurag; Mukherjee, Keya; Choudhary, Kumari Sonal; Kumar, Vikas; Arora, Anshula; Munusamy, Prabhakaran; Subramanian, Ahalyaa; Venkatachalam, Aparna; S, Gayathri; Raj, Sweety; Chitra, Vijaya; Verma, Kaveri; Zaheer, Salman; J, Balaganesh; Gurusamy, Malarvizhi; Razeeth, Mohammed; Raja, Ilamathi; Thandapani, Madhumohan; Mevada, Vishal; Soni, Raviraj; Rana, Shruti; Ramanna, Girish Muthagadhalli; Raghavan, Swetha; Subramanya, Sunil N.; Kholia, Trupti; Patel, Rajesh; Bhavnani, Varsha; Chiranjeevi, Lakavath; Sengupta, Soumi; Singh, Pankaj Kumar; Atray, Naresh; Gandhi, Swati; Avasthi, Tiruvayipati Suma; Nisthar, Shefin; Anurag, Meenakshi; Sharma, Pratibha; Hasija, Yasha; Dash, Debasis; Sharma, Arun; Scaria, Vinod; Thomas, Zakir; Chandra, Nagasuma; Brahmachari, Samir K.; Bhardwaj, Anshu

    2012-01-01

    A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative ‘Connect to Decode’ (C2D) to generate the first and largest manually curated interactome of Mtb termed ‘interactome pathway’ (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach. PMID:22808064

  10. Identification and characterization of carprofen as a multi-target FAAH/COX inhibitor

    Science.gov (United States)

    Favia, Angelo D.; Habrant, Damien; Scarpelli, Rita; Migliore, Marco; Albani, Clara; Bertozzi, Sine Mandrup; Dionisi, Mauro; Tarozzo, Glauco; Piomelli, Daniele; Cavalli, Andrea; De Vivo, Marco

    2013-01-01

    Pain and inflammation are major therapeutic areas for drug discovery. Current drugs for these pathologies have limited efficacy, however, and often cause a number of unwanted side effects. In the present study, we identify the non-steroid anti-inflammatory drug, carprofen, as a multi-target-directed ligand that simultaneously inhibits cyclooxygenase-1 (COX-1), COX-2 and fatty acid amide hydrolase (FAAH). Additionally, we synthesized and tested several racemic derivatives of carprofen, sharing this multi-target activity. This may result in improved analgesic efficacy and reduced side effects (Naidu, et al (2009) J Pharmacol Exp Ther 329, 48-56; Fowler, C.J. et al. (2012) J Enzym Inhib Med Chem Jan 6; Sasso, et al (2012) Pharmacol Res 65, 553). The new compounds are among the most potent multi-target FAAH/COXs inhibitors reported so far in the literature, and thus may represent promising starting points for the discovery of new analgesic and anti-inflammatory drugs. PMID:23043222

  11. Identification of the feedforward component in manual control with predictable target signals.

    Science.gov (United States)

    Drop, Frank M; Pool, Daan M; Damveld, Herman J; van Paassen, Marinus M; Mulder, Max

    2013-12-01

    In the manual control of a dynamic system, the human controller (HC) often follows a visible and predictable reference path. Compared with a purely feedback control strategy, performance can be improved by making use of this knowledge of the reference. The operator could effectively introduce feedforward control in conjunction with a feedback path to compensate for errors, as hypothesized in literature. However, feedforward behavior has never been identified from experimental data, nor have the hypothesized models been validated. This paper investigates human control behavior in pursuit tracking of a predictable reference signal while being perturbed by a quasi-random multisine disturbance signal. An experiment was done in which the relative strength of the target and disturbance signals were systematically varied. The anticipated changes in control behavior were studied by means of an ARX model analysis and by fitting three parametric HC models: two different feedback models and a combined feedforward and feedback model. The ARX analysis shows that the experiment participants employed control action on both the error and the target signal. The control action on the target was similar to the inverse of the system dynamics. Model fits show that this behavior can be modeled best by the combined feedforward and feedback model.

  12. Crowd sourcing a new paradigm for interactome driven drug target identification in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Rohit Vashisht

    Full Text Available A decade since the availability of Mycobacterium tuberculosis (Mtb genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative 'Connect to Decode' (C2D to generate the first and largest manually curated interactome of Mtb termed 'interactome pathway' (IPW, encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.

  13. Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.

    Directory of Open Access Journals (Sweden)

    Keerthi Krishnan

    Full Text Available Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. Here, we identify direct target genes of Spt5 using fog(sk8 zebrafish mutant, which disrupts the foggy/spt5 gene. We identified that fog(sk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, we identified a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together our findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development.

  14. Identification of novel targets for PGC-1α and histone deacetylase inhibitors in neuroblastoma cells

    International Nuclear Information System (INIS)

    Cowell, Rita M.; Talati, Pratik; Blake, Kathryn R.; Meador-Woodruff, James H.; Russell, James W.

    2009-01-01

    Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1α express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1α in neuronal cells and whether there are ways to pharmacologically target PGC-1α in neurons. Here, PGC-1α overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3 activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1α and glucose transporter 4 (GLUT4). These results suggest that PGC-1α regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1α and GLUT4 in HD and other neurological disorders.

  15. RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

    Directory of Open Access Journals (Sweden)

    Johanna Meier-Soelch

    2018-04-01

    Full Text Available The potent proinflammatory cytokine interleukin (IL-1 triggers gene expression through the NF-κB signaling pathway. Here, we investigated the cofactor requirements of strongly regulated IL-1 target genes whose expression is impaired in p65 NF-κB-deficient murine embryonic fibroblasts. By two independent small-hairpin (shRNA screens, we examined 170 genes annotated to encode nuclear cofactors for their role in Cxcl2 mRNA expression and identified 22 factors that modulated basal or IL-1-inducible Cxcl2 levels. The functions of 16 of these factors were validated for Cxcl2 and further analyzed for their role in regulation of 10 additional IL-1 target genes by RT-qPCR. These data reveal that each inducible gene has its own (quantitative requirement of cofactors to maintain basal levels and to respond to IL-1. Twelve factors (Epc1, H2afz, Kdm2b, Kdm6a, Mbd3, Mta2, Phf21a, Ruvbl1, Sin3b, Suv420h1, Taf1, and Ube3a have not been previously implicated in inflammatory cytokine functions. Bioinformatics analysis indicates that they are components of complex nuclear protein networks that regulate chromatin functions and gene transcription. Collectively, these data suggest that downstream from the essential NF-κB signal each cytokine-inducible target gene has further subtle requirements for individual sets of nuclear cofactors that shape its transcriptional activation profile.

  16. Identification of Treatment Targets in a Genetic Mouse Model of Voluntary Methamphetamine Drinking.

    Science.gov (United States)

    Phillips, T J; Mootz, J R K; Reed, C

    2016-01-01

    Methamphetamine has powerful stimulant and euphoric effects that are experienced as rewarding and encourage use. Methamphetamine addiction is associated with debilitating illnesses, destroyed relationships, child neglect, violence, and crime; but after many years of research, broadly effective medications have not been identified. Individual differences that may impact not only risk for developing a methamphetamine use disorder but also affect treatment response have not been fully considered. Human studies have identified candidate genes that may be relevant, but lack of control over drug history, the common use or coabuse of multiple addictive drugs, and restrictions on the types of data that can be collected in humans are barriers to progress. To overcome some of these issues, a genetic animal model comprised of lines of mice selectively bred for high and low voluntary methamphetamine intake was developed to identify risk and protective alleles for methamphetamine consumption, and identify therapeutic targets. The mu opioid receptor gene was supported as a target for genes within a top-ranked transcription factor network associated with level of methamphetamine intake. In addition, mice that consume high levels of methamphetamine were found to possess a nonfunctional form of the trace amine-associated receptor 1 (TAAR1). The Taar1 gene is within a mouse chromosome 10 quantitative trait locus for methamphetamine consumption, and TAAR1 function determines sensitivity to aversive effects of methamphetamine that may curb intake. The genes, gene interaction partners, and protein products identified in this genetic mouse model represent treatment target candidates for methamphetamine addiction. © 2016 Elsevier Inc. All rights reserved.

  17. Identification of CELF1 RNA targets by CLIP-seq in human HeLa cells

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    Olivier Le Tonquèze

    2016-06-01

    Full Text Available The specific interactions between RNA-binding proteins and their target RNAs are an essential level to control gene expression. By combining ultra-violet cross-linking and immunoprecipitation (CLIP and massive SoliD sequencing we identified the RNAs bound by the RNA-binding protein CELF1, in human HeLa cells. The CELF1 binding sites deduced from the sequence data allow characterizing specific features of CELF1-RNA association. We present therefore the first map of CELF1 binding sites in human cells.

  18. Identification of novel targets for HIV-1: Molecular dynamics simulation and binding energy calculations

    Science.gov (United States)

    Pandey, Vishnudatt; Tiwari, Gargi; Mall, Vijaya Shri; Tiwari, Rakesh Kumar; Ojha, R. P.

    2018-05-01

    HIV-1 envelope glycoprotein-mediated fusion is managed by the concerted coalescence of the HIV-1 gp41 N- and C- helical regions, which is a product in the formation of 6-helix bundles. These two regions are considered prime targets for peptides and antibodies that inhibit HIV-1 entry. There are so many rational method aimed to attach a rationally designed artificial tail to the C-terminus of HIV-1 fusion inhibitors to increase their antiviral potency. Here M. D. simulation was performed to go insight for study of C-terminal tail of Ile-Asp-Leu (IDL).

  19. Identification and profiling of microRNAs and their target genes from developing caprine skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Yanhong Wang

    Full Text Available Goat is an important agricultural animal for meat production. Functional studies have demonstrated that microRNAs (miRNAs regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although studies on miRNAs expression profiles have been performed in various animals, relatively limited information about goat muscle miRNAs has been reported. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for expression profiles of caprine miRNAs during two developmental stages of skeletal muscles: fetal stage and six month-old stage. As a result, 15,627,457 and 15,593,721 clean reads were obtained from the fetal goat library (FC and the six month old goat library (SMC, respectively. 464 known miRNAs and 83 novel miRNA candidates were identified. Furthermore, by comparing the miRNA profile, 336 differentially expressed miRNAs were identified and then the potential targets of the differentially expressed miRNAs were predicted. To understand the regulatory network of miRNAs during muscle development, the mRNA expression profiles for the two development stages were characterized and 7322 differentially expressed genes (DEGs were identified. Then the potential targets of miRNAs were compared to the DEGs, the intersection of the two gene sets were screened out and called differentially expressed targets (DE-targets, which were involved in 231 pathways. Ten of the 231 pathways that have smallest P-value were shown as network figures. Based on the analysis of pathways and networks, we found that miR-424-5p and miR-29a might have important regulatory effect on muscle development, which needed to be further studied. This study provided the first global view of the miRNAs in caprine muscle tissues. Our results help elucidation of complex regulatory networks between miRNAs and mRNAs and for the study of muscle

  20. Identification of treatment response predictors and potential molecular targets for chemo preventive and antiangiogenic therapies

    International Nuclear Information System (INIS)

    Pfeffer, U.; Albini, A.

    2009-01-01

    The aims of the project were: To evaluate the cellular responses to anti-inflammatory and anti-angiogenic natural or synthetic compounds (chemo preventives, inhibitors of cell survival and inflammation related signal transduction). To identify bio markers for treatment response through the selection of targets that are common to or specific for anti-inflammatory and anti-angiogenic activities. To analyze the regulation of the key tumor-promotion pathways Akt, HIF1α, NFκB. We focused our studies on the antiapoptotic role of the AKT survival pathway, which is involved in prostate tumor progression to an androgen-independent phenotype

  1. Identification of conserved, centrosome-targeting ASH domains in TRAPPII complex subunits and TRAPPC8

    DEFF Research Database (Denmark)

    Schou, Kenneth Bødtker; Morthorst, Stine Kjær; Christensen, Søren Tvorup

    2014-01-01

    , the Rab8 guanine nucleotide exchange factor Rabin8, and the transport protein particle (TRAPP) components TRAPPC3, -C9, and -C10, which physically interact with each other and function together with Bardet Biedl syndrome (BBS) proteins in ciliary membrane biogenesis. However, despite recent advances...... confer targeting to the centrosome and cilia, and that TRAPPC8 has cilia-related functions. Further, we propose that the yeast TRAPPII complex and its mammalian counterpart are evolutionarily related to the bacterial periplasmic trafficking chaperone PapD of the usher pili assembly machinery....

  2. REAL TIME PCR IDENTIFICATION FOR TARGET ADJUNCTIVE ANTIBIOTIC THERAPY OF SEVERE CHRONIC PERIODONTITIS. PART II - MICROBIOLOGICAL EFFECTIVENESS.

    Directory of Open Access Journals (Sweden)

    Kamen Kotsilkov

    2014-10-01

    Full Text Available INTRODUCTION: Antibiotic use in chronic periodontitis may result in improvement in periodontal status, although many questions regarding the indications for this therapy remain unanswered. The polymicrobial etiology of the periodontal infection hinders the choice of the proper antibiotic agent. Furthermore the indiscriminate use of antibiotics could lead to high levels of resistance and to various adverse reactions. In the recent years a various molecular diagnostics protocols were proposed in order to facilitate the decision for adjunctive antibiotic administration. OBJECTIVE: The aim of this study is to compare the microbiological effectiveness of adjunctive antibiotic administration with the mechanical periodontal therapy. METHODS: 30 patients with severe chronic periodontitis were enrolled in this study and were divided in 3 groups: Control group – with mechanical debridement only. Test group 1 – with combined adjunctive antibiotic administration using Amoxicillin+ Metronidazole. Test group 2 – with target antibiotic administration according to the resuts from the Real Time PCR identification. RESULTS: The prevalence of all the isolated microorganisms (exept. E.nodatum and C.gingivalis in Test Group 2 demonstrates statistically significant reduction compared with the other treatment approaches. Almost complete elimination was registered for the consensus pathogens from the red and orange complexes (above 99% and 100% for P.intemedia. CONCLUSION: The adjunct antibiotic treatment targeted with Real-Time PCR identification demonstrates almost complete elimination of the putative periodontal pathogens in the deep periodontal pockets in patients with severe chronic periodontitis. This result suggests slower recolonisation of these habitats thus limiting the risk for progression of the periodontal destruction.

  3. High-throughput identification of off-targets for the mechanistic study of severe adverse drug reactions induced by analgesics

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Jian-Bo [Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian 361005 (China); Ji, Nan; Pan, Wen; Hong, Ru [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102 (China); Wang, Hao [Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian 361005 (China); Ji, Zhi-Liang, E-mail: appo@xmu.edu.cn [State Key Laboratory of Stress Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102 (China); Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian 361005 (China)

    2014-01-01

    Drugs may induce adverse drug reactions (ADRs) when they unexpectedly bind to proteins other than their therapeutic targets. Identification of these undesired protein binding partners, called off-targets, can facilitate toxicity assessment in the early stages of drug development. In this study, a computational framework was introduced for the exploration of idiosyncratic mechanisms underlying analgesic-induced severe adverse drug reactions (SADRs). The putative analgesic-target interactions were predicted by performing reverse docking of analgesics or their active metabolites against human/mammal protein structures in a high-throughput manner. Subsequently, bioinformatics analyses were undertaken to identify ADR-associated proteins (ADRAPs) and pathways. Using the pathways and ADRAPs that this analysis identified, the mechanisms of SADRs such as cardiac disorders were explored. For instance, 53 putative ADRAPs and 24 pathways were linked with cardiac disorders, of which 10 ADRAPs were confirmed by previous experiments. Moreover, it was inferred that pathways such as base excision repair, glycolysis/glyconeogenesis, ErbB signaling, calcium signaling, and phosphatidyl inositol signaling likely play pivotal roles in drug-induced cardiac disorders. In conclusion, our framework offers an opportunity to globally understand SADRs at the molecular level, which has been difficult to realize through experiments. It also provides some valuable clues for drug repurposing. - Highlights: • A novel computational framework was developed for mechanistic study of SADRs. • Off-targets of drugs were identified in large scale and in a high-throughput manner. • SADRs like cardiac disorders were systematically explored in molecular networks. • A number of ADR-associated proteins were identified.

  4. High-throughput identification of off-targets for the mechanistic study of severe adverse drug reactions induced by analgesics

    International Nuclear Information System (INIS)

    Pan, Jian-Bo; Ji, Nan; Pan, Wen; Hong, Ru; Wang, Hao; Ji, Zhi-Liang

    2014-01-01

    Drugs may induce adverse drug reactions (ADRs) when they unexpectedly bind to proteins other than their therapeutic targets. Identification of these undesired protein binding partners, called off-targets, can facilitate toxicity assessment in the early stages of drug development. In this study, a computational framework was introduced for the exploration of idiosyncratic mechanisms underlying analgesic-induced severe adverse drug reactions (SADRs). The putative analgesic-target interactions were predicted by performing reverse docking of analgesics or their active metabolites against human/mammal protein structures in a high-throughput manner. Subsequently, bioinformatics analyses were undertaken to identify ADR-associated proteins (ADRAPs) and pathways. Using the pathways and ADRAPs that this analysis identified, the mechanisms of SADRs such as cardiac disorders were explored. For instance, 53 putative ADRAPs and 24 pathways were linked with cardiac disorders, of which 10 ADRAPs were confirmed by previous experiments. Moreover, it was inferred that pathways such as base excision repair, glycolysis/glyconeogenesis, ErbB signaling, calcium signaling, and phosphatidyl inositol signaling likely play pivotal roles in drug-induced cardiac disorders. In conclusion, our framework offers an opportunity to globally understand SADRs at the molecular level, which has been difficult to realize through experiments. It also provides some valuable clues for drug repurposing. - Highlights: • A novel computational framework was developed for mechanistic study of SADRs. • Off-targets of drugs were identified in large scale and in a high-throughput manner. • SADRs like cardiac disorders were systematically explored in molecular networks. • A number of ADR-associated proteins were identified

  5. Identification of novel human damage response proteins targeted through yeast orthology.

    Directory of Open Access Journals (Sweden)

    J Peter Svensson

    Full Text Available Studies in Saccharomyces cerevisiae show that many proteins influence cellular survival upon exposure to DNA damaging agents. We hypothesized that human orthologs of these S. cerevisiae proteins would also be required for cellular survival after treatment with DNA damaging agents. For this purpose, human homologs of S. cerevisiae proteins were identified and mapped onto the human protein-protein interaction network. The resulting human network was highly modular and a series of selection rules were implemented to identify 45 candidates for human toxicity-modulating proteins. The corresponding transcripts were targeted by RNA interference in human cells. The cell lines with depleted target expression were challenged with three DNA damaging agents: the alkylating agents MMS and 4-NQO, and the oxidizing agent t-BuOOH. A comparison of the survival revealed that the majority (74% of proteins conferred either sensitivity or resistance. The identified human toxicity-modulating proteins represent a variety of biological functions: autophagy, chromatin modifications, RNA and protein metabolism, and telomere maintenance. Further studies revealed that MMS-induced autophagy increase the survival of cells treated with DNA damaging agents. In summary, we show that damage recovery proteins in humans can be identified through homology to S. cerevisiae and that many of the same pathways are represented among the toxicity modulators.

  6. Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

    Science.gov (United States)

    Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

    2015-12-15

    MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

    Science.gov (United States)

    Mor, Visesato; Rella, Antonella; Farnoud, Amir M.; Singh, Ashutosh; Munshi, Mansa; Bryan, Arielle; Naseem, Shamoon; Konopka, James B.; Ojima, Iwao; Bullesbach, Erika; Ashbaugh, Alan; Linke, Michael J.; Cushion, Melanie; Collins, Margaret; Ananthula, Hari Krishna; Sallans, Larry; Desai, Pankaj B.; Wiederhold, Nathan P.; Fothergill, Annette W.; Kirkpatrick, William R.; Patterson, Thomas; Wong, Lai Hong; Sinha, Sunita; Giaever, Guri; Nislow, Corey; Flaherty, Patrick; Pan, Xuewen; Cesar, Gabriele Vargas; de Melo Tavares, Patricia; Frases, Susana; Miranda, Kildare; Rodrigues, Marcio L.; Luberto, Chiara; Nimrichter, Leonardo

    2015-01-01

    ABSTRACT Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. PMID:26106079

  8. A trigger for the identification of pions stopped in an active target

    International Nuclear Information System (INIS)

    Raywood, K.J.; Ambardar, A.R.; Lange, J.B.; Sevior, M.E.

    1995-03-01

    The total cross sections of the π + p→ π + π + n and π - p→ π + π - n reactions threshold can be used to obtain scattering lengths which are directly comparable to predictions of chiral perturbation theory. Data for these reactions were taken at TRIUMF using a segmented active scintillator target. The signature of a π + in an active target segment was a prompt pulse caused by the particle stopping, followed by a second pulse due to the π + to μ + decay. TRIUMF 500 Mhz transient digitisers were used to record the scintillator outputs in 2 ns steps so that the double pulses could be identified with high efficiency in off-line data analysis. A second level trigger able to reject events in less than 10 μs was necessary to avoid a prohibitively high deadtime due to the long read-out time of the digitisers. It was implemented with fast ECLine electronics and increased the useful event acquisition rate by a factor of more than forty. (author)

  9. Identification of the epigenetic reader CBX2 as a potential drug target in advanced prostate cancer.

    Science.gov (United States)

    Clermont, Pier-Luc; Crea, Francesco; Chiang, Yan Ting; Lin, Dong; Zhang, Amy; Wang, James Z L; Parolia, Abhijit; Wu, Rebecca; Xue, Hui; Wang, Yuwei; Ding, Jiarui; Thu, Kelsie L; Lam, Wan L; Shah, Sohrab P; Collins, Colin C; Wang, Yuzhuo; Helgason, Cheryl D

    2016-01-01

    While localized prostate cancer (PCa) can be effectively cured, metastatic disease inevitably progresses to a lethal state called castration-resistant prostate cancer (CRPC). Emerging evidence suggests that aberrant epigenetic repression by the polycomb group (PcG) complexes fuels PCa progression, providing novel therapeutic opportunities. In the search for potential epigenetic drivers of CRPC, we analyzed the molecular profile of PcG members in patient-derived xenografts and clinical samples. Overall, our results identify the PcG protein and methyl-lysine reader CBX2 as a potential therapeutic target in advanced PCa. We report that CBX2 was recurrently up-regulated in metastatic CRPC and that elevated CBX2 expression was correlated with poor clinical outcome in PCa cohorts. Furthermore, CBX2 depletion abrogated cell viability and induced caspase 3-mediated apoptosis in metastatic PCa cell lines. Mechanistically explaining this phenotype, microarray analysis in CBX2-depleted cells revealed that CBX2 controls the expression of many key regulators of cell proliferation and metastasis. Taken together, this study provides the first evidence that CBX2 inhibition induces cancer cell death, positioning CBX2 as an attractive drug target in lethal CRPC.

  10. Global Identification of EVI1 Target Genes in Acute Myeloid Leukemia.

    Directory of Open Access Journals (Sweden)

    Carolyn Glass

    Full Text Available The ecotropic virus integration site 1 (EVI1 transcription factor is associated with human myeloid malignancy of poor prognosis and is overexpressed in 8-10% of adult AML and strikingly up to 27% of pediatric MLL-rearranged leukemias. For the first time, we report comprehensive genomewide EVI1 binding and whole transcriptome gene deregulation in leukemic cells using a combination of ChIP-Seq and RNA-Seq expression profiling. We found disruption of terminal myeloid differentiation and cell cycle regulation to be prominent in EVI-induced leukemogenesis. Specifically, we identified EVI1 directly binds to and downregulates the master myeloid differentiation gene Cebpe and several of its downstream gene targets critical for terminal myeloid differentiation. We also found EVI1 binds to and downregulates Serpinb2 as well as numerous genes involved in the Jak-Stat signaling pathway. Finally, we identified decreased expression of several ATP-dependent P2X purinoreceptors genes involved in apoptosis mechanisms. These findings provide a foundation for future study of potential therapeutic gene targets for EVI1-induced leukemia.

  11. Identification of Novel Targets for Lung Cancer Therapy Using an Induced Pluripotent Stem Cell Model.

    Science.gov (United States)

    Shukla, Vivek; Rao, Mahadev; Zhang, Hongen; Beers, Jeanette; Wangsa, Darawalee; Wangsa, Danny; Buishand, Floryne O; Wang, Yonghong; Yu, Zhiya; Stevenson, Holly; Reardon, Emily; McLoughlin, Kaitlin C; Kaufman, Andrew; Payabyab, Eden; Hong, Julie A; Zhang, Mary; Davis, Sean R; Edelman, Daniel C; Chen, Guokai; Miettinen, Markku; Restifo, Nicholas; Ried, Thomas; Meltzer, Paul S; Schrump, David S

    2018-04-01

    Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human

  12. Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis

    Directory of Open Access Journals (Sweden)

    Chowdhury MRH

    2014-11-01

    Full Text Available Md Rabiul Hossain Chowdhury,1 Md IqbalKaiser Bhuiyan,2 Ayan Saha,2 Ivan MHAI Mosleh,2 Sobuj Mondol,2 C M Sabbir Ahmed3 1Department of Pharmacy, University of Science and Technology Chittagong, Chittagong, Bangladesh; 2Department of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong, Bangladesh; 3Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna, Bangladesh Purpose: Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim–sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. Materials and methods: In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein–protein interaction. Results: In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic

  13. Can we improve the identification of cold homes for targeted home energy-efficiency improvements?

    International Nuclear Information System (INIS)

    Hutchinson, Emma J.; Wilkinson, Paul; Hong, Sung H.; Oreszczyn, Tadj

    2006-01-01

    Objective: To investigate the extent to which homes with low indoor-temperatures can be identified from dwelling and household characteristics. Design: Analysis of data from a national survey of dwellings, occupied by low-income households, scheduled for home energy-efficiency improvements. Setting: Five urban areas of England: Birmingham, Liverpool, Manchester, Newcastle and Southampton. Methods: Half-hourly living-room temperatures were recorded for two to four weeks in dwellings over the winter periods November to April 2001-2002 and 2002-2003. Regression of indoor on outdoor temperatures was used to identify cold-homes in which standardized daytime living-room and/or nighttime bedroom-temperatures were o C (when the outdoor temperature was 5 o C). Tabulation and logistic regression were used to examine the extent to which these cold-homes can be identified from dwelling and household characteristics. Results: Overall, 21.0% of dwellings had standardized daytime living-room temperatures o C, and 46.4% had standardized nighttime bedroom-temperatures below the same temperature. Standardized indoor-temperatures were influenced by a wide range of household and dwelling characteristics, but most strongly by the energy efficiency (SAP) rating and by standardized heating costs. However, even using these variables, along with other dwelling and household characteristics in a multi-variable prediction model, it would be necessary to target more than half of all dwellings in our sample to ensure at least 80% sensitivity for identifying dwellings with cold living-room temperatures. An even higher proportion would have to be targeted to ensure 80% sensitivity for identifying dwellings with cold-bedroom temperatures. Conclusion: Property and household characteristics provide only limited potential for identifying dwellings where winter indoor temperatures are likely to be low, presumably because of the multiple influences on home heating, including personal choice and

  14. Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer

    International Nuclear Information System (INIS)

    McGoldrick, Christopher A; Jiang, Yu-Lin; Paromov, Victor; Brannon, Marianne; Krishnan, Koyamangalath; Stone, William L

    2014-01-01

    Esterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells. Cellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results. The major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 3.4.19.1) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells. These data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH

  15. Assertive outreach in Slovenia; identification of target group and goals of treatment in a new program.

    Science.gov (United States)

    Furlan, Mirjana

    2009-09-01

    A team from the Rehabilitation unit of Ljubljana psychiatric clinic attended a course on community care in London in October and November 2005. Because we decided that the methods presented to us could be of great use in Slovenia where the Health system is lacking such services we decided to implement them after our return. Immediately after we returned we started to carry out our plan. We designated our target group which were patients who poorly participated in treatment or had multiple and severe difficulties functioning and retaining their progress after discharge. Our goals were to improve patient participation in treatment before and after discharge, less and shorter hospitalizations and better integration of patients into society. Initial results are very positive, which leaves me much hope for further implementation of assertive outreach and community care in Slovenia.

  16. Functional Analysis of Barley Powdery Mildew Effector Candidates and Identification of their Barley Targets

    DEFF Research Database (Denmark)

    Ahmed, Ali Abdurehim

    The genome of barley powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh) encodes around 500 Candidate Secreted Effector Proteins (CSEPs), which are believed to be delivered to the barley cells either to interfere with plant defence and/or promote nutrient uptake. So far, little is known...... about the function of many CSEPs in virulence and the identities of their host targets. In this PhD study, we investigated the function of nine CSEPs and found that CSEP0081, CSEP0105, CSEP0162 and CSEP0254 act as effectors by promoting the Bgh infection success. Independent silencing of these CSEPs...... proteins (sHsps), Hsp16.9 and Hsp17.5, were identified as interactors for both CSEP0105 and CSEP0162. These interactions were confirmed in planta by BiFC and co-localization studies. Small heat shock proteins are highly conserved ATP-independent chaperones that protect the cell from stress-induced protein...

  17. Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

    Science.gov (United States)

    Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

    2012-04-25

    A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

  18. Identification of conserved microRNAs and their targets in chickpea (Cicer arietinum L.).

    Science.gov (United States)

    Hu, Jihong; Sun, Lulu; Ding, Yi

    2013-04-01

    The microRNAs (miRNAs) are a new class of non-protein coding small RNAs that regulate gene expression at the post-transcriptional level in plants. Although thousands of miRNAs have been identified in many plant species, little studies have been reported about chickpea microRNAs. In this study, 28 potential miRNA candidates belonging to 20 families were identified from 16 ESTs and 12 GSSs in chickpea using a comparative genome-based computational analysis. A total of 664 miRNA targets were predicted and some of them encoded transcription factors as well as genes that function in stress response, signal transduction, methylation and a variety of other metabolic processes. These findings lay the foundation for further understanding of miRNA function in the development of chickpea.

  19. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

    Directory of Open Access Journals (Sweden)

    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  20. Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

    Directory of Open Access Journals (Sweden)

    Ruixin Hao

    Full Text Available Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2 or vehicle from 3 hours to 4 days post fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

  1. Identification of novel candidate compounds targeting TrkB to induce apoptosis in neuroblastoma

    International Nuclear Information System (INIS)

    Nakamura, Yohko; Suganami, Akiko; Fukuda, Mayu; Hasan, Md Kamrul; Yokochi, Tomoki; Takatori, Atsushi; Satoh, Shunpei; Hoshino, Tyuji; Tamura, Yutaka; Nakagawara, Akira

    2014-01-01

    Neuroblastoma (NB) is one of the most frequent solid tumors in children and its prognosis is still poor. The neurotrophin receptor TrkB and its ligand brain-derived neurotrophic factor (BDNF) are expressed at high levels in high-risk NBs and are involved in defining the poor prognosis of the patients. However, the TrkB targeting therapy has never been realized in the clinic. We performed an in silico screening procedure utilizing an AutoDock/grid computing technology in order to identify novel small chemical compounds targeting the BDNF-binding domain of TrkB. For the first screening, a library of three million synthetic compounds was screened in silico and was ranked according to the Docking energy. The top-ranked 37 compounds were further functionally screened for cytotoxicity by using NB cell lines. We have finally identified seven compounds that kill NB cells with the IC 50 values of 0.07–4.6 μmol/L. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that these molecules induce apoptosis accompanied by p53 activation in NB cell lines. The candidate compounds and BDNF demonstrated an antagonistic effect on cell growth, invasion, and colony formation, possibly suggesting competition at the BDNF-binding site of TrkB. The candidate compounds had tumor-suppressive activity in xenograft and in vivo toxicity tests (oral and intravenous administrations) using mice, and did not show any abnormal signs. Using in silico Docking screening we have found new candidate TrkB inhibitors against high-risk NBs, which could lead to new anti-cancer drugs

  2. Identification of the Consistently Altered Metabolic Targets in Human Hepatocellular Carcinoma.

    Science.gov (United States)

    Nwosu, Zeribe Chike; Megger, Dominik Andre; Hammad, Seddik; Sitek, Barbara; Roessler, Stephanie; Ebert, Matthias Philip; Meyer, Christoph; Dooley, Steven

    2017-09-01

    Cancer cells rely on metabolic alterations to enhance proliferation and survival. Metabolic gene alterations that repeatedly occur in liver cancer are largely unknown. We aimed to identify metabolic genes that are consistently deregulated, and are of potential clinical significance in human hepatocellular carcinoma (HCC). We studied the expression of 2,761 metabolic genes in 8 microarray datasets comprising 521 human HCC tissues. Genes exclusively up-regulated or down-regulated in 6 or more datasets were defined as consistently deregulated. The consistent genes that correlated with tumor progression markers ( ECM2 and MMP9) (Pearson correlation P < .05) were used for Kaplan-Meier overall survival analysis in a patient cohort. We further compared proteomic expression of metabolic genes in 19 tumors vs adjacent normal liver tissues. We identified 634 consistent metabolic genes, ∼60% of which are not yet described in HCC. The down-regulated genes (n = 350) are mostly involved in physiologic hepatocyte metabolic functions (eg, xenobiotic, fatty acid, and amino acid metabolism). In contrast, among consistently up-regulated metabolic genes (n = 284) are those involved in glycolysis, pentose phosphate pathway, nucleotide biosynthesis, tricarboxylic acid cycle, oxidative phosphorylation, proton transport, membrane lipid, and glycan metabolism. Several metabolic genes (n = 434) correlated with progression markers, and of these, 201 predicted overall survival outcome in the patient cohort analyzed. Over 90% of the metabolic targets significantly altered at the protein level were similarly up- or down-regulated as in genomic profile. We provide the first exposition of the consistently altered metabolic genes in HCC and show that these genes are potentially relevant targets for onward studies in preclinical and clinical contexts.

  3. Identification of the Consistently Altered Metabolic Targets in Human Hepatocellular CarcinomaSummary

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    Zeribe Chike Nwosu

    2017-09-01

    Full Text Available Background & Aims: Cancer cells rely on metabolic alterations to enhance proliferation and survival. Metabolic gene alterations that repeatedly occur in liver cancer are largely unknown. We aimed to identify metabolic genes that are consistently deregulated, and are of potential clinical significance in human hepatocellular carcinoma (HCC. Methods: We studied the expression of 2,761 metabolic genes in 8 microarray datasets comprising 521 human HCC tissues. Genes exclusively up-regulated or down-regulated in 6 or more datasets were defined as consistently deregulated. The consistent genes that correlated with tumor progression markers (ECM2 and MMP9 (Pearson correlation P < .05 were used for Kaplan-Meier overall survival analysis in a patient cohort. We further compared proteomic expression of metabolic genes in 19 tumors vs adjacent normal liver tissues. Results: We identified 634 consistent metabolic genes, ∼60% of which are not yet described in HCC. The down-regulated genes (n = 350 are mostly involved in physiologic hepatocyte metabolic functions (eg, xenobiotic, fatty acid, and amino acid metabolism. In contrast, among consistently up-regulated metabolic genes (n = 284 are those involved in glycolysis, pentose phosphate pathway, nucleotide biosynthesis, tricarboxylic acid cycle, oxidative phosphorylation, proton transport, membrane lipid, and glycan metabolism. Several metabolic genes (n = 434 correlated with progression markers, and of these, 201 predicted overall survival outcome in the patient cohort analyzed. Over 90% of the metabolic targets significantly altered at the protein level were similarly up- or down-regulated as in genomic profile. Conclusions: We provide the first exposition of the consistently altered metabolic genes in HCC and show that these genes are potentially relevant targets for onward studies in preclinical and clinical contexts. Keywords: Liver Cancer, HCC, Tumor Metabolism

  4. Identification of thioredoxin-interacting protein (TXNIP) as a downstream target for IGF1 action.

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    Nagaraj, Karthik; Lapkina-Gendler, Lena; Sarfstein, Rive; Gurwitz, David; Pasmanik-Chor, Metsada; Laron, Zvi; Yakar, Shoshana; Werner, Haim

    2018-01-30

    Laron syndrome (LS), or primary growth hormone (GH) insensitivity, is the best-characterized entity among the congenital insulin-like growth factor 1 (IGF1) deficiencies. Life-long exposure to minute endogenous IGF1 levels is linked to low stature as well as a number of endocrine and metabolic abnormalities. While elevated IGF1 is correlated with increased cancer incidence, epidemiological studies revealed that patients with LS do not develop tumors. The mechanisms associated with cancer protection in LS are yet to be discovered. Recent genomic analyses identified a series of metabolic genes that are overrepresented in patients with LS. Given the augmented expression of these genes in a low IGF1 milieu, we hypothesized that they may constitute targets for IGF1 action. Thioredoxin-interacting protein (TXNIP) plays a critical role in cellular redox control by thioredoxin. TXNIP serves as a glucose and oxidative stress sensor, being commonly silenced by genetic or epigenetic events in cancer cells. Consistent with its enhanced expression in LS, we provide evidence that TXNIP gene expression is negatively regulated by IGF1. These results were corroborated in animal studies. In addition, we show that oxidative and glucose stresses led to marked increases in TXNIP expression. Supplementation of IGF1 attenuated TXNIP levels, suggesting that IGF1 exerts its antiapoptotic effect via inhibition of TXNIP Augmented TXNIP expression in LS may account for cancer protection in this condition. Finally, TXNIP levels could be potentially useful in the clinic as a predictive or diagnostic biomarker for IGF1R-targeted therapies.

  5. Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

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    Yan Wusheng

    2012-01-01

    Full Text Available Abstract Background Esophageal squamous cell carcinoma (ESCC, the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB, normal differentiated squamous epithelium (ND, and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets. Results As a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA were verified to be dysregulated in the same pattern at both the mRNA and protein levels. Conclusions These data reveal insight into genes and

  6. Identification and target prediction of miRNAs specifically expressed in rat neural tissue

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    Tu Kang

    2009-05-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a large group of RNAs that play important roles in regulating gene expression and protein translation. Several studies have indicated that some miRNAs are specifically expressed in human, mouse and zebrafish tissues. For example, miR-1 and miR-133 are specifically expressed in muscles. Tissue-specific miRNAs may have particular functions. Although previous studies have reported the presence of human, mouse and zebrafish tissue-specific miRNAs, there have been no detailed reports of rat tissue-specific miRNAs. In this study, Home-made rat miRNA microarrays which established in our previous study were used to investigate rat neural tissue-specific miRNAs, and mapped their target genes in rat tissues. This study will provide information for the functional analysis of these miRNAs. Results In order to obtain as complete a picture of specific miRNA expression in rat neural tissues as possible, customized miRNA microarrays with 152 selected miRNAs from miRBase were used to detect miRNA expression in 14 rat tissues. After a general clustering analysis, 14 rat tissues could be clearly classified into neural and non-neural tissues based on the obtained expression profiles with p values Conclusion Our work provides a global view of rat neural tissue-specific miRNA profiles and a target map of miRNAs, which is expected to contribute to future investigations of miRNA regulatory mechanisms in neural systems.

  7. Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis.

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    Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan Mhai; Mondol, Sobuj; Ahmed, C M Sabbir

    2014-01-01

    Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim-sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein-protein interaction. In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to develop and discover more effective and specific

  8. Identification of CDC25 as a Common Therapeutic Target for Triple-Negative Breast Cancer

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    Jeff C. Liu

    2018-04-01

    Full Text Available Summary: CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC, RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs. : Liu et al. report that inhibition of the protein phosphatase CDC25 kills diverse triple-negative breast cancer (TNBC cells. Moreover, CDC25 antagonists cooperate with other drugs, such as PI3K inhibitors, to efficiently suppress growth of human TNBC engrafted into mice. Keywords: triple negative breast cancer, basal-like breast cancer, therapy, RB1, PTEN, TP53, CDC25, WEE1, CHK1, checkpoint control

  9. Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis

    Science.gov (United States)

    Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan MHAI; Mondol, Sobuj; Ahmed, C M Sabbir

    2014-01-01

    Purpose Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim–sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. Materials and methods In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein–protein interaction. Results In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Conclusion Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to

  10. Identification of cell surface targets for HIV-1 therapeutics using genetic screens

    International Nuclear Information System (INIS)

    Dunn, Stephen J.; Khan, Imran H.; Chan, Ursula A.; Scearce, Robin L.; Melara, Claudia L.; Paul, Amber M.; Sharma, Vikram; Bih, Fong-Yih; Holzmayer, Tanya A.; Luciw, Paul A.; Abo, Arie

    2004-01-01

    Human immunodeficiency virus (HIV) drugs designed to interfere with obligatory utilization of certain host cell factors by virus are less likely to encounter development of resistant strains than drugs directed against viral components. Several cellular genes required for productive infection by HIV were identified by the use of genetic suppressor element (GSE) technology as potential targets for anti-HIV drug development. Fragmented cDNA libraries from various pools of human peripheral blood mononuclear cells (PBMC) were expressed in vitro in human immunodeficiency virus type 1 (HIV-1)-susceptible cell lines and subjected to genetic screens to identify GSEs that interfered with viral replication. After three rounds of selection, more than 15 000 GSEs were sequenced, and the cognate genes were identified. The GSEs that inhibited the virus were derived from a diverse set of genes including cell surface receptors, cytokines, signaling proteins, transcription factors, as well as genes with unknown function. Approximately 2.5% of the identified genes were previously shown to play a role in the HIV-1 life cycle; this finding supports the biological relevance of the assay. GSEs were derived from the following 12 cell surface proteins: CXCR4, CCR4, CCR7, CD11C, CD44, CD47, CD68, CD69, CD74, CSF3R, GABBR1, and TNFR2. Requirement of some of these genes for viral infection was also investigated by using RNA interference (RNAi) technology; accordingly, 10 genes were implicated in early events of the viral life cycle, before viral DNA synthesis. Thus, these cell surface proteins represent novel targets for the development of therapeutics against HIV-1 infection and AIDS

  11. Identification of Arx targets unveils new candidates for controlling cortical interneuron migration and differentiation

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    Gaelle M Friocourt

    2011-12-01

    Full Text Available Mutations in the homeobox transcription factor ARX have been found to be responsible for a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of intellectual disabilities without apparent brain abnormalities, but with associated features of dystonia and epilepsy. Arx expression is mainly restricted to populations of GABA-containing neurons. Studies of the effects of ARX loss of function, either in humans or mutant mice, revealed varying defects, suggesting multiple roles of this gene in brain patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. However, to date, little is known about how Arx functions as a transcription factor or which genes it binds and regulates. Recently, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified approximately 1000 gene promoters bound by Arx in transfected neuroblastoma N2a cells and mouse embryonic brain. To narrow the analysis of Arx targets to those most likely to control cortical interneuron migration and/or differentiation, we compare here our data to previously published studies searching for genes enriched or down-regulated in cortical interneurons between E13.5 and E15.5. We thus identified 14 Arx-target genes enriched (Cxcr7, Meis1, Ppap2a, Slc12a5, Ets2, Phlda1, Zif268, Igf1, Lmo3, Sema6, Lgi1, Alk, Tgfb3, Napb and 5 genes specifically down-regulated (Hmgn3, Lmo1, Ebf3, Rasgef1b and Slit2 in cortical migrating neurons. In this review, we present these genes and discuss how their possible regulation by Arx may lead to the dysfunction of GABAergic neurons, resulting in mental retardation and epilepsy.

  12. Identification and Characterization of a Chloroplast-Targeted Obg GTPase in Dendrobium officinale.

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    Chen, Ji; Deng, Feng; Deng, Mengsheng; Han, Jincheng; Chen, Jianbin; Wang, Li; Yan, Shen; Tong, Kai; Liu, Fan; Tian, Mengliang

    2016-12-01

    Bacterial homologous chloroplast-targeted Obg GTPases (ObgCs) belong to the plant-typical Obg group, which is involved in diverse physiological processes during chloroplast development. However, the evolutionarily conserved function of ObgC in plants remains elusive and requires further investigation. In this study, we identified DoObgC from an epiphytic plant Dendrobium officinale and demonstrated the characteristics of DoObgC. Sequence analysis indicated that DoObgC is highly conserved with other plant ObgCs, which contain the chloroplast transit peptide (cTP), Obg fold, G domain, and OCT regions. The C terminus of DoObgC lacking the chloroplast-targeting cTP region, DoObgC Δ1-160 , showed strong similarity to ObgE and other bacterial Obgs. Overexpression of DoObgC Δ1-160 in Escherichia coli caused slow cell growth and an increased number of elongated cells. This phenotype was consistent with the phenotype of cells overexpressing ObgE. Furthermore, the expression of recombinant DoObgC Δ1-160 enhanced the cell persistence of E. coli to streptomycin. Results of transient expression assays revealed that DoObgC was localized to chloroplasts. Moreover, we demonstrated that DoObgC could rescue the embryotic lethal phenotype of the Arabidopsis obgc-t mutant, suggesting that DoObgC is a functional homolog to Arabidopsis AtObgC in D. officinale. Gene expression profiles showed that DoObgC was expressed in leaf-specific and light-dependent patterns and that DoObgC responded to wounding treatments. Our previous and present studies reveal that ObgC has an evolutionarily conserved role in ribosome biogenesis to adapt chloroplast development to the environment.

  13. Comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane between normal and preeclampsia pregnancies with high-resolution mass spectrometry.

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    Fuqiang Wang

    Full Text Available Preeclampsia is a serious complication of pregnancy, which affects 2-8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane.

  14. Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor.

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    Izumi Kaneko

    2015-05-01

    Full Text Available Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

  15. Insights into Integrated Lead Generation and Target Identification in Malaria and Tuberculosis Drug Discovery.

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    Okombo, John; Chibale, Kelly

    2017-07-18

    New, safe and effective drugs are urgently needed to treat and control malaria and tuberculosis, which affect millions of people annually. However, financial return on investment in the poor settings where these diseases are mostly prevalent is very minimal to support market-driven drug discovery and development. Moreover, the imminent loss of therapeutic lifespan of existing therapies due to evolution and spread of drug resistance further compounds the urgency to identify novel effective drugs. However, the advent of new public-private partnerships focused on tropical diseases and the recent release of large data sets by pharmaceutical companies on antimalarial and antituberculosis compounds derived from phenotypic whole cell high throughput screening have spurred renewed interest and opened new frontiers in malaria and tuberculosis drug discovery. This Account recaps the existing challenges facing antimalarial and antituberculosis drug discovery, including limitations associated with experimental animal models as well as biological complexities intrinsic to the causative pathogens. We enlist various highlights from a body of work within our research group aimed at identifying and characterizing new chemical leads, and navigating these challenges to contribute toward the global drug discovery and development pipeline in malaria and tuberculosis. We describe a catalogue of in-house efforts toward deriving safe and efficacious preclinical drug development candidates via cell-based medicinal chemistry optimization of phenotypic whole-cell medium and high throughput screening hits sourced from various small molecule chemical libraries. We also provide an appraisal of target-based screening, as invoked in our laboratory for mechanistic evaluation of the hits generated, with particular focus on the enzymes within the de novo pyrimidine biosynthetic and hemoglobin degradation pathways, the latter constituting a heme detoxification process and an associated cysteine

  16. Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples.

    Science.gov (United States)

    Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K

    2011-11-01

    In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

    Energy Technology Data Exchange (ETDEWEB)

    Suyari, Osamu; Ida, Hiroyuki [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Venture Laboratory, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Yamaguchi, Masamitsu, E-mail: myamaguc@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan)

    2009-05-01

    The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-{beta}-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

  18. Transcription Factors Expressed in Lateral Organ Boundaries: Identification of Downstream Targets

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Patricia S

    2010-07-12

    The processes of lateral organ initiation and patterning are central to the generation of mature plant form. Characterization of the molecular mechanisms underlying these processes is essential to our understanding of plant development. Communication between the shoot apical meristem and initiating organ primordia is important both for functioning of the meristem and for proper organ patterning, and very little is known about this process. In particular, the boundary between meristem and leaf is emerging as a critical region that is important for SAM maintenance and regulation of organogenesis. The goal of this project was to characterize three boundary-expressed genes that encode predicted transcription factors. Specifically, we have studied LATERAL ORGAN BOUNDARIES (LOB), LATERAL ORGAN FUSION1 (LOF1), and LATERAL ORGAN FUSION2 (LOF2). LOB encodes the founding member of the LOB-DOMAIN (LBD) plant-specific DNA binding transcription factor family and LOF1 and LOF2 encode paralogous MYB-domain transcription factors. We characterized the genetic relationship between these three genes and other boundary and meristem genes. We also used an ectopic inducible expression system to identify direct targets of LOB.

  19. Pancreatic polypeptide: Identification of target tissues and investigation of possible physiologic significance

    International Nuclear Information System (INIS)

    Shetzline, M.A.

    1988-01-01

    Pancreatic Polypeptide (PP) is a 36 amino acid peptide with hormonal properties but whose physiologic function remains unknown. In order to determine the function of this peptide we investigated potential target tissues using an in vivo radioreceptor assay. In vitro high concentrations of unlabeled hormone competitively inhibit low concentrations of labeled hormone from binding to receptors. Our in vivo studies indicate that, in the presence of concentrated unlabeled peptide, labeled PP distributes between the plasma and interstitial fluid. When saline rather than excess unlabeled PP is injected, the labeled peptide appears to distribute in a volume exceeding the combined plasma and interstitial fluid volume of tissue. The distribution volume which exceeds the anatomic extracellular volume and which is not present with excess unlabeled peptide, is the receptor compartment. With this assay we demonstrated in the rat specific and displaceable PP binding to the ductus choledochus, duodenum, ileum, and adrenal gland. In vitro rat adrenal cell membranes showed specific I-125 labeled PP binding. Specific binding also occurred in bovine cortical and medullary membranes

  20. Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF

    International Nuclear Information System (INIS)

    Suyari, Osamu; Ida, Hiroyuki; Yoshioka, Yasuhide; Kato, Yasuko; Hashimoto, Reina; Yamaguchi, Masamitsu

    2009-01-01

    The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-β-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

  1. Identification of Pathways in Liver Repair Potentially Targeted by Secretory Proteins from Human Mesenchymal Stem Cells

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    Sandra Winkler

    2016-07-01

    Full Text Available Background: The beneficial impact of mesenchymal stem cells (MSC on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1 increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor signalling pathways as relevant networks. Relationships to transforming growth factor β (TGF-β and hypoxia-inducible factor 1-α (HIF1-α signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

  2. Identification of potential pathway mediation targets in Toll-like receptor signaling.

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    Fan Li

    2009-02-01

    Full Text Available Recent advances in reconstruction and analytical methods for signaling networks have spurred the development of large-scale models that incorporate fully functional and biologically relevant features. An extended reconstruction of the human Toll-like receptor signaling network is presented herein. This reconstruction contains an extensive complement of kinases, phosphatases, and other associated proteins that mediate the signaling cascade along with a delineation of their associated chemical reactions. A computational framework based on the methods of large-scale convex analysis was developed and applied to this network to characterize input-output relationships. The input-output relationships enabled significant modularization of the network into ten pathways. The analysis identified potential candidates for inhibitory mediation of TLR signaling with respect to their specificity and potency. Subsequently, we were able to identify eight novel inhibition targets through constraint-based modeling methods. The results of this study are expected to yield meaningful avenues for further research in the task of mediating the Toll-like receptor signaling network and its effects.

  3. Revised annotation of Plutella xylostella microRNAs and their genome-wide target identification.

    Science.gov (United States)

    Etebari, K; Asgari, S

    2016-12-01

    The diamondback moth, Plutella xylostella, is the most devastating pest of brassica crops worldwide. Although 128 mature microRNAs (miRNAs) have been annotated from this species in miRBase, there is a need to extend and correct the current P. xylostella miRNA repertoire as a result of its recently improved genome assembly and more available small RNA sequence data. We used our new ultra-deep sequence data and bioinformatics to re-annotate the P. xylostella genome for high confidence miRNAs with the correct 5p and 3p arm features. Furthermore, all the P. xylostella annotated genes were also screened to identify potential miRNA binding sites using three target-predicting algorithms. In total, 203 mature miRNAs were annotated, including 33 novel miRNAs. We identified 7691 highly confident binding sites for 160 pxy-miRNAs. The data provided here will facilitate future studies involving functional analyses of P. xylostella miRNAs as a platform to introduce novel approaches for sustainable management of this destructive pest. © 2016 The Royal Entomological Society.

  4. Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis.

    Science.gov (United States)

    Galdiero, Francesca; Bello, Anna Maria; Spina, Anna; Capiluongo, Anna; Liuu, Sophie; De Marco, Margot; Rosati, Alessandra; Capunzo, Mario; Napolitano, Maria; Vuttariello, Emilia; Monaco, Mario; Califano, Daniela; Turco, Maria Caterina; Chiappetta, Gennaro; Vinh, Joëlle; Chiappetta, Giovanni

    2018-01-30

    BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.

  5. Identification of the human ApoAV gene as a novel RORα target gene

    International Nuclear Information System (INIS)

    Lind, Ulrika; Nilsson, Tina; McPheat, Jane; Stroemstedt, Per-Erik; Bamberg, Krister; Balendran, Clare; Kang, Daiwu

    2005-01-01

    Retinoic acid receptor-related orphan receptor-α (RORα) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that RORα regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that RORα also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of RORα increased the endogenous expression of ApoAV in HepG2 cells and RORα also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate RORα transactivation, one of which overlaps with a previously identified binding site for PPARα. Together, these results suggest a novel mechanism whereby RORα modulates lipid metabolism and implies RORα as a potential target for the treatment of dyslipidemia and atherosclerosis

  6. Identification of the human ApoAV gene as a novel ROR{alpha} target gene

    Energy Technology Data Exchange (ETDEWEB)

    Lind, Ulrika [Department of Molecular Pharmacology, AstraZeneca R and D Moelndal (Sweden); Nilsson, Tina [Department of Molecular Pharmacology, AstraZeneca R and D Moelndal (Sweden); McPheat, Jane [Department of Molecular Pharmacology, AstraZeneca R and D Moelndal (Sweden); Stroemstedt, Per-Erik [Department of Molecular Pharmacology, AstraZeneca R and D Moelndal (Sweden); Bamberg, Krister [Department of Molecular Pharmacology, AstraZeneca R and D Moelndal (Sweden); Balendran, Clare [Department of Molecular Pharmacology, AstraZeneca R and D Moelndal (Sweden); Kang, Daiwu [Department of Molecular Pharmacology, AstraZeneca R and D Moelndal (Sweden)

    2005-04-29

    Retinoic acid receptor-related orphan receptor-{alpha} (ROR{alpha}) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that ROR{alpha} regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that ROR{alpha} also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of ROR{alpha} increased the endogenous expression of ApoAV in HepG2 cells and ROR{alpha} also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate ROR{alpha} transactivation, one of which overlaps with a previously identified binding site for PPAR{alpha}. Together, these results suggest a novel mechanism whereby ROR{alpha} modulates lipid metabolism and implies ROR{alpha} as a potential target for the treatment of dyslipidemia and atherosclerosis.

  7. Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

    Science.gov (United States)

    Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

    2018-04-25

    Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones

  8. Combining the Post-Cue Task and the Perceptual Identification Task to Assess Parallel Activation and Mutual Facilitation of Related Primes and Targets.

    Science.gov (United States)

    Scherer, Demian; Wentura, Dirk

    2018-03-01

    Recent theories assume a mutual facilitation in case of semantic overlap for concepts being activated simultaneously. We provide evidence for this claim using a semantic priming paradigm. To test for mutual facilitation of related concepts, a perceptual identification task was employed, presenting prime-target pairs briefly and masked, with an SOA of 0 ms (i.e., prime and target were presented concurrently, one above the other). Participants were instructed to identify the target. In Experiment 1, a cue defining the target was presented at stimulus onset, whereas in Experiment 2 the cue was not presented before the offset of stimuli. Accordingly, in Experiment 2, a post-cue task was merged with the perceptual identification task. We obtained significant semantic priming effects in both experiments. This result is compatible with the view that two concepts can both be activated in parallel and can mutually facilitate each other if they are related.

  9. Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach.

    Science.gov (United States)

    Floegel, Anna; Stefan, Norbert; Yu, Zhonghao; Mühlenbruch, Kristin; Drogan, Dagmar; Joost, Hans-Georg; Fritsche, Andreas; Häring, Hans-Ulrich; Hrabě de Angelis, Martin; Peters, Annette; Roden, Michael; Prehn, Cornelia; Wang-Sattler, Rui; Illig, Thomas; Schulze, Matthias B; Adamski, Jerzy; Boeing, Heiner; Pischon, Tobias

    2013-02-01

    Metabolomic discovery of biomarkers of type 2 diabetes (T2D) risk may reveal etiological pathways and help to identify individuals at risk for disease. We prospectively investigated the association between serum metabolites measured by targeted metabolomics and risk of T2D in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam (27,548 adults) among all incident cases of T2D (n = 800, mean follow-up 7 years) and a randomly drawn subcohort (n = 2,282). Flow injection analysis tandem mass spectrometry was used to quantify 163 metabolites, including acylcarnitines, amino acids, hexose, and phospholipids, in baseline serum samples. Serum hexose; phenylalanine; and diacyl-phosphatidylcholines C32:1, C36:1, C38:3, and C40:5 were independently associated with increased risk of T2D and serum glycine; sphingomyelin C16:1; acyl-alkyl-phosphatidylcholines C34:3, C40:6, C42:5, C44:4, and C44:5; and lysophosphatidylcholine C18:2 with decreased risk. Variance of the metabolites was largely explained by two metabolite factors with opposing risk associations (factor 1 relative risk in extreme quintiles 0.31 [95% CI 0.21-0.44], factor 2 3.82 [2.64-5.52]). The metabolites significantly improved T2D prediction compared with established risk factors. They were further linked to insulin sensitivity and secretion in the Tübingen Family study and were partly replicated in the independent KORA (Cooperative Health Research in the Region of Augsburg) cohort. The data indicate that metabolic alterations, including sugar metabolites, amino acids, and choline-containing phospholipids, are associated early on with a higher risk of T2D.

  10. Identification of compounds that selectively target highly chemotherapy refractory neuroblastoma cancer stem cells.

    Science.gov (United States)

    Díaz-Carballo, David; Acikelli, Ali Haydar; Bardenheuer, Walter; Gustmann, Sebastian; Malak, Sascha; Stoll, Raphael; Kedziorski, Thorsten; Nazif, Mhd Ali; Jastrow, Holger; Wennemuth, Gunter; Dammann, Philip; Feigel, Martin; Strumberg, Dirk

    2014-09-01

    Relapse of cancer months or years after an apparently successful therapy is probably caused by cancer stem cells (CSCs) due to their intrinsic features like dormant periods, radiorefraction, and acquired multidrug resistance (MDR) phenotypes, among other mechanisms of cellular drug evasiveness. Thus, the lack of currently efficacious interventions remains a major problem in the treatment of malignancies, together with the inability of existing drugs to destroy specifically CSCs. Neuroblastomas per se are highly chemotherapy-refractory extracranial tumors in infants with very low survival rates. So far, no effective cytostatics against this kind of tumors are clinically available. Therefore, we have put much effort into the development of agents to efficiently combat this malignancy. For this purpose, we tested several compounds isolated from Cuban propolis on induced CSCs (iCSC) derived from LAN-1 neuroblastoma cells which expressed several characteristics of tumor-initiating cells both in in-vitro and in-vivo models. Some small molecules such as flavonoids and polycyclic polyprenylated acylphloroglucinols (PPAP) were isolated using successive RT-HPLC cycles and identified employing mass spectrometry and NMR spectroscopic techniques. Their cytotoxicity was first screened in sensitive cell systems by MTT proliferation assays and afterwards studied in less sensitive neuroblastoma iCSC models. We found several compounds with considerable anti-iCSC activity, most of them belonging to the PPAP class. The majority of the compounds act in a pleiotropic manner on the molecular biology of tumors although their specific targets remain unclear. Nevertheless, two substances, one of them a flavonoid, induced a strong disruption of tubulin polymerization. In addition, an unknown compound strongly inhibited replicative enzymes like toposimerases I/II and DNA polymerase. Here, we report for the first time cytotoxic activities of small molecules isolated from Caribbean propolis

  11. Identification of Rorβ targets in cultured osteoblasts and in human bone

    Energy Technology Data Exchange (ETDEWEB)

    Roforth, Matthew M., E-mail: roforth.matthew@mayo.edu; Khosla, Sundeep, E-mail: khosla.sundeep@mayo.edu; Monroe, David G., E-mail: monroe.david@mayo.edu

    2013-11-01

    Highlights: •We examine the gene expression patterns controlled by Rorβ in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Rorβ mRNA levels increase in aged, human bone biopsies. •Rorβ may affect osteoblast activity by modulation of these pathways. -- Abstract: Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

  12. Construction of a rice glycoside hydrolase phylogenomic database and identification of targets for biofuel research

    Directory of Open Access Journals (Sweden)

    Rita eSharma

    2013-08-01

    Full Text Available Glycoside hydrolases (GH catalyze the hydrolysis of glycosidic bonds in cell wall polymers and can have major effects on cell wall architecture. Taking advantage of the massive datasets available in public databases, we have constructed a rice phylogenomic database of GHs (http://ricephylogenomics.ucdavis.edu/cellwalls/gh/. This database integrates multiple data types including the structural features, orthologous relationships, mutant availability and gene expression patterns for each GH family in a phylogenomic context. The rice genome encodes 437 GH genes classified into 34 families. Based on pairwise comparison with eight dicot and four monocot genomes, we identified 138 GH genes that are highly diverged between monocots and dicots, 57 of which have diverged further in rice as compared with four monocot genomes scanned in this study. Chromosomal localization and expression analysis suggest a role for both whole-genome and localized gene duplications in expansion and diversification of GH families in rice. We examined the meta-profiles of expression patterns of GH genes in twenty different anatomical tissues of rice. Transcripts of 51 genes exhibit tissue or developmental stage-preferential expression, whereas, seventeen other genes preferentially accumulate in actively growing tissues. When queried in RiceNet, a probabilistic functional gene network that facilitates functional gene predictions, nine out of seventeen genes form a regulatory network with the well-characterized genes involved in biosynthesis of cell wall polymers including cellulose synthase and cellulose synthase-like genes of rice. Two-thirds of the GH genes in rice are up regulated in response to biotic and abiotic stress treatments indicating a role in stress adaptation. Our analyses identify potential GH targets for cell wall modification.

  13. Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets.

    Science.gov (United States)

    Marimuthu, Srinivasan; Chivukula, Raghavender S V; Alfonso, Lloyd F; Moridani, Majid; Hagen, Fred K; Bhat, G Jayarama

    2011-11-01

    Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immuno-affinity purification, mass spectrometry and immuno-blotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeleton proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiment showed that aspirin also acetylated glucose-6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6 phosphate dehydrogenase activity. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

  14. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors.

    Directory of Open Access Journals (Sweden)

    Susanne Pfefferle

    2011-10-01

    Full Text Available Coronaviruses (CoVs are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS in 2002/2003 has demonstrated human vulnerability to (Coronavirus CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B as interaction partners of the CoV non-structural protein 1 (Nsp1. These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

  15. Identification of the interleukin 4 receptor alpha gene as a direct target for p73.

    Science.gov (United States)

    Sasaki, Yasushi; Mita, Hiroaki; Toyota, Minoru; Ishida, Setsuko; Morimoto, Ichiro; Yamashita, Toshiharu; Tanaka, Toshihiro; Imai, Kohzoh; Nakamura, Yusuke; Tokino, Takashi

    2003-12-01

    p73 has a high degree of structural homology to p53 and can activate transcription of p53-responsive genes. However, analysis of p73-deficient mice revealed a marked divergence in the physiological activities of p53 family genes and distinguishes p73 from p53. Mice deficient for p73 exhibit profound defects, including hippocampal dysgenesis, chronic infection, and inflammation, as well as abnormalities in pheromone sensory pathways. p73 plays important roles in neurogenesis, sensory pathways, and homeostatic regulation. Here, we found that the interleukin 4 receptor alpha (IL-4Ralpha) gene is up-regulated by p73 but not significantly by p53 in several human cancer cell lines. IL-4Ralphatranscription is also activated in response to cisplatin, a DNA-damaging agent known to induce p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abrogates the induction of the IL-4Ralpha gene after cisplatin treatment. Furthermore, we identified a p73-binding site in the first intron of the IL-4Ralpha gene that can directly interact with the p73 protein in vivo. This p73-binding site consists of eight copies of a 10-bp consensus p53-binding motif and is a functional response element that is relatively specific for p73 among the p53 family. p73beta promoted localized nucleosomal acetylation through recruitment of coactivator p300, indicating that p73 regulates transcription of IL-4Ralpha through the unique p73-binding site. We also found that p73beta-transfected tumor cells are sensitive to IL-4-mediated apoptosis. Our data suggest that IL-4Ralpha could mediate, in part, certain immune responses and p73-dependent cell death.

  16. Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation

    Directory of Open Access Journals (Sweden)

    Nakano Toshitsugu

    2011-01-01

    Full Text Available Abstract Background During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Results Using immunoprecipitated (IPed DNA fragments recovered by chromatin immunoprecipitation (ChIP with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. Conclusions The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes

  17. Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation.

    Science.gov (United States)

    Fujisawa, Masaki; Nakano, Toshitsugu; Ito, Yasuhiro

    2011-01-30

    During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Using immunoprecipitated (IPed) DNA fragments recovered by chromatin immunoprecipitation (ChIP) with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites) in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes during ripening. Moreover, the binding of RIN to its own

  18. Identification of RegIV as a novel GLI1 target gene in human pancreatic cancer.

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    Feng Wang

    2011-04-01

    Full Text Available GLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1.GLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC, and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP and electrophoretic mobility shift assays (EMSA.The results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001. RegIV expression correlated with GLI1 expression in these tissues (R = 0.795, p<0.0001. These results were verified for protein (R = 0.939, p = 0.018 and mRNA expression (R = 0.959, p = 0.011 in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively. Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA in pancreatic cancer cells.GLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer.

  19. Identification of genomic variants putatively targeted by selection during dog domestication.

    Science.gov (United States)

    Cagan, Alex; Blass, Torsten

    2016-01-12

    Dogs [Canis lupus familiaris] were the first animal species to be domesticated and continue to occupy an important place in human societies. Recent studies have begun to reveal when and where dog domestication occurred. While much progress has been made in identifying the genetic basis of phenotypic differences between dog breeds we still know relatively little about the genetic changes underlying the phenotypes that differentiate all dogs from their wild progenitors, wolves [Canis lupus]. In particular, dogs generally show reduced aggression and fear towards humans compared to wolves. Therefore, selection for tameness was likely a necessary prerequisite for dog domestication. With the increasing availability of whole-genome sequence data it is possible to try and directly identify the genetic variants contributing to the phenotypic differences between dogs and wolves. We analyse the largest available database of genome-wide polymorphism data in a global sample of dogs 69 and wolves 7. We perform a scan to identify regions of the genome that are highly differentiated between dogs and wolves. We identify putatively functional genomic variants that are segregating or at high frequency [> = 0.75 Fst] for alternative alleles between dogs and wolves. A biological pathways analysis of the genes containing these variants suggests that there has been selection on the 'adrenaline and noradrenaline biosynthesis pathway', well known for its involvement in the fight-or-flight response. We identify 11 genes with putatively functional variants fixed for alternative alleles between dogs and wolves. The segregating variants in these genes are strong candidates for having been targets of selection during early dog domestication. We present the first genome-wide analysis of the different categories of putatively functional variants that are fixed or segregating at high frequency between a global sampling of dogs and wolves. We find evidence that selection has been strongest

  20. Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

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    Hernández-Moneo Jose-Luis

    2006-09-01

    Full Text Available Abstract Background Conventional cytogenetic and comparative genomic hybridization (CGH studies in brain malignancies have shown that glioblastoma multiforme (GBM is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations. Results We performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR, 7q21 (CDK6, 4q12 (PDGFRA, and 12q13-15 (MDM2 and CDK4. In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15, and TNFSF13B and COL4A2 (13q32-34. Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs. Conclusion This high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development

  1. Identification and Initial Characterization of the Effectors of an Anther Smut Fungus and Potential Host Target Proteins

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    Venkata S. Kuppireddy

    2017-11-01

    Full Text Available (1 Background: Plant pathogenic fungi often display high levels of host specificity and biotrophic fungi; in particular, they must manipulate their hosts to avoid detection and to complete their obligate pathogenic lifecycles. One important strategy of such fungi is the secretion of small proteins that serve as effectors in this process. Microbotryum violaceum is a species complex whose members infect members of the Caryophyllaceae; M. lychnidis-dioicae, a parasite on Silene latifolia, is one of the best studied interactions. We are interested in identifying and characterizing effectors of the fungus and possible corresponding host targets; (2 Methods: In silico analysis of the M. lychnidis-dioicae genome and transcriptomes allowed us to predict a pool of small secreted proteins (SSPs with the hallmarks of effectors, including a lack of conserved protein family (PFAM domains and also localized regions of disorder. Putative SSPs were tested for secretion using a yeast secretion trap method. We then used yeast two-hybrid analyses for candidate-secreted effectors to probe a cDNA library from a range of growth conditions of the fungus, including infected plants; (3 Results: Roughly 50 SSPs were identified by in silico analysis. Of these, 4 were studied further and shown to be secreted, as well as examined for potential host interactors. One of the putative effectors, MVLG_01732, was found to interact with Arabidopsis thaliana calcium-dependent lipid binding protein (AtCLB and with cellulose synthase interactive protein 1 orthologues; and (4 Conclusions: The identification of a pool of putative effectors provides a resource for functional characterization of fungal proteins that mediate the delicate interaction between pathogen and host. The candidate targets of effectors, e.g., AtCLB, involved in pollen germination suggest tantalizing insights that could drive future studies.

  2. RAS - Target Identification - Informatics

    Science.gov (United States)

    The RAS Informatics lab group develops tools to track and analyze “big data” from the RAS Initiative, as well as analyzes data from external projects. By integrating internal and external data, this group helps improve understanding of RAS-driven cancers.

  3. MALDI based identification of soybean protein markers--possible analytical targets for allergen detection in processed foods.

    Science.gov (United States)

    Cucu, Tatiana; De Meulenaer, Bruno; Devreese, Bart

    2012-02-01

    Soybean (Glycine max) is extensively used all over the world due to its nutritional qualities. However, soybean is included in the "big eight" list of food allergens. According to the EU directive 2007/68/EC, food products containing soybeans have to be labeled in order to protect the allergic consumers. Nevertheless, soybeans can still inadvertently be present in food products. The development of analytical methods for the detection of traces of allergens is important for the protection of allergic consumers. Mass spectrometry of marker proteolytical fragments of protein allergens is growingly recognized as a detection method in food control. However, quantification of soybean at the peptide level is hindered due to limited information regarding specific stable markers derived after proteolytic digestion. The aim of this study was to use MALDI-TOF/MS and MS/MS as a fast screening tool for the identification of stable soybean derived tryptic markers which were still identifiable even if the proteins were subjected to various changes at the molecular level through a number of reactions typically occurring during food processing (denaturation, the Maillard reaction and oxidation). The peptides (401)Val-Arg(410) from the G1 glycinin (Gly m 6) and the (518)Gln-Arg(528) from the α' chain of the β-conglycinin (Gly m 5) proved to be the most stable. These peptides hold potential to be used as targets for the development of new analytical methods for the detection of soybean protein traces in processed foods. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Targeted genotyping-by-sequencing permits cost-effective identification and discrimination of pasture grass species and cultivars.

    Science.gov (United States)

    Pembleton, Luke W; Drayton, Michelle C; Bain, Melissa; Baillie, Rebecca C; Inch, Courtney; Spangenberg, German C; Wang, Junping; Forster, John W; Cogan, Noel O I

    2016-05-01

    A targeted amplicon-based genotyping-by-sequencing approach has permitted cost-effective and accurate discrimination between ryegrass species (perennial, Italian and inter-species hybrid), and identification of cultivars based on bulked samples. Perennial ryegrass and Italian ryegrass are the most important temperate forage species for global agriculture, and are represented in the commercial pasture seed market by numerous cultivars each composed of multiple highly heterozygous individuals. Previous studies have identified difficulties in the use of morphophysiological criteria to discriminate between these two closely related taxa. Recently, a highly multiplexed single nucleotide polymorphism (SNP)-based genotyping assay has been developed that permits accurate differentiation between both species and cultivars of ryegrasses at the genetic level. This assay has since been further developed into an amplicon-based genotyping-by-sequencing (GBS) approach implemented on a second-generation sequencing platform, allowing accelerated throughput and ca. sixfold reduction in cost. Using the GBS approach, 63 cultivars of perennial, Italian and interspecific hybrid ryegrasses, as well as intergeneric Festulolium hybrids, were genotyped. The genetic relationships between cultivars were interpreted in terms of known breeding histories and indistinct species boundaries within the Lolium genus, as well as suitability of current cultivar registration methodologies. An example of applicability to quality assurance and control (QA/QC) of seed purity is also described. Rapid, low-cost genotypic assays provide new opportunities for breeders to more fully explore genetic diversity within breeding programs, allowing the combination of novel unique genetic backgrounds. Such tools also offer the potential to more accurately define cultivar identities, allowing protection of varieties in the commercial market and supporting processes of cultivar accreditation and quality assurance.

  5. Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation.

    Directory of Open Access Journals (Sweden)

    Kishore Polireddy

    Full Text Available Pancreatic cancer has an enrichment of stem-like cancer cells (CSCs that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition.An immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay.Initial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl indoline (BSI, Compound #38 significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities.This study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be developed as anti-EMT and

  6. Evaluating the forensic application of 19 target microRNAs as biomarkers in body fluid and tissue identification.

    Science.gov (United States)

    Sirker, M; Fimmers, R; Schneider, P M; Gomes, I

    2017-03-01

    RNA-based body fluid and tissue identification has evolved as a promising and reliable new technique to classify type and source of biological evidence in crime cases. In particular, mRNA-based approaches are currently on the rise to replace conventional protein-based methods and are increasingly implemented into forensic casework. However, degradation of these nucleic acid molecules can cause issues on laboratory scale and need to be considered for a credible investigation. For this reason, the analysis of miRNAs using qPCR has been proposed to be a sensitive and specific approach to identify the origin of a biological trace taking advantage of their small size and resistance to degradation. Despite the straightforward workflow of this method, suitable endogenous controls are inevitable when performing real-time PCR to ensure accurate normalization of gene expression data in order to allow a meaningful interpretation. In this regard, we have validated reference genes for a set of forensically relevant body fluids and tissues (blood, saliva, semen, vaginal secretions, menstrual blood and skin) and tested 15 target genes aiming to identify abovementioned sample types. Our data showed that preselected endogenous controls (miR26b, miR92 and miR484) and miR144, initially selected as potential marker for the detection of menstrual blood, were the most stable expressed genes among our set of samples. Normalizing qPCR data with these four validated references revealed that only five miRNA markers are necessary to differentiate between the six different cell types selected in this study. Nevertheless, our observations in the present study indicate that miRNA analysis methods may not provide straightforward data interpretation strategies required for an implementation in forensic casework. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

    Science.gov (United States)

    Huang, Jiqing; Kast, Juergen

    2015-08-07

    Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.

  8. Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing.

    Directory of Open Access Journals (Sweden)

    Hansaim Lim

    2016-10-01

    Full Text Available Target-based screening is one of the major approaches in drug discovery. Besides the intended target, unexpected drug off-target interactions often occur, and many of them have not been recognized and characterized. The off-target interactions can be responsible for either therapeutic or side effects. Thus, identifying the genome-wide off-targets of lead compounds or existing drugs will be critical for designing effective and safe drugs, and providing new opportunities for drug repurposing. Although many computational methods have been developed to predict drug-target interactions, they are either less accurate than the one that we are proposing here or computationally too intensive, thereby limiting their capability for large-scale off-target identification. In addition, the performances of most machine learning based algorithms have been mainly evaluated to predict off-target interactions in the same gene family for hundreds of chemicals. It is not clear how these algorithms perform in terms of detecting off-targets across gene families on a proteome scale. Here, we are presenting a fast and accurate off-target prediction method, REMAP, which is based on a dual regularized one-class collaborative filtering algorithm, to explore continuous chemical space, protein space, and their interactome on a large scale. When tested in a reliable, extensive, and cross-gene family benchmark, REMAP outperforms the state-of-the-art methods. Furthermore, REMAP is highly scalable. It can screen a dataset of 200 thousands chemicals against 20 thousands proteins within 2 hours. Using the reconstructed genome-wide target profile as the fingerprint of a chemical compound, we predicted that seven FDA-approved drugs can be repurposed as novel anti-cancer therapies. The anti-cancer activity of six of them is supported by experimental evidences. Thus, REMAP is a valuable addition to the existing in silico toolbox for drug target identification, drug repurposing

  9. Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing.

    Science.gov (United States)

    Lim, Hansaim; Poleksic, Aleksandar; Yao, Yuan; Tong, Hanghang; He, Di; Zhuang, Luke; Meng, Patrick; Xie, Lei

    2016-10-01

    Target-based screening is one of the major approaches in drug discovery. Besides the intended target, unexpected drug off-target interactions often occur, and many of them have not been recognized and characterized. The off-target interactions can be responsible for either therapeutic or side effects. Thus, identifying the genome-wide off-targets of lead compounds or existing drugs will be critical for designing effective and safe drugs, and providing new opportunities for drug repurposing. Although many computational methods have been developed to predict drug-target interactions, they are either less accurate than the one that we are proposing here or computationally too intensive, thereby limiting their capability for large-scale off-target identification. In addition, the performances of most machine learning based algorithms have been mainly evaluated to predict off-target interactions in the same gene family for hundreds of chemicals. It is not clear how these algorithms perform in terms of detecting off-targets across gene families on a proteome scale. Here, we are presenting a fast and accurate off-target prediction method, REMAP, which is based on a dual regularized one-class collaborative filtering algorithm, to explore continuous chemical space, protein space, and their interactome on a large scale. When tested in a reliable, extensive, and cross-gene family benchmark, REMAP outperforms the state-of-the-art methods. Furthermore, REMAP is highly scalable. It can screen a dataset of 200 thousands chemicals against 20 thousands proteins within 2 hours. Using the reconstructed genome-wide target profile as the fingerprint of a chemical compound, we predicted that seven FDA-approved drugs can be repurposed as novel anti-cancer therapies. The anti-cancer activity of six of them is supported by experimental evidences. Thus, REMAP is a valuable addition to the existing in silico toolbox for drug target identification, drug repurposing, phenotypic screening, and

  10. A multidisciplinary approach to the identification and evaluation of novel concepts for deeply buried hardened target defeat

    Science.gov (United States)

    Branscome, Ewell Caleb

    During the Cold War, Deeply Buried Hardened Targets (DBHTs) and the assets they protected were of great strategic and tactical concern to the Department of Defense. Megaton-class nuclear warheads were the only viable means of attacking many of these facilities, and even so, a small subset of DBHTs was anticipated to be robust even in the face of such an attack. Post Cold War, the threat posed by DBHTs has not disappeared. Rather, the conventional warfare advantages of the United States have led to an increasing emphasis by potential adversaries on the construction and use of hardened facilities such as DBHTs for protection of both conventional and unconventional assets. Further, the shift in perceived relative risk to the United States' national security from large scale all-out nuclear attack towards very limited attack by Weapons of Mass Destruction (WMD) has led some to hypothesize that "self-deterrence" may diminish the strategic value of current inventory nuclear weapons. The objective of the work described was to identify and explore a paradigm shifting solution that could offer leap-ahead capabilities to counter current and future DBHT threats while mitigating or eliminating the "self-deterrence" issue. Systematic evaluation of DHBT defeat alternatives lead to the selection of a thermal subterrene as a hypothetical means of providing such a capability. A number of possible implementation alternatives for a thermal subterrene were investigated, resulting in the identification of the RadioIsotope Powered Thermal Penetrator (RIPTP) concept for providing an effectively unlimited hard rock penetration capability using near-term technologies. However, the proposed approach was novel and thus required formulation and application of a physics based multidisciplinary analysis code to enable evaluation of lv design alternatives and analysis of performance. Technical considerations identified as important to the feasibility of a RIPTP for DBHT defeat included: packing

  11. A novel neural network based image reconstruction model with scale and rotation invariance for target identification and classification for Active millimetre wave imaging

    Science.gov (United States)

    Agarwal, Smriti; Bisht, Amit Singh; Singh, Dharmendra; Pathak, Nagendra Prasad

    2014-12-01

    Millimetre wave imaging (MMW) is gaining tremendous interest among researchers, which has potential applications for security check, standoff personal screening, automotive collision-avoidance, and lot more. Current state-of-art imaging techniques viz. microwave and X-ray imaging suffers from lower resolution and harmful ionizing radiation, respectively. In contrast, MMW imaging operates at lower power and is non-ionizing, hence, medically safe. Despite these favourable attributes, MMW imaging encounters various challenges as; still it is very less explored area and lacks suitable imaging methodology for extracting complete target information. Keeping in view of these challenges, a MMW active imaging radar system at 60 GHz was designed for standoff imaging application. A C-scan (horizontal and vertical scanning) methodology was developed that provides cross-range resolution of 8.59 mm. The paper further details a suitable target identification and classification methodology. For identification of regular shape targets: mean-standard deviation based segmentation technique was formulated and further validated using a different target shape. For classification: probability density function based target material discrimination methodology was proposed and further validated on different dataset. Lastly, a novel artificial neural network based scale and rotation invariant, image reconstruction methodology has been proposed to counter the distortions in the image caused due to noise, rotation or scale variations. The designed neural network once trained with sample images, automatically takes care of these deformations and successfully reconstructs the corrected image for the test targets. Techniques developed in this paper are tested and validated using four different regular shapes viz. rectangle, square, triangle and circle.

  12. A novel recombinantly produced banana lectin isoform is a valuable tool for glycoproteomics and a potent modulator of the proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs

    DEFF Research Database (Denmark)

    Gavrovic-Jankulovic, M; Poulsen, Knud; Brckalo, T

    2008-01-01

    Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal......, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3(+), CD4(+), and CD8(+) populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics...

  13. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

    2015-01-01

    Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells often...... target diverse regions in highly variable viral pathogens and this diversity may need to be addressed through redefinition of suitable peptide targets. Methods: We have developed a method for antigen assessment and target selection for polyvalent vaccines, with which we identified immune epitopes from...... variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results: We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased...

  14. Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching.

    Science.gov (United States)

    Dresen, S; Ferreirós, N; Gnann, H; Zimmermann, R; Weinmann, W

    2010-04-01

    The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

  15. Validating the TOD method with identification of real targets : Effects of aspect angle, dynamic imaging and signal processing

    NARCIS (Netherlands)

    Beintema, J.A.; Hogervorst, M.A.; Dijk, J.; Bijl, P.

    2008-01-01

    How far the eye reaches via camera is quantifiable and modeled with the TOD (triangle orientation discrimination) methodology (Bijl and Hogervorst, 2008 Perception 37 Supplement, this issue). For validation and extension purposes, we studied identification of real objects in several conditions.

  16. The Confidence-Accuracy Relationship in Eyewitness Identification: Effects of Lineup Instructions, Foil Similarity, and Target-Absent Base Rates

    Science.gov (United States)

    Brewer, Neil; Wells, Gary L.

    2006-01-01

    Discriminating accurate from mistaken eyewitness identifications is a major issue facing criminal justice systems. This study examined whether eyewitness confidence assists such decisions under a variety of conditions using a confidence-accuracy (CA) calibration approach. Participants (N = 1,200) viewed a simulated crime and attempted 2 separate…

  17. Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

    Science.gov (United States)

    Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

    2015-05-01

    Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

  18. N-glycoproteome analysis of the secretome of human metastatic hepatocellular carcinoma cell lines combining hydrazide chemistry, HILIC enrichment and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Xianyu Li

    Full Text Available Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC cell lines with low (MHCC97L or high (HCCLM3 metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC, with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.

  19. Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network

    Science.gov (United States)

    Li, Wan; Wei, Wenqing; Li, Yiran; Xie, Ruiqiang; Guo, Shanshan; Wang, Yahui; Jiang, Jing; Chen, Binbin; Lv, Junjie; Zhang, Nana; Chen, Lina; He, Weiming

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biological background, 1 PPDT-Module and 22 PCOS potential drug targets were identified, 21 of which were verified by literatures to be associated with the pathogenesis of PCOS. 42 drugs targeting to 13 PCOS potential drug targets were investigated experimentally or clinically for PCOS. Evaluated by independent datasets, the whole PPDT-Module and 22 PCOS potential drug targets could not only reveal the drug response, but also distinguish the statuses between normal and disease. Our identified PPDT-Module and PCOS potential drug targets would shed light on the treatment of PCOS. And our approach would provide valuable insights to research on the pathogenesis and drug response of other diseases. PMID:27191267

  20. [Identification of Clonorchis sinensis metacercariae based on PCR targeting ribosomal DNA ITS regions and COX1 gene].

    Science.gov (United States)

    Yang, Qing-Li; Shen, Ji-Qing; Jiang, Zhi-Hua; Yang, Yi-Chao; Li, Hong-Mei; Chen, Ying-Dan; Zhou, Xiao-Nong

    2014-06-01

    To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene. Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed. C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification. C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

  1. Strand Analysis, a free online program for the computational identification of the best RNA interference (RNAi targets based on Gibbs free energy

    Directory of Open Access Journals (Sweden)

    Tiago Campos Pereira

    2007-01-01

    Full Text Available The RNA interference (RNAi technique is a recent technology that uses double-stranded RNA molecules to promote potent and specific gene silencing. The application of this technique to molecular biology has increased considerably, from gene function identification to disease treatment. However, not all small interfering RNAs (siRNAs are equally efficient, making target selection an essential procedure. Here we present Strand Analysis (SA, a free online software tool able to identify and classify the best RNAi targets based on Gibbs free energy (deltaG. Furthermore, particular features of the software, such as the free energy landscape and deltaG gradient, may be used to shed light on RNA-induced silencing complex (RISC activity and RNAi mechanisms, which makes the SA software a distinct and innovative tool.

  2. Identification of targets of miR-200b by a SILAC-based quantitative proteomic approach

    Directory of Open Access Journals (Sweden)

    Arivusudar Marimuthu

    2014-09-01

    Full Text Available miRNAs regulate gene expression by binding to cognate mRNAs causing mRNA degradation or translational repression. Mass spectrometry-based proteomic analysis is being widely used to identify miRNA targets. The miR-200b miRNA cluster is often overexpressed in multiple cancer types, but the identity of the targets remains elusive. Using SILAC-based analysis, we examined the effects of overexpression of a miR-200b mimic or a control miRNA in fibrosarcoma cells. We identified around 300 potential targets of miR-200b based on a change in the expression of protein levels. We validated a subset of potential targets at the transcript level using quantitative PCR.

  3. Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2008-01-01

    Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

  4. Identification of Pro-Differentiation P53 Target Genes and Evaluation of Expression in Normal and Malignant Mammary Gland

    National Research Council Canada - National Science Library

    Li, Hua; Cherukuri, Pratima; Pho, Alissa; Cowling, Victoria; Cole, Michael; Godwin, Andrew K; Wells, Wendy; Direnzo, James

    2006-01-01

    ... molecular targets such as ER alpha, PR and Her2- overexpression. These tumors display a high degree of cellular heterogeneity suggesting that they may arise as the result of unregulated self-renewal in a multipotent cell...

  5. Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network

    OpenAIRE

    Huang, Hao; He, Yuehan; Li, Wan; Wei, Wenqing; Li, Yiran; Xie, Ruiqiang; Guo, Shanshan; Wang, Yahui; Jiang, Jing; Chen, Binbin; Lv, Junjie; Zhang, Nana; Chen, Lina; He, Weiming

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biologi...

  6. Systematic Identification and Assessment of Therapeutic Targets for Breast Cancer Based on Genome-Wide RNA Interference Transcriptomes

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2017-02-01

    Full Text Available With accumulating public omics data, great efforts have been made to characterize the genetic heterogeneity of breast cancer. However, identifying novel targets and selecting the best from the sizeable lists of candidate targets is still a key challenge for targeted therapy, largely owing to the lack of economical, efficient and systematic discovery and assessment to prioritize potential therapeutic targets. Here, we describe an approach that combines the computational evaluation and objective, multifaceted assessment to systematically identify and prioritize targets for biological validation and therapeutic exploration. We first establish the reference gene expression profiles from breast cancer cell line MCF7 upon genome-wide RNA interference (RNAi of a total of 3689 genes, and the breast cancer query signatures using RNA-seq data generated from tissue samples of clinical breast cancer patients in the Cancer Genome Atlas (TCGA. Based on gene set enrichment analysis, we identified a set of 510 genes that when knocked down could significantly reverse the transcriptome of breast cancer state. We then perform multifaceted assessment to analyze the gene set to prioritize potential targets for gene therapy. We also propose drug repurposing opportunities and identify potentially druggable proteins that have been poorly explored with regard to the discovery of small-molecule modulators. Finally, we obtained a small list of candidate therapeutic targets for four major breast cancer subtypes, i.e., luminal A, luminal B, HER2+ and triple negative breast cancer. This RNAi transcriptome-based approach can be a helpful paradigm for relevant researches to identify and prioritize candidate targets for experimental validation.

  7. Genome-Wide Identification of Polycomb Target Genes Reveals a Functional Association of Pho with Scm in Bombyx mori

    OpenAIRE

    Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Tatsuke, Tsuneyuki; Zhu, Li; Xu, Jian; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

    2012-01-01

    Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers and act together in three multimeric complexes, Polycomb repressive complex 1 (PRC1), Polycomb repressive complex 2 (PRC2), and Pleiohomeotic repressive complex (PhoRC), to repress transcription of the target genes. Here, we identified Polycomb target genes in Bombyx mori with holocentric centromere using genome-wide expression screening based on the knockdown of BmSCE, BmESC, BmPHO, or BmSCM gene, which represent ...

  8. Interventions targeted at primary care practitioners to improve the identification and referral of patients with co-morbid obesity: a realist review protocol.

    Science.gov (United States)

    Blane, David N; Macdonald, Sara; Morrison, David; O'Donnell, Catherine A

    2015-05-01

    Obesity is one of the most significant public health challenges in the developed world. Recent policy has suggested that more can be done in primary care to support adults with obesity. In particular, general practitioners (GPs) and practice nurses (PNs) could improve the identification and referral of adults with obesity to appropriate weight management services. Previous interventions targeted at primary care practitioners in this area have had mixed results, suggesting a more complex interplay between patients, practitioners, and systems. The objectives of this review are (i) to identify the underlying 'programme theory' of interventions targeted at primary care practitioners to improve the identification and referral of adults with obesity and (ii) to explore how and why GPs and PNs identify and refer individuals with obesity, particularly in the context of weight-related co-morbidity. This protocol will explain the rationale for using a realist review approach and outline the key steps in this process. Realist review is a theory-led approach to knowledge synthesis that provides an explanatory analysis aimed at discerning what works, for whom, in what circumstances, how, and why. In this review, scoping interviews with key stakeholders involved in the planning and delivery of adult weight management services in Scotland helped to inform the identification of formal theories - from psychology, sociology, and implementation science - that will be tested as the review progresses. A comprehensive search strategy is described, including scope for iterative searching. Data analysis is outlined in three stages (describing context-mechanism-outcome configurations, exploring patterns in these configurations, and developing and testing middle-range theories, informed by the formal theories previously identified), culminating in the production of explanatory programme theory that considers individual, interpersonal, and institutional/systems-level components. This is the

  9. Understanding the magnitude of emergent contaminant releases through target screening and metabolite identification using high resolution mass spectrometry: Illicit drugs in raw sewage influents.

    Science.gov (United States)

    Heuett, Nubia V; Batchu, Sudha Rani; Gardinali, Piero R

    2015-01-23

    A QExactive Orbitrap was used for the identification of phase I and II transformation products (TPs) of illicit drugs in raw sewage influents. Two operating modes (targeted MS(2) and Data-dependent screening) were used for data acquisition. Even though, data-dependent scan is a faster route towards the potential identification of metabolites, it suffered from its limitation to provide enough data points across the chromatographic peak during the MS(2) cycle in contrast to targeted MS(2). Therefore, the later technique was implemented as the method of choice in this study for the positive confirmation and quantitation of TPs (n=54). The vast majority of the identified TPs were products of phase I transformation reactions, with the latter being more prevalent in the nature. Estimated mole fractions showed that for a large number of the analytes, TPs must also be monitored in order to fully understand their environmental fate and calculate potential consumption. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

    Science.gov (United States)

    Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

    2013-08-01

    Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

  11. Identification of putative drug targets in Vancomycin-resistant Staphylococcus aureus (VRSA) using computer aided protein data analysis.

    Science.gov (United States)

    Hasan, Md Anayet; Khan, Md Arif; Sharmin, Tahmina; Hasan Mazumder, Md Habibul; Chowdhury, Afrin Sultana

    2016-01-01

    Vancomycin-resistant Staphylococcus aureus (VRSA) is a Gram-positive, facultative aerobic bacterium which is evolved from the extensive exposure of Vancomycin to Methicillin resistant S. aureus (MRSA) that had become the most common cause of hospital and community-acquired infections. Due to the emergence of different antibiotic resistance strains, there is an exigency to develop novel drug targets to address the provocation of multidrug-resistant bacteria. In this study, in-silico genome subtraction methodology was used to design potential and pathogen specific drug targets against VRSA. Our study divulged 1987 proteins from the proteome of 34,549 proteins, which have no homologues in human genome after sequential analysis through CD-HIT and BLASTp. The high stringency analysis of the remaining proteins against database of essential genes (DEG) resulted in 169 proteins which are essential for S. aureus. Metabolic pathway analysis of human host and pathogen by KAAS at the KEGG server sorted out 19 proteins involved in unique metabolic pathways. 26 human non-homologous membrane-bound essential proteins including 4 which were also involved in unique metabolic pathway were deduced through PSORTb, CELLO v.2.5, ngLOC. Functional classification of uncharacterized proteins through SVMprot derived 7 human non-homologous membrane-bound hypothetical essential proteins. Study of potential drug target against Drug Bank revealed pbpA-penicillin-binding protein 1 and hypothetical protein MQW_01796 as the best drug target candidate. 2D structure was predicted by PRED-TMBB, 3D structure and functional analysis was also performed. Protein-protein interaction network of potential drug target proteins was analyzed by using STRING. The identified drug targets are expected to have great potential for designing novel drugs against VRSA infections and further screening of the compounds against these new targets may result in the discovery of novel therapeutic compounds that can be

  12. Identification of Forensically Important Calliphoridae and Sarcophagidae Species Collected in Korea Using SNaPshot Multiplex System Targeting the Cytochrome c Oxidase Subunit I Gene

    Directory of Open Access Journals (Sweden)

    Ji Hye Park

    2018-01-01

    Full Text Available Estimation of postmortem interval (PMI is paramount in modern forensic investigation. After the disappearance of the early postmortem phenomena conventionally used to estimate PMI, entomologic evidence provides important indicators for PMI estimation. The age of the oldest fly larvae or pupae can be estimated to pinpoint the time of oviposition, which is considered the minimum PMI (PMImin. The development rate of insects is usually temperature dependent and species specific. Therefore, species identification is mandatory for PMImin estimation using entomological evidence. The classical morphological identification method cannot be applied when specimens are damaged or have not yet matured. To overcome this limitation, some investigators employ molecular identification using mitochondrial cytochrome c oxidase subunit I (COI nucleotide sequences. The molecular identification method commonly uses Sanger’s nucleotide sequencing and molecular phylogeny, which are complex and time consuming and constitute another obstacle for forensic investigators. In this study, instead of using conventional Sanger’s nucleotide sequencing, single-nucleotide polymorphisms (SNPs in the COI gene region, which are unique between fly species, were selected and targeted for single-base extension (SBE technology. These SNPs were genotyped using a SNaPshot® kit. Eleven Calliphoridae and seven Sarcophagidae species were covered. To validate this genotyping, fly DNA samples (103 adults, 84 larvae, and 4 pupae previously confirmed by DNA barcoding were used. This method worked quickly with minimal DNA, providing a potential alternative to conventional DNA barcoding. Consisting of only a few simple electropherogram peaks, the results were more straightforward compared with those of the conventional DNA barcoding produced by Sanger’s nucleotide sequencing.

  13. Genome wide identification of cotton (Gossypium hirsutum)-encoded microRNA targets against Cotton leaf curl Burewala virus.

    Science.gov (United States)

    Shweta; Akhter, Yusuf; Khan, Jawaid Ahmad

    2018-01-05

    Cotton leaf curl Burewala virus (CLCuBV, genus Begomovirus) causes devastating cotton leaf curl disease. Among various known virus controlling strategies, RNAi-mediated one has shown potential to protect host crop plants. Micro(mi) RNAs, are the endogenous small RNAs and play a key role in plant development and stress resistance. In the present study we have identified cotton (Gossypium hirsutum)-encoded miRNAs targeting the CLCuBV. Based on threshold free energy and maximum complementarity scores of host miRNA-viral mRNA target pairs, a number of potential miRNAs were annotated. Among them, ghr-miR168 was selected as the most potent candidate, capable of targeting several vital genes namely C1, C3, C4, V1 and V2 of CLCuBV genome. In addition, ghr-miR395a and ghr-miR395d were observed to target the overlapping transcripts of C1 and C4 genes. We have verified the efficacy of these miRNA targets against CLCuBV following suppression of RNAi-mediated virus control through translational inhibition or cleavage of viral mRNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Identification of broadly reactive epitopes targeting major glycoproteins of Herpes simplex virus (HSV) 1 and 2 - An immunoinformatics analysis.

    Science.gov (United States)

    Chauhan, Varun; Goyal, Kapil; Singh, Mini P

    2018-07-01

    Infections due to both HSV-1 and HSV-2 constitute an enormous health burden worldwide. Development of vaccine against herpes infections is a WHO supported public health priority. The viral glycoproteins have always been the major hotspots for vaccine designing. The present study was aimed to identify the conserved T and B cell epitopes in the major glycoproteins of both HSV-1 and HSV-2 via rigorous computational approaches. Identification of promiscuous T cell epitopes is of utmost importance in vaccine designing as such epitopes are capable of binding to several allelic forms of HLA and could generate effective immune response in the host. The criteria designed for identification of T and B cell epitopes was that it should be conserved in both HSV-1 and 2, promiscuous, have high affinity towards HLA alleles, should be located on the surface of glycoproteins and not be present in the glycosylation sites. This study led to the identification of 17 HLA Class II and 26 HLA Class I T cell epitopes, 9 linear and some conformational B cell epitopes. The identified T cell epitopes were further subjected to molecular docking analysis to analyze their binding patterns. Altogether we have identified 4 most promising regions in glycoproteins (2-gB, 1-gD, 1-gH) of HSV-1 and 2 which are promiscuous to HLA Class II alleles and have overlapping HLA Class I and B cell epitopes, which could be very useful in generating both arms of immune response in the host i.e. adaptive as well as humoral immunity. Further the authors propose the cross-validation of the identified epitopes in experimental settings for confirming their immunogenicity to support the present findings. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Identification of Novel Molecular Targets for Pleckstrin Homology (PH) Domains Found in Oncogenes Implicated in Breast Cancer

    Science.gov (United States)

    2008-03-01

    roles as a positive regulator of IL- 2 gene induction in T cells (Deckert et al, 1998), NK cell-mediated cytotoxicity (Jevremovic et al, 2001), and FcRI...Identification of a gene encoding a human oxysterol-binding protein-homologue: a potential general molecular marker for blood dissemination of solid...Biol. 6(5), 384-385. Scanlan, MJ, Gout , I, Gordon, CM, Williamson, B, Stockert, E, Gure, AO, Jager, D, Chen, YT, Mackay, A, O’Hare, MJ, and Old LJ

  16. Modeling with a view to target identification in metabolic engineering: a critical evaluation of the available tools.

    Science.gov (United States)

    Maertens, Jo; Vanrolleghem, Peter A

    2010-01-01

    The state of the art tools for modeling metabolism, typically used in the domain of metabolic engineering, were reviewed. The tools considered are stoichiometric network analysis (elementary modes and extreme pathways), stoichiometric modeling (metabolic flux analysis, flux balance analysis, and carbon modeling), mechanistic and approximative modeling, cybernetic modeling, and multivariate statistics. In the context of metabolic engineering, one should be aware that the usefulness of these tools to optimize microbial metabolism for overproducing a target compound depends predominantly on the characteristic properties of that compound. Because of their shortcomings not all tools are suitable for every kind of optimization; issues like the dependence of the target compound's synthesis on severe (redox) constraints, the characteristics of its formation pathway, and the achievable/desired flux towards the target compound should play a role when choosing the optimization strategy.

  17. Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

    Directory of Open Access Journals (Sweden)

    Bin Tang

    Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

  18. Identification of Biomarkers of Exposure to FTOHs and PAPs in Humans Using a Targeted and Nontargeted Analysis Approach.

    Science.gov (United States)

    Dagnino, Sonia; Strynar, Mark J; McMahen, Rebecca L; Lau, Christopher S; Ball, Carol; Garantziotis, Stavros; Webster, Thomas F; McClean, Michael D; Lindstrom, Andrew B

    2016-09-20

    Although historic perfluorinated compounds are currently under scrutiny and growing regulatory control in the world, little is known about human exposure to other polyfluorinated compounds presently in use. Fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) are known to degrade to terminal perfluorinated acids and toxic reactive intermediates through metabolic pathways. Therefore, it is important to characterize their human exposure by the identification of unique biomarkers. With the use of liquid chromatography-mass spectrometry-time-of-flight analysis (LC-MS-TOF), we developed a workflow for the identification of metabolites for the 8:2 FTOH and 8:2 diPAP. Analysis of serum and urine of dosed rats indicated the 8:2 FTOH-sulfate and the 8:2 diPAP as potential biomarkers. These compounds, as well as 25 other fluorinated compounds and metabolites, were analyzed in human serum and urine samples from the general population (n = 100) and office workers (n = 30). The 8:2 FTOH-sulfate was measured for the first time in human samples in 5 to 10% of the serum samples, ranging from 50 to 80 pg/mL. The 8:2 diPAP was measured in 58% of the samples, ranging from 100 to 800 pg/mL. This study indicates the FTOH-sulfate conjugate as a biomarker of exposure to FTOHs and PAPs in humans.

  19. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Sun Xiaonan [Department of Radiation Oncology, Sir Run Run Shaw Hospital, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou (China); Yang Chunying [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Liu Hai; Wang Qi [Department of Radiation Oncology, Sir Run Run Shaw Hospital, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou (China); Wu Shixiu [Department of Radiation Oncology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou (China); Li Xia; Xie Tian [Research Center of Biomedicine and Health, Hangzhou Normal University, Hangzhou (China); Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Xu Bo, E-mail: bxu@tmhs.org [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Zheng, Shu, E-mail: zhengshu@zju.edu.cn [Cancer Institute, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou (China)

    2012-12-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged {gamma}-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  20. Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

    NARCIS (Netherlands)

    Schmidt, Marco F; Groves, Matthew R; Rademann, Jörg

    2011-01-01

    Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic

  1. Identification of a disease-causing mutation in a Chinese patient with retinitis pigmentosa by targeted next-generation sequencing

    DEFF Research Database (Denmark)

    Xiao, Jianping; Guo, Xueqin; Wang, Yong

    2017-01-01

    Purpose: To identify disease-causing mutations in a Chinese patient with retinitis pigmentosa (RP). Methods: A detailed clinical examination was performed on the proband. Targeted next-generation sequencing (NGS) combined with bioinformatics analysis was performed on the proband to detect candidate...

  2. Computational identification of miRNAs, their targets and functions in three-spined stickleback (Gasterosteus aculeatus).

    Science.gov (United States)

    Chaturvedi, Anurag; Raeymaekers, Joost A M; Volckaert, Filip A M

    2014-07-01

    An intriguing question in biology is how the evolution of gene regulation is shaped by natural selection in natural populations. Among the many known regulatory mechanisms, regulation of gene expression by microRNAs (miRNAs) is of critical importance. However, our understanding of their evolution in natural populations is limited. Studying the role of miRNAs in three-spined stickleback, an important natural model for speciation research, may provide new insights into adaptive polymorphisms. However, lack of annotation of miRNA genes in its genome is a bottleneck. To fill this research gap, we used the genome of three-spined stickleback to predict miRNAs and their targets. We predicted 1486 mature miRNAs using the homology-based miRNA prediction approach. We then performed functional annotation and enrichment analysis of these targets, which identified over-represented motifs. Further, a database resource (GAmiRdb) has been developed for dynamically searching miRNAs and their targets exclusively in three-spined stickleback. Finally, the database was used in two case studies focusing on freshwater adaptation in natural populations. In the first study, we found 44 genomic regions overlapping with predicted miRNA targets. In the second study, we identified two SNPs altering the MRE seed site of sperm-specific glyceraldehyde-3-phosphate gene. These findings highlight the importance of the GAmiRdb knowledge base in understanding adaptive evolution. © 2014 John Wiley & Sons Ltd.

  3. Identification of target genes of the p16INK4A-pRB-E2F pathway

    DEFF Research Database (Denmark)

    Vernell, Richard; Helin, Kristian; Müller, Heiko

    2003-01-01

    as physiological targets of the pRB pathway, and the further characterization of these genes should provide insights into how this pathway controls proliferation. We show that Gibbs sampling detects enrichment of several sequence motifs, including E2F consensus binding sites, in the upstream regions of these genes...

  4. Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation

    DEFF Research Database (Denmark)

    Hartman, Hassan B.; Fell, David A.; Rossell, Sergio

    2014-01-01

    Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of ...

  5. Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

    Energy Technology Data Exchange (ETDEWEB)

    Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko [Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 (Japan); Umezawa, Kazuo, E-mail: umezawa@aichi-med-u.ac.jp [Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195 (Japan)

    2016-08-05

    15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. -- Highlights: •Fifteen-deoxyspergualin (DSG) is an immunosuppressive agent clinically used. •We have identified PCBP2, an RNA-binding protein, as a molecular target of DSG. •Alteration of PCBP2 activity may explain the immunosuppressive activity of DSG.

  6. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    International Nuclear Information System (INIS)

    Sun Xiaonan; Yang Chunying; Liu Hai; Wang Qi; Wu Shixiu; Li Xia; Xie Tian; Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian; Xu Bo; Zheng, Shu

    2012-01-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged γ-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  7. Equal opportunity for low-degree network nodes: a PageRank-based method for protein target identification in metabolic graphs.

    Directory of Open Access Journals (Sweden)

    Dániel Bánky

    Full Text Available Biological network data, such as metabolic-, signaling- or physical interaction graphs of proteins are increasingly available in public repositories for important species. Tools for the quantitative analysis of these networks are being developed today. Protein network-based drug target identification methods usually return protein hubs with large degrees in the networks as potentially important targets. Some known, important protein targets, however, are not hubs at all, and perturbing protein hubs in these networks may have several unwanted physiological effects, due to their interaction with numerous partners. Here, we show a novel method applicable in networks with directed edges (such as metabolic networks that compensates for the low degree (non-hub vertices in the network, and identifies important nodes, regardless of their hub properties. Our method computes the PageRank for the nodes of the network, and divides the PageRank by the in-degree (i.e., the number of incoming edges of the node. This quotient is the same in all nodes in an undirected graph (even for large- and low-degree nodes, that is, for hubs and non-hubs as well, but may differ significantly from node to node in directed graphs. We suggest to assign importance to non-hub nodes with large PageRank/in-degree quotient. Consequently, our method gives high scores to nodes with large PageRank, relative to their degrees: therefore non-hub important nodes can easily be identified in large networks. We demonstrate that these relatively high PageRank scores have biological relevance: the method correctly finds numerous already validated drug targets in distinct organisms (Mycobacterium tuberculosis, Plasmodium falciparum and MRSA Staphylococcus aureus, and consequently, it may suggest new possible protein targets as well. Additionally, our scoring method was not chosen arbitrarily: its value for all nodes of all undirected graphs is constant; therefore its high value captures

  8. Equal opportunity for low-degree network nodes: a PageRank-based method for protein target identification in metabolic graphs.

    Science.gov (United States)

    Bánky, Dániel; Iván, Gábor; Grolmusz, Vince

    2013-01-01

    Biological network data, such as metabolic-, signaling- or physical interaction graphs of proteins are increasingly available in public repositories for important species. Tools for the quantitative analysis of these networks are being developed today. Protein network-based drug target identification methods usually return protein hubs with large degrees in the networks as potentially important targets. Some known, important protein targets, however, are not hubs at all, and perturbing protein hubs in these networks may have several unwanted physiological effects, due to their interaction with numerous partners. Here, we show a novel method applicable in networks with directed edges (such as metabolic networks) that compensates for the low degree (non-hub) vertices in the network, and identifies important nodes, regardless of their hub properties. Our method computes the PageRank for the nodes of the network, and divides the PageRank by the in-degree (i.e., the number of incoming edges) of the node. This quotient is the same in all nodes in an undirected graph (even for large- and low-degree nodes, that is, for hubs and non-hubs as well), but may differ significantly from node to node in directed graphs. We suggest to assign importance to non-hub nodes with large PageRank/in-degree quotient. Consequently, our method gives high scores to nodes with large PageRank, relative to their degrees: therefore non-hub important nodes can easily be identified in large networks. We demonstrate that these relatively high PageRank scores have biological relevance: the method correctly finds numerous already validated drug targets in distinct organisms (Mycobacterium tuberculosis, Plasmodium falciparum and MRSA Staphylococcus aureus), and consequently, it may suggest new possible protein targets as well. Additionally, our scoring method was not chosen arbitrarily: its value for all nodes of all undirected graphs is constant; therefore its high value captures importance in the

  9. Identification and characterization of miRNAs and targets in flax (Linum usitatissimum) under saline, alkaline, and saline-alkaline stresses.

    Science.gov (United States)

    Yu, Ying; Wu, Guangwen; Yuan, Hongmei; Cheng, Lili; Zhao, Dongsheng; Huang, Wengong; Zhang, Shuquan; Zhang, Liguo; Chen, Hongyu; Zhang, Jian; Guan, Fengzhi

    2016-05-27

    MicroRNAs (miRNAs) play a critical role in responses to biotic and abiotic stress and have been characterized in a large number of plant species. Although flax (Linum usitatissimum L.) is one of the most important fiber and oil crops worldwide, no reports have been published describing flax miRNAs (Lus-miRNAs) induced in response to saline, alkaline, and saline-alkaline stresses. In this work, combined small RNA and degradome deep sequencing was used to analyze flax libraries constructed after alkaline-salt stress (AS2), neutral salt stress (NSS), alkaline stress (AS), and the non-stressed control (CK). From the CK, AS, AS2, and NSS libraries, a total of 118, 119, 122, and 120 known Lus-miRNAs and 233, 213, 211, and 212 novel Lus-miRNAs were isolated, respectively. After assessment of differential expression profiles, 17 known Lus-miRNAs and 36 novel Lus-miRNAs were selected and used to predict putative target genes. Gene ontology term enrichment analysis revealed target genes that were involved in responses to stimuli, including signaling and catalytic activity. Eight Lus-miRNAs were selected for analysis using qRT-PCR to confirm the accuracy and reliability of the miRNA-seq results. The qRT-PCR results showed that changes in stress-induced expression profiles of these miRNAs mirrored expression trends observed using miRNA-seq. Degradome sequencing and transcriptome profiling showed that expression of 29 miRNA-target pairs displayed inverse expression patterns under saline, alkaline, and saline-alkaline stresses. From the target prediction analysis, the miR398a-targeted gene codes for a copper/zinc superoxide dismutase, and the miR530 has been shown to explicitly target WRKY family transcription factors, which suggesting that these two micRNAs and their targets may significant involve in the saline, alkaline, and saline-alkaline stress response in flax. Identification and characterization of flax miRNAs, their target genes, functional annotations, and gene

  10. 2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets.

    Science.gov (United States)

    Orsatti, Laura; Forte, Eleonora; Tomei, Licia; Caterino, Marianna; Pessi, Antonello; Talamo, Fabio

    2009-07-01

    The protein tyrosine phosphatase PRL-3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL-3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over-expressing PRL-3. We used 2-D difference gel electrophoresis combined with the fluorescence staining Pro-Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL-3 over-expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL-3-induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [Biochim. Biophys. Acta 2008, 1783, 334-344]. The combination of 2-D difference in gel electrophoresis and Pro-Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.

  11. Case Report Identification of a novel SLC45A2 mutation in albinism by targeted next-generation sequencing.

    Science.gov (United States)

    Xue, J J; Xue, J F; Xue, H Q; Guo, Y Y; Liu, Y; Ouyang, N

    2016-09-19

    Albinism is a diverse group of hypopigmentary disorders caused by multiple-genetic defects. The genetic diagnosis of patients affected with albinism by Sanger sequencing is often complex, expensive, and time-consuming. In this study, we performed targeted next-generation sequencing to screen for 16 genes in a patient with albinism, and identified 21 genetic variants, including 19 known single nucleotide polymorphisms, one novel missense mutation (c.1456 G>A), and one disease-causing mutation (c.478 G>C). The novel mutation was not observed in 100 controls, and was predicted to be a damaging mutation by SIFT and Polyphen. Thus, we identified a novel mutation in SLC45A2 in a Chinese family, expanding the mutational spectrum of albinism. Our results also demonstrate that targeted next-generation sequencing is an effective genetic test for albinism.

  12. Development of a qualitative real-time PCR method to detect 19 targets for identification of genetically modified organisms.

    Science.gov (United States)

    Peng, Cheng; Wang, Pengfei; Xu, Xiaoli; Wang, Xiaofu; Wei, Wei; Chen, Xiaoyun; Xu, Junfeng

    2016-01-01

    As the amount of commercially available genetically modified organisms (GMOs) grows recent years, the diversity of target sequences for molecular detection techniques are eagerly needed. Considered as the gold standard for GMO analysis, the real-time PCR technology was optimized to produce a high-throughput GMO screening method. With this method we can detect 19 transgenic targets. The specificity of the assays was demonstrated to be 100 % by the specific amplification of DNA derived from reference material from 20 genetically modified crops and 4 non modified crops. Furthermore, most assays showed a very sensitive detection, reaching the limit of ten copies. The 19 assays are the most frequently used genetic elements present in GM crops and theoretically enable the screening of the known GMO described in Chinese markets. Easy to use, fast and cost efficient, this method approach fits the purpose of GMO testing laboratories.

  13. Technological advances and proteomic applications in drug discovery and target deconvolution: identification of the pleiotropic effects of statins.

    Science.gov (United States)

    Banfi, Cristina; Baetta, Roberta; Gianazza, Erica; Tremoli, Elena

    2017-06-01

    Proteomic-based techniques provide a powerful tool for identifying the full spectrum of protein targets of a drug, elucidating its mechanism(s) of action, and identifying biomarkers of its efficacy and safety. Herein, we outline the technological advancements in the field, and illustrate the contribution of proteomics to the definition of the pharmacological profile of statins, which represent the cornerstone of the prevention and treatment of cardiovascular diseases (CVDs). Statins act by inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, thus reducing cholesterol biosynthesis and consequently enhancing the clearance of low-density lipoproteins from the blood; however, HMG-CoA reductase inhibition can result in a multitude of additional effects beyond lipid lowering, known as 'pleiotropic effects'. The case of statins highlights the unique contribution of proteomics to the target profiling of a drug molecule. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Studies on the Roles of PDGFRA and EGFR in the Classification and Identification of Therapeutic Targets for Human Gliomas

    OpenAIRE

    Chen, Dongfeng

    2013-01-01

    Glioma is the most common type of primary tumor in the adult central nervous system (CNS). However, the current classification of gliomas is highly subjective and even inaccurate in some cases, which leads to clinical confusion and hinders the development of targeted therapies. EGFR and PDGFRA play crucial roles in glia development and glioma pathogenesis. In this thesis we aim to establish a glial genesis-guided molecular classification scheme for gliomas based on the genes co-expressed with...

  15. Targeted dual-color silica nanoparticles provide univocal identification of micrometastases in preclinical models of colorectal cancer

    Directory of Open Access Journals (Sweden)

    Soster M

    2012-09-01

    Full Text Available Marco Soster,1,* Riccardo Juris,2,* Sara Bonacchi,2 Damiano Genovese,2 Marco Montalti,2 Enrico Rampazzo,2 Nelsi Zaccheroni,2 Paolo Garagnani,2 Federico Bussolino,3,4 Luca Prodi,2 Serena Marchiò1,4 1Institute for Cancer Research and Treatment, Laboratory of Tumor Microenvironment and University of Torino, Department of Oncological Sciences, Candiolo, 2University of Bologna, Department of Chemistry "G.Ciamician", Bologna, 3Institute for Cancer Research and Treatment, Laboratory of Vascular Oncology and University of Torino, Department of Oncological Sciences, Candiolo, 4APAvadis Biotechnologies, BioIndustry Park S Fumero, Colleretto Giacosa, Italy*These authors equally contributed to the workBackground and methods: Despite the recent introduction of targeted bio-drugs, the scarcity of successful therapeutic options for advanced colorectal cancer remains a limiting factor in patient management. The efficacy of curative surgical interventions can only be extended through earlier detection of metastatic foci, which is dependent on both the sensitivity and specificity of the diagnostic tools.Results: We propose a high-performance imaging platform based on silica-poly(ethylene glycol nanoparticles doped with rhodamine B and cyanine 5. Simultaneous detection of these dyes is the basis for background subtraction and signal amplification, thus providing high-sensitivity imaging. The functionalization of poly(ethylene glycol tails on the external face of the nanoparticles with metastasis-specific peptides guarantees their homing to and accumulation at target tissues, resulting in specific visualization, even of submillimetric metastases.Conclusions: The results reported here demonstrate that our rationally designed modular nanosystems have the ability to produce a breakthrough in the detection of micrometastases for subsequent translation to clinics in the immediate future.Keywords: colorectal cancer, imaging platform, luminescent targeting

  16. Dana-Farber Cancer Institute: Identification of Therapeutic Targets in KRAS Driven Lung Cancer | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Dana Farber Cancer Institute focuses on the use of high-throughput genetic and bioinformatic approaches to identify and credential oncogenes and co-dependencies in cancers. This Center aims to provide the cancer research community with information that will facilitate the prioritization of targets based on both genomic and functional evidence, inform the most appropriate genetic context for downstream mechanistic and validation studies, and enable the translation of this information into therapeutics and diagnostics.

  17. Pathway-based identification of biomarkers for targeted therapeutics: personalized oncology with PI3K pathway inhibitors.

    Science.gov (United States)

    Andersen, Jannik N; Sathyanarayanan, Sriram; Di Bacco, Alessandra; Chi, An; Zhang, Theresa; Chen, Albert H; Dolinski, Brian; Kraus, Manfred; Roberts, Brian; Arthur, William; Klinghoffer, Rich A; Gargano, Diana; Li, Lixia; Feldman, Igor; Lynch, Bethany; Rush, John; Hendrickson, Ronald C; Blume-Jensen, Peter; Paweletz, Cloud P

    2010-08-04

    Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K-mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway-relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40(Thr246). Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)-deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40(Thr246) positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKT(Thr308), the phospho-PRAS40(Thr246) epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug

  18. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

    2015-01-01

    Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells ofte...... or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/....

  19. Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

    Science.gov (United States)

    Crawford, Lisa J; Anderson, Gordon; Johnston, Cliona K; Irvine, Alexandra E

    2016-10-25

    Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

  20. Identification of MALT1 as both a prognostic factor and a potential therapeutic target of regorafenib in cholangiocarcinoma patients.

    Science.gov (United States)

    Yeh, Chun-Nan; Chang, Yu-Chan; Su, Yeu; Shin-Shian Hsu, Dennis; Cheng, Chi-Tung; Wu, Ren-Chin; Chung, Yi-Hsiu; Chiang, Kun-Chun; Yeh, Ta-Sen; Lu, Meng-Lun; Liu, Chun-Yu; Mu-Hsin Chang, Peter; Chen, Ming-Han; Huang, Chi-Ying F; Hsiao, Michael; Chen, Ming-Huang

    2017-12-26

    Intrahepatic cholangiocarcinoma (CCA) is an aggressive cancer that lacks an effective targeted therapy. Here, we assessed the therapeutic efficacy of regorafenib in CCA, as well as elucidated its underlying mechanism. We first demonstrated that regorafenib not only inhibited growth but also induced apoptosis in human CCA cells. Subsequently, we used in silico approaches to identify MALT1 (Mucosa-associated lymphoid tissue protein 1), which plays an important role in activating NF-κB, as a potential target of regorafenib. Overexpression of Elk-1, but not Ets-1, in HuCCT1 cells markedly reduced their sensitivity to regorafenib, which might be attributed to a significant increase in MALT1 levels. Our results further demonstrated that this drug drastically inhibited MALT1 expression by suppressing the Raf/Erk/Elk-1 pathway. The efficacy of regorafenib in decreasing in vivo CCA growth was confirmed in animal models. Regorafenib efficacy was observed in two MALT1-positive CCA patients who failed to respond to several other lines of therapy. Finally, MALT1 was also identified as an independent poor prognostic factor for patients with intrahepatic CCA. In conclusion, our study identified MALT1 to be a downstream mediator of the Raf/Erk/Elk-1 pathway and suggested that MALT1 may be a new therapeutic target for successful treatment of CCA by regorafenib.

  1. Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT--relationship to newly identified mitochondrial pyruvate carrier proteins.

    Directory of Open Access Journals (Sweden)

    Jerry R Colca

    Full Text Available Thiazolidinedione (TZD insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44 and BRP44 Like (BRP44L, which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

  2. Network motif-based identification of transcription factor-target gene relationships by integrating multi-source biological data

    Directory of Open Access Journals (Sweden)

    de los Reyes Benildo G

    2008-04-01

    Full Text Available Abstract Background Integrating data from multiple global assays and curated databases is essential to understand the spatio-temporal interactions within cells. Different experiments measure cellular processes at various widths and depths, while databases contain biological information based on established facts or published data. Integrating these complementary datasets helps infer a mutually consistent transcriptional regulatory network (TRN with strong similarity to the structure of the underlying genetic regulatory modules. Decomposing the TRN into a small set of recurring regulatory patterns, called network motifs (NM, facilitates the inference. Identifying NMs defined by specific transcription factors (TF establishes the framework structure of a TRN and allows the inference of TF-target gene relationship. This paper introduces a computational framework for utilizing data from multiple sources to infer TF-target gene relationships on the basis of NMs. The data include time course gene expression profiles, genome-wide location analysis data, binding sequence data, and gene ontology (GO information. Results The proposed computational framework was tested using gene expression data associated with cell cycle progression in yeast. Among 800 cell cycle related genes, 85 were identified as candidate TFs and classified into four previously defined NMs. The NMs for a subset of TFs are obtained from literature. Support vector machine (SVM classifiers were used to estimate NMs for the remaining TFs. The potential downstream target genes for the TFs were clustered into 34 biologically significant groups. The relationships between TFs and potential target gene clusters were examined by training recurrent neural networks whose topologies mimic the NMs to which the TFs are classified. The identified relationships between TFs and gene clusters were evaluated using the following biological validation and statistical analyses: (1 Gene set enrichment

  3. A functional glycoproteomics approach identifies CD13 as a novel E-selectin ligand in breast cancer.

    Science.gov (United States)

    Carrascal, M A; Silva, M; Ferreira, J A; Azevedo, R; Ferreira, D; Silva, A M N; Ligeiro, D; Santos, L L; Sackstein, R; Videira, P A

    2018-05-17

    The glycan moieties sialyl-Lewis-X and/or -A (sLe X/A ) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. We observed that the CF1_T cell line expressed sLe X , but not sLe A and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLe X -CD44 and sLe X -CD13 was confirmed in clinical breast cancer tissue samples. Both CD44 and CD13 glycoforms display sLe X in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. A genome-wide RNAi screen identifies novel targets of neratinib resistance leading to identification of potential drug resistant genetic markers.

    Science.gov (United States)

    Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Wei; Ryan, Terence E

    2012-04-01

    Neratinib (HKI-272) is a small molecule tyrosine kinase inhibitor of the ErbB receptor family currently in Phase III clinical trials. Despite its efficacy, the mechanism of potential cellular resistance to neratinib and genes involved with it remains unknown. We have used a pool-based lentiviral genome-wide functional RNAi screen combined with a lethal dose of neratinib to discover chemoresistant interactions with neratinib. Our screen has identified a collection of genes whose inhibition by RNAi led to neratinib resistance including genes involved in oncogenesis (e.g. RAB33A, RAB6A and BCL2L14), transcription factors (e.g. FOXP4, TFEC, ZNF), cellular ion transport (e.g. CLIC3, TRAPPC2P1, P2RX2), protein ubiquitination (e.g. UBL5), cell cycle (e.g. CCNF), and genes known to interact with breast cancer-associated genes (e.g. CCNF, FOXP4, TFEC, several ZNF factors, GNA13, IGFBP1, PMEPA1, SOX5, RAB33A, RAB6A, FXR1, DDO, TFEC, OLFM2). The identification of novel mediators of cellular resistance to neratinib could lead to the identification of new or neoadjuvant drug targets. Their use as patient or treatment selection biomarkers could make the application of anti-ErbB therapeutics more clinically effective.

  5. Proteotranscriptomic Profiling of 231-BR Breast Cancer Cells: Identification of Potential Biomarkers and Therapeutic Targets for Brain Metastasis*

    Science.gov (United States)

    Dun, Matthew D.; Chalkley, Robert J.; Faulkner, Sam; Keene, Sheridan; Avery-Kiejda, Kelly A.; Scott, Rodney J.; Falkenby, Lasse G.; Cairns, Murray J.; Larsen, Martin R.; Bradshaw, Ralph A.; Hondermarck, Hubert

    2015-01-01

    Brain metastases are a devastating consequence of cancer and currently there are no specific biomarkers or therapeutic targets for risk prediction, diagnosis, and treatment. Here the proteome of the brain metastatic breast cancer cell line 231-BR has been compared with that of the parental cell line MDA-MB-231, which is also metastatic but has no organ selectivity. Using SILAC and nanoLC-MS/MS, 1957 proteins were identified in reciprocal labeling experiments and 1584 were quantified in the two cell lines. A total of 152 proteins were confidently determined to be up- or down-regulated by more than twofold in 231-BR. Of note, 112/152 proteins were decreased as compared with only 40/152 that were increased, suggesting that down-regulation of specific proteins is an important part of the mechanism underlying the ability of breast cancer cells to metastasize to the brain. When matched against transcriptomic data, 43% of individual protein changes were associated with corresponding changes in mRNA, indicating that the transcript level is a limited predictor of protein level. In addition, differential miRNA analyses showed that most miRNA changes in 231-BR were up- (36/45) as compared with down-regulations (9/45). Pathway analysis revealed that proteome changes were mostly related to cell signaling and cell cycle, metabolism and extracellular matrix remodeling. The major protein changes in 231-BR were confirmed by parallel reaction monitoring mass spectrometry and consisted in increases (by more than fivefold) in the matrix metalloproteinase-1, ephrin-B1, stomatin, myc target-1, and decreases (by more than 10-fold) in transglutaminase-2, the S100 calcium-binding protein A4, and l-plastin. The clinicopathological significance of these major proteomic changes to predict the occurrence of brain metastases, and their potential value as therapeutic targets, warrants further investigation. PMID:26041846

  6. Specification of Change Mechanisms in Pregnant Smokers for Malleable Target Identification: A Novel Approach to a Tenacious Public Health Problem

    Directory of Open Access Journals (Sweden)

    Suena H. Massey

    2017-09-01

    Full Text Available Maternal smoking during pregnancy (MSDP continues to be a leading modifiable risk factor for perinatal complications and a range of neurodevelopmental and cardio-metabolic outcomes across the lifespan. Despite 40 years of intervention research less than one in five pregnant smokers who receive an intervention quit by delivery. Within this context, recognition of pregnancy is commonly associated with abrupt suspension or reduction of smoking in the absence of intervention, yet has not been investigated as a volitional target. The goal of this article is to provide the empirical foundation for a novel direction of research aimed at identifying malleable targets for intervention through the specification of behavior change mechanisms specific to pregnant women. To do so, we: (1 summarize progress on MSDP in the United States generated from conventional empirical approaches to health behavior change; (2 discuss the phenomenon of spontaneous change in the absence of intervention among pregnant smokers to illustrate the need for mechanistic specification of behavior change motivated by concern for fetal well-being; (3 summarize component processes in neurobiological models of parental and non-parental social behaviors as a conceptual framework for understanding change mechanisms during pregnancy; (4 discuss the evidence for the malleability of these processes to support their translational relevance for preventive interventions; and (5 propose a roadmap for validating the proposed change mechanism using an experimental medicine approach. A greater understanding of social and interpersonal processes that facilitate health behavior change among expectant mothers and how these processes differ interindividually could yield novel volitional targets for prenatal interventions. More broadly, explicating other-oriented mechanisms of behavior change during pregnancy could serve as a paradigm for understanding how social and interpersonal processes

  7. Genome-wide identification of polycomb target genes reveals a functional association of Pho with Scm in Bombyx mori.

    Science.gov (United States)

    Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Tatsuke, Tsuneyuki; Zhu, Li; Xu, Jian; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

    2012-01-01

    Polycomb group (PcG) proteins are evolutionarily conserved chromatin modifiers and act together in three multimeric complexes, Polycomb repressive complex 1 (PRC1), Polycomb repressive complex 2 (PRC2), and Pleiohomeotic repressive complex (PhoRC), to repress transcription of the target genes. Here, we identified Polycomb target genes in Bombyx mori with holocentric centromere using genome-wide expression screening based on the knockdown of BmSCE, BmESC, BmPHO, or BmSCM gene, which represent the distinct complexes. As a result, the expressions of 29 genes were up-regulated after knocking down 4 PcG genes. Particularly, there is a significant overlap between targets of BmPho (331 out of 524) and BmScm (331 out of 532), and among these, 190 genes function as regulator factors playing important roles in development. We also found that BmPho, as well as BmScm, can interact with other Polycomb components examined in this study. Further detailed analysis revealed that the C-terminus of BmPho containing zinc finger domain is involved in the interaction between BmPho and BmScm. Moreover, the zinc finger domain in BmPho contributes to its inhibitory function and ectopic overexpression of BmScm is able to promote transcriptional repression by Gal4-Pho fusions including BmScm-interacting domain. Loss of BmPho expression causes relocalization of BmScm into the cytoplasm. Collectively, we provide evidence of a functional link between BmPho and BmScm, and propose two Polycomb-related repression mechanisms requiring only BmPho associated with BmScm or a whole set of PcG complexes.

  8. Identification of chemokines associated with the recruitment of decidual leukocytes in human labour: potential novel targets for preterm labour.

    Directory of Open Access Journals (Sweden)

    Sarah A Hamilton

    Full Text Available Current therapies for preterm labour (PTL focus on arresting myometrial contractions but are largely ineffective, thus alternative therapeutic targets need to be identified. Leukocytes infiltrate the uterus around the time of labour, and are in particularly abundant in decidua (maternal-fetal interface. Moreover, decidual inflammation precedes labour in rat pregnancies and thus may contribute to initiation of labour. We hypothesized that chemokines mediate decidual leukocyte trafficking during preterm labour (PTL and term labour (TL, thus representing potential targets for preventing PTL. Women were recruited into 4 groups: TL, term not in labour (TNL, idiopathic PTL and PTL with infection (PTLI. Choriodecidual RNA was subjected to a pathway-specific PCR array for chemokines. Differential expression of 12 candidate chemokines was validated by real time RT-PCR and Bioplex assay, with immunohistochemistry to confirm cellular origin. 25 chemokines were upregulated in choriodecidua from TL compared to TNL. A similar pattern was detected in PTL, however a distinct profile was observed in PTLI consistent with differences in leukocyte infiltration. Upregulation of CCL2, CCL4, CCL5, CXCL8 and CXCL10 mRNA and protein was confirmed in TL, with CCL8 upregulated in PTL. Significant correlations were detected between these chemokines and decidual leukocyte abundance previously assessed by immunohistochemical and image analysis. Chemokines were primarily expressed by decidual stromal cells. In addition, CXCL8 and CCL5 were significantly elevated in maternal plasma during labour, suggesting chemokines contribute to peripheral inflammatory events during labour. Differences in chemokine expression patterns between TL and idiopathic PTL may be attributable to suppression of chemokine expression by betamethasone administered to women in PTL; this was supported by in vitro evidence of chemokine downregulation by clinically relevant concentrations of the steroid

  9. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

    Directory of Open Access Journals (Sweden)

    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  10. Genome-wide identification of polycomb target genes reveals a functional association of Pho with Scm in Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Zhiqing Li

    Full Text Available Polycomb group (PcG proteins are evolutionarily conserved chromatin modifiers and act together in three multimeric complexes, Polycomb repressive complex 1 (PRC1, Polycomb repressive complex 2 (PRC2, and Pleiohomeotic repressive complex (PhoRC, to repress transcription of the target genes. Here, we identified Polycomb target genes in Bombyx mori with holocentric centromere using genome-wide expression screening based on the knockdown of BmSCE, BmESC, BmPHO, or BmSCM gene, which represent the distinct complexes. As a result, the expressions of 29 genes were up-regulated after knocking down 4 PcG genes. Particularly, there is a significant overlap between targets of BmPho (331 out of 524 and BmScm (331 out of 532, and among these, 190 genes function as regulator factors playing important roles in development. We also found that BmPho, as well as BmScm, can interact with other Polycomb components examined in this study. Further detailed analysis revealed that the C-terminus of BmPho containing zinc finger domain is involved in the interaction between BmPho and BmScm. Moreover, the zinc finger domain in BmPho contributes to its inhibitory function and ectopic overexpression of BmScm is able to promote transcriptional repression by Gal4-Pho fusions including BmScm-interacting domain. Loss of BmPho expression causes relocalization of BmScm into the cytoplasm. Collectively, we provide evidence of a functional link between BmPho and BmScm, and propose two Polycomb-related repression mechanisms requiring only BmPho associated with BmScm or a whole set of PcG complexes.

  11. Identification, Expression Analysis, and Target Prediction of Flax Genotroph MicroRNAs Under Normal and Nutrient Stress Conditions

    Science.gov (United States)

    Melnikova, Nataliya V.; Dmitriev, Alexey A.; Belenikin, Maxim S.; Koroban, Nadezhda V.; Speranskaya, Anna S.; Krinitsina, Anastasia A.; Krasnov, George S.; Lakunina, Valentina A.; Snezhkina, Anastasiya V.; Sadritdinova, Asiya F.; Kishlyan, Natalya V.; Rozhmina, Tatiana A.; Klimina, Kseniya M.; Amosova, Alexandra V.; Zelenin, Alexander V.; Muravenko, Olga V.; Bolsheva, Nadezhda L.; Kudryavtseva, Anna V.

    2016-01-01

    Cultivated flax (Linum usitatissimum L.) is an important plant valuable for industry. Some flax lines can undergo heritable phenotypic and genotypic changes (LIS-1 insertion being the most common) in response to nutrient stress and are called plastic lines. Offspring of plastic lines, which stably inherit the changes, are called genotrophs. MicroRNAs (miRNAs) are involved in a crucial regulatory mechanism of gene expression. They have previously been assumed to take part in nutrient stress response and can, therefore, participate in genotroph formation. In the present study, we performed high-throughput sequencing of small RNAs (sRNAs) extracted from flax plants grown under normal, phosphate deficient and nutrient excess conditions to identify miRNAs and evaluate their expression. Our analysis revealed expression of 96 conserved miRNAs from 21 families in flax. Moreover, 475 novel potential miRNAs were identified for the first time, and their targets were predicted. However, none of the identified miRNAs were transcribed from LIS-1. Expression of seven miRNAs (miR168, miR169, miR395, miR398, miR399, miR408, and lus-miR-N1) with up- or down-regulation under nutrient stress (on the basis of high-throughput sequencing data) was evaluated on extended sampling using qPCR. Reference gene search identified ETIF3H and ETIF3E genes as most suitable for this purpose. Down-regulation of novel potential lus-miR-N1 and up-regulation of conserved miR399 were revealed under the phosphate deficient conditions. In addition, the negative correlation of expression of lus-miR-N1 and its predicted target, ubiquitin-activating enzyme E1 gene, as well as, miR399 and its predicted target, ubiquitin-conjugating enzyme E2 gene, was observed. Thus, in our study, miRNAs expressed in flax plastic lines and genotrophs were identified and their expression and expression of their targets was evaluated using high-throughput sequencing and qPCR for the first time. These data provide new insights

  12. Phosphoproteomic analysis for the identification of predictive molecular factors and new molecular targets in breast, colorectal, ovary, and lung cancer

    International Nuclear Information System (INIS)

    Belluco, C.; De Paoli, P.

    2009-01-01

    Neoadjuvant chemotherapy is a standard therapeutic approach for several types of locally advanced and metastatic tumors. Molecular factors predictive of response to therapy are highly needed for exploiting the potential benefit of properative chemotherapy. Moreover, neoadjuvant chemotherapy represents an ideal clinical model for studying predictive factors since it allows to obtain pre-treatment tissue biopsies which can be analyzed and the results compared to clinical and pathological response. Protein kinases represent some of the most important drug targets in medicine today and their aberrant activation are involved in many disease processes including cancer development and progression

  13. In silico identification of miRNAs and their target genes and analysis of gene co-expression network in saffron (Crocus sativus L.) stigma

    Science.gov (United States)

    Zinati, Zahra; Shamloo-Dashtpagerdi, Roohollah; Behpouri, Ali

    2016-01-01

    As an aromatic and colorful plant of substantive taste, saffron (Crocus sativus L.) owes such properties of matter to growing class of the secondary metabolites derived from the carotenoids, apocarotenoids. Regarding the critical role of microRNAs in secondary metabolic synthesis and the limited number of identified miRNAs in C. sativus, on the other hand, one may see the point how the characterization of miRNAs along with the corresponding target genes in C. sativus might expand our perspectives on the roles of miRNAs in carotenoid/apocarotenoid biosynthetic pathway. A computational analysis was used to identify miRNAs and their targets using EST (Expressed Sequence Tag) library from mature saffron stigmas. Then, a gene co- expression network was constructed to identify genes which are potentially involved in carotenoid/apocarotenoid biosynthetic pathways. EST analysis led to the identification of two putative miRNAs (miR414 and miR837-5p) along with the corresponding stem- looped precursors. To our knowledge, this is the first report on miR414 and miR837-5p in C. sativus. Co-expression network analysis indicated that miR414 and miR837-5p may play roles in C. sativus metabolic pathways and led to identification of candidate genes including six transcription factors and one protein kinase probably involved in carotenoid/apocarotenoid biosynthetic pathway. Presence of transcription factors, miRNAs and protein kinase in the network indicated multiple layers of regulation in saffron stigma. The candidate genes from this study may help unraveling regulatory networks underlying the carotenoid/apocarotenoid biosynthesis in saffron and designing metabolic engineering for enhanced secondary metabolites. PMID:28261627

  14. Identification of specific bovine blood biomarkers with a non-targeted approach using HPLC ESI tandem mass spectrometry.

    Science.gov (United States)

    Lecrenier, M C; Marbaix, H; Dieu, M; Veys, P; Saegerman, C; Raes, M; Baeten, V

    2016-12-15

    Animal by-products are valuable protein sources in animal nutrition. Among them are blood products and blood meal, which are used as high-quality material for their beneficial effects on growth and health. Within the framework of the feed ban relaxation, the development of complementary methods in order to refine the identification of processed animal proteins remains challenging. The aim of this study was to identify specific biomarkers that would allow the detection of bovine blood products and processed animal proteins using tandem mass spectrometry. Seventeen biomarkers were identified: nine peptides for bovine plasma powder; seven peptides for bovine haemoglobin powder, including six peptides for bovine blood meal; and one peptide for porcine blood. They were not detected in several commercial compound feed or feed materials, such as blood by-products of other animal origins, milk-derived products and fish meal. These biomarkers could be used for developing a species-specific and blood-specific detection method. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies

    Science.gov (United States)

    Lehmann, Brian D.; Bauer, Joshua A.; Chen, Xi; Sanders, Melinda E.; Chakravarthy, A. Bapsi; Shyr, Yu; Pietenpol, Jennifer A.

    2011-01-01

    Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and subtyping is necessary to better identify molecular-based therapies. In this study, we analyzed gene expression (GE) profiles from 21 breast cancer data sets and identified 587 TNBC cases. Cluster analysis identified 6 TNBC subtypes displaying unique GE and ontologies, including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem–like (MSL), and a luminal androgen receptor (LAR) subtype. Further, GE analysis allowed us to identify TNBC cell line models representative of these subtypes. Predicted “driver” signaling pathways were pharmacologically targeted in these cell line models as proof of concept that analysis of distinct GE signatures can inform therapy selection. BL1 and BL2 subtypes had higher expression of cell cycle and DNA damage response genes, and representative cell lines preferentially responded to cisplatin. M and MSL subtypes were enriched in GE for epithelial-mesenchymal transition, and growth factor pathways and cell models responded to NVP-BEZ235 (a PI3K/mTOR inhibitor) and dasatinib (an abl/src inhibitor). The LAR subtype includes patients with decreased relapse-free survival and was characterized by androgen receptor (AR) signaling. LAR cell lines were uniquely sensitive to bicalutamide (an AR antagonist). These data may be useful in biomarker selection, drug discovery, and clinical trial design that will enable alignment of TNBC patients to appropriate targeted therapies. PMID:21633166

  16. Simultaneous analysis of proteome, phospho- and glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction chromatography.

    Directory of Open Access Journals (Sweden)

    Piliang Hao

    Full Text Available Protein post-translational modifications (PTMs are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.

  17. Identification of pathogenic Nocardia species by reverse line blot hybridization targeting the 16S rRNA and 16S-23S rRNA gene spacer regions.

    Science.gov (United States)

    Xiao, Meng; Kong, Fanrong; Sorrell, Tania C; Cao, Yongyan; Lee, Ok Cha; Liu, Ying; Sintchenko, Vitali; Chen, Sharon C A

    2010-02-01

    Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n = 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

  18. Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2β in development.

    Directory of Open Access Journals (Sweden)

    Sushma Grellscheid

    2011-12-01

    Full Text Available Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2β (Sfrs10 is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2β is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2β binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl; Nestin-Cre(tg/+. This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2β regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2β binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2β protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2β. Versions of Tra2β lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2β protein.

  19. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

    Science.gov (United States)

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M Michael; Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A Mary; Velmurugan, D; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-11-26

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

  20. Epigenetics of prostate cancer and the prospect of identification of novel drug targets by RNAi screening of epigenetic enzymes.

    Science.gov (United States)

    Björkman, Mari; Rantala, Juha; Nees, Matthias; Kallioniemi, Olli

    2010-10-01

    Alterations in epigenetic processes probably underlie most human malignancies. Novel genome-wide techniques, such as chromatin immunoprecipitation and high-throughput sequencing, have become state-of-the-art methods to map the epigenomic landscape of development and disease, such as in cancers. Despite these advances, the functional significance of epigenetic enzymes in cancer progression, such as prostate cancer, remain incompletely understood. A comprehensive mapping and functional understanding of the cancer epigenome will hopefully help to facilitate development of novel cancer therapy targets and improve future diagnostics. The authors have developed a novel cell microarray-based high-content siRNA screening technique suitable to address the putative functional role and impact of all known putative and novel epigenetic enzymes in cancer, including prostate cancer.

  1. Identification of functionally important amino acid residues in the mitochondria targeting sequence of Hepatitis B virus X protein

    International Nuclear Information System (INIS)

    Li, Sai Kam; Ho, Sai Fan; Tsui, Kwok Wing; Fung, Kwok Pui; Waye, M.Y. Mary

    2008-01-01

    Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma (HCC) and the X protein (HBx) is thought to mediate the cellular changes associated with carcinogenesis. Recently, isolation of the hepatitis B virus integrants from HCC tissue by others have established the fact that the X gene is often truncated at its C-terminus. Expression of the GFP fusion proteins of HBx and its truncation mutants with a GFP tag in human liver cell-lines in this study revealed that the C-terminus of HBx is indispensable for its specific localization in the mitochondria. A crucial region of seven amino acids at the C-terminus has been mapped out in which the cysteine residue at position 115 serves as the most important residue for the subcellular localization. When cysteine 115 of HBx is mutated to alanine the mitochondria targeting property of HBx is abrogated

  2. Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein-Protein Interaction.

    Science.gov (United States)

    Sail, Vibhavari; Rizzo, Alessandro A; Chatterjee, Nimrat; Dash, Radha C; Ozen, Zuleyha; Walker, Graham C; Korzhnev, Dmitry M; Hadden, M Kyle

    2017-07-21

    Translesion synthesis (TLS) is an important mechanism through which proliferating cells tolerate DNA damage during replication. The mutagenic Rev1/Polζ-dependent branch of TLS helps cancer cells survive first-line genotoxic chemotherapy and introduces mutations that can contribute to the acquired resistance so often observed with standard anticancer regimens. As such, inhibition of Rev1/Polζ-dependent TLS has recently emerged as a strategy to enhance the efficacy of first-line chemotherapy and reduce the acquisition of chemoresistance by decreasing tumor mutation rate. The TLS DNA polymerase Rev1 serves as an integral scaffolding protein that mediates the assembly of the active multiprotein TLS complexes. Protein-protein interactions (PPIs) between the C-terminal domain of Rev1 (Rev1-CT) and the Rev1-interacting region (RIR) of other TLS DNA polymerases play an essential role in regulating TLS activity. To probe whether disrupting the Rev1-CT/RIR PPI is a valid approach for developing a new class of targeted anticancer agents, we designed a fluorescence polarization-based assay that was utilized in a pilot screen for small molecule inhibitors of this PPI. Two small molecule scaffolds that disrupt this interaction were identified, and secondary validation assays confirmed that compound 5 binds to Rev1-CT at the RIR interface. Finally, survival and mutagenesis assays in mouse embryonic fibroblasts and human fibrosarcoma HT1080 cells treated with cisplatin and ultraviolet light indicate that these compounds inhibit mutagenic Rev1/Polζ-dependent TLS in cells, validating the Rev1-CT/RIR PPI for future anticancer drug discovery and identifying the first small molecule inhibitors of TLS that target Rev1-CT.

  3. Evidence-based identification of key beliefs explaining adult male circumcision motivation in Zimbabwe: targets for behavior change messaging.

    Science.gov (United States)

    Montaño, Daniel E; Kasprzyk, Danuta; Hamilton, Deven T; Tshimanga, Mufuta; Gorn, Gerald

    2014-05-01

    Male circumcision (MC) reduces HIV acquisition among men, leading WHO/UNAIDS to recommend a goal to circumcise 80 % of men in high HIV prevalence countries. Significant investment to increase MC capacity in priority countries was made, yet only 5 % of the goal has been achieved in Zimbabwe. The integrated behavioral model (IBM) was used as a framework to investigate the factors affecting MC motivation among men in Zimbabwe. A survey instrument was designed based on elicitation study results, and administered to a representative household-based sample of 1,201 men aged 18-30 from two urban and two rural areas in Zimbabwe. Multiple regression analysis found all five IBM constructs significantly explained MC Intention. Nearly all beliefs underlying the IBM constructs were significantly correlated with MC Intention. Stepwise regression analysis of beliefs underlying each construct respectively found that 13 behavioral beliefs, 5 normative beliefs, 4 descriptive norm beliefs, 6 efficacy beliefs, and 10 control beliefs were significant in explaining MC Intention. A final stepwise regression of the five sets of significant IBM construct beliefs identified 14 key beliefs that best explain Intention. Similar analyses were carried out with subgroups of men by urban-rural and age. Different sets of behavioral, normative, efficacy, and control beliefs were significant for each sub-group, suggesting communication messages need to be targeted to be most effective for sub-groups. Implications for the design of effective MC demand creation messages are discussed. This study demonstrates the application of theory-driven research to identify evidence-based targets for intervention messages to increase men's motivation to get circumcised and thereby improve demand for male circumcision.

  4. Proteomic Identification of Target Proteins of Thiodigalactoside in White Adipose Tissue from Diet-Induced Obese Rats

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    Hilal Ahmad Parray

    2015-06-01

    Full Text Available Previously, galectin-1 (GAL1 was found to be up-regulated in obesity-prone subjects, suggesting that use of a GAL1 inhibitor could be a novel therapeutic approach for treatment of obesity. We evaluated thiodigalactoside (TDG as a potent inhibitor of GAL1 and identified target proteins of TDG by performing comparative proteome analysis of white adipose tissue (WAT from control and TDG-treated rats fed a high fat diet (HFD using two dimensional gel electrophoresis (2-DE combined with MALDI-TOF-MS. Thirty-two spots from a total of 356 matched spots showed differential expression between control and TDG-treated rats, as identified by peptide mass fingerprinting. These proteins were categorized into groups such as carbohydrate metabolism, tricarboxylic acid (TCA cycle, signal transduction, cytoskeletal, and mitochondrial proteins based on functional analysis using Protein Annotation Through Evolutionary Relationship (PANTHER and Database for Annotation, Visualization, Integrated Discovery (DAVID classification. One of the most striking findings of this study was significant changes in Carbonic anhydrase 3 (CA3, Voltage-dependent anion channel 1 (VDAC1, phosphatidylethanolamine-binding protein 1 (PEBP1, annexin A2 (ANXA2 and lactate dehydrogenase A chain (LDHA protein levels between WAT from control and TDG-treated groups. In addition, we confirmed increased expression of thermogenic proteins as well as reduced expression of lipogenic proteins in response to TDG treatment. These results suggest that TDG may effectively prevent obesity, and TDG-responsive proteins can be used as novel target proteins for obesity treatment.

  5. Identification of type IV collagen exposure as a molecular imaging target for early detection of thoracic aortic dissection

    Science.gov (United States)

    Xu, Ke; Xu, Chen; Zhang, Yanzhenzi; Qi, Feiran; Yu, Bingran; Li, Ping; Jia, Lixin; Li, Yulin; Xu, Fu-jian; Du, Jie

    2018-01-01

    Thoracic aortic dissection (TAD) is an aggressive and life-threatening vascular disease and there is no effective means of early diagnosis of dissection. Type IV collagen (Col-IV) is a major component of the sub-endothelial basement membrane, which is initially exposed followed by endothelial injury as early-stage event of TAD. So, we want to build a noninvasive diagnostic method to detect early dissection by identifying the exposed Col-IV via MRI. Methods: Col-IV-targeted magnetic resonance/ fluorescence dual probe (Col-IV-DOTA-Gd-rhodamine B; CDR) was synthesized by amide reaction and coordination reaction. Flow cytometry analysis was used to evaluate the cell viability of SMC treated with CDR and fluorescence assays were used to assess the Col-IV targeting ability of CDR in vitro. We then examined the sensitivity and specificity of CDR at different stages of TAD via MRI and bioluminescence imaging in vivo. Results: The localization of Col-IV (under the intima) was observed by histology images. CDR bound specifically to Col-IV-expressing vascular smooth muscle cells and BAPN-induced dissected aorta. The CDR signal was co-detected by magnetic resonance imaging (MRI) and bioluminescence imaging as early as 2 weeks after BAPN administration (pre-dissection stage). The ability to detect rupture of dissected aorta was indicated by a strong normalized signal enhancement (NSE) in vivo. Moreover, NSE was negatively correlated with the time of dissection rupture after BAPN administration (r2 = 0.8482). Conclusion: As confirmed by in vivo studies, the CDR can identify the exposed Col-IV in degenerated aorta to monitor the progress of aortic dissection from the early stage to the rupture via MRI. Thus, CDR-enhanced MRI proposes a potential method for dissection screening, and for monitoring disease progression and therapeutic response. PMID:29290819

  6. Identification of Five Novel Salmonella Typhi-Specific Genes as Markers for Diagnosis of Typhoid Fever Using Single-Gene Target PCR Assays

    Directory of Open Access Journals (Sweden)

    Yuan Xin Goay

    2016-01-01

    Full Text Available Salmonella Typhi (S. Typhi causes typhoid fever which is a disease characterised by high mortality and morbidity worldwide. In order to curtail the transmission of this highly infectious disease, identification of new markers that can detect the pathogen is needed for development of sensitive and specific diagnostic tests. In this study, genomic comparison of S. Typhi with other enteric pathogens was performed, and 6 S. Typhi genes, that is, STY0201, STY0307, STY0322, STY0326, STY2020, and STY2021, were found to be specific in silico. Six PCR assays each targeting a unique gene were developed to test the specificity of these genes in vitro. The diagnostic sensitivities and specificities of each assay were determined using 39 S. Typhi, 62 non-Typhi Salmonella, and 10 non-Salmonella clinical isolates. The results showed that 5 of these genes, that is, STY0307, STY0322, STY0326, STY2020, and STY2021, demonstrated 100% sensitivity (39/39 and 100% specificity (0/72. The detection limit of the 5 PCR assays was 32 pg for STY0322, 6.4 pg for STY0326, STY2020, and STY2021, and 1.28 pg for STY0307. In conclusion, 5 PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution for diagnosis of typhoid fever.

  7. Identification of Five Novel Salmonella Typhi-Specific Genes as Markers for Diagnosis of Typhoid Fever Using Single-Gene Target PCR Assays.

    Science.gov (United States)

    Goay, Yuan Xin; Chin, Kai Ling; Tan, Clarissa Ling Ling; Yeoh, Chiann Ying; Ja'afar, Ja'afar Nuhu; Zaidah, Abdul Rahman; Chinni, Suresh Venkata; Phua, Kia Kien

    2016-01-01

    Salmonella Typhi ( S . Typhi) causes typhoid fever which is a disease characterised by high mortality and morbidity worldwide. In order to curtail the transmission of this highly infectious disease, identification of new markers that can detect the pathogen is needed for development of sensitive and specific diagnostic tests. In this study, genomic comparison of S . Typhi with other enteric pathogens was performed, and 6 S . Typhi genes, that is, STY0201, STY0307, STY0322, STY0326, STY2020, and STY2021, were found to be specific in silico . Six PCR assays each targeting a unique gene were developed to test the specificity of these genes in vitro . The diagnostic sensitivities and specificities of each assay were determined using 39 S . Typhi, 62 non-Typhi Salmonella , and 10 non- Salmonella clinical isolates. The results showed that 5 of these genes, that is, STY0307, STY0322, STY0326, STY2020, and STY2021, demonstrated 100% sensitivity (39/39) and 100% specificity (0/72). The detection limit of the 5 PCR assays was 32 pg for STY0322, 6.4 pg for STY0326, STY2020, and STY2021, and 1.28 pg for STY0307. In conclusion, 5 PCR assays using STY0307, STY0322, STY0326, STY2020, and STY2021 were developed and found to be highly specific at single-gene target resolution for diagnosis of typhoid fever.

  8. Comparative genomics study for the identification of drug and vaccine targets in Staphylococcus aureus: MurA ligase enzyme as a proposed candidate.

    Science.gov (United States)

    Ghosh, Soma; Prava, Jyoti; Samal, Himanshu Bhusan; Suar, Mrutyunjay; Mahapatra, Rajani Kanta

    2014-06-01

    Now-a-days increasing emergence of antibiotic-resistant pathogenic microorganisms is one of the biggest challenges for management of disease. In the present study comparative genomics, metabolic pathways analysis and additional parameters were defined for the identification of 94 non-homologous essential proteins in Staphylococcus aureus genome. Further study prioritized 19 proteins as vaccine candidates where as druggability study reports 34 proteins suitable as drug targets. Enzymes from peptidoglycan biosynthesis, folate biosynthesis were identified as candidates for drug development. Furthermore, bacterial secretory proteins and few hypothetical proteins identified in our analysis fulfill the criteria of vaccine candidates. As a case study, we built a homology model of one of the potential drug target, MurA ligase, using MODELLER (9v12) software. The model has been further selected for in silico docking study with inhibitors from the DrugBank database. Results from this study could facilitate selection of proteins for entry into drug design and vaccine production pipelines. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Comparative Profiling of Triple-Negative Breast Carcinomas Tissue Glycoproteome by Sequential Purification of Glycoproteins and Stable Isotope Labeling

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    Xiang Chen

    2016-01-01

    Full Text Available Background: Women with triple negative breast cancers (TNBCs have a poor prognosis due to lack of suitable targeted therapies. Changes in the protein glycosylation are increasingly being recognized as an important modification associated with cancer etiology. Methods: In an attempt to identify TNBC biomarkers with greater diagnostic and prognostic capabilities, hydrazide- based chemistry method combined with LC-MS/MS were used to purify and identify N-linked glycopeptides or glycoproteins of tissues from TNBC patients. Results: A total of 550 unique N-linked glycoproteins were identified, among these proteins, 72 unique N-linked glycoproteins were significantly regulated in tumor tissues, of which 56 proteins were upregulated and 16 proteins were downregulated. To assess the validity of the results, three selected proteins including Vascular endothelial growth factor receptor 1, Insulin receptor, Tissue factor pathway inhibitor were selected for western blot analysis, and these proteins were found as potential biomarkers of TNBC. The top three pathways of differentially expressed glycoproteins participated in were caveolar-mediated endocytosis signaling, agrin interactions at neuromuscular junction and LXR/RXR activation. Conclusion: This work provides potential glycoprotein markers to function as a novel tissue-based biomarker for TNBC.

  10. Comparative profiling of miRNAs and target gene identification in distant-grafting between tomato and Lycium (goji berry

    Directory of Open Access Journals (Sweden)

    A B M Khaldun

    2016-10-01

    Full Text Available Local translocation of small RNAs between cells is proved. Long distance translocation between rootstock and scion is also well documented in the homo-grafting system, but the process in distant-grafting is widely unexplored where rootstock and scion belonging to different genera. Micro RNAs are a class of small, endogenous, noncoding, gene silencing RNAs that regulate target genes of a wide range of important biological pathways in plants. In this study, tomato was grafted onto goji (Lycium chinense Mill. to reveal the insight of miRNAs regulation and expression patterns within a distant-grafting system. Goji is an important traditional Chinese medicinal plant with enriched phytochemicals. Illumina sequencing technology has identified 68 evolutionary known miRNAs of 37 miRNA families. Moreover, 168 putative novel miRNAs were also identified. Compared with control tomato, 43 (11 known and 32 novels and 163 (33 known and 130 novels miRNAs were expressed significantly different in shoot and fruit of grafted tomato, respectively. The fruiting stage was identified as the most responsive in the distant-grafting approach and 123 miRNAs were found as up-regulating in the grafted fruit which is remarkably higher compare to the grafted shoot tip (28. Potential targets of differentially expressed miRNAs were found to be involved in diverse metabolic and regulatory pathways. ADP binding activities, molybdopterin synthase complex and RNA helicase activity were found as enriched terms in GO (Gene Ontology analysis. Additionally, ‘metabolic pathways’ was revealed as the most significant pathway in KEGG (Kyoto Encyclopedia of Genes and Genomes analysis. The information of the small RNA transcriptomes that are obtained from this study might be the first miRNAs elucidation for a distant-grafting system, particularly between goji and tomato. The results from this study will provide the insights into the molecular aspects of miRNA-mediated regulation in the

  11. Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.

    Science.gov (United States)

    Varela-M, Rubén E; Ochoa, Rodrigo; Muskus, Carlos E; Muro, Antonio; Mollinedo, Faustino

    2017-10-10

    Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and

  12. Identification of novel low-dose bisphenol a targets in human foreskin fibroblast cells derived from hypospadias patients.

    Directory of Open Access Journals (Sweden)

    Xian-Yang Qin

    Full Text Available The effect of low-dose bisphenol A (BPA exposure on human reproductive health is still controversial. To better understand the molecular basis of the effect of BPA on human reproductive health, a genome-wide screen was performed using human foreskin fibroblast cells (hFFCs derived from child hypospadias (HS patients to identify novel targets of low-dose BPA exposure.Gene expression profiles of hFFCs were measured after exposure to 10 nM BPA, 0.01 nM 17β-estradiol (E2 or 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD for 24 h. Differentially expressed genes were identified using an unpaired Student's t test with P value cut off at 0.05 and fold change of more than 1.2. These genes were selected for network generation and pathway analysis using Ingenuity Pathways Analysis, Pathway Express and KegArray. Seventy-one genes (42 downregulated and 29 upregulated were identified as significantly differentially expressed in response to BPA, among which 43 genes were found to be affected exclusively by BPA compared with E2 and TCDD. Of particular interest, real-time PCR analysis revealed that the expression of matrix metallopeptidase 11 (MMP11, a well-known effector of development and normal physiology, was found to be inhibited by BPA (0.47-fold and 0.37-fold at 10 nM and 100 nM, respectively. Furthermore, study of hFFCs derived from HS and cryptorchidism (CO patients (n = 23 and 11, respectively indicated that MMP11 expression was significantly lower in the HS group than in the CO group (0.25-fold, P = 0.0027.This present study suggests that an involvement of BPA in the etiology of HS might be associated with the downregulation of MMP11. Further study to elucidate the function of the novel target genes identified in this study during genital tubercle development might increase our knowledge of the effects of low-dose BPA exposure on human reproductive health.

  13. Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF

    International Nuclear Information System (INIS)

    Dang Thi Phuong Thao; Ida, Hiroyuki; Yoshida, Hideki; Yamaguchi, Masamitsu

    2006-01-01

    SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5'-TATCGATA) and DRE-like (5'-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter-luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter-lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF

  14. Genome-wide computational identification of microRNAs and their targets in the deep-branching eukaryote Giardia lamblia.

    Science.gov (United States)

    Zhang, Yan-Qiong; Chen, Dong-Liang; Tian, Hai-Feng; Zhang, Bao-Hong; Wen, Jian-Fan

    2009-10-01

    Using a combined computational program, we identified 50 potential microRNAs (miRNAs) in Giardia lamblia, one of the most primitive unicellular eukaryotes. These miRNAs are unique to G. lamblia and no homologues have been found in other organisms; miRNAs, currently known in other species, were not found in G. lamblia. This suggests that miRNA biogenesis and miRNA-mediated gene regulation pathway may evolve independently, especially in evolutionarily distant lineages. A majority (43) of the predicted miRNAs are located at one single locus; however, some miRNAs have two or more copies in the genome. Among the 58 miRNA genes, 28 are located in the intergenic regions whereas 30 are present in the anti-sense strands of the protein-coding sequences. Five predicted miRNAs are expressed in G. lamblia trophozoite cells evidenced by expressed sequence tags or RT-PCR. Thirty-seven identified miRNAs may target 50 protein-coding genes, including seven variant-specific surface proteins (VSPs). Our findings provide a clue that miRNA-mediated gene regulation may exist in the early stage of eukaryotic evolution, suggesting that it is an important regulation system ubiquitous in eukaryotes.

  15. Learning-based adaptive prescribed performance control of postcapture space robot-target combination without inertia identifications

    Science.gov (United States)

    Wei, Caisheng; Luo, Jianjun; Dai, Honghua; Bian, Zilin; Yuan, Jianping

    2018-05-01

    In this paper, a novel learning-based adaptive attitude takeover control method is investigated for the postcapture space robot-target combination with guaranteed prescribed performance in the presence of unknown inertial properties and external disturbance. First, a new static prescribed performance controller is developed to guarantee that all the involved attitude tracking errors are uniformly ultimately bounded by quantitatively characterizing the transient and steady-state performance of the combination. Then, a learning-based supplementary adaptive strategy based on adaptive dynamic programming is introduced to improve the tracking performance of static controller in terms of robustness and adaptiveness only utilizing the input/output data of the combination. Compared with the existing works, the prominent advantage is that the unknown inertial properties are not required to identify in the development of learning-based adaptive control law, which dramatically decreases the complexity and difficulty of the relevant controller design. Moreover, the transient and steady-state performance is guaranteed a priori by designer-specialized performance functions without resorting to repeated regulations of the controller parameters. Finally, the three groups of illustrative examples are employed to verify the effectiveness of the proposed control method.

  16. Identification of the functional binding pocket for compounds targeting small-conductance Ca²⁺-activated potassium channels.

    Science.gov (United States)

    Zhang, Miao; Pascal, John M; Schumann, Marcel; Armen, Roger S; Zhang, Ji-Fang

    2012-01-01

    Small- and intermediate-conductance Ca(2+)-activated potassium channels, activated by Ca(2+)-bound calmodulin, have an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potential for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-ethyl-2-benzimidazolinone (1-EBIO) class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class.

  17. Identification of the functional binding pocket for compounds targeting small-conductance Ca2+-activated potassium channels

    Science.gov (United States)

    Zhang, Miao; Pascal, John M.; Schumann, Marcel; Armen, Roger S.; Zhang, Ji-fang

    2012-01-01

    Small- and intermediate-conductance Ca2+-activated potassium channels, activated by Ca2+-bound calmodulin, play an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potentials for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-EBIO class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class. PMID:22929778

  18. Targeted identification of short interspersed nuclear element families shows their widespread existence and extreme heterogeneity in plant genomes.

    Science.gov (United States)

    Wenke, Torsten; Döbel, Thomas; Sörensen, Thomas Rosleff; Junghans, Holger; Weisshaar, Bernd; Schmidt, Thomas

    2011-09-01

    Short interspersed nuclear elements (SINEs) are non-long terminal repeat retrotransposons that are highly abundant, heterogeneous, and mostly not annotated in eukaryotic genomes. We developed a tool designated SINE-Finder for the targeted discovery of tRNA-derived SINEs. We analyzed sequence data of 16 plant genomes, including 13 angiosperms and three gymnosperms and identified 17,829 full-length and truncated SINEs falling into 31 families showing the widespread occurrence of SINEs in higher plants. The investigation focused on potato (Solanum tuberosum), resulting in the detection of seven different SolS SINE families consisting of 1489 full-length and 870 5' truncated copies. Consensus sequences of full-length members range in size from 106 to 244 bp depending on the SINE family. SolS SINEs populated related species and evolved separately, which led to some distinct subfamilies. Solanaceae SINEs are dispersed along chromosomes and distributed without clustering but with preferred integration into short A-rich motifs. They emerged more than 23 million years ago and were species specifically amplified during the radiation of potato, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). We show that tobacco TS retrotransposons are composite SINEs consisting of the 3' end of a long interspersed nuclear element integrated downstream of a nonhomologous SINE family followed by successfully colonization of the genome. We propose an evolutionary scenario for the formation of TS as a spontaneous event, which could be typical for the emergence of SINE families.

  19. Targeted Identification of Short Interspersed Nuclear Element Families Shows Their Widespread Existence and Extreme Heterogeneity in Plant Genomes[W

    Science.gov (United States)

    Wenke, Torsten; Döbel, Thomas; Sörensen, Thomas Rosleff; Junghans, Holger; Weisshaar, Bernd; Schmidt, Thomas

    2011-01-01

    Short interspersed nuclear elements (SINEs) are non-long terminal repeat retrotransposons that are highly abundant, heterogeneous, and mostly not annotated in eukaryotic genomes. We developed a tool designated SINE-Finder for the targeted discovery of tRNA-derived SINEs. We analyzed sequence data of 16 plant genomes, including 13 angiosperms and three gymnosperms and identified 17,829 full-length and truncated SINEs falling into 31 families showing the widespread occurrence of SINEs in higher plants. The investigation focused on potato (Solanum tuberosum), resulting in the detection of seven different SolS SINE families consisting of 1489 full-length and 870 5′ truncated copies. Consensus sequences of full-length members range in size from 106 to 244 bp depending on the SINE family. SolS SINEs populated related species and evolved separately, which led to some distinct subfamilies. Solanaceae SINEs are dispersed along chromosomes and distributed without clustering but with preferred integration into short A-rich motifs. They emerged more than 23 million years ago and were species specifically amplified during the radiation of potato, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). We show that tobacco TS retrotransposons are composite SINEs consisting of the 3′ end of a long interspersed nuclear element integrated downstream of a nonhomologous SINE family followed by successfully colonization of the genome. We propose an evolutionary scenario for the formation of TS as a spontaneous event, which could be typical for the emergence of SINE families. PMID:21908723

  20. Proteomic characterization of Aspergillus fumigatus treated with an antifungal coumarin for identification of novel target molecules of key pathways.

    Science.gov (United States)

    Singh, Seema; Gupta, Shilpi; Singh, Bharat; Sharma, Sunil K; Gupta, Vijay K; Sharma, Gainda L

    2012-06-01

    A synthetic coumarin, N,N,N-triethyl-11-(4-methyl-2-oxo-2H-chromen-7-yloxy)-11-oxoundecan-1-aminium bromide (SCD-1), having potent activity against pathogenic Aspergilli (MIC90 15.62 μg/mL), was investigated to identify its molecular targets in the pathogen. The proteome of Aspergillus fumigatus was developed after treatment with sublethal doses of compound and analyzed. The results demonstrated 143 differentially expressed proteins on treatment with SCD-1. The expression of four proteins, namely cell division control protein, ubiquitin-like activating enzyme, vacuolar ATP synthase catalytic subunit A, and UTP-glucose-1-phosphate uridylyltransferase of A. fumigatus, was completely inhibited, whereas there were 13 newly expressed and 96 overexpressed proteins, mainly belonging to stress pathway. The treatment of A. fumigatus with SCD-1 also led to attenuation of proteins involved in cell replication and other important biosynthetic processes, including riboflavin biosynthesis, which has been pathogen-specific. In addition to key enzymatic players and antioxidants, nine hypothetical proteins were also identified, seven of which have been novel, being described for the first time. As no cellular functions have yet been described for these hypothetical proteins, their alteration in response to SCD-1 provides significant information about their putative roles in pathogen defense.

  1. Identification of the cognate peptide-MHC target of T cell receptors using molecular modeling and force field scoring

    DEFF Research Database (Denmark)

    Lanzarotti, Esteban; Marcatili, Paolo; Nielsen, Morten

    2018-01-01

    Interactions of T cell receptors (TCR) to peptides in complex with MHC (p:MHC) are key features that mediate cellular immune responses. While MHC binding is required for a peptide to be presented to T cells, not all MHC binders are immunogenic. The interaction of a TCR to the p:MHC complex holds...... terms. Building a benchmark of TCR:p:MHC complexes where epitopes and non-epitopes are modelled using state-of-the-art molecular modelling tools, scoring p:MHC to a given TCR using force-fields, optimized in a cross-validation setup to evaluate TCR inter atomic interactions involved with each p:MHC, we...... and model the TCR:p:MHC complex structure. In summary, we conclude that it is possible to identify the TCR cognate target among different candidate peptides by using a force-field based model, and believe this works could lay the foundation for future work within prediction of TCR:p:MHC interactions....

  2. Literature mining, gene-set enrichment and pathway analysis for target identification in Behçet's disease.

    Science.gov (United States)

    Wilson, Paul; Larminie, Christopher; Smith, Rona

    2016-01-01

    To use literature mining to catalogue Behçet's associated genes, and advanced computational methods to improve the understanding of the pathways and signalling mechanisms that lead to the typical clinical characteristics of Behçet's patients. To extend this technique to identify potential treatment targets for further experimental validation. Text mining methods combined with gene enrichment tools, pathway analysis and causal analysis algorithms. This approach identified 247 human genes associated with Behçet's disease and the resulting disease map, comprising 644 nodes and 19220 edges, captured important details of the relationships between these genes and their associated pathways, as described in diverse data repositories. Pathway analysis has identified how Behçet's associated genes are likely to participate in innate and adaptive immune responses. Causal analysis algorithms have identified a number of potential therapeutic strategies for further investigation. Computational methods have captured pertinent features of the prominent disease characteristics presented in Behçet's disease and have highlighted NOD2, ICOS and IL18 signalling as potential therapeutic strategies.

  3. Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

    Directory of Open Access Journals (Sweden)

    Caselle Michele

    2007-09-01

    methodology to sequence-based methods for the identification of DNA-binding protein target sequences.

  4. Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) – a class-I KNOX gene in potato

    Science.gov (United States)

    Mahajan, Ameya S.; Kondhare, Kirtikumar R.; Rajabhoj, Mohit P.; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K.

    2016-01-01

    Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

  5. Identification of Phytochemicals Targeting c-Met Kinase Domain using Consensus Docking and Molecular Dynamics Simulation Studies.

    Science.gov (United States)

    Aliebrahimi, Shima; Montasser Kouhsari, Shideh; Ostad, Seyed Nasser; Arab, Seyed Shahriar; Karami, Leila

    2018-06-01

    c-Met receptor tyrosine kinase is a proto-oncogene whose aberrant activation is attributed to a lower rate of survival in most cancers. Natural product-derived inhibitors known as "fourth generation inhibitors" constitute more than 60% of anticancer drugs. Furthermore, consensus docking approach has recently been introduced to augment docking accuracy and reduce false positives during a virtual screening. In order to obtain novel small-molecule Met inhibitors, consensus docking approach was performed using Autodock Vina and Autodock 4.2 to virtual screen Naturally Occurring Plant-based Anti-cancer Compound-Activity-Target database against active and inactive conformation of c-Met kinase domain structure. Two hit molecules that were in line with drug-likeness criteria, desired docking score, and binding pose were subjected to molecular dynamics simulations to elucidate intermolecular contacts in protein-ligand complexes. Analysis of molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area studies showed that ZINC08234189 is a plausible inhibitor for the active state of c-Met, whereas ZINC03871891 may be more effective toward active c-Met kinase domain compared to the inactive form due to higher binding energy. Our analysis showed that both the hit molecules formed hydrogen bonds with key residues of the hinge region (P1158, M1160) in the active form, which is a hallmark of kinase domain inhibitors. Considering the pivotal role of HGF/c-Met signaling in carcinogenesis, our results propose ZINC08234189 and ZINC03871891 as the therapeutic options to surmount Met-dependent cancers.

  6. Identification of Key Beliefs Explaining Male Circumcision Motivation Among Adolescent Boys in Zimbabwe: Targets for Behavior Change Communication.

    Science.gov (United States)

    Kasprzyk, Danuta; Tshimanga, Mufuta; Hamilton, Deven T; Gorn, Gerald J; Montaño, Daniel E

    2018-02-01

    Male circumcision (MC) significantly reduces HIV acquisition among men, leading WHO/UNAIDS to recommend high HIV and low MC prevalence countries circumcise 80% of adolescents and men age 15-49. Despite significant investment to increase MC capacity only 27% of the goal has been achieved in Zimbabwe. To increase adoption, research to create evidence-based messages is greatly needed. The Integrated Behavioral Model (IBM) was used to investigate factors affecting MC motivation among adolescents. Based on qualitative elicitation study results a survey was designed and administered to a representative sample of 802 adolescent boys aged 13-17 in two urban and two rural areas in Zimbabwe. Multiple regression analysis found all six IBM constructs (2 attitude, 2 social influence, 2 personal agency) significantly explained MC intention (R 2  = 0.55). Stepwise regression analysis of beliefs underlying each IBM belief-based construct found 9 behavioral, 6 injunctive norm, 2 descriptive norm, 5 efficacy, and 8 control beliefs significantly explained MC intention. A final stepwise regression of all the significant IBM construct beliefs identified 12 key beliefs best explaining intention. Similar analyses were carried out with subgroups of adolescents by urban-rural and age. Different sets of behavioral, normative, efficacy, and control beliefs were significant for each sub-group. This study demonstrates the application of theory-driven research to identify evidence-based targets for the design of effective MC messages for interventions to increase adolescents' motivation. Incorporating these findings into communication campaigns is likely to improve demand for MC.

  7. Effective identification of Lactobacillus casei group species: genome-based selection of the gene mutL as the target of a novel multiplex PCR assay.

    Science.gov (United States)

    Bottari, Benedetta; Felis, Giovanna E; Salvetti, Elisa; Castioni, Anna; Campedelli, Ilenia; Torriani, Sandra; Bernini, Valentina; Gatti, Monica

    2017-07-01

    Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

  8. Identification of cellular targets involved in cardiac failure caused by PKI in oncology: an approach combining pharmacovigilance and pharmacodynamics.

    Science.gov (United States)

    Patras de Campaigno, Emilie; Bondon-Guitton, Emmanuelle; Laurent, Guy; Montastruc, Francois; Montastruc, Jean-Louis; Lapeyre-Mestre, Maryse; Despas, Fabien

    2017-07-01

    The aims of the present study were to evaluate the risk of cardiac failure (CF) associated with 15 anticancer protein kinase inhibitors (PKIs) through a case/noncase analysis and to identify which PK(s) and pathways are involved in PKI-induced CF. In order to evaluate the risk of CF, adjusted reporting odds ratios (aRORs) were calculated for the 15 anticancer PKIs in the World Health Organization safety report database (VigiBase®). We realised a literature review to identify 21 protein kinases (PKs) that were possibly involved in CF caused by PKIs. Pearson correlation coefficients (r) between aRORs and affinity data of the 15 PKIs for the 21 PKs were calculated to identify the cellular target most likely to be involved in PKI-induced CF. A total of 141 601 individual case safety reports (ICSRs) were extracted from VigiBase® for the following PKIs: afatinib, axitinib, bosutinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ruxolitinib, sorafenib, sunitinib and vandetanib. Among them, 2594 ICSRs concerned CF. The disproportionality analysis revealed that, for dasatinib, imatinib, bosutinib, sunitinib and nilotinib, disproportionality for CF was significantly higher than for other PKIs, with aRORs of 2.52 [95% CI 2.26, 2.82], 1.79 (95% CI 1.57, 2.03), 1.73 (95% CI 1.18, 2.54), 1.67 (95% CI 1.51, 1.84) and 1.38 (95% CI 1.18, 1.61), respectively. Significant values for correlation coefficients between the product of dissociation constant (pKd) and aROR were observed for two non-receptor protein kinases: ABL1 (non-phosphorylated and phosphorylated forms) and ABL2 protein kinases, with values of r = 0.83 (P = 0.0001), r = 0.75 (P = 0.0014) and r = 0.78 (P = 0.0006), respectively. We observed a higher disproportionality for CF with dasatinib, imatinib, bosutinib, sunitinib and nilotinib than with other PKIs. In addition, the study highlighted the role of ABL tyrosine kinases in CF caused by anticancer PKIs. © 2017 The British

  9. Targeting safety improvements through identification of incident origination and detection in a near-miss incident learning system

    International Nuclear Information System (INIS)

    Novak, Avrey; Nyflot, Matthew J.; Ermoian, Ralph P.; Jordan, Loucille E.; Sponseller, Patricia A.; Kane, Gabrielle M.; Ford, Eric C.; Zeng, Jing

    2016-01-01

    during the documentation of patient positioning and localization of the patient. Incidents were most frequently detected during treatment delivery (30%), and incidents identified at this point also had higher severity scores than other workflow areas (NMRI = 1.6). Incidents identified during on-treatment quality management were also more severe (NMRI = 1.7), and the specific process steps of reviewing portal and CBCT images tended to catch highest-severity incidents. On average, safety barriers caught 46% of all incidents, most frequently at physics chart review, therapist’s chart check, and the review of portal images; however, most of the incidents that pass through a particular safety barrier are not designed to be capable of being captured at that barrier. Conclusions: Incident learning systems can be used to assess the most common points of error origination and detection in radiation oncology. This can help tailor safety improvement efforts and target the highest impact portions of the workflow. The most severe near-miss events tend to originate during simulation, with the most severe near-miss events detected at the time of patient treatment. Safety barriers can be improved to allow earlier detection of near-miss events.

  10. Targeting safety improvements through identification of incident origination and detection in a near-miss incident learning system

    Energy Technology Data Exchange (ETDEWEB)

    Novak, Avrey; Nyflot, Matthew J.; Ermoian, Ralph P.; Jordan, Loucille E.; Sponseller, Patricia A.; Kane, Gabrielle M.; Ford, Eric C.; Zeng, Jing, E-mail: jzeng13@uw.edu [Department of Radiation Oncology, University of Washington Medical Center, 1959 NE Pacific Street, Campus Box 356043, Seattle, Washington 98195 (United States)

    2016-05-15

    during the documentation of patient positioning and localization of the patient. Incidents were most frequently detected during treatment delivery (30%), and incidents identified at this point also had higher severity scores than other workflow areas (NMRI = 1.6). Incidents identified during on-treatment quality management were also more severe (NMRI = 1.7), and the specific process steps of reviewing portal and CBCT images tended to catch highest-severity incidents. On average, safety barriers caught 46% of all incidents, most frequently at physics chart review, therapist’s chart check, and the review of portal images; however, most of the incidents that pass through a particular safety barrier are not designed to be capable of being captured at that barrier. Conclusions: Incident learning systems can be used to assess the most common points of error origination and detection in radiation oncology. This can help tailor safety improvement efforts and target the highest impact portions of the workflow. The most severe near-miss events tend to originate during simulation, with the most severe near-miss events detected at the time of patient treatment. Safety barriers can be improved to allow earlier detection of near-miss events.

  11. Nuclear Security: Target Analysis-rev

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Surinder Paul [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Gibbs, Philip W. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Bultz, Garl A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2014-03-01

    The objectives of this presentation are to understand target identification, including roll-up and protracted theft; evaluate target identification in the SNRI; recognize the target characteristics and consequence levels; and understand graded safeguards.

  12. Identification and validation of reference genes for quantification of target gene expression with quantitative real-time PCR for tall fescue under four abiotic stresses.

    Directory of Open Access Journals (Sweden)

    Zhimin Yang

    Full Text Available Tall fescue (Festuca arundinacea Schreb. is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41 was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.

  13. Use of tuf as a target for sequence-based identification of Gram-positive cocci of the genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus

    Directory of Open Access Journals (Sweden)

    Li Xuerui

    2012-11-01

    Full Text Available Abstract Background Accurate identification of isolates belonging to genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus at the species level is necessary to provide a better understanding of their pathogenic potential, to aid in making clinical decisions, and to conduct epidemiologic investigations,especially when large blind samples must be analyzed. It is useful to simultaneously identify species in different genera using a single primer pair. Methods We developed a primer pair based on the tuf gene (encoding elongation factor sequence to identify 56 Gram-positive cocci isolates. Results The target sequences were amplified from all 56 samples. The sequencing results and the phylogenetic tree derived from the partial tuf gene sequences identified the isolates as three enterococcal species, two lactococcal species, two staphylococcal species, and six streptococcal species, as well as eight isolates that were novel species of the genus Streptococcus. Partial gene sequence analysis of the sodA, dnaK, and 16S RNA genes confirmed the results obtained by tuf gene sequencing. Conclusion Based on the uniform amplification of the tuf gene from all samples and the ability to identify all isolates at both the genus and species levels, we conclude that the primer pair developed in this research provides a powerful tool for identifying these organisms in clinical laboratories where large blind samples are used.

  14. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    Science.gov (United States)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

  15. A targeted genotyping approach enhances identification of variants in taste receptor and appetite/reward genes of potential functional importance for obesity-related porcine traits.

    Science.gov (United States)

    Cirera, S; Clop, A; Jacobsen, M J; Guerin, M; Lesnik, P; Jørgensen, C B; Fredholm, M; Karlskov-Mortensen, P

    2018-04-01

    Taste receptors (TASRs) and appetite and reward (AR) mechanisms influence eating behaviour, which in turn affects food intake and risk of obesity. In a previous study, we used next generation sequencing to identify potentially functional mutations in TASR and AR genes and found indications for genetic associations between identified variants and growth and fat deposition in a subgroup of animals (n = 38) from the UNIK resource pig population. This population was created for studying obesity and obesity-related diseases. In the present study we validated results from our previous study by investigating genetic associations between 24 selected single nucleotide variants in TASR and AR gene variants and 35 phenotypes describing obesity and metabolism in the entire UNIK population (n = 564). Fifteen variants showed significant association with specific obesity-related phenotypes after Bonferroni correction. Six of the 15 genes, namely SIM1, FOS, TAS2R4, TAS2R9, MCHR2 and LEPR, showed good correlation between known biological function and associated phenotype. We verified a genetic association between potentially functional variants in TASR/AR genes and growth/obesity and conclude that the combination of identification of potentially functional variants by next generation sequencing followed by targeted genotyping and association studies is a powerful and cost-effective approach for increasing the power of genetic association studies. © 2018 Stichting International Foundation for Animal Genetics.

  16. Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly

    Energy Technology Data Exchange (ETDEWEB)

    Tannukit, Sissada [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Crabb, Tara L.; Hertel, Klemens J. [Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA 92697-4025 (United States); Wen, Xin [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Jans, David A. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, Clayton, Victoria 3800 (Australia); Paine, Michael L., E-mail: paine@usc.edu [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States)

    2009-12-18

    Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701-706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711-735 and defines TFIP11's distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle-targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function.

  17. Nondestructive Testing and Target Identification

    Science.gov (United States)

    2016-12-21

    inverse algorithm for the simultaneous reconstruction of both the shape of the inclusion and the two impedance functions via a boundary integral... Simultaneous reconstruction of shape and general- ized impedance functions in electrostatic imaging, Inverse Problems, 30, paper 105010 (2014) 3. F. Cakoni...important role in inverse scattering theory and a survey of recent results in this area can be found in the CBMS-NSF sponsored monograph [18]. In a

  18. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

    Science.gov (United States)

    Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

    1997-04-01

    A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.

  19. Metabolism of the synthetic cannabinoid 5F-PY-PICA by human and rat hepatocytes and identification of biliary analytical targets by directional efflux in sandwich-cultured rat hepatocytes using UHPLC-HR-MS/MS

    DEFF Research Database (Denmark)

    Mardal, Marie; Annaert, Pieter; Noble, Carolina

    2018-01-01

    Analytical strategies for detecting drugs in biological samples rely on information on metabolism and elimination. 5F-PY-PICA belongs to the group of synthetic cannabinoids that are known to undergo excretion into the bile. The aims of this study were the in vitro identification of metabolites of 5......F-PY-PICA and to determine which analytical targets are excreted into the bile and urine. Metabolites identified after incubation of 5F-PY-PICA with pooled human liver microsomes (pHLM), pooled human hepatocytes (pHH), or suspended and sandwich-cultured rat hepatocytes (SCRH). Rat hepatocytes were......-PY-PICA, M4, and M22 are proposed as analytical targets for bile analysis in forensic screening protocols, whereas M6 should be one of the main urinary targets for 5F-PY-PICA analysis....

  20. Mistaken identity of a PCR target proposed for identification of Mycoplasma bovis and the effect of sequence variation on assay performance

    Science.gov (United States)

    Background. Mycoplasma bovis is an important cause of disease in cattle and has recently emerged as a primary disease agent in bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories use PCR to replace or complement traditional isolation and identification ...

  1. Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, HCD and ETD-MS applied to the N-linked glycoproteome of Campylobacter jejuni

    DEFF Research Database (Denmark)

    Scott, Nichollas E; Parker, Benjamin L; Connolly, Angela M

    2011-01-01

    by the resistance of the glycan-peptide bond to enzymatic digestion or ss-elimination, and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction......-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled...

  2. Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets

    NARCIS (Netherlands)

    Farazi, Thalia A.; ten Hoeve, Jelle J.; Brown, Miguel; Mihailovic, Aleksandra; Horlings, Hugo M.; van de Vijver, Marc J.; Tuschl, Thomas; Wessels, Lodewyk F. A.

    2014-01-01

    Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to

  3. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms

    DEFF Research Database (Denmark)

    Maeda, K.; Finnie, Christine; Svensson, Birte

    2004-01-01

    search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase...

  4. Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana.

    Science.gov (United States)

    Zhang, Weixiong; Ruan, Jianhua; Ho, Tuan-Hua David; You, Youngsook; Yu, Taotao; Quatrano, Ralph S

    2005-07-15

    A fundamental problem of computational genomics is identifying the genes that respond to certain endogenous cues and environmental stimuli. This problem can be referred to as targeted gene finding. Since gene regulation is mainly determined by the binding of transcription factors and cis-regulatory DNA sequences, most existing gene annotation methods, which exploit the conservation of open reading frames, are not effective in finding target genes. A viable approach to targeted gene finding is to exploit the cis-regulatory elements that are known to be responsible for the transcription of target genes. Given such cis-elements, putative target genes whose promoters contain the elements can be identified. As a case study, we apply the above approach to predict the genes in model plant Arabidopsis thaliana which are inducible by a phytohormone, abscisic acid (ABA), and abiotic stress, such as drought, cold and salinity. We first construct and analyze two ABA specific cis-elements, ABA-responsive element (ABRE) and its coupling element (CE), in A.thaliana, based on their conservation in rice and other cereal plants. We then use the ABRE-CE module to identify putative ABA-responsive genes in A.thaliana. Based on RT-PCR verification and the results from literature, this method has an accuracy rate of 67.5% for the top 40 predictions. The cis-element based targeted gene finding approach is expected to be widely applicable since a large number of cis-elements in many species are available.

  5. In Silico Identification of Proteins Associated with Drug-induced Liver Injury Based on the Prediction of Drug-target Interactions.

    Science.gov (United States)

    Ivanov, Sergey; Semin, Maxim; Lagunin, Alexey; Filimonov, Dmitry; Poroikov, Vladimir

    2017-07-01

    Drug-induced liver injury (DILI) is the leading cause of acute liver failure as well as one of the major reasons for drug withdrawal from clinical trials and the market. Elucidation of molecular interactions associated with DILI may help to detect potentially hazardous pharmacological agents at the early stages of drug development. The purpose of our study is to investigate which interactions with specific human protein targets may cause DILI. Prediction of interactions with 1534 human proteins was performed for the dataset with information about 699 drugs, which were divided into three categories of DILI: severe (178 drugs), moderate (310 drugs) and without DILI (211 drugs). Based on the comparison of drug-target interactions predicted for different drugs' categories and interpretation of those results using clustering, Gene Ontology, pathway and gene expression analysis, we identified 61 protein targets associated with DILI. Most of the revealed proteins were linked with hepatocytes' death caused by disruption of vital cellular processes, as well as the emergence of inflammation in the liver. It was found that interaction of a drug with the identified targets is the essential molecular mechanism of the severe DILI for the most of the considered pharmaceuticals. Thus, pharmaceutical agents interacting with many of the identified targets may be considered as candidates for filtering out at the early stages of drug research. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Identification of the target areas to be resected in patients with non-bullous emphysema by using gas and perfusion SPECT images

    International Nuclear Information System (INIS)

    Sugi, Kazuro; Ueda, Kazuhiro; Fujita, Nobuhiro; Nawata, Kouichi; Kaneda, Yoshikazu; Nawata, Sumihiko; Esato, Kensuke

    1997-01-01

    Significant improvement of pulmonary function after the volume reduction surgery has been reported in patients with bullous emphysema. However, there has been no successful report in patients with non-bullous emphysema. The reason of failure in patients with non-bullous emphysema should lie mainly on the difficulty to identify the target areas to be resected. We describe how to identify the target areas in the patients with non-bullous emphysema by using gas and perfusion single-photon emission computed tomography (SPECT). Twelve patients with non-bullous emphysema were studied by 133 Xe gas and 99m Tc MAA SPECT, which revealed that abnormal retention and low perfusion areas were located in the apex of the upper lobe and the basal segment of the lower lobe. The resections were performed thoracoscopically focused on the target areas by hockey-stick shape resection in the upper lobe and spiral shape resection in the lower lobe without deformity formation of the residual lung. Slight but significant improvement in the pulmonary functions was observed after the surgery in patients with non-bullous emphysema. The Xe gas and Tc MAA SPECT were useful procedures to identify the target areas to be resected in patients with non-bullous emphysema. (author)

  7. Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise

    DEFF Research Database (Denmark)

    Ingold, S; Dørum, G; Hanson, E

    2018-01-01

    to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values...

  8. Identification of a novel FGFRL1 MicroRNA target site polymorphism for bone mineral density in meta-analyses of genome-wide association studies

    NARCIS (Netherlands)

    T. Niu (Tianhua); N. Liu (Ning); M. Zhao (Ming); G. Xie (Guie); L. Zhang (Lei); J. Li (Jian); Y.-F. Pei (Yu-Fang); H. Shen (Hui); X. Fu (Xiaoying); H. He (Hao); S. Lu (Shan); X. Chen (Xiangding); L. Tan (Lijun); T.-L. Yang (Tie-Lin); Y. Guo (Yan); P.J. Leo (Paul); E.L. Duncan (Emma); J. Shen (Jie); Y.-F. Guo (Yan-fang); G.C. Nicholson (Geoffrey); R.L. Prince (Richard L.); J.A. Eisman (John); G. Jones (Graeme); P.N. Sambrook (Philip); X. Hu (Xiang); P.M. Das (Partha M.); Q. Tian (Qing); X.-Z. Zhu (Xue-Zhen); C.J. Papasian (Christopher J.); M.A. Brown (Matthew); A.G. Uitterlinden (André); Y.-P. Wang (Yu-Ping); S. Xiang (Shuanglin); H.-W. Deng

    2015-01-01

    textabstractMicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNAs' Target Sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)-, and femoral neck

  9. Target Immobilization as a Strategy for NMR-Based Fragment Screening: Comparison of TINS, STD, and SPR for Fragment Hit Identification

    NARCIS (Netherlands)

    Kobayashi, M.; Retra, K.; Figaroa, F.; Hollander, J.G.; Ab, E.; Heetebrij, R.J.; Irth, H.; Siegal, G.

    2010-01-01

    Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the

  10. Identification of Functional Variants for Cleft Lip with or without Cleft Palate in or near PAX7, FGFR2, and NOG by Targeted Sequencing of GWAS Loci

    DEFF Research Database (Denmark)

    Leslie, Elizabeth J; Taub, Margaret A; Liu, Huan

    2015-01-01

    Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European...

  11. ProSelection: A Novel Algorithm to Select Proper Protein Structure Subsets for in Silico Target Identification and Drug Discovery Research.

    Science.gov (United States)

    Wang, Nanyi; Wang, Lirong; Xie, Xiang-Qun

    2017-11-27

    Molecular docking is widely applied to computer-aided drug design and has become relatively mature in the recent decades. Application of docking in modeling varies from single lead compound optimization to large-scale virtual screening. The performance of molecular docking is highly dependent on the protein structures selected. It is especially challenging for large-scale target prediction research when multiple structures are available for a single target. Therefore, we have established ProSelection, a docking preferred-protein selection algorithm, in order to generate the proper structure subset(s). By the ProSelection algorithm, protein structures of "weak selectors" are filtered out whereas structures of "strong selectors" are kept. Specifically, the structure which has a good statistical performance of distinguishing active ligands from inactive ligands is defined as a strong selector. In this study, 249 protein structures of 14 autophagy-related targets are investigated. Surflex-dock was used as the docking engine to distinguish active and inactive compounds against these protein structures. Both t test and Mann-Whitney U test were used to distinguish the strong from the weak selectors based on the normality of the docking score distribution. The suggested docking score threshold for active ligands (SDA) was generated for each strong selector structure according to the receiver operating characteristic (ROC) curve. The performance of ProSelection was further validated by predicting the potential off-targets of 43 U.S. Federal Drug Administration approved small molecule antineoplastic drugs. Overall, ProSelection will accelerate the computational work in protein structure selection and could be a useful tool for molecular docking, target prediction, and protein-chemical database establishment research.

  12. Identification of V-ATPase as a molecular sensor of SOX11-levels and potential therapeutic target for mantle cell lymphoma

    International Nuclear Information System (INIS)

    Emruli, Venera Kuci; Olsson, Roger; Ek, Fredrik; Ek, Sara

    2016-01-01

    Mantle cell lymphoma (MCL) is an aggressive disease with short median survival. Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene. However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/β-catenin pathway that is of importance for proliferation in MCL. Therefore, we evaluated a compound library focused on the Wnt pathway with the aim of identifying Wnt-related targets that regulate growth and survival in MCL, with particular focus on SOX11-dependent growth regulation. An inducible SOX11 knock-down system was used to functionally screen a library of compounds (n = 75) targeting the Wnt signaling pathway. A functionally interesting target, vacuolar-type H + -ATPase (V-ATPase), was further evaluated by western blot, siRNA-mediated gene silencing, immunofluorescence, and flow cytometry. We show that 15 out of 75 compounds targeting the Wnt pathway reduce proliferation in all three MCL cell lines tested. Furthermore, three substances targeting two different targets (V-ATPase and Dkk1) showed SOX11-dependent activity. Further validation analyses were focused on V-ATPase and showed that two independent V-ATPase inhibitors (bafilomycin A1 and concanamycin A) are sensitive to SOX11 levels, causing reduced anti-proliferative response in SOX11 low cells. We further show, using fluorescence imaging and flow cytometry, that V-ATPase is mainly localized to the plasma membrane in primary and MCL cell lines. We show that SOX11 status affect V-ATPase dependent pathways, and thus may be involved in regulating pH in intracellular and extracellular compartments. The plasma membrane localization of V-ATPase indicates that pH regulation of the immediate extracellular compartment may be of importance for receptor functionality and potentially invasiveness in vivo. The online version of this article (doi:10

  13. Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress.

    Science.gov (United States)

    Zhu, Jianfeng; Li, Wanfeng; Yang, Wenhua; Qi, Liwang; Han, Suying

    2013-09-01

    142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5' RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

  14. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    Science.gov (United States)

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  15. Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.

    Science.gov (United States)

    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.

  16. Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.

    Directory of Open Access Journals (Sweden)

    Pablo Armas

    Full Text Available 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.

  17. From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors

    Science.gov (United States)

    Cibiel, Agnes; Nguyen Quang, Nam; Gombert, Karine; Thézé, Benoit; Garofalakis, Anikitos; Ducongé, Frédéric

    2014-01-01

    Background Cell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged. Methodology/Principal Findings Cell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence. Conclusions/Significance Although aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application. PMID:24489826

  18. Identification of miRNAs and Their Targets in Cotton Inoculated with Verticillium dahliae by High-Throughput Sequencing and Degradome Analysis

    Directory of Open Access Journals (Sweden)

    Yujuan Zhang

    2015-06-01

    Full Text Available MicroRNAs (miRNAs are a group of endogenous small non-coding RNAs that play important roles in plant growth, development, and stress response processes. Verticillium wilt is a vascular disease in plants mainly caused by Verticillium dahliae Kleb., the soil-borne fungal pathogen. However, the role of miRNAs in the regulation of Verticillium defense responses is mostly unknown. This study aimed to identify new miRNAs and their potential targets that are involved in the regulation of Verticillium defense responses. Four small RNA libraries and two degradome libraries from mock-infected and infected roots of cotton (both Gossypium hirsutum L. and Gossypium barbadense L. were constructed for deep sequencing. A total of 140 known miRNAs and 58 novel miRNAs were identified. Among the identified miRNAs, many were differentially expressed between libraries. Degradome analysis showed that a total of 83 and 24 genes were the targets of 31 known and 14 novel miRNA families, respectively. Gene Ontology analysis indicated that many of the identified miRNA targets may function in controlling root development and the regulation of Verticillium defense responses in cotton. Our findings provide an overview of potential miRNAs involved in the regulation of Verticillium defense responses in cotton and the interactions between miRNAs and their corresponding targets. The profiling of these miRNAs lays the foundation for further understanding of the function of small RNAs in regulating plant response to fungal infection and Verticillium wilt in particular.

  19. Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining.

    Directory of Open Access Journals (Sweden)

    Etienne G J Danchin

    2013-10-01

    Full Text Available Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when

  20. Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network.

    Science.gov (United States)

    Varrault, Annie; Dantec, Christelle; Le Digarcher, Anne; Chotard, Laëtitia; Bilanges, Benoit; Parrinello, Hugues; Dubois, Emeric; Rialle, Stéphanie; Severac, Dany; Bouschet, Tristan; Journot, Laurent

    2017-10-13

    PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes.

    Science.gov (United States)

    Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Qiu, Shuqing; Rollins, Meaghen; Datla, Raju; Gupta, Vidya S; Kadoo, Narendra Y

    2013-04-01

    MicroRNAs (miRNAs) are small (20-24 nucleotide long) endogenous regulatory RNAs that play important roles in plant growth and development. They regulate gene expression at the post-transcriptional level by translational repression or target degradation and gene silencing. In this study, we identified 116 conserved miRNAs belonging to 23 families from the flax (Linum usitatissimum L.) genome using a computational approach. The precursor miRNAs varied in length; while most of the mature miRNAs were 21 nucleotide long, intergenic and showed conserved signatures of RNA polymerase II transcripts in their upstream regions. Promoter region analysis of the flax miRNA genes indicated prevalence of MYB transcription factor binding sites. Four miRNA gene clusters containing members of three phylogenetic groups were identified. Further, 142 target genes were predicted for these miRNAs and most of these represent transcriptional regulators. The miRNA encoding genes were expressed in diverse tissues as determined by digital expression analysis as well as real-time PCR. The expression of fourteen miRNAs and nine target genes was independently validated using the quantitative reverse transcription PCR (qRT-PCR). This study suggests that a large number of conserved plant miRNAs are also found in flax and these may play important roles in growth and development of flax.

  2. Application of Combination High-Throughput Phenotypic Screening and Target Identification Methods for the Discovery of Natural Product-Based Combination Drugs.

    Science.gov (United States)

    Isgut, Monica; Rao, Mukkavilli; Yang, Chunhua; Subrahmanyam, Vangala; Rida, Padmashree C G; Aneja, Ritu

    2018-03-01

    Modern drug discovery efforts have had mediocre success rates with increasing developmental costs, and this has encouraged pharmaceutical scientists to seek innovative approaches. Recently with the rise of the fields of systems biology and metabolomics, network pharmacology (NP) has begun to emerge as a new paradigm in drug discovery, with a focus on multiple targets and drug combinations for treating disease. Studies on the benefits of drug combinations lay the groundwork for a renewed focus on natural products in drug discovery. Natural products consist of a multitude of constituents that can act on a variety of targets in the body to induce pharmacodynamic responses that may together culminate in an additive or synergistic therapeutic effect. Although natural products cannot be patented, they can be used as starting points in the discovery of potent combination therapeutics. The optimal mix of bioactive ingredients in natural products can be determined via phenotypic screening. The targets and molecular mechanisms of action of these active ingredients can then be determined using chemical proteomics, and by implementing a reverse pharmacokinetics approach. This review article provides evidence supporting the potential benefits of natural product-based combination drugs, and summarizes drug discovery methods that can be applied to this class of drugs. © 2017 Wiley Periodicals, Inc.

  3. In silico identification of a therapeutic target for photo-activated disinfection with indocyanine green: Modeling and virtual screening analysis of Arg-gingipain from Porphyromonas gingivalis.

    Science.gov (United States)

    Pourhajibagher, Maryam; Bahador, Abbas

    2017-06-01

    Porphyromonas gingivalis is a momentous bacterial etiological agent associated with periodontitis, peri-implantitis as well as endodontic infections. The potential advantage of Photo-activated disinfection (PAD) as a promising novel approach is the choice of a suitable target site, specific photosensitizer, and wavelength of light for delivery of the light from source to target. Since Arg-gingipain is a cysteine proteinase that is involved in the virulence of P. gingivalis, it was evaluated as a target site for PAD with indocyanine green (ICG) as a photosensitizer. In this study, we used a range of in silico strategies, bioinformatics tools, biological databases, and computer simulation molecular modeling to evaluate the capacity of Arg-gingipain. The predicted structure of Arg-gingipain indicated that it is located outside the cell and has nine domains and 17 ligands, including two calcium ions and three sodium ions with positive charges which can be a site of interaction for anionic ICG. Based on the results of this study, anionic ICG desires to bind and interact with residues of Arg-gingipain during PAD as a main site to enhance the yield of treatment of endo-periodontal lesions. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Identification of genes highly downregulated in pancreatic cancer through a meta-analysis of microarray datasets: implications for discovery of novel tumor-suppressor genes and therapeutic targets.

    Science.gov (United States)

    Goonesekere, Nalin C W; Andersen, Wyatt; Smith, Alex; Wang, Xiaosheng

    2018-02-01

    The lack of specific symptoms at early tumor stages, together with a high biological aggressiveness of the tumor contribute to the high mortality rate for pancreatic cancer (PC), which has a 5-year survival rate of about 7%. Recent failures of targeted therapies inhibiting kinase activity in clinical trials have highlighted the need for new approaches towards combating this deadly disease. In this study, we have identified genes that are significantly downregulated in PC, through a meta-analysis of large number of microarray datasets. We have used qRT-PCR to confirm the downregulation of selected genes in a panel of PC cell lines. This study has yielded several novel candidate tumor-suppressor genes (TSGs) including GNMT, CEL, PLA2G1B and SERPINI2. We highlight the role of GNMT, a methyl transferase associated with the methylation potential of the cell, and CEL, a lipase, as potential therapeutic targets. We have uncovered genetic links to risk factors associated with PC such as smoking and obesity. Genes important for patient survival and prognosis are also discussed, and we confirm the dysregulation of metabolic pathways previously observed in PC. While many of the genes downregulated in our dataset are associated with protein products normally produced by the pancreas for excretion, we have uncovered some genes whose downregulation appear to play a more causal role in PC. These genes will assist in providing a better understanding of the disease etiology of PC, and in the search for new therapeutic targets and biomarkers.

  5. Target immobilization as a strategy for NMR-based fragment screening: comparison of TINS, STD, and SPR for fragment hit identification.

    Science.gov (United States)

    Kobayashi, Masakazu; Retra, Kim; Figaroa, Francis; Hollander, Johan G; Ab, Eiso; Heetebrij, Robert J; Irth, Hubertus; Siegal, Gregg

    2010-09-01

    Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM K(D) is limited. Similarly, they find that SPR detection is most suited to ligands that bind with K(D) better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.

  6. Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

    Science.gov (United States)

    Lipchina, Inna; Elkabetz, Yechiel; Hafner, Markus; Sheridan, Robert; Mihailovic, Aleksandra; Tuschl, Thomas; Sander, Chris; Studer, Lorenz; Betel, Doron

    2011-01-01

    MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area. PMID:22012620

  7. Knowledge-based identification of the ERK2/STAT3 signal pathway as a therapeutic target for type 2 diabetes and drug discovery.

    Science.gov (United States)

    Kinoshita, Takayoshi; Doi, Kentaro; Sugiyama, Hajime; Kinoshita, Shuhei; Wada, Mutsuyo; Naruto, Shuji; Tomonaga, Atsushi

    2011-09-01

    Many existing agents for diabetes therapy are unable to restore or maintain normal glucose homeostasis or prevent the eventual emergence of hyperglycemia-related complication. Therefore, agents based on novel mechanisms are sought to complement and extend the current therapeutic approaches. Based on the initial paper research, we focused on active STAT3 as an attractive pharmacological target for type 2 diabetes. The subsequent text mining with a unique query to identify suppressors but not activators of STAT3 revealed the ERK2/STAT3 pathway as a novel diabetes target. The description of ERK2 inhibitors as diabetes target had not been found in our text mining research at present. The mechanism-based peptide inhibitor for ERK2 was identified using the knowledge of the KIM sequence, which has an important role in the recognition of cognate kinases, phosphatases, scaffold proteins, and substrates. The peptide inhibitor was confirmed to exert effects in vitro and in vivo. The peptide inhibitor conferred a significant decrease in HOMA-IR levels on Day 28 compared with that in the vehicle group. Besides lowering the fasting blood glucose level, the peptide inhibitor also attenuated the blood glucose increment in the fed state, as compared with the vehicle group. © 2011 John Wiley & Sons A/S.

  8. Identification of the first homozygous 1-bp deletion in GDF9 gene leading to primary ovarian insufficiency by using targeted massively parallel sequencing.

    Science.gov (United States)

    França, M M; Funari, M F A; Nishi, M Y; Narcizo, A M; Domenice, S; Costa, E M F; Lerario, A M; Mendonca, B B

    2018-02-01

    Targeted massively parallel sequencing (TMPS) has been used in genetic diagnosis for Mendelian disorders. In the past few years, the TMPS has identified new and already described genes associated with primary ovarian insufficiency (POI) phenotype. Here, we performed a targeted gene sequencing to find a genetic diagnosis in idiopathic cases of Brazilian POI cohort. A custom SureSelect XT DNA target enrichment panel was designed and the sequencing was performed on Illumina NextSeq sequencer. We identified 1 homozygous 1-bp deletion variant (c.783delC) in the GDF9 gene in 1 patient with POI. The variant was confirmed and segregated using Sanger sequencing. The c.783delC GDF9 variant changed an amino acid creating a premature termination codon (p.Ser262Hisfs*2). This variant was not present in all public databases (ExAC/gnomAD, NHLBI/EVS and 1000Genomes). Moreover, it was absent in 400 alleles from fertile Brazilian women screened by Sanger sequencing. The patient's mother and her unaffected sister carried the c.783delC variant in a heterozygous state, as expected for an autosomal recessive inheritance. Here, the TMPS identified the first homozygous 1-bp deletion variant in GDF9. This finding reveals a novel inheritance pattern of pathogenic variant in GDF9 associated with POI, thus improving the genetic diagnosis of this disorder. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Regularities in eyewitness identification.

    Science.gov (United States)

    Clark, Steven E; Howell, Ryan T; Davey, Sherrie L

    2008-06-01

    What do eyewitness identification experiments typically show? We address this question through a meta-analysis of 94 comparisons between target-present and target-absent lineups. The analyses showed that: (a) correct identifications and correct-nonidentifications were uncorrelated, (b) suspect identifications were more diagnostic with respect to the suspect's guilt or innocence than any other response, (c) nonidentifications were diagnostic of the suspect's innocence, (d) the diagnosticity of foil identifications depended on lineup composition, and (e) don't know responses were nondiagnostic with respect to guilt or innocence. Results of diagnosticity analyses for simultaneous and sequential lineups varied for full-sample versus direct-comparison analyses. Diagnosticity patterns also varied as a function of lineup composition. Theoretical, forensic, and legal implications are discussed.

  10. Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

    Science.gov (United States)

    Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

    2015-06-05

    The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

  11. Identification of Achaete-scute complex-like 1 (ASCL1) target genes and evaluation of DKK1 and TPH1 expression in pancreatic endocrine tumours

    International Nuclear Information System (INIS)

    Johansson, Térèse A; Westin, Gunnar; Skogseid, Britt

    2009-01-01

    ASCL1 role in pancreatic endocrine tumourigenesis has not been established. Recently it was suggested that ASCL1 negatively controls expression of the Wnt signalling antagonist DKK1. Notch signalling regulates expression of TPH1, the rate limiting enzyme in the biosyntesis of serotonin. Understanding the development and proliferation of pancreatic endocrine tumours (PETs) is essential for the development of new therapies. ASCL1 target genes in the pancreatic endocrine tumour cell line BON1 were identified by RNA interference and microarray expression analysis. Protein expressions of selected target genes in PETs were evaluated by immunohistochemistry. 158 annotated ASCL1 target genes were identified in BON1 cells, among them DKK1 and TPH1 that were negatively regulated by ASCL1. An inverse relation of ASCL1 to DKK1 protein expression was observed for 15 out of 22 tumours (68%). Nine tumours displayed low ASCL1/high DKK1 and six tumours high ASCL1/low DKK1 expression. Remaining PETs showed high ASCL1/high DKK1 (n = 4) or low ASCL1/low DKK1 (n = 3) expression. Nine of twelve analysed PETs (75%) showed TPH1 expression with no relation to ASCL1. A number of genes with potential importance for PET tumourigenesis have been identified. ASCL1 negatively regulated the Wnt signalling antagonist DKK1, and TPH1 expression in BON1 cells. In concordance with these findings DKK1 showed an inverse relation to ASCL1 expression in a subset of PETs, which may affect growth control by the Wnt signalling pathway

  12. Targets Fishing and Identification of Calenduloside E as Hsp90AB1: Design, Synthesis, and Evaluation of Clickable Activity-Based Probe

    Science.gov (United States)

    Wang, Shan; Tian, Yu; Zhang, Jing-Yi; Xu, Hui-Bo; Zhou, Ping; Wang, Min; Lu, Sen-Bao; Luo, Yun; Wang, Min; Sun, Gui-Bo; Xu, Xu-Dong; Sun, Xiao-Bo

    2018-01-01

    Calenduloside E (CE), a natural triterpenoid compound isolated from Aralia elata, can protect against ox-LDL-induced human umbilical vein endothelial cell (HUVEC) injury in our previous reports. However, the exact targets and mechanisms of CE remain elusive. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. Based on the previous studies of the structure-activity relationship (SAR), we introduced an alkyne moiety at the C-28 carboxylic group of CE, which kept the protective and anti-apoptosis activity. Via proteomic approach, one of the potential proteins bound to CE-P was identified as Hsp90AB1, and further verification was performed by pure recombinant Hsp90AB1 and competitive assay. These results demonstrated that CE could bind to Hsp90AB1. We also found that CE could reverse the Hsp90AB1 decrease after ox-LDL treatment. To make our results more convincing, we performed SPR analysis and the affinity kinetic assay showed that CE/CE-P could bind to Hsp90AB1 in a dose-dependent manner. Taken together, our research showed CE could probably bind to Hsp90AB1 to protect the cell injury, which might provide the basis for the further exploration of its cardiovascular protective mechanisms. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. PMID:29875664

  13. Identification and Target Prediction of MicroRNAs in Ulmus pumila L. Seedling Roots under Salt Stress by High-Throughput Sequencing

    Directory of Open Access Journals (Sweden)

    Jianfeng Zhu

    2016-12-01

    Full Text Available MicroRNAs (miRNAs are a class of endogenous small RNAs with important roles in plant growth, development, and environmental stress responses. Ulmus pumila L., a deciduous broadleaved tree species of northern temperate regions, is widely distributed in central and northern Asia and has important economic and ecological value. With the spread and aggravation of soil salinization, salt stress has become a major abiotic stress affecting the normal growth and development of U. pumila. However, the influence of salt stress on U. pumila miRNA expression has not been investigated. To identify miRNAs and predict their target mRNA genes under salt stress, three small RNA libraries were generated and sequenced from roots of U. pumila seedlings treated with various concentrations of NaCl corresponding to no salt stress, light short-term salt stress, and medium-heavy long-term salt stress. Integrative analysis identified 254 conserved miRNAs representing 29 families and 49 novel miRNAs; 232 potential targets of the miRNAs were also predicted. Expression profiling of miRNAs between libraries was performed, and the expression of six miRNAs was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR. Our findings provide an overview of potential miRNAs and corresponding targets involved in regulating U. pumila salt defense responses. These results lay the foundation for further research into molecular mechanisms involved in salt stress resistance in U. pumila and other Ulmaceae species.

  14. Identification of Ideal Multi-targeting Bioactive Compounds Against Mur Ligases of Enterobacter aerogenes and Its Binding Mechanism in Comparison with Chemical Inhibitors.

    Science.gov (United States)

    Chakkyarath, Vijina; Natarajan, Jeyakumar

    2017-10-31

    Enterobacter aerogenes have been reported as important opportunistic and multi-resistant bacterial pathogens for humans during the last three decades in hospital wards. The emergence of drug-resistant E. aerogenes demands the need for developing new drugs. Peptidoglycan is an important component of the cell wall of bacteria and the peptidoglycan biochemical pathway is considered as the best source of antibacterial targets. Within this pathway, four Mur ligases MurC, MurD, MurE, and MurF are responsible for the successive additions of L-alanine and suitable targets for developing novel antibacterial drugs. As an inference from this fact, we modeled the three-dimensional structure of above Mur ligases using best template structures available in PDB and analyzed its common binding features. Structural refinement and energy minimization of the predicted Mur ligases models is also being done using molecular dynamics studies. The models of Mur ligases were further investigated for in silico docking studies using bioactive plant compounds from the literature. Interestingly, these results indicate that four plant compounds Isojuripidine, Atroviolacegenin, Porrigenin B, and Nummularogenin showing better docking results in terms of binding energy and number of hydrogen bonds. All these four compounds are spirostan-based compounds with differences in side chains and the amino acid such as ASN, LYS, THR, HIS, ARG (polar) and PHE, GLY, VAL, ALA, MET (non-polar) playing active role in binding site of all four Mur ligases. Overall, in the predicted model, the four plant compounds with its binding features could pave way to design novel multi-targeted antibacterial plant-based bioactive compounds specific to Mur ligases for the treatment of Enterobacter infections.

  15. Identification of aryl hydrocarbon receptor binding targets in mouse hepatic tissue treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

    International Nuclear Information System (INIS)

    Lo, Raymond; Celius, Trine; Forgacs, Agnes L.; Dere, Edward; MacPherson, Laura; Harper, Patricia; Zacharewski, Timothy; Matthews, Jason

    2011-01-01

    Genome-wide, promoter-focused ChIP-chip analysis of hepatic aryl hydrocarbon receptor (AHR) binding sites was conducted in 8-week old female C57BL/6 treated with 30 μg/kg/body weight 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 2 h and 24 h. These studies identified 1642 and 508 AHR-bound regions at 2 h and 24 h, respectively. A total of 430 AHR-bound regions were common between the two time points, corresponding to 403 unique genes. Comparison with previous AHR ChIP-chip studies in mouse hepatoma cells revealed that only 62 of the putative target genes overlapped with the 2 h AHR-bound regions in vivo. Transcription factor binding site analysis revealed an over-representation of aryl hydrocarbon response elements (AHREs) in AHR-bound regions with 53% (2 h) and 68% (24 h) of them containing at least one AHRE. In addition to AHREs, E2f-Myc activator motifs previously implicated in AHR function, as well as a number of other motifs, including Sp1, nuclear receptor subfamily 2 factor, and early growth response factor motifs were also identified. Expression microarray studies identified 133 unique genes differentially regulated after 4 h treatment with TCDD. Of which, 39 were identified as AHR-bound genes at 2 h. Ingenuity Pathway Analysis on the 39 AHR-bound TCDD responsive genes identified potential perturbation in biological processes such as lipid metabolism, drug metabolism, and endocrine system development as a result of TCDD-mediated AHR activation. Our findings identify direct AHR target genes in vivo, highlight in vitro and in vivo differences in AHR signaling and show that AHR recruitment does not necessarily result in changes in target gene expression. -- Highlights: ► ChIP-chip analysis of hepatic AHR binding after 2 h and 24 h of TCDD. ► We identified 1642 and 508 AHR-bound regions at 2 h and 24 h. ► 430 regions were common to both time points and highly enriched with AHREs. ► Only 62 putative target regions overlapped AHR-bound regions in

  16. Identification of aryl hydrocarbon receptor binding targets in mouse hepatic tissue treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Raymond; Celius, Trine [Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario (Canada); Forgacs, Agnes L. [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI (United States); Dere, Edward [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI (United States); MacPherson, Laura [Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario (Canada); Harper, Patricia [Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario (Canada); Research Institute, The Hospital for Sick Children, Toronto, Ontario (Canada); Zacharewski, Timothy [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI (United States); Matthews, Jason, E-mail: jason.matthews@utoronto.ca [Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario (Canada)

    2011-11-15

    Genome-wide, promoter-focused ChIP-chip analysis of hepatic aryl hydrocarbon receptor (AHR) binding sites was conducted in 8-week old female C57BL/6 treated with 30 {mu}g/kg/body weight 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 2 h and 24 h. These studies identified 1642 and 508 AHR-bound regions at 2 h and 24 h, respectively. A total of 430 AHR-bound regions were common between the two time points, corresponding to 403 unique genes. Comparison with previous AHR ChIP-chip studies in mouse hepatoma cells revealed that only 62 of the putative target genes overlapped with the 2 h AHR-bound regions in vivo. Transcription factor binding site analysis revealed an over-representation of aryl hydrocarbon response elements (AHREs) in AHR-bound regions with 53% (2 h) and 68% (24 h) of them containing at least one AHRE. In addition to AHREs, E2f-Myc activator motifs previously implicated in AHR function, as well as a number of other motifs, including Sp1, nuclear receptor subfamily 2 factor, and early growth response factor motifs were also identified. Expression microarray studies identified 133 unique genes differentially regulated after 4 h treatment with TCDD. Of which, 39 were identified as AHR-bound genes at 2 h. Ingenuity Pathway Analysis on the 39 AHR-bound TCDD responsive genes identified potential perturbation in biological processes such as lipid metabolism, drug metabolism, and endocrine system development as a result of TCDD-mediated AHR activation. Our findings identify direct AHR target genes in vivo, highlight in vitro and in vivo differences in AHR signaling and show that AHR recruitment does not necessarily result in changes in target gene expression. -- Highlights: Black-Right-Pointing-Pointer ChIP-chip analysis of hepatic AHR binding after 2 h and 24 h of TCDD. Black-Right-Pointing-Pointer We identified 1642 and 508 AHR-bound regions at 2 h and 24 h. Black-Right-Pointing-Pointer 430 regions were common to both time points and highly enriched with

  17. Identification of pre-erythrocytic malaria antigens that target hepatocytes for killing in vivo and contribute to protection elicited by whole-parasite vaccination.

    Directory of Open Access Journals (Sweden)

    Lin Chen

    Full Text Available Pre-erythrocytic malaria vaccines, including those based on whole-parasite approaches, have shown protective efficacy in animal and human studies. However few pre-erythocytic antigens other than the immunodominant circumsporozoite protein (CSP have been studied in depth with the goal of developing potent subunit malaria vaccines that are suited for use in endemic areas. Here we describe a novel technique to identify pre-erythrocytic malaria antigens that contribute to protection elicited by whole-parasite vaccination in the mouse model. Our approach combines immunization with genetically attenuated parasites and challenge with DNA plasmids encoding for potential protective pre-erythrocytic malaria antigens as luciferase fusions by hydrodynamic tail vein injection. After optimizing the technique, we first showed that immunization with Pyfabb/f-, a P. yoelii genetically attenuated parasite, induces killing of CSP-presenting hepatocytes. Depletion of CD8+ but not CD4+ T cells diminished the killing of CSP-expressing hepatocytes, indicating that killing is CD8+ T cell-dependent. Finally we showed that the use of heterologous prime/boost immunization strategies that use genetically attenuated parasites and DNA vaccines enabled the characterization of a novel pre-erythrocytic antigen, Tmp21, as a contributor to Pyfabb/f- induced protection. This technique will be valuable for identification of potentially protective liver stage antigens and has the potential to contribute to the understanding of immunity elicited by whole parasite vaccination, as well as the development of effective subunit malaria vaccines.

  18. Identification and characterization of microRNAs and their targets in high-altitude stress-adaptive plant maca (Lepidium meyenii Walp).

    Science.gov (United States)

    Paul, Sujay

    2017-06-01

    MicroRNAs (miRNAs) are endogenous, short (~21-nucleotide), non-coding RNA molecules that play pivotal roles in plant growth, development, and stress response signaling. In this study using recently published draft genome sequence of a high-altitude plant maca (Lepidium meyenii Walp) and applying genome-wide computational-based approaches, a total of 62 potentially conserved miRNAs belonging to 28 families were identified and four (lme-miR160a, lme-miR164c, lme-miR 166a, and lme-miR 319a) of them further validated by RT-PCR. Deploying psRNATarget tool a total of 99 potential miRNA target transcripts were also identified in maca. Targets include a number of transcription factors like Squamosa promoter-binding, NAC, MYB, auxin response factor, APETALA, WRKY, and F-box protein. To the best of my knowledge, this is the first genome-based miRNA profiling of a high-altitude plant.

  19. Evidence-Based Identification of Key Beliefs Explaining Infant Male Circumcision Motivation Among Expectant Parents in Zimbabwe: Targets for Behavior Change Messaging.

    Science.gov (United States)

    Montaño, Daniel E; Tshimanga, Mufuta; Hamilton, Deven T; Gorn, Gerald; Kasprzyk, Danuta

    2018-02-01

    Slow adult male circumcision uptake is one factor leading some to recommend increased priority for infant male circumcision (IMC) in sub-Saharan African countries. This research, guided by the integrated behavioral model (IBM), was carried out to identify key beliefs that best explain Zimbabwean parents' motivation to have their infant sons circumcised. A quantitative survey, designed from qualitative elicitation study results, was administered to independent representative samples of 800 expectant mothers and 795 expectant fathers in two urban and two rural areas in Zimbabwe. Multiple regression analyses found IMC motivation among fathers was explained by instrumental attitude, descriptive norm and self-efficacy; while motivation among mothers was explained by instrumental attitude, injunctive norm, descriptive norm, self-efficacy, and perceived control. Regression analyses of beliefs underlying IBM constructs found some overlap but many differences in key beliefs explaining IMC motivation among mothers and fathers. We found differences in key beliefs among urban and rural parents. Urban fathers' IMC motivation was explained best by behavioral beliefs, while rural fathers' motivation was explained by both behavioral and efficacy beliefs. Urban mothers' IMC motivation was explained primarily by behavioral and normative beliefs, while rural mothers' motivation was explained mostly by behavioral beliefs. The key beliefs we identified should serve as targets for developing messages to improve demand and maximize parent uptake as IMC programs are rolled out. These targets need to be different among urban and rural expectant mothers and fathers.

  20. Identification of the Geographic Origin of Parmigiano Reggiano (P.D.O.) Cheeses Deploying Non-Targeted Mass Spectrometry and Chemometrics.

    Science.gov (United States)

    Popping, Bert; De Dominicis, Emiliano; Dante, Mario; Nocetti, Marco

    2017-02-16

    Parmigiano Reggiano is an Italian product with a protected designation of origin (P.D.O.). It is an aged hard cheese made from raw milk. P.D.O. products are protected by European regulations. Approximately 3 million wheels are produced each year, and the product attracts a relevant premium price due to its quality and all around the world well known typicity. Due to the high demand that exceeds the production, several fraudulent products can be found on the market. The rate of fraud is estimated between 20% and 40%, the latter predominantly in the grated form. We have developed a non-target method based on Liquid Chomatography-High Resolution Mass Spectrometry (LC-HRMS) that allows the discrimination of Parmigiano Reggiano from non-authentic products with milk from different geographical origins or products, where other aspects of the production process do not comply with the rules laid down in the production specifications for Parmeggiano Reggiano. Based on a database created with authentic samples provided by the Consortium of Parmigiano Reggiano Cheese, a reliable classification model was built. The overall classification capabilities of this non-targeted method was verified on 32 grated cheese samples. The classification was 87.5% accurate.

  1. Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

    Science.gov (United States)

    de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

    2003-01-10

    The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

  2. Identification of Protein Targets of 12/15-Lipoxygenase-Derived Lipid Electrophiles in Mouse Peritoneal Macrophages Using Omega-Alkynyl Fatty Acid.

    Science.gov (United States)

    Isobe, Yosuke; Kawashima, Yusuke; Ishihara, Tomoaki; Watanabe, Kenji; Ohara, Osamu; Arita, Makoto

    2018-04-20

    The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of highest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Here, we describe a chemoproteomic method to identify the targets of enzymatically generated LDE. By treating mouse peritoneal macrophages with omega-alkynyl arachidonic acid (aAA), we identified a series of proteins adducted by LDE generated through a 12/15-LOX catalyzed reaction. Pathway analysis revealed a dramatic enrichment of proteins involved in energy metabolism and found that glycolytic flux and mitochondrial respiration were significantly affected by the expression of 12/15-LOX. Our findings thus highlight the utility of chemoproteomics using aAA for identifying intracellular targets of enzymatically generated LDE.

  3. RNA-seq based identification and mutant validation of gene targets related to ethanol resistance in cyanobacterial Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Wang Jiangxin

    2012-12-01

    Full Text Available Abstract Background Fermentation production of biofuel ethanol consumes agricultural crops, which will compete directly with the food supply. As an alternative, photosynthetic cyanobacteria have been proposed as microbial factories to produce ethanol directly from solar energy and CO2. However, the ethanol productivity from photoautotrophic cyanobacteria is still very low, mostly due to the low tolerance of cyanobacterial systems to ethanol stress. Results To build a foundation necessary to engineer robust ethanol-producing cyanobacterial hosts, in this study we applied a quantitative transcriptomics approach with a next-generation sequencing technology, combined with quantitative reverse-transcript PCR (RT-PCR analysis, to reveal the global metabolic responses to ethanol in model cyanobacterial Synechocystis sp. PCC 6803. The results showed that ethanol exposure induced genes involved in common stress responses, transporting and cell envelope modification. In addition, the cells can also utilize enhanced polyhydroxyalkanoates (PHA accumulation and glyoxalase detoxication pathway as means against ethanol stress. The up-regulation of photosynthesis by ethanol was also further confirmed at transcriptional level. Finally, we used gene knockout strains to validate the potential target genes related to ethanol tolerance. Conclusion RNA-Seq based global transcriptomic analysis provided a comprehensive view of cellular response to ethanol exposure. The analysis provided a list of gene targets for engineering ethanol tolerance in cyanobacterium Synechocystis.

  4. Identification of miR-2400 gene as a novel regulator in skeletal muscle satellite cells proliferation by targeting MYOG gene

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wei Wei [The Laboratory of Cell and Development, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); College of Life Sciences and Agriculture & Forestry, Qiqihar University, Qiqihar, Heilongjiang 161006 (China); Tong, Hui Li; Sun, Xiao Feng; Hu, Qian; Yang, Yu; Li, Shu Feng [The Laboratory of Cell and Development, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Yan, Yun Qin, E-mail: yanyunqin@sohu.com [The Laboratory of Cell and Development, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Li, Guang Peng [The Key Laboratory of Mammal Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Hohhot 010021 (China)

    2015-08-07

    MicroRNAs play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. Previous study in our laboratory showed that the expression level of miR-2400, a novel and unique miRNA from bovine, had significantly changed in skeletal muscle-derived satellite cells (MDSCs) during differentiation, however, the function and expression pattern for miR-2400 in MDSCs has not been fully understood. In this report, we firstly identified that the expression levels of miR-2400 were down-regulated during MDSCs differentiation by stem-loop RT-PCR. Over-expression and inhibition studies demonstrated that miR-2400 promoted MDSCs proliferation by EdU (5-ethynyl-2′ deoxyuridine) incorporation assay and immunofluorescence staining of Proliferating cell nuclear antigen (PCNA). Luciferase reporter assays showed that miR-2400 directly targeted the 3′ untranslated regions (UTRs) of myogenin (MYOG) mRNA. These data suggested that miR-2400 could promote MDSCs proliferation through targeting MYOG. Furthermore, we found that miR-2400, which was located within the eighth intron of the Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) gene, was down-regulated in MDSCs in a direct correlation with the WHSC1L1 transcript by Clustered regularly interspaced palindromic repeats interference (CRISPRi). In addition, these observations not only provided supporting evidence for the codependent expression of intronic miRNAs and their host genes in vitro, but also gave insight into the role of miR-2400 in MDSCs proliferation. - Highlights: • miR-2400 is a novel and unique miRNA from bovine. • miR-2400 could promote skeletal muscle satellite cells proliferation. • miR-2400 directly targeted the 3′ untranslated regions of MYOG mRNA. • miR-2400 could be coexpressed together with its host gene WHSC1L1.

  5. Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis.

    Science.gov (United States)

    Chen, Jingli; Zheng, Yi; Qin, Li; Wang, Yan; Chen, Lifei; He, Yanjun; Fei, Zhangjun; Lu, Gang

    2016-04-12

    MicroRNAs (miRNAs), a class of non-coding small RNAs (sRNAs), regulate various biological processes. Although miRNAs have been identified and characterized in several plant species, miRNAs in Asparagus officinalis have not been reported. As a dioecious plant with homomorphic sex chromosomes, asparagus is regarded as an important model system for studying mechanisms of plant sex determination. Two independent sRNA libraries from male and female asparagus plants were sequenced with Illumina sequencing, thereby generating 4.13 and 5.88 million final clean reads, respectively. Both libraries predominantly contained 24-nt sRNAs, followed by 21-nt sRNAs. Further analysis identified 154 conserved miRNAs, which belong to 26 families, and 39 novel miRNA candidates seemed to be specific to asparagus. Comparative profiling revealed that 63 miRNAs exhibited significant differential expression between male and female plants, which was confirmed by real-time quantitative PCR analysis. Among them, 37 miRNAs were significantly up-regulated in the female library, whereas the others were preferentially expressed in the male library. Furthermore, 40 target mRNAs representing 44 conserved and seven novel miRNAs were identified in asparagus through high-throughput degradome sequencing. Functional annotation showed that these target mRNAs were involved in a wide range of developmental and metabolic processes. We identified a large set of conserved and specific miRNAs and compared their expression levels between male and female asparagus plants. Several asparagus miRNAs, which belong to the miR159, miR167, and miR172 families involved in reproductive organ development, were differentially expressed between male and female plants, as well as during flower development. Consistently, several predicted targets of asparagus miRNAs were associated with floral organ development. These findings suggest the potential roles of miRNAs in sex determination and reproductive developmental processes in

  6. Antisense sequences and antagomiR 155 in therapy for B lymphomas over expressing miR-155: preclinical models and identification of target mRNAs

    International Nuclear Information System (INIS)

    Marziali, G.; Peschle, C.

    2009-01-01

    Micro RNAs (miRNAs) are a conserved class of small noncoding RNAs (22-25 nucleotides), which modulate gene expression at post-transcriptional level by base pairing to the 3'UTR of the target mRNAs, thus causing messenger degradation or inhibiting its translation. Experimental evidence indicate that several miRNAs are deregulated in human tumors. MIRN155 has been shown to be highly expressed in a variety of human B cell lymphomas, especially diffuse large B cells, Hodgkin, and a subset of Burkitt lymphomas. Its expression is physiologically increased in activated B and T cells and it plays a key role in regulating the homeostasis and function of the immune system

  7. Identification of anti-HBV activities in Paeonia suffruticosa Andr. using GRP78 as a drug target on Herbochip®.

    Science.gov (United States)

    Lam, Iao-Fai; Huang, Min; Chang, Margaret Dah-Tysr; Yao, Pei-Wun; Chou, Yu-Ting; Ng, Sim-Kun; Tsai, Ying-Lin; Lin, Yu-Chang; Zhang, Yun-Feng; Yang, Xiao-Yuan; Lai, Yiu-Kay

    2017-01-01

    Herbochip ® technology is a high throughput drug screening platform in a reverse screening manner, in which potential chemical leads in herbal extracts are immobilized and drug target proteins can be used as probes for screening process [BMC Complementary and Alternative Medicine (2015) 15:146]. While herbal medicines represent an ideal reservoir for drug screenings, here a molecular chaperone GRP78 is demonstrated to serve as a potential target for antiviral drug discovery. We cloned and expressed a truncated but fully functional form of human GRP78 (hGRP78 1-508 ) and used it as a probe for anti-HBV drug screening on herbochips. In vitro cytotoxicity and in vitro anti-HBV activity of the herbal extracts were evaluated by MTT and ELISA assays, respectively. Finally, anti-HBV activity was confirmed by in vivo assay using DHBV DNA levels in DHBV-infected ducklings as a model. Primary screenings using GRP78 on 40 herbochips revealed 11 positives. Four of the positives, namely Dioscorea bulbifera , Lasiosphaera fenzlii , Paeonia suffruticosa and Polygonum cuspidatum were subjected to subsequent assays. None of the above extracts was cytotoxic to AML12 cells, but P. cuspidatum extract (PCE) was found to be cytotoxic to HepG2 2.2.15 cells. Both PCE and P. suffruticosa extract (PSE) suppressed secretion of HBsAg and HBeAg in HepG2 2.2.15 cells. The anti-HBV activity of PSE was further confirmed in vivo. We have demonstrated that GRP78 is a valid probe for anti-HBV drug screening on herbochips. We have also shown that PSE, while being non-cytotoxic, possesses in vitro and in vivo anti-HBV activities. Taken together, our data suggest that PSE may be a potential anti-HBV agent for therapeutic use.

  8. Identification of ERdj3 and OBF-1/BOB-1/OCA-B as direct targets of XBP-1 during plasma cell differentiation.

    Science.gov (United States)

    Shen, Ying; Hendershot, Linda M

    2007-09-01

    Plasma cell differentiation is accompanied by a modified unfolded protein response (UPR), which involves activation of the Ire1 and activating transcription factor 6 branches, but not the PKR-like endoplasmic reticulum kinase branch. Ire1-mediated splicing of XBP-1 (XBP-1(S)) is required for terminal differentiation, although the direct targets of XBP-1(S) in this process have not been identified. We demonstrate that XBP-1(S) binds to the promoter of ERdj3 in plasmacytoma cells and in LPS-stimulated primary splenic B cells, which corresponds to increased expression of ERdj3 transcripts in both cases. When small hairpin RNA was used to decrease XBP-1 expression in plasmacytoma lines, ERdj3 transcripts were concomitantly reduced. The accumulation of Ig gamma H chain protein was also diminished, but unexpectedly this occurred at the transcriptional level as opposed to effects on H chain stability. The decrease in H chain transcripts correlated with a reduction in mRNA encoding the H chain transcription factor, OBF-1/BOB-1/OCA-B. Chromatin immunoprecipitation experiments revealed that XBP-1(S) binds to the OBF-1/BOB-1/OCA-B promoter in the plasmacytoma line and in primary B cells not only during plasma cell differentiation, but also in response to classical UPR activation. Gel shift assays suggest that XBP-1(S) binding occurs through a UPR element conserved in both murine and human OBF-1/BOB-1/OCA-B promoters as opposed to endoplasmic reticulum stress response elements. Our studies are the first to identify direct downstream targets of XBP-1(S) during either plasma cell differentiation or the UPR. In addition, our data further define the XBP-1(S)-binding sequence and provide yet another role for this protein as a master regulator of plasma cell differentiation.

  9. Selection, Identification, and Binding Mechanism Studies of an ssDNA Aptamer Targeted to Different Stages of E. coli O157:H7.

    Science.gov (United States)

    Zou, Ying; Duan, Nuo; Wu, Shijia; Shen, Mofei; Wang, Zhouping

    2018-06-06

    Enterohemorrhagic Escherichia coli O157:H7 ( E. coli O157:H7) is known as an important food-borne pathogen related to public health. In this study, aptamers which could bind to different stages of E. coli O157:H7 (adjustment phase, log phase, and stationary phase) with high affinity and specificity were obtained by the whole cell-SELEX method through 14 selection rounds including three counter-selection rounds. Altogether, 32 sequences were obtained, and nine families were classified to select the optimal aptamer. To analyze affinity and specificity by flow cytometer, an ssDNA aptamer named Apt-5 was picked out as the optimal aptamer that recognizes different stages of E. coli O157:H7 specifically with the K d value of 9.04 ± 2.80 nM. In addition, in order to study the binding mechanism, target bacteria were treated by proteinase K and trypsin, indicating that the specific binding site is not protein on the cell membrane. Furthermore, when we treated E. coli O157:H7 with EDTA, the result showed that the binding site might be lipopolysaccharide (LPS) on the outer membrane of E. coli O157:H7.

  10. A Genome-Wide Identification of the WRKY Family Genes and a Survey of Potential WRKY Target Genes in Dendrobium officinale.

    Science.gov (United States)

    He, Chunmei; Teixeira da Silva, Jaime A; Tan, Jianwen; Zhang, Jianxia; Pan, Xiaoping; Li, Mingzhi; Luo, Jianping; Duan, Jun

    2017-08-23

    The WRKY family, one of the largest families of transcription factors, plays important roles in the regulation of various biological processes, including growth, development and stress responses in plants. In the present study, 63 DoWRKY genes were identified from the Dendrobium officinale genome. These were classified into groups I, II, III and a non-group, each with 14, 28, 10 and 11 members, respectively. ABA-responsive, sulfur-responsive and low temperature-responsive elements were identified in the 1-k upstream regulatory region of DoWRKY genes. Subsequently, the expression of the 63 DoWRKY genes under cold stress was assessed, and the expression profiles of a large number of these genes were regulated by low temperature in roots and stems. To further understand the regulatory mechanism of DoWRKY genes in biological processes, potential WRKY target genes were investigated. Among them, most stress-related genes contained multiple W-box elements in their promoters. In addition, the genes involved in polysaccharide synthesis and hydrolysis contained W-box elements in their 1-k upstream regulatory regions, suggesting that DoWRKY genes may play a role in polysaccharide metabolism. These results provide a basis for investigating the function of WRKY genes and help to understand the downstream regulation network in plants within the Orchidaceae.

  11. Screening In Vitro Targets Related to Diabetes in Herbal Extracts from Peru: Identification of Active Compounds in Hypericum laricifolium Juss. by Offline High-Performance Liquid Chromatography.

    Science.gov (United States)

    Quispe, Yanymee N. Guillen; Hwang, Seung Hwan; Wang, Zhiqiang; Zuo, Guanglei; Lim, Soon Sung

    2017-11-24

    This study investigates in vitro targets related to diabetes in 30 herbal extracts from Peru, for the first time, using α-glucosidase, aldose reductase (AR) inhibitory assays and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging assays. Among the 30 herbal extracts, Hypericum laricifolium Juss. (HL) was the herb which showed more than 50% inhibition in all assays, presenting 97.2 ± 2.0%, 56.9 ± 5.6%, 81.9 ± 2.5%, and 58.8 ± 4.6% inhibition for the α-glucosidase, AR, DPPH, and ABTS assays, respectively. Finally, six bioactive compounds, namely, protocatechuic acid, chlorogenic acid, caffeic acid, kaempferol 3- O -glucuronide, quercetin, and kaempferol were identified in HL by offline high-performance liquid chromatography (HPLC). Quercetin exhibited the strongest inhibition in all enzyme assays and the strongest antioxidant activity. The results suggest that HL shows great potential for the complementary treatment of diabetes and its complications.

  12. Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target.

    Directory of Open Access Journals (Sweden)

    Mariëlle I Gallegos Ruiz

    Full Text Available BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%, which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008, survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04. Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

  13. Identification and quantification of 56 targeted phenols in wines, spirits, and vinegars by online solid-phase extraction - ultrahigh-performance liquid chromatography - quadrupole-orbitrap mass spectrometry.

    Science.gov (United States)

    Barnaba, C; Dellacassa, E; Nicolini, G; Nardin, T; Malacarne, M; Larcher, R

    2015-12-04

    Phenolic compounds seriously affect the sensory and nutritional qualities of food products, both through the positive contribution of wood transfer in barrel-aged products and as off-flavours. A new targeted analytical approach combining on-line solid-phase extraction (SPE) clean-up to reduce matrix interference and rapid chromatographic detection performed with ultrahigh-performance liquid chromatography coupled with quadrupole/high-resolution mass spectrometry (Q-Orbitrap), was developed for the quantification of 56 simple phenols. Considering the advantages of using on-line SPE and a resolving power of 140,000, the proposed method was applied to define phenolic content in red (N=8) and white (8) wines, spirits (8), common (8) and balsamic (8) vinegars. The final method was linear from the limits of quantification (0.0001-0.001μgmL(-1)) up to 10μgmL(-1) with R(2) of at least 0.99. Recovery, used to define method accuracy, ranged from 80 to 120% for 89% of compounds. The method was suitable for analytical requirements in the tested matrices being able to analyse 46 phenols in red wines, 41 phenols in white wines and in spirits, 42 phenols in common vinegars and 44 phenols in balsamic vinegars. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Identification of a New Target slr0946 of the Response Regulator Sll0649 Involving Cadmium Tolerance in Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Tao Sun

    2017-08-01

    Full Text Available Survival of photosynthetic cyanobacteria is challenged by environmental contaminations like heavy metals. Among them, deciphering the regulatory mechanisms for cadmium (Cd in cyanobacteria would facilitate the construction of Cd-resistant strains. In this study, the DNA-Affinity-Purified-chromatin immunoprecipitation assay was employed to identify the direct targets of Sll0649, which was a Cd2+-related response regulator identified in our previous work in model cyanobacteria Synechocystis sp. PCC 6803. As a result, the promoter region of slr0946 encoding the arsenate reductase was enriched fourfolds by quantitative real time PCR analysis. Further, deletion of slr0946 led to a sensitive phenotype to Cd2+ stress compared with the wild type (WT and the sensitive phenotype of Δslr0946 could be rescued by complementation assay via introducing slr0946 back into Δslr0946. Finally, individually overexpression of slr0946 as well as two Cd2+-related genes identified priviously (i.e., sll1598 and slr0798 in WT could significantly improve the tolerance of Synechocystis sp. PCC 6803 to Cd2+. This study provided a better understanding of the tolerance mechanism to Cd2+ in cyanobacteria and also feasible strategies for tolerance modifications to heavy metals in the future.

  15. Identification of Novel Gene Targets and Putative Regulators of Arsenic-Associated DNA Methylation in Human Urothelial Cells and Bladder Cancer

    Science.gov (United States)

    Rager, Julia E.; Miller, Sloane; Tulenko, Samantha E.; Smeester, Lisa; Ray, Paul D.; Yosim, Andrew; Currier, Jenna M.; Ishida, María C.; González-Horta, Maria del Carmen; Sánchez-Ramírez, Blanca; Ballinas-Casarrubias, Lourdes; Gutiérrez-Torres, Daniela S.; Drobná, Zuzana; Del Razo, Luz M.; García-Vargas, Gonzalo G.; Kim, William Y.; Zhou, Yi-Hui; Wright, Fred A.; Stýblo, Miroslav; Fry, Rebecca C.

    2016-01-01

    There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to a myriad of adverse health effects, including cancer of the bladder. The present study set out to identify DNA methylation patterns associated with iAs and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic (As)-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total As (tAs) and As species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 As-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the As- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer. PMID:26039340

  16. Screening In Vitro Targets Related to Diabetes in Herbal Extracts from Peru: Identification of Active Compounds in Hypericum laricifolium Juss. by Offline High-Performance Liquid Chromatography

    Science.gov (United States)

    Guillen Quispe, Yanymee N.; Hwang, Seung Hwan; Wang, Zhiqiang

    2017-01-01

    This study investigates in vitro targets related to diabetes in 30 herbal extracts from Peru, for the first time, using α-glucosidase, aldose reductase (AR) inhibitory assays and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging assays. Among the 30 herbal extracts, Hypericum laricifolium Juss. (HL) was the herb which showed more than 50% inhibition in all assays, presenting 97.2 ± 2.0%, 56.9 ± 5.6%, 81.9 ± 2.5%, and 58.8 ± 4.6% inhibition for the α-glucosidase, AR, DPPH, and ABTS assays, respectively. Finally, six bioactive compounds, namely, protocatechuic acid, chlorogenic acid, caffeic acid, kaempferol 3-O-glucuronide, quercetin, and kaempferol were identified in HL by offline high-performance liquid chromatography (HPLC). Quercetin exhibited the strongest inhibition in all enzyme assays and the strongest antioxidant activity. The results suggest that HL shows great potential for the complementary treatment of diabetes and its complications. PMID:29186785

  17. Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans.

    Science.gov (United States)

    Noborn, Fredrik; Gomez Toledo, Alejandro; Green, Anders; Nasir, Waqas; Sihlbom, Carina; Nilsson, Jonas; Larson, Göran

    2016-10-03

    Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans.

  18. Identification of novel mutations including a double mutation in patients with inherited cardiomyopathy by a targeted sequencing approach using the Ion Torrent PGM system.

    Science.gov (United States)

    Zhao, Yue; Cao, Hong; Song, Yindi; Feng, Yue; Ding, Xiaoxue; Pang, Mingjie; Zhang, Yunmei; Zhang, Hong; Ding, Jiahuan; Xia, Xueshan

    2016-06-01

    knowledge. This patient provided us with more information regarding the genotype-phenotype correlation between mutations of MYH7 and PRKAG2. Taken together, these findings provide insight into the molecular mechanisms underlying inherited cardiomyopathy. The mutations identified in this study may be further investigated in the future in order to improve the diagnosis and treatment of patients with inherited cardiomyopathy. Furthermore, our findings indicated that sequencing using the Ion Torrent PGM system is a useful approach for the identification of pathogenic mutations associated with inherited cardiomyopathy, and it may be used for the risk evaluation of individuals with a possible susceptibility to inherited cardiomyopathy.

  19. Identification of new therapeutic targets by genome-wide analysis of gene expression in the ipsilateral cortex of aged rats after stroke.

    Directory of Open Access Journals (Sweden)

    Ana-Maria Buga

    Full Text Available Because most human stroke victims are elderly, studies of experimental stroke in the aged rather than the young rat model may be optimal for identifying clinically relevant cellular responses, as well for pinpointing beneficial interventions.We employed the Affymetrix platform to analyze the whole-gene transcriptome following temporary ligation of the middle cerebral artery in aged and young rats. The correspondence, heat map, and dendrogram analyses independently suggest a differential, age-group-specific behaviour of major gene clusters after stroke. Overall, the pattern of gene expression strongly suggests that the response of the aged rat brain is qualitatively rather than quantitatively different from the young, i.e. the total number of regulated genes is comparable in the two age groups, but the aged rats had great difficulty in mounting a timely response to stroke. Our study indicates that four genes related to neuropathic syndrome, stress, anxiety disorders and depression (Acvr1c, Cort, Htr2b and Pnoc may have impaired response to stroke in aged rats. New therapeutic options in aged rats may also include Calcrl, Cyp11b1, Prcp, Cebpa, Cfd, Gpnmb, Fcgr2b, Fcgr3a, Tnfrsf26, Adam 17 and Mmp14. An unexpected target is the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 in aged rats, a key enzyme in the cholesterol synthesis pathway. Post-stroke axonal growth was compromised in both age groups.We suggest that a multi-stage, multimodal treatment in aged animals may be more likely to produce positive results. Such a therapeutic approach should be focused on tissue restoration but should also address other aspects of patient post-stroke therapy such as neuropathic syndrome, stress, anxiety disorders, depression, neurotransmission and blood pressure.

  20. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine.

    Directory of Open Access Journals (Sweden)

    Yaffa Mizrachi Nebenzahl

    Full Text Available In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

  1. Identification of Splice Variants, Targeted MicroRNAs and Functional Single Nucleotide Polymorphisms of the BOLA-DQA2 Gene in Dairy Cattle

    Science.gov (United States)

    Hou, Qinlei; Huang, Jinming; Ju, Zhihua; Li, Qiuling; Li, Liming; Wang, Changfa; Sun, Tao; Wang, Lingling; Hou, Minghai

    2012-01-01

    Major histocompatibility complex, class II, DQ alpha 2, also named BOLA-DQA2, belongs to the Bovine Leukocyte Antigen (BOLA) class II genes which are involved in the immune response. To explore the variability of the BOLA-DQA2 gene and resistance to mastitis in cows, the splice variants (SV), targeted microRNAs (miRNAs), and single nucleotide polymorphisms (SNPs) were identified in this study. A new SV (BOLA-DQA2-SV1) lacking part of exon 3 (195 bp) and two 3′-untranslated regions (UTR) (52 bp+167 bp) of the BOLA-DQA2 gene was found in the healthy and mastitis-infected mammary gland tissues. Four of 13 new SNPs and multiple nucleotide polymorphisms resulted in amino acid changes in the protein and SNP (c. +1283 C>T) may affect the binding to the seed sequence of bta-miR-2318. Further, we detected the relative expressions of two BOLA-DQA2 transcripts and five candidated microRNAs binding to the 3′-UTR of two transcripts in the mammary gland tissues in dairy cattle by using the quantitative real-time polymerase chain reaction. The result showed that expression of the BOLA-DQA2-SV1 mRNA was significantly upregulated 2.67-fold (pmastitis-infected mammary tissues (n=5) compared with the healthy mammary gland mammary tissues (n=5). Except for bta-miR-1777a, miRNA expression (bta-miR-296, miR-2430, and miR-671) was upregulated 1.75 to 2.59-fold (pmastitis cows. Our findings reveal that BOLA-DQA2-SV1 may play an important role in the mastitis resistance in dairy cattle. Whether the SNPs affect the structure of the BOLA-DQA2 gene or association with mastitis resistance is unknown and warrants further investigation. PMID:22084936

  2. First time identification of Acanthamoeba genotypes in the cornea samples of wild birds; Is Acanthamoeba keratitis making the predatory birds a target?

    Science.gov (United States)

    Karakavuk, Muhammet; Aykur, Mehmet; Şahar, Esra Atalay; Karakuş, Mehmet; Aldemir, Duygu; Döndüren, Ömer; Özdemir, Hüseyin Gökhan; Can, Hüseyin; Gürüz, Adnan Yüksel; Dağcı, Hande; Döşkaya, Mert

    2017-12-01

    Acanthamoeba is a free-living amoeba which can be isolated from environment and among others well known as an opportunist protozoan parasite causing infections in humans and animals. Eyes are extremely important for the wild birds and losing sight ability due to Acanthamoeba can be dangerous. The studies on Acanthamoeba infection in wild birds is very few in world and Turkey therefore we aimed to screen deceased wild birds found in İzmir and Manisa provinces located in western Turkey using PCR and non-nutrition agar (NNA) plate method. Cornea samples were obtained from 18 deceased wild birds. During the external examination, signs of keratitis were observed in two Eurasian sparrowhawks (Accipiter nisus). All of the corneal samples were analyzed by two PCR methods and NNA plate. According to results, the Acanthamoeba positivity in corneal samples was 16.6% and 5.5% by PCR and plate method, respectively. According to sequencing data, two of isolates belonged to genotype T5 and one was genotype T4. In conclusion, Acanthamoeba infection was detected in wild bird cornea samples with/without keratitis for the first time in the world. The result of this study also show that Acanthamoeba can be a cause of keratitis in wild birds of Turkey and thus these predator birds can be a target of other wild animals due to loss of sight ability. In terms of public health, these results show the importance of wild birds as a source of Acanthamoeba infection in nature. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising target for combination therapy.

    Science.gov (United States)

    Yu, Huan; Ma, Meng; Yan, Junya; Xu, Longwen; Yu, Jiayi; Dai, Jie; Xu, Tianxiao; Tang, Huan; Wu, Xiaowen; Li, Siming; Lian, Bin; Mao, Lili; Chi, Zhihong; Cui, Chuanliang; Guo, Jun; Kong, Yan

    2017-12-04

    Coexistence of enhancer of zeste homolog 2 (EZH2) and BRAF gene aberrations has been described in many cancer types. In this study, we aim to explore the coexistence status of BRAF V600E mutation and the copy number variation of EZH2 and explore the potential of this combination as a therapeutic target. A total of 138 cases of melanoma samples harboring BRAF V600E mutation were included, and EZH2 copy numbers were examined by QuantiGenePlex DNA Assays. Clinical pathological distinction between patient groups with or without EZH2 amplification (hereafter referred to as EZH2 gain) was statistically analyzed. The sensitivity of melanoma cell lines and patient-derived xenograft (PDX) models containing BRAF V600E mutation with or without EZH2 gain to vemurafenib (BRAF inhibitor), GSK2816126 (EZH2 inhibitor) and a combination of both agents was evaluated. In our cohort, the coexistence rate of BRAF V600E mutation and EZH2 gain was up to 29.0%, and significant differences in overall survival and disease-free survival were found between no EZH2 copy number gain and gain groups (P = 0.038, P = 0.030), gain and high EZH2 copy number gain groups (P = 0.006, P = 0.010). Combination with BRAF and EZH2 inhibition showed better inhibitory efficacy in melanoma prevention compared with vemurafenib monotherapy. More importantly, this improved therapeutic effect was observed especially in melanoma cell lines and PDX models containing concurrently BRAF V600E mutation and EZH2 gain. Coexistence of BRAF V600E mutation and EZH2 gain is rather prevalent in melanoma. Our findings provided evidence for the feasibility of combination therapy with EZH2 and BRAF inhibitors in melanoma with concurrent BRAF V600E mutation and EZH2 gain.

  4. High-speed counter-current chromatography coupled online to high performance liquid chromatography-diode array detector-mass spectrometry for purification, analysis and identification of target compounds from natural products.

    Science.gov (United States)

    Liang, Xuejuan; Zhang, Yuping; Chen, Wei; Cai, Ping; Zhang, Shuihan; Chen, Xiaoqin; Shi, Shuyun

    2015-03-13

    A challenge in coupling high-speed counter-current chromatography (HSCCC) online with high performance liquid chromatography (HPLC) for purity analysis was their time incompatibility. Consequently, HSCCC-HPLC was conducted by either controlling HPLC analysis time and HSCCC flow rate or using stop-and-go scheme. For natural products containing compounds with a wide range of polarities, the former would optimize experimental conditions, while the latter required more time. Here, a novel HSCCC-HPLC-diode array detector-mass spectrometry (HSCCC-HPLC-DAD-MS) was developed for undisrupted purification, analysis and identification of multi-compounds from natural products. Two six-port injection valves and a six-port switching valve were used as interface for collecting key HSCCC effluents alternatively for HPLC-DAD-MS analysis and identification. The ethyl acetate extract of Malus doumeri was performed on the hyphenated system to verify its efficacy. Five main flavonoids, 3-hydroxyphloridzin (1), phloridzin (2), 4',6'-dihydroxyhydrochalcone-2'-O-β-D-glucopyranoside (3, first found in M. doumeri), phloretin (4), and chrysin (5), were purified with purities over 99% by extrusion elution and/or stepwise elution mode in two-step HSCCC, and 25mM ammonium acetate solution was selected instead of water to depress emulsification in the first HSCCC. The online system shortened manipulation time largely compared with off-line analysis procedure and stop-and-go scheme. The results indicated that the present method could serve as a simple, rapid and effective way to achieve target compounds with high purity from natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Identification of effective treatment criteria for use in targeted selective treatment programs to control haemonchosis in periparturient ewes in Ontario, Canada.

    Science.gov (United States)

    Westers, T; Jones-Bitton, A; Menzies, P; VanLeeuwen, J; Poljak, Z; Peregrine, A S

    2016-11-01

    Haemonchosis is often associated with late gestation and parturition in ewes in Canada. Due to widespread concerns about development of anthelmintic resistance (AR), targeted selective treatment (TST), where individual animals are treated with an anthelmintic rather than the entire flock, is a possible strategy to control clinical signs in recently lambed ewes while still maintaining parasite refugia. Performing fecal egg counts (FEC) on individual animals is often cost-prohibitive, so indicators that identify ewes with high FEC are essential for TST programs. The study objectives were to: a) evaluate the ability of four TST indicators to identify periparturient ewes with high Haemonchus sp. FEC and b) determine appropriate treatment thresholds for statistically-significant indicators. A field study was conducted during the 2013 and 2014 lambing seasons (February-May) on three client-owned farms in Ontario with documented AR and problems with haemonchosis in ewes. Ewes were examined within three days of lambing and selected for treatment with oral closantel (10mg/kg body weight), a novel anthelmintic to Canada, if they met at least one of four criteria: a) the last grazing season was their first grazing season; b) body condition score ≤2; c) Faffa Malan Chart (FAMACHA © ) score ≥3; and/or d) three or more nursing lambs. Fecal samples were collected per rectum on the treatment day from each of 20 randomly selected treated and untreated ewes on each farm. Haemonchus sp. percentages on each farm, as determined by coproculture, ranged from 53% to 92% of total fecal trichostrongyle-type egg counts. Mean Haemonchus sp. FECs were significantly higher in treated ewes (n=136) than in untreated ewes (n=103) on the day of treatment in both years (p=0.001), suggesting the indicators were suitable for identifying animals with high Haemonchus sp. FEC. A linear mixed model was fit with logarithmic-transformed Haemonchus sp. FEC as the outcome variable, the four indicators

  6. Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

    International Nuclear Information System (INIS)

    Josse, Rozenn; Dumont, Julie; Fautrel, Alain; Robin, Marie-Anne; Guillouzo, André

    2012-01-01

    Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds. -- Highlights: ► Gene expression profile changes induced by aflatoxin B1 in human hepatocytes. ► AFB1 modulates various genes including tumor suppressor genes and proto-oncogenes. ► Important inter-individual variations in the response to AFB1. ► Some genes also altered by other

  7. Bioinformatics for cancer immunotherapy target discovery

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Campos, Benito; Barnkob, Mike Stein

    2014-01-01

    therapy target discovery in a bioinformatics analysis pipeline. We describe specialized bioinformatics tools and databases for three main bottlenecks in immunotherapy target discovery: the cataloging of potentially antigenic proteins, the identification of potential HLA binders, and the selection epitopes...

  8. Targeted isolation and identification of bioactive compounds lowering cholesterol in the crude extracts of crabapples using UPLC-DAD-MS-SPE/NMR based on pharmacology-guided PLS-DA.

    Science.gov (United States)

    Wen, Chao; Wang, Dongshan; Li, Xing; Huang, Tao; Huang, Cheng; Hu, Kaifeng

    2018-02-20

    The anti-hyperlipidemic effects of crude crabapple extracts derived from Malus 'Red jade', Malus hupehensis (Pamp.) Rehd. and Malus prunifolia (Willd.) Borkh. were evaluated on high-fat diet induced obese (HF DIO) mice. The results revealed that some of these extracts could lower serum cholesterol levels in HF DIO mice. The same extracts were also parallelly analyzed by LC-MS in both positive and negative ionization modes. Based on the pharmacological results, 22 LC-MS variables were identified to be correlated with the anti-hyperlipidemic effects using partial least square discriminant analysis (PLS-DA) and independent samples t-test. Further, under the guidance of the bioactivity-correlated LC-MS signals, 10 compounds were targetedly isolated and enriched using UPLC-DAD-MS-SPE and identified/elucidated by NMR together with MS/MS as citric acid(1), p-coumaric acid(2), hyperoside(3), myricetin(4), naringenin(5), quercetin(6), kaempferol(7), gentiopicroside(8), ursolic acid(9) and 8-epiloganic acid(10). Among these 10 compounds, 6 compounds, hyperoside(3), myricetin(4), naringenin(5), quercetin(6), kaempferol(7) and ursolic acid(9), were individually studied and reported to indeed have effects on lowering the serum lipid levels. These results demonstrated the efficiency of this strategy for drug discovery. In contrast to traditional routes to discover bioactive compounds in the plant extracts, targeted isolation and identification of bioactive compounds in the crude plant extracts using UPLC-DAD-MS-SPE/NMR based on pharmacology-guided PLS-DA of LC-MS data brings forward a new efficient dereplicated approach to natural products research for drug discovery. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway

    Directory of Open Access Journals (Sweden)

    Jessica L. Bell

    2013-05-01

    Full Text Available CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identification, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain biological activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active competitor. Future work will focus on identifying the labeled protein(s.

  10. Targeted identification of Schistosoma mansoni egg glycans

    NARCIS (Netherlands)

    Robijn, Marjolein Louise Maria

    2008-01-01

    Schistosomiasis, also known as bilharzia, is a disease that still occurs in many parts of Africa, the Middle East and Southern America, mainly Brasil and the Caribbean. Schistosomiasis is, after malaria, the second most common parasitic infection. Currently 200 million people are infected with the

  11. Identification of the base-pairing requirements for repression of hctA translation by the small RNA IhtA leads to the discovery of a new mRNA target in Chlamydia trachomatis.

    Directory of Open Access Journals (Sweden)

    Nicole A Grieshaber

    Full Text Available The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen Chlamydia trachomatis modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of hctA, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of hctA. Two additional genes in the chlamydial genome, CTL0322 and CTL0097, contained some elements of the hctA:IhtA recognition sequence. The mRNA of both CTL0322and CTL0097 interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of CTL0322. The proposed IhtA recognition site in the CTL0322 message contains significant G/C base pairing on either side of the initiation codon while CTL0097 only contains G/C base pairing 3' to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle.

  12. CERN: Fixed target targets

    Energy Technology Data Exchange (ETDEWEB)

    Anon.

    1993-03-15

    Full text: While the immediate priority of CERN's research programme is to exploit to the full the world's largest accelerator, the LEP electron-positron collider and its concomitant LEP200 energy upgrade (January, page 1), CERN is also mindful of its long tradition of diversified research. Away from LEP and preparations for the LHC proton-proton collider to be built above LEP in the same 27-kilometre tunnel, CERN is also preparing for a new generation of heavy ion experiments using a new source, providing heavier ions (April 1992, page 8), with first physics expected next year. CERN's smallest accelerator, the LEAR Low Energy Antiproton Ring continues to cover a wide range of research topics, and saw a record number of hours of operation in 1992. The new ISOLDE on-line isotope separator was inaugurated last year (July, page 5) and physics is already underway. The remaining effort concentrates around fixed target experiments at the SPS synchrotron, which formed the main thrust of CERN's research during the late 1970s. With the SPS and LEAR now approaching middle age, their research future was extensively studied last year. Broadly, a vigorous SPS programme looks assured until at least the end of 1995. Decisions for the longer term future of the West Experimental Area of the SPS will have to take into account the heavy demand for test beams from work towards experiments at big colliders, both at CERN and elsewhere. The North Experimental Area is the scene of larger experiments with longer lead times. Several more years of LEAR exploitation are already in the pipeline, but for the longer term, the ambitious Superlear project for a superconducting ring (January 1992, page 7) did not catch on. Neutrino physics has a long tradition at CERN, and this continues with the preparations for two major projects, the Chorus and Nomad experiments (November 1991, page 7), to start next year in the West Area. Delicate neutrino oscillation effects could become visible for the first

  13. Bi-Spectral Method for Radar Target Recognition

    National Research Council Canada - National Science Library

    Yeo, Jiunn W

    2006-01-01

    .... Since World War II, the Identification of Friend or Foe (IFF) systems installed in radar systems have served as the primary cooperative target identification techniques based on the "question and answer" interrogation loop of unidentified aircraft...

  14. Automatic and rapid identification of glycopeptides by nano-UPLC-LTQ-FT-MS and proteomic search engine.

    Science.gov (United States)

    Giménez, Estela; Gay, Marina; Vilaseca, Marta

    2017-01-30

    Here we demonstrate the potential of nano-UPLC-LTQ-FT-MS and the Byonic™ proteomic search engine for the separation, detection, and identification of N- and O-glycopeptide glycoforms in standard glycoproteins. The use of a BEH C18 nanoACQUITY column allowed the separation of the glycopeptides present in the glycoprotein digest and a baseline-resolution of the glycoforms of the same glycopeptide on the basis of the number of sialic acids. Moreover, we evaluated several acquisition strategies in order to improve the detection and characterization of glycopeptide glycoforms with the maximum number of identification percentages. The proposed strategy is simple to set up with the technology platforms commonly used in proteomic labs. The method allows the straightforward and rapid obtention of a general glycosylated map of a given protein, including glycosites and their corresponding glycosylated structures. The MS strategy selected in this work, based on a gas phase fractionation approach, led to 136 unique peptides from four standard proteins, which represented 78% of the total number of peptides identified. Moreover, the method does not require an extra glycopeptide enrichment step, thus preventing the bias that this step could cause towards certain glycopeptide species. Data are available via ProteomeXchange with identifier PXD003578. We propose a simple and high-throughput glycoproteomics-based methodology that allows the separation of glycopeptide glycoforms on the basis of the number of sialic acids, and their automatic and rapid identification without prior knowledge of protein glycosites or type and structure of the glycans. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. CERN: Fixed target targets

    International Nuclear Information System (INIS)

    Anon.

    1993-01-01

    Full text: While the immediate priority of CERN's research programme is to exploit to the full the world's largest accelerator, the LEP electron-positron collider and its concomitant LEP200 energy upgrade (January, page 1), CERN is also mindful of its long tradition of diversified research. Away from LEP and preparations for the LHC proton-proton collider to be built above LEP in the same 27-kilometre tunnel, CERN is also preparing for a new generation of heavy ion experiments using a new source, providing heavier ions (April 1992, page 8), with first physics expected next year. CERN's smallest accelerator, the LEAR Low Energy Antiproton Ring continues to cover a wide range of research topics, and saw a record number of hours of operation in 1992. The new ISOLDE on-line isotope separator was inaugurated last year (July, page 5) and physics is already underway. The remaining effort concentrates around fixed target experiments at the SPS synchrotron, which formed the main thrust of CERN's research during the late 1970s. With the SPS and LEAR now approaching middle age, their research future was extensively studied last year. Broadly, a vigorous SPS program