Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition.

PubMed

Bahal, Raman; Quijano, Elias; McNeer, Nicole A; Liu, Yanfeng; Bhunia, Dinesh C; Lopez-Giraldez, Francesco; Fields, Rachel J; Saltzman, William M; Ly, Danith H; Glazer, Peter M

2014-01-01

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

A robust recognition and accurate locating method for circular coded diagonal target

NASA Astrophysics Data System (ADS)

Bao, Yunna; Shang, Yang; Sun, Xiaoliang; Zhou, Jiexin

2017-10-01

As a category of special control points which can be automatically identified, artificial coded targets have been widely developed in the field of computer vision, photogrammetry, augmented reality, etc. In this paper, a new circular coded target designed by RockeTech technology Corp. Ltd is analyzed and studied, which is called circular coded diagonal target (CCDT). A novel detection and recognition method with good robustness is proposed in the paper, and implemented on Visual Studio. In this algorithm, firstly, the ellipse features of the center circle are used for rough positioning. Then, according to the characteristics of the center diagonal target, a circular frequency filter is designed to choose the correct center circle and eliminates non-target noise. The precise positioning of the coded target is done by the correlation coefficient fitting extreme value method. Finally, the coded target recognition is achieved by decoding the binary sequence in the outer ring of the extracted target. To test the proposed algorithm, this paper has carried out simulation experiments and real experiments. The results show that the CCDT recognition and accurate locating method proposed in this paper can robustly recognize and accurately locate the targets in complex and noisy background.

A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

PubMed

Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

2016-06-15

The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

A universal entropy-driven mechanism for thioredoxin–target recognition

PubMed Central

Palde, Prakash B.; Carroll, Kate S.

2015-01-01

Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx–protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx–target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein–protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx–target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition. PMID:26080424

Toward a General Approach for RNA-Templated Hierarchical Assembly of Split-Proteins

PubMed Central

Furman, Jennifer L.; Badran, Ahmed H.; Ajulo, Oluyomi; Porter, Jason R.; Stains, Cliff I.; Segal, David J.; Ghosh, Indraneel

2010-01-01

The ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ssRNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3' overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target. PMID:20681585

Creation of a type IIS restriction endonuclease with a long recognition sequence

PubMed Central

Lippow, Shaun M.; Aha, Patti M.; Parker, Matthew H.; Blake, William J.; Baynes, Brian M.; Lipovšek, Daša

2009-01-01

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases. PMID:19304757

Attention-Based Recurrent Temporal Restricted Boltzmann Machine for Radar High Resolution Range Profile Sequence Recognition.

PubMed

Zhang, Yifan; Gao, Xunzhang; Peng, Xuan; Ye, Jiaqi; Li, Xiang

2018-05-16

The High Resolution Range Profile (HRRP) recognition has attracted great concern in the field of Radar Automatic Target Recognition (RATR). However, traditional HRRP recognition methods failed to model high dimensional sequential data efficiently and have a poor anti-noise ability. To deal with these problems, a novel stochastic neural network model named Attention-based Recurrent Temporal Restricted Boltzmann Machine (ARTRBM) is proposed in this paper. RTRBM is utilized to extract discriminative features and the attention mechanism is adopted to select major features. RTRBM is efficient to model high dimensional HRRP sequences because it can extract the information of temporal and spatial correlation between adjacent HRRPs. The attention mechanism is used in sequential data recognition tasks including machine translation and relation classification, which makes the model pay more attention to the major features of recognition. Therefore, the combination of RTRBM and the attention mechanism makes our model effective for extracting more internal related features and choose the important parts of the extracted features. Additionally, the model performs well with the noise corrupted HRRP data. Experimental results on the Moving and Stationary Target Acquisition and Recognition (MSTAR) dataset show that our proposed model outperforms other traditional methods, which indicates that ARTRBM extracts, selects, and utilizes the correlation information between adjacent HRRPs effectively and is suitable for high dimensional data or noise corrupted data.

Structural basis for microRNA targeting

DOE PAGES

Schirle, Nicole T.; Sheu-Gruttadauria, Jessica; MacRae, Ian J.

2014-10-31

MicroRNAs (miRNAs) control expression of thousands of genes in plants and animals. miRNAs function by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. In this paper, we determined crystal structures of human Argonaute-2 (Ago2) bound to a defined guide RNA with and without target RNAs representing miRNA recognition sites. These structures suggest a stepwise mechanism, in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing. Pairing to nt 2 to 5 promotes conformational changes that expose nt 2 to 8 and 13 to 16 for further target recognition. Interactions withmore » the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner, whereas an adenosine binding-pocket opposite guide nt 1 further facilitates target recognition. Spurious slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion. Finally, these results explain the conserved nucleotide-pairing patterns in animal miRNA target sites first observed over two decades ago.« less

Exploring the sequence-structure protein landscape in the glycosyltransferase family

PubMed Central

Zhang, Ziding; Kochhar, Sunil; Grigorov, Martin

2003-01-01

To understand the molecular basis of glycosyltransferases’ (GTFs) catalytic mechanism, extensive structural information is required. Here, fold recognition methods were employed to assign 3D protein shapes (folds) to the currently known GTF sequences, available in public databases such as GenBank and Swissprot. First, GTF sequences were retrieved and classified into clusters, based on sequence similarity only. Intracluster sequence similarity was chosen sufficiently high to ensure that the same fold is found within a given cluster. Then, a representative sequence from each cluster was selected to compose a subset of GTF sequences. The members of this reduced set were processed by three different fold recognition methods: 3D-PSSM, FUGUE, and GeneFold. Finally, the results from different fold recognition methods were analyzed and compared to sequence-similarity search methods (i.e., BLAST and PSI-BLAST). It was established that the folds of about 70% of all currently known GTF sequences can be confidently assigned by fold recognition methods, a value which is higher than the fold identification rate based on sequence comparison alone (48% for BLAST and 64% for PSI-BLAST). The identified folds were submitted to 3D clustering, and we found that most of the GTF sequences adopt the typical GTF A or GTF B folds. Our results indicate a lack of evidence that new GTF folds (i.e., folds other than GTF A and B) exist. Based on cases where fold identification was not possible, we suggest several sequences as the most promising targets for a structural genomics initiative focused on the GTF protein family. PMID:14500887

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

PubMed Central

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

2010-01-01

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

Recognition of Double Stranded RNA by Guanidine-Modified Peptide Nucleic Acids (GPNA)

PubMed Central

Gupta, Pankaj; Muse, Oluwatoyosi; Rozners, Eriks

2011-01-01

Double helical RNA has become an attractive target for molecular recognition because many non-coding RNAs play important roles in control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double helical RNA via triple helix formation. Herein we tested if the molecular recognition of RNA can be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-workers that the guanidine modification greatly enhances the cellular delivery of PNA. Isothermal titration calorimetry showed that the guanidine-modified PNA (GPNA) had reduced affinity and sequence selectivity for triple helical recognition of RNA. The data suggested that in contrast to unmodified PNA, which formed a 1:1 PNA-RNA triple helix, GPNA preferred a 2:1 GPNA-RNA triplex-invasion complex. Nevertheless, promising results were obtained for recognition of biologically relevant double helical RNA. Consistent with enhanced strand invasion ability, GPNA derived from D-arginine recognized the transactivation response element (TAR) of HIV-1 with high affinity and sequence selectivity, presumably via Watson-Crick duplex formation. On the other hand, strong and sequence selective triple helices were formed by unmodified and nucelobase-modified PNAs and the purine rich strand of bacterial A-site. These results suggest that appropriate chemical modifications of PNA may enhance molecular recognition of complex non-coding RNAs. PMID:22146072

Combining high-speed SVM learning with CNN feature encoding for real-time target recognition in high-definition video for ISR missions

NASA Astrophysics Data System (ADS)

Kroll, Christine; von der Werth, Monika; Leuck, Holger; Stahl, Christoph; Schertler, Klaus

2017-05-01

For Intelligence, Surveillance, Reconnaissance (ISR) missions of manned and unmanned air systems typical electrooptical payloads provide high-definition video data which has to be exploited with respect to relevant ground targets in real-time by automatic/assisted target recognition software. Airbus Defence and Space is developing required technologies for real-time sensor exploitation since years and has combined the latest advances of Deep Convolutional Neural Networks (CNN) with a proprietary high-speed Support Vector Machine (SVM) learning method into a powerful object recognition system with impressive results on relevant high-definition video scenes compared to conventional target recognition approaches. This paper describes the principal requirements for real-time target recognition in high-definition video for ISR missions and the Airbus approach of combining an invariant feature extraction using pre-trained CNNs and the high-speed training and classification ability of a novel frequency-domain SVM training method. The frequency-domain approach allows for a highly optimized implementation for General Purpose Computation on a Graphics Processing Unit (GPGPU) and also an efficient training of large training samples. The selected CNN which is pre-trained only once on domain-extrinsic data reveals a highly invariant feature extraction. This allows for a significantly reduced adaptation and training of the target recognition method for new target classes and mission scenarios. A comprehensive training and test dataset was defined and prepared using relevant high-definition airborne video sequences. The assessment concept is explained and performance results are given using the established precision-recall diagrams, average precision and runtime figures on representative test data. A comparison to legacy target recognition approaches shows the impressive performance increase by the proposed CNN+SVM machine-learning approach and the capability of real-time high-definition video exploitation.

TIA-1 RRM23 binding and recognition of target oligonucleotides

PubMed Central

Waris, Saboora; García-Mauriño, Sofía M.; Sivakumaran, Andrew; Beckham, Simone A.; Loughlin, Fionna E.; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C.J.

2017-01-01

Abstract TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. PMID:28184449

TIA-1 RRM23 binding and recognition of target oligonucleotides.

PubMed

Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

2017-05-05

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

One-Shot Learning of Human Activity With an MAP Adapted GMM and Simplex-HMM.

PubMed

Rodriguez, Mario; Orrite, Carlos; Medrano, Carlos; Makris, Dimitrios

2016-05-10

This paper presents a novel activity class representation using a single sequence for training. The contribution of this representation lays on the ability to train an one-shot learning recognition system, useful in new scenarios where capturing and labeling sequences is expensive or impractical. The method uses a universal background model of local descriptors obtained from source databases available on-line and adapts it to a new sequence in the target scenario through a maximum a posteriori adaptation. Each activity sample is encoded in a sequence of normalized bag of features and modeled by a new hidden Markov model formulation, where the expectation-maximization algorithm for training is modified to deal with observations consisting in vectors in a unit simplex. Extensive experiments in recognition have been performed using one-shot learning over the public datasets Weizmann, KTH, and IXMAS. These experiments demonstrate the discriminative properties of the representation and the validity of application in recognition systems, achieving state-of-the-art results.

Dynamic Gesture Recognition with a Terahertz Radar Based on Range Profile Sequences and Doppler Signatures

PubMed Central

Pi, Yiming

2017-01-01

The frequency of terahertz radar ranges from 0.1 THz to 10 THz, which is higher than that of microwaves. Multi-modal signals, including high-resolution range profile (HRRP) and Doppler signatures, can be acquired by the terahertz radar system. These two kinds of information are commonly used in automatic target recognition; however, dynamic gesture recognition is rarely discussed in the terahertz regime. In this paper, a dynamic gesture recognition system using a terahertz radar is proposed, based on multi-modal signals. The HRRP sequences and Doppler signatures were first achieved from the radar echoes. Considering the electromagnetic scattering characteristics, a feature extraction model is designed using location parameter estimation of scattering centers. Dynamic Time Warping (DTW) extended to multi-modal signals is used to accomplish the classifications. Ten types of gesture signals, collected from a terahertz radar, are applied to validate the analysis and the recognition system. The results of the experiment indicate that the recognition rate reaches more than 91%. This research verifies the potential applications of dynamic gesture recognition using a terahertz radar. PMID:29267249

Dynamic Gesture Recognition with a Terahertz Radar Based on Range Profile Sequences and Doppler Signatures.

PubMed

Zhou, Zhi; Cao, Zongjie; Pi, Yiming

2017-12-21

The frequency of terahertz radar ranges from 0.1 THz to 10 THz, which is higher than that of microwaves. Multi-modal signals, including high-resolution range profile (HRRP) and Doppler signatures, can be acquired by the terahertz radar system. These two kinds of information are commonly used in automatic target recognition; however, dynamic gesture recognition is rarely discussed in the terahertz regime. In this paper, a dynamic gesture recognition system using a terahertz radar is proposed, based on multi-modal signals. The HRRP sequences and Doppler signatures were first achieved from the radar echoes. Considering the electromagnetic scattering characteristics, a feature extraction model is designed using location parameter estimation of scattering centers. Dynamic Time Warping (DTW) extended to multi-modal signals is used to accomplish the classifications. Ten types of gesture signals, collected from a terahertz radar, are applied to validate the analysis and the recognition system. The results of the experiment indicate that the recognition rate reaches more than 91%. This research verifies the potential applications of dynamic gesture recognition using a terahertz radar.

A bottom-up model of spatial attention predicts human error patterns in rapid scene recognition.

PubMed

Einhäuser, Wolfgang; Mundhenk, T Nathan; Baldi, Pierre; Koch, Christof; Itti, Laurent

2007-07-20

Humans demonstrate a peculiar ability to detect complex targets in rapidly presented natural scenes. Recent studies suggest that (nearly) no focal attention is required for overall performance in such tasks. Little is known, however, of how detection performance varies from trial to trial and which stages in the processing hierarchy limit performance: bottom-up visual processing (attentional selection and/or recognition) or top-down factors (e.g., decision-making, memory, or alertness fluctuations)? To investigate the relative contribution of these factors, eight human observers performed an animal detection task in natural scenes presented at 20 Hz. Trial-by-trial performance was highly consistent across observers, far exceeding the prediction of independent errors. This consistency demonstrates that performance is not primarily limited by idiosyncratic factors but by visual processing. Two statistical stimulus properties, contrast variation in the target image and the information-theoretical measure of "surprise" in adjacent images, predict performance on a trial-by-trial basis. These measures are tightly related to spatial attention, demonstrating that spatial attention and rapid target detection share common mechanisms. To isolate the causal contribution of the surprise measure, eight additional observers performed the animal detection task in sequences that were reordered versions of those all subjects had correctly recognized in the first experiment. Reordering increased surprise before and/or after the target while keeping the target and distractors themselves unchanged. Surprise enhancement impaired target detection in all observers. Consequently, and contrary to several previously published findings, our results demonstrate that attentional limitations, rather than target recognition alone, affect the detection of targets in rapidly presented visual sequences.

CRISPRTarget

PubMed Central

Biswas, Ambarish; Gagnon, Joshua N.; Brouns, Stan J.J.; Fineran, Peter C.; Brown, Chris M.

2013-01-01

The bacterial and archaeal CRISPR/Cas adaptive immune system targets specific protospacer nucleotide sequences in invading organisms. This requires base pairing between processed CRISPR RNA and the target protospacer. For type I and II CRISPR/Cas systems, protospacer adjacent motifs (PAM) are essential for target recognition, and for type III, mismatches in the flanking sequences are important in the antiviral response. In this study, we examine the properties of each class of CRISPR. We use this information to provide a tool (CRISPRTarget) that predicts the most likely targets of CRISPR RNAs (http://bioanalysis.otago.ac.nz/CRISPRTarget). This can be used to discover targets in newly sequenced genomic or metagenomic data. To test its utility, we discover features and targets of well-characterized Streptococcus thermophilus and Sulfolobus solfataricus type II and III CRISPR/Cas systems. Finally, in Pectobacterium species, we identify new CRISPR targets and propose a model of temperate phage exposure and subsequent inhibition by the type I CRISPR/Cas systems. PMID:23492433

PNA containing isocytidine nucleobase: synthesis and recognition of double helical RNA

PubMed Central

Zengeya, Thomas; Li, Ming; Rozners, Eriks

2011-01-01

Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Triple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was studied using isothermal titration calorimetry. The iC nucleobase recognized the proposed target, C-G inversion in polypurine tract of RNA, with slightly higher affinity than the natural nucleobases, though the sequence selectivity of recognition was low. Compared to non-modified PNA, PNA1 had lower affinity for its RNA target. PMID:21333533

Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

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

Joshi, Rakesh; Ho, Kwok Ki; Tenney, Kristen

2013-09-18

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{submore » +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.« less

p53 Specifically Binds Triplex DNA In Vitro and in Cells

PubMed Central

Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L.; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

2016-01-01

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed. PMID:27907175

Reconsidering the role of temporal order in spoken word recognition.

PubMed

Toscano, Joseph C; Anderson, Nathaniel D; McMurray, Bob

2013-10-01

Models of spoken word recognition assume that words are represented as sequences of phonemes. We evaluated this assumption by examining phonemic anadromes, words that share the same phonemes but differ in their order (e.g., sub and bus). Using the visual-world paradigm, we found that listeners show more fixations to anadromes (e.g., sub when bus is the target) than to unrelated words (well) and to words that share the same vowel but not the same set of phonemes (sun). This contrasts with the predictions of existing models and suggests that words are not defined as strict sequences of phonemes.

Fibronectin tetrapeptide is target for syphilis spirochete cytadherence

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

Thomas, D.D.; Baseman, J.B.; Alderete, J.F.

1985-11-01

The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with SVI-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitismmore » of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.« less

Enzyme-triggered Gelation: Targeting Proteases with Internal Cleavage Sites

PubMed Central

Bremmer, Steven C.

2014-01-01

A generalizable method for detecting protease activity via gelation is described. A recognition sequence is used to target the protease of interest while a second protease is used to remove the residual residues from the gelator scaffold. Using this approach, selective assays for both MMP-9 and PSA are demonstrated. PMID:24394494

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

PubMed

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

NASA Astrophysics Data System (ADS)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Methods and compositions for controlling gene expression by RNA processing

DOEpatents

Doudna, Jennifer A.; Qi, Lei S.; Haurwitz, Rachel E.; Arkin, Adam P.

2017-08-29

The present disclosure provides nucleic acids encoding an RNA recognition sequence positioned proximal to an insertion site for the insertion of a sequence of interest; and host cells genetically modified with the nucleic acids. The present disclosure also provides methods of modifying the activity of a target RNA, and kits and compositions for carrying out the methods.

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition.

PubMed Central

Barnes, W M; Bevan, M

1983-01-01

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels. Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA. Images PMID:6298723

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI

PubMed Central

Kasarjian, Julie K. A.; Hidaka, Masumi; Horiuchi, Takashi; Iida, Masatake; Ryu, Junichi

2004-01-01

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT). PMID:15199175

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

PubMed

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

2013-04-03

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

Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation

PubMed Central

Li, Ming; Wang, Rui; Xiang, Hua

2014-01-01

The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring ‘self’ DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition. PMID:24803673

Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes.

PubMed Central

Matter, A

1979-01-01

A study was carried out to determine the sequence of events of T-cell mediated target cell lysis in microcinematography and electron microscopy. Highly efficient cytotoxic T lymphocytes (CTL) were generated in vivo and in vitro using preimmunized spleen cells and purification procedures. Such CTL were highly specific. This specificity correlated well with the number of adhesions formed between CTL and targets and this criterion was used to study killer-target cell interaction. Microcinematography showed that target cell lysis at the single cell level, despite time variations, could be clearly separated into three phases: (a) a recognition phase, visible by random crawling of CTL over the target cell surface until firm contact was established; (b) a post-recognition phase, during which firm contact between CTL and target was maintained without gross modification of either cell; (c) a phase of target cell disintegration, mainly characterized by vigorous blebbing of the cell membrane resulting in a motionless carcass of the target cell but not in its total dissolution. Only later this carcass decayed and formed a necrotic ghost. Electron microscopic observations were put into sequence according to microcinematography. Post-recognition phase was characterized by a tight apposition of the membranes of CTL and target cell. No gap junctions could be observed. During target cell disintegration, profound cytoplasmic and nuclear changes occurred simultaneous with surface blebbing. Most noticeable were extensive internal vacuolization, mitochondrial swelling, nuclear pycnosis and dissolution of the nucleolus. These observations suggested that target cell lysis does not start with a surface phenomenon similar to complement lysis, but a process involving practically the whole cell simultaneously. It is conceivable, therefore, that the signal from the CTL is transmitted across the target cell, and that the switch to sudden cell death is manipulated deep inside the cell. Images Figure 3 Figures 4-7 Figures 8-11 Figure 12 Figures 13-14 Figure 15 PMID:312256

TCRmodel: high resolution modeling of T cell receptors from sequence.

PubMed

Gowthaman, Ragul; Pierce, Brian G

2018-05-22

T cell receptors (TCRs), along with antibodies, are responsible for specific antigen recognition in the adaptive immune response, and millions of unique TCRs are estimated to be present in each individual. Understanding the structural basis of TCR targeting has implications in vaccine design, autoimmunity, as well as T cell therapies for cancer. Given advances in deep sequencing leading to immune repertoire-level TCR sequence data, fast and accurate modeling methods are needed to elucidate shared and unique 3D structural features of these molecules which lead to their antigen targeting and cross-reactivity. We developed a new algorithm in the program Rosetta to model TCRs from sequence, and implemented this functionality in a web server, TCRmodel. This web server provides an easy to use interface, and models are generated quickly that users can investigate in the browser and download. Benchmarking of this method using a set of nonredundant recently released TCR crystal structures shows that models are accurate and compare favorably to models from another available modeling method. This server enables the community to obtain insights into TCRs of interest, and can be combined with methods to model and design TCR recognition of antigens. The TCRmodel server is available at: http://tcrmodel.ibbr.umd.edu/.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Helix Unwinding and Base Flipping Enable Human MTERF1 to Terminate Mitochondrial Transcription

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

Yakubovskaya, E.; Mejia, E; Byrnes, J

2010-01-01

Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that themore » mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.« less

Simian T Lymphotropic Virus 1 Infection of Papio anubis: tax Sequence Heterogeneity and T Cell Recognition.

PubMed

Termini, James M; Magnani, Diogo M; Maxwell, Helen S; Lauer, William; Castro, Iris; Pecotte, Jerilyn; Barber, Glen N; Watkins, David I; Desrosiers, Ronald C

2017-10-15

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8 + T cell recognition were not observed, premature stop codons were observed in 7% and 56% of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752, respectively. IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts. Copyright © 2017 American Society for Microbiology.

LiveBench-1: continuous benchmarking of protein structure prediction servers.

PubMed

Bujnicki, J M; Elofsson, A; Fischer, D; Rychlewski, L

2001-02-01

We present a novel, continuous approach aimed at the large-scale assessment of the performance of available fold-recognition servers. Six popular servers were investigated: PDB-Blast, FFAS, T98-lib, GenTHREADER, 3D-PSSM, and INBGU. The assessment was conducted using as prediction targets a large number of selected protein structures released from October 1999 to April 2000. A target was selected if its sequence showed no significant similarity to any of the proteins previously available in the structural database. Overall, the servers were able to produce structurally similar models for one-half of the targets, but significantly accurate sequence-structure alignments were produced for only one-third of the targets. We further classified the targets into two sets: easy and hard. We found that all servers were able to find the correct answer for the vast majority of the easy targets if a structurally similar fold was present in the server's fold libraries. However, among the hard targets--where standard methods such as PSI-BLAST fail--the most sensitive fold-recognition servers were able to produce similar models for only 40% of the cases, half of which had a significantly accurate sequence-structure alignment. Among the hard targets, the presence of updated libraries appeared to be less critical for the ranking. An "ideally combined consensus" prediction, where the results of all servers are considered, would increase the percentage of correct assignments by 50%. Each server had a number of cases with a correct assignment, where the assignments of all the other servers were wrong. This emphasizes the benefits of considering more than one server in difficult prediction tasks. The LiveBench program (http://BioInfo.PL/LiveBench) is being continued, and all interested developers are cordially invited to join.

Prediction of TF target sites based on atomistic models of protein-DNA complexes

PubMed Central

Angarica, Vladimir Espinosa; Pérez, Abel González; Vasconcelos, Ana T; Collado-Vides, Julio; Contreras-Moreira, Bruno

2008-01-01

Background The specific recognition of genomic cis-regulatory elements by transcription factors (TFs) plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence. Results Here we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models. Conclusion Our results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition. PMID:18922190

Trends in Correlation-Based Pattern Recognition and Tracking in Forward-Looking Infrared Imagery

PubMed Central

Alam, Mohammad S.; Bhuiyan, Sharif M. A.

2014-01-01

In this paper, we review the recent trends and advancements on correlation-based pattern recognition and tracking in forward-looking infrared (FLIR) imagery. In particular, we discuss matched filter-based correlation techniques for target detection and tracking which are widely used for various real time applications. We analyze and present test results involving recently reported matched filters such as the maximum average correlation height (MACH) filter and its variants, and distance classifier correlation filter (DCCF) and its variants. Test results are presented for both single/multiple target detection and tracking using various real-life FLIR image sequences. PMID:25061840

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

PubMed Central

Ullers, Ronald S.; Houben, Edith N.G.; Raine, Amanda; ten Hagen-Jongman, Corinne M.; Ehrenberg, Måns; Brunner, Joseph; Oudega, Bauke; Harms, Nellie; Luirink, Joen

2003-01-01

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain. PMID:12756233

Factors influencing the specific interaction of Neisseria gonorrhoeae with transforming DNA.

PubMed Central

Goodman, S D; Scocca, J J

1991-01-01

The specific interaction of transformable Neisseria gonorrhoeae with DNA depends on the recognition of specific 10-residue target sequences. The relative affinity for DNA between 3 and 17 kb in size appears to be linearly related to the frequency of targets on the segment and is unaffected by absolute size. The average frequency of targets in chromosomal DNA of N. gonorrhoeae appears to be approximately one per 1,000 bp. PMID:1909325

An SRY mutation causing human sex reversal resolves a general mechanism of structure-specific DNA recognition: application to the four-way DNA junction.

PubMed

Peters, R; King, C Y; Ukiyama, E; Falsafi, S; Donahoe, P K; Weiss, M A

1995-04-11

SRY, a genetic "master switch" for male development in mammals, exhibits two biochemical activities: sequence-specific recognition of duplex DNA and sequence-independent binding to the sharp angles of four-way DNA junctions. Here, we distinguish between these activities by analysis of a mutant SRY associated with human sex reversal (46, XY female with pure gonadal dysgenesis). The substitution (168T in human SRY) alters a nonpolar side chain in the minor-groove DNA recognition alpha-helix of the HMG box [Haqq, C.M., King, C.-Y., Ukiyama, E., Haqq, T.N., Falsalfi, S., Donahoe, P.K., & Weiss, M.A. (1994) Science 266, 1494-1500]. The native (but not mutant) side chain inserts between specific base pairs in duplex DNA, interrupting base stacking at a site of induced DNA bending. Isotope-aided 1H-NMR spectroscopy demonstrates that analogous side-chain insertion occurs on binding of SRY to a four-way junction, establishing a shared mechanism of sequence- and structure-specific DNA binding. Although the mutant DNA-binding domain exhibits > 50-fold reduction in sequence-specific DNA recognition, near wild-type affinity for four-way junctions is retained. Our results (i) identify a shared SRY-DNA contact at a site of either induced or intrinsic DNA bending, (ii) demonstrate that this contact is not required to bind an intrinsically bent DNA target, and (iii) rationalize patterns of sequence conservation or diversity among HMG boxes. Clinical association of the I68T mutation with human sex reversal supports the hypothesis that specific DNA recognition by SRY is required for male sex determination.

PHYSICAL MODEL FOR RECOGNITION TUNNELING

PubMed Central

Krstić, Predrag; Ashcroft, Brian; Lindsay, Stuart

2015-01-01

Recognition tunneling (RT) identifies target molecules trapped between tunneling electrodes functionalized with recognition molecules that serve as specific chemical linkages between the metal electrodes and the trapped target molecule. Possible applications include single molecule DNA and protein sequencing. This paper addresses several fundamental aspects of RT by multiscale theory, applying both all-atom and coarse-grained DNA models: (1) We show that the magnitude of the observed currents are consistent with the results of non-equilibrium Green's function calculations carried out on a solvated all-atom model. (2) Brownian fluctuations in hydrogen bond-lengths lead to current spikes that are similar to what is observed experimentally. (3) The frequency characteristics of these fluctuations can be used to identify the trapped molecules with a machine-learning algorithm, giving a theoretical underpinning to this new method of identifying single molecule signals. PMID:25650375

Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

PubMed

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Bruhn, Laurakay; Dellinger, Douglas J

2018-01-25

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

TALE-PvuII fusion proteins--novel tools for gene targeting.

PubMed

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

PubMed Central

Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

2016-01-01

CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

Identification of novel antibody-reactive detection sites for comprehensive gluten monitoring.

PubMed

Röckendorf, Niels; Meckelein, Barbara; Scherf, Katharina A; Schalk, Kathrin; Koehler, Peter; Frey, Andreas

2017-01-01

Certain cereals like wheat, rye or barley contain gluten, a protein mixture that can trigger celiac disease (CD). To make gluten-free diets available for affected individuals the gluten content of foodstuff must be monitored. For this purpose, antibody-based assays exist which rely on the recognition of certain linear gluten sequence motifs. Yet, not all CD-active gluten constituents and fragments formed during food processing/fermentation may be covered by those tests. In this study, we therefore assayed the coverage of reportedly CD-active gluten components by currently available detection antibodies and determined the antibody-inducing capacity of wheat gluten constituents in order to provide novel diagnostic targets for comprehensive gluten quantitation. Immunizations of outbred mice with purified gliadins and glutenins were conducted and the linear target recognition profile of the sera was recorded using synthetic peptide arrays that covered the sequence space of gluten constituents present in those preparations. The resulting murine immunorecognition profile of gluten demonstrated that further linear binding sites beyond those recognized by the monoclonal antibodies α20, R5 and G12 exist and may be exploitable as diagnostic targets. We conclude that the safety of foodstuffs for CD patients can be further improved by complementing current tests with antibodies directed against additional CD-active gluten components. Currently unrepresented linear gluten detection sites in glutenins and α-gliadins suggest sequences QQQYPS, PQQSFP, QPGQGQQG and QQPPFS as novel targets for antibody generation.

Identification of novel antibody-reactive detection sites for comprehensive gluten monitoring

PubMed Central

Röckendorf, Niels; Meckelein, Barbara; Scherf, Katharina A.; Schalk, Kathrin; Koehler, Peter

2017-01-01

Certain cereals like wheat, rye or barley contain gluten, a protein mixture that can trigger celiac disease (CD). To make gluten-free diets available for affected individuals the gluten content of foodstuff must be monitored. For this purpose, antibody-based assays exist which rely on the recognition of certain linear gluten sequence motifs. Yet, not all CD-active gluten constituents and fragments formed during food processing/fermentation may be covered by those tests. In this study, we therefore assayed the coverage of reportedly CD-active gluten components by currently available detection antibodies and determined the antibody-inducing capacity of wheat gluten constituents in order to provide novel diagnostic targets for comprehensive gluten quantitation. Immunizations of outbred mice with purified gliadins and glutenins were conducted and the linear target recognition profile of the sera was recorded using synthetic peptide arrays that covered the sequence space of gluten constituents present in those preparations. The resulting murine immunorecognition profile of gluten demonstrated that further linear binding sites beyond those recognized by the monoclonal antibodies α20, R5 and G12 exist and may be exploitable as diagnostic targets. We conclude that the safety of foodstuffs for CD patients can be further improved by complementing current tests with antibodies directed against additional CD-active gluten components. Currently unrepresented linear gluten detection sites in glutenins and α-gliadins suggest sequences QQQYPS, PQQSFP, QPGQGQQG and QQPPFS as novel targets for antibody generation. PMID:28759621

SOVEREIGN: An autonomous neural system for incrementally learning planned action sequences to navigate towards a rewarded goal.

PubMed

Gnadt, William; Grossberg, Stephen

2008-06-01

How do reactive and planned behaviors interact in real time? How are sequences of such behaviors released at appropriate times during autonomous navigation to realize valued goals? Controllers for both animals and mobile robots, or animats, need reactive mechanisms for exploration, and learned plans to reach goal objects once an environment becomes familiar. The SOVEREIGN (Self-Organizing, Vision, Expectation, Recognition, Emotion, Intelligent, Goal-oriented Navigation) animat model embodies these capabilities, and is tested in a 3D virtual reality environment. SOVEREIGN includes several interacting subsystems which model complementary properties of cortical What and Where processing streams and which clarify similarities between mechanisms for navigation and arm movement control. As the animat explores an environment, visual inputs are processed by networks that are sensitive to visual form and motion in the What and Where streams, respectively. Position-invariant and size-invariant recognition categories are learned by real-time incremental learning in the What stream. Estimates of target position relative to the animat are computed in the Where stream, and can activate approach movements toward the target. Motion cues from animat locomotion can elicit head-orienting movements to bring a new target into view. Approach and orienting movements are alternately performed during animat navigation. Cumulative estimates of each movement are derived from interacting proprioceptive and visual cues. Movement sequences are stored within a motor working memory. Sequences of visual categories are stored in a sensory working memory. These working memories trigger learning of sensory and motor sequence categories, or plans, which together control planned movements. Predictively effective chunk combinations are selectively enhanced via reinforcement learning when the animat is rewarded. Selected planning chunks effect a gradual transition from variable reactive exploratory movements to efficient goal-oriented planned movement sequences. Volitional signals gate interactions between model subsystems and the release of overt behaviors. The model can control different motor sequences under different motivational states and learns more efficient sequences to rewarded goals as exploration proceeds.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli.

PubMed

Bernheim, Aude G; Libis, Vincent K; Lindner, Ariel B; Wintermute, Edwin H

2016-03-20

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Deep sequencing methods for protein engineering and design.

PubMed

Wrenbeck, Emily E; Faber, Matthew S; Whitehead, Timothy A

2017-08-01

The advent of next-generation sequencing (NGS) has revolutionized protein science, and the development of complementary methods enabling NGS-driven protein engineering have followed. In general, these experiments address the functional consequences of thousands of protein variants in a massively parallel manner using genotype-phenotype linked high-throughput functional screens followed by DNA counting via deep sequencing. We highlight the use of information rich datasets to engineer protein molecular recognition. Examples include the creation of multiple dual-affinity Fabs targeting structurally dissimilar epitopes and engineering of a broad germline-targeted anti-HIV-1 immunogen. Additionally, we highlight the generation of enzyme fitness landscapes for conducting fundamental studies of protein behavior and evolution. We conclude with discussion of technological advances. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

PubMed Central

Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

Bio-recognitive photonics of a DNA-guided organic semiconductor

PubMed Central

Back, Seung Hyuk; Park, Jin Hyuk; Cui, Chunzhi; Ahn, Dong June

2016-01-01

Incorporation of duplex DNA with higher molecular weights has attracted attention for a new opportunity towards a better organic light-emitting diode (OLED) capability. However, biological recognition by OLED materials is yet to be addressed. In this study, specific oligomeric DNA–DNA recognition is successfully achieved by tri (8-hydroxyquinoline) aluminium (Alq3), an organic semiconductor. Alq3 rods crystallized with guidance from single-strand DNA molecules show, strikingly, a unique distribution of the DNA molecules with a shape of an ‘inverted' hourglass. The crystal's luminescent intensity is enhanced by 1.6-fold upon recognition of the perfect-matched target DNA sequence, but not in the case of a single-base mismatched one. The DNA–DNA recognition forming double-helix structure is identified to occur only in the rod's outer periphery. This study opens up new opportunities of Alq3, one of the most widely used OLED materials, enabling biological recognition. PMID:26725969

Bio-recognitive photonics of a DNA-guided organic semiconductor.

PubMed

Back, Seung Hyuk; Park, Jin Hyuk; Cui, Chunzhi; Ahn, Dong June

2016-01-04

Incorporation of duplex DNA with higher molecular weights has attracted attention for a new opportunity towards a better organic light-emitting diode (OLED) capability. However, biological recognition by OLED materials is yet to be addressed. In this study, specific oligomeric DNA-DNA recognition is successfully achieved by tri (8-hydroxyquinoline) aluminium (Alq3), an organic semiconductor. Alq3 rods crystallized with guidance from single-strand DNA molecules show, strikingly, a unique distribution of the DNA molecules with a shape of an 'inverted' hourglass. The crystal's luminescent intensity is enhanced by 1.6-fold upon recognition of the perfect-matched target DNA sequence, but not in the case of a single-base mismatched one. The DNA-DNA recognition forming double-helix structure is identified to occur only in the rod's outer periphery. This study opens up new opportunities of Alq3, one of the most widely used OLED materials, enabling biological recognition.

Bio-recognitive photonics of a DNA-guided organic semiconductor

NASA Astrophysics Data System (ADS)

Back, Seung Hyuk; Park, Jin Hyuk; Cui, Chunzhi; Ahn, Dong June

2016-01-01

Incorporation of duplex DNA with higher molecular weights has attracted attention for a new opportunity towards a better organic light-emitting diode (OLED) capability. However, biological recognition by OLED materials is yet to be addressed. In this study, specific oligomeric DNA-DNA recognition is successfully achieved by tri (8-hydroxyquinoline) aluminium (Alq3), an organic semiconductor. Alq3 rods crystallized with guidance from single-strand DNA molecules show, strikingly, a unique distribution of the DNA molecules with a shape of an `inverted' hourglass. The crystal's luminescent intensity is enhanced by 1.6-fold upon recognition of the perfect-matched target DNA sequence, but not in the case of a single-base mismatched one. The DNA-DNA recognition forming double-helix structure is identified to occur only in the rod's outer periphery. This study opens up new opportunities of Alq3, one of the most widely used OLED materials, enabling biological recognition.

Mechanism of foreign DNA selection in a bacterial adaptive immune system

PubMed Central

Sashital, Dipali G.; Wiedenheft, Blake; Doudna, Jennifer A.

2012-01-01

Summary In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary “non-self” DNA sequences for destruction, while avoiding binding to “self” sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for non-self target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved 3-base pair motif that is required for non-self target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade. PMID:22521690

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

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

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

PubMed

Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

2014-04-01

The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

Thermodynamics of DNA target site recognition by homing endonucleases

PubMed Central

Eastberg, Jennifer H.; Smith, Audrey McConnell; Zhao, Lei; Ashworth, Justin; Shen, Betty W.; Stoddard, Barry L.

2007-01-01

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of ΔH and TΔS are not strongly correlated with the overall extent of DNA bending, unfavorable ΔHbinding is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific ΔSbinding. PMID:17947319

TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

PubMed Central

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

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

2011-01-01

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

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site.

PubMed

Wu, Fei; Shao, Yong; Ma, Kun; Cui, Qinghua; Liu, Guiying; Xu, Shujuan

2012-04-28

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe. This journal is © The Royal Society of Chemistry 2012

Interactions between the R2R3-MYB Transcription Factor, AtMYB61, and Target DNA Binding Sites

PubMed Central

Prouse, Michael B.; Campbell, Malcolm M.

2013-01-01

Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators. PMID:23741471

Enzyme-free homogeneous electrochemical biosensor for DNA assay using toehold-triggered strand displacement reaction coupled with host-guest recognition of Fe3O4@SiO2@β-CD nanocomposites.

PubMed

Jiang, Jingjing; Lin, Xinyi; Ding, Dong; Diao, Guowang

2018-04-17

Taking advantages of the toehold-triggered strand displacement reaction (TSDR) and host-guest interaction of β-cyclodextrin (β-CD), a facile enzyme-free and homogeneous electrochemical sensing strategy was designed for the sensitive assay of target DNA using Fe 3 O 4 @SiO 2 @β-CD nanocomposites and ferrocene-labeled hairpin DNA (H-1) as the capture and electrochemical probes, respectively. Upon addition of target molecule, the initiated TSDR process induced the conformational change of H-1, and subsequently stimulated the dynamic assembly of assist probes (A-1 and A-2) to generate H-1:A-1:A-2 duplex along with the release of target sequence. The released target could drive the next TSDR recycling and finally result in the formation of numerous DNA duplex. After the molecular recognition of Fe 3 O 4 @SiO 2 @β-CD nanocomposites, a large number of duplex were easily separated from the supernatant solution under an external magnetic field, which led to a decreased H-1 concentration in residual solution, concomitant with a remarkable reduction of peak current. Under the optimized conditions, wide linear range (1-5000 pM), low detection limit (0.3 pM), desirable reproducibility, good selectivity, and satisfactory practical analysis were obtained by the combination of the superior recognition capability of β-CD, TSDR-induced signal amplification, and homogeneous electroanalytical method. The proposed detection strategy could offer a universal approach for the monitoring of various biological analytes via the rational design of probe sequences. Copyright © 2018 Elsevier B.V. All rights reserved.

Divided attention enhances the recognition of emotional stimuli: evidence from the attentional boost effect.

PubMed

Rossi-Arnaud, Clelia; Spataro, Pietro; Costanzi, Marco; Saraulli, Daniele; Cestari, Vincenzo

2018-01-01

The present study examined predictions of the early-phase-elevated-attention hypothesis of the attentional boost effect (ABE), which suggests that transient increases in attention at encoding, as instantiated in the ABE paradigm, should enhance the recognition of neutral and positive items (whose encoding is mostly based on controlled processes), while having small or null effects on the recognition of negative items (whose encoding is primarily based on automatic processes). Participants were presented a sequence of negative, neutral and positive stimuli (pictures in Experiment 1, words in Experiment 2) associated to target (red) squares, distractor (green) squares or no squares (baseline condition). They were told to attend to the pictures/words and simultaneously press the spacebar of the computer when a red square appeared. In a later recognition task, stimuli associated to target squares were recognised better than stimuli associated to distractor squares, replicating the standard ABE. More importantly, we also found that: (a) the memory enhancement following target detection occurred with all types of stimuli (neutral, negative and positive) and (b) the advantage of negative stimuli over neutral stimuli was intact in the DA condition. These findings suggest that the encoding of negative stimuli depends on both controlled (attention-dependent) and automatic (attention-independent) processes.

Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes

PubMed Central

Dodds, Peter N.; Lawrence, Gregory J.; Catanzariti, Ann-Maree; Teh, Trazel; Wang, Ching-I. A.; Ayliffe, Michael A.; Kobe, Bostjan; Ellis, Jeffrey G.

2006-01-01

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R–Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R–Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R–Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant–pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes. PMID:16731621

Assembly and analysis of eukaryotic Argonaute–RNA complexes in microRNA-target recognition

PubMed Central

Gan, Hin Hark; Gunsalus, Kristin C.

2015-01-01

Experimental studies have uncovered a variety of microRNA (miRNA)–target duplex structures that include perfect, imperfect and seedless duplexes. However, non-canonical binding modes from imperfect/seedless duplexes are not well predicted by computational approaches, which rely primarily on sequence and secondary structural features, nor have their tertiary structures been characterized because solved structures to date are limited to near perfect, straight duplexes in Argonautes (Agos). Here, we use structural modeling to examine the role of Ago dynamics in assembling viable eukaryotic miRNA-induced silencing complexes (miRISCs). We show that combinations of low-frequency, global modes of motion of Ago domains are required to accommodate RNA duplexes in model human and C. elegans Ago structures. Models of viable miRISCs imply that Ago adopts variable conformations at distinct target sites that generate distorted, imperfect miRNA-target duplexes. Ago's ability to accommodate a duplex is dependent on the region where structural distortions occur: distortions in solvent-exposed seed and 3′-end regions are less likely to produce steric clashes than those in the central duplex region. Energetic analyses of assembled miRISCs indicate that target recognition is also driven by favorable Ago-duplex interactions. Such structural insights into Ago loading and target recognition mechanisms may provide a more accurate assessment of miRNA function. PMID:26432829

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

PubMed

Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui; Falconieri, Veronica; Rao, Prashant; Merk, Alan; Eng, Edward T; Raczkowski, Ashleigh M; Fox, Tara; Earl, Lesley A; Patel, Dinshaw J; Subramaniam, Sriram

2017-10-05

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. Published by Elsevier Inc.

Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease.

PubMed

Repanas, Kostas; Zingler, Nora; Layer, Liliana E; Schumann, Gerald G; Perrakis, Anastassis; Weichenrieder, Oliver

2007-01-01

The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for approximately 28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. Based on the crystal structure, we generated L1-EN mutants to analyze and manipulate DNA target site recognition. Crystal structures and their dynamic and functional analysis show entire loop grafts to be feasible, resulting in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. Structural parameters of the DNA target seem more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons.

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

PubMed Central

Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

2014-01-01

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

Identification and Targeting of Tyrosine Kinase Activity in Prostate Cancer Initiation, Progression, and Metastasis

DTIC Science & Technology

2013-12-01

University) "Effectors of the DNA damage and radiotherapy response in cancer" 9:20 pm - 9:30 pm Discussion TUESDAY 7:30 am - 8:30 am Breakfast 9:00...M. Morris , Hideki Onagi, Timothy M. Altamore, Allan B. Gamble, Christopher J. Easton Prohormone-substrate peptide sequence recognition by

A novel probabilistic framework for event-based speech recognition

NASA Astrophysics Data System (ADS)

Juneja, Amit; Espy-Wilson, Carol

2003-10-01

One of the reasons for unsatisfactory performance of the state-of-the-art automatic speech recognition (ASR) systems is the inferior acoustic modeling of low-level acoustic-phonetic information in the speech signal. An acoustic-phonetic approach to ASR, on the other hand, explicitly targets linguistic information in the speech signal, but such a system for continuous speech recognition (CSR) is not known to exist. A probabilistic and statistical framework for CSR based on the idea of the representation of speech sounds by bundles of binary valued articulatory phonetic features is proposed. Multiple probabilistic sequences of linguistically motivated landmarks are obtained using binary classifiers of manner phonetic features-syllabic, sonorant and continuant-and the knowledge-based acoustic parameters (APs) that are acoustic correlates of those features. The landmarks are then used for the extraction of knowledge-based APs for source and place phonetic features and their binary classification. Probabilistic landmark sequences are constrained using manner class language models for isolated or connected word recognition. The proposed method could overcome the disadvantages encountered by the early acoustic-phonetic knowledge-based systems that led the ASR community to switch to systems highly dependent on statistical pattern analysis methods and probabilistic language or grammar models.

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

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

Li, T; Huang, S; Zhao, XF

Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs tomore » target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.« less

Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

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

Hudson, William H.; Pickard, Mark R.; de Vera, Ian Mitchelle S.

2014-12-23

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5more » lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.« less

Stability of miRNA 5′terminal and seed regions is correlated with experimentally observed miRNA-mediated silencing efficacy

PubMed Central

Hibio, Naoki; Hino, Kimihiro; Shimizu, Eigo; Nagata, Yoshiro; Ui-Tei, Kumiko

2012-01-01

MicroRNAs (miRNAs) are key regulators of sequence-specific gene silencing. However, crucial factors that determine the efficacy of miRNA-mediated target gene silencing are poorly understood. Here we mathematized base-pairing stability and showed that miRNAs with an unstable 5′ terminal duplex and stable seed-target duplex exhibit strong silencing activity. The results are consistent with the previous findings that an RNA strand with unstable 5′ terminal in miRNA duplex easily loads onto the RNA-induced silencing complex (RISC), and miRNA recognizes target mRNAs with seed-complementary sequences to direct posttranscriptional repression. Our results suggested that both the unwinding and target recognition processes of miRNAs could be proficiently controlled by the thermodynamics of base-pairing in protein-free condition. Interestingly, such thermodynamic parameters might be evolutionarily well adapted to the body temperatures of various species. PMID:23251782

Structural impact of complete CpG methylation within target DNA on specific complex formation of the inducible transcription factor Egr-1.

PubMed

Zandarashvili, Levani; White, Mark A; Esadze, Alexandre; Iwahara, Junji

2015-07-08

The inducible transcription factor Egr-1 binds specifically to 9-bp target sequences containing two CpG sites that can potentially be methylated at four cytosine bases. Although it appears that complete CpG methylation would make an unfavorable steric clash in the previous crystal structures of the complexes with unmethylated or partially methylated DNA, our affinity data suggest that DNA recognition by Egr-1 is insensitive to CpG methylation. We have determined, at a 1.4-Å resolution, the crystal structure of the Egr-1 zinc-finger complex with completely methylated target DNA. Structural comparison of the three different methylation states reveals why Egr-1 can recognize the target sequences regardless of CpG methylation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification of Biomolecular Building Blocks by Recognition Tunneling: Stride towards Nanopore Sequencing of Biomolecules

NASA Astrophysics Data System (ADS)

Sen, Suman

DNA, RNA and Protein are three pivotal biomolecules in human and other organisms, playing decisive roles in functionality, appearance, diseases development and other physiological phenomena. Hence, sequencing of these biomolecules acquires the prime interest in the scientific community. Single molecular identification of their building blocks can be done by a technique called Recognition Tunneling (RT) based on Scanning Tunneling Microscope (STM). A single layer of specially designed recognition molecule is attached to the STM electrodes, which trap the targeted molecules (DNA nucleoside monophosphates, RNA nucleoside monophosphates or amino acids) inside the STM nanogap. Depending on their different binding interactions with the recognition molecules, the analyte molecules generate stochastic signal trains accommodating their "electronic fingerprints". Signal features are used to detect the molecules using a machine learning algorithm and different molecules can be identified with significantly high accuracy. This, in turn, paves the way for rapid, economical nanopore sequencing platform, overcoming the drawbacks of Next Generation Sequencing (NGS) techniques. To read DNA nucleotides with high accuracy in an STM tunnel junction a series of nitrogen-based heterocycles were designed and examined to check their capabilities to interact with naturally occurring DNA nucleotides by hydrogen bonding in the tunnel junction. These recognition molecules are Benzimidazole, Imidazole, Triazole and Pyrrole. Benzimidazole proved to be best among them showing DNA nucleotide classification accuracy close to 99%. Also, Imidazole reader can read an abasic monophosphate (AP), a product from depurination or depyrimidination that occurs 10,000 times per human cell per day. In another study, I have investigated a new universal reader, 1-(2-mercaptoethyl)pyrene (Pyrene reader) based on stacking interactions, which should be more specific to the canonical DNA nucleosides. In addition, Pyrene reader showed higher DNA base-calling accuracy compare to Imidazole reader, the workhorse in our previous projects. In my other projects, various amino acids and RNA nucleoside monophosphates were also classified with significantly high accuracy using RT. Twenty naturally occurring amino acids and various RNA nucleosides (four canonical and two modified) were successfully identified. Thus, we envision nanopore sequencing biomolecules using Recognition Tunneling (RT) that should provide comprehensive betterment over current technologies in terms of time, chemical and instrumental cost and capability of de novo sequencing.

Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA.

PubMed

Dayeh, Daniel M; Cantara, William A; Kitzrow, Jonathan P; Musier-Forsyth, Karin; Nakanishi, Kotaro

2018-06-12

The recent identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. Here, we introduce a programmable RNA restriction enzyme, based on a budding yeast Argonaute (AGO), programmed with cost-effective 23-nucleotide (nt) single-stranded DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with 11 different guide DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5'-untranslated region. This assay, coupled with primer extension and capillary electrophoresis, allows detection and relative quantification of all DISC-cleavage sites simultaneously in a single reaction. Comparison between DISC cleavage and RNase H cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs.

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9.

PubMed

Singh, Digvijay; Sternberg, Samuel H; Fei, Jingyi; Doudna, Jennifer A; Ha, Taekjip

2016-09-14

Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s(-1) to >2 s(-1) upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s(-1)), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

PubMed Central

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-01-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity. PMID:26646790

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

PubMed

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-12-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.

Conformation of Tax-response elements in the human T-cell leukemia virus type I promoter.

PubMed

Cox, J M; Sloan, L S; Schepartz, A

1995-12-01

HTLV-I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CREB) and increasing their affinities for proviral TRE target sites. Each 21 bp TRE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation. Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax. The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CREB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.

GeneSilico protein structure prediction meta-server.

PubMed

Kurowski, Michal A; Bujnicki, Janusz M

2003-07-01

Rigorous assessments of protein structure prediction have demonstrated that fold recognition methods can identify remote similarities between proteins when standard sequence search methods fail. It has been shown that the accuracy of predictions is improved when refined multiple sequence alignments are used instead of single sequences and if different methods are combined to generate a consensus model. There are several meta-servers available that integrate protein structure predictions performed by various methods, but they do not allow for submission of user-defined multiple sequence alignments and they seldom offer confidentiality of the results. We developed a novel WWW gateway for protein structure prediction, which combines the useful features of other meta-servers available, but with much greater flexibility of the input. The user may submit an amino acid sequence or a multiple sequence alignment to a set of methods for primary, secondary and tertiary structure prediction. Fold-recognition results (target-template alignments) are converted into full-atom 3D models and the quality of these models is uniformly assessed. A consensus between different FR methods is also inferred. The results are conveniently presented on-line on a single web page over a secure, password-protected connection. The GeneSilico protein structure prediction meta-server is freely available for academic users at http://genesilico.pl/meta.

GeneSilico protein structure prediction meta-server

PubMed Central

Kurowski, Michal A.; Bujnicki, Janusz M.

2003-01-01

Rigorous assessments of protein structure prediction have demonstrated that fold recognition methods can identify remote similarities between proteins when standard sequence search methods fail. It has been shown that the accuracy of predictions is improved when refined multiple sequence alignments are used instead of single sequences and if different methods are combined to generate a consensus model. There are several meta-servers available that integrate protein structure predictions performed by various methods, but they do not allow for submission of user-defined multiple sequence alignments and they seldom offer confidentiality of the results. We developed a novel WWW gateway for protein structure prediction, which combines the useful features of other meta-servers available, but with much greater flexibility of the input. The user may submit an amino acid sequence or a multiple sequence alignment to a set of methods for primary, secondary and tertiary structure prediction. Fold-recognition results (target-template alignments) are converted into full-atom 3D models and the quality of these models is uniformly assessed. A consensus between different FR methods is also inferred. The results are conveniently presented on-line on a single web page over a secure, password-protected connection. The GeneSilico protein structure prediction meta-server is freely available for academic users at http://genesilico.pl/meta. PMID:12824313

Classification of video sequences into chosen generalized use classes of target size and lighting level.

PubMed

Leszczuk, Mikołaj; Dudek, Łukasz; Witkowski, Marcin

The VQiPS (Video Quality in Public Safety) Working Group, supported by the U.S. Department of Homeland Security, has been developing a user guide for public safety video applications. According to VQiPS, five parameters have particular importance influencing the ability to achieve a recognition task. They are: usage time-frame, discrimination level, target size, lighting level, and level of motion. These parameters form what are referred to as Generalized Use Classes (GUCs). The aim of our research was to develop algorithms that would automatically assist classification of input sequences into one of the GUCs. Target size and lighting level parameters were approached. The experiment described reveals the experts' ambiguity and hesitation during the manual target size determination process. However, the automatic methods developed for target size classification make it possible to determine GUC parameters with 70 % compliance to the end-users' opinion. Lighting levels of the entire sequence can be classified with an efficiency reaching 93 %. To make the algorithms available for use, a test application has been developed. It is able to process video files and display classification results, the user interface being very simple and requiring only minimal user interaction.

Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1.

PubMed

Nishimasu, Hiroshi; Yamano, Takashi; Gao, Linyi; Zhang, Feng; Ishitani, Ryuichiro; Nureki, Osamu

2017-07-06

The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA recognition by an RNA-guided bacterial Argonaute

PubMed Central

Doudna, Jennifer A.

2017-01-01

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins. PMID:28520746

Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs

PubMed Central

Ryan, Daniel E; Taussig, David; Steinfeld, Israel; Phadnis, Smruti M; Lunstad, Benjamin D; Singh, Madhurima; Vuong, Xuan; Okochi, Kenji D; McCaffrey, Ryan; Olesiak, Magdalena; Roy, Subhadeep; Yung, Chong Wing; Curry, Bo; Sampson, Jeffrey R; Dellinger, Douglas J

2018-01-01

Abstract CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications. PMID:29216382

Simultaneous Binding of Hybrid Molecules Constructed with Dual DNA-Binding Components to a G-Quadruplex and Its Proximal Duplex.

PubMed

Asamitsu, Sefan; Obata, Shunsuke; Phan, Anh Tuân; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2018-03-20

A G-quadruplex (quadruplex) is a nucleic acid secondary structure adopted by guanine-rich sequences and is considered to be relevant to various pharmacological and biological contexts. Although a number of researchers have endeavored to discover and develop quadruplex-interactive molecules, poor ligand designability originating from topological similarity of the skeleton of diverse quadruplexes has remained a bottleneck for gaining specificity for individual quadruplexes. This work reports on hybrid molecules that were constructed with dual DNA-binding components, a cyclic imidazole/lysine polyamide (cIKP), and a hairpin pyrrole/imidazole polyamide (hPIP), with the aim toward specific quadruplex targeting by reading out the local duplex DNA sequence adjacent to designated quadruplexes in the genome. By means of circular dichroism (CD), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and NMR techniques, we showed the dual and simultaneous recognition of the respective segment via hybrid molecules, and the synergistic and mutual effect of each binding component that was appropriately linked on higher binding affinity and modest sequence specificity. Monitoring quadruplex and duplex imino protons of the quadruplex/duplex motif titrated with hybrid molecules clearly revealed distinct features of the binding of hybrid molecules to the respective segments upon their simultaneous recognition. A series of the systematic and detailed binding assays described here showed that the concept of simultaneous recognition of quadruplex and its proximal duplex by hybrid molecules constructed with the dual DNA-binding components may provide a new strategy for ligand design, enabling targeting of a large variety of designated quadruplexes at specific genome locations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recognition of Human Histocompatibility Leukocyte Antigen (HLA)-E Complexed with HLA Class I Signal Sequence–derived Peptides by CD94/NKG2 Confers Protection from Natural Killer Cell–mediated Lysis

PubMed Central

Borrego, Francisco; Ulbrecht, Matthias; Weiss, Elisabeth H.; Coligan, John E.; Brooks, Andrew G.

1998-01-01

Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical HLA class I molecule, the gene for which is transcribed in most tissues. It has recently been reported that this molecule binds peptides derived from the signal sequence of HLA class I proteins; however, no function for HLA-E has yet been described. We show that natural killer (NK) cells can recognize target cells expressing HLA-E molecules on the cell surface and this interaction results in inhibition of the lytic process. Furthermore, HLA-E recognition is mediated primarily through the CD94/NKG2-A heterodimer, as CD94-specific, but not killer cell inhibitory receptor (KIR)–specific mAbs block HLA-E–mediated protection of target cells. Cell surface HLA-E could be increased by incubation with synthetic peptides corresponding to residues 3–11 from the signal sequences of a number of HLA class I molecules; however, only peptides which contained a Met at position 2 were capable of conferring resistance to NK-mediated lysis, whereas those having Thr at position 2 had no effect. Interestingly, HLA class I molecules previously correlated with CD94/NKG2 recognition all have Met at residue 4 of the signal sequence (position 2 of the HLA-E binding peptide), whereas those which have been reported not to interact with CD94/NKG2 have Thr at this position. Thus, these data show a function for HLA-E and suggest an alternative explanation for the apparent broad reactivity of CD94/NKG2 with HLA class I molecules; that CD94/NKG2 interacts with HLA-E complexed with signal sequence peptides derived from “protective” HLA class I alleles rather than directly interacting with classical HLA class I proteins. PMID:9480992

Structural and sequencing analysis of local target DNA recognition by MLV integrase.

PubMed

Aiyer, Sriram; Rossi, Paolo; Malani, Nirav; Schneider, William M; Chandar, Ashwin; Bushman, Frederic D; Montelione, Gaetano T; Roth, Monica J

2015-06-23

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Human β-glucuronidase: structure, function, and application in enzyme replacement therapy.

PubMed

Naz, Huma; Islam, Asimul; Waheed, Abdul; Sly, William S; Ahmad, Faizan; Hassan, Imtaiyaz

2013-10-01

Lysosomal storage diseases occur due to incomplete metabolic degradation of macromolecules by various hydrolytic enzymes in the lysosome. Despite structural differences, most of the lysosomal enzymes share many common features including a lysosomal targeting motif and phosphotransferase recognition sites. β-Glucuronidase (GUSB) is an important lysosomal enzyme involved in the degradation of glucuronate-containing glycosaminoglycan. The deficiency of GUSB causes mucopolysaccharidosis type VII (MPSVII), leading to lysosomal storage in the brain. GUSB is a well-studied protein for its expression, sequence, structure, and function. The purpose of this review is to summarize our current understanding of sequence, structure, function, and evolution of GUSB and its lysosomal enzyme targeting. Enzyme replacement therapy reported for this protein is also discussed.

[Screening specific recognition motif of RNA-binding proteins by SELEX in combination with next-generation sequencing technique].

PubMed

Zhang, Lu; Xu, Jinhao; Ma, Jinbiao

2016-07-25

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.

Individual microRNAs (miRNAs) display distinct mRNA targeting "rules".

PubMed

Wang, Wang-Xia; Wilfred, Bernard R; Xie, Kevin; Jennings, Mary H; Hu, Yanling Hu; Stromberg, Arnold J; Nelson, Peter T

2010-01-01

MicroRNAs (miRNAs) guide Argonaute (AGO)-containing microribonucleoprotein (miRNP) complexes to target mRNAs.It has been assumed that miRNAs behave similarly to each other with regard to mRNA target recognition. The usual assumptions, which are based on prior studies, are that miRNAs target preferentially sequences in the 3'UTR of mRNAs,guided by the 5' "seed" portion of the miRNAs. Here we isolated AGO- and miRNA-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with anti-AGO antibody. Cells were transfected with miR-107, miR-124,miR-128, miR-320, or a negative control miRNA. Co-IPed RNAs were subjected to downstream high-density Affymetrix Human Gene 1.0 ST microarray analyses using an assay we validated previously-a "RIP-Chip" experimental design. RIP-Chip data provided a list of mRNAs recruited into the AGO-miRNP in correlation to each miRNA. These experimentally identified miRNA targets were analyzed for complementary six nucleotide "seed" sequences within the transfected miRNAs. We found that miR-124 targets tended to have sequences in the 3'UTR that would be recognized by the 5' seed of miR-124, as described in previous studies. By contrast, miR-107 targets tended to have 'seed' sequences in the mRNA open reading frame, but not the 3' UTR. Further, mRNA targets of miR-128 and miR-320 are less enriched for 6-mer seed sequences in comparison to miR-107 and miR-124. In sum, our data support the importance of the 5' seed in determining binding characteristics for some miRNAs; however, the "binding rules" are complex, and individual miRNAs can have distinct sequence determinants that lead to mRNA targeting.

Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

PubMed Central

Hong, Ka Lok

2015-01-01

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. PMID:26199940

Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

PubMed Central

Kuhn, Patrick; Draycheva, Albena; Vogt, Andreas; Petriman, Narcis-Adrian; Sturm, Lukas; Drepper, Friedel; Warscheid, Bettina; Wintermeyer, Wolfgang

2015-01-01

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex. PMID:26459600

Extending the language of DNA molecular recognition by polyamides: unexpected influence of imidazole and pyrrole arrangement on binding affinity and specificity.

PubMed

Buchmueller, Karen L; Staples, Andrew M; Howard, Cameron M; Horick, Sarah M; Uthe, Peter B; Le, N Minh; Cox, Kari K; Nguyen, Binh; Pacheco, Kimberly A O; Wilson, W David; Lee, Moses

2005-01-19

Pyrrole (Py) and imidazole (Im) polyamides can be designed to target specific DNA sequences. The effect that the pyrrole and imidazole arrangement, plus DNA sequence, have on sequence specificity and binding affinity has been investigated using DNA melting (DeltaT(M)), circular dichroism (CD), and surface plasmon resonance (SPR) studies. SPR results obtained from a complete set of triheterocyclic polyamides show a dramatic difference in the affinity of f-ImPyIm for its cognate DNA (K(eq) = 1.9 x 10(8) M(-1)) and f-PyPyIm for its cognate DNA (K(eq) = 5.9 x 10(5) M(-1)), which could not have been anticipated prior to characterization of these compounds. Moreover, f-ImPyIm has a 10-fold greater affinity for CGCG than distamycin A has for its cognate, AATT. To understand this difference, the triamide dimers are divided into two structural groupings: central and terminal pairings. The four possible central pairings show decreasing selectivity and affinity for their respective cognate sequences: -ImPy > -PyPy- > -PyIm- approximately -ImIm-. These results extend the language of current design motifs for polyamide sequence recognition to include the use of "words" for recognizing two adjacent base pairs, rather than "letters" for binding to single base pairs. Thus, polyamides designed to target Watson-Crick base pairs should utilize the strength of -ImPy- and -PyPy- central pairings. The f/Im and f/Py terminal groups yielded no advantage for their respective C/G or T/A base pairs. The exception is with the -ImPy- central pairing, for which f/Im has a 10-fold greater affinity for C/G than f/Py has for T/A.

Transcription Factors Bind Thousands of Active and InactiveRegions in the Drosophila Blastoderm

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

Li, Xiao-Yong; MacArthur, Stewart; Bourgon, Richard

2008-01-10

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. Here, we use whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched inmore » bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over forty well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly-bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets.« less

Context influences on TALE–DNA binding revealed by quantitative profiling

PubMed Central

Rogers, Julia M.; Barrera, Luis A.; Reyon, Deepak; Sander, Jeffry D.; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L.

2015-01-01

Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE–DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000–20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE–DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design. PMID:26067805

Context influences on TALE-DNA binding revealed by quantitative profiling.

PubMed

Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

2015-06-11

Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

Identifying mRNA sequence elements for target recognition by human Argonaute proteins

PubMed Central

Li, Jingjing; Kim, TaeHyung; Nutiu, Razvan; Ray, Debashish; Hughes, Timothy R.; Zhang, Zhaolei

2014-01-01

It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO–mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity. PMID:24663241

Role for ribosome-associated complex and stress-seventy subfamily B (RAC-Ssb) in integral membrane protein translation.

PubMed

Acosta-Sampson, Ligia; Döring, Kristina; Lin, Yuping; Yu, Vivian Y; Bukau, Bernd; Kramer, Günter; Cate, Jamie H D

2017-12-01

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus

PubMed Central

Cao, Xiaoxiao; Li, Shaohua; Chen, Liucun; Ding, Hongmei; Xu, Hua; Huang, Yanping; Li, Jie; Liu, Nongle; Cao, Weihong; Zhu, Yanjun; Shen, Beifen; Shao, Ningsheng

2009-01-01

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer. PMID:19498077

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus.

PubMed

Cao, Xiaoxiao; Li, Shaohua; Chen, Liucun; Ding, Hongmei; Xu, Hua; Huang, Yanping; Li, Jie; Liu, Nongle; Cao, Weihong; Zhu, Yanjun; Shen, Beifen; Shao, Ningsheng

2009-08-01

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer.

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

PubMed Central

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

2013-01-01

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

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

PubMed

Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

2015-03-12

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

SMARTIV: combined sequence and structure de-novo motif discovery for in-vivo RNA binding data.

PubMed

Polishchuk, Maya; Paz, Inbal; Yakhini, Zohar; Mandel-Gutfreund, Yael

2018-05-25

Gene expression regulation is highly dependent on binding of RNA-binding proteins (RBPs) to their RNA targets. Growing evidence supports the notion that both RNA primary sequence and its local secondary structure play a role in specific Protein-RNA recognition and binding. Despite the great advance in high-throughput experimental methods for identifying sequence targets of RBPs, predicting the specific sequence and structure binding preferences of RBPs remains a major challenge. We present a novel webserver, SMARTIV, designed for discovering and visualizing combined RNA sequence and structure motifs from high-throughput RNA-binding data, generated from in-vivo experiments. The uniqueness of SMARTIV is that it predicts motifs from enriched k-mers that combine information from ranked RNA sequences and their predicted secondary structure, obtained using various folding methods. Consequently, SMARTIV generates Position Weight Matrices (PWMs) in a combined sequence and structure alphabet with assigned P-values. SMARTIV concisely represents the sequence and structure motif content as a single graphical logo, which is informative and easy for visual perception. SMARTIV was examined extensively on a variety of high-throughput binding experiments for RBPs from different families, generated from different technologies, showing consistent and accurate results. Finally, SMARTIV is a user-friendly webserver, highly efficient in run-time and freely accessible via http://smartiv.technion.ac.il/.

Electrochemical detection of sequence-specific DNA based on formation of G-quadruplex-hemin through continuous hybridization chain reaction.

PubMed

Sun, Xiaofan; Chen, Haohan; Wang, Shuling; Zhang, Yiping; Tian, Yaping; Zhou, Nandi

2018-08-27

A high-sensitive detection of sequence-specific DNA was established based on the formation of G-quadruplex-hemin complex through continuous hybridization chain reaction (HCR). Taking HIV DNA sequence as an example, a capture probe complementary to part of HIV DNA was firstly self-assembled onto the surface of Au electrode. Then a specially designed assistant probe with both terminals complementary to the target DNA and a G-quadruplex-forming sequence in the center was introduced into the detection solution. In the presence of both the target DNA and the assistant probe, the target DNA can be captured on the electrode surface and then a continuous HCR can be conducted due to the mutual recognition of the target DNA and the assistant probe, leading to the formation of a large number of G-quadruplex on the electrode surface. With the help of hemin, a pronounced electrochemical signal can be observed in differential pulse voltammetry (DPV), due to the formation of G-quadruplex-hemin complex. The peak current is linearly related with the logarithm of the concentration of the target DNA in the range from 10 fM to 10 pM. The electrochemical sensor has high selectivity to clearly discriminate single-base mismatched and three-base mismatched sequences from the original HIV DNA sequence. Moreover, the established DNA sensor was challenged by detection of HIV DNA in human serum samples, which showed the low detection limit of 6.3 fM. Thus it has great application prospect in the field of clinical diagnosis and environmental monitoring. Copyright © 2018 Elsevier B.V. All rights reserved.

Analytical applications of aptamers

NASA Astrophysics Data System (ADS)

Tombelli, S.; Minunni, M.; Mascini, M.

2007-05-01

Aptamers are single stranded DNA or RNA ligands which can be selected for different targets starting from a library of molecules containing randomly created sequences. Aptamers have been selected to bind very different targets, from proteins to small organic dyes. Aptamers are proposed as alternatives to antibodies as biorecognition elements in analytical devices with ever increasing frequency. This in order to satisfy the demand for quick, cheap, simple and highly reproducible analytical devices, especially for protein detection in the medical field or for the detection of smaller molecules in environmental and food analysis. In our recent experience, DNA and RNA aptamers, specific for three different proteins (Tat, IgE and thrombin), have been exploited as bio-recognition elements to develop specific biosensors (aptasensors). These recognition elements have been coupled to piezoelectric quartz crystals and surface plasmon resonance (SPR) devices as transducers where the aptamers have been immobilized on the gold surface of the crystals electrodes or on SPR chips, respectively.

Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases.

PubMed

Boomsma, Wouter; Nielsen, Sofie V; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus; Ellgaard, Lars

2016-01-01

The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is not a unique case, and that several other yeast and human E3 ligases have sequence properties that may allow them to recognize substrates by a similar mechanism as San1.

A Dual-Specific Targeting Approach Based on the Simultaneous Recognition of Duplex and Quadruplex Motifs.

PubMed

Nguyen, Thi Quynh Ngoc; Lim, Kah Wai; Phan, Anh Tuân

2017-09-20

Small-molecule ligands targeting nucleic acids have been explored as potential therapeutic agents. Duplex groove-binding ligands have been shown to recognize DNA in a sequence-specific manner. On the other hand, quadruplex-binding ligands exhibit high selectivity between quadruplex and duplex, but show limited discrimination between different quadruplex structures. Here we propose a dual-specific approach through the simultaneous application of duplex- and quadruplex-binders. We demonstrated that a quadruplex-specific ligand and a duplex-specific ligand can simultaneously interact at two separate binding sites of a quadruplex-duplex hybrid harbouring both quadruplex and duplex structural elements. Such a dual-specific targeting strategy would combine the sequence specificity of duplex-binders and the strong binding affinity of quadruplex-binders, potentially allowing the specific targeting of unique quadruplex structures. Future research can be directed towards the development of conjugated compounds targeting specific genomic quadruplex-duplex sites, for which the linker would be highly context-dependent in terms of length and flexibility, as well as the attachment points onto both ligands.

Fluorescence self-quenching assay for the detection of target collagen sequences using a short probe peptide.

PubMed

Nian, Linge; Hu, Yue; Fu, Caihong; Song, Chen; Wang, Jie; Xiao, Jianxi

2018-01-01

The development of novel assays to detect collagen fragments is of utmost importance for diagnostic, prognostic and therapeutic decisions in various collagen-related diseases, and one essential question is to discover probe peptides that can specifically recognize target collagen sequences. Herein we have developed the fluorescence self-quenching assay as a convenient tool to screen the capability of a series of fluorescent probe peptides of variable lengths to bind with target collagen peptides. We have revealed that the targeting ability of probe peptides is length-dependent, and have discovered a relatively short probe peptide FAM-G(POG) 8 capable to identify the target peptide. We have further demonstrated that fluorescence self-quenching assay together with this short probe peptide can be applied to specifically detect the desired collagen fragment in complex biological media. Fluorescence self-quenching assay provides a powerful new tool to discover effective peptides for the recognition of collagen biomarkers, and it may have great potential to identify probe peptides for various protein biomarkers involved in pathological conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

Ultrafast scene detection and recognition with limited visual information

PubMed Central

Hagmann, Carl Erick; Potter, Mary C.

2016-01-01

Humans can detect target color pictures of scenes depicting concepts like picnic or harbor in sequences of six or twelve pictures presented as briefly as 13 ms, even when the target is named after the sequence (Potter, Wyble, Hagmann, & McCourt, 2014). Such rapid detection suggests that feedforward processing alone enabled detection without recurrent cortical feedback. There is debate about whether coarse, global, low spatial frequencies (LSFs) provide predictive information to high cortical levels through the rapid magnocellular (M) projection of the visual path, enabling top-down prediction of possible object identities. To test the “Fast M” hypothesis, we compared detection of a named target across five stimulus conditions: unaltered color, blurred color, grayscale, thresholded monochrome, and LSF pictures. The pictures were presented for 13–80 ms in six-picture rapid serial visual presentation (RSVP) sequences. Blurred, monochrome, and LSF pictures were detected less accurately than normal color or grayscale pictures. When the target was named before the sequence, all picture types except LSF resulted in above-chance detection at all durations. Crucially, when the name was given only after the sequence, performance dropped and the monochrome and LSF pictures (but not the blurred pictures) were at or near chance. Thus, without advance information, monochrome and LSF pictures were rarely understood. The results offer only limited support for the Fast M hypothesis, suggesting instead that feedforward processing is able to activate conceptual representations without complementary reentrant processing. PMID:28255263

Sequence-Specific Targeting of Dosage Compensation in Drosophila Favors an Active Chromatin Context

PubMed Central

Gelbart, Marnie; Tolstorukov, Michael Y.; Plachetka, Annette; Kharchenko, Peter V.; Jung, Youngsook L.; Gorchakov, Andrey A.; Larschan, Erica; Gu, Tingting; Minoda, Aki; Riddle, Nicole C.; Schwartz, Yuri B.; Elgin, Sarah C. R.; Karpen, Gary H.; Pirrotta, Vincenzo; Kuroda, Mitzi I.; Park, Peter J.

2012-01-01

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at “entry sites” that contain a consensus sequence motif (“MSL recognition element” or MRE). However, this motif is only ∼2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome. PMID:22570616

A conserved mechanism for replication origin recognition and binding in archaea.

PubMed

Majerník, Alan I; Chong, James P J

2008-01-15

To date, methanogens are the only group within the archaea where firing DNA replication origins have not been demonstrated in vivo. In the present study we show that a previously identified cluster of ORB (origin recognition box) sequences do indeed function as an origin of replication in vivo in the archaeon Methanothermobacter thermautotrophicus. Although the consensus sequence of ORBs in M. thermautotrophicus is somewhat conserved when compared with ORB sequences in other archaea, the Cdc6-1 protein from M. thermautotrophicus (termed MthCdc6-1) displays sequence-specific binding that is selective for the MthORB sequence and does not recognize ORBs from other archaeal species. Stabilization of in vitro MthORB DNA binding by MthCdc6-1 requires additional conserved sequences 3' to those originally described for M. thermautotrophicus. By testing synthetic sequences bearing mutations in the MthORB consensus sequence, we show that Cdc6/ORB binding is critically dependent on the presence of an invariant guanine found in all archaeal ORB sequences. Mutation of a universally conserved arginine residue in the recognition helix of the winged helix domain of archaeal Cdc6-1 shows that specific origin sequence recognition is dependent on the interaction of this arginine residue with the invariant guanine. Recognition of a mutated origin sequence can be achieved by mutation of the conserved arginine residue to a lysine or glutamine residue. Thus despite a number of differences in protein and DNA sequences between species, the mechanism of origin recognition and binding appears to be conserved throughout the archaea.

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

PubMed

Warnatz, Hans-Jörg; Schmidt, Dominic; Manke, Thomas; Piccini, Ilaria; Sultan, Marc; Borodina, Tatiana; Balzereit, Daniela; Wruck, Wasco; Soldatov, Alexey; Vingron, Martin; Lehrach, Hans; Yaspo, Marie-Laure

2011-07-01

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

Loop nucleotides control primary and mature miRNA function in target recognition and repression

PubMed Central

Yue, Si-Biao; Deis Trujillo, Robin; Tang, Yujie; O'Gorman, William E

2011-01-01

MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5′ end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs' sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation. PMID:22142974

Influence of motion on face recognition.

PubMed

Bonfiglio, Natale S; Manfredi, Valentina; Pessa, Eliano

2012-02-01

The influence of motion information and temporal associations on recognition of non-familiar faces was investigated using two groups which performed a face recognition task. One group was presented with regular temporal sequences of face views designed to produce the impression of motion of the face rotating in depth, the other group with random sequences of the same views. In one condition, participants viewed the sequences of the views in rapid succession with a negligible interstimulus interval (ISI). This condition was characterized by three different presentation times. In another condition, participants were presented a sequence with a 1-sec. ISI among the views. That regular sequences of views with a negligible ISI and a shorter presentation time were hypothesized to give rise to better recognition, related to a stronger impression of face rotation. Analysis of data from 45 participants showed a shorter presentation time was associated with significantly better accuracy on the recognition task; however, differences between performances associated with regular and random sequences were not significant.

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

PDNAsite: Identification of DNA-binding Site from Protein Sequence by Incorporating Spatial and Sequence Context

PubMed Central

Zhou, Jiyun; Xu, Ruifeng; He, Yulan; Lu, Qin; Wang, Hongpeng; Kong, Bing

2016-01-01

Protein-DNA interactions are involved in many fundamental biological processes essential for cellular function. Most of the existing computational approaches employed only the sequence context of the target residue for its prediction. In the present study, for each target residue, we applied both the spatial context and the sequence context to construct the feature space. Subsequently, Latent Semantic Analysis (LSA) was applied to remove the redundancies in the feature space. Finally, a predictor (PDNAsite) was developed through the integration of the support vector machines (SVM) classifier and ensemble learning. Results on the PDNA-62 and the PDNA-224 datasets demonstrate that features extracted from spatial context provide more information than those from sequence context and the combination of them gives more performance gain. An analysis of the number of binding sites in the spatial context of the target site indicates that the interactions between binding sites next to each other are important for protein-DNA recognition and their binding ability. The comparison between our proposed PDNAsite method and the existing methods indicate that PDNAsite outperforms most of the existing methods and is a useful tool for DNA-binding site identification. A web-server of our predictor (http://hlt.hitsz.edu.cn:8080/PDNAsite/) is made available for free public accessible to the biological research community. PMID:27282833

Nucleic acid constructs containing orthogonal site selective recombinases (OSSRs)

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

Gilmore, Joshua M.; Anderson, J. Christopher; Dueber, John E.

The present invention provides for a recombinant nucleic acid comprising a nucleotide sequence comprising a plurality of constructs, wherein each construct independently comprises a nucleotide sequence of interest flanked by a pair of recombinase recognition sequences. Each pair of recombinase recognition sequences is recognized by a distinct recombinase. Optionally, each construct can, independently, further comprise one or more genes encoding a recombinase capable of recognizing the pair of recombinase recognition sequences of the construct. The recombinase can be an orthogonal (non-cross reacting), site-selective recombinase (OSSR).

Function-based classification of carbohydrate-active enzymes by recognition of short, conserved peptide motifs.

PubMed

Busk, Peter Kamp; Lange, Lene

2013-06-01

Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision.

A bacterial Argonaute with noncanonical guide RNA specificity

PubMed Central

Kaya, Emine; Doxzen, Kevin W.; Knoll, Kilian R.; Wilson, Ross C.; Strutt, Steven C.; Kranzusch, Philip J.; Doudna, Jennifer A.

2016-01-01

Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo–RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPR-cas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide. PMID:27035975

Synthesis of mouse centromere-targeted polyamides and physico-chemical studies of their interaction with the target double-stranded DNA.

PubMed

Nozeret, Karine; Bonan, Marc; Yarmoluk, Serguiy M; Novopashina, Darya S; Boutorine, Alexandre S

2015-09-01

Synthetic minor groove-binding pyrrole-imidazole polyamides labeled by fluorophores are promising candidates for fluorescence imaging of double-stranded DNA in isolated chromosomes or fixed and living cells. We synthesized nine hairpin and two head-to-head tandem polyamides targeting repeated sequences from mouse major satellites. Their interaction with synthetic target dsDNA has been studied by physico-chemical methods in vitro before and after coupling to various fluorophores. Great variability in affinities and fluorescence properties reveals a conclusion that these properties do not only rely on recognition rules, but also on other known and unknown structural factors. Individual testing of each probe is needed before cellular applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular dynamics studies on the DNA-binding process of ERG.

PubMed

Beuerle, Matthias G; Dufton, Neil P; Randi, Anna M; Gould, Ian R

2016-11-15

The ETS family of transcription factors regulate gene targets by binding to a core GGAA DNA-sequence. The ETS factor ERG is required for homeostasis and lineage-specific functions in endothelial cells, some subset of haemopoietic cells and chondrocytes; its ectopic expression is linked to oncogenesis in multiple tissues. To date details of the DNA-binding process of ERG including DNA-sequence recognition outside the core GGAA-sequence are largely unknown. We combined available structural and experimental data to perform molecular dynamics simulations to study the DNA-binding process of ERG. In particular we were able to reproduce the ERG DNA-complex with a DNA-binding simulation starting in an unbound configuration with a final root-mean-square-deviation (RMSD) of 2.1 Å to the core ETS domain DNA-complex crystal structure. This allowed us to elucidate the relevance of amino acids involved in the formation of the ERG DNA-complex and to identify Arg385 as a novel key residue in the DNA-binding process. Moreover we were able to show that water-mediated hydrogen bonds are present between ERG and DNA in our simulations and that those interactions have the potential to achieve sequence recognition outside the GGAA core DNA-sequence. The methodology employed in this study shows the promising capabilities of modern molecular dynamics simulations in the field of protein DNA-interactions.

Comparison of Methods of Detection of Exceptional Sequences in Prokaryotic Genomes.

PubMed

Rusinov, I S; Ershova, A S; Karyagina, A S; Spirin, S A; Alexeevski, A V

2018-02-01

Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.

The ARTT motif and a unified structural understanding of substraterecognition in ADP ribosylating bacterial toxins and eukaryotic ADPribosyltransferases

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

Han, S.; Tainer, J.A.

2001-08-01

ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division. The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release. A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins. Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing NAD-binding pocket formed by the two perpendicular b-sheet core hasmore » been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases. The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues. In contrast, Diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosyltransferases are characterized by conserved Arg and catalytic Glu residues. The NAD-binding core of a binary toxin and a C3-like toxin family identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition by structural and mutagenic studies. Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD binding cleft as characterized by conserved Arg and catalytic Glu residues. Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD binding mechanisms but differing mechanisms of target protein binding via sequence variations within the ARTT motif structural framework. Thus, we propose here that the ARTT motif represents an experimentally testable general recognition motif region for many ADP-ribosyltransferases and thereby potentially provides a unified structural understanding of substrate recognition in ADP-ribosylation processes.« less

Temporal identity in axonal target layer recognition.

PubMed

Petrovic, Milan; Hummel, Thomas

2008-12-11

The segregation of axon and dendrite projections into distinct synaptic layers is a fundamental principle of nervous system organization and the structural basis for information processing in the brain. Layer-specific recognition molecules that allow projecting neurons to stabilize transient contacts and initiate synaptogenesis have been identified. However, most of the neuronal cell-surface molecules critical for layer organization are expressed broadly in the developing nervous system, raising the question of how these so-called permissive adhesion molecules support synaptic specificity. Here we show that the temporal expression dynamics of the zinc-finger protein sequoia is the major determinant of Drosophila photoreceptor connectivity into distinct synaptic layers. Neighbouring R8 and R7 photoreceptors show consecutive peaks of elevated sequoia expression, which correspond to their sequential target-layer innervation. Loss of sequoia in R7 leads to a projection switch into the R8 recipient layer, whereas a prolonged expression in R8 induces a redirection of their axons into the R7 layer. The sequoia-induced axon targeting is mediated through the ubiquitously expressed Cadherin-N cell adhesion molecule. Our data support a model in which recognition specificity during synaptic layer formation is generated through a temporally restricted axonal competence to respond to broadly expressed adhesion molecules. Because developing neurons innervating the same target area often project in a distinct, birth-order-dependent sequence, temporal identity seems to contain crucial information in generating not only cell type diversity during neuronal division but also connection diversity of projecting neurons.

Sequence-controlled methacrylic multiblock copolymers via sulfur-free RAFT emulsion polymerization

NASA Astrophysics Data System (ADS)

Engelis, Nikolaos G.; Anastasaki, Athina; Nurumbetov, Gabit; Truong, Nghia P.; Nikolaou, Vasiliki; Shegiwal, Ataulla; Whittaker, Michael R.; Davis, Thomas P.; Haddleton, David M.

2017-02-01

Translating the precise monomer sequence control achieved in nature over macromolecular structure (for example, DNA) to whole synthetic systems has been limited due to the lack of efficient synthetic methodologies. So far, chemists have only been able to synthesize monomer sequence-controlled macromolecules by means of complex, time-consuming and iterative chemical strategies such as solid-state Merrifield-type approaches or molecularly dissolved solution-phase systems. Here, we report a rapid and quantitative synthesis of sequence-controlled multiblock polymers in discrete stable nanoscale compartments via an emulsion polymerization approach in which a vinyl-terminated macromolecule is used as an efficient chain-transfer agent. This approach is environmentally friendly, fully translatable to industry and thus represents a significant advance in the development of complex macromolecule synthesis, where a high level of molecular precision or monomer sequence control confers potential for molecular targeting, recognition and biocatalysis, as well as molecular information storage.

Molecular mechanisms of floral organ specification by MADS domain proteins.

PubMed

Yan, Wenhao; Chen, Dijun; Kaufmann, Kerstin

2016-02-01

Flower development is a model system to understand organ specification in plants. The identities of different types of floral organs are specified by homeotic MADS transcription factors that interact in a combinatorial fashion. Systematic identification of DNA-binding sites and target genes of these key regulators show that they have shared and unique sets of target genes. DNA binding by MADS proteins is not based on 'simple' recognition of a specific DNA sequence, but depends on DNA structure and combinatorial interactions. Homeotic MADS proteins regulate gene expression via alternative mechanisms, one of which may be to modulate chromatin structure and accessibility in their target gene promoters. Copyright © 2015 Elsevier Ltd. All rights reserved.

Autonomous replication of nucleic acids by polymerization/nicking enzyme/DNAzyme cascades for the amplified detection of DNA and the aptamer-cocaine complex.

PubMed

Wang, Fuan; Freage, Lina; Orbach, Ron; Willner, Itamar

2013-09-03

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

PubMed Central

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-01-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. PMID:26674414

Facial Affect Recognition in Violent and Nonviolent Antisocial Behavior Subtypes.

PubMed

Schönenberg, Michael; Mayer, Sarah Verena; Christian, Sandra; Louis, Katharina; Jusyte, Aiste

2016-10-01

Prior studies provide evidence for impaired recognition of distress cues in individuals exhibiting antisocial behavior. However, it remains unclear whether this deficit is generally associated with antisociality or may be specific to violent behavior only. To examine whether there are meaningful differences between the two behavioral dimensions rule-breaking and aggression, violent and nonviolent incarcerated offenders as well as control participants were presented with an animated face recognition task in which a video sequence of a neutral face changed into an expression of one of the six basic emotions. The participants were instructed to press a button as soon as they were able to identify the emotional expression, allowing for an assessment of the perceived emotion onset. Both aggressive and nonaggressive offenders demonstrated a delayed perception of primarily fearful facial cues as compared to controls. These results suggest the importance of targeting impaired emotional processing in both types of antisocial behavior.

The Bxb1 recombination system demonstrates heritable transmission of site-specific excision in Arabidopsis

PubMed Central

2012-01-01

Background The mycobacteriophage large serine recombinase Bxb1 catalyzes site-specific recombination between its corresponding attP and attB recognition sites. Previously, we and others have shown that Bxb1 has catalytic activity in various eukaryotic species including Nicotiana tabacum, Schizosaccharomyces pombe, insects and mammalian cells. Results In this work, the Bxb1 recombinase gene was transformed and constitutively expressed in Arabidopsis thaliana plants harboring a chromosomally integrated attP and attB-flanked target sequence. The Bxb1 recombinase successfully excised the target sequence in a conservative manner and the resulting recombination event was heritably transmitted to subsequent generations in the absence of the recombinase transgene. In addition, we also show that Bxb1 recombinase expressing plants can be manually crossed with att-flanked target transgenic plants to generate excised progeny. Conclusion The Bxb1 large serine recombinase performs site-specific recombination in Arabidopsis thaliana germinal tissue, producing stable lines free of unwanted DNA. The precise site-specific deletion produced by Bxb1 in planta demonstrates that this enzyme can be a useful tool for the genetic engineering of plants without selectable marker transgenes or other undesirable exogenous sequences. PMID:22436504

Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: a new Rapid Catch and Signal (RCS) technology.

PubMed

Dragan, Anatoliy I; Golberg, Karina; Elbaz, Amit; Marks, Robert; Zhang, Yongxia; Geddes, Chris D

2011-03-07

For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new "Catch and Signal" technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis. Copyright © 2010 Elsevier B.V. All rights reserved.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

PubMed Central

Martin, Jennifer A.; Smith, Joshua E.; Warren, Mercedes; Chávez, Jorge L.; Hagen, Joshua A.; Kelley-Loughnane, Nancy

2015-01-01

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids. PMID:25870978

Face recognition based on matching of local features on 3D dynamic range sequences

NASA Astrophysics Data System (ADS)

Echeagaray-Patrón, B. A.; Kober, Vitaly

2016-09-01

3D face recognition has attracted attention in the last decade due to improvement of technology of 3D image acquisition and its wide range of applications such as access control, surveillance, human-computer interaction and biometric identification systems. Most research on 3D face recognition has focused on analysis of 3D still data. In this work, a new method for face recognition using dynamic 3D range sequences is proposed. Experimental results are presented and discussed using 3D sequences in the presence of pose variation. The performance of the proposed method is compared with that of conventional face recognition algorithms based on descriptors.

Learning Enterprise Malware Triage from Automatic Dynamic Analysis

DTIC Science & Technology

2013-03-01

Kolter and Maloof n-gram method, Dube’s malware target recognition (MaTR) static method performs significantly more accurately at the 95% confidence...from the static method as in Kolter and Maloof. The MIST approach with behavior sequences 9 allows researchers to tailor the level of analysis to the...citations, none publish work that implements it. Only Kolter and Maloof use nearly as long gram structures, although that research uses static grams rather

Application of Sequence Comparison Methods to Multisensor Data Fusion and Target Recognition

DTIC Science & Technology

1993-06-18

lin- ear comparison). A particularly attractive aspect of the proposed fusion scheme is that it has the potential to work for any object with (1...radar sensing is a historical custom - however, the reader should keep in mind that the fundamental issue in this research is to explore and exploit...reduce the computationally expensive need to compute partial derivatives. In usual practice, the computationally more attractive filter design is

The chaperonin-60 universal target is a barcode for bacteria that enables de novo assembly of metagenomic sequence data.

PubMed

Links, Matthew G; Dumonceaux, Tim J; Hemmingsen, Sean M; Hill, Janet E

2012-01-01

Barcoding with molecular sequences is widely used to catalogue eukaryotic biodiversity. Studies investigating the community dynamics of microbes have relied heavily on gene-centric metagenomic profiling using two genes (16S rRNA and cpn60) to identify and track Bacteria. While there have been criteria formalized for barcoding of eukaryotes, these criteria have not been used to evaluate gene targets for other domains of life. Using the framework of the International Barcode of Life we evaluated DNA barcodes for Bacteria. Candidates from the 16S rRNA gene and the protein coding cpn60 gene were evaluated. Within complete bacterial genomes in the public domain representing 983 species from 21 phyla, the largest difference between median pairwise inter- and intra-specific distances ("barcode gap") was found from cpn60. Distribution of sequence diversity along the ∼555 bp cpn60 target region was remarkably uniform. The barcode gap of the cpn60 universal target facilitated the faithful de novo assembly of full-length operational taxonomic units from pyrosequencing data from a synthetic microbial community. Analysis supported the recognition of both 16S rRNA and cpn60 as DNA barcodes for Bacteria. The cpn60 universal target was found to have a much larger barcode gap than 16S rRNA suggesting cpn60 as a preferred barcode for Bacteria. A large barcode gap for cpn60 provided a robust target for species-level characterization of data. The assembly of consensus sequences for barcodes was shown to be a reliable method for the identification and tracking of novel microbes in metagenomic studies.

Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system.

PubMed

Hogan, Daniel J; Riordan, Daniel P; Gerber, André P; Herschlag, Daniel; Brown, Patrick O

2008-10-28

RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3'-untranslated regions, others in 5'-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate.

The multi-zinc finger protein ZNF217 contacts DNA through a two-finger domain.

PubMed

Nunez, Noelia; Clifton, Molly M K; Funnell, Alister P W; Artuz, Crisbel; Hallal, Samantha; Quinlan, Kate G R; Font, Josep; Vandevenne, Marylène; Setiyaputra, Surya; Pearson, Richard C M; Mackay, Joel P; Crossley, Merlin

2011-11-04

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.

The Multi-zinc Finger Protein ZNF217 Contacts DNA through a Two-finger Domain*

PubMed Central

Nunez, Noelia; Clifton, Molly M. K.; Funnell, Alister P. W.; Artuz, Crisbel; Hallal, Samantha; Quinlan, Kate G. R.; Font, Josep; Vandevenne, Marylène; Setiyaputra, Surya; Pearson, Richard C. M.; Mackay, Joel P.; Crossley, Merlin

2011-01-01

Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif. PMID:21908891

High-speed railway real-time localization auxiliary method based on deep neural network

NASA Astrophysics Data System (ADS)

Chen, Dongjie; Zhang, Wensheng; Yang, Yang

2017-11-01

High-speed railway intelligent monitoring and management system is composed of schedule integration, geographic information, location services, and data mining technology for integration of time and space data. Assistant localization is a significant submodule of the intelligent monitoring system. In practical application, the general access is to capture the image sequences of the components by using a high-definition camera, digital image processing technique and target detection, tracking and even behavior analysis method. In this paper, we present an end-to-end character recognition method based on a deep CNN network called YOLO-toc for high-speed railway pillar plate number. Different from other deep CNNs, YOLO-toc is an end-to-end multi-target detection framework, furthermore, it exhibits a state-of-art performance on real-time detection with a nearly 50fps achieved on GPU (GTX960). Finally, we realize a real-time but high-accuracy pillar plate number recognition system and integrate natural scene OCR into a dedicated classification YOLO-toc model.

Learned Non-Rigid Object Motion is a View-Invariant Cue to Recognizing Novel Objects

PubMed Central

Chuang, Lewis L.; Vuong, Quoc C.; Bülthoff, Heinrich H.

2012-01-01

There is evidence that observers use learned object motion to recognize objects. For instance, studies have shown that reversing the learned direction in which a rigid object rotated in depth impaired recognition accuracy. This motion reversal can be achieved by playing animation sequences of moving objects in reverse frame order. In the current study, we used this sequence-reversal manipulation to investigate whether observers encode the motion of dynamic objects in visual memory, and whether such dynamic representations are encoded in a way that is dependent on the viewing conditions. Participants first learned dynamic novel objects, presented as animation sequences. Following learning, they were then tested on their ability to recognize these learned objects when their animation sequence was shown in the same sequence order as during learning or in the reverse sequence order. In Experiment 1, we found that non-rigid motion contributed to recognition performance; that is, sequence-reversal decreased sensitivity across different tasks. In subsequent experiments, we tested the recognition of non-rigidly deforming (Experiment 2) and rigidly rotating (Experiment 3) objects across novel viewpoints. Recognition performance was affected by viewpoint changes for both experiments. Learned non-rigid motion continued to contribute to recognition performance and this benefit was the same across all viewpoint changes. By comparison, learned rigid motion did not contribute to recognition performance. These results suggest that non-rigid motion provides a source of information for recognizing dynamic objects, which is not affected by changes to viewpoint. PMID:22661939

Noise-robust speech recognition through auditory feature detection and spike sequence decoding.

PubMed

Schafer, Phillip B; Jin, Dezhe Z

2014-03-01

Speech recognition in noisy conditions is a major challenge for computer systems, but the human brain performs it routinely and accurately. Automatic speech recognition (ASR) systems that are inspired by neuroscience can potentially bridge the performance gap between humans and machines. We present a system for noise-robust isolated word recognition that works by decoding sequences of spikes from a population of simulated auditory feature-detecting neurons. Each neuron is trained to respond selectively to a brief spectrotemporal pattern, or feature, drawn from the simulated auditory nerve response to speech. The neural population conveys the time-dependent structure of a sound by its sequence of spikes. We compare two methods for decoding the spike sequences--one using a hidden Markov model-based recognizer, the other using a novel template-based recognition scheme. In the latter case, words are recognized by comparing their spike sequences to template sequences obtained from clean training data, using a similarity measure based on the length of the longest common sub-sequence. Using isolated spoken digits from the AURORA-2 database, we show that our combined system outperforms a state-of-the-art robust speech recognizer at low signal-to-noise ratios. Both the spike-based encoding scheme and the template-based decoding offer gains in noise robustness over traditional speech recognition methods. Our system highlights potential advantages of spike-based acoustic coding and provides a biologically motivated framework for robust ASR development.

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope

PubMed Central

1992-01-01

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202- 221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site. PMID:1383384

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

PubMed

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

A bimetallic nanocomposite modified genosensor for recognition and determination of thalassemia gene.

PubMed

Hamidi-Asl, Ezat; Raoof, Jahan Bakhsh; Naghizadeh, Nahid; Akhavan-Niaki, Haleh; Ojani, Reza; Banihashemi, Ali

2016-10-01

The main roles of DNA in the cells are to maintain and properly express genetic information. It is important to have analytical methods capable of fast and sensitive detection of DNA damage. DNA hybridization sensors are well suited for diagnostics and other purposes, including determination of bacteria and viruses. Beta thalassemias (βth) are due to mutations in the β-globin gene. In this study, an electrochemical biosensor which detects the sequences related to the β-globin gene issued from real samples amplified by polymerase chain reaction (PCR) is described for the first time. The biosensor relies on the immobilization of 20-mer single stranded oligonucleotide (probe) related to βth sequence on the carbon paste electrode (CPE) modified by 15% silver (Ag) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode and hybridization of this oligonucleotide with its complementary sequence (target). The extent of hybridization between the probe and target sequences was shown by using linear sweep voltammetry (LSV) with methylene blue (MB) as hybridization indicator. The selectivity of sensor was investigated using PCR samples containing non-complementary oligonucleotides. The detection limit of biosensor was calculated about 470.0pg/μL. Copyright © 2016 Elsevier B.V. All rights reserved.

Terahertz spectroscopy for the isothermal detection of bacterial DNA by magnetic bead-based rolling circle amplification.

PubMed

Yang, Xiang; Yang, Ke; Zhao, Xiang; Lin, Zhongquan; Liu, Zhiyong; Luo, Sha; Zhang, Yang; Wang, Yunxia; Fu, Weiling

2017-12-04

The demand for rapid and sensitive bacterial detection is continuously increasing due to the significant requirements of various applications. In this study, a terahertz (THz) biosensor based on rolling circle amplification (RCA) was developed for the isothermal detection of bacterial DNA. The synthetic bacterium-specific sequence of 16S rDNA hybridized with a padlock probe (PLP) that contains a sequence fully complementary to the target sequence at the 5' and 3' ends. The linear PLP was circularized by ligation to form a circular PLP upon recognition of the target sequence; then the capture probe (CP) immobilized on magnetic beads (MBs) acted as a primer to initialize RCA. As DNA molecules are much less absorptive than water molecules in the THz range, the RCA products on the surface of the MBs cause a significant decrease in THz absorption, which can be sensitively probed by THz spectroscopy. Our results showed that 0.12 fmol of synthetic bacterial DNA and 0.05 ng μL -1 of genomic DNA could be effectively detected using this assay. In addition, the specificity of this strategy was demonstrated by its low signal response to interfering bacteria. The proposed strategy not only represents a new method for the isothermal detection of the target bacterial DNA but also provides a general methodology for sensitive and specific DNA biosensing using THz spectroscopy.

The consequences of sequence erosion in the evolution of recombination hotspots.

PubMed

Tiemann-Boege, Irene; Schwarz, Theresa; Striedner, Yasmin; Heissl, Angelika

2017-12-19

Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans -acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion.This article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'. © 2017 The Authors.

The consequences of sequence erosion in the evolution of recombination hotspots

PubMed Central

Schwarz, Theresa; Heissl, Angelika

2017-01-01

Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans-acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro. Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’. PMID:29109225

Recognition of Yeast Species from Gene Sequence Comparisons

USDA-ARS?s Scientific Manuscript database

This review discusses recognition of yeast species from gene sequence comparisons, which have been responsible for doubling the number of known yeasts over the past decade. The resolution provided by various single gene sequences is examined for both ascomycetous and basidiomycetous species, and th...

Structural basis of DNA target recognition by the B3 domain of Arabidopsis epigenome reader VAL1

PubMed Central

Sasnauskas, Giedrius; Kauneckaitė, Kotryna; Siksnys, Virginijus

2018-01-01

Abstract Arabidopsis thaliana requires a prolonged period of cold exposure during winter to initiate flowering in a process termed vernalization. Exposure to cold induces epigenetic silencing of the FLOWERING LOCUS C (FLC) gene by Polycomb group (PcG) proteins. A key role in this epigenetic switch is played by transcriptional repressors VAL1 and VAL2, which specifically recognize Sph/RY DNA sequences within FLC via B3 DNA binding domains, and mediate recruitment of PcG silencing machinery. To understand the structural mechanism of site-specific DNA recognition by VAL1, we have solved the crystal structure of VAL1 B3 domain (VAL1-B3) bound to a 12 bp oligoduplex containing the canonical Sph/RY DNA sequence 5′-CATGCA-3′/5′-TGCATG-3′. We find that VAL1-B3 makes H-bonds and van der Waals contacts to DNA bases of all six positions of the canonical Sph/RY element. In agreement with the structure, in vitro DNA binding studies show that VAL1-B3 does not tolerate substitutions at any position of the 5′-TGCATG-3′ sequence. The VAL1-B3–DNA structure presented here provides a structural model for understanding the specificity of plant B3 domains interacting with the Sph/RY and other DNA sequences. PMID:29660015

Influence of time and length size feature selections for human activity sequences recognition.

PubMed

Fang, Hongqing; Chen, Long; Srinivasan, Raghavendiran

2014-01-01

In this paper, Viterbi algorithm based on a hidden Markov model is applied to recognize activity sequences from observed sensors events. Alternative features selections of time feature values of sensors events and activity length size feature values are tested, respectively, and then the results of activity sequences recognition performances of Viterbi algorithm are evaluated. The results show that the selection of larger time feature values of sensor events and/or smaller activity length size feature values will generate relatively better results on the activity sequences recognition performances. © 2013 ISA Published by ISA All rights reserved.

Correlation Time of Ocean Ambient Noise Intensity in San Diego Bay and Target Recognition in Acoustic Daylight Images

NASA Astrophysics Data System (ADS)

Wadsworth, Adam J.

A method for passively detecting and imaging underwater targets using ambient noise as the sole source of illumination (named acoustic daylight) was successfully implemented in the form of the Acoustic Daylight Ocean Noise Imaging System (ADONIS). In a series of imaging experiments conducted in San Diego Bay, where the dominant source of high-frequency ambient noise is snapping shrimp, a large quantity of ambient noise intensity data was collected with the ADONIS (Epifanio, 1997). In a subset of the experimental data sets, fluctuations of time-averaged ambient noise intensity exhibited a diurnal pattern consistent with the increase in frequency of shrimp snapping near dawn and dusk. The same subset of experimental data is revisited here and the correlation time is estimated and analysed for sequences of ambient noise data several minutes in length, with the aim of detecting possible periodicities or other trends in the fluctuation of the shrimp-dominated ambient noise field. Using videos formed from sequences of acoustic daylight images along with other experimental information, candidate segments of static-configuration ADONIS raw ambient noise data were isolated. For each segment, the normalized intensity auto-correlation closely resembled the delta function, the auto-correlation of white noise. No intensity fluctuation patterns at timescales smaller than a few minutes were discernible, suggesting that the shrimp do not communicate, synchronise, or exhibit any periodicities in their snapping. Also presented here is a ADONIS-specific target recognition algorithm based on principal component analysis, along with basic experimental results using a database of acoustic daylight images.

Exploring 3D Human Action Recognition: from Offline to Online.

PubMed

Liu, Zhenyu; Li, Rui; Tan, Jianrong

2018-02-20

With the introduction of cost-effective depth sensors, a tremendous amount of research has been devoted to studying human action recognition using 3D motion data. However, most existing methods work in an offline fashion, i.e., they operate on a segmented sequence. There are a few methods specifically designed for online action recognition, which continually predicts action labels as a stream sequence proceeds. In view of this fact, we propose a question: can we draw inspirations and borrow techniques or descriptors from existing offline methods, and then apply these to online action recognition? Note that extending offline techniques or descriptors to online applications is not straightforward, since at least two problems-including real-time performance and sequence segmentation-are usually not considered in offline action recognition. In this paper, we give a positive answer to the question. To develop applicable online action recognition methods, we carefully explore feature extraction, sequence segmentation, computational costs, and classifier selection. The effectiveness of the developed methods is validated on the MSR 3D Online Action dataset and the MSR Daily Activity 3D dataset.

Exploring 3D Human Action Recognition: from Offline to Online

PubMed Central

Li, Rui; Liu, Zhenyu; Tan, Jianrong

2018-01-01

With the introduction of cost-effective depth sensors, a tremendous amount of research has been devoted to studying human action recognition using 3D motion data. However, most existing methods work in an offline fashion, i.e., they operate on a segmented sequence. There are a few methods specifically designed for online action recognition, which continually predicts action labels as a stream sequence proceeds. In view of this fact, we propose a question: can we draw inspirations and borrow techniques or descriptors from existing offline methods, and then apply these to online action recognition? Note that extending offline techniques or descriptors to online applications is not straightforward, since at least two problems—including real-time performance and sequence segmentation—are usually not considered in offline action recognition. In this paper, we give a positive answer to the question. To develop applicable online action recognition methods, we carefully explore feature extraction, sequence segmentation, computational costs, and classifier selection. The effectiveness of the developed methods is validated on the MSR 3D Online Action dataset and the MSR Daily Activity 3D dataset. PMID:29461502

Fast cat-eye effect target recognition based on saliency extraction

NASA Astrophysics Data System (ADS)

Li, Li; Ren, Jianlin; Wang, Xingbin

2015-09-01

Background complexity is a main reason that results in false detection in cat-eye target recognition. Human vision has selective attention property which can help search the salient target from complex unknown scenes quickly and precisely. In the paper, we propose a novel cat-eye effect target recognition method named Multi-channel Saliency Processing before Fusion (MSPF). This method combines traditional cat-eye target recognition with the selective characters of visual attention. Furthermore, parallel processing enables it to achieve fast recognition. Experimental results show that the proposed method performs better in accuracy, robustness and speed compared to other methods.

psRNATarget: a plant small RNA target analysis server

PubMed Central

Dai, Xinbin; Zhao, Patrick Xuechun

2011-01-01

Plant endogenous non-coding short small RNAs (20–24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to ‘open’ secondary structure around small RNA’s target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/. PMID:21622958

The role of conformational selection in the molecular recognition of the wild type and mutants XPA67-80 peptides by ERCC1.

PubMed

Fadda, Elisa

2015-07-01

Molecular recognition is a fundamental step in the coordination of biomolecular pathways. Understanding how recognition and binding occur between highly flexible protein domains is a complex task. The conformational selection theory provides an elegant rationalization of the recognition mechanism, especially valid in cases when unstructured protein regions are involved. The recognition of a poorly structured peptide, namely XPA67-80 , by its target receptor ERCC1, falls in this challenging study category. The microsecond molecular dynamics (MD) simulations, discussed in this work, show that the conformational propensity of the wild type XPA67-80 peptide in solution supports conformational selection as the key mechanism driving its molecular recognition by ERCC1. Moreover, all the mutations of the XPA67-80 peptide studied here cause a significant increase of its conformational disorder, relative to the wild type. Comparison to experimental data suggests that the loss of the recognized structural motifs at the microscopic time scale can contribute to the critical decrease in binding observed for one of the mutants, further substantiating the key role of conformational selection in recognition. Ultimately, because of the high sequence identity and analogy in binding, it is conceivable that the conclusions of this study on the XPA67-80 peptide also apply to the ERCC1-binding domain of the XPA protein. © 2015 Wiley Periodicals, Inc.

[Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris].

PubMed

Yu, Ai-Ping; Shi, Bing-Xing; Dong, Chun-Na; Jiang, Zhong-Hua; Wu, Zu-Ze

2005-07-01

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.

A mass spectrometry-based multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification.

PubMed

Park, Jung Hun; Jang, Hyowon; Jung, Yun Kyung; Jung, Ye Lim; Shin, Inkyung; Cho, Dae-Yeon; Park, Hyun Gyu

2017-05-15

We herein describe a new mass spectrometry-based method for multiplex SNP genotyping by utilizing allele-specific ligation and strand displacement amplification (SDA) reaction. In this method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site within the PCR-amplified target DNA. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific allele in the target DNA and the resulting ligated product serves as a template to promote the SDA reaction using a universal primer. This process isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. By varying the sizes of the mass markers, we successfully demonstrated the multiplex SNP genotyping capability of this method by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry. Copyright © 2016 Elsevier B.V. All rights reserved.

Binding-induced DNA walker for signal amplification in highly selective electrochemical detection of protein.

PubMed

Ji, Yuhang; Zhang, Lei; Zhu, Longyi; Lei, Jianping; Wu, Jie; Ju, Huangxian

2017-10-15

A binding-induced DNA walker-assisted signal amplification was developed for highly selective electrochemical detection of protein. Firstly, the track of DNA walker was constructed by self-assembly of the high density ferrocene (Fc)-labeled anchor DNA and aptamer 1 on the gold electrode surface. Sequentially, a long swing-arm chain containing aptamer 2 and walking strand DNA was introduced onto gold electrode through aptamers-target specific recognition, and thus initiated walker strand sequences to hybridize with anchor DNA. Then, the DNA walker was activated by the stepwise cleavage of the hybridized anchor DNA by nicking endonuclease to release multiple Fc molecules for signal amplification. Taking thrombin as the model target, the Fc-generated electrochemical signal decreased linearly with logarithm value of thrombin concentration ranging from 10pM to 100nM with a detection limit of 2.5pM under the optimal conditions. By integrating the specific recognition of aptamers to target with the enzymatic cleavage of nicking endonuclease, the aptasensor showed the high selectivity. The binding-induced DNA walker provides a promising strategy for signal amplification in electrochemical biosensor, and has the extensive applications in sensitive and selective detection of the various targets. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

PubMed

Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

2018-01-01

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biosensors for DNA sequence detection

NASA Technical Reports Server (NTRS)

Vercoutere, Wenonah; Akeson, Mark

2002-01-01

DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

Hypotheses on the evolution of hyaluronan: A highly ironic acid

PubMed Central

Csoka, Antonei B; Stern, Robert

2013-01-01

Hyaluronan is a high-molecular-weight glycosaminoglycan (GAG) prominent in the extracellular matrix. Emerging relatively late in evolution, it may have evolved to evade immune recognition. Chondroitin is a more ancient GAG and a possible hyaluronan precursor. Epimerization of a 4-hydroxyl in N-acetylgalactosamine in chondroitin to N-acetylglucosamine of hyaluronan is the only structural difference other than chain length between these two polymers. The axial 4-hydroxyl group extends out perpendicular from the equatorial plane of N-acetylgalactosamine in chondroitin. We suspect that this hydroxyl is a prime target for immune recognition. Conversion of a thumbs-up hydroxyl group into a thumbs-down position in the plane of the sugar endows hyaluronan with the ability to avoid immune recognition. Chitin is another potential precursor to hyaluronan. But regardless whether of chondroitin or of chitin origin, an ancient chondroitinase enzyme sequence seems to have been commandeered to catalyze the cleavage of the new hyaluronan substrate. The evolution of six hyaluronidase-like sequences in the human genome from a single chondroitinase as found in Caenorhabditis elegans can now be traced. Confirming our previous predictions, two duplication events occurred, with three hyaluronidase-like sequences occurring in the genome of Ciona intestinalis (sea squirt), the earliest known chordate. This was probably followed by en masse duplication, with six such genes present in the genome of zebra fish onwards. These events occurred, however, much earlier than predicted. It is also apparent on an evolutionary time scale that in several species, this gene family is continuing to evolve. PMID:23315448

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP.

PubMed

Lin, C H; Patel, D J

1997-11-01

Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP) with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution. The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G.A mismatches are flanked by sheared G.A and reversed Hoogsteen G.G mismatch pairs. The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G.A mismatch formation. The recognition G.A mismatch stacks with a reversed Hoogsteen G.G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10(14) molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.

Automatic Target Recognition Based on Cross-Plot

PubMed Central

Wong, Kelvin Kian Loong; Abbott, Derek

2011-01-01

Automatic target recognition that relies on rapid feature extraction of real-time target from photo-realistic imaging will enable efficient identification of target patterns. To achieve this objective, Cross-plots of binary patterns are explored as potential signatures for the observed target by high-speed capture of the crucial spatial features using minimal computational resources. Target recognition was implemented based on the proposed pattern recognition concept and tested rigorously for its precision and recall performance. We conclude that Cross-plotting is able to produce a digital fingerprint of a target that correlates efficiently and effectively to signatures of patterns having its identity in a target repository. PMID:21980508

Mechanistic insights into phosphoprotein-binding FHA domains.

PubMed

Liang, Xiangyang; Van Doren, Steven R

2008-08-01

[Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

A graphene-based biosensing platform based on the release of DNA probes and rolling circle amplification.

PubMed

Liu, Meng; Song, Jinping; Shuang, Shaomin; Dong, Chuan; Brennan, John D; Li, Yingfu

2014-06-24

We report a versatile biosensing platform capable of achieving ultrasensitive detection of both small-molecule and macromolecular targets. The system features three components: reduced graphene oxide for its ability to adsorb single-stranded DNA molecules nonspecifically, DNA aptamers for their ability to bind reduced graphene oxide but undergo target-induced conformational changes that facilitate their release from the reduced graphene oxide surface, and rolling circle amplification (RCA) for its ability to amplify a primer-template recognition event into repetitive sequence units that can be easily detected. The key to the design is the tagging of a short primer to an aptamer sequence, which results in a small DNA probe that allows for both effective probe adsorption onto the reduced graphene oxide surface to mask the primer domain in the absence of the target, as well as efficient probe release in the presence of the target to make the primer available for template binding and RCA. We also made an observation that the circular template, which on its own does not cause a detectable level of probe release from the reduced graphene oxide, augments target-induced probe release. The synergistic release of DNA probes is interpreted to be a contributing factor for the high detection sensitivity. The broad utility of the platform is illustrated though engineering three different sensors that are capable of achieving ultrasensitive detection of a protein target, a DNA sequence and a small-molecule analyte. We envision that the approach described herein will find useful applications in the biological, medical, and environmental fields.

Hetero-enzyme-based two-round signal amplification strategy for trace detection of aflatoxin B1 using an electrochemical aptasensor.

PubMed

Zheng, Wanli; Teng, Jun; Cheng, Lin; Ye, Yingwang; Pan, Daodong; Wu, Jingjing; Xue, Feng; Liu, Guodong; Chen, Wei

2016-06-15

An electrochemical aptasensor for trace detection of aflatoxin B1 (AFB1) was developed by using an aptamer as the recognition unit while adopting the telomerase and EXO III based two-round signal amplification strategy as the signal enhancement units. The telomerase amplification was used to elongate the ssDNA probes on the surface of gold nanoparticles, by which the signal response range of the signal-off model electrochemical aptasensor could be correspondingly enlarged. Then, the EXO III amplification was used to hydrolyze the 3'-end of the dsDNA after the recognition of target AFB1, which caused the release of bounded AFB1 into the sensing system, where it participated in the next recognition-sensing cycle. With this two-round signal amplified electrochemical aptasensor, target AFB1 was successfully measured at trace concentrations with excellent detection limit of 0.6*10(-4)ppt and satisfied specificity due to the excellent affinity of the aptamer against AFB1. Based on this designed two-round signal amplification strategy, both the sensing range and detection limit were greatly improved. This proposed ultrasensitive electrochemical aptasensor method was also validated by comparison with the classic instrumental methods. Importantly, this hetero-enzyme based two-round signal amplified electrochemical aptasensor offers a great promising protocol for ultrasensitive detection of AFB1 and other mycotoxins by replacing the core recognition sequence of the aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of HMGA2 binding to DNA by netropsin

PubMed Central

Miao, Yi; Cui, Tengjiao; Leng, Fenfei; Wilson, W. David

2008-01-01

The design of small synthetic molecules that can be used to affect gene expression is an area of active interest for development of agents in therapeutic and biotechnology applications. Many compounds that target the minor groove in AT sequences in DNA are well characterized and are promising reagents for use as modulators of protein-DNA complexes. The mammalian high mobility group transcriptional factor, HMGA2, also targets the DNA minor groove and plays critical roles in disease processes from cancer to obesity. Biosensor-surface plasmon resonance methods were used to monitor HMGA2 binding to target sites on immobilized DNA and a competition assay for inhibition of the HMGA2-DNA complex was designed. HMGA2 binds strongly to the DNA through AT hook domains with KD values of 20 - 30 nM depending on the DNA sequence. The well-characterized minor groove binder, netropsin, was used to develop and test the assay. The compound has two binding sites in the protein-DNA interaction sequence and this provides an advantage for inhibition. An equation for analysis of results when the inhibitor has two binding sites in the biopolymer recognition surface is presented with the results. The assay provides a platform for discovery of HMGA2 inhibitors. PMID:18023407

Quantification of DNA cleavage specificity in Hi-C experiments.

PubMed

Meluzzi, Dario; Arya, Gaurav

2016-01-08

Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effective evaluation of privacy protection techniques in visible and thermal imagery

NASA Astrophysics Data System (ADS)

Nawaz, Tahir; Berg, Amanda; Ferryman, James; Ahlberg, Jörgen; Felsberg, Michael

2017-09-01

Privacy protection may be defined as replacing the original content in an image region with a (less intrusive) content having modified target appearance information to make it less recognizable by applying a privacy protection technique. Indeed, the development of privacy protection techniques also needs to be complemented with an established objective evaluation method to facilitate their assessment and comparison. Generally, existing evaluation methods rely on the use of subjective judgments or assume a specific target type in image data and use target detection and recognition accuracies to assess privacy protection. An annotation-free evaluation method that is neither subjective nor assumes a specific target type is proposed. It assesses two key aspects of privacy protection: "protection" and "utility." Protection is quantified as an appearance similarity, and utility is measured as a structural similarity between original and privacy-protected image regions. We performed an extensive experimentation using six challenging datasets (having 12 video sequences), including a new dataset (having six sequences) that contains visible and thermal imagery. The new dataset is made available online for the community. We demonstrate effectiveness of the proposed method by evaluating six image-based privacy protection techniques and also show comparisons of the proposed method over existing methods.

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

PubMed

Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

2015-12-01

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. © 2015 The Authors.

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.

PubMed

Xiao, Yibei; Luo, Min; Hayes, Robert P; Kim, Jonathan; Ng, Sherwin; Ding, Fang; Liao, Maofu; Ke, Ailong

2017-06-29

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Research on application of LADAR in ground vehicle recognition

NASA Astrophysics Data System (ADS)

Lan, Jinhui; Shen, Zhuoxun

2009-11-01

For the requirement of many practical applications in the field of military, the research of 3D target recognition is active. The representation that captures the salient attributes of a 3D target independent of the viewing angle will be especially useful to the automatic 3D target recognition system. This paper presents a new approach of image generation based on Laser Detection and Ranging (LADAR) data. Range image of target is obtained by transformation of point cloud. In order to extract features of different ground vehicle targets and to recognize targets, zernike moment properties of typical ground vehicle targets are researched in this paper. A technique of support vector machine is applied to the classification and recognition of target. The new method of image generation and feature representation has been applied to the outdoor experiments. Through outdoor experiments, it can be proven that the method of image generation is stability, the moments are effective to be used as features for recognition, and the LADAR can be applied to the field of 3D target recognition.

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy

PubMed Central

Matkovich, Scot J.; Dorn, Gerald W.

2018-01-01

Summary MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicates purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses. PMID:25836573

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy.

PubMed

Matkovich, Scot J; Dorn, Gerald W

2015-01-01

MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicate purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses.

Specific minor groove solvation is a crucial determinant of DNA binding site recognition

PubMed Central

Harris, Lydia-Ann; Williams, Loren Dean; Koudelka, Gerald B.

2014-01-01

The DNA sequence preferences of nearly all sequence specific DNA binding proteins are influenced by the identities of bases that are not directly contacted by protein. Discrimination between non-contacted base sequences is commonly based on the differential abilities of DNA sequences to allow narrowing of the DNA minor groove. However, the factors that govern the propensity of minor groove narrowing are not completely understood. Here we show that the differential abilities of various DNA sequences to support formation of a highly ordered and stable minor groove solvation network are a key determinant of non-contacted base recognition by a sequence-specific binding protein. In addition, disrupting the solvent network in the non-contacted region of the binding site alters the protein's ability to recognize contacted base sequences at positions 5–6 bases away. This observation suggests that DNA solvent interactions link contacted and non-contacted base recognition by the protein. PMID:25429976

An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

PubMed

Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

2013-10-15

Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. © 2013 Elsevier B.V. All rights reserved.

Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity*

PubMed Central

Jaeger, Alex M.; Makley, Leah N.; Gestwicki, Jason E.; Thiele, Dennis J.

2014-01-01

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer. PMID:25204655

Research on autonomous identification of airport targets based on Gabor filtering and Radon transform

NASA Astrophysics Data System (ADS)

Yi, Juan; Du, Qingyu; Zhang, Hong jiang; Zhang, Yao lei

2017-11-01

Target recognition is a leading key technology in intelligent image processing and application development at present, with the enhancement of computer processing ability, autonomous target recognition algorithm, gradually improve intelligence, and showed good adaptability. Taking the airport target as the research object, analysis the airport layout characteristics, construction of knowledge model, Gabor filter and Radon transform based on the target recognition algorithm of independent design, image processing and feature extraction of the airport, the algorithm was verified, and achieved better recognition results.

Deep kernel learning method for SAR image target recognition

NASA Astrophysics Data System (ADS)

Chen, Xiuyuan; Peng, Xiyuan; Duan, Ran; Li, Junbao

2017-10-01

With the development of deep learning, research on image target recognition has made great progress in recent years. Remote sensing detection urgently requires target recognition for military, geographic, and other scientific research. This paper aims to solve the synthetic aperture radar image target recognition problem by combining deep and kernel learning. The model, which has a multilayer multiple kernel structure, is optimized layer by layer with the parameters of Support Vector Machine and a gradient descent algorithm. This new deep kernel learning method improves accuracy and achieves competitive recognition results compared with other learning methods.

The effect of letter string length and report condition on letter recognition accuracy.

PubMed

Raghunandan, Avesh; Karmazinaite, Berta; Rossow, Andrea S

Letter sequence recognition accuracy has been postulated to be limited primarily by low-level visual factors. The influence of high level factors such as visual memory (load and decay) has been largely overlooked. This study provides insight into the role of these factors by investigating the interaction between letter sequence recognition accuracy, letter string length and report condition. Letter sequence recognition accuracy for trigrams and pentagrams were measured in 10 adult subjects for two report conditions. In the complete report condition subjects reported all 3 or all 5 letters comprising trigrams and pentagrams, respectively. In the partial report condition, subjects reported only a single letter in the trigram or pentagram. Letters were presented for 100ms and rendered in high contrast, using black lowercase Courier font that subtended 0.4° at the fixation distance of 0.57m. Letter sequence recognition accuracy was consistently higher for trigrams compared to pentagrams especially for letter positions away from fixation. While partial report increased recognition accuracy in both string length conditions, the effect was larger for pentagrams, and most evident for the final letter positions within trigrams and pentagrams. The effect of partial report on recognition accuracy for the final letter positions increased as eccentricity increased away from fixation, and was independent of the inner/outer position of a letter. Higher-level visual memory functions (memory load and decay) play a role in letter sequence recognition accuracy. There is also suggestion of additional delays imposed on memory encoding by crowded letter elements. Copyright © 2016 Spanish General Council of Optometry. Published by Elsevier España, S.L.U. All rights reserved.

Segmentation and Recognition of Continuous Human Activity

DTIC Science & Technology

2001-01-01

This paper presents a methodology for automatic segmentation and recognition of continuous human activity . We segment a continuous human activity into...commencement or termination. We use single action sequences for the training data set. The test sequences, on the other hand, are continuous sequences of human ... activity that consist of three or more actions in succession. The system has been tested on continuous activity sequences containing actions such as

Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

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

Shevtsov, M. B.; Streeter, S. D.; Thresh, S.-J.

2015-02-01

The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers. In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class ofmore » controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.« less

Structure and immune recognition of trimeric pre-fusion HIV-1 Env

DOE PAGES

Pancera, Marie; Zhou, Tongqing; Druz, Aliaksandr; ...

2014-10-08

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed formore » fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. In conclusion, N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.« less

Structure and immune recognition of trimeric pre-fusion HIV-1 Env

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

Pancera, Marie; Zhou, Tongqing; Druz, Aliaksandr

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed formore » fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. In conclusion, N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.« less

TRACX: A Recognition-Based Connectionist Framework for Sequence Segmentation and Chunk Extraction

ERIC Educational Resources Information Center

French, Robert M.; Addyman, Caspar; Mareschal, Denis

2011-01-01

Individuals of all ages extract structure from the sequences of patterns they encounter in their environment, an ability that is at the very heart of cognition. Exactly what underlies this ability has been the subject of much debate over the years. A novel mechanism, implicit chunk recognition (ICR), is proposed for sequence segmentation and chunk…

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

PubMed Central

Rubtsova, Myroslava; Kumlehn, Jochen; Gils, Mario

2012-01-01

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity. PMID:23024817

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA*

PubMed Central

Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

2012-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum. PMID:22767603

Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

PubMed

Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

2013-11-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

Direct Sequence Detection of Structured H5 Influenza Viral RNA

PubMed Central

Kerby, Matthew B.; Freeman, Sarah; Prachanronarong, Kristina; Artenstein, Andrew W.; Opal, Steven M.; Tripathi, Anubhav

2008-01-01

We describe the development of sequence-specific molecular beacons (dual-labeled DNA probes) for identification of the H5 influenza subtype, cleavage motif, and receptor specificity when hybridized directly with in vitro transcribed viral RNA (vRNA). The cloned hemagglutinin segment from a highly pathogenic H5N1 strain, A/Hanoi/30408/2005(H5N1), isolated from humans was used as template for in vitro transcription of sense-strand vRNA. The hybridization behavior of vRNA and a conserved subtype probe was characterized experimentally by varying conditions of time, temperature, and Mg2+ to optimize detection. Comparison of the hybridization rates of probe to DNA and RNA targets indicates that conformational switching of influenza RNA structure is a rate-limiting step and that the secondary structure of vRNA dominates the binding kinetics. The sensitivity and specificity of probe recognition of other H5 strains was calculated from sequence matches to the National Center for Biotechnology Information influenza database. The hybridization specificity of the subtype probes was experimentally verified with point mutations within the probe loop at five locations corresponding to the other human H5 strains. The abundance frequencies of the hemagglutinin cleavage motif and sialic acid recognition sequences were experimentally tested for H5 in all host viral species. Although the detection assay must be coupled with isothermal amplification on the chip, the new probes form the basis of a portable point-of-care diagnostic device for influenza subtyping. PMID:18403607

Polymorphism in Human Cytomegalovirus UL40 Impacts on Recognition of Human Leukocyte Antigen-E (HLA-E) by Natural Killer Cells*

PubMed Central

Heatley, Susan L.; Pietra, Gabriella; Lin, Jie; Widjaja, Jacqueline M. L.; Harpur, Christopher M.; Lester, Sue; Rossjohn, Jamie; Szer, Jeff; Schwarer, Anthony; Bradstock, Kenneth; Bardy, Peter G.; Mingari, Maria Cristina; Moretta, Lorenzo; Sullivan, Lucy C.; Brooks, Andrew G.

2013-01-01

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a “mimic” of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a “polymorphic hot spot” within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors. PMID:23335510

Polymorphism in human cytomegalovirus UL40 impacts on recognition of human leukocyte antigen-E (HLA-E) by natural killer cells.

PubMed

Heatley, Susan L; Pietra, Gabriella; Lin, Jie; Widjaja, Jacqueline M L; Harpur, Christopher M; Lester, Sue; Rossjohn, Jamie; Szer, Jeff; Schwarer, Anthony; Bradstock, Kenneth; Bardy, Peter G; Mingari, Maria Cristina; Moretta, Lorenzo; Sullivan, Lucy C; Brooks, Andrew G

2013-03-22

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.

Detection and recognition of targets by using signal polarization properties

NASA Astrophysics Data System (ADS)

Ponomaryov, Volodymyr I.; Peralta-Fabi, Ricardo; Popov, Anatoly V.; Babakov, Mikhail F.

1999-08-01

The quality of radar target recognition can be enhanced by exploiting its polarization signatures. A specialized X-band polarimetric radar was used for target recognition in experimental investigations. The following polarization characteristics connected to the object geometrical properties were investigated: the amplitudes of the polarization matrix elements; an anisotropy coefficient; depolarization coefficient; asymmetry coefficient; the energy of a backscattering signal; object shape factor. A large quantity of polarimetric radar data was measured and processed to form a database of different object and different weather conditions. The histograms of polarization signatures were approximated by a Nakagami distribution, then used for real- time target recognition. The Neyman-Pearson criterion was used for the target detection, and the criterion of the maximum of a posterior probability was used for recognition problem. Some results of experimental verification of pattern recognition and detection of objects with different electrophysical and geometrical characteristics urban in clutter are presented in this paper.

Active Multimodal Sensor System for Target Recognition and Tracking

PubMed Central

Zhang, Guirong; Zou, Zhaofan; Liu, Ziyue; Mao, Jiansen

2017-01-01

High accuracy target recognition and tracking systems using a single sensor or a passive multisensor set are susceptible to external interferences and exhibit environmental dependencies. These difficulties stem mainly from limitations to the available imaging frequency bands, and a general lack of coherent diversity of the available target-related data. This paper proposes an active multimodal sensor system for target recognition and tracking, consisting of a visible, an infrared, and a hyperspectral sensor. The system makes full use of its multisensor information collection abilities; furthermore, it can actively control different sensors to collect additional data, according to the needs of the real-time target recognition and tracking processes. This level of integration between hardware collection control and data processing is experimentally shown to effectively improve the accuracy and robustness of the target recognition and tracking system. PMID:28657609

Association of a Platinum Complex to a G-Quadruplex Ligand Enhances Telomere Disruption.

PubMed

Charif, Razan; Granotier-Beckers, Christine; Bertrand, Hélène Charlotte; Poupon, Joël; Ségal-Bendirdjian, Evelyne; Teulade-Fichou, Marie-Paule; Boussin, François D; Bombard, Sophie

2017-08-21

Telomeres protect the ends of chromosomes against illegitimate recombination and repair. They can be targets for G-quadruplex ligands and platinum complexes due to their repeated G-rich sequences. Protection of telomeres is ensured by a complex of six proteins, including TRF2, which inhibits the DNA damage response pathway. We analyzed telomere modifications induced in cancer cells by the experimental hybrid platinum complex, Pt-MPQ, comprising both an ethylene diamine monofunctional platinum complex and a G-quadruplex recognition moiety (MPQ). Pt-MPQ promotes the displacement of two telomeric proteins (TRF2 and TRF1) from telomeres, as well as the formation of telomere damage and telomere sister losses, whereas the control compound MPQ does not. This suggests that the platinum moiety potentiates the targeting of the G-quadruplex ligand to telomeres, opening a new perspective for telomere biology and anticancer therapy. Interestingly, the chemotherapy drug cisplatin, which has no specific affinity for G-quadruplex structures, partially induces the TRF2 delocalization from telomeres but produces less telomeric DNA damage, suggesting that this TRF2 displacement could be independent of G-quadruplex recognition.

CRISPR-Cas9 nuclear dynamics and target recognition in living cells

PubMed Central

Ma, Hanhui; Tu, Li-Chun; Zhang, Shaojie; Grunwald, David

2016-01-01

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)–associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9–guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time. PMID:27551060

Method of passive ranging from infrared image sequence based on equivalent area

NASA Astrophysics Data System (ADS)

Yang, Weiping; Shen, Zhenkang

2007-11-01

The information of range between missile and targets is important not only to missile controlling component, but also to automatic target recognition, so studying the technique of passive ranging from infrared images has important theoretic and practical meanings. Here we tried to get the range between guided missile and target and help to identify targets or dodge a hit. The issue of distance between missile and target is currently a hot and difficult research content. As all know, infrared imaging detector can not range so that it restricts the functions of the guided information processing system based on infrared images. In order to break through the technical puzzle, we investigated the principle of the infrared imaging, after analysing the imaging geometric relationship between the guided missile and the target, we brought forward the method of passive ranging based on equivalent area and provided mathematical analytic formulas. Validating Experiments demonstrate that the presented method has good effect, the lowest relative error can reach 10% in some circumstances.

Cooperative DNA binding and sequence discrimination by the Opaque2 bZIP factor.

PubMed Central

Yunes, J A; Vettore, A L; da Silva, M J; Leite, A; Arruda, P

1998-01-01

The maize Opaque2 (O2) protein is a basic leucine zipper transcription factor that controls the expression of distinct classes of endosperm genes through the recognition of different cis-acting elements in their promoters. The O2 target region in the promoter of the alpha-coixin gene was analyzed in detail and shown to comprise two closely adjacent binding sites, named O2u and O2d, which are related in sequence to the GCN4 binding site. Quantitative DNase footprint analysis indicated that O2 binding to alpha-coixin target sites is best described by a cooperative model. Transient expression assays showed that the two adjacent sites act synergistically. This synergy is mediated in part by cooperative DNA binding. In tobacco protoplasts, O2 binding at the O2u site is more important for enhancer activity than is binding at the O2d site, suggesting that the architecture of the O2-DNA complex is important for interaction with the transcriptional machinery. PMID:9811800

Cooperative DNA binding and sequence discrimination by the Opaque2 bZIP factor.

PubMed

Yunes, J A; Vettore, A L; da Silva, M J; Leite, A; Arruda, P

1998-11-01

The maize Opaque2 (O2) protein is a basic leucine zipper transcription factor that controls the expression of distinct classes of endosperm genes through the recognition of different cis-acting elements in their promoters. The O2 target region in the promoter of the alpha-coixin gene was analyzed in detail and shown to comprise two closely adjacent binding sites, named O2u and O2d, which are related in sequence to the GCN4 binding site. Quantitative DNase footprint analysis indicated that O2 binding to alpha-coixin target sites is best described by a cooperative model. Transient expression assays showed that the two adjacent sites act synergistically. This synergy is mediated in part by cooperative DNA binding. In tobacco protoplasts, O2 binding at the O2u site is more important for enhancer activity than is binding at the O2d site, suggesting that the architecture of the O2-DNA complex is important for interaction with the transcriptional machinery.

Recognition of Local DNA Structures by p53 Protein

PubMed Central

Brázda, Václav; Coufal, Jan

2017-01-01

p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells. PMID:28208646

Digital video steganalysis exploiting collusion sensitivity

NASA Astrophysics Data System (ADS)

Budhia, Udit; Kundur, Deepa

2004-09-01

In this paper we present an effective steganalyis technique for digital video sequences based on the collusion attack. Steganalysis is the process of detecting with a high probability and low complexity the presence of covert data in multimedia. Existing algorithms for steganalysis target detecting covert information in still images. When applied directly to video sequences these approaches are suboptimal. In this paper, we present a method that overcomes this limitation by using redundant information present in the temporal domain to detect covert messages in the form of Gaussian watermarks. Our gains are achieved by exploiting the collusion attack that has recently been studied in the field of digital video watermarking, and more sophisticated pattern recognition tools. Applications of our scheme include cybersecurity and cyberforensics.

Widespread Site-Dependent Buffering of Human Regulatory Polymorphism

PubMed Central

Kutyavin, Tanya; Stamatoyannopoulos, John A.

2012-01-01

The average individual is expected to harbor thousands of variants within non-coding genomic regions involved in gene regulation. However, it is currently not possible to interpret reliably the functional consequences of genetic variation within any given transcription factor recognition sequence. To address this, we comprehensively analyzed heritable genome-wide binding patterns of a major sequence-specific regulator (CTCF) in relation to genetic variability in binding site sequences across a multi-generational pedigree. We localized and quantified CTCF occupancy by ChIP-seq in 12 related and unrelated individuals spanning three generations, followed by comprehensive targeted resequencing of the entire CTCF–binding landscape across all individuals. We identified hundreds of variants with reproducible quantitative effects on CTCF occupancy (both positive and negative). While these effects paralleled protein–DNA recognition energetics when averaged, they were extensively buffered by striking local context dependencies. In the significant majority of cases buffering was complete, resulting in silent variants spanning every position within the DNA recognition interface irrespective of level of binding energy or evolutionary constraint. The prevalence of complex partial or complete buffering effects severely constrained the ability to predict reliably the impact of variation within any given binding site instance. Surprisingly, 40% of variants that increased CTCF occupancy occurred at positions of human–chimp divergence, challenging the expectation that the vast majority of functional regulatory variants should be deleterious. Our results suggest that, even in the presence of “perfect” genetic information afforded by resequencing and parallel studies in multiple related individuals, genomic site-specific prediction of the consequences of individual variation in regulatory DNA will require systematic coupling with empirical functional genomic measurements. PMID:22457641

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

PubMed

Han, S; Arvai, A S; Clancy, S B; Tainer, J A

2001-01-05

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework. Copyright 2001 Academic Press.

Road sign recognition with fuzzy adaptive pre-processing models.

PubMed

Lin, Chien-Chuan; Wang, Ming-Shi

2012-01-01

A road sign recognition system based on adaptive image pre-processing models using two fuzzy inference schemes has been proposed. The first fuzzy inference scheme is to check the changes of the light illumination and rich red color of a frame image by the checking areas. The other is to check the variance of vehicle's speed and angle of steering wheel to select an adaptive size and position of the detection area. The Adaboost classifier was employed to detect the road sign candidates from an image and the support vector machine technique was employed to recognize the content of the road sign candidates. The prohibitory and warning road traffic signs are the processing targets in this research. The detection rate in the detection phase is 97.42%. In the recognition phase, the recognition rate is 93.04%. The total accuracy rate of the system is 92.47%. For video sequences, the best accuracy rate is 90.54%, and the average accuracy rate is 80.17%. The average computing time is 51.86 milliseconds per frame. The proposed system can not only overcome low illumination and rich red color around the road sign problems but also offer high detection rates and high computing performance.

Road Sign Recognition with Fuzzy Adaptive Pre-Processing Models

PubMed Central

Lin, Chien-Chuan; Wang, Ming-Shi

2012-01-01

A road sign recognition system based on adaptive image pre-processing models using two fuzzy inference schemes has been proposed. The first fuzzy inference scheme is to check the changes of the light illumination and rich red color of a frame image by the checking areas. The other is to check the variance of vehicle's speed and angle of steering wheel to select an adaptive size and position of the detection area. The Adaboost classifier was employed to detect the road sign candidates from an image and the support vector machine technique was employed to recognize the content of the road sign candidates. The prohibitory and warning road traffic signs are the processing targets in this research. The detection rate in the detection phase is 97.42%. In the recognition phase, the recognition rate is 93.04%. The total accuracy rate of the system is 92.47%. For video sequences, the best accuracy rate is 90.54%, and the average accuracy rate is 80.17%. The average computing time is 51.86 milliseconds per frame. The proposed system can not only overcome low illumination and rich red color around the road sign problems but also offer high detection rates and high computing performance. PMID:22778650

Phosphotyrosine recognition domains: the typical, the atypical and the versatile

PubMed Central

2012-01-01

SH2 domains are long known prominent players in the field of phosphotyrosine recognition within signaling protein networks. However, over the years they have been joined by an increasing number of other protein domain families that can, at least with some of their members, also recognise pTyr residues in a sequence-specific context. This superfamily of pTyr recognition modules, which includes substantial fractions of the PTB domains, as well as much smaller, or even single member fractions like the HYB domain, the PKCδ and PKCθ C2 domains and RKIP, represents a fascinating, medically relevant and hence intensely studied part of the cellular signaling architecture of metazoans. Protein tyrosine phosphorylation clearly serves a plethora of functions and pTyr recognition domains are used in a similarly wide range of interaction modes, which encompass, for example, partner protein switching, tandem recognition functionalities and the interaction with catalytically active protein domains. If looked upon closely enough, virtually no pTyr recognition and regulation event is an exact mirror image of another one in the same cell. Thus, the more we learn about the biology and ultrastructural details of pTyr recognition domains, the more does it become apparent that nature cleverly combines and varies a few basic principles to generate a sheer endless number of sophisticated and highly effective recognition/regulation events that are, under normal conditions, elegantly orchestrated in time and space. This knowledge is also valuable when exploring pTyr reader domains as diagnostic tools, drug targets or therapeutic reagents to combat human diseases. PMID:23134684

Hemispheric asymmetries of a motor memory in a recognition test after learning a movement sequence.

PubMed

Leinen, Peter; Panzer, Stefan; Shea, Charles H

2016-11-01

Two experiments utilizing a spatial-temporal movement sequence were designed to determine if the memory of the sequence is lateralized in the left or right hemisphere. In Experiment 1, dominant right-handers were randomly assigned to one of two acquisition groups: a left-hand starter and a right-hand starter group. After an acquisition phase, reaction time (RT) was measured in a recognition test by providing the learned sequential pattern in the left or right visual half-field for 150ms. In a retention test and two transfer tests the dominant coordinate system for sequence production was evaluated. In Experiment 2 dominant left-handers and dominant right-handers had to acquire the sequence with their dominant limb. The results of Experiment 1 indicated that RT was significantly shorter when the acquired sequence was provided in the right visual field during the recognition test. The same results occurred in Experiment 2 for dominant right-handers and left-handers. These results indicated a right visual field left hemisphere advantage in the recognition test for the practiced stimulus for dominant left and right-handers, when the task was practiced with the dominant limb. Copyright © 2016 Elsevier B.V. All rights reserved.

24/7 security system: 60-FPS color EMCCD camera with integral human recognition

NASA Astrophysics Data System (ADS)

Vogelsong, T. L.; Boult, T. E.; Gardner, D. W.; Woodworth, R.; Johnson, R. C.; Heflin, B.

2007-04-01

An advanced surveillance/security system is being developed for unattended 24/7 image acquisition and automated detection, discrimination, and tracking of humans and vehicles. The low-light video camera incorporates an electron multiplying CCD sensor with a programmable on-chip gain of up to 1000:1, providing effective noise levels of less than 1 electron. The EMCCD camera operates in full color mode under sunlit and moonlit conditions, and monochrome under quarter-moonlight to overcast starlight illumination. Sixty frame per second operation and progressive scanning minimizes motion artifacts. The acquired image sequences are processed with FPGA-compatible real-time algorithms, to detect/localize/track targets and reject non-targets due to clutter under a broad range of illumination conditions and viewing angles. The object detectors that are used are trained from actual image data. Detectors have been developed and demonstrated for faces, upright humans, crawling humans, large animals, cars and trucks. Detection and tracking of targets too small for template-based detection is achieved. For face and vehicle targets the results of the detection are passed to secondary processing to extract recognition templates, which are then compared with a database for identification. When combined with pan-tilt-zoom (PTZ) optics, the resulting system provides a reliable wide-area 24/7 surveillance system that avoids the high life-cycle cost of infrared cameras and image intensifiers.

Next generation sequencing--implications for clinical practice.

PubMed

Raffan, Eleanor; Semple, Robert K

2011-01-01

Genetic testing in inherited disease has traditionally relied upon recognition of the presenting clinical syndrome and targeted analysis of genes known to be linked to that syndrome. Consequently, many patients with genetic syndromes remain without a specific diagnosis. New 'next-generation' sequencing (NGS) techniques permit simultaneous sequencing of enormous amounts of DNA. A slew of research publications have recently demonstrated the tremendous power of these technologies in increasing understanding of human genetic disease. These approaches are likely to be increasingly employed in routine diagnostic practice, but the scale of the genetic information yielded about individuals means that caution must be exercised to avoid net harm in this setting. Use of NGS in a research setting will increasingly have a major but indirect beneficial impact on clinical practice. However, important technical, ethical and social challenges need to be addressed through informed professional and public dialogue before it finds its mature niche as a direct tool in the clinical diagnostic armoury.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

PubMed

Wu, Yushu; Yan, Ping; Xu, Xiaowen; Jiang, Wei

2016-03-07

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases.

PubMed

Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit; Pagès, Hervé; Kucukural, Alper; Garber, Manuel; Wolfe, Scot A

2017-05-15

Genome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed. Here, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction algorithm permitting the identification of genomic sequences with unexpected cleavage activity. The GUIDEseq package enables analysis of GUIDE-data from various nuclease platforms for any species with a defined genomic sequence. This software package has been used successfully to analyze several GUIDE-seq datasets. The software, source code and documentation are freely available at http://www.bioconductor.org/packages/release/bioc/html/GUIDEseq.html .

Triggered optical biosensor

DOEpatents

Song, Xuedong; Swanson, Basil I.

2001-10-02

An optical biosensor is provided for the detection of a multivalent target biomolecule, the biosensor including a substrate having a bilayer membrane thereon, a recognition molecule situated at the surface, the recognition molecule capable of binding with the multivalent target biomolecule, the recognition molecule further characterized as including a fluorescence label thereon and as being movable at the surface and a device for measuring a fluorescence change in response to binding between the recognition molecule and the multivalent target biomolecule.

Quantitative analysis of TALE-DNA interactions suggests polarity effects.

PubMed

Meckler, Joshua F; Bhakta, Mital S; Kim, Moon-Soo; Ovadia, Robert; Habrian, Chris H; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David J; Baldwin, Enoch P

2013-04-01

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN > NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

Targeted exome sequencing for the identification of a protective variant against Internet gaming disorder at rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3): A pilot study

PubMed Central

Kim, Jeong-Yu; Jeong, Jo-Eun; Rhee, Je-Keun; Cho, Hyun; Chun, Ji-Won; Kim, Tae-Min; Choi, Sam-Wook; Choi, Jung-Seok; Kim, Dai-Jin

2016-01-01

Background and aims Internet gaming disorder (IGD) has gained recognition as a potential new diagnosis in the fifth revision of the Diagnostic and Statistical Manual of Mental Disorders, but genetic evidence supporting this disorder remains scarce. Methods In this study, targeted exome sequencing was conducted in 30 IGD patients and 30 control subjects with a focus on genes linked to various neurotransmitters associated with substance and non-substance addictions, depression, and attention deficit hyperactivity disorder. Results rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3) was the only single nucleotide polymorphism (SNP) that exhibited a significantly different minor allele frequency in IGD subjects compared to controls (p = .01932), suggesting that this SNP has a protective effect against IGD (odds ratio = 0.1541). The presence of this potentially protective allele was also associated with less time spent on Internet gaming and lower scores on the Young’s Internet Addiction Test and Korean Internet Addiction Proneness Scale for Adults. Conclusions The results of this first targeted exome sequencing study of IGD subjects indicate that rs2229910 of NTRK3 is a genetic variant that is significantly related to IGD. These findings may have significant implications for future research investigating the genetics of IGD and other behavioral addictions. PMID:27826991

Cistrome of the aldosterone-activated mineralocorticoid receptor in human renal cells.

PubMed

Le Billan, Florian; Khan, Junaid A; Lamribet, Khadija; Viengchareun, Say; Bouligand, Jérôme; Fagart, Jérôme; Lombès, Marc

2015-09-01

Aldosterone exerts its effects mainly by activating the mineralocorticoid receptor (MR), a transcription factor that regulates gene expression through complex and dynamic interactions with coregulators and transcriptional machinery, leading to fine-tuned control of vectorial ionic transport in the distal nephron. To identify genome-wide aldosterone-regulated MR targets in human renal cells, we set up a chromatin immunoprecipitation (ChIP) assay by using a specific anti-MR antibody in a differentiated human renal cell line expressing green fluorescent protein (GFP)-MR. This approach, coupled with high-throughput sequencing, allowed identification of 974 genomic MR targets. Computational analysis identified an MR response element (MRE) including single or multiple half-sites and palindromic motifs in which the AGtACAgxatGTtCt sequence was the most prevalent motif. Most genomic MR-binding sites (MBSs) are located >10 kb from the transcriptional start sites of target genes (84%). Specific aldosterone-induced recruitment of MR on the first most relevant genomic sequences was further validated by ChIP-quantitative (q)PCR and correlated with concomitant and positive aldosterone-activated transcriptional regulation of the corresponding gene, as assayed by RT-qPCR. It was notable that most MBSs lacked MREs but harbored DNA recognition motifs for other transcription factors (FOX, EGR1, AP1, PAX5) suggesting functional interaction. This work provides new insights into aldosterone MR-mediated renal signaling and opens relevant perspectives for mineralocorticoid-related pathophysiology. © FASEB.

A variable DNA recognition site organization establishes the LiaR-mediated cell envelope stress response of enterococci to daptomycin

DOE PAGES

Davlieva, Milya; Shi, Yiwen; Leonard, Paul G.; ...

2015-04-19

LiaR is a ‘master regulator’ of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structuralmore » basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR D191N increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. The crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.« less

Songbirds use spectral shape, not pitch, for sound pattern recognition

PubMed Central

Bregman, Micah R.; Patel, Aniruddh D.; Gentner, Timothy Q.

2016-01-01

Humans easily recognize “transposed” musical melodies shifted up or down in log frequency. Surprisingly, songbirds seem to lack this capacity, although they can learn to recognize human melodies and use complex acoustic sequences for communication. Decades of research have led to the widespread belief that songbirds, unlike humans, are strongly biased to use absolute pitch (AP) in melody recognition. This work relies almost exclusively on acoustically simple stimuli that may belie sensitivities to more complex spectral features. Here, we investigate melody recognition in a species of songbird, the European Starling (Sturnus vulgaris), using tone sequences that vary in both pitch and timbre. We find that small manipulations altering either pitch or timbre independently can drive melody recognition to chance, suggesting that both percepts are poor descriptors of the perceptual cues used by birds for this task. Instead we show that melody recognition can generalize even in the absence of pitch, as long as the spectral shapes of the constituent tones are preserved. These results challenge conventional views regarding the use of pitch cues in nonhuman auditory sequence recognition. PMID:26811447

Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

PubMed

Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

2013-04-01

Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

PubMed Central

Lorenz, Felix K. M.; Wilde, Susanne; Voigt, Katrin; Kieback, Elisa; Mosetter, Barbara; Schendel, Dolores J.; Uckert, Wolfgang

2015-01-01

Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. PMID:25799237

Interactive object recognition assistance: an approach to recognition starting from target objects

NASA Astrophysics Data System (ADS)

Geisler, Juergen; Littfass, Michael

1999-07-01

Recognition of target objects in remotely sensed imagery required detailed knowledge about the target object domain as well as about mapping properties of the sensing system. The art of object recognition is to combine both worlds appropriately and to provide models of target appearance with respect to sensor characteristics. Common approaches to support interactive object recognition are either driven from the sensor point of view and address the problem of displaying images in a manner adequate to the sensing system. Or they focus on target objects and provide exhaustive encyclopedic information about this domain. Our paper discusses an approach to assist interactive object recognition based on knowledge about target objects and taking into account the significance of object features with respect to characteristics of the sensed imagery, e.g. spatial and spectral resolution. An `interactive recognition assistant' takes the image analyst through the interpretation process by indicating step-by-step the respectively most significant features of objects in an actual set of candidates. The significance of object features is expressed by pregenerated trees of significance, and by the dynamic computation of decision relevance for every feature at each step of the recognition process. In the context of this approach we discuss the question of modeling and storing the multisensorial/multispectral appearances of target objects and object classes as well as the problem of an adequate dynamic human-machine-interface that takes into account various mental models of human image interpretation.

Promoter scanning of the Human COX-2 gene with 8-ring polyamides: unexpected weakening of polyamide-DNA binding and selectivity by replacing an internal N-Me-pyrrole with β-alanine

PubMed Central

Aston, Karl; Ramos, Joseph P.; Koeller, Kevin J.; Nanjunda, Rupesh; He, Gaofei

2012-01-01

Rules for polyamide DNA recognition have proved invaluable for the design of sequence-selective DNA-binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal β-alanines as polyamide building blocks in place of N-methyl pyrrole; β-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327–339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with β-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with β-alanine to generate a β/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that β-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition. PMID:23023196

The chemical structure of DNA sequence signals for RNA transcription

NASA Technical Reports Server (NTRS)

George, D. G.; Dayhoff, M. O.

1982-01-01

The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

Doppler-Only Synthetic Aperture Radar

DTIC Science & Technology

2006-12-01

5 B. TARGET RECOGNITION TECHNIQUES .................................................6 1. Cooperative Targets...6 3. Techniques ............................................................................................6 C. TARGET RECOGNITION...3. Implementation of High Range Resolution Techniques .................12 F. TWO-DIMENSIONAL IMAGING

REBASE--a database for DNA restriction and modification: enzymes, genes and genomes.

PubMed

Roberts, Richard J; Vincze, Tamas; Posfai, Janos; Macelis, Dana

2015-01-01

REBASE is a comprehensive and fully curated database of information about the components of restriction-modification (RM) systems. It contains fully referenced information about recognition and cleavage sites for both restriction enzymes and methyltransferases as well as commercial availability, methylation sensitivity, crystal and sequence data. All genomes that are completely sequenced are analyzed for RM system components, and with the advent of PacBio sequencing, the recognition sequences of DNA methyltransferases (MTases) are appearing rapidly. Thus, Type I and Type III systems can now be characterized in terms of recognition specificity merely by DNA sequencing. The contents of REBASE may be browsed from the web http://rebase.neb.com and selected compilations can be downloaded by FTP (ftp.neb.com). Monthly updates are also available via email. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans

PubMed Central

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-01-01

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans. PMID:22508728

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

PubMed

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-04-15

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Recognition of A. thaliana centromeres by heterologous CENH3 requires high similarity to the endogenous protein.

PubMed

Moraes, Izabel C R; Lermontova, Inna; Schubert, Ingo

2011-02-01

The centromere is an essential chromosomal component assembling the kinetochore for chromosome attachment to the spindle microtubules and for directing the chromosome segregation during nuclear division. Kinetochore assembly requires deposition of the centromeric histone H3 variant (CENH3) into centromeric nucleosomes. CENH3 has a variable N-terminal and a more conserved C-terminal part, including the loop1 region of the histone fold domain, which is considered to be critical for centromere targeting. To investigate the structural requirements for centromere targeting, constructs for EYFP-tagged CENH3 of A. lyrata, A. arenosa, Capsella bursa-pastoris, Zea mays and Luzula nivea (the latter with holocentric chromosomes) were transformed into A. thaliana. Except for LnCENH3, all recombinant CENH3 proteins targeted A. thaliana centromeres, but the more distantly related the heterologous protein is, the lower is the efficiency of targeting. Alignment of CENH3 sequences revealed that the tested species share only three amino acids at loop1 region: threonine2, arginine12 and alanine15. These three amino acids were substituted by asparagine, proline and valine encoding sequences within a recombinant EYFP-AtCENH3 construct via PCR mutagenesis prior to transformation of A. thaliana. After transformation, immunostaining of root tip nuclei with anti-GFP antibodies yielded only diffuse signals, indicating that the original three amino acids are necessary but not sufficient for targeting A. thaliana centromeres.

Emerging branches of the N-end rule pathways are revealing the sequence complexities of N-termini dependent protein degradation.

PubMed

Eldeeb, Mohamed A; Leitao, Luana C A; Fahlman, Richard P

2018-06-01

The N-end rule links the identity of the N-terminal amino acid of a protein to its in vivo half-life, as some N-terminal residues confer metabolic instability to a protein via their recognition by the cellular machinery that targets them for degradation. Since its discovery, the N-end rule has generally been defined as set of rules of whether an N-terminal residue is stabilizing or not. However, recent studies are revealing that the N-terminal code of amino acids conferring protein instability is more complex than previously appreciated, as recent investigations are revealing that the identity of adjoining downstream residues can also influence the metabolic stability of N-end rule substrate. This is exemplified by the recent discovery of a new branch of N-end rule pathways that target proteins bearing N-terminal proline. In addition, recent investigations are demonstrating that the molecular machinery in N-termini dependent protein degradation may also target proteins for lysosomal degradation, in addition to proteasome-dependent degradation. Herein, we describe some of the recent advances in N-end rule pathways and discuss some of the implications regarding the emerging additional sequence requirements.

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

PubMed Central

Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

2015-01-01

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

Forecasting Occurrences of Activities.

PubMed

Minor, Bryan; Cook, Diane J

2017-07-01

While activity recognition has been shown to be valuable for pervasive computing applications, less work has focused on techniques for forecasting the future occurrence of activities. We present an activity forecasting method to predict the time that will elapse until a target activity occurs. This method generates an activity forecast using a regression tree classifier and offers an advantage over sequence prediction methods in that it can predict expected time until an activity occurs. We evaluate this algorithm on real-world smart home datasets and provide evidence that our proposed approach is most effective at predicting activity timings.

Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis.

PubMed

He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

2016-04-15

The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors.

Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

NASA Astrophysics Data System (ADS)

Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

1988-08-01

Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

Modeling guidance and recognition in categorical search: bridging human and computer object detection.

PubMed

Zelinsky, Gregory J; Peng, Yifan; Berg, Alexander C; Samaras, Dimitris

2013-10-08

Search is commonly described as a repeating cycle of guidance to target-like objects, followed by the recognition of these objects as targets or distractors. Are these indeed separate processes using different visual features? We addressed this question by comparing observer behavior to that of support vector machine (SVM) models trained on guidance and recognition tasks. Observers searched for a categorically defined teddy bear target in four-object arrays. Target-absent trials consisted of random category distractors rated in their visual similarity to teddy bears. Guidance, quantified as first-fixated objects during search, was strongest for targets, followed by target-similar, medium-similarity, and target-dissimilar distractors. False positive errors to first-fixated distractors also decreased with increasing dissimilarity to the target category. To model guidance, nine teddy bear detectors, using features ranging in biological plausibility, were trained on unblurred bears then tested on blurred versions of the same objects appearing in each search display. Guidance estimates were based on target probabilities obtained from these detectors. To model recognition, nine bear/nonbear classifiers, trained and tested on unblurred objects, were used to classify the object that would be fixated first (based on the detector estimates) as a teddy bear or a distractor. Patterns of categorical guidance and recognition accuracy were modeled almost perfectly by an HMAX model in combination with a color histogram feature. We conclude that guidance and recognition in the context of search are not separate processes mediated by different features, and that what the literature knows as guidance is really recognition performed on blurred objects viewed in the visual periphery.

Modeling guidance and recognition in categorical search: Bridging human and computer object detection

PubMed Central

Zelinsky, Gregory J.; Peng, Yifan; Berg, Alexander C.; Samaras, Dimitris

2013-01-01

Search is commonly described as a repeating cycle of guidance to target-like objects, followed by the recognition of these objects as targets or distractors. Are these indeed separate processes using different visual features? We addressed this question by comparing observer behavior to that of support vector machine (SVM) models trained on guidance and recognition tasks. Observers searched for a categorically defined teddy bear target in four-object arrays. Target-absent trials consisted of random category distractors rated in their visual similarity to teddy bears. Guidance, quantified as first-fixated objects during search, was strongest for targets, followed by target-similar, medium-similarity, and target-dissimilar distractors. False positive errors to first-fixated distractors also decreased with increasing dissimilarity to the target category. To model guidance, nine teddy bear detectors, using features ranging in biological plausibility, were trained on unblurred bears then tested on blurred versions of the same objects appearing in each search display. Guidance estimates were based on target probabilities obtained from these detectors. To model recognition, nine bear/nonbear classifiers, trained and tested on unblurred objects, were used to classify the object that would be fixated first (based on the detector estimates) as a teddy bear or a distractor. Patterns of categorical guidance and recognition accuracy were modeled almost perfectly by an HMAX model in combination with a color histogram feature. We conclude that guidance and recognition in the context of search are not separate processes mediated by different features, and that what the literature knows as guidance is really recognition performed on blurred objects viewed in the visual periphery. PMID:24105460

Learning of goal-relevant and -irrelevant complex visual sequences in human V1.

PubMed

Rosenthal, Clive R; Mallik, Indira; Caballero-Gaudes, Cesar; Sereno, Martin I; Soto, David

2018-06-12

Learning and memory are supported by a network involving the medial temporal lobe and linked neocortical regions. Emerging evidence indicates that primary visual cortex (i.e., V1) may contribute to recognition memory, but this has been tested only with a single visuospatial sequence as the target memorandum. The present study used functional magnetic resonance imaging to investigate whether human V1 can support the learning of multiple, concurrent complex visual sequences involving discontinous (second-order) associations. Two peripheral, goal-irrelevant but structured sequences of orientated gratings appeared simultaneously in fixed locations of the right and left visual fields alongside a central, goal-relevant sequence that was in the focus of spatial attention. Pseudorandom sequences were introduced at multiple intervals during the presentation of the three structured visual sequences to provide an online measure of sequence-specific knowledge at each retinotopic location. We found that a network involving the precuneus and V1 was involved in learning the structured sequence presented at central fixation, whereas right V1 was modulated by repeated exposure to the concurrent structured sequence presented in the left visual field. The same result was not found in left V1. These results indicate for the first time that human V1 can support the learning of multiple concurrent sequences involving complex discontinuous inter-item associations, even peripheral sequences that are goal-irrelevant. Copyright © 2018. Published by Elsevier Inc.

Evaluation and control of miRNA-like off-target repression for RNA interference.

PubMed

Seok, Heeyoung; Lee, Haejeong; Jang, Eun-Sook; Chi, Sung Wook

2018-03-01

RNA interference (RNAi) has been widely adopted to repress specific gene expression and is easily achieved by designing small interfering RNAs (siRNAs) with perfect sequence complementarity to the intended target mRNAs. Although siRNAs direct Argonaute (Ago), a core component of the RNA-induced silencing complex (RISC), to recognize and silence target mRNAs, they also inevitably function as microRNAs (miRNAs) and suppress hundreds of off-targets. Such miRNA-like off-target repression is potentially detrimental, resulting in unwanted toxicity and phenotypes. Despite early recognition of the severity of miRNA-like off-target repression, this effect has often been overlooked because of difficulties in recognizing and avoiding off-targets. However, recent advances in genome-wide methods and knowledge of Ago-miRNA target interactions have set the stage for properly evaluating and controlling miRNA-like off-target repression. Here, we describe the intrinsic problems of miRNA-like off-target effects caused by canonical and noncanonical interactions. We particularly focus on various genome-wide approaches and chemical modifications for the evaluation and prevention of off-target repression to facilitate the use of RNAi with secured specificity.

Test Sequence Priming in Recognition Memory

ERIC Educational Resources Information Center

Johns, Elizabeth E.; Mewhort, D. J. K.

2009-01-01

The authors examined priming within the test sequence in 3 recognition memory experiments. A probe primed its successor whenever both probes shared a feature with the same studied item ("interjacent priming"), indicating that the study item like the probe is central to the decision. Interjacent priming occurred even when the 2 probes did…

Immediate-Early Transactivator Rta of Epstein-Barr Virus (EBV) Shows Multiple Epitopes Recognized by EBV-Specific Cytotoxic T Lymphocytes

PubMed Central

Pepperl, Sandra; Benninger-Döring, Gerlinde; Modrow, Susanne; Wolf, Hans; Jilg, Wolfgang

1998-01-01

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells. PMID:9765404

Beta carbonic anhydrases: novel targets for pesticides and anti-parasitic agents in agriculture and livestock husbandry.

PubMed

Zolfaghari Emameh, Reza; Barker, Harlan; Hytönen, Vesa P; Tolvanen, Martti E E; Parkkila, Seppo

2014-08-29

The genomes of many insect and parasite species contain beta carbonic anhydrase (β-CA) protein coding sequences. The lack of β-CA proteins in mammals makes them interesting target proteins for inhibition in treatment of some infectious diseases and pests. Many insects and parasites represent important pests for agriculture and cause enormous economic damage worldwide. Meanwhile, pollution of the environment by old pesticides, emergence of strains resistant to them, and their off-target effects are major challenges for agriculture and society. In this study, we analyzed a multiple sequence alignment of 31 β-CAs from insects, some parasites, and selected plant species relevant to agriculture and livestock husbandry. Using bioinformatics tools a phylogenetic tree was generated and the subcellular localizations and antigenic sites of each protein were predicted. Structural models for β-CAs of Ancylostoma caninum, Ascaris suum, Trichinella spiralis, and Entamoeba histolytica, were built using Pisum sativum and Mycobacterium tuberculosis β-CAs as templates. Six β-CAs of insects and parasites and six β-CAs of plants are predicted to be mitochondrial and chloroplastic, respectively, and thus may be involved in important metabolic functions. All 31 sequences showed the presence of the highly conserved β-CA active site sequence motifs, CXDXR and HXXC (C: cysteine, D: aspartic acid, R: arginine, H: histidine, X: any residue). We discovered that these two motifs are more antigenic than others. Homology models suggested that these motifs are mostly buried and thus not well accessible for recognition by antibodies. The predicted mitochondrial localization of several β-CAs and hidden antigenic epitopes within the protein molecule, suggest that they may not be considered major targets for vaccines. Instead, they are promising candidate enzymes for small-molecule inhibitors which can easily penetrate the cell membrane. Based on current knowledge, we conclude that β-CAs are potential targets for development of small molecule pesticides or anti-parasitic agents with minimal side effects on vertebrates.

Molecular evolution of candidate male reproductive genes in the brown algal model Ectocarpus.

PubMed

Lipinska, Agnieszka P; Van Damme, Els J M; De Clerck, Olivier

2016-01-05

Evolutionary studies of genes that mediate recognition between sperm and egg contribute to our understanding of reproductive isolation and speciation. Surface receptors involved in fertilization are targets of sexual selection, reinforcement, and other evolutionary forces including positive selection. This observation was made across different lineages of the eukaryotic tree from land plants to mammals, and is particularly evident in free-spawning animals. Here we use the brown algal model species Ectocarpus (Phaeophyceae) to investigate the evolution of candidate gamete recognition proteins in a distant major phylogenetic group of eukaryotes. Male gamete specific genes were identified by comparing transcriptome data covering different stages of the Ectocarpus life cycle and screened for characteristics expected from gamete recognition receptors. Selected genes were sequenced in a representative number of strains from distant geographical locations and varying stages of reproductive isolation, to search for signatures of adaptive evolution. One of the genes (Esi0130_0068) showed evidence of selective pressure. Interestingly, that gene displayed domain similarities to the receptor for egg jelly (REJ) protein involved in sperm-egg recognition in sea urchins. We have identified a male gamete specific gene with similarity to known gamete recognition receptors and signatures of adaptation. Altogether, this gene could contribute to gamete interaction during reproduction as well as reproductive isolation in Ectocarpus and is therefore a good candidate for further functional evaluation.

The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition.

PubMed

Tamayo, Joel V; Teramoto, Takamasa; Chatterjee, Seema; Hall, Traci M Tanaka; Gavis, Elizabeth R

2017-04-04

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo's RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subset of Glo's functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Drosophila hnRNP F/H homolog glorund uses two distinct RNA-binding modes to diversify target recognition

DOE PAGES

Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema; ...

2017-04-04

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subsetmore » of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Lastly, our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.« less

The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition

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

Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subsetmore » of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.« less

The Drosophila hnRNP F/H homolog glorund uses two distinct RNA-binding modes to diversify target recognition

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

Tamayo, Joel V.; Teramoto, Takamasa; Chatterjee, Seema

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo’s RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subsetmore » of Glo’s functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Lastly, our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.« less

Investigation of the mechanism of meiotic DNA cleavage by VMA1-derived endonuclease uncovers a meiotic alteration in chromatin structure around the target site.

PubMed

Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

2006-06-01

VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation.

Investigation of the Mechanism of Meiotic DNA Cleavage by VMA1-Derived Endonuclease Uncovers a Meiotic Alteration in Chromatin Structure around the Target Site

PubMed Central

Fukuda, Tomoyuki; Ohta, Kunihiro; Ohya, Yoshikazu

2006-01-01

VMA1-derived endonuclease (VDE), a homing endonuclease in Saccharomyces cerevisiae, is encoded by the mobile intein-coding sequence within the nuclear VMA1 gene. VDE recognizes and cleaves DNA at the 31-bp VDE recognition sequence (VRS) in the VMA1 gene lacking the intein-coding sequence during meiosis to insert a copy of the intein-coding sequence at the cleaved site. The mechanism underlying the meiosis specificity of VMA1 intein-coding sequence homing remains unclear. We studied various factors that might influence the cleavage activity in vivo and found that VDE binding to the VRS can be detected only when DNA cleavage by VDE takes place, implying that meiosis-specific DNA cleavage is regulated by the accessibility of VDE to its target site. As a possible candidate for the determinant of this accessibility, we analyzed chromatin structure around the VRS and revealed that local chromatin structure near the VRS is altered during meiosis. Although the meiotic chromatin alteration exhibits correlations with DNA binding and cleavage by VDE at the VMA1 locus, such a chromatin alteration is not necessarily observed when the VRS is embedded in ectopic gene loci. This suggests that nucleosome positioning or occupancy around the VRS by itself is not the sole mechanism for the regulation of meiosis-specific DNA cleavage by VDE and that other mechanisms are involved in the regulation. PMID:16757746

Directed evolution of the TALE N-terminal domain for recognition of all 5′ bases

PubMed Central

Lamb, Brian M.; Mercer, Andrew C.; Barbas, Carlos F.

2013-01-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5′-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5′ T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5′ T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes. PMID:23980031

Model-based recognition of 3D articulated target using ladar range data.

PubMed

Lv, Dan; Sun, Jian-Feng; Li, Qi; Wang, Qi

2015-06-10

Ladar is suitable for 3D target recognition because ladar range images can provide rich 3D geometric surface information of targets. In this paper, we propose a part-based 3D model matching technique to recognize articulated ground military vehicles in ladar range images. The key of this approach is to solve the decomposition and pose estimation of articulated parts of targets. The articulated components were decomposed into isolate parts based on 3D geometric properties of targets, such as surface point normals, data histogram distribution, and data distance relationships. The corresponding poses of these separate parts were estimated through the linear characteristics of barrels. According to these pose parameters, all parts of the target were roughly aligned to 3D point cloud models in a library and fine matching was finally performed to accomplish 3D articulated target recognition. The recognition performance was evaluated with 1728 ladar range images of eight different articulated military vehicles with various part types and orientations. Experimental results demonstrated that the proposed approach achieved a high recognition rate.

Research on gait-based human identification

NASA Astrophysics Data System (ADS)

Li, Youguo

Gait recognition refers to automatic identification of individual based on his/her style of walking. This paper proposes a gait recognition method based on Continuous Hidden Markov Model with Mixture of Gaussians(G-CHMM). First, we initialize a Gaussian mix model for training image sequence with K-means algorithm, then train the HMM parameters using a Baum-Welch algorithm. These gait feature sequences can be trained and obtain a Continuous HMM for every person, therefore, the 7 key frames and the obtained HMM can represent each person's gait sequence. Finally, the recognition is achieved by Front algorithm. The experiments made on CASIA gait databases obtain comparatively high correction identification ratio and comparatively strong robustness for variety of bodily angle.

Research and Implementation of Tibetan Word Segmentation Based on Syllable Methods

NASA Astrophysics Data System (ADS)

Jiang, Jing; Li, Yachao; Jiang, Tao; Yu, Hongzhi

2018-03-01

Tibetan word segmentation (TWS) is an important problem in Tibetan information processing, while abbreviated word recognition is one of the key and most difficult problems in TWS. Most of the existing methods of Tibetan abbreviated word recognition are rule-based approaches, which need vocabulary support. In this paper, we propose a method based on sequence tagging model for abbreviated word recognition, and then implement in TWS systems with sequence labeling models. The experimental results show that our abbreviated word recognition method is fast and effective and can be combined easily with the segmentation model. This significantly increases the effect of the Tibetan word segmentation.

Pattern recognition of electronic bit-sequences using a semiconductor mode-locked laser and spatial light modulators

NASA Astrophysics Data System (ADS)

Bhooplapur, Sharad; Akbulut, Mehmetkan; Quinlan, Franklyn; Delfyett, Peter J.

2010-04-01

A novel scheme for recognition of electronic bit-sequences is demonstrated. Two electronic bit-sequences that are to be compared are each mapped to a unique code from a set of Walsh-Hadamard codes. The codes are then encoded in parallel on the spectral phase of the frequency comb lines from a frequency-stabilized mode-locked semiconductor laser. Phase encoding is achieved by using two independent spatial light modulators based on liquid crystal arrays. Encoded pulses are compared using interferometric pulse detection and differential balanced photodetection. Orthogonal codes eight bits long are compared, and matched codes are successfully distinguished from mismatched codes with very low error rates, of around 10-18. This technique has potential for high-speed, high accuracy recognition of bit-sequences, with applications in keyword searches and internet protocol packet routing.

Deep Learning Improves Antimicrobial Peptide Recognition.

PubMed

Veltri, Daniel; Kamath, Uday; Shehu, Amarda

2018-03-24

Bacterial resistance to antibiotics is a growing concern. Antimicrobial peptides (AMPs), natural components of innate immunity, are popular targets for developing new drugs. Machine learning methods are now commonly adopted by wet-laboratory researchers to screen for promising candidates. In this work we utilize deep learning to recognize antimicrobial activity. We propose a neural network model with convolutional and recurrent layers that leverage primary sequence composition. Results show that the proposed model outperforms state-of-the-art classification models on a comprehensive data set. By utilizing the embedding weights, we also present a reduced-alphabet representation and show that reasonable AMP recognition can be maintained using nine amino-acid types. Models and data sets are made freely available through the Antimicrobial Peptide Scanner vr.2 web server at: www.ampscanner.com. amarda@gmu.edu for general inquiries and dan.veltri@gmail.com for web server information. Supplementary data are available at Bioinformatics online.

Recognition Tunneling

PubMed Central

Lindsay, Stuart; He, Jin; Sankey, Otto; Hapala, Prokop; Jelinek, Pavel; Zhang, Peiming; Chang, Shuai; Huang, Shuo

2010-01-01

Single molecules in a tunnel junction can now be interrogated reliably using chemically-functionalized electrodes. Monitoring stochastic bonding fluctuations between a ligand bound to one electrode and its target bound to a second electrode (“tethered molecule-pair” configuration) gives insight into the nature of the intermolecular bonding at a single molecule-pair level, and defines the requirements for reproducible tunneling data. Simulations show that there is an instability in the tunnel gap at large currents, and this results in a multiplicity of contacts with a corresponding spread in the measured currents. At small currents (i.e. large gaps) the gap is stable, and functionalizing a pair of electrodes with recognition reagents (the “free analyte” configuration) can generate a distinct tunneling signal when an analyte molecule is trapped in the gap. This opens up a new interface between chemistry and electronics with immediate implications for rapid sequencing of single DNA molecules. PMID:20522930

Targeting RNA in mammalian systems with small molecules.

PubMed

Donlic, Anita; Hargrove, Amanda E

2018-05-03

The recognition of RNA functions beyond canonical protein synthesis has challenged the central dogma of molecular biology. Indeed, RNA is now known to directly regulate many important cellular processes, including transcription, splicing, translation, and epigenetic modifications. The misregulation of these processes in disease has led to an appreciation of RNA as a therapeutic target. This potential was first recognized in bacteria and viruses, but discoveries of new RNA classes following the sequencing of the human genome have invigorated exploration of its disease-related functions in mammals. As stable structure formation is evolving as a hallmark of mammalian RNAs, the prospect of utilizing small molecules to specifically probe the function of RNA structural domains and their interactions is gaining increased recognition. To date, researchers have discovered bioactive small molecules that modulate phenotypes by binding to expanded repeats, microRNAs, G-quadruplex structures, and RNA splice sites in neurological disorders, cancers, and other diseases. The lessons learned from achieving these successes both call for additional studies and encourage exploration of the plethora of mammalian RNAs whose precise mechanisms of action remain to be elucidated. Efforts toward understanding fundamental principles of small molecule-RNA recognition combined with advances in methodology development should pave the way toward targeting emerging RNA classes such as long noncoding RNAs. Together, these endeavors can unlock the full potential of small molecule-based probing of RNA-regulated processes and enable us to discover new biology and underexplored avenues for therapeutic intervention in human disease. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA in Disease and Development > RNA in Disease. © 2018 Wiley Periodicals, Inc.

Structural Insights Into the Recognition of Peroxisomal Targeting Signal 1 By Trypanosoma Brucei Peroxin 5

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

Sampathkumar, P.; Roach, C.; Michels, P.A.M.

2009-05-27

Glycosomes are peroxisome-like organelles essential for trypanosomatid parasites. Glycosome biogenesis is mediated by proteins called 'peroxins,' which are considered to be promising drug targets in pathogenic Trypanosomatidae. The first step during protein translocation across the glycosomal membrane of peroxisomal targeting signal 1 (PTS1)-harboring proteins is signal recognition by the cytosolic receptor peroxin 5 (PEX5). The C-terminal PTS1 motifs interact with the PTS1 binding domain (P1BD) of PEX5, which is made up of seven tetratricopeptide repeats. Obtaining diffraction-quality crystals of the P1BD of Trypanosoma brucei PEX5 (TbPEX5) required surface entropy reduction mutagenesis. Each of the seven tetratricopeptide repeats appears to havemore » a residue in the alpha(L) conformation in the loop connecting helices A and B. Five crystal structures of the P1BD of TbPEX5 were determined, each in complex with a hepta- or decapeptide corresponding to a natural or nonnatural PTS1 sequence. The PTS1 peptides are bound between the two subdomains of the P1BD. These structures indicate precise recognition of the C-terminal Leu of the PTS1 motif and important interactions between the PTS1 peptide main chain and up to five invariant Asn side chains of PEX5. The TbPEX5 structures reported here reveal a unique hydrophobic pocket in the subdomain interface that might be explored to obtain compounds that prevent relative motions of the subdomains and interfere selectively with PTS1 motif binding or release in trypanosomatids, and would therefore disrupt glycosome biogenesis and prevent parasite growth.« less

TALE: a tale of genome editing.

PubMed

Zhang, Mingjie; Wang, Feng; Li, Shifei; Wang, Yan; Bai, Yun; Xu, Xueqing

2014-01-01

Transcription activator-like effectors (TALEs), first identified in Xanthomonas bacteria, are naturally occurring or artificially designed proteins that modulate gene transcription. These proteins recognize and bind DNA sequences based on a variable numbers of tandem repeats. Each repeat is comprised of a set of ∼ 34 conserved amino acids; within this conserved domain, there are usually two amino acids that distinguish one TALE from another. Interestingly, TALEs have revealed a simple cipher for the one-to-one recognition of proteins for DNA bases. Synthetic TALEs have been used to successfully target genes in a variety of species, including humans. Depending on the type of functional domain that is fused to the TALE of interest, these proteins can have diverse biological effects. For example, after binding DNA, TALEs fused to transcriptional activation domains can function as robust transcription factors (TALE-TFs), while fused to restriction endonucleases (TALENs) can cut DNA. Targeted genome editing, in theory, is capable of modifying any endogenous gene sequence of interest; this can be performed in cells or organisms, and may be applied to clinical gene-based therapies in the future. With current technologies, highly accurate, specific, and reliable gene editing cannot be achieved. Thus, recognition and binding mechanisms governing TALE biology are currently hot research areas. In this review, we summarize the major advances in TALE technology over the past several years with a focus on the interaction between TALEs and DNA, TALE design and construction, potential applications for this technology, and unique characteristics that make TALEs superior to zinc finger endonucleases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Deep Recurrent Neural Networks for Human Activity Recognition

PubMed Central

Murad, Abdulmajid

2017-01-01

Adopting deep learning methods for human activity recognition has been effective in extracting discriminative features from raw input sequences acquired from body-worn sensors. Although human movements are encoded in a sequence of successive samples in time, typical machine learning methods perform recognition tasks without exploiting the temporal correlations between input data samples. Convolutional neural networks (CNNs) address this issue by using convolutions across a one-dimensional temporal sequence to capture dependencies among input data. However, the size of convolutional kernels restricts the captured range of dependencies between data samples. As a result, typical models are unadaptable to a wide range of activity-recognition configurations and require fixed-length input windows. In this paper, we propose the use of deep recurrent neural networks (DRNNs) for building recognition models that are capable of capturing long-range dependencies in variable-length input sequences. We present unidirectional, bidirectional, and cascaded architectures based on long short-term memory (LSTM) DRNNs and evaluate their effectiveness on miscellaneous benchmark datasets. Experimental results show that our proposed models outperform methods employing conventional machine learning, such as support vector machine (SVM) and k-nearest neighbors (KNN). Additionally, the proposed models yield better performance than other deep learning techniques, such as deep believe networks (DBNs) and CNNs. PMID:29113103

Deep Recurrent Neural Networks for Human Activity Recognition.

PubMed

Murad, Abdulmajid; Pyun, Jae-Young

2017-11-06

Adopting deep learning methods for human activity recognition has been effective in extracting discriminative features from raw input sequences acquired from body-worn sensors. Although human movements are encoded in a sequence of successive samples in time, typical machine learning methods perform recognition tasks without exploiting the temporal correlations between input data samples. Convolutional neural networks (CNNs) address this issue by using convolutions across a one-dimensional temporal sequence to capture dependencies among input data. However, the size of convolutional kernels restricts the captured range of dependencies between data samples. As a result, typical models are unadaptable to a wide range of activity-recognition configurations and require fixed-length input windows. In this paper, we propose the use of deep recurrent neural networks (DRNNs) for building recognition models that are capable of capturing long-range dependencies in variable-length input sequences. We present unidirectional, bidirectional, and cascaded architectures based on long short-term memory (LSTM) DRNNs and evaluate their effectiveness on miscellaneous benchmark datasets. Experimental results show that our proposed models outperform methods employing conventional machine learning, such as support vector machine (SVM) and k-nearest neighbors (KNN). Additionally, the proposed models yield better performance than other deep learning techniques, such as deep believe networks (DBNs) and CNNs.

Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid β-related Behavioral Deficits in a Mouse Model of Alzheimer Disease

PubMed Central

Basi, Guriqbal S.; Feinberg, Hadar; Oshidari, Farshid; Anderson, John; Barbour, Robin; Baker, Jeanne; Comery, Thomas A.; Diep, Linnea; Gill, Davinder; Johnson-Wood, Kelly; Goel, Amita; Grantcharova, Katerina; Lee, Mike; Li, Jingzhi; Partridge, Anthony; Griswold-Prenner, Irene; Piot, Nicolas; Walker, Don; Widom, Angela; Pangalos, Menelas N.; Seubert, Peter; Jacobsen, J. Steven; Schenk, Dale; Weis, William I.

2010-01-01

Immunotherapy targeting of amyloid β (Aβ) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Aβ-targeted immunotherapy has also been demonstrated to reverse Aβ-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Aβ, Aβ3–7, differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Aβ in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Aβ. The conformation of the Aβ peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Aβ:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Aβ species postulated to underlie cognitive deficits in AD. PMID:19923222

Ground target recognition using rectangle estimation.

PubMed

Grönwall, Christina; Gustafsson, Fredrik; Millnert, Mille

2006-11-01

We propose a ground target recognition method based on 3-D laser radar data. The method handles general 3-D scattered data. It is based on the fact that man-made objects of complex shape can be decomposed to a set of rectangles. The ground target recognition method consists of four steps; 3-D size and orientation estimation, target segmentation into parts of approximately rectangular shape, identification of segments that represent the target's functional/main parts, and target matching with CAD models. The core in this approach is rectangle estimation. The performance of the rectangle estimation method is evaluated statistically using Monte Carlo simulations. A case study on tank recognition is shown, where 3-D data from four fundamentally different types of laser radar systems are used. Although the approach is tested on rather few examples, we believe that the approach is promising.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

PubMed

Miller, Jeffrey C; Zhang, Lei; Xia, Danny F; Campo, John J; Ankoudinova, Irina V; Guschin, Dmitry Y; Babiarz, Joshua E; Meng, Xiangdong; Hinkley, Sarah J; Lam, Stephen C; Paschon, David E; Vincent, Anna I; Dulay, Gladys P; Barlow, Kyle A; Shivak, David A; Leung, Elo; Kim, Jinwon D; Amora, Rainier; Urnov, Fyodor D; Gregory, Philip D; Rebar, Edward J

2015-05-01

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

Mucin-based targeted pancreatic cancer therapy.

PubMed

Torres, Maria P; Chakraborty, Subhankar; Souchek, Joshua; Batra, Surinder K

2012-01-01

The prognosis of pancreatic cancer (PC) patients is very poor with a five-year survival of less than 5%. One of the major challenges in developing new therapies for PC is the lack of expression of specific markers by pancreatic tumor cells. Mucins are heavily Oglycosylated proteins characterized by the presence of short stretches of amino acid sequences repeated several times in tandem. The expression of several mucins including MUC1, MUC4, MUC5AC, and MUC16 is strongly upregulated in PC. Recent studies have also demonstrated a link between the aberrant expression and differential overexpression of mucin glycoproteins to the initiation, progression, and poor prognosis of the disease. These studies have led to increasing recognition of mucins as potential diagnostic markers and therapeutic targets in PC. In this focused review we present an overview of the therapies targeting mucins in PC, including immunotherapy (i.e. vaccines, antibodies, and radioimmunoconjugates), gene therapy, and other novel therapeutic strategies.

Minimal effects of visual memory training on the auditory performance of adult cochlear implant users

PubMed Central

Oba, Sandra I.; Galvin, John J.; Fu, Qian-Jie

2014-01-01

Auditory training has been shown to significantly improve cochlear implant (CI) users’ speech and music perception. However, it is unclear whether post-training gains in performance were due to improved auditory perception or to generally improved attention, memory and/or cognitive processing. In this study, speech and music perception, as well as auditory and visual memory were assessed in ten CI users before, during, and after training with a non-auditory task. A visual digit span (VDS) task was used for training, in which subjects recalled sequences of digits presented visually. After the VDS training, VDS performance significantly improved. However, there were no significant improvements for most auditory outcome measures (auditory digit span, phoneme recognition, sentence recognition in noise, digit recognition in noise), except for small (but significant) improvements in vocal emotion recognition and melodic contour identification. Post-training gains were much smaller with the non-auditory VDS training than observed in previous auditory training studies with CI users. The results suggest that post-training gains observed in previous studies were not solely attributable to improved attention or memory, and were more likely due to improved auditory perception. The results also suggest that CI users may require targeted auditory training to improve speech and music perception. PMID:23516087

Shape and texture fused recognition of flying targets

NASA Astrophysics Data System (ADS)

Kovács, Levente; Utasi, Ákos; Kovács, Andrea; Szirányi, Tamás

2011-06-01

This paper presents visual detection and recognition of flying targets (e.g. planes, missiles) based on automatically extracted shape and object texture information, for application areas like alerting, recognition and tracking. Targets are extracted based on robust background modeling and a novel contour extraction approach, and object recognition is done by comparisons to shape and texture based query results on a previously gathered real life object dataset. Application areas involve passive defense scenarios, including automatic object detection and tracking with cheap commodity hardware components (CPU, camera and GPS).

Target recognitions in multiple-camera closed-circuit television using color constancy

NASA Astrophysics Data System (ADS)

Soori, Umair; Yuen, Peter; Han, Ji Wen; Ibrahim, Izzati; Chen, Wentao; Hong, Kan; Merfort, Christian; James, David; Richardson, Mark

2013-04-01

People tracking in crowded scenes from closed-circuit television (CCTV) footage has been a popular and challenging task in computer vision. Due to the limited spatial resolution in the CCTV footage, the color of people's dress may offer an alternative feature for their recognition and tracking. However, there are many factors, such as variable illumination conditions, viewing angles, and camera calibration, that may induce illusive modification of intrinsic color signatures of the target. Our objective is to recognize and track targets in multiple camera views using color as the detection feature, and to understand if a color constancy (CC) approach may help to reduce these color illusions due to illumination and camera artifacts and thereby improve target recognition performance. We have tested a number of CC algorithms using various color descriptors to assess the efficiency of target recognition from a real multicamera Imagery Library for Intelligent Detection Systems (i-LIDS) data set. Various classifiers have been used for target detection, and the figure of merit to assess the efficiency of target recognition is achieved through the area under the receiver operating characteristics (AUROC). We have proposed two modifications of luminance-based CC algorithms: one with a color transfer mechanism and the other using a pixel-wise sigmoid function for an adaptive dynamic range compression, a method termed enhanced luminance reflectance CC (ELRCC). We found that both algorithms improve the efficiency of target recognitions substantially better than that of the raw data without CC treatment, and in some cases the ELRCC improves target tracking by over 100% within the AUROC assessment metric. The performance of the ELRCC has been assessed over 10 selected targets from three different camera views of the i-LIDS footage, and the averaged target recognition efficiency over all these targets is found to be improved by about 54% in AUROC after the data are processed by the proposed ELRCC algorithm. This amount of improvement represents a reduction of probability of false alarm by about a factor of 5 at the probability of detection of 0.5. Our study concerns mainly the detection of colored targets; and issues for the recognition of white or gray targets will be addressed in a forthcoming study.

On-chip learning of hyper-spectral data for real time target recognition

NASA Technical Reports Server (NTRS)

Duong, T. A.; Daud, T.; Thakoor, A.

2000-01-01

As the focus of our present paper, we have used the cascade error projection (CEP) learning algorithm (shown to be hardware-implementable) with on-chip learning (OCL) scheme to obtain three orders of magnitude speed-up in target recognition compared to software-based learning schemes. Thus, it is shown, real time learning as well as data processing for target recognition can be achieved.

Detection of possible restriction sites for type II restriction enzymes in DNA sequences.

PubMed

Gagniuc, P; Cimponeriu, D; Ionescu-Tîrgovişte, C; Mihai, Andrada; Stavarachi, Monica; Mihai, T; Gavrilă, L

2011-01-01

In order to make a step forward in the knowledge of the mechanism operating in complex polygenic disorders such as diabetes and obesity, this paper proposes a new algorithm (PRSD -possible restriction site detection) and its implementation in Applied Genetics software. This software can be used for in silico detection of potential (hidden) recognition sites for endonucleases and for nucleotide repeats identification. The recognition sites for endonucleases may result from hidden sequences through deletion or insertion of a specific number of nucleotides. Tests were conducted on DNA sequences downloaded from NCBI servers using specific recognition sites for common type II restriction enzymes introduced in the software database (n = 126). Each possible recognition site indicated by the PRSD algorithm implemented in Applied Genetics was checked and confirmed by NEBcutter V2.0 and Webcutter 2.0 software. In the sequence NG_008724.1 (which includes 63632 nucleotides) we found a high number of potential restriction sites for ECO R1 that may be produced by deletion (n = 43 sites) or insertion (n = 591 sites) of one nucleotide. The second module of Applied Genetics has been designed to find simple repeats sizes with a real future in understanding the role of SNPs (Single Nucleotide Polymorphisms) in the pathogenesis of the complex metabolic disorders. We have tested the presence of simple repetitive sequences in five DNA sequence. The software indicated exact position of each repeats detected in the tested sequences. Future development of Applied Genetics can provide an alternative for powerful tools used to search for restriction sites or repetitive sequences or to improve genotyping methods.

Event-related potential correlates of declarative and non-declarative sequence knowledge.

PubMed

Ferdinand, Nicola K; Rünger, Dennis; Frensch, Peter A; Mecklinger, Axel

2010-07-01

The goal of the present study was to demonstrate that declarative and non-declarative knowledge acquired in an incidental sequence learning task contributes differentially to memory retrieval and leads to dissociable ERP signatures in a recognition memory task. For this purpose, participants performed a sequence learning task and were classified as verbalizers, partial verbalizers, or nonverbalizers according to their ability to verbally report the systematic response sequence. Thereafter, ERPs were recorded in a recognition memory task time-locked to sequence triplets that were either part of the previously learned sequence or not. Although all three groups executed old sequence triplets faster than new triplets in the recognition memory task, qualitatively distinct ERP patterns were found for participants with and without reportable knowledge. Verbalizers and, to a lesser extent, partial verbalizers showed an ERP correlate of recollection for parts of the incidentally learned sequence. In contrast, nonverbalizers showed a different ERP effect with a reverse polarity that might reflect priming. This indicates that an ensemble of qualitatively different processes is at work when declarative and non-declarative sequence knowledge is retrieved. By this, our findings favor a multiple-systems view postulating that explicit and implicit learning are supported by different and functionally independent systems. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Emotionally conditioning the target-speech voice enhances recognition of the target speech under "cocktail-party" listening conditions.

PubMed

Lu, Lingxi; Bao, Xiaohan; Chen, Jing; Qu, Tianshu; Wu, Xihong; Li, Liang

2018-05-01

Under a noisy "cocktail-party" listening condition with multiple people talking, listeners can use various perceptual/cognitive unmasking cues to improve recognition of the target speech against informational speech-on-speech masking. One potential unmasking cue is the emotion expressed in a speech voice, by means of certain acoustical features. However, it was unclear whether emotionally conditioning a target-speech voice that has none of the typical acoustical features of emotions (i.e., an emotionally neutral voice) can be used by listeners for enhancing target-speech recognition under speech-on-speech masking conditions. In this study we examined the recognition of target speech against a two-talker speech masker both before and after the emotionally neutral target voice was paired with a loud female screaming sound that has a marked negative emotional valence. The results showed that recognition of the target speech (especially the first keyword in a target sentence) was significantly improved by emotionally conditioning the target speaker's voice. Moreover, the emotional unmasking effect was independent of the unmasking effect of the perceived spatial separation between the target speech and the masker. Also, (skin conductance) electrodermal responses became stronger after emotional learning when the target speech and masker were perceptually co-located, suggesting an increase of listening efforts when the target speech was informationally masked. These results indicate that emotionally conditioning the target speaker's voice does not change the acoustical parameters of the target-speech stimuli, but the emotionally conditioned vocal features can be used as cues for unmasking target speech.

Crosslinking transcription factors to their recognition sequences with PtII complexes

NASA Technical Reports Server (NTRS)

Chu, B. C.; Orgel, L. E.

1992-01-01

We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.

Target recognition of ladar range images using even-order Zernike moments.

PubMed

Liu, Zheng-Jun; Li, Qi; Xia, Zhi-Wei; Wang, Qi

2012-11-01

Ladar range images have attracted considerable attention in automatic target recognition fields. In this paper, Zernike moments (ZMs) are applied to classify the target of the range image from an arbitrary azimuth angle. However, ZMs suffer from high computational costs. To improve the performance of target recognition based on small samples, even-order ZMs with serial-parallel backpropagation neural networks (BPNNs) are applied to recognize the target of the range image. It is found that the rotation invariance and classified performance of the even-order ZMs are both better than for odd-order moments and for moments compressed by principal component analysis. The experimental results demonstrate that combining the even-order ZMs with serial-parallel BPNNs can significantly improve the recognition rate for small samples.

Mapping a disordered portion of the Brz2001-binding site on a plant monooxygenase, DWARF4, using a quartz-crystal microbalance biosensor-based T7 phage display.

PubMed

Takakusagi, Yoichi; Manita, Daisuke; Kusayanagi, Tomoe; Izaguirre-Carbonell, Jesus; Takakusagi, Kaori; Kuramochi, Kouji; Iwabata, Kazuki; Kanai, Yoshihiro; Sakaguchi, Kengo; Sugawara, Fumio

2013-04-01

In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.

Views of Translational Research from a Somewhat Translational Scientist

PubMed Central

Talman, William T.

2013-01-01

This review arose from a talk entitled “Identifying Targets” and given by the author at EB2011 at the invitation of the American Federation for Medical Research (AFMR). The presentation was part of the AFMR workshop entitled “Keys for Translation: Science and Strategy” and focused on identifying clinically relevant targets as a result of observations made during basic scientific studies. The review emphasizes that targets do not have to be the aim that drives basic discovery, but communication between the basic scientist and clinical investigators may aid recognition of such targets and their translation to clinical applications. Using one line of investigator-initiated research from his own laboratory as an example, the author emphasizes that basic discovery must be hypothesis driven and allowed to follow its logical sequence. Finding treatments, while always an aim of biomedical research, may arise as a result of basic studies that were not originally aimed at a target of translational research. PMID:22781556

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

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

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE PAGES

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; ...

2017-09-11

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

In vivo Discovery of Immunotherapy Targets in the Tumor Microenvironment

PubMed Central

Zhou, Penghui; Shaffer, Donald R.; Arias, Diana A. Alvarez; Nakazaki, Yukoh; Pos, Wouter; Torres, Alexis J.; Cremasco, Viviana; Dougan, Stephanie K.; Cowley, Glenn S.; Elpek, Kutlu; Brogdon, Jennifer; Lamb, John; Turley, Shannon; Ploegh, Hidde L.; Root, David E.; Love, J. Christopher; Dranoff, Glenn; Hacohen, Nir; Cantor, Harvey; Wucherpfennig, Kai W.

2014-01-01

Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T cell function in immunosuppressive tumors are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumor microenvironment. We devised an in vivo pooled shRNA screen in which shRNAs targeting negative regulators became highly enriched in tumors by releasing a block on T cell proliferation upon tumor antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumor or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family: In tumors, Ppp2r2d knockdown inhibited T cell apoptosis and enhanced T cell proliferation as well as cytokine production. Key regulators of immune function can thus be discovered in relevant tissue microenvironments. PMID:24476824

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

PubMed Central

Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

2003-01-01

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

Unsupervised learning in persistent sensing for target recognition by wireless ad hoc networks of ground-based sensors

NASA Astrophysics Data System (ADS)

Hortos, William S.

2008-04-01

In previous work by the author, effective persistent and pervasive sensing for recognition and tracking of battlefield targets were seen to be achieved, using intelligent algorithms implemented by distributed mobile agents over a composite system of unmanned aerial vehicles (UAVs) for persistence and a wireless network of unattended ground sensors for pervasive coverage of the mission environment. While simulated performance results for the supervised algorithms of the composite system are shown to provide satisfactory target recognition over relatively brief periods of system operation, this performance can degrade by as much as 50% as target dynamics in the environment evolve beyond the period of system operation in which the training data are representative. To overcome this limitation, this paper applies the distributed approach using mobile agents to the network of ground-based wireless sensors alone, without the UAV subsystem, to provide persistent as well as pervasive sensing for target recognition and tracking. The supervised algorithms used in the earlier work are supplanted by unsupervised routines, including competitive-learning neural networks (CLNNs) and new versions of support vector machines (SVMs) for characterization of an unknown target environment. To capture the same physical phenomena from battlefield targets as the composite system, the suite of ground-based sensors can be expanded to include imaging and video capabilities. The spatial density of deployed sensor nodes is increased to allow more precise ground-based location and tracking of detected targets by active nodes. The "swarm" mobile agents enabling WSN intelligence are organized in a three processing stages: detection, recognition and sustained tracking of ground targets. Features formed from the compressed sensor data are down-selected according to an information-theoretic algorithm that reduces redundancy within the feature set, reducing the dimension of samples used in the target recognition and tracking routines. Target tracking is based on simplified versions of Kalman filtration. Accuracy of recognition and tracking of implemented versions of the proposed suite of unsupervised algorithms is somewhat degraded from the ideal. Target recognition and tracking by supervised routines and by unsupervised SVM and CLNN routines in the ground-based WSN is evaluated in simulations using published system values and sensor data from vehicular targets in ground-surveillance scenarios. Results are compared with previously published performance for the system of the ground-based sensor network (GSN) and UAV swarm.

Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation

PubMed Central

Huska, Matthew R.; Jurk, Marcel; Schöpflin, Robert; Starick, Stephan R.; Schwahn, Kevin; Cooper, Samantha B.; Yamamoto, Keith R.; Thomas-Chollier, Morgane; Vingron, Martin

2017-01-01

Abstract The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter–proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined. PMID:27903902

Changes in behavior and salivary cortisol after targeted cognitive training in typical 12-month-old infants.

PubMed

Wass, Sam V; Cook, Clare; Clackson, Kaili

2017-05-01

Previous research has suggested that early development may be an optimal period to implement cognitive training interventions, particularly those relating to attention control, a basic ability that is essential for the development of other cognitive skills. In the present study, we administered gaze-contingent training (95 min across 2 weeks) targeted at voluntary attention control to a cohort of typical 12-month-old children (N = 24) and sham training to a control group (N = 24). We assessed training effects on (a) tasks involving nontrained aspects of attention control: visual sustained attention, habituation speed, visual recognition memory, sequence learning, and reversal learning; (b) general attentiveness (on-task behaviors during testing); and (c) salivary cortisol levels. Assessments were administered immediately after the cessation of training and at a 6-week follow-up. On the immediate posttest infants showed significantly more sustained visual attention, faster habituation, and improved sequence learning. Significant effects were also found for increased general attentiveness and decreased salivary cortisol. Some of these effects were still evident at the 6-week follow-up (significantly improved sequence learning and marginally improved sustained attention). These findings extend the emerging literature showing that attention training is possible in infancy. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Gaussian mixture models-based ship target recognition algorithm in remote sensing infrared images

NASA Astrophysics Data System (ADS)

Yao, Shoukui; Qin, Xiaojuan

2018-02-01

Since the resolution of remote sensing infrared images is low, the features of ship targets become unstable. The issue of how to recognize ships with fuzzy features is an open problem. In this paper, we propose a novel ship target recognition algorithm based on Gaussian mixture models (GMMs). In the proposed algorithm, there are mainly two steps. At the first step, the Hu moments of these ship target images are calculated, and the GMMs are trained on the moment features of ships. At the second step, the moment feature of each ship image is assigned to the trained GMMs for recognition. Because of the scale, rotation, translation invariance property of Hu moments and the power feature-space description ability of GMMs, the GMMs-based ship target recognition algorithm can recognize ship reliably. Experimental results of a large simulating image set show that our approach is effective in distinguishing different ship types, and obtains a satisfactory ship recognition performance.

Constraints in distortion-invariant target recognition system simulation

NASA Astrophysics Data System (ADS)

Iftekharuddin, Khan M.; Razzaque, Md A.

2000-11-01

Automatic target recognition (ATR) is a mature but active research area. In an earlier paper, we proposed a novel ATR approach for recognition of targets varying in fine details, rotation, and translation using a Learning Vector Quantization (LVQ) Neural Network (NN). The proposed approach performed segmentation of multiple objects and the identification of the objects using LVQNN. In this current paper, we extend the previous approach for recognition of targets varying in rotation, translation, scale, and combination of all three distortions. We obtain the analytical results of the system level design to show that the approach performs well with some constraints. The first constraint determines the size of the input images and input filters. The second constraint shows the limits on amount of rotation, translation, and scale of input objects. We present the simulation verification of the constraints using DARPA's Moving and Stationary Target Recognition (MSTAR) images with different depression and pose angles. The simulation results using MSTAR images verify the analytical constraints of the system level design.

FRankenstein becomes a cyborg: the automatic recombination and realignment of fold recognition models in CASP6.

PubMed

Kosinski, Jan; Gajda, Michal J; Cymerman, Iwona A; Kurowski, Michal A; Pawlowski, Marcin; Boniecki, Michal; Obarska, Agnieszka; Papaj, Grzegorz; Sroczynska-Obuchowicz, Paulina; Tkaczuk, Karolina L; Sniezynska, Paulina; Sasin, Joanna M; Augustyn, Anna; Bujnicki, Janusz M; Feder, Marcin

2005-01-01

In the course of CASP6, we generated models for all targets using a new version of the "FRankenstein's monster approach." Previously (in CASP5) we were able to build many very accurate full-atom models by selection and recombination of well-folded fragments obtained from crude fold recognition (FR) results, followed by optimization of the sequence-structure fit and assessment of alternative alignments on the structural level. This procedure was however very arduous, as most of the steps required extensive visual and manual input from the human modeler. Now, we have automated the most tedious steps, such as superposition of alternative models, extraction of best-scoring fragments, and construction of a hybrid "monster" structure, as well as generation of alternative alignments in the regions that remain poorly scored in the refined hybrid model. We have also included the ROSETTA method to construct those parts of the target for which no reasonable structures were generated by FR methods (such as long insertions and terminal extensions). The analysis of successes and failures of the current version of the FRankenstein approach in modeling of CASP6 targets reveals that the considerably streamlined and automated method performs almost as well as the initial, mostly manual version, which suggests that it may be a useful tool for accurate protein structure prediction even in the hands of nonexperts. 2005 Wiley-Liss, Inc.

Target recognition and phase acquisition by using incoherent digital holographic imaging

NASA Astrophysics Data System (ADS)

Lee, Munseob; Lee, Byung-Tak

2017-05-01

In this study, we proposed the Incoherent Digital Holographic Imaging (IDHI) for recognition and phase information of dedicated target. Although recent development of a number of target recognition techniques such as LIDAR, there have limited success in target discrimination, in part due to low-resolution, low scanning speed, and computation power. In the paper, the proposed system consists of the incoherent light source, such as LED, Michelson interferometer, and digital CCD for acquisition of four phase shifting image. First of all, to compare with relative coherence, we used a source as laser and LED, respectively. Through numerical reconstruction by using the four phase shifting method and Fresnel diffraction method, we recovered the intensity and phase image of USAF resolution target apart from about 1.0m distance. In this experiment, we show 1.2 times improvement in resolution compared to conventional imaging. Finally, to confirm the recognition result of camouflaged targets with the same color from background, we carry out to test holographic imaging in incoherent light. In this result, we showed the possibility of a target detection and recognition that used three dimensional shape and size signatures, numerical distance from phase information of obtained holographic image.

G-Quadruplex Induction by the Hairpin Pyrrole-Imidazole Polyamide Dimer.

PubMed

Obata, Shunsuke; Asamitsu, Sefan; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2018-02-06

The G-quadruplex (G4) is one type of higher-order structure of nucleic acids and is thought to play important roles in various biological events such as regulation of transcription and inhibition of DNA replication. Pyrrole-imidazole polyamides (PIPs) are programmable small molecules that can sequence-specifically bind with high affinity to the minor groove of double-stranded DNA (dsDNA). Herein, we designed head-to-head hairpin PIP dimers and their target dsDNA in a model G4-forming sequence. Using an electrophoresis mobility shift assay and transcription arrest assay, we found that PIP dimers could induce the structural change to G4 DNA from dsDNA through the recognition by one PIP dimer molecule of two duplex-binding sites flanking both ends of the G4-forming sequence. This induction ability was dependent on linker length. This is the first study to induce G4 formation using PIPs, which are known to be dsDNA binders. The results reported here suggest that selective G4 induction in native sequences may be achieved with PIP dimers by applying the same design strategy.

Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve

PubMed Central

Bottacini, Francesca; Morrissey, Ruth; Roberts, Richard John; James, Kieran; van Breen, Justin; Egan, Muireann; Lambert, Jolanda; van Limpt, Kees; Knol, Jan; Motherway, Mary O’Connell; van Sinderen, Douwe

2018-01-01

Abstract Bifidobacterium breve represents one of the most abundant bifidobacterial species in the gastro-intestinal tract of breast-fed infants, where their presence is believed to exert beneficial effects. In the present study whole genome sequencing, employing the PacBio Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that genes encoding Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variable gene content (or variome). Using the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BS-seq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, we show that R/M systems typically impose a very significant barrier to genetic accessibility of B. breve strains, and that cloning of a methyltransferase-encoding gene may overcome such a barrier, thus allowing future functional investigations of members of this species. PMID:29294107

The design of strain-specific polymerase chain reactions for discrimination of the racoon rabies virus strain from indigenous rabies viruses of Ontario.

PubMed

Nadin-Davis, S A; Huang, W; Wandeler, A I

1996-03-01

Since its recognition as a discrete epizootic in Florida in the early 1950s, the raccoon strain of rabies virus (RV) has spread over almost the entire eastern seaboard of the US and now threatens to enter the southernmost regions of Canada. To characterise this RV strain in more detail, nucleotide sequencing of the N and G genes, encoding the nucleoprotein and glycoprotein, respectively, of representative isolates has been undertaken. This sequence information generated a conserved restriction map of the N gene, thereby permitting unequivocal identification of this strain by molecular techniques. Comparisons of the predicted nucleoprotein and glycoprotein products with those of other RV strains identified a number of amino acid sequence variations conserved only in the raccoon strain. This information was used to design strain-specific primers targeted to the N gene sequences encoding these residues. The incorporation of these primers into a multiplex polymerase chain reaction (PCR) protocol permitted easy and rapid discrimination between the raccoon RV strain and indigenous Ontario RVs.

Serial position effects in recognition memory for odors: a reexamination.

PubMed

Miles, Christopher; Hodder, Kathryn

2005-10-01

Seven experiments examined recognition memory for sequentially presented odors. Following Reed (2000), participants were presented with a sequence of odors and then required to identify an odor from the sequence in a test probe comprising 2 odors. The pattern of results obtained by Reed (2000, although statistically marginal) demonstrated enhanced recognition for odors presented at the start (primacy) and end (recency) of the sequence: a result that we failed to replicate in any of the experiments reported here. Experiments 1 and 3 were designed to replicate Reed (2000), employing five-item and seven-item sequences, respectively, and each demonstrated significant recency, with evidence of primacy in Experiment 3 only. Experiment 2 replicated Experiment 1, with reduced interstimulus intervals, and produced a null effect of serial position. The ease with which the odors could be verbally labeled was manipulated in Experiments 4 and 5. Nameable odors produced a null effect of serial position (Experiment 4), and hard-to-name odors produced a pronounced recency effect (Experiment 5); nevertheless, overall rates of recognition were remarkably similar for the two experiments at around 70%. Articulatory suppression reduced recognition accuracy (Experiment 6), but recency was again present in the absence of primacy. Odor recognition performance was immune to the effects of an interleaved odor (Experiment 7), and, again, both primacy and recency effects were absent. There was no evidence of olfactory fatigue: Recognition accuracy improved across trials (Experiment 1). It is argued that the results of the experiments reported here are generally consistent with that body of work employing hard-to-name visual stimuli, where recency is obtained in the absence of primacy when the retention interval is short.

Using eye movements as an index of implicit face recognition in autism spectrum disorder.

PubMed

Hedley, Darren; Young, Robyn; Brewer, Neil

2012-10-01

Individuals with an autism spectrum disorder (ASD) typically show impairment on face recognition tasks. Performance has usually been assessed using overt, explicit recognition tasks. Here, a complementary method involving eye tracking was used to examine implicit face recognition in participants with ASD and in an intelligence quotient-matched non-ASD control group. Differences in eye movement indices between target and foil faces were used as an indicator of implicit face recognition. Explicit face recognition was assessed using old-new discrimination and reaction time measures. Stimuli were faces of studied (target) or unfamiliar (foil) persons. Target images at test were either identical to the images presented at study or altered by changing the lighting, pose, or by masking with visual noise. Participants with ASD performed worse than controls on the explicit recognition task. Eye movement-based measures, however, indicated that implicit recognition may not be affected to the same degree as explicit recognition. Autism Res 2012, 5: 363-379. © 2012 International Society for Autism Research, Wiley Periodicals, Inc. © 2012 International Society for Autism Research, Wiley Periodicals, Inc.

Coding visual features extracted from video sequences.

PubMed

Baroffio, Luca; Cesana, Matteo; Redondi, Alessandro; Tagliasacchi, Marco; Tubaro, Stefano

2014-05-01

Visual features are successfully exploited in several applications (e.g., visual search, object recognition and tracking, etc.) due to their ability to efficiently represent image content. Several visual analysis tasks require features to be transmitted over a bandwidth-limited network, thus calling for coding techniques to reduce the required bit budget, while attaining a target level of efficiency. In this paper, we propose, for the first time, a coding architecture designed for local features (e.g., SIFT, SURF) extracted from video sequences. To achieve high coding efficiency, we exploit both spatial and temporal redundancy by means of intraframe and interframe coding modes. In addition, we propose a coding mode decision based on rate-distortion optimization. The proposed coding scheme can be conveniently adopted to implement the analyze-then-compress (ATC) paradigm in the context of visual sensor networks. That is, sets of visual features are extracted from video frames, encoded at remote nodes, and finally transmitted to a central controller that performs visual analysis. This is in contrast to the traditional compress-then-analyze (CTA) paradigm, in which video sequences acquired at a node are compressed and then sent to a central unit for further processing. In this paper, we compare these coding paradigms using metrics that are routinely adopted to evaluate the suitability of visual features in the context of content-based retrieval, object recognition, and tracking. Experimental results demonstrate that, thanks to the significant coding gains achieved by the proposed coding scheme, ATC outperforms CTA with respect to all evaluation metrics.

Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site

PubMed Central

Watzinger, Franz; Hörth, Elfriede; Lion, Thomas

2001-01-01

Despite the recent introduction of real-time PCR methods, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. Here we describe a shifted restriction-site competitive PCR (SRS-cPCR) assay based on a modified type of competitor. The competitor fragments are designed to contain a recognition site for a restriction endonuclease that is also present in the target sequence to be quantified, but in a different position. Upon completion of the PCR, the amplicons are digested in the same tube with a single restriction enzyme, without the need to purify PCR products. The generated competitor- and target-specific restriction fragments display different sizes, and can be readily separated by electrophoresis and quantified by image analysis. Suboptimal digestion affects competitor- and target-derived amplicons to the same extent, thus eliminating the problem of incorrect quantification as a result of incomplete digestion of PCR products. We have established optimized conditions for a panel of 20 common restriction endonucleases permitting efficient digestion in PCR buffer. It is possible, therefore, to find a suitable restriction site for competitive PCR in virtually any sequence of interest. The assay presented is inexpensive, widely applicable, and permits reliable and accurate quantification of nucleic acid targets. PMID:11376164

PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

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

Yang, Hui; Gao, Pu; Rajashankar, Kanagalaghatta R.

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering upmore » of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.« less

Action Recognition in a Crowded Environment

PubMed Central

Nieuwenhuis, Judith; Bülthoff, Isabelle; Barraclough, Nick; de la Rosa, Stephan

2017-01-01

So far, action recognition has been mainly examined with small point-light human stimuli presented alone within a narrow central area of the observer’s visual field. Yet, we need to recognize the actions of life-size humans viewed alone or surrounded by bystanders, whether they are seen in central or peripheral vision. Here, we examined the mechanisms in central vision and far periphery (40° eccentricity) involved in the recognition of the actions of a life-size actor (target) and their sensitivity to the presence of a crowd surrounding the target. In Experiment 1, we used an action adaptation paradigm to probe whether static or idly moving crowds might interfere with the recognition of a target’s action (hug or clap). We found that this type of crowds whose movements were dissimilar to the target action hardly affected action recognition in central and peripheral vision. In Experiment 2, we examined whether crowd actions that were more similar to the target actions affected action recognition. Indeed, the presence of that crowd diminished adaptation aftereffects in central vision as wells as in the periphery. We replicated Experiment 2 using a recognition task instead of an adaptation paradigm. With this task, we found evidence of decreased action recognition accuracy, but this was significant in peripheral vision only. Our results suggest that the presence of a crowd carrying out actions similar to that of the target affects its recognition. We outline how these results can be understood in terms of high-level crowding effects that operate on action-sensitive perceptual channels. PMID:29308177

Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection.

PubMed

Kaewkascholkul, Napol; Somboonviwat, Kulwadee; Asakawa, Shuichi; Hirono, Ikuo; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

2016-07-01

MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response. Copyright © 2016. Published by Elsevier Ltd.

Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

NASA Astrophysics Data System (ADS)

Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

Application of automatic threshold in dynamic target recognition with low contrast

NASA Astrophysics Data System (ADS)

Miao, Hua; Guo, Xiaoming; Chen, Yu

2014-11-01

Hybrid photoelectric joint transform correlator can realize automatic real-time recognition with high precision through the combination of optical devices and electronic devices. When recognizing targets with low contrast using photoelectric joint transform correlator, because of the difference of attitude, brightness and grayscale between target and template, only four to five frames of dynamic targets can be recognized without any processing. CCD camera is used to capture the dynamic target images and the capturing speed of CCD is 25 frames per second. Automatic threshold has many advantages like fast processing speed, effectively shielding noise interference, enhancing diffraction energy of useful information and better reserving outline of target and template, so this method plays a very important role in target recognition with optical correlation method. However, the automatic obtained threshold by program can not achieve the best recognition results for dynamic targets. The reason is that outline information is broken to some extent. Optimal threshold is obtained by manual intervention in most cases. Aiming at the characteristics of dynamic targets, the processing program of improved automatic threshold is finished by multiplying OTSU threshold of target and template by scale coefficient of the processed image, and combining with mathematical morphology. The optimal threshold can be achieved automatically by improved automatic threshold processing for dynamic low contrast target images. The recognition rate of dynamic targets is improved through decreased background noise effect and increased correlation information. A series of dynamic tank images with the speed about 70 km/h are adapted as target images. The 1st frame of this series of tanks can correlate only with the 3rd frame without any processing. Through OTSU threshold, the 80th frame can be recognized. By automatic threshold processing of the joint images, this number can be increased to 89 frames. Experimental results show that the improved automatic threshold processing has special application value for the recognition of dynamic target with low contrast.

Advances in image compression and automatic target recognition; Proceedings of the Meeting, Orlando, FL, Mar. 30, 31, 1989

NASA Technical Reports Server (NTRS)

Tescher, Andrew G. (Editor)

1989-01-01

Various papers on image compression and automatic target recognition are presented. Individual topics addressed include: target cluster detection in cluttered SAR imagery, model-based target recognition using laser radar imagery, Smart Sensor front-end processor for feature extraction of images, object attitude estimation and tracking from a single video sensor, symmetry detection in human vision, analysis of high resolution aerial images for object detection, obscured object recognition for an ATR application, neural networks for adaptive shape tracking, statistical mechanics and pattern recognition, detection of cylinders in aerial range images, moving object tracking using local windows, new transform method for image data compression, quad-tree product vector quantization of images, predictive trellis encoding of imagery, reduced generalized chain code for contour description, compact architecture for a real-time vision system, use of human visibility functions in segmentation coding, color texture analysis and synthesis using Gibbs random fields.

Programming mRNA decay to modulate synthetic circuit resource allocation

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

Venturelli, Ophelia S.; Tei, Mika; Bauer, Stefan

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstratesmore » that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.« less

Programming mRNA decay to modulate synthetic circuit resource allocation

DOE PAGES

Venturelli, Ophelia S.; Tei, Mika; Bauer, Stefan; ...

2017-04-26

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstratesmore » that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.« less

DNA sequence alignment by microhomology sampling during homologous recombination

PubMed Central

Qi, Zhi; Redding, Sy; Lee, Ja Yil; Gibb, Bryan; Kwon, YoungHo; Niu, Hengyao; Gaines, William A.; Sung, Patrick

2015-01-01

Summary Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair ssDNA with a homologous dsDNA template. Here we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real-time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a 9th nucleotide coincides with an additional reduction in binding free energy and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination. PMID:25684365

Sequence, Structure, and Context Preferences of Human RNA Binding Proteins.

PubMed

Dominguez, Daniel; Freese, Peter; Alexis, Maria S; Su, Amanda; Hochman, Myles; Palden, Tsultrim; Bazile, Cassandra; Lambert, Nicole J; Van Nostrand, Eric L; Pratt, Gabriel A; Yeo, Gene W; Graveley, Brenton R; Burge, Christopher B

2018-06-07

RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Study of cnidarian-algal symbiosis in the "omics" age.

PubMed

Meyer, Eli; Weis, Virginia M

2012-08-01

The symbiotic associations between cnidarians and dinoflagellate algae (Symbiodinium) support productive and diverse ecosystems in coral reefs. Many aspects of this association, including the mechanistic basis of host-symbiont recognition and metabolic interaction, remain poorly understood. The first completed genome sequence for a symbiotic anthozoan is now available (the coral Acropora digitifera), and extensive expressed sequence tag resources are available for a variety of other symbiotic corals and anemones. These resources make it possible to profile gene expression, protein abundance, and protein localization associated with the symbiotic state. Here we review the history of "omics" studies of cnidarian-algal symbiosis and the current availability of sequence resources for corals and anemones, identifying genes putatively involved in symbiosis across 10 anthozoan species. The public availability of candidate symbiosis-associated genes leaves the field of cnidarian-algal symbiosis poised for in-depth comparative studies of sequence diversity and gene expression and for targeted functional studies of genes associated with symbiosis. Reviewing the progress to date suggests directions for future investigations of cnidarian-algal symbiosis that include (i) sequencing of Symbiodinium, (ii) proteomic analysis of the symbiosome membrane complex, (iii) glycomic analysis of Symbiodinium cell surfaces, and (iv) expression profiling of the gastrodermal cells hosting Symbiodinium.

Noise Suppression Based on Multi-Model Compositions Using Multi-Pass Search with Multi-Label N-gram Models

NASA Astrophysics Data System (ADS)

Jitsuhiro, Takatoshi; Toriyama, Tomoji; Kogure, Kiyoshi

We propose a noise suppression method based on multi-model compositions and multi-pass search. In real environments, input speech for speech recognition includes many kinds of noise signals. To obtain good recognized candidates, suppressing many kinds of noise signals at once and finding target speech is important. Before noise suppression, to find speech and noise label sequences, we introduce multi-pass search with acoustic models including many kinds of noise models and their compositions, their n-gram models, and their lexicon. Noise suppression is frame-synchronously performed using the multiple models selected by recognized label sequences with time alignments. We evaluated this method using the E-Nightingale task, which contains voice memoranda spoken by nurses during actual work at hospitals. The proposed method obtained higher performance than the conventional method.

Display of adenoregulin with a novel Pichia pastoris cell surface display system.

PubMed

Ren, Ren; Jiang, Zhengbing; Liu, Meiyun; Tao, Xinyi; Ma, Yushu; Wei, Dongzhi

2007-02-01

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo1p with its own secretion signal sequence or the alpha-factor secretion signal sequence, a polyhistidine (6xHis) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

Mutations altering the cleavage specificity of a homing endonuclease

PubMed Central

Seligman, Lenny M.; Chisholm, Karen M.; Chevalier, Brett S.; Chadsey, Meggen S.; Edwards, Samuel T.; Savage, Jeremiah H.; Veillet, Adeline L.

2002-01-01

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences. PMID:12202772

Target recognition of log-polar ladar range images using moment invariants

NASA Astrophysics Data System (ADS)

Xia, Wenze; Han, Shaokun; Cao, Jie; Yu, Haoyong

2017-01-01

The ladar range image has received considerable attentions in the automatic target recognition field. However, previous research does not cover target recognition using log-polar ladar range images. Therefore, we construct a target recognition system based on log-polar ladar range images in this paper. In this system combined moment invariants and backpropagation neural network are selected as shape descriptor and shape classifier, respectively. In order to fully analyze the effect of log-polar sampling pattern on recognition result, several comparative experiments based on simulated and real range images are carried out. Eventually, several important conclusions are drawn: (i) if combined moments are computed directly by log-polar range images, translation, rotation and scaling invariant properties of combined moments will be invalid (ii) when object is located in the center of field of view, recognition rate of log-polar range images is less sensitive to the changing of field of view (iii) as object position changes from center to edge of field of view, recognition performance of log-polar range images will decline dramatically (iv) log-polar range images has a better noise robustness than Cartesian range images. Finally, we give a suggestion that it is better to divide field of view into recognition area and searching area in the real application.

Adaptive optics to enhance target recognition

NASA Astrophysics Data System (ADS)

McAulay, Alastair D.

2012-06-01

Target recognition can be enhanced by reducing image degradation due to atmospheric turbulence. This is accomplished by an adaptive optic system. We discuss the forms of degradation when a target is viewed through the atmosphere1: scintillation from ground targets on a hot day in visible or infrared light; beam spreading and wavering around in time; atmospheric turbulence caused by motion of the target or by weather. In the case of targets we can use a beacon laser that reflects back from the target into a wavefront detector to measure the effects of turbulence on propagation to and from the target before imaging.1 A deformable mirror then corrects the wavefront shape of the transmitted, reflected or scattered data for enhanced imaging. Further, recognition of targets is enhanced by performing accurate distance measurements to localized parts of the target using lidar. Distance is obtained by sending a short pulse to the target and measuring the time for the pulse to return. There is inadequate time to scan the complete field of view so that the beam must be steered to regions of interest such as extremities of the image during image recognition. Distance is particularly valuable to recognize fine features in range along the target or when segmentation is required to separate a target from background or from other targets. We discuss the issues involved.

Engineering a switch-on peptide to ricin A chain for increasing its specificity towards HIV-infected cells.

PubMed

Au, Ka-Yee; Wang, Rui-Rui; Wong, Yuen-Ting; Wong, Kam-Bo; Zheng, Yong-Tang; Shaw, Pang-Chui

2014-03-01

Ricin is a type II ribosome-inactivating protein (RIP) that potently inactivates eukaryotic ribosomes by removing a specific adenine residue at the conserved α-sarcin/ricin loop of 28S ribosomal RNA (rRNA). Here, we try to increase the specificity of the enzymatically active ricin A chain (RTA) towards human immunodeficiency virus type 1 (HIV-1) by adding a loop with HIV protease recognition site to RTA. HIV-specific RTA variants were constructed by inserting a peptide with HIV-protease recognition site either internally or at the C-terminal region of wild type RTA. Cleavability of variants by viral protease was tested in vitro and in HIV-infected cells. The production of viral p24 antigen and syncytium in the presence of C-terminal variants was measured to examine the anti-HIV activities of the variants. C-terminal RTA variants were specifically cleaved by HIV-1 protease both in vitro and in HIV-infected cells. Upon proteolysis, the processed variants showed enhanced antiviral effect with low cytotoxicity towards uninfected cells. RTA variants with HIV protease recognition sequence engineered at the C-terminus were cleaved and the products mediated specific inhibitory effect towards HIV replication. Current cocktail treatment of HIV infection fails to eradicate the virus from patients. Here we illustrate the feasibility of targeting an RIP towards HIV-infected cells by incorporation of HIV protease cleavage sequence. This approach may be generalized to other RIPs and is promising in drug design for combating HIV. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors

PubMed Central

Wan, Hua; Hu, Jian-ping; Li, Kang-shun; Tian, Xu-hong; Chang, Shan

2013-01-01

TAL (transcriptional activator-like) effectors (TALEs) are DNA-binding proteins, containing a modular central domain that recognizes specific DNA sequences. Recently, the crystallographic studies of TALEs revealed the structure of DNA-recognition domain. In this article, molecular dynamics (MD) simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues) with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA), the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL). The conformational analysis of DNA indicates that the 5′ end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism. PMID:24130757

Individual differences in language and working memory affect children's speech recognition in noise.

PubMed

McCreery, Ryan W; Spratford, Meredith; Kirby, Benjamin; Brennan, Marc

2017-05-01

We examined how cognitive and linguistic skills affect speech recognition in noise for children with normal hearing. Children with better working memory and language abilities were expected to have better speech recognition in noise than peers with poorer skills in these domains. As part of a prospective, cross-sectional study, children with normal hearing completed speech recognition in noise for three types of stimuli: (1) monosyllabic words, (2) syntactically correct but semantically anomalous sentences and (3) semantically and syntactically anomalous word sequences. Measures of vocabulary, syntax and working memory were used to predict individual differences in speech recognition in noise. Ninety-six children with normal hearing, who were between 5 and 12 years of age. Higher working memory was associated with better speech recognition in noise for all three stimulus types. Higher vocabulary abilities were associated with better recognition in noise for sentences and word sequences, but not for words. Working memory and language both influence children's speech recognition in noise, but the relationships vary across types of stimuli. These findings suggest that clinical assessment of speech recognition is likely to reflect underlying cognitive and linguistic abilities, in addition to a child's auditory skills, consistent with the Ease of Language Understanding model.

Perceptual fluency and affect without recognition.

PubMed

Anand, P; Sternthal, B

1991-05-01

A dichotic listening task was used to investigate the affect-without-recognition phenomenon. Subjects performed a distractor task by responding to the information presented in one ear while ignoring the target information presented in the other ear. The subjects' recognition of and affect toward the target information as well as toward foils was measured. The results offer evidence for the affect-without-recognition phenomenon. Furthermore, the data suggest that the subjects' affect toward the stimuli depended primarily on the extent to which the stimuli were perceived as familiar (i.e., subjective familiarity), and this perception was influenced by the ear in which the distractor or the target information was presented. These data are interpreted in terms of current models of recognition memory and hemispheric lateralization.

Enantiospecific recognition of DNA sequences by a proflavine Tröger base.

PubMed

Bailly, C; Laine, W; Demeunynck, M; Lhomme, J

2000-07-05

The DNA interaction of a chiral Tröger base derived from proflavine was investigated by DNA melting temperature measurements and complementary biochemical assays. DNase I footprinting experiments demonstrate that the binding of the proflavine-based Tröger base is both enantio- and sequence-specific. The (+)-isomer poorly interacts with DNA in a non-sequence-selective fashion. In sharp contrast, the corresponding (-)-isomer recognizes preferentially certain DNA sequences containing both A. T and G. C base pairs, such as the motifs 5'-GTT. AAC and 5'-ATGA. TCAT. This is the first experimental demonstration that acridine-type Tröger bases can be used for enantiospecific recognition of DNA sequences. Copyright 2000 Academic Press.

A promoter recognition mechanism common to yeast mitochondrial and phage t7 RNA polymerases.

PubMed

Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

2009-05-15

Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family.

A Single Electrochemical Probe Used for Analysis of Multiple Nucleic Acid Sequences

PubMed Central

Mills, Dawn M.; Calvo-Marzal, Percy; Pinzon, Jeffer M.; Armas, Stephanie; Kolpashchikov, Dmitry M.; Chumbimuni-Torres, Karin Y.

2017-01-01

Electrochemical hybridization sensors have been explored extensively for analysis of specific nucleic acids. However, commercialization of the platform is hindered by the need for attachment of separate oligonucleotide probes complementary to a RNA or DNA target to an electrode’s surface. Here we demonstrate that a single probe can be used to analyze several nucleic acid targets with high selectivity and low cost. The universal electrochemical four-way junction (4J)-forming (UE4J) sensor consists of a universal DNA stem-loop (USL) probe attached to the electrode’s surface and two adaptor strands (m and f) which hybridize to the USL probe and the analyte to form a 4J associate. The m adaptor strand was conjugated with a methylene blue redox marker for signal ON sensing and monitored using square wave voltammetry. We demonstrated that a single sensor can be used for detection of several different DNA/RNA sequences and can be regenerated in 30 seconds by a simple water rinse. The UE4J sensor enables a high selectivity by recognition of a single base substitution, even at room temperature. The UE4J sensor opens a venue for a re-useable universal platform that can be adopted at low cost for the analysis of DNA or RNA targets. PMID:29371782

Molecular profiling of childhood cancer: Biomarkers and novel therapies.

PubMed

Saletta, Federica; Wadham, Carol; Ziegler, David S; Marshall, Glenn M; Haber, Michelle; McCowage, Geoffrey; Norris, Murray D; Byrne, Jennifer A

2014-06-01

Technological advances including high-throughput sequencing have identified numerous tumor-specific genetic changes in pediatric and adolescent cancers that can be exploited as targets for novel therapies. This review provides a detailed overview of recent advances in the application of target-specific therapies for childhood cancers, either as single agents or in combination with other therapies. The review summarizes preclinical evidence on which clinical trials are based, early phase clinical trial results, and the incorporation of predictive biomarkers into clinical practice, according to cancer type. There is growing evidence that molecularly targeted therapies can valuably add to the arsenal available for treating childhood cancers, particularly when used in combination with other therapies. Nonetheless the introduction of molecularly targeted agents into practice remains challenging, due to the use of unselected populations in some clinical trials, inadequate methods to evaluate efficacy, and the need for improved preclinical models to both evaluate dosing and safety of combination therapies. The increasing recognition of the heterogeneity of molecular causes of cancer favors the continued development of molecularly targeted agents, and their transfer to pediatric and adolescent populations.

A Low-Dimensional Radial Silhouette-Based Feature for Fast Human Action Recognition Fusing Multiple Views.

PubMed

Chaaraoui, Alexandros Andre; Flórez-Revuelta, Francisco

2014-01-01

This paper presents a novel silhouette-based feature for vision-based human action recognition, which relies on the contour of the silhouette and a radial scheme. Its low-dimensionality and ease of extraction result in an outstanding proficiency for real-time scenarios. This feature is used in a learning algorithm that by means of model fusion of multiple camera streams builds a bag of key poses, which serves as a dictionary of known poses and allows converting the training sequences into sequences of key poses. These are used in order to perform action recognition by means of a sequence matching algorithm. Experimentation on three different datasets returns high and stable recognition rates. To the best of our knowledge, this paper presents the highest results so far on the MuHAVi-MAS dataset. Real-time suitability is given, since the method easily performs above video frequency. Therefore, the related requirements that applications as ambient-assisted living services impose are successfully fulfilled.

ZifBASE: a database of zinc finger proteins and associated resources.

PubMed

Jayakanthan, Mannu; Muthukumaran, Jayaraman; Chandrasekar, Sanniyasi; Chawla, Konika; Punetha, Ankita; Sundar, Durai

2009-09-09

Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP) to identify the potential binding sites and its location, which reduce the time of in vivo task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence. ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed citations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more informative. A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface http://web.iitd.ac.in/~sundar/zifbase.

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

PubMed

Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

2000-04-01

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Method of determining the necessary number of observations for video stream documents recognition

NASA Astrophysics Data System (ADS)

Arlazarov, Vladimir V.; Bulatov, Konstantin; Manzhikov, Temudzhin; Slavin, Oleg; Janiszewski, Igor

2018-04-01

This paper discusses a task of document recognition on a sequence of video frames. In order to optimize the processing speed an estimation is performed of stability of recognition results obtained from several video frames. Considering identity document (Russian internal passport) recognition on a mobile device it is shown that significant decrease is possible of the number of observations necessary for obtaining precise recognition result.

Oxytocin increases bias, but not accuracy, in face recognition line-ups.

PubMed

Bate, Sarah; Bennetts, Rachel; Parris, Benjamin A; Bindemann, Markus; Udale, Robert; Bussunt, Amanda

2015-07-01

Previous work indicates that intranasal inhalation of oxytocin improves face recognition skills, raising the possibility that it may be used in security settings. However, it is unclear whether oxytocin directly acts upon the core face-processing system itself or indirectly improves face recognition via affective or social salience mechanisms. In a double-blind procedure, 60 participants received either an oxytocin or placebo nasal spray before completing the One-in-Ten task-a standardized test of unfamiliar face recognition containing target-present and target-absent line-ups. Participants in the oxytocin condition outperformed those in the placebo condition on target-present trials, yet were more likely to make false-positive errors on target-absent trials. Signal detection analyses indicated that oxytocin induced a more liberal response bias, rather than increasing accuracy per se. These findings support a social salience account of the effects of oxytocin on face recognition and indicate that oxytocin may impede face recognition in certain scenarios. © The Author (2014). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi.

PubMed

Schoch, Conrad L; Robbertse, Barbara; Robert, Vincent; Vu, Duong; Cardinali, Gianluigi; Irinyi, Laszlo; Meyer, Wieland; Nilsson, R Henrik; Hughes, Karen; Miller, Andrew N; Kirk, Paul M; Abarenkov, Kessy; Aime, M Catherine; Ariyawansa, Hiran A; Bidartondo, Martin; Boekhout, Teun; Buyck, Bart; Cai, Qing; Chen, Jie; Crespo, Ana; Crous, Pedro W; Damm, Ulrike; De Beer, Z Wilhelm; Dentinger, Bryn T M; Divakar, Pradeep K; Dueñas, Margarita; Feau, Nicolas; Fliegerova, Katerina; García, Miguel A; Ge, Zai-Wei; Griffith, Gareth W; Groenewald, Johannes Z; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Gueidan, Cécile; Guo, Liangdong; Hambleton, Sarah; Hamelin, Richard; Hansen, Karen; Hofstetter, Valérie; Hong, Seung-Beom; Houbraken, Jos; Hyde, Kevin D; Inderbitzin, Patrik; Johnston, Peter R; Karunarathna, Samantha C; Kõljalg, Urmas; Kovács, Gábor M; Kraichak, Ekaphan; Krizsan, Krisztina; Kurtzman, Cletus P; Larsson, Karl-Henrik; Leavitt, Steven; Letcher, Peter M; Liimatainen, Kare; Liu, Jian-Kui; Lodge, D Jean; Luangsa-ard, Janet Jennifer; Lumbsch, H Thorsten; Maharachchikumbura, Sajeewa S N; Manamgoda, Dimuthu; Martín, María P; Minnis, Andrew M; Moncalvo, Jean-Marc; Mulè, Giuseppina; Nakasone, Karen K; Niskanen, Tuula; Olariaga, Ibai; Papp, Tamás; Petkovits, Tamás; Pino-Bodas, Raquel; Powell, Martha J; Raja, Huzefa A; Redecker, Dirk; Sarmiento-Ramirez, J M; Seifert, Keith A; Shrestha, Bhushan; Stenroos, Soili; Stielow, Benjamin; Suh, Sung-Oui; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M Teresa; Udayanga, Dhanushka; Untereiner, Wendy A; Diéguez Uribeondo, Javier; Subbarao, Krishna V; Vágvölgyi, Csaba; Visagie, Cobus; Voigt, Kerstin; Walker, Donald M; Weir, Bevan S; Weiß, Michael; Wijayawardene, Nalin N; Wingfield, Michael J; Xu, J P; Yang, Zhu L; Zhang, Ning; Zhuang, Wen-Ying; Federhen, Scott

2014-01-01

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi. Database URL: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353. Published by Oxford University Press 2013. This work is written by US Government employees and is in the public domain in the US.

SAR image dataset of military ground targets with multiple poses for ATR

NASA Astrophysics Data System (ADS)

Belloni, Carole; Balleri, Alessio; Aouf, Nabil; Merlet, Thomas; Le Caillec, Jean-Marc

2017-10-01

Automatic Target Recognition (ATR) is the task of automatically detecting and classifying targets. Recognition using Synthetic Aperture Radar (SAR) images is interesting because SAR images can be acquired at night and under any weather conditions, whereas optical sensors operating in the visible band do not have this capability. Existing SAR ATR algorithms have mostly been evaluated using the MSTAR dataset.1 The problem with the MSTAR is that some of the proposed ATR methods have shown good classification performance even when targets were hidden,2 suggesting the presence of a bias in the dataset. Evaluations of SAR ATR techniques are currently challenging due to the lack of publicly available data in the SAR domain. In this paper, we present a high resolution SAR dataset consisting of images of a set of ground military target models taken at various aspect angles, The dataset can be used for a fair evaluation and comparison of SAR ATR algorithms. We applied the Inverse Synthetic Aperture Radar (ISAR) technique to echoes from targets rotating on a turntable and illuminated with a stepped frequency waveform. The targets in the database consist of four variants of two 1.7m-long models of T-64 and T-72 tanks. The gun, the turret position and the depression angle are varied to form 26 different sequences of images. The emitted signal spanned the frequency range from 13 GHz to 18 GHz to achieve a bandwidth of 5 GHz sampled with 4001 frequency points. The resolution obtained with respect to the size of the model targets is comparable to typical values obtained using SAR airborne systems. Single polarized images (Horizontal-Horizontal) are generated using the backprojection algorithm.3 A total of 1480 images are produced using a 20° integration angle. The images in the dataset are organized in a suggested training and testing set to facilitate a standard evaluation of SAR ATR algorithms.

The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.

PubMed

Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

2014-01-01

T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.

The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain

PubMed Central

Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J; Sivakumaran, Andrew; Yoon, Je-Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, B Göran; Wilce, Jacqueline A; Díaz-Moreno, Irene

2014-01-01

T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein–nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5′ TOPs (5′ terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations. PMID:24824036

Wireless sensor

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

Lamberti, Vincent E.; Howell, JR, Layton N.; Mee, David K.

Disclosed is a sensor for detecting a target material. The sensor includes a ferromagnetic metal and a molecular recognition reagent coupled to the ferromagnetic metal. The molecular recognition reagent is operable to expand upon exposure to vapor or liquid from the target material such that the molecular recognition reagent changes a tensile stress upon the ferromagnetic metal. The target material is detected based on changes in the magnetic switching characteristics of the ferromagnetic metal caused by the changes in the tensile stress.

Component-based target recognition inspired by human vision

NASA Astrophysics Data System (ADS)

Zheng, Yufeng; Agyepong, Kwabena

2009-05-01

In contrast with machine vision, human can recognize an object from complex background with great flexibility. For example, given the task of finding and circling all cars (no further information) in a picture, you may build a virtual image in mind from the task (or target) description before looking at the picture. Specifically, the virtual car image may be composed of the key components such as driver cabin and wheels. In this paper, we propose a component-based target recognition method by simulating the human recognition process. The component templates (equivalent to the virtual image in mind) of the target (car) are manually decomposed from the target feature image. Meanwhile, the edges of the testing image can be extracted by using a difference of Gaussian (DOG) model that simulates the spatiotemporal response in visual process. A phase correlation matching algorithm is then applied to match the templates with the testing edge image. If all key component templates are matched with the examining object, then this object is recognized as the target. Besides the recognition accuracy, we will also investigate if this method works with part targets (half cars). In our experiments, several natural pictures taken on streets were used to test the proposed method. The preliminary results show that the component-based recognition method is very promising.

Transferring the Characteristics of Naturally Occurring and Biased Antibody Repertoires to Human Antibody Libraries by Trapping CDRH3 Sequences

PubMed Central

Venet, Sophie; Ravn, Ulla; Buatois, Vanessa; Gueneau, Franck; Calloud, Sébastien; Kosco-Vilbois, Marie; Fischer, Nicolas

2012-01-01

Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications. PMID:22937053

Efficient Processing of the Immunodominant, HLA-A*0201-Restricted Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitope despite Multiple Variations in the Epitope Flanking Sequences

PubMed Central

Brander, Christian; Yang, Otto O.; Jones, Norman G.; Lee, Yun; Goulder, Philip; Johnson, R. Paul; Trocha, Alicja; Colbert, David; Hay, Christine; Buchbinder, Susan; Bergmann, Cornelia C.; Zweerink, Hans J.; Wolinsky, Steven; Blattner, William A.; Kalams, Spyros A.; Walker, Bruce D.

1999-01-01

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences. PMID:10559335

The writer independent online handwriting recognition system frog on hand and cluster generative statistical dynamic time warping.

PubMed

Bahlmann, Claus; Burkhardt, Hans

2004-03-01

In this paper, we give a comprehensive description of our writer-independent online handwriting recognition system frog on hand. The focus of this work concerns the presentation of the classification/training approach, which we call cluster generative statistical dynamic time warping (CSDTW). CSDTW is a general, scalable, HMM-based method for variable-sized, sequential data that holistically combines cluster analysis and statistical sequence modeling. It can handle general classification problems that rely on this sequential type of data, e.g., speech recognition, genome processing, robotics, etc. Contrary to previous attempts, clustering and statistical sequence modeling are embedded in a single feature space and use a closely related distance measure. We show character recognition experiments of frog on hand using CSDTW on the UNIPEN online handwriting database. The recognition accuracy is significantly higher than reported results of other handwriting recognition systems. Finally, we describe the real-time implementation of frog on hand on a Linux Compaq iPAQ embedded device.

[Recognition of visual objects under forward masking. Effects of cathegorial similarity of test and masking stimuli].

PubMed

Gerasimenko, N Iu; Slavutskaia, A V; Kalinin, S A; Kulikov, M A; Mikhaĭlova, E S

2013-01-01

In 38 healthy subjects accuracy and response time were examined during recognition of two categories of images--animals andnonliving objects--under forward masking. We revealed new data that masking effects depended of categorical similarity of target and masking stimuli. The recognition accuracy was the lowest and the response time was the most slow, when the target and masking stimuli belongs to the same category, that was combined with high dispersion of response times. The revealed effects were more clear in the task of animal recognition in comparison with the recognition of nonliving objects. We supposed that the revealed effects connected with interference between cortical representations of the target and masking stimuli and discussed our results in context of cortical interference and negative priming.

Type III restriction-modification enzymes: a historical perspective.

PubMed

Rao, Desirazu N; Dryden, David T F; Bheemanaik, Shivakumara

2014-01-01

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

A novel rotational invariants target recognition method for rotating motion blurred images

NASA Astrophysics Data System (ADS)

Lan, Jinhui; Gong, Meiling; Dong, Mingwei; Zeng, Yiliang; Zhang, Yuzhen

2017-11-01

The imaging of the image sensor is blurred due to the rotational motion of the carrier and reducing the target recognition rate greatly. Although the traditional mode that restores the image first and then identifies the target can improve the recognition rate, it takes a long time to recognize. In order to solve this problem, a rotating fuzzy invariants extracted model was constructed that recognizes target directly. The model includes three metric layers. The object description capability of metric algorithms that contain gray value statistical algorithm, improved round projection transformation algorithm and rotation-convolution moment invariants in the three metric layers ranges from low to high, and the metric layer with the lowest description ability among them is as the input which can eliminate non pixel points of target region from degenerate image gradually. Experimental results show that the proposed model can improve the correct target recognition rate of blurred image and optimum allocation between the computational complexity and function of region.

Composite Wavelet Filters for Enhanced Automated Target Recognition

NASA Technical Reports Server (NTRS)

Chiang, Jeffrey N.; Zhang, Yuhan; Lu, Thomas T.; Chao, Tien-Hsin

2012-01-01

Automated Target Recognition (ATR) systems aim to automate target detection, recognition, and tracking. The current project applies a JPL ATR system to low-resolution sonar and camera videos taken from unmanned vehicles. These sonar images are inherently noisy and difficult to interpret, and pictures taken underwater are unreliable due to murkiness and inconsistent lighting. The ATR system breaks target recognition into three stages: 1) Videos of both sonar and camera footage are broken into frames and preprocessed to enhance images and detect Regions of Interest (ROIs). 2) Features are extracted from these ROIs in preparation for classification. 3) ROIs are classified as true or false positives using a standard Neural Network based on the extracted features. Several preprocessing, feature extraction, and training methods are tested and discussed in this paper.

Ab initio DNA synthesis by Bst polymerase in the presence of nicking endonucleases Nt.AlwI, Nb.BbvCI, and Nb.BsmI.

PubMed

Antipova, Valeriya N; Zheleznaya, Lyudmila A; Zyrina, Nadezhda V

2014-08-01

In the absence of added DNA, thermophilic DNA polymerases synthesize double-stranded DNA from free dNTPs, which consist of numerous repetitive units (ab initio DNA synthesis). The addition of thermophilic restriction endonuclease (REase), or nicking endonuclease (NEase), effectively stimulates ab initio DNA synthesis and determines the nucleotide sequence of reaction products. We have found that NEases Nt.AlwI, Nb.BbvCI, and Nb.BsmI with non-palindromic recognition sites stimulate the synthesis of sequences organized mainly as palindromes. Moreover, the nucleotide sequence of the palindromes appeared to be dependent on NEase recognition/cleavage modes. Thus, the heterodimeric Nb.BbvCI stimulated the synthesis of palindromes composed of two recognition sites of this NEase, which were separated by AT-reach sequences or (A)n (T)m spacers. Palindromic DNA sequences obtained in the ab initio DNA synthesis with the monomeric NEases Nb.BsmI and Nt.AlwI contained, along with the sites of these NEases, randomly synthesized sequences consisted of blocks of short repeats. These findings could help investigation of the potential abilities of highly productive ab initio DNA synthesis for the creation of DNA molecules with desirable sequence. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Structure of the PSD-95/MAP1A complex reveals a unique target recognition mode of the MAGUK GK domain.

PubMed

Xia, Yitian; Shang, Yuan; Zhang, Rongguang; Zhu, Jinwei

2017-08-10

The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the 'hydrophobic site' of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the 'phosphor-site' of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Textual emotion recognition for enhancing enterprise computing

NASA Astrophysics Data System (ADS)

Quan, Changqin; Ren, Fuji

2016-05-01

The growing interest in affective computing (AC) brings a lot of valuable research topics that can meet different application demands in enterprise systems. The present study explores a sub area of AC techniques - textual emotion recognition for enhancing enterprise computing. Multi-label emotion recognition in text is able to provide a more comprehensive understanding of emotions than single label emotion recognition. A representation of 'emotion state in text' is proposed to encompass the multidimensional emotions in text. It ensures the description in a formal way of the configurations of basic emotions as well as of the relations between them. Our method allows recognition of the emotions for the words bear indirect emotions, emotion ambiguity and multiple emotions. We further investigate the effect of word order for emotional expression by comparing the performances of bag-of-words model and sequence model for multi-label sentence emotion recognition. The experiments show that the classification results under sequence model are better than under bag-of-words model. And homogeneous Markov model showed promising results of multi-label sentence emotion recognition. This emotion recognition system is able to provide a convenient way to acquire valuable emotion information and to improve enterprise competitive ability in many aspects.

High throughput profile-profile based fold recognition for the entire human proteome.

PubMed

McGuffin, Liam J; Smith, Richard T; Bryson, Kevin; Sørensen, Søren-Aksel; Jones, David T

2006-06-07

In order to maintain the most comprehensive structural annotation databases we must carry out regular updates for each proteome using the latest profile-profile fold recognition methods. The ability to carry out these updates on demand is necessary to keep pace with the regular updates of sequence and structure databases. Providing the highest quality structural models requires the most intensive profile-profile fold recognition methods running with the very latest available sequence databases and fold libraries. However, running these methods on such a regular basis for every sequenced proteome requires large amounts of processing power. In this paper we describe and benchmark the JYDE (Job Yield Distribution Environment) system, which is a meta-scheduler designed to work above cluster schedulers, such as Sun Grid Engine (SGE) or Condor. We demonstrate the ability of JYDE to distribute the load of genomic-scale fold recognition across multiple independent Grid domains. We use the most recent profile-profile version of our mGenTHREADER software in order to annotate the latest version of the Human proteome against the latest sequence and structure databases in as short a time as possible. We show that our JYDE system is able to scale to large numbers of intensive fold recognition jobs running across several independent computer clusters. Using our JYDE system we have been able to annotate 99.9% of the protein sequences within the Human proteome in less than 24 hours, by harnessing over 500 CPUs from 3 independent Grid domains. This study clearly demonstrates the feasibility of carrying out on demand high quality structural annotations for the proteomes of major eukaryotic organisms. Specifically, we have shown that it is now possible to provide complete regular updates of profile-profile based fold recognition models for entire eukaryotic proteomes, through the use of Grid middleware such as JYDE.

Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80.

PubMed

Anamika; Spyracopoulos, Leo

2016-02-26

Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys(63)-linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2·phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Personal glucose meters for detection and quantification of a broad range of analytes

DOEpatents

Lu, Yi; Xiang, Yu

2015-02-03

A general methodology for the development of highly sensitive and selective sensors that can achieve portable, low-cost and quantitative detection of a broad range of targets using only a personal glucose meter (PGM) is disclosed. The method uses recognition molecules that are specific for a target agent, enzymes that can convert an enzyme substrate into glucose, and PGM. Also provided are sensors, which can include a solid support to which is attached a recognition molecule that permits detection of a target agent, wherein the recognition molecule specifically binds to the target agent in the presence of the target agent but not significantly to other agents as well as an enzyme that can catalyze the conversion of a substance into glucose, wherein the enzyme is attached directly or indirectly to the recognition molecule, and wherein in the presence of the target agent the enzyme can convert the substance into glucose. The disclosed sensors can be part of a lateral flow device. Methods of using such sensors for detecting target agents are also provided.

Under what conditions is recognition spared relative to recall after selective hippocampal damage in humans?

PubMed

Holdstock, J S; Mayes, A R; Roberts, N; Cezayirli, E; Isaac, C L; O'Reilly, R C; Norman, K A

2002-01-01

The claim that recognition memory is spared relative to recall after focal hippocampal damage has been disputed in the literature. We examined this claim by investigating object and object-location recall and recognition memory in a patient, YR, who has adult-onset selective hippocampal damage. Our aim was to identify the conditions under which recognition was spared relative to recall in this patient. She showed unimpaired forced-choice object recognition but clearly impaired recall, even when her control subjects found the object recognition task to be numerically harder than the object recall task. However, on two other recognition tests, YR's performance was not relatively spared. First, she was clearly impaired at an equivalently difficult yes/no object recognition task, but only when targets and foils were very similar. Second, YR was clearly impaired at forced-choice recognition of object-location associations. This impairment was also unrelated to difficulty because this task was no more difficult than the forced-choice object recognition task for control subjects. The clear impairment of yes/no, but not of forced-choice, object recognition after focal hippocampal damage, when targets and foils are very similar, is predicted by the neural network-based Complementary Learning Systems model of recognition. This model postulates that recognition is mediated by hippocampally dependent recollection and cortically dependent familiarity; thus hippocampal damage should not impair item familiarity. The model postulates that familiarity is ineffective when very similar targets and foils are shown one at a time and subjects have to identify which items are old (yes/no recognition). In contrast, familiarity is effective in discriminating which of similar targets and foils, seen together, is old (forced-choice recognition). Independent evidence from the remember/know procedure also indicates that YR's familiarity is normal. The Complementary Learning Systems model can also accommodate the clear impairment of forced-choice object-location recognition memory if it incorporates the view that the most complete convergence of spatial and object information, represented in different cortical regions, occurs in the hippocampus.

Distinguishing highly confident accurate and inaccurate memory: insights about relevant and irrelevant influences on memory confidence

PubMed Central

Chua, Elizabeth F.; Hannula, Deborah E.; Ranganath, Charan

2012-01-01

It is generally believed that accuracy and confidence in one’s memory are related, but there are many instances when they diverge. Accordingly, it is important to disentangle the factors which contribute to memory accuracy and confidence, especially those factors that contribute to confidence, but not accuracy. We used eye movements to separately measure fluent cue processing, the target recognition experience, and relative evidence assessment on recognition confidence and accuracy. Eye movements were monitored during a face-scene associative recognition task, in which participants first saw a scene cue, followed by a forced-choice recognition test for the associated face, with confidence ratings. Eye movement indices of the target recognition experience were largely indicative of accuracy, and showed a relationship to confidence for accurate decisions. In contrast, eye movements during the scene cue raised the possibility that more fluent cue processing was related to higher confidence for both accurate and inaccurate recognition decisions. In a second experiment, we manipulated cue familiarity, and therefore cue fluency. Participants showed higher confidence for cue-target associations for when the cue was more familiar, especially for incorrect responses. These results suggest that over-reliance on cue familiarity and under-reliance on the target recognition experience may lead to erroneous confidence. PMID:22171810

Distinguishing highly confident accurate and inaccurate memory: insights about relevant and irrelevant influences on memory confidence.

PubMed

Chua, Elizabeth F; Hannula, Deborah E; Ranganath, Charan

2012-01-01

It is generally believed that accuracy and confidence in one's memory are related, but there are many instances when they diverge. Accordingly it is important to disentangle the factors that contribute to memory accuracy and confidence, especially those factors that contribute to confidence, but not accuracy. We used eye movements to separately measure fluent cue processing, the target recognition experience, and relative evidence assessment on recognition confidence and accuracy. Eye movements were monitored during a face-scene associative recognition task, in which participants first saw a scene cue, followed by a forced-choice recognition test for the associated face, with confidence ratings. Eye movement indices of the target recognition experience were largely indicative of accuracy, and showed a relationship to confidence for accurate decisions. In contrast, eye movements during the scene cue raised the possibility that more fluent cue processing was related to higher confidence for both accurate and inaccurate recognition decisions. In a second experiment we manipulated cue familiarity, and therefore cue fluency. Participants showed higher confidence for cue-target associations for when the cue was more familiar, especially for incorrect responses. These results suggest that over-reliance on cue familiarity and under-reliance on the target recognition experience may lead to erroneous confidence.

Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template*

PubMed Central

Lee, Seung-Joo; Zhu, Bin; Akabayov, Barak; Richardson, Charles C.

2012-01-01

The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis. PMID:23024359

Invariant recognition drives neural representations of action sequences

PubMed Central

Poggio, Tomaso

2017-01-01

Recognizing the actions of others from visual stimuli is a crucial aspect of human perception that allows individuals to respond to social cues. Humans are able to discriminate between similar actions despite transformations, like changes in viewpoint or actor, that substantially alter the visual appearance of a scene. This ability to generalize across complex transformations is a hallmark of human visual intelligence. Advances in understanding action recognition at the neural level have not always translated into precise accounts of the computational principles underlying what representations of action sequences are constructed by human visual cortex. Here we test the hypothesis that invariant action discrimination might fill this gap. Recently, the study of artificial systems for static object perception has produced models, Convolutional Neural Networks (CNNs), that achieve human level performance in complex discriminative tasks. Within this class, architectures that better support invariant object recognition also produce image representations that better match those implied by human and primate neural data. However, whether these models produce representations of action sequences that support recognition across complex transformations and closely follow neural representations of actions remains unknown. Here we show that spatiotemporal CNNs accurately categorize video stimuli into action classes, and that deliberate model modifications that improve performance on an invariant action recognition task lead to data representations that better match human neural recordings. Our results support our hypothesis that performance on invariant discrimination dictates the neural representations of actions computed in the brain. These results broaden the scope of the invariant recognition framework for understanding visual intelligence from perception of inanimate objects and faces in static images to the study of human perception of action sequences. PMID:29253864

Characterizing protein domain associations by Small-molecule ligand binding

PubMed Central

Li, Qingliang; Cheng, Tiejun; Wang, Yanli; Bryant, Stephen H.

2012-01-01

Background Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to small-molecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing. PMID:23745168

Human recognition based on head-shoulder contour extraction and BP neural network

NASA Astrophysics Data System (ADS)

Kong, Xiao-fang; Wang, Xiu-qin; Gu, Guohua; Chen, Qian; Qian, Wei-xian

2014-11-01

In practical application scenarios like video surveillance and human-computer interaction, human body movements are uncertain because the human body is a non-rigid object. Based on the fact that the head-shoulder part of human body can be less affected by the movement, and will seldom be obscured by other objects, in human detection and recognition, a head-shoulder model with its stable characteristics can be applied as a detection feature to describe the human body. In order to extract the head-shoulder contour accurately, a head-shoulder model establish method with combination of edge detection and the mean-shift algorithm in image clustering has been proposed in this paper. First, an adaptive method of mixture Gaussian background update has been used to extract targets from the video sequence. Second, edge detection has been used to extract the contour of moving objects, and the mean-shift algorithm has been combined to cluster parts of target's contour. Third, the head-shoulder model can be established, according to the width and height ratio of human head-shoulder combined with the projection histogram of the binary image, and the eigenvectors of the head-shoulder contour can be acquired. Finally, the relationship between head-shoulder contour eigenvectors and the moving objects will be formed by the training of back-propagation (BP) neural network classifier, and the human head-shoulder model can be clustered for human detection and recognition. Experiments have shown that the method combined with edge detection and mean-shift algorithm proposed in this paper can extract the complete head-shoulder contour, with low calculating complexity and high efficiency.

Evidence of automatic processing in sequence learning using process-dissociation

PubMed Central

Mong, Heather M.; McCabe, David P.; Clegg, Benjamin A.

2012-01-01

This paper proposes a way to apply process-dissociation to sequence learning in addition and extension to the approach used by Destrebecqz and Cleeremans (2001). Participants were trained on two sequences separated from each other by a short break. Following training, participants self-reported their knowledge of the sequences. A recognition test was then performed which required discrimination of two trained sequences, either under the instructions to call any sequence encountered in the experiment “old” (the inclusion condition), or only sequence fragments from one half of the experiment “old” (the exclusion condition). The recognition test elicited automatic and controlled process estimates using the process dissociation procedure, and suggested both processes were involved. Examining the underlying processes supporting performance may provide more information on the fundamental aspects of the implicit and explicit constructs than has been attainable through awareness testing. PMID:22679465

A label-free, fluorescence based assay for microarray

NASA Astrophysics Data System (ADS)

Niu, Sanjun

DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same intensity of excitation light. The fluorescence contrast is used to quantify the amount of probe-target hybridization. A mathematical model that considers multiple reflections and scattering is developed to explain the mechanism of the fluorescence contrast which depends on the thickness of the PS film. Scattering is the dominant factor that contributes to the contrast. The potential of this assay to detect single nucleotide polymorphism is also tested.

Molecular evolution of pentatricopeptide repeat genes reveals truncation in species lacking an editing target and structural domains under distinct selective pressures.

PubMed

Hayes, Michael L; Giang, Karolyn; Mulligan, R Michael

2012-05-14

Pentatricopeptide repeat (PPR) proteins are required for numerous RNA processing events in plant organelles including C-to-U editing, splicing, stabilization, and cleavage. Fifteen PPR proteins are known to be required for RNA editing at 21 sites in Arabidopsis chloroplasts, and belong to the PLS class of PPR proteins. In this study, we investigate the co-evolution of four PPR genes (CRR4, CRR21, CLB19, and OTP82) and their six editing targets in Brassicaceae species. PPR genes are composed of approximately 10 to 20 tandem repeats and each repeat has two α-helical regions, helix A and helix B, that are separated by short coil regions. Each repeat and structural feature was examined to determine the selective pressures on these regions. All of the PPR genes examined are under strong negative selection. Multiple independent losses of editing site targets are observed for both CRR21 and OTP82. In several species lacking the known editing target for CRR21, PPR genes are truncated near the 17th PPR repeat. The coding sequences of the truncated CRR21 genes are maintained under strong negative selection; however, the 3' UTR sequences beyond the truncation site have substantially diverged. Phylogenetic analyses of four PPR genes show that sequences corresponding to helix A are high compared to helix B sequences. Differential evolutionary selection of helix A versus helix B is observed in both plant and mammalian PPR genes. PPR genes and their cognate editing sites are mutually constrained in evolution. Editing sites are frequently lost by replacement of an edited C with a genomic T. After the loss of an editing site, the PPR genes are observed with three outcomes: first, few changes are detected in some cases; second, the PPR gene is present as a pseudogene; and third, the PPR gene is present but truncated in the C-terminal region. The retention of truncated forms of CRR21 that are maintained under strong negative selection even in the absence of an editing site target suggests that unrecognized function(s) might exist for this PPR protein. PPR gene sequences that encode helix A are under strong selection, and could be involved in RNA substrate recognition.

Metasecretome-selective phage display approach for mining the functional potential of a rumen microbial community.

PubMed

Ciric, Milica; Moon, Christina D; Leahy, Sinead C; Creevey, Christopher J; Altermann, Eric; Attwood, Graeme T; Rakonjac, Jasna; Gagic, Dragana

2014-05-12

In silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects. By combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cell-surface complexes specialised for recognition and degradation of the plant fibre. As a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis.

Testing of a Composite Wavelet Filter to Enhance Automated Target Recognition in SONAR

NASA Technical Reports Server (NTRS)

Chiang, Jeffrey N.

2011-01-01

Automated Target Recognition (ATR) systems aim to automate target detection, recognition, and tracking. The current project applies a JPL ATR system to low resolution SONAR and camera videos taken from Unmanned Underwater Vehicles (UUVs). These SONAR images are inherently noisy and difficult to interpret, and pictures taken underwater are unreliable due to murkiness and inconsistent lighting. The ATR system breaks target recognition into three stages: 1) Videos of both SONAR and camera footage are broken into frames and preprocessed to enhance images and detect Regions of Interest (ROIs). 2) Features are extracted from these ROIs in preparation for classification. 3) ROIs are classified as true or false positives using a standard Neural Network based on the extracted features. Several preprocessing, feature extraction, and training methods are tested and discussed in this report.

Examination of soldier target recognition with direct view optics

NASA Astrophysics Data System (ADS)

Long, Frederick H.; Larkin, Gabriella; Bisordi, Danielle; Dorsey, Shauna; Marianucci, Damien; Goss, Lashawnta; Bastawros, Michael; Misiuda, Paul; Rodgers, Glenn; Mazz, John P.

2017-10-01

Target recognition and identification is a problem of great military and scientific importance. To examine the correlation between target recognition and optical magnification, ten U.S. Army soldiers were tasked with identifying letters on targets at 800 and 1300 meters away. Letters were used since they are a standard method for measuring visual acuity. The letters were approximately 90 cm high, which is the size of a well-known rifle. Four direct view optics with angular magnifications of 1.5x, 4x, 6x, and 9x were used. The goal of this approach was to measure actual probabilities for correct target identification. Previous scientific literature suggests that target recognition can be modeled as a linear response problem in angular frequency space using the established values for the contrast sensitivity function for a healthy human eye and the experimentally measured modulation transfer function of the optic. At the 9x magnification, the soldiers could identify the letters with almost no errors (i.e., 97% probability of correct identification). At lower magnification, errors in letter identification were more frequent. The identification errors were not random but occurred most frequently with a few pairs of letters (e.g., O and Q), which is consistent with the literature for letter recognition. In addition, in the small subject sample of ten soldiers, there was considerable variation in the observer recognition capability at 1.5x and a range of 800 meters. This can be directly attributed to the variation in the observer visual acuity.

A rule of seven in Watson-Crick base-pairing of mismatched sequences.

PubMed

Cisse, Ibrahim I; Kim, Hajin; Ha, Taekjip

2012-05-13

Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid-based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.

A multiplexable TALE-based binary expression system for in vivo cellular interaction studies.

PubMed

Toegel, Markus; Azzam, Ghows; Lee, Eunice Y; Knapp, David J H F; Tan, Ying; Fa, Ming; Fulga, Tudor A

2017-11-21

Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE-VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.

Conserved binding of GCAC motifs by MEC-8, couch potato, and the RBPMS protein family

PubMed Central

Soufari, Heddy

2017-01-01

Precise regulation of mRNA processing, translation, localization, and stability relies on specific interactions with RNA-binding proteins whose biological function and target preference are dictated by their preferred RNA motifs. The RBPMS family of RNA-binding proteins is defined by a conserved RNA recognition motif (RRM) domain found in metazoan RBPMS/Hermes and RBPMS2, Drosophila couch potato, and MEC-8 from Caenorhabditis elegans. In order to determine the parameters of RNA sequence recognition by the RBPMS family, we have first used the N-terminal domain from MEC-8 in binding assays and have demonstrated a preference for two GCAC motifs optimally separated by >6 nucleotides (nt). We have also determined the crystal structure of the dimeric N-terminal RRM domain from MEC-8 in the unbound form, and in complex with an oligonucleotide harboring two copies of the optimal GCAC motif. The atomic details reveal the molecular network that provides specificity to all four bases in the motif, including multiple hydrogen bonds to the initial guanine. Further studies with human RBPMS, as well as Drosophila couch potato, confirm a general preference for this double GCAC motif by other members of the protein family and the presence of this motif in known targets. PMID:28003515

Single-Molecule View of Small RNA-Guided Target Search and Recognition.

PubMed

Globyte, Viktorija; Kim, Sung Hyun; Joo, Chirlmin

2018-05-20

Most everyday processes in life involve a necessity for an entity to locate its target. On a cellular level, many proteins have to find their target to perform their function. From gene-expression regulation to DNA repair to host defense, numerous nucleic acid-interacting proteins use distinct target search mechanisms. Several proteins achieve that with the help of short RNA strands known as guides. This review focuses on single-molecule advances studying the target search and recognition mechanism of Argonaute and CRISPR (clustered regularly interspaced short palindromic repeats) systems. We discuss different steps involved in search and recognition, from the initial complex prearrangement into the target-search competent state to the final proofreading steps. We focus on target search mechanisms that range from weak interactions, to one- and three-dimensional diffusion, to conformational proofreading. We compare the mechanisms of Argonaute and CRISPR with a well-studied target search system, RecA.

Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

PubMed Central

Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

2011-01-01

HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

Role of the chromatin landscape and sequence in determining cell type-specific genomic glucocorticoid receptor binding and gene regulation.

PubMed

Love, Michael I; Huska, Matthew R; Jurk, Marcel; Schöpflin, Robert; Starick, Stephan R; Schwahn, Kevin; Cooper, Samantha B; Yamamoto, Keith R; Thomas-Chollier, Morgane; Vingron, Martin; Meijsing, Sebastiaan H

2017-02-28

The genomic loci bound by the glucocorticoid receptor (GR), a hormone-activated transcription factor, show little overlap between cell types. To study the role of chromatin and sequence in specifying where GR binds, we used Bayesian modeling within the universe of accessible chromatin. Taken together, our results uncovered that although GR preferentially binds accessible chromatin, its binding is biased against accessible chromatin located at promoter regions. This bias can only be explained partially by the presence of fewer GR recognition sequences, arguing for the existence of additional mechanisms that interfere with GR binding at promoters. Therefore, we tested the role of H3K9ac, the chromatin feature with the strongest negative association with GR binding, but found that this correlation does not reflect a causative link. Finally, we find a higher percentage of promoter-proximal GR binding for genes regulated by GR across cell types than for cell type-specific target genes. Given that GR almost exclusively binds accessible chromatin, we propose that cell type-specific regulation by GR preferentially occurs via distal enhancers, whose chromatin accessibility is typically cell type-specific, whereas ubiquitous target gene regulation is more likely to result from binding to promoter regions, which are often accessible regardless of cell type examined. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of structure switching aptamer assay for detection of aflatoxin M1 in milk sample.

PubMed

Sharma, Atul; Catanante, Gaëlle; Hayat, Akhtar; Istamboulie, Georges; Ben Rejeb, Ines; Bhand, Sunil; Marty, Jean Louis

2016-09-01

The discovery of in-vitro systematic evolution of ligands by exponential enrichment (SELEX) process has considerably broaden the utility of aptamer as bio-recognition element, providing the high binding affinity and specificity against the target analytes. Recent research has focused on the development of structure switching signaling aptamer assay, transducing the aptamer- target recognition event into an easily detectable signal. In this paper, we demonstrate the development of structure switching aptamer assay for determination of aflatoxin M1 (AFM1) employing the quenching-dequenching mechanism. Hybridization of fluorescein labelled anti-AFM1 aptamer (F-aptamer) with TAMRA labelled complementary sequences (Q-aptamer) brings the fluorophore and the quencher into close proximity, which results in maximum fluorescence quenching. On addition of AFM1, the target induced conformational formation of antiparallel G-quadruplex aptamer-AFM1 complex results in fluorescence recovery. Under optimized experimental conditions, the developed method showed the good linearity with limit of detection (LOD) at 5.0ngkg(-1) for AFM1. The specificity of the sensing platform was carefully investigated against aflatoxin B1 (AFB1) and ochratoxin A (OTA). The developed assay platform showed the high specificity towards AFM1. The practical application of the developed aptamer assay was verified for detection of AFM1 in spiked milk samples. Good recoveries were obtained in the range from 94.40% to 95.28% (n=3) from AFM1 spiked milk sample. Copyright © 2016. Published by Elsevier B.V.

Using Metadynamics to Understand the Mechanism of Calmodulin/Target Recognition at Atomic Detail

PubMed Central

Fiorin, G.; Pastore, A.; Carloni, P.; Parrinello, M.

2006-01-01

The ability of calcium-bound calmodulin (CaM) to recognize most of its target peptides is caused by its binding to two hydrophobic residues (‘anchors’). In most of the CaM complexes, the anchors pack against the hydrophobic pockets of the CaM domains and are surrounded by fully conserved Met side chains. Here, by using metadynamics simulations, we investigate quantitatively the energetics of the final step of this process using the M13 peptide, which has a high affinity and spans the sequence of the skeletal myosin light chain kinase, an important natural CaM target. We established the accuracy of our calculations by a comparison between calculated and NMR-derived structural and dynamical properties. Our calculations provide novel insights into the mechanism of protein/peptide recognition: we show that the process is associated with a free energy gain similar to that experimentally measured for the CaM complex with the homologous smooth muscle MLCK peptide (Ehrhardt et al., 1995, Biochemistry 34, 2731). We suggest that binding is dominated by the entropic effect, in agreement with previous proposals. Furthermore, we explain the role of conserved methionines by showing that the large flexibility of these side chains is a key feature of the binding mechanism. Finally, we provide a rationale for the experimental observation that in all CaM complexes the C-terminal domain seems to be hierarchically more important in establishing the interaction. PMID:16877506

AzaHx, a novel fluorescent, DNA minor groove and G·C recognition element: Synthesis and DNA binding properties of a p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (azaHx-PI) polyamide.

PubMed

Satam, Vijay; Babu, Balaji; Patil, Pravin; Brien, Kimberly A; Olson, Kevin; Savagian, Mia; Lee, Megan; Mepham, Andrew; Jobe, Laura Beth; Bingham, John P; Pett, Luke; Wang, Shuo; Ferrara, Maddi; Bruce, Chrystal D; Wilson, W David; Lee, Moses; Hartley, John A; Kiakos, Konstantinos

2015-09-01

The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔT(M), and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C-G doublet. AzaHx-PI was found to target 5'-ACGCGT-3', the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (K(eq) 4.0±0.2×10(7) M(-1)). Copyright © 2015 Elsevier Ltd. All rights reserved.

Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

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

T Wang; K Heran Darwin; H Li

2011-12-31

Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Ourmore » work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.« less

Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

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

Wang, T.; Li, H.; Darwin, K. H.

2010-11-01

Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Ourmore » work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.« less

FPGA design of correlation-based pattern recognition

NASA Astrophysics Data System (ADS)

Jridi, Maher; Alfalou, Ayman

2017-05-01

Optical/Digital pattern recognition and tracking based on optical/digital correlation are a well-known techniques to detect, identify and localize a target object in a scene. Despite the limited number of treatments required by the correlation scheme, computational time and resources are relatively high. The most computational intensive treatment required by the correlation is the transformation from spatial to spectral domain and then from spectral to spatial domain. Furthermore, these transformations are used on optical/digital encryption schemes like the double random phase encryption (DRPE). In this paper, we present a VLSI architecture for the correlation scheme based on the fast Fourier transform (FFT). One interesting feature of the proposed scheme is its ability to stream image processing in order to perform correlation for video sequences. A trade-off between the hardware consumption and the robustness of the correlation can be made in order to understand the limitations of the correlation implementation in reconfigurable and portable platforms. Experimental results obtained from HDL simulations and FPGA prototype have demonstrated the advantages of the proposed scheme.

Visual performance with sport-tinted contact lenses in natural sunlight.

PubMed

Erickson, Graham B; Horn, Fraser C; Barney, Tyler; Pexton, Brett; Baird, Richard Y

2009-05-01

The use of tinted and clear contact lenses (CLs) in all aspects of life is becoming a more popular occurrence, particularly in athletic activities. This study broadens previous research regarding performance-tinted CLs and their effects on measures of visual performance. Thirty-three subjects (14 male, 19 female) were fitted with clear B&L Optima 38, 50% visible light transmission Amber and 36% visible light transmission Gray-Green Nike Maxsight CLs in an individualized randomized sequence. Subjects were dark-adapted with welding goggles before testing and in between subtests involving a Bailey-Lovie chart and the Haynes Distance Rock test. The sequence of testing was repeated for each lens modality. The Amber and Gray-Green lenses enabled subjects to recover vision faster in bright sunlight compared with clear lenses. Also, subjects were able to achieve better visual recognition in bright sunlight when compared with clear lenses. Additionally, the lenses allowed the subjects to alternate fixation between a bright and shaded target at a more rapid rate in bright sunlight as compared with clear lenses. Subjects preferred both the Amber and Gray-Green lenses over clear lenses in the bright and shadowed target conditions. The results of the current study show that Maxsight Amber and Gray-Green lenses provide better contrast discrimination in bright sunlight, better contrast discrimination when alternating between bright and shaded target conditions, better speed of visual recovery in bright sunlight, and better overall visual performance in bright and shaded target conditions compared with clear lenses.

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits.

PubMed

Lebar, Tina; Jerala, Roman

2016-10-21

Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.

Progress towards the development of SH2 domain inhibitors.

PubMed

Kraskouskaya, Dziyana; Duodu, Eugenia; Arpin, Carolynn C; Gunning, Patrick T

2013-04-21

Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein-protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR.

PubMed

Song, Weiling; Yin, Wenshuo; Sun, Wenbo; Guo, Xiaoyan; He, Peng; Yang, Xiaoyan; Zhang, Xiaoru

2018-04-24

Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier Inc. All rights reserved.

Recent Progress in Aptamer-Based Functional Probes for Bioanalysis and Biomedicine.

PubMed

Zhang, Huimin; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong

2016-07-11

Nucleic acid aptamers are short synthetic DNA or RNA sequences that can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have attracted increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal recognition ligands for bioanalysis, disease diagnosis, and cancer therapy. This review highlights the recent progress in aptamer selection and the latest applications of aptamer-based functional probes in the fields of bioanalysis and biomedicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanism of protein import across the chloroplast envelope.

PubMed

Chen, K; Chen, X; Schnell, D J

2000-01-01

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

A split recognition mode combined with cascade signal amplification strategy for highly specific, sensitive detection of microRNA.

PubMed

Wang, Rui; Wang, Lei; Zhao, Haiyan; Jiang, Wei

2016-12-15

MicroRNAs (miRNAs) are vital for many biological processes and have been regarded as cancer biomarkers. Specific and sensitive detection of miRNAs is essential for cancer diagnosis and therapy. Herein, a split recognition mode combined with cascade signal amplification strategy is developed for highly specific and sensitive detection of miRNA. The split recognition mode possesses two specific recognition processes, which are based on toehold-mediated strand displacement reaction (TSDR) and direct hybridization reaction. Two recognition probes, hairpin probe (HP) with overhanging toehold domain and assistant probe (AP), are specially designed. Firstly, the toehold domain of HP and AP recognize part of miRNA simultaneously, accompanied with TSDR to unfold the HP and form the stable DNA Y-shaped junction structure (YJS). Then, the AP in YJS can further act as primer to initiate strand displacement amplification, releasing numerous trigger sequences. Finally, the trigger sequences hybridize with padlock DNA to initiate circular rolling circle amplification and generate enhanced fluorescence responses. In this strategy, the dual recognition effect of split recognition mode guarantees the excellent selectivity to discriminate let-7b from high-homology sequences. Furthermore, the high amplification efficiency of cascade signal amplification guarantees a high sensitivity with the detection limit of 3.2 pM and the concentration of let-7b in total RNA sample extracted from Hela cells is determined. These results indicate our strategy will be a promising miRNA detection strategy in clinical diagnosis and disease treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Learning target masks in infrared linescan imagery

NASA Astrophysics Data System (ADS)

Fechner, Thomas; Rockinger, Oliver; Vogler, Axel; Knappe, Peter

1997-04-01

In this paper we propose a neural network based method for the automatic detection of ground targets in airborne infrared linescan imagery. Instead of using a dedicated feature extraction stage followed by a classification procedure, we propose the following three step scheme: In the first step of the recognition process, the input image is decomposed into its pyramid representation, thus obtaining a multiresolution signal representation. At the lowest three levels of the Laplacian pyramid a neural network filter of moderate size is trained to indicate the target location. The last step consists of a fusion process of the several neural network filters to obtain the final result. To perform this fusion we use a belief network to combine the various filter outputs in a statistical meaningful way. In addition, the belief network allows the integration of further knowledge about the image domain. By applying this multiresolution recognition scheme, we obtain a nearly scale- and rotational invariant target recognition with a significantly decreased false alarm rate compared with a single resolution target recognition scheme.

Prediction of glycolipid-binding domains from the amino acid sequence of lipid raft-associated proteins: application to HpaA, a protein involved in the adhesion of Helicobacter pylori to gastrointestinal cells.

PubMed

Fantini, Jacques; Garmy, Nicolas; Yahi, Nouara

2006-09-12

Protein-glycolipid interactions mediate the attachment of various pathogens to the host cell surface as well as the association of numerous cellular proteins with lipid rafts. Thus, it is of primary importance to identify the protein domains involved in glycolipid recognition. Using structure similarity searches, we could identify a common glycolipid-binding domain in the three-dimensional structure of several proteins known to interact with lipid rafts. Yet the three-dimensional structure of most raft-targeted proteins is still unknown. In the present study, we have identified a glycolipid-binding domain in the amino acid sequence of a bacterial adhesin (Helicobacter pylori adhesin A, HpaA). The prediction was based on the major properties of the glycolipid-binding domains previously characterized by structural searches. A short (15-mer) synthetic peptide corresponding to this putative glycolipid-binding domain was synthesized, and we studied its interaction with glycolipid monolayers at the air-water interface. The synthetic HpaA peptide recognized LacCer but not Gb3. This glycolipid specificity was in line with that of the whole bacterium. Molecular modeling studies gave some insights into this high selectivity of interaction. It also suggested that Phe147 in HpaA played a key role in LacCer recognition, through sugar-aromatic CH-pi stacking interactions with the hydrophobic side of the galactose ring of LacCer. Correspondingly, the replacement of Phe147 with Ala strongly affected LacCer recognition, whereas substitution with Trp did not. Our method could be used to identify glycolipid-binding domains in microbial and cellular proteins interacting with lipid shells, rafts, and other specialized membrane microdomains.

Molecular determinants for recognition of divergent SAMHD1 proteins by the lentiviral accessory protein Vpx.

PubMed

Schwefel, David; Boucherit, Virginie C; Christodoulou, Evangelos; Walker, Philip A; Stoye, Jonathan P; Bishop, Kate N; Taylor, Ian A

2015-04-08

The SAMHD1 triphosphohydrolase inhibits HIV-1 infection of myeloid and resting T cells by depleting dNTPs. To overcome SAMHD1, HIV-2 and some SIVs encode either of two lineages of the accessory protein Vpx that bind the SAMHD1 N or C terminus and redirect the host cullin-4 ubiquitin ligase to target SAMHD1 for proteasomal degradation. We present the ternary complex of Vpx from SIV that infects mandrills (SIVmnd-2) with the cullin-4 substrate receptor, DCAF1, and N-terminal and SAM domains from mandrill SAMHD1. The structure reveals details of Vpx lineage-specific targeting of SAMHD1 N-terminal "degron" sequences. Comparison with Vpx from SIV that infects sooty mangabeys (SIVsmm) complexed with SAMHD1-DCAF1 identifies molecular determinants directing Vpx lineages to N- or C-terminal SAMHD1 sequences. Inspection of the Vpx-DCAF1 interface also reveals conservation of Vpx with the evolutionally related HIV-1/SIV accessory protein Vpr. These data suggest a unified model for how Vpx and Vpr exploit DCAF1 to promote viral replication. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A novel visual saliency detection method for infrared video sequences

NASA Astrophysics Data System (ADS)

Wang, Xin; Zhang, Yuzhen; Ning, Chen

2017-12-01

Infrared video applications such as target detection and recognition, moving target tracking, and so forth can benefit a lot from visual saliency detection, which is essentially a method to automatically localize the ;important; content in videos. In this paper, a novel visual saliency detection method for infrared video sequences is proposed. Specifically, for infrared video saliency detection, both the spatial saliency and temporal saliency are considered. For spatial saliency, we adopt a mutual consistency-guided spatial cues combination-based method to capture the regions with obvious luminance contrast and contour features. For temporal saliency, a multi-frame symmetric difference approach is proposed to discriminate salient moving regions of interest from background motions. Then, the spatial saliency and temporal saliency are combined to compute the spatiotemporal saliency using an adaptive fusion strategy. Besides, to highlight the spatiotemporal salient regions uniformly, a multi-scale fusion approach is embedded into the spatiotemporal saliency model. Finally, a Gestalt theory-inspired optimization algorithm is designed to further improve the reliability of the final saliency map. Experimental results demonstrate that our method outperforms many state-of-the-art saliency detection approaches for infrared videos under various backgrounds.

Codon-Anticodon Recognition in the Bacillus subtilis glyQS T Box Riboswitch

PubMed Central

Caserta, Enrico; Liu, Liang-Chun; Grundy, Frank J.; Henkin, Tina M.

2015-01-01

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNAGly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system. PMID:26229106

Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.

PubMed

Boivin, Vincent; Deschamps-Francoeur, Gabrielle; Couture, Sonia; Nottingham, Ryan M; Bouchard-Bourelle, Philia; Lambowitz, Alan M; Scott, Michelle S; Abou-Elela, Sherif

2018-07-01

Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. S tructured n on c oding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. © 2018 Boivin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Tomiyoshi, Keisuke; Hoshida, Hisashi; Akada, Rinji

2015-12-01

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

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

Valley, Cary T.; Porter, Douglas F.; Qiu, Chen

2012-06-28

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites.more » Despite this conserved 'two-handed' pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.« less

Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme

PubMed Central

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; Holton, James M.; Charles, Emeric; O’Connell, Mitchell R.; Doudna, Jennifer A.

2018-01-01

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated ribonuclease (RNase) capable of crRNA processing and single-stranded RNA degradation upon target transcript binding. Here we present the 2.0 Å resolution crystal structure of a crRNA-bound L. bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define for the first time the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked prior to target RNA recognition, with implications for both bacterial immunity and diagnostic applications. PMID:28892041

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.

PubMed

Yang, Hui; Patel, Dinshaw J

2017-07-06

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing. Published by Elsevier Inc.

Molecular dynamics simulations of Ago silencing complexes reveal a large repertoire of admissible ‘seed-less’ targets

PubMed Central

Xia, Zhen; Clark, Peter; Huynh, Tien; Loher, Phillipe; Zhao, Yue; Chen, Huang-Wen; Rigoutsos, Isidore; Zhou, Ruhong

2012-01-01

To better understand the recognition mechanism of RISC and the repertoire of guide-target interactions we introduced G:U wobbles and mismatches at various positions of the microRNA (miRNA) ‘seed’ region and performed all-atom molecular dynamics simulations of the resulting Ago-miRNA:mRNA ternary complexes. Our simulations reveal that many modifications, including combinations of multiple G:U wobbles and mismatches in the seed region, are admissible and result in only minor structural fluctuations that do not affect overall complex stability. These results are further supported by analyses of HITS-CLIP data. Lastly, introduction of disruptive mutations revealed a bending motion of the PAZ domain along the L1/L2 ‘hinge’ and a subsequent opening of the nucleic-acid-binding channel. Our findings suggest that the spectrum of a miRNA's admissible targets is different from what is currently anticipated by the canonical seed-model. Moreover, they provide a likely explanation for the previously reported sequence-dependent regulation of unintended targeting by siRNAs. PMID:22888400

Engineering a Cell-surface Aptamer Circuit for Targeted and Amplified Photodynamic Cancer Therapy

PubMed Central

Han, Da; Zhu, Guizhi; Wu, Cuichen; Zhu, Zhi; Chen, Tao; Zhang, Xiaobing

2013-01-01

Photodynamic therapy (PDT) is one of the most promising and noninvasive methods for clinical treatment of different malignant diseases. Here, we present a novel strategy of designing an aptamer-based DNA nanocircuit capable of the selective recognition of cancer cells, controllable activation of photosensitizer and amplification of photodynamic therapeutic effect. The aptamers can selectively recognize target cancer cells and bind to the specific proteins on cell membranes. Then the overhanging catalyst sequence on aptamer can trigger a toehold-mediated catalytic strand displacement to activate photosensitizer and achieve amplified therapeutic effect. The specific binding-induced activation allows the DNA circuit to distinguish diseased cells from healthy cells, reducing damage to nearby healthy cells. Moreover, the catalytic amplification reaction will only take place close to the target cancer cells, resulting in a high local concentration of singlet oxygen to selectively kill the target cells. The principle employed in this study demonstrated the feasibility of assembling a DNA circuit on cell membranes and could further broaden the utility of DNA circuits for applications in biology, biotechnology, and biomedicine. PMID:23397942

Assessing the performance of a motion tracking system based on optical joint transform correlation

NASA Astrophysics Data System (ADS)

Elbouz, M.; Alfalou, A.; Brosseau, C.; Ben Haj Yahia, N.; Alam, M. S.

2015-08-01

We present an optimized system specially designed for the tracking and recognition of moving subjects in a confined environment (such as an elderly remaining at home). In the first step of our study, we use a VanderLugt correlator (VLC) with an adapted pre-processing treatment of the input plane and a postprocessing of the correlation plane via a nonlinear function allowing us to make a robust decision. The second step is based on an optical joint transform correlation (JTC)-based system (NZ-NL-correlation JTC) for achieving improved detection and tracking of moving persons in a confined space. The proposed system has been found to have significantly superior discrimination and robustness capabilities allowing to detect an unknown target in an input scene and to determine the target's trajectory when this target is in motion. This system offers robust tracking performance of a moving target in several scenarios, such as rotational variation of input faces. Test results obtained using various real life video sequences show that the proposed system is particularly suitable for real-time detection and tracking of moving objects.

The utility of multiple synthesized views in the recognition of unfamiliar faces.

PubMed

Jones, Scott P; Dwyer, Dominic M; Lewis, Michael B

2017-05-01

The ability to recognize an unfamiliar individual on the basis of prior exposure to a photograph is notoriously poor and prone to errors, but recognition accuracy is improved when multiple photographs are available. In applied situations, when only limited real images are available (e.g., from a mugshot or CCTV image), the generation of new images might provide a technological prosthesis for otherwise fallible human recognition. We report two experiments examining the effects of providing computer-generated additional views of a target face. In Experiment 1, provision of computer-generated views supported better target face recognition than exposure to the target image alone and equivalent performance to that for exposure of multiple photograph views. Experiment 2 replicated the advantage of providing generated views, but also indicated an advantage for multiple viewings of the single target photograph. These results strengthen the claim that identifying a target face can be improved by providing multiple synthesized views based on a single target image. In addition, our results suggest that the degree of advantage provided by synthesized views may be affected by the quality of synthesized material.

Detection, recognition, identification, and tracking of military vehicles using biomimetic intelligence

NASA Astrophysics Data System (ADS)

Pace, Paul W.; Sutherland, John

2001-10-01

This project is aimed at analyzing EO/IR images to provide automatic target detection/recognition/identification (ATR/D/I) of militarily relevant land targets. An increase in performance was accomplished using a biomimetic intelligence system functioning on low-cost, commercially available processing chips. Biomimetic intelligence has demonstrated advanced capabilities in the areas of hand- printed character recognition, real-time detection/identification of multiple faces in full 3D perspectives in cluttered environments, advanced capabilities in classification of ground-based military vehicles from SAR, and real-time ATR/D/I of ground-based military vehicles from EO/IR/HRR data in cluttered environments. The investigation applied these tools to real data sets and examined the parameters such as the minimum resolution for target recognition, the effect of target size, rotation, line-of-sight changes, contrast, partial obscuring, background clutter etc. The results demonstrated a real-time ATR/D/I capability against a subset of militarily relevant land targets operating in a realistic scenario. Typical results on the initial EO/IR data indicate probabilities of correct classification of resolved targets to be greater than 95 percent.

Iterative cross section sequence graph for handwritten character segmentation.

PubMed

Dawoud, Amer

2007-08-01

The iterative cross section sequence graph (ICSSG) is an algorithm for handwritten character segmentation. It expands the cross section sequence graph concept by applying it iteratively at equally spaced thresholds. The iterative thresholding reduces the effect of information loss associated with image binarization. ICSSG preserves the characters' skeletal structure by preventing the interference of pixels that causes flooding of adjacent characters' segments. Improving the structural quality of the characters' skeleton facilitates better feature extraction and classification, which improves the overall performance of optical character recognition (OCR). Experimental results showed significant improvements in OCR recognition rates compared to other well-established segmentation algorithms.

Training the max-margin sequence model with the relaxed slack variables.

PubMed

Niu, Lingfeng; Wu, Jianmin; Shi, Yong

2012-09-01

Sequence models are widely used in many applications such as natural language processing, information extraction and optical character recognition, etc. We propose a new approach to train the max-margin based sequence model by relaxing the slack variables in this paper. With the canonical feature mapping definition, the relaxed problem is solved by training a multiclass Support Vector Machine (SVM). Compared with the state-of-the-art solutions for the sequence learning, the new method has the following advantages: firstly, the sequence training problem is transformed into a multiclassification problem, which is more widely studied and already has quite a few off-the-shelf training packages; secondly, this new approach reduces the complexity of training significantly and achieves comparable prediction performance compared with the existing sequence models; thirdly, when the size of training data is limited, by assigning different slack variables to different microlabel pairs, the new method can use the discriminative information more frugally and produces more reliable model; last but not least, by employing kernels in the intermediate multiclass SVM, nonlinear feature space can be easily explored. Experimental results on the task of named entity recognition, information extraction and handwritten letter recognition with the public datasets illustrate the efficiency and effectiveness of our method. Copyright © 2012 Elsevier Ltd. All rights reserved.

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

PubMed

Liu, Tina Y; Iavarone, Anthony T; Doudna, Jennifer A

2017-01-01

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

Novel proteases from the genome of the carnivorous plant Drosera capensis: structural prediction and comparative analysis

PubMed Central

Butts, Carter T.; Bierma, Jan C.; Martin, Rachel W.

2016-01-01

In his 1875 monograph on insectivorous plants, Darwin described the feeding reactions of Drosera flypaper traps and predicted that their secretions contained a “ferment” similar to mammalian pepsin, an aspartic protease. Here we report a high-quality draft genome sequence for the cape sundew, Drosera capensis, the first genome of a carnivorous plant from order Caryophyllales, which also includes the Venus flytrap (Dionaea) and the tropical pitcher plants (Nepenthes). This species was selected in part for its hardiness and ease of cultivation, making it an excellent model organism for further investigations of plant carnivory. Analysis of predicted protein sequences yields genes encoding proteases homologous to those found in other plants, some of which display sequence and structural features that suggest novel functionalities. Because the sequence similarity to proteins of known structure is in most cases too low for traditional homology modeling, 3D structures of representative proteases are predicted using comparative modeling with all-atom refinement. Although the overall folds and active residues for these proteins are conserved, we find structural and sequence differences consistent with a diversity of substrate recognition patterns. Finally, we predict differences in substrate specificities using in silico experiments, providing targets for structure/function studies of novel enzymes with biological and technological significance. PMID:27353064

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

NASA Technical Reports Server (NTRS)

Pitulle, C.; Hedenstierna, K. O.; Fox, G. E.

1995-01-01

Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

Birth and death of genes linked to chromosomal inversion

PubMed Central

Furuta, Yoshikazu; Kawai, Mikihiko; Yahara, Koji; Takahashi, Noriko; Handa, Naofumi; Tsuru, Takeshi; Oshima, Kenshiro; Yoshida, Masaru; Azuma, Takeshi; Hattori, Masahira; Uchiyama, Ikuo; Kobayashi, Ichizo

2011-01-01

The birth and death of genes is central to adaptive evolution, yet the underlying genome dynamics remain elusive. The availability of closely related complete genome sequences helps to follow changes in gene contents and clarify their relationship to overall genome organization. Helicobacter pylori, bacteria in our stomach, are known for their extreme genome plasticity through mutation and recombination and will make a good target for such an analysis. In comparing their complete genome sequences, we found that gain and loss of genes (loci) for outer membrane proteins, which mediate host interaction, occurred at breakpoints of chromosomal inversions. Sequence comparison there revealed a unique mechanism of DNA duplication: DNA duplication associated with inversion. In this process, a DNA segment at one chromosomal locus is copied and inserted, in an inverted orientation, into a distant locus on the same chromosome, while the entire region between these two loci is also inverted. Recognition of this and three more inversion modes, which occur through reciprocal recombination between long or short sequence similarity or adjacent to a mobile element, allowed reconstruction of synteny evolution through inversion events in this species. These results will guide the interpretation of extensive DNA sequencing results for understanding long- and short-term genome evolution in various organisms and in cancer cells. PMID:21212362

Variability in the impairment of recognition memory in patients with frontal lobe lesions.

PubMed

Bastin, Christine; Van der Linden, Martial; Lekeu, Françoise; Andrés, Pilar; Salmon, Eric

2006-10-01

Fourteen patients with frontal lobe lesions and 14 normal subjects were tested on a recognition memory task that required discriminating between target words, new words that are synonyms of the targets and unrelated distractors. A deficit was found in 12 of the patients. Moreover, three different patterns of recognition impairment were identified: (I) poor memory for targets, (II) normal hits but increased false recognitions for both types of distractors, (III) normal hit rates, but increased false recognitions for synonyms only. Differences in terms of location of the damage and behavioral characteristics between these subgroups were examined. An encoding deficit was proposed to explain the performance of patients in subgroup I. The behavioral patterns of the patients in subgroups II and III could be interpreted as deficient post-retrieval verification processes and an inability to recollect item-specific information, respectively.

The nonverbal expression of pride: evidence for cross-cultural recognition.

PubMed

Tracy, Jessica L; Robins, Richard W

2008-03-01

The present research tests whether recognition for the nonverbal expression of pride generalizes across cultures. Study 1 provided the first evidence for cross-cultural recognition of pride, demonstrating that the expression generalizes across Italy and the United States. Study 2 found that the pride expression generalizes beyond Western cultures; individuals from a preliterate, highly isolated tribe in Burkina Faso, West Africa, reliably recognized pride, regardless of whether it was displayed by African or American targets. These Burkinabe participants were unlikely to have learned the pride expression through cross-cultural transmission, so their recognition suggests that pride may be a human universal. Studies 3 and 4 used drawn figures to systematically manipulate the ethnicity and gender of targets showing the expression, and demonstrated that pride recognition generalizes across male and female targets of African, Asian, and Caucasian descent. Discussion focuses on the implications of the findings for the universality of the pride expression.

View-invariant gait recognition method by three-dimensional convolutional neural network

NASA Astrophysics Data System (ADS)

Xing, Weiwei; Li, Ying; Zhang, Shunli

2018-01-01

Gait as an important biometric feature can identify a human at a long distance. View change is one of the most challenging factors for gait recognition. To address the cross view issues in gait recognition, we propose a view-invariant gait recognition method by three-dimensional (3-D) convolutional neural network. First, 3-D convolutional neural network (3DCNN) is introduced to learn view-invariant feature, which can capture the spatial information and temporal information simultaneously on normalized silhouette sequences. Second, a network training method based on cross-domain transfer learning is proposed to solve the problem of the limited gait training samples. We choose the C3D as the basic model, which is pretrained on the Sports-1M and then fine-tune C3D model to adapt gait recognition. In the recognition stage, we use the fine-tuned model to extract gait features and use Euclidean distance to measure the similarity of gait sequences. Sufficient experiments are carried out on the CASIA-B dataset and the experimental results demonstrate that our method outperforms many other methods.

Multiview human activity recognition system based on spatiotemporal template for video surveillance system

NASA Astrophysics Data System (ADS)

Kushwaha, Alok Kumar Singh; Srivastava, Rajeev

2015-09-01

An efficient view invariant framework for the recognition of human activities from an input video sequence is presented. The proposed framework is composed of three consecutive modules: (i) detect and locate people by background subtraction, (ii) view invariant spatiotemporal template creation for different activities, (iii) and finally, template matching is performed for view invariant activity recognition. The foreground objects present in a scene are extracted using change detection and background modeling. The view invariant templates are constructed using the motion history images and object shape information for different human activities in a video sequence. For matching the spatiotemporal templates for various activities, the moment invariants and Mahalanobis distance are used. The proposed approach is tested successfully on our own viewpoint dataset, KTH action recognition dataset, i3DPost multiview dataset, MSR viewpoint action dataset, VideoWeb multiview dataset, and WVU multiview human action recognition dataset. From the experimental results and analysis over the chosen datasets, it is observed that the proposed framework is robust, flexible, and efficient with respect to multiple views activity recognition, scale, and phase variations.

Identification and location of catenary insulator in complex background based on machine vision

NASA Astrophysics Data System (ADS)

Yao, Xiaotong; Pan, Yingli; Liu, Li; Cheng, Xiao

2018-04-01

It is an important premise to locate insulator precisely for fault detection. Current location algorithms for insulator under catenary checking images are not accurate, a target recognition and localization method based on binocular vision combined with SURF features is proposed. First of all, because of the location of the insulator in complex environment, using SURF features to achieve the coarse positioning of target recognition; then Using binocular vision principle to calculate the 3D coordinates of the object which has been coarsely located, realization of target object recognition and fine location; Finally, Finally, the key is to preserve the 3D coordinate of the object's center of mass, transfer to the inspection robot to control the detection position of the robot. Experimental results demonstrate that the proposed method has better recognition efficiency and accuracy, can successfully identify the target and has a define application value.

Foundations for a syntatic pattern recognition system for genomic DNA sequences

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

Searles, D.B.

1993-03-01

The goal of the proposed work is the creation of a software system that will perform sophisticated pattern recognition and related functions at a level of abstraction and with expressive power beyond current general-purpose pattern-matching systems for biological sequences; and with a more uniform language, environment, and graphical user interface, and with greater flexibility, extensibility, embeddability, and ability to incorporate other algorithms, than current special-purpose analytic software.

Functional specificity of a Hox protein mediated by the recognition of minor groove structure.

PubMed

Joshi, Rohit; Passner, Jonathan M; Rohs, Remo; Jain, Rinku; Sosinsky, Alona; Crickmore, Michael A; Jacob, Vinitha; Aggarwal, Aneel K; Honig, Barry; Mann, Richard S

2007-11-02

The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.

Mining sequential patterns for protein fold recognition.

PubMed

Exarchos, Themis P; Papaloukas, Costas; Lampros, Christos; Fotiadis, Dimitrios I

2008-02-01

Protein data contain discriminative patterns that can be used in many beneficial applications if they are defined correctly. In this work sequential pattern mining (SPM) is utilized for sequence-based fold recognition. Protein classification in terms of fold recognition plays an important role in computational protein analysis, since it can contribute to the determination of the function of a protein whose structure is unknown. Specifically, one of the most efficient SPM algorithms, cSPADE, is employed for the analysis of protein sequence. A classifier uses the extracted sequential patterns to classify proteins in the appropriate fold category. For training and evaluating the proposed method we used the protein sequences from the Protein Data Bank and the annotation of the SCOP database. The method exhibited an overall accuracy of 25% in a classification problem with 36 candidate categories. The classification performance reaches up to 56% when the five most probable protein folds are considered.

Intact suppression of increased false recognition in schizophrenia.

PubMed

Weiss, Anthony P; Dodson, Chad S; Goff, Donald C; Schacter, Daniel L; Heckers, Stephan

2002-09-01

Recognition memory is impaired in patients with schizophrenia, as they rely largely on item familiarity, rather than conscious recollection, to make mnemonic decisions. False recognition of novel items (foils) is increased in schizophrenia and may relate to this deficit in conscious recollection. By studying pictures of the target word during encoding, healthy adults can suppress false recognition. This study examined the effect of pictorial encoding on subsequent recognition of repeated foils in patients with schizophrenia. The study included 40 patients with schizophrenia and 32 healthy comparison subjects. After incidental encoding of 60 words or pictures, subjects were tested for recognition of target items intermixed with 60 new foils. These new foils were subsequently repeated following either a two- or 24-word delay. Subjects were instructed to label these repeated foils as new and not to mistake them for old target words. Schizophrenic patients showed greater overall false recognition of repeated foils. The rate of false recognition of repeated foils was lower after picture encoding than after word encoding. Despite higher levels of false recognition of repeated new items, patients and comparison subjects demonstrated a similar degree of false recognition suppression after picture, as compared to word, encoding. Patients with schizophrenia displayed greater false recognition of repeated foils than comparison subjects, suggesting both a decrement of item- (or source-) specific recollection and a consequent reliance on familiarity in schizophrenia. Despite these deficits, presenting pictorial information at encoding allowed schizophrenic subjects to suppress false recognition to a similar degree as the comparison group, implying the intact use of a high-level cognitive strategy in this population.

A Pathogenic Nematode Targets Recognition Proteins to Avoid Insect Defenses

PubMed Central

Toubarro, Duarte; Avila, Mónica Martinez; Montiel, Rafael; Simões, Nelson

2013-01-01

Steinernema carpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with Ki values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S . carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions. PMID:24098715

Use of designed sequences in protein structure recognition.

PubMed

Kumar, Gayatri; Mudgal, Richa; Srinivasan, Narayanaswamy; Sandhya, Sankaran

2018-05-09

Knowledge of the protein structure is a pre-requisite for improved understanding of molecular function. The gap in the sequence-structure space has increased in the post-genomic era. Grouping related protein sequences into families can aid in narrowing the gap. In the Pfam database, structure description is provided for part or full-length proteins of 7726 families. For the remaining 52% of the families, information on 3-D structure is not yet available. We use the computationally designed sequences that are intermediately related to two protein domain families, which are already known to share the same fold. These strategically designed sequences enable detection of distant relationships and here, we have employed them for the purpose of structure recognition of protein families of yet unknown structure. We first measured the success rate of our approach using a dataset of protein families of known fold and achieved a success rate of 88%. Next, for 1392 families of yet unknown structure, we made structural assignments for part/full length of the proteins. Fold association for 423 domains of unknown function (DUFs) are provided as a step towards functional annotation. The results indicate that knowledge-based filling of gaps in protein sequence space is a lucrative approach for structure recognition. Such sequences assist in traversal through protein sequence space and effectively function as 'linkers', where natural linkers between distant proteins are unavailable. This article was reviewed by Oliviero Carugo, Christine Orengo and Srikrishna Subramanian.

Non-Cooperative Target Recognition by Means of Singular Value Decomposition Applied to Radar High Resolution Range Profiles †

PubMed Central

López-Rodríguez, Patricia; Escot-Bocanegra, David; Fernández-Recio, Raúl; Bravo, Ignacio

2015-01-01

Radar high resolution range profiles are widely used among the target recognition community for the detection and identification of flying targets. In this paper, singular value decomposition is applied to extract the relevant information and to model each aircraft as a subspace. The identification algorithm is based on angle between subspaces and takes place in a transformed domain. In order to have a wide database of radar signatures and evaluate the performance, simulated range profiles are used as the recognition database while the test samples comprise data of actual range profiles collected in a measurement campaign. Thanks to the modeling of aircraft as subspaces only the valuable information of each target is used in the recognition process. Thus, one of the main advantages of using singular value decomposition, is that it helps to overcome the notable dissimilarities found in the shape and signal-to-noise ratio between actual and simulated profiles due to their difference in nature. Despite these differences, the recognition rates obtained with the algorithm are quite promising. PMID:25551484

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

PubMed

Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

2018-05-31

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

Domain Regeneration for Cross-Database Micro-Expression Recognition

NASA Astrophysics Data System (ADS)

Zong, Yuan; Zheng, Wenming; Huang, Xiaohua; Shi, Jingang; Cui, Zhen; Zhao, Guoying

2018-05-01

In this paper, we investigate the cross-database micro-expression recognition problem, where the training and testing samples are from two different micro-expression databases. Under this setting, the training and testing samples would have different feature distributions and hence the performance of most existing micro-expression recognition methods may decrease greatly. To solve this problem, we propose a simple yet effective method called Target Sample Re-Generator (TSRG) in this paper. By using TSRG, we are able to re-generate the samples from target micro-expression database and the re-generated target samples would share same or similar feature distributions with the original source samples. For this reason, we can then use the classifier learned based on the labeled source samples to accurately predict the micro-expression categories of the unlabeled target samples. To evaluate the performance of the proposed TSRG method, extensive cross-database micro-expression recognition experiments designed based on SMIC and CASME II databases are conducted. Compared with recent state-of-the-art cross-database emotion recognition methods, the proposed TSRG achieves more promising results.

Object recognition of real targets using modelled SAR images

NASA Astrophysics Data System (ADS)

Zherdev, D. A.

2017-12-01

In this work the problem of recognition is studied using SAR images. The algorithm of recognition is based on the computation of conjugation indices with vectors of class. The support subspaces for each class are constructed by exception of the most and the less correlated vectors in a class. In the study we examine the ability of a significant feature vector size reduce that leads to recognition time decrease. The images of targets form the feature vectors that are transformed using pre-trained convolutional neural network (CNN).

Newer Gene Editing Technologies toward HIV Gene Therapy

PubMed Central

Manjunath, N.; Yi, Guohua; Dang, Ying; Shankar, Premlata

2013-01-01

Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy. PMID:24284874

Effect of colour pop-out on the recognition of letters in crowding conditions.

PubMed

Põder, Endel

2007-11-01

The crowding effect of adjacent objects on the recognition of a target can be reduced when target and flankers differ in some feature, that is irrelevant to the recognition task. In this study, the mechanisms of this effect were explored using targets and flankers of the same and different colours. It was found that facilitation nearly equal to that of differently coloured targets and flankers can be observed with a differently coloured background blob in the location of the target. The different-colour effect does not require advance knowledge of the target and flanker colours, but the effect increases in the course of three trials with constant mapping of colours. The results are consistent with the notion of exogenous attention that facilitates the processing at the most salient locations in the visual field.

A bio-inspired system for spatio-temporal recognition in static and video imagery

NASA Astrophysics Data System (ADS)

Khosla, Deepak; Moore, Christopher K.; Chelian, Suhas

2007-04-01

This paper presents a bio-inspired method for spatio-temporal recognition in static and video imagery. It builds upon and extends our previous work on a bio-inspired Visual Attention and object Recognition System (VARS). The VARS approach locates and recognizes objects in a single frame. This work presents two extensions of VARS. The first extension is a Scene Recognition Engine (SCE) that learns to recognize spatial relationships between objects that compose a particular scene category in static imagery. This could be used for recognizing the category of a scene, e.g., office vs. kitchen scene. The second extension is the Event Recognition Engine (ERE) that recognizes spatio-temporal sequences or events in sequences. This extension uses a working memory model to recognize events and behaviors in video imagery by maintaining and recognizing ordered spatio-temporal sequences. The working memory model is based on an ARTSTORE1 neural network that combines an ART-based neural network with a cascade of sustained temporal order recurrent (STORE)1 neural networks. A series of Default ARTMAP classifiers ascribes event labels to these sequences. Our preliminary studies have shown that this extension is robust to variations in an object's motion profile. We evaluated the performance of the SCE and ERE on real datasets. The SCE module was tested on a visual scene classification task using the LabelMe2 dataset. The ERE was tested on real world video footage of vehicles and pedestrians in a street scene. Our system is able to recognize the events in this footage involving vehicles and pedestrians.

Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication.

PubMed

Peixoto, Paul; Liu, Yang; Depauw, Sabine; Hildebrand, Marie-Paule; Boykin, David W; Bailly, Christian; Wilson, W David; David-Cordonnier, Marie-Hélène

2008-06-01

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim, a phenyl-furan-benzimidazole dication DB293 binds AT-rich sites as a monomer and 5'-ATGA sequence as a stacked dimer, both in the minor groove. Here, we used a protein/DNA array approach to evaluate the ability of DB293 to specifically inhibit transcription factors DNA-binding in a single-step, competitive mode. DB293 inhibits two POU-domain transcription factors Pit-1 and Brn-3 but not IRF-1, despite the presence of an ATGA and AT-rich sites within all three consensus sequences. EMSA, DNase I footprinting and surface-plasmon-resonance experiments determined the precise binding site, affinity and stoichiometry of DB293 interaction to the consensus targets. Binding of DB293 occurred as a cooperative dimer on the ATGA part of Brn-3 site but as two monomers on AT-rich sites of IRF-1 sequence. For Pit-1 site, ATGA or AT-rich mutated sequences identified the contribution of both sites for DB293 recognition. In conclusion, DB293 is a strong inhibitor of two POU-domain transcription factors through a cooperative binding to ATGA. These findings are the first to show that heterocyclic dications can inhibit major groove transcription factors and they open the door to the control of transcription factors activity by those compounds.

Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6.

PubMed

Stamos, Jennifer L; Chu, Matthew Ling-Hon; Enos, Michael D; Shah, Niket; Weis, William I

2014-03-18

Glycogen synthase kinase-3 (GSK-3) is a key regulator of many cellular signaling pathways. Unlike most kinases, GSK-3 is controlled by inhibition rather than by specific activation. In the insulin and several other signaling pathways, phosphorylation of a serine present in a conserved sequence near the amino terminus of GSK-3 generates an auto-inhibitory peptide. In contrast, Wnt/β-catenin signal transduction requires phosphorylation of Ser/Pro rich sequences present in the Wnt co-receptors LRP5/6, and these motifs inhibit GSK-3 activity. We present crystal structures of GSK-3 bound to its phosphorylated N-terminus and to two of the phosphorylated LRP6 motifs. A conserved loop unique to GSK-3 undergoes a dramatic conformational change that clamps the bound pseudo-substrate peptides, and reveals the mechanism of primed substrate recognition. The structures rationalize target sequence preferences and suggest avenues for the design of inhibitors selective for a subset of pathways regulated by GSK-3. DOI: http://dx.doi.org/10.7554/eLife.01998.001.

Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

PubMed

Rampello, Anthony J; Glynn, Steven E

2017-03-24

The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rolling chain amplification based signal-enhanced electrochemical aptasensor for ultrasensitive detection of ochratoxin A.

PubMed

Huang, Lin; Wu, Jingjing; Zheng, Lei; Qian, Haisheng; Xue, Feng; Wu, Yucheng; Pan, Daodong; Adeloju, Samuel B; Chen, Wei

2013-11-19

A novel electrochemical aptasensor is described for rapid and ultrasensitive detection of ochratoxin A (OTA) based on signal enhancement with rolling circle amplification (RCA). The primer for RCA was designed to compose of a two-part sequence, one part of the aptamer sequence directed against OTA while the other part was complementary to the capture probe on the electrode surface. In the presence of target OTA, the primer, originally hybridized with the RCA padlock, is replaced to combine with OTA. This induces the inhibition of RCA and decreases the OTA sensing signal obtained with the electrochemical aptasensor. Under the optimized conditions, ultrasensitive detection of OTA was achieved with a limit of detection (LOD) of 0.065 ppt (pg/mL), which is much lower than previously reported. The electrochemical aptasensor was also successfully applied to the determination of OTA in wine samples. This ultrasensitive electrochemical aptasensor is of great practical importance in food safety and could be widely extended to the detection of other toxins by replacing the sequence of the recognition aptamer.

Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features.

PubMed

Carapito, Raphael; Konantz, Martina; Paillard, Catherine; Miao, Zhichao; Pichot, Angélique; Leduc, Magalie S; Yang, Yaping; Bergstrom, Katie L; Mahoney, Donald H; Shardy, Deborah L; Alsaleh, Ghada; Naegely, Lydie; Kolmer, Aline; Paul, Nicodème; Hanauer, Antoine; Rolli, Véronique; Müller, Joëlle S; Alghisi, Elisa; Sauteur, Loïc; Macquin, Cécile; Morlon, Aurore; Sancho, Consuelo Sebastia; Amati-Bonneau, Patrizia; Procaccio, Vincent; Mosca-Boidron, Anne-Laure; Marle, Nathalie; Osmani, Naël; Lefebvre, Olivier; Goetz, Jacky G; Unal, Sule; Akarsu, Nurten A; Radosavljevic, Mirjana; Chenard, Marie-Pierre; Rialland, Fanny; Grain, Audrey; Béné, Marie-Christine; Eveillard, Marion; Vincent, Marie; Guy, Julien; Faivre, Laurence; Thauvin-Robinet, Christel; Thevenon, Julien; Myers, Kasiani; Fleming, Mark D; Shimamura, Akiko; Bottollier-Lemallaz, Elodie; Westhof, Eric; Lengerke, Claudia; Isidor, Bertrand; Bahram, Seiamak

2017-11-01

Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond-like phenotype.

Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy

PubMed Central

Tochio, Naoya; Umehara, Kohei; Uewaki, Jun-ichi; Flechsig, Holger; Kondo, Masaharu; Dewa, Takehisa; Sakuma, Tetsushi; Yamamoto, Takashi; Saitoh, Takashi; Togashi, Yuichi; Tate, Shin-ichi

2016-01-01

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats. PMID:27883072

Local structure preserving sparse coding for infrared target recognition

PubMed Central

Han, Jing; Yue, Jiang; Zhang, Yi; Bai, Lianfa

2017-01-01

Sparse coding performs well in image classification. However, robust target recognition requires a lot of comprehensive template images and the sparse learning process is complex. We incorporate sparsity into a template matching concept to construct a local sparse structure matching (LSSM) model for general infrared target recognition. A local structure preserving sparse coding (LSPSc) formulation is proposed to simultaneously preserve the local sparse and structural information of objects. By adding a spatial local structure constraint into the classical sparse coding algorithm, LSPSc can improve the stability of sparse representation for targets and inhibit background interference in infrared images. Furthermore, a kernel LSPSc (K-LSPSc) formulation is proposed, which extends LSPSc to the kernel space to weaken the influence of the linear structure constraint in nonlinear natural data. Because of the anti-interference and fault-tolerant capabilities, both LSPSc- and K-LSPSc-based LSSM can implement target identification based on a simple template set, which just needs several images containing enough local sparse structures to learn a sufficient sparse structure dictionary of a target class. Specifically, this LSSM approach has stable performance in the target detection with scene, shape and occlusions variations. High performance is demonstrated on several datasets, indicating robust infrared target recognition in diverse environments and imaging conditions. PMID:28323824

Cat-eye effect target recognition with single-pixel detectors

NASA Astrophysics Data System (ADS)

Jian, Weijian; Li, Li; Zhang, Xiaoyue

2015-12-01

A prototype of cat-eye effect target recognition with single-pixel detectors is proposed. Based on the framework of compressive sensing, it is possible to recognize cat-eye effect targets by projecting a series of known random patterns and measuring the backscattered light with three single-pixel detectors in different locations. The prototype only requires simpler, less expensive detectors and extends well beyond the visible spectrum. The simulations are accomplished to evaluate the feasibility of the proposed prototype. We compared our results to that obtained from conventional cat-eye effect target recognition methods using area array sensor. The experimental results show that this method is feasible and superior to the conventional method in dynamic and complicated backgrounds.

Increase in Speech Recognition Due to Linguistic Mismatch between Target and Masker Speech: Monolingual and Simultaneous Bilingual Performance

ERIC Educational Resources Information Center

Calandruccio, Lauren; Zhou, Haibo

2014-01-01

Purpose: To examine whether improved speech recognition during linguistically mismatched target-masker experiments is due to linguistic unfamiliarity of the masker speech or linguistic dissimilarity between the target and masker speech. Method: Monolingual English speakers (n = 20) and English-Greek simultaneous bilinguals (n = 20) listened to…

Anodal tDCS targeting the right orbitofrontal cortex enhances facial expression recognition

PubMed Central

Murphy, Jillian M.; Ridley, Nicole J.; Vercammen, Ans

2015-01-01

The orbitofrontal cortex (OFC) has been implicated in the capacity to accurately recognise facial expressions. The aim of the current study was to determine if anodal transcranial direct current stimulation (tDCS) targeting the right OFC in healthy adults would enhance facial expression recognition, compared with a sham condition. Across two counterbalanced sessions of tDCS (i.e. anodal and sham), 20 undergraduate participants (18 female) completed a facial expression labelling task comprising angry, disgusted, fearful, happy, sad and neutral expressions, and a control (social judgement) task comprising the same expressions. Responses on the labelling task were scored for accuracy, median reaction time and overall efficiency (i.e. combined accuracy and reaction time). Anodal tDCS targeting the right OFC enhanced facial expression recognition, reflected in greater efficiency and speed of recognition across emotions, relative to the sham condition. In contrast, there was no effect of tDCS to responses on the control task. This is the first study to demonstrate that anodal tDCS targeting the right OFC boosts facial expression recognition. This finding provides a solid foundation for future research to examine the efficacy of this technique as a means to treat facial expression recognition deficits, particularly in individuals with OFC damage or dysfunction. PMID:25971602

Individual differences in forced-choice recognition memory: partitioning contributions of recollection and familiarity.

PubMed

Migo, Ellen M; Quamme, Joel R; Holmes, Selina; Bendell, Andrew; Norman, Kenneth A; Mayes, Andrew R; Montaldi, Daniela

2014-01-01

In forced-choice recognition memory, two different testing formats are possible under conditions of high target-foil similarity: Each target can be presented alongside foils similar to itself (forced-choice corresponding; FCC), or alongside foils similar to other targets (forced-choice noncorresponding; FCNC). Recent behavioural and neuropsychological studies suggest that FCC performance can be supported by familiarity whereas FCNC performance is supported primarily by recollection. In this paper, we corroborate this finding from an individual differences perspective. A group of older adults were given a test of FCC and FCNC recognition for object pictures, as well as standardized tests of recall, recognition, and IQ. Recall measures were found to predict FCNC, but not FCC performance, consistent with a critical role for recollection in FCNC only. After the common influence of recall was removed, standardized tests of recognition predicted FCC, but not FCNC performance. This is consistent with a contribution of only familiarity in FCC. Simulations show that a two-process model, where familiarity and recollection make separate contributions to recognition, is 10 times more likely to give these results than a single-process model. This evidence highlights the importance of recognition memory test design when examining the involvement of recollection and familiarity.

Recognition of visual stimuli and memory for spatial context in schizophrenic patients and healthy volunteers.

PubMed

Brébion, Gildas; David, Anthony S; Pilowsky, Lyn S; Jones, Hugh

2004-11-01

Verbal and visual recognition tasks were administered to 40 patients with schizophrenia and 40 healthy comparison subjects. The verbal recognition task consisted of discriminating between 16 target words and 16 new words. The visual recognition task consisted of discriminating between 16 target pictures (8 black-and-white and 8 color) and 16 new pictures (8 black-and-white and 8 color). Visual recognition was followed by a spatial context discrimination task in which subjects were required to remember the spatial location of the target pictures at encoding. Results showed that recognition deficit in patients was similar for verbal and visual material. In both schizophrenic and healthy groups, men, but not women, obtained better recognition scores for the colored than for the black-and-white pictures. However, men and women similarly benefited from color to reduce spatial context discrimination errors. Patients showed a significant deficit in remembering the spatial location of the pictures, independently of accuracy in remembering the pictures themselves. These data suggest that patients are impaired in the amount of visual information that they can encode. With regards to the perceptual attributes of the stimuli, memory for spatial information appears to be affected, but not processing of color information.

Sequence-Specific Recognition of DNA by Proteins: Binding Motifs Discovered Using a Novel Statistical/Computational Analysis

PubMed Central

Jakubec, David; Laskowski, Roman A.; Vondrasek, Jiri

2016-01-01

Decades of intensive experimental studies of the recognition of DNA sequences by proteins have provided us with a view of a diverse and complicated world in which few to no features are shared between individual DNA-binding protein families. The originally conceived direct readout of DNA residue sequences by amino acid side chains offers very limited capacity for sequence recognition, while the effects of the dynamic properties of the interacting partners remain difficult to quantify and almost impossible to generalise. In this work we investigated the energetic characteristics of all DNA residue—amino acid side chain combinations in the conformations found at the interaction interface in a very large set of protein—DNA complexes by the means of empirical potential-based calculations. General specificity-defining criteria were derived and utilised to look beyond the binding motifs considered in previous studies. Linking energetic favourability to the observed geometrical preferences, our approach reveals several additional amino acid motifs which can distinguish between individual DNA bases. Our results remained valid in environments with various dielectric properties. PMID:27384774

Induced oligomerization targets Golgi proteins for degradation in lysosomes.

PubMed

Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

2015-12-01

Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. © 2015 Tewari et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Ligation-rolling circle amplification combined with γ-cyclodextrin mediated stemless molecular beacon for sensitive and specific genotyping of single-nucleotide polymorphism.

PubMed

Zou, Zhen; Qing, Zhihe; He, Xiaoxiao; Wang, Kemin; He, Dinggeng; Shi, Hui; Yang, Xue; Qing, Taiping; Yang, Xiaoxiao

2014-07-01

A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of β-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Processing Stages Underlying Word Recognition in the Anteroventral Temporal Lobe

PubMed Central

Halgren, Eric; Wang, Chunmao; Schomer, Donald L.; Knake, Susanne; Marinkovic, Ksenija; Wu, Julian; Ulbert, Istvan

2006-01-01

The anteroventral temporal lobe integrates visual, lexical, semantic and mnestic aspects of word-processing, through its reciprocal connections with the ventral visual stream, language areas, and the hippocampal formation. We used linear microelectrode arrays to probe population synaptic currents and neuronal firing in different cortical layers of the anteroventral temporal lobe, during semantic judgments with implicit priming, and overt word recognition. Since different extrinsic and associative inputs preferentially target different cortical layers, this method can help reveal the sequence and nature of local processing stages at a higher resolution than was previously possible. The initial response in inferotemporal and perirhinal cortices is a brief current sink beginning at ~120ms, and peaking at ~170ms. Localization of this initial sink to middle layers suggests that it represents feedforward input from lower visual areas, and simultaneously increased firing implies that it represents excitatory synaptic currents. Until ~800ms, the main focus of transmembrane current sinks alternates between middle and superficial layers, with the superficial focus becoming increasingly dominant after ~550ms. Since superficial layers are the target of local and feedback associative inputs, this suggests an alternation in predominant synaptic input between feedforward and feedback modes. Word repetition does not affect the initial perirhinal and inferotemporal middle layer sink, but does decrease later activity. Entorhinal activity begins later (~200ms), with greater apparent excitatory postsynaptic currents and multiunit activity in neocortically-projecting than hippocampal-projecting layers. In contrast to perirhinal and entorhinal responses, entorhinal responses are larger to repeated words during memory retrieval. These results identify a sequence of physiological activation, beginning with a sharp activation from lower level visual areas carrying specific information to middle layers. This is followed by feedback and associative interactions involving upper cortical layers, which are abbreviated to repeated words. Following bottom-up and associative stages, top-down recollective processes may be driven by entorhinal cortex. Word processing involves a systematic sequence of fast feedforward information transfer from visual areas to anteroventral temporal cortex, followed by prolonged interactions of this feedforward information with local associations, and feedback mnestic information from the medial temporal lobe. PMID:16488158

Combining functionalised nanoparticles and SERS for the detection of DNA relating to disease.

PubMed

Graham, Duncan; Stevenson, Ross; Thompson, David G; Barrett, Lee; Dalton, Colette; Faulds, Karen

2011-01-01

DNA functionalised nanoparticle probes offer new opportunities in analyte detection. Ultrasensitive, molecularly specific targeting of analytes is possible through the use of metallic nanoparticles and their ability to generate a surface enhanced Raman scattering (SERS) response. This is leading to a new range of diagnostic clinical probes based on SERS detection. Our approaches have shown how such probes can detect specific DNA sequences by using a biomolecular recognition event to 'turn on' a SERS response through a controlled assembly process of the DNA functionalised nanoparticles. Further, we have prepared DNA aptamer functionalised SERS probes and demonstrated how introduction of a protein target can change the aggregation state of the nanoparticles in a dose-dependant manner. These approaches are being used as methods to detect biomolecules that indicate a specific disease being present with a view to improving disease management.

Efficient DNA binding and nuclear uptake by distamycin derivatives conjugated to octa-arginine sequences.

PubMed

Vázquez, Olalla; Blanco-Canosa, Juan B; Vázquez, M Eugenio; Martínez-Costas, Jose; Castedo, Luis; Mascareñas, José L

2008-11-24

Efficient targeting of DNA by designed molecules requires not only careful fine-tuning of their DNA-recognition properties, but also appropriate cell internalization of the compounds so that they can reach the cell nucleus in a short period of time. Previous observations in our group on the relatively high affinity displayed by conjugates between distamycin derivatives and bZIP basic regions for A-rich DNA sites, led us to investigate whether the covalent attachment of a positively charged cell-penetrating peptide to a distamycin-like tripyrrole might yield high affinity DNA binders with improved cell internalization properties. Our work has led to the discovery of synthetic tripyrrole-octa-arginine conjugates that are capable of targeting specific DNA sites that contain A-rich tracts with low nanomolar affinity; they simultaneously exhibit excellent membrane and nuclear translocation properties in living HeLa cells.

Emerging strategies for RNA interference (RNAi) applications in insects.

PubMed

Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

2015-01-01

RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response.

A molecular beacon based on DNA-templated silver nanoclusters for the highly sensitive and selective multiplexed detection of virulence genes.

PubMed

Han, Dan; Wei, Chunying

2018-05-01

In this work, we develop a fluorescent molecular beacon based on the DNA-templated silver nanoclusters (DNA-Ag NCs). The skillfully designed molecular beacon can be conveniently used for detection of diverse virulence genes as long as the corresponding recognition sequences are embedded. Importantly, the constructed detection system allows simultaneous detection of multiple nucleic acids, which is attributed to non-overlapping emission spectra of the as-synthesized silver nanoclusters. Based on the target-induced fluorescence enhancement, three infectious disease-related genes HIV, H1N1, and H5N1 are detected, and the corresponding detection limits are 3.53, 0.12 and 3.95nM, respectively. This design allows specific, versatile and simultaneous detection of diverse targets with easy operation and low cost. Copyright © 2017 Elsevier B.V. All rights reserved.

Advanced miniature processing handware for ATR applications

NASA Technical Reports Server (NTRS)

Chao, Tien-Hsin (Inventor); Daud, Taher (Inventor); Thakoor, Anikumar (Inventor)

2003-01-01

A Hybrid Optoelectronic Neural Object Recognition System (HONORS), is disclosed, comprising two major building blocks: (1) an advanced grayscale optical correlator (OC) and (2) a massively parallel three-dimensional neural-processor. The optical correlator, with its inherent advantages in parallel processing and shift invariance, is used for target of interest (TOI) detection and segmentation. The three-dimensional neural-processor, with its robust neural learning capability, is used for target classification and identification. The hybrid optoelectronic neural object recognition system, with its powerful combination of optical processing and neural networks, enables real-time, large frame, automatic target recognition (ATR).

Target recognition based on convolutional neural network

NASA Astrophysics Data System (ADS)

Wang, Liqiang; Wang, Xin; Xi, Fubiao; Dong, Jian

2017-11-01

One of the important part of object target recognition is the feature extraction, which can be classified into feature extraction and automatic feature extraction. The traditional neural network is one of the automatic feature extraction methods, while it causes high possibility of over-fitting due to the global connection. The deep learning algorithm used in this paper is a hierarchical automatic feature extraction method, trained with the layer-by-layer convolutional neural network (CNN), which can extract the features from lower layers to higher layers. The features are more discriminative and it is beneficial to the object target recognition.

Automated target recognition and tracking using an optical pattern recognition neural network

NASA Technical Reports Server (NTRS)

Chao, Tien-Hsin

1991-01-01

The on-going development of an automatic target recognition and tracking system at the Jet Propulsion Laboratory is presented. This system is an optical pattern recognition neural network (OPRNN) that is an integration of an innovative optical parallel processor and a feature extraction based neural net training algorithm. The parallel optical processor provides high speed and vast parallelism as well as full shift invariance. The neural network algorithm enables simultaneous discrimination of multiple noisy targets in spite of their scales, rotations, perspectives, and various deformations. This fully developed OPRNN system can be effectively utilized for the automated spacecraft recognition and tracking that will lead to success in the Automated Rendezvous and Capture (AR&C) of the unmanned Cargo Transfer Vehicle (CTV). One of the most powerful optical parallel processors for automatic target recognition is the multichannel correlator. With the inherent advantages of parallel processing capability and shift invariance, multiple objects can be simultaneously recognized and tracked using this multichannel correlator. This target tracking capability can be greatly enhanced by utilizing a powerful feature extraction based neural network training algorithm such as the neocognitron. The OPRNN, currently under investigation at JPL, is constructed with an optical multichannel correlator where holographic filters have been prepared using the neocognitron training algorithm. The computation speed of the neocognitron-type OPRNN is up to 10(exp 14) analog connections/sec that enabling the OPRNN to outperform its state-of-the-art electronics counterpart by at least two orders of magnitude.

Target Recognition Using Neural Networks for Model Deformation Measurements

NASA Technical Reports Server (NTRS)

Ross, Richard W.; Hibler, David L.

1999-01-01

Optical measurements provide a non-invasive method for measuring deformation of wind tunnel models. Model deformation systems use targets mounted or painted on the surface of the model to identify known positions, and photogrammetric methods are used to calculate 3-D positions of the targets on the model from digital 2-D images. Under ideal conditions, the reflective targets are placed against a dark background and provide high-contrast images, aiding in target recognition. However, glints of light reflecting from the model surface, or reduced contrast caused by light source or model smoothness constraints, can compromise accurate target determination using current algorithmic methods. This paper describes a technique using a neural network and image processing technologies which increases the reliability of target recognition systems. Unlike algorithmic methods, the neural network can be trained to identify the characteristic patterns that distinguish targets from other objects of similar size and appearance and can adapt to changes in lighting and environmental conditions.

Learning and Recognition of a Non-conscious Sequence of Events in Human Primary Visual Cortex.

PubMed

Rosenthal, Clive R; Andrews, Samantha K; Antoniades, Chrystalina A; Kennard, Christopher; Soto, David

2016-03-21

Human primary visual cortex (V1) has long been associated with learning simple low-level visual discriminations [1] and is classically considered outside of neural systems that support high-level cognitive behavior in contexts that differ from the original conditions of learning, such as recognition memory [2, 3]. Here, we used a novel fMRI-based dichoptic masking protocol-designed to induce activity in V1, without modulation from visual awareness-to test whether human V1 is implicated in human observers rapidly learning and then later (15-20 min) recognizing a non-conscious and complex (second-order) visuospatial sequence. Learning was associated with a change in V1 activity, as part of a temporo-occipital and basal ganglia network, which is at variance with the cortico-cerebellar network identified in prior studies of "implicit" sequence learning that involved motor responses and visible stimuli (e.g., [4]). Recognition memory was associated with V1 activity, as part of a temporo-occipital network involving the hippocampus, under conditions that were not imputable to mechanisms associated with conscious retrieval. Notably, the V1 responses during learning and recognition separately predicted non-conscious recognition memory, and functional coupling between V1 and the hippocampus was enhanced for old retrieval cues. The results provide a basis for novel hypotheses about the signals that can drive recognition memory, because these data (1) identify human V1 with a memory network that can code complex associative serial visuospatial information and support later non-conscious recognition memory-guided behavior (cf. [5]) and (2) align with mouse models of experience-dependent V1 plasticity in learning and memory [6]. Copyright © 2016 Elsevier Ltd. All rights reserved.

Algorithm, applications and evaluation for protein comparison by Ramanujan Fourier transform.

PubMed

Zhao, Jian; Wang, Jiasong; Hua, Wei; Ouyang, Pingkai

2015-12-01

The amino acid sequence of a protein determines its chemical properties, chain conformation and biological functions. Protein sequence comparison is of great importance to identify similarities of protein structures and infer their functions. Many properties of a protein correspond to the low-frequency signals within the sequence. Low frequency modes in protein sequences are linked to the secondary structures, membrane protein types, and sub-cellular localizations of the proteins. In this paper, we present Ramanujan Fourier transform (RFT) with a fast algorithm to analyze the low-frequency signals of protein sequences. The RFT method is applied to similarity analysis of protein sequences with the Resonant Recognition Model (RRM). The results show that the proposed fast RFT method on protein comparison is more efficient than commonly used discrete Fourier transform (DFT). RFT can detect common frequencies as significant feature for specific protein families, and the RFT spectrum heat-map of protein sequences demonstrates the information conservation in the sequence comparison. The proposed method offers a new tool for pattern recognition, feature extraction and structural analysis on protein sequences. Copyright © 2015 Elsevier Ltd. All rights reserved.

[The Amsterdam Dementia Screening Test in cognitively healthy and clinical samples. An update of normative data].

PubMed

van Toutert, Meta; Diesfeldt, Han; Hoek, Dirk

2016-10-01

The six tests in the Amsterdam Dementia Screening Test (ADST) examine the cognitive domains of episodic memory (delayed picture recognition, word learning), orientation, category fluency (animals and occupations), constructional ability (figure copying) and executive function (alternating sequences). New normative data were collected in a sample of 102 elderly volunteers (aged 65-94), including subjects with medical or other health conditions, except dementia or frank cognitive impairment (MMSE > 24). Included subjects were independent in complex instrumental activities of daily living.Fluency, not the other tests, needed adjustment for age and education. A deficit score (0-1) was computed for each test. Summation (range 0-6) proved useful in differentiating patients with dementia (N = 741) from normal elderly (N = 102).Positive and negative predictive power across a range of summed deficit scores and base rates are displayed in Bayesian probability tables.In the normal elderly, delayed recall for eight words was tested and adjusted for initial recall. A recognition test mixed the target words with eight distractors. Delayed recognition was adjusted for immediate and delayed recall.The ADST and the normative data in this paper help the clinical neuropsychologist to make decisions concerning the presence or absence of neurocognitive disorder in individual elderly examinees.

Wavelet-Based Signal and Image Processing for Target Recognition

NASA Astrophysics Data System (ADS)

Sherlock, Barry G.

2002-11-01

The PI visited NSWC Dahlgren, VA, for six weeks in May-June 2002 and collaborated with scientists in the G33 TEAMS facility, and with Marilyn Rudzinsky of T44 Technology and Photonic Systems Branch. During this visit the PI also presented six educational seminars to NSWC scientists on various aspects of signal processing. Several items from the grant proposal were completed, including (1) wavelet-based algorithms for interpolation of 1-d signals and 2-d images; (2) Discrete Wavelet Transform domain based algorithms for filtering of image data; (3) wavelet-based smoothing of image sequence data originally obtained for the CRITTIR (Clutter Rejection Involving Temporal Techniques in the Infra-Red) project. The PI visited the University of Stellenbosch, South Africa to collaborate with colleagues Prof. B.M. Herbst and Prof. J. du Preez on the use of wavelet image processing in conjunction with pattern recognition techniques. The University of Stellenbosch has offered the PI partial funding to support a sabbatical visit in Fall 2003, the primary purpose of which is to enable the PI to develop and enhance his expertise in Pattern Recognition. During the first year, the grant supported publication of 3 referred papers, presentation of 9 seminars and an intensive two-day course on wavelet theory. The grant supported the work of two students who functioned as research assistants.

Neural Dynamics Underlying Target Detection in the Human Brain

PubMed Central

Bansal, Arjun K.; Madhavan, Radhika; Agam, Yigal; Golby, Alexandra; Madsen, Joseph R.

2014-01-01

Sensory signals must be interpreted in the context of goals and tasks. To detect a target in an image, the brain compares input signals and goals to elicit the correct behavior. We examined how target detection modulates visual recognition signals by recording intracranial field potential responses from 776 electrodes in 10 epileptic human subjects. We observed reliable differences in the physiological responses to stimuli when a cued target was present versus absent. Goal-related modulation was particularly strong in the inferior temporal and fusiform gyri, two areas important for object recognition. Target modulation started after 250 ms post stimulus, considerably after the onset of visual recognition signals. While broadband signals exhibited increased or decreased power, gamma frequency power showed predominantly increases during target presence. These observations support models where task goals interact with sensory inputs via top-down signals that influence the highest echelons of visual processing after the onset of selective responses. PMID:24553944

Repetition priming across distinct contexts: effects of lexical status, word frequency, and retrieval test.

PubMed

Coane, Jennifer H; Balota, David A

2010-12-01

Repetition priming, the facilitation observed when a target is preceded by an identity prime, is a robust phenomenon that occurs across a variety of conditions. Oliphant (1983), however, failed to observe repetition priming for targets embedded in the instructions to an experiment in a subsequent lexical decision task. In the present experiments, we examined the roles of priming context (list or instructions), target lexicality, and target frequency in both lexical decision and episodic recognition performance. Initial encoding context did not modulate priming in lexical decision or recognition memory for low-frequency targets or nonwords, whereas context strongly modulated episodic recognition for high-frequency targets. The results indicate that priming across contexts is sensitive to the distinctiveness of the trace and the reliance on episodic retrieval mechanisms. These results also shed light on the influence of event boundaries, such that priming occurs across different events for relatively distinct (low-frequency) items.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

Human target acquisition performance

NASA Astrophysics Data System (ADS)

Teaney, Brian P.; Du Bosq, Todd W.; Reynolds, Joseph P.; Thompson, Roger; Aghera, Sameer; Moyer, Steven K.; Flug, Eric; Espinola, Richard; Hixson, Jonathan

2012-06-01

The battlefield has shifted from armored vehicles to armed insurgents. Target acquisition (identification, recognition, and detection) range performance involving humans as targets is vital for modern warfare. The acquisition and neutralization of armed insurgents while at the same time minimizing fratricide and civilian casualties is a mounting concern. U.S. Army RDECOM CERDEC NVESD has conducted many experiments involving human targets for infrared and reflective band sensors. The target sets include human activities, hand-held objects, uniforms & armament, and other tactically relevant targets. This paper will define a set of standard task difficulty values for identification and recognition associated with human target acquisition performance.

Diversification of a single ancestral gene into a successful toxin superfamily in highly venomous Australian funnel-web spiders.

PubMed

Pineda, Sandy S; Sollod, Brianna L; Wilson, David; Darling, Aaron; Sunagar, Kartik; Undheim, Eivind A B; Kely, Laurence; Antunes, Agostinho; Fry, Bryan G; King, Glenn F

2014-03-05

Spiders have evolved pharmacologically complex venoms that serve to rapidly subdue prey and deter predators. The major toxic factors in most spider venoms are small, disulfide-rich peptides. While there is abundant evidence that snake venoms evolved by recruitment of genes encoding normal body proteins followed by extensive gene duplication accompanied by explosive structural and functional diversification, the evolutionary trajectory of spider-venom peptides is less clear. Here we present evidence of a spider-toxin superfamily encoding a high degree of sequence and functional diversity that has evolved via accelerated duplication and diversification of a single ancestral gene. The peptides within this toxin superfamily are translated as prepropeptides that are posttranslationally processed to yield the mature toxin. The N-terminal signal sequence, as well as the protease recognition site at the junction of the propeptide and mature toxin are conserved, whereas the remainder of the propeptide and mature toxin sequences are variable. All toxin transcripts within this superfamily exhibit a striking cysteine codon bias. We show that different pharmacological classes of toxins within this peptide superfamily evolved under different evolutionary selection pressures. Overall, this study reinforces the hypothesis that spiders use a combinatorial peptide library strategy to evolve a complex cocktail of peptide toxins that target neuronal receptors and ion channels in prey and predators. We show that the ω-hexatoxins that target insect voltage-gated calcium channels evolved under the influence of positive Darwinian selection in an episodic fashion, whereas the κ-hexatoxins that target insect calcium-activated potassium channels appear to be under negative selection. A majority of the diversifying sites in the ω-hexatoxins are concentrated on the molecular surface of the toxins, thereby facilitating neofunctionalisation leading to new toxin pharmacology.

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

PubMed Central

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

2014-01-01

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

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

PubMed

Hodgens, Charles; Nimchuk, Zachary L; Kieber, Joseph J

2017-01-01

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

Codebook-based electrooculography data analysis towards cognitive activity recognition.

PubMed

Lagodzinski, P; Shirahama, K; Grzegorzek, M

2018-04-01

With the advancement in mobile/wearable technology, people started to use a variety of sensing devices to track their daily activities as well as health and fitness conditions in order to improve the quality of life. This work addresses an idea of eye movement analysis, which due to the strong correlation with cognitive tasks can be successfully utilized in activity recognition. Eye movements are recorded using an electrooculographic (EOG) system built into the frames of glasses, which can be worn more unobtrusively and comfortably than other devices. Since the obtained information is low-level sensor data expressed as a sequence representing values in constant intervals (100 Hz), the cognitive activity recognition problem is formulated as sequence classification. However, it is unclear what kind of features are useful for accurate cognitive activity recognition. Thus, a machine learning algorithm like a codebook approach is applied, which instead of focusing on feature engineering is using a distribution of characteristic subsequences (codewords) to describe sequences of recorded EOG data, where the codewords are obtained by clustering a large number of subsequences. Further, statistical analysis of the codeword distribution results in discovering features which are characteristic to a certain activity class. Experimental results demonstrate good accuracy of the codebook-based cognitive activity recognition reflecting the effective usage of the codewords. Copyright © 2017 Elsevier Ltd. All rights reserved.

Do pattern recognition skills transfer across sports? A preliminary analysis.

PubMed

Smeeton, Nicholas J; Ward, Paul; Williams, A Mark

2004-02-01

The ability to recognize patterns of play is fundamental to performance in team sports. While typically assumed to be domain-specific, pattern recognition skills may transfer from one sport to another if similarities exist in the perceptual features and their relations and/or the strategies used to encode and retrieve relevant information. A transfer paradigm was employed to compare skilled and less skilled soccer, field hockey and volleyball players' pattern recognition skills. Participants viewed structured and unstructured action sequences from each sport, half of which were randomly represented with clips not previously seen. The task was to identify previously viewed action sequences quickly and accurately. Transfer of pattern recognition skill was dependent on the participant's skill, sport practised, nature of the task and degree of structure. The skilled soccer and hockey players were quicker than the skilled volleyball players at recognizing structured soccer and hockey action sequences. Performance differences were not observed on the structured volleyball trials between the skilled soccer, field hockey and volleyball players. The skilled field hockey and soccer players were able to transfer perceptual information or strategies between their respective sports. The less skilled participants' results were less clear. Implications for domain-specific expertise, transfer and diversity across domains are discussed.

Continuous Human Action Recognition Using Depth-MHI-HOG and a Spotter Model

PubMed Central

Eum, Hyukmin; Yoon, Changyong; Lee, Heejin; Park, Mignon

2015-01-01

In this paper, we propose a new method for spotting and recognizing continuous human actions using a vision sensor. The method is comprised of depth-MHI-HOG (DMH), action modeling, action spotting, and recognition. First, to effectively separate the foreground from background, we propose a method called DMH. It includes a standard structure for segmenting images and extracting features by using depth information, MHI, and HOG. Second, action modeling is performed to model various actions using extracted features. The modeling of actions is performed by creating sequences of actions through k-means clustering; these sequences constitute HMM input. Third, a method of action spotting is proposed to filter meaningless actions from continuous actions and to identify precise start and end points of actions. By employing the spotter model, the proposed method improves action recognition performance. Finally, the proposed method recognizes actions based on start and end points. We evaluate recognition performance by employing the proposed method to obtain and compare probabilities by applying input sequences in action models and the spotter model. Through various experiments, we demonstrate that the proposed method is efficient for recognizing continuous human actions in real environments. PMID:25742172

Extended target recognition in cognitive radar networks.

PubMed

Wei, Yimin; Meng, Huadong; Liu, Yimin; Wang, Xiqin

2010-01-01

We address the problem of adaptive waveform design for extended target recognition in cognitive radar networks. A closed-loop active target recognition radar system is extended to the case of a centralized cognitive radar network, in which a generalized likelihood ratio (GLR) based sequential hypothesis testing (SHT) framework is employed. Using Doppler velocities measured by multiple radars, the target aspect angle for each radar is calculated. The joint probability of each target hypothesis is then updated using observations from different radar line of sights (LOS). Based on these probabilities, a minimum correlation algorithm is proposed to adaptively design the transmit waveform for each radar in an amplitude fluctuation situation. Simulation results demonstrate performance improvements due to the cognitive radar network and adaptive waveform design. Our minimum correlation algorithm outperforms the eigen-waveform solution and other non-cognitive waveform design approaches.

Bioinspired Pollen-Like Hierarchical Surface for Efficient Recognition of Target Cancer Cells.

PubMed

Wang, Wenshuo; Yang, Gao; Cui, Haijun; Meng, Jingxin; Wang, Shutao; Jiang, Lei

2017-08-01

The efficient recognition and isolation of rare cancer cells holds great promise for cancer diagnosis and prognosis. In nature, pollens exploit spiky structures to realize recognition and adhesion to stigma. Herein, a bioinspired pollen-like hierarchical surface is developed by replicating the assembly of pollen grains, and efficient and specific recognition to target cancer cells is achieved. The pollen-like surface is fabricated by combining filtering-assisted assembly and soft lithography-based replication of pollen grains of wild chrysanthemum. After modification with a capture agent specific to cancer cells, the pollen-like surface enables the capture of target cancer cells with high efficiency and specificity. In addition, the pollen-like surface not only assures high viability of captured cells but also performs well in cell mixture system and at low cell density. This study represents a good example of constructing cell recognition biointerfaces inspired by pollen-stigma adhesion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Eyes on crowding: crowding is preserved when responding by eye and similarly affects identity and position accuracy.

PubMed

Yildirim, Funda; Meyer, Vincent; Cornelissen, Frans W

2015-02-16

Peripheral vision guides recognition and selection of targets for eye movements. Crowding—a decline in recognition performance that occurs when a potential target is surrounded by other, similar, objects—influences peripheral object recognition. A recent model study suggests that crowding may be due to increased uncertainty about both the identity and the location of peripheral target objects, but very few studies have assessed these properties in tandem. Eye tracking can integrally provide information on both the perceived identity and the position of a target and therefore could become an important approach in crowding studies. However, recent reports suggest that around the moment of saccade preparation crowding may be significantly modified. If these effects were to generalize to regular crowding tasks, it would complicate the interpretation of results obtained with eye tracking and the comparison to results obtained using manual responses. For this reason, we first assessed whether the manner by which participants responded—manually or by eye—affected their performance. We found that neither recognition performance nor response time was affected by the response type. Hence, we conclude that crowding magnitude was preserved when observers responded by eye. In our main experiment, observers made eye movements to the location of a tilted Gabor target while we varied flanker tilt to manipulate target-flanker similarity. The results indicate that this similarly affected the accuracy of peripheral recognition and saccadic target localization. Our results inform about the importance of both location and identity uncertainty in crowding. © 2015 ARVO.

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen

PubMed Central

Lu, Xunli; Kracher, Barbara; Saur, Isabel M. L.; Bauer, Saskia; Ellwood, Simon R.; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

2016-01-01

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1. Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation. PMID:27702901

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

PubMed

Lu, Xunli; Kracher, Barbara; Saur, Isabel M L; Bauer, Saskia; Ellwood, Simon R; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

2016-10-18

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVR a gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVR a genes and identified AVR a1 and AVR a13 , encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVR a1 and AVR a13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVR A1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVR A1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVR A1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

Structural basis of UGUA recognition by the Nudix protein CFIm25 and implications for a regulatory role in mRNA 3′ processing

PubMed Central

Yang, Qin; Gilmartin, Gregory M.; Doublié, Sylvie

2010-01-01

Human Cleavage Factor Im (CFIm) is an essential component of the pre-mRNA 3′ processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFIm25) of the CFIm complex possesses a characteristic α/β/α Nudix fold, CFIm25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFIm25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFIm25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson–Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap4A (diadenosine tetraphosphate) by CFIm25 suggests a potential role for small molecules in the regulation of mRNA 3′ processing. PMID:20479262

Structural basis of UGUA recognition by the Nudix protein CFI(m)25 and implications for a regulatory role in mRNA 3' processing.

PubMed

Yang, Qin; Gilmartin, Gregory M; Doublié, Sylvie

2010-06-01

Human Cleavage Factor Im (CFI(m)) is an essential component of the pre-mRNA 3' processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI(m)25) of the CFI(m) complex possesses a characteristic alpha/beta/alpha Nudix fold, CFI(m)25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI(m)25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI(m)25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap(4)A (diadenosine tetraphosphate) by CFI(m)25 suggests a potential role for small molecules in the regulation of mRNA 3' processing.

A hierarchical, automated target recognition algorithm for a parallel analog processor

NASA Technical Reports Server (NTRS)

Woodward, Gail; Padgett, Curtis

1997-01-01

A hierarchical approach is described for an automated target recognition (ATR) system, VIGILANTE, that uses a massively parallel, analog processor (3DANN). The 3DANN processor is capable of performing 64 concurrent inner products of size 1x4096 every 250 nanoseconds.

Foundations for a syntatic pattern recognition system for genomic DNA sequences. [Annual] report, 1 December 1991--31 March 1993

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

Searles, D.B.

1993-03-01

The goal of the proposed work is the creation of a software system that will perform sophisticated pattern recognition and related functions at a level of abstraction and with expressive power beyond current general-purpose pattern-matching systems for biological sequences; and with a more uniform language, environment, and graphical user interface, and with greater flexibility, extensibility, embeddability, and ability to incorporate other algorithms, than current special-purpose analytic software.

Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute

PubMed Central

Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

2016-01-01

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson–Crick base-pairing even in the 3′-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5′ base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region. PMID:27325485

Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

PubMed

Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

2016-06-21

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

Crowding by a single bar: probing pattern recognition mechanisms in the visual periphery.

PubMed

Põder, Endel

2014-11-06

Whereas visual crowding does not greatly affect the detection of the presence of simple visual features, it heavily inhibits combining them into recognizable objects. Still, crowding effects have rarely been directly related to general pattern recognition mechanisms. In this study, pattern recognition mechanisms in visual periphery were probed using a single crowding feature. Observers had to identify the orientation of a rotated T presented briefly in a peripheral location. Adjacent to the target, a single bar was presented. The bar was either horizontal or vertical and located in a random direction from the target. It appears that such a crowding bar has very strong and regular effects on the identification of the target orientation. The observer's responses are determined by approximate relative positions of basic visual features; exact image-based similarity to the target is not important. A version of the "standard model" of object recognition with second-order features explains the main regularities of the data. © 2014 ARVO.

SAR target recognition and posture estimation using spatial pyramid pooling within CNN

NASA Astrophysics Data System (ADS)

Peng, Lijiang; Liu, Xiaohua; Liu, Ming; Dong, Liquan; Hui, Mei; Zhao, Yuejin

2018-01-01

Many convolution neural networks(CNN) architectures have been proposed to strengthen the performance on synthetic aperture radar automatic target recognition (SAR-ATR) and obtained state-of-art results on targets classification on MSTAR database, but few methods concern about the estimation of depression angle and azimuth angle of targets. To get better effect on learning representation of hierarchies of features on both 10-class target classification task and target posture estimation tasks, we propose a new CNN architecture with spatial pyramid pooling(SPP) which can build high hierarchy of features map by dividing the convolved feature maps from finer to coarser levels to aggregate local features of SAR images. Experimental results on MSTAR database show that the proposed architecture can get high recognition accuracy as 99.57% on 10-class target classification task as the most current state-of-art methods, and also get excellent performance on target posture estimation tasks which pays attention to depression angle variety and azimuth angle variety. What's more, the results inspire us the application of deep learning on SAR target posture description.

Strategic deployment of orthographic knowledge in phoneme detection.

PubMed

Cutler, Anne; Treiman, Rebecca; van Ooijen, Brit

2010-01-01

The phoneme detection task is widely used in spoken-word recognition research. Alphabetically literate participants, however, are more used to explicit representations of letters than of phonemes. The present study explored whether phoneme detection is sensitive to how target phonemes are, or may be, orthographically realized. Listeners detected the target sounds [b, m, t, f, s, k] in word-initial position in sequences of isolated English words. Response times were faster to the targets [b, m, t], which have consistent word-initial spelling, than to the targets [f, s, k], which are inconsistently spelled, but only when spelling was rendered salient by the presence in the experiment of many irregularly spelled filler words. Within the inconsistent targets [f, s, k], there was no significant difference between responses to targets in words with more usual (foam, seed, cattle) versus less usual (phone, cede, kettle) spellings. Phoneme detection is thus not necessarily sensitive to orthographic effects; knowledge of spelling stored in the lexical representations of words does not automatically become available as word candidates are activated. However, salient orthographic manipulations in experimental input can induce such sensitivity. We attribute this to listeners' experience of the value of spelling in everyday situations that encourage phonemic decisions (such as learning new names).

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Skeleton-based human action recognition using multiple sequence alignment

NASA Astrophysics Data System (ADS)

Ding, Wenwen; Liu, Kai; Cheng, Fei; Zhang, Jin; Li, YunSong

2015-05-01

Human action recognition and analysis is an active research topic in computer vision for many years. This paper presents a method to represent human actions based on trajectories consisting of 3D joint positions. This method first decompose action into a sequence of meaningful atomic actions (actionlets), and then label actionlets with English alphabets according to the Davies-Bouldin index value. Therefore, an action can be represented using a sequence of actionlet symbols, which will preserve the temporal order of occurrence of each of the actionlets. Finally, we employ sequence comparison to classify multiple actions through using string matching algorithms (Needleman-Wunsch). The effectiveness of the proposed method is evaluated on datasets captured by commodity depth cameras. Experiments of the proposed method on three challenging 3D action datasets show promising results.

Extrinsic Cognitive Load Impairs Spoken Word Recognition in High- and Low-Predictability Sentences.

PubMed

Hunter, Cynthia R; Pisoni, David B

Listening effort (LE) induced by speech degradation reduces performance on concurrent cognitive tasks. However, a converse effect of extrinsic cognitive load on recognition of spoken words in sentences has not been shown. The aims of the present study were to (a) examine the impact of extrinsic cognitive load on spoken word recognition in a sentence recognition task and (b) determine whether cognitive load and/or LE needed to understand spectrally degraded speech would differentially affect word recognition in high- and low-predictability sentences. Downstream effects of speech degradation and sentence predictability on the cognitive load task were also examined. One hundred twenty young adults identified sentence-final spoken words in high- and low-predictability Speech Perception in Noise sentences. Cognitive load consisted of a preload of short (low-load) or long (high-load) sequences of digits, presented visually before each spoken sentence and reported either before or after identification of the sentence-final word. LE was varied by spectrally degrading sentences with four-, six-, or eight-channel noise vocoding. Level of spectral degradation and order of report (digits first or words first) were between-participants variables. Effects of cognitive load, sentence predictability, and speech degradation on accuracy of sentence-final word identification as well as recall of preload digit sequences were examined. In addition to anticipated main effects of sentence predictability and spectral degradation on word recognition, we found an effect of cognitive load, such that words were identified more accurately under low load than high load. However, load differentially affected word identification in high- and low-predictability sentences depending on the level of sentence degradation. Under severe spectral degradation (four-channel vocoding), the effect of cognitive load on word identification was present for high-predictability sentences but not for low-predictability sentences. Under mild spectral degradation (eight-channel vocoding), the effect of load was present for low-predictability sentences but not for high-predictability sentences. There were also reliable downstream effects of speech degradation and sentence predictability on recall of the preload digit sequences. Long digit sequences were more easily recalled following spoken sentences that were less spectrally degraded. When digits were reported after identification of sentence-final words, short digit sequences were recalled more accurately when the spoken sentences were predictable. Extrinsic cognitive load can impair recognition of spectrally degraded spoken words in a sentence recognition task. Cognitive load affected word identification in both high- and low-predictability sentences, suggesting that load may impact both context use and lower-level perceptual processes. Consistent with prior work, LE also had downstream effects on memory for visual digit sequences. Results support the proposal that extrinsic cognitive load and LE induced by signal degradation both draw on a central, limited pool of cognitive resources that is used to recognize spoken words in sentences under adverse listening conditions.

Thermodynamic basis for engineering high-affinity, high-specificity binding-induced DNA clamp nanoswitches.

PubMed

Idili, Andrea; Plaxco, Kevin W; Vallée-Bélisle, Alexis; Ricci, Francesco

2013-12-23

Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the "specificity window" of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain*

PubMed Central

Podolnikova, Nataly P.; Yakovlev, Sergiy; Yakubenko, Valentin P.; Wang, Xu; Gorkun, Oleg V.; Ugarova, Tatiana P.

2014-01-01

The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen. PMID:24338009

Recognition mechanism of p63 by the E3 ligase Itch

PubMed Central

Bellomaria, Alessia; Barbato, Gaetano; Melino, Gerry; Paci, Maurizio; Melino, Sonia

2012-01-01

The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer’s or Huntington’s diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly ¹³C-¹⁵N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534–551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase. PMID:22935697

Colocalization recognition-activated cascade signal amplification strategy for ultrasensitive detection of transcription factors.

PubMed

Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

2017-03-15

Transcription factors (TFs) bind to specific double-stranded DNA (dsDNA) sequences in the regulatory regions of genes to regulate the process of gene transcription. Their expression levels sensitively reflect cell developmental situation and disease state. TFs have become potential diagnostic markers and therapeutic targets of cancers and some other diseases. Hence, high sensitive detection of TFs is of vital importance for early diagnosis of diseases and drugs development. The traditional exonucleases-assisted signal amplification methods suffered from the false positives caused by incomplete digestion of excess recognition probes. Herein, based on a new recognition way-colocalization recognition (CR)-activated dual signal amplification, an ultrasensitive fluorescent detection strategy for TFs was developed. TFs-induced the colocalization of three split recognition components resulted in noticeable increases of local effective concentrations and hybridization of three split components, which activated the subsequent cascade signal amplification including strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). This strategy eliminated the false positive influence and achieved ultra-high sensitivity towards the purified NF-κB p50 with detection limit of 2.0×10 -13 M. Moreover, NF-κB p50 can be detected in as low as 0.21ngμL -1 HeLa cell nuclear extracts. In addition, this proposed strategy could be used for the screening of NF-κB p50 activity inhibitors and potential anti-NF-κB p50 drugs. Finally, our proposed strategy offered a potential method for reliable detection of TFs in medical diagnosis and treatment research of cancers and other related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Glucose enhancement of a facial recognition task in young adults.

PubMed

Metzger, M M

2000-02-01

Numerous studies have reported that glucose administration enhances memory processes in both elderly and young adult subjects. Although these studies have utilized a variety of procedures and paradigms, investigations of both young and elderly subjects have typically used verbal tasks (word list recall, paragraph recall, etc.). In the present study, the effect of glucose consumption on a nonverbal, facial recognition task in young adults was examined. Lemonade sweetened with either glucose (50 g) or saccharin (23.7 mg) was consumed by college students (mean age of 21.1 years) 15 min prior to a facial recognition task. The task consisted of a familiarization phase in which subjects were presented with "target" faces, followed immediately by a recognition phase in which subjects had to identify the targets among a random array of familiar target and novel "distractor" faces. Statistical analysis indicated that there were no differences on hit rate (target identification) for subjects who consumed either saccharin or glucose prior to the test. However, further analyses revealed that subjects who consumed glucose committed significantly fewer false alarms and had (marginally) higher d-prime scores (a signal detection measure) compared to subjects who consumed saccharin prior to the test. These results parallel a previous report demonstrating glucose enhancement of a facial recognition task in probable Alzheimer's patients; however, this is believed to be the first demonstration of glucose enhancement for a facial recognition task in healthy, young adults.

A fusion approach for coarse-to-fine target recognition

NASA Astrophysics Data System (ADS)

Folkesson, Martin; Grönwall, Christina; Jungert, Erland

2006-04-01

A fusion approach in a query based information system is presented. The system is designed for querying multimedia data bases, and here applied to target recognition using heterogeneous data sources. The recognition process is coarse-to-fine, with an initial attribute estimation step and a following matching step. Several sensor types and algorithms are involved in each of these two steps. An independence of the matching results, on the origin of the estimation results, is observed. It allows for distribution of data between algorithms in an intermediate fusion step, without risk of data incest. This increases the overall chance of recognising the target. An implementation of the system is described.

The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation.

PubMed

Klug, Aaron

2010-02-01

A long-standing goal of molecular biologists has been to construct DNA-binding proteins for the control of gene expression. The classical Cys2His2 (C2H2) zinc finger design is ideally suited for such purposes. Discriminating between closely related DNA sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically. Zinc fingers were discovered in 1985, arising from the interpretation of our biochemical studies on the interaction of the Xenopus protein transcription factor IIIA (TFIIIA) with 5S RNA. Subsequent structural studies revealed its three-dimensional structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligated to a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome. The zinc finger design can be used to construct DNA-binding proteins for specific intervention in gene expression. By fusing selected zinc finger peptides to repression or activation domains, genes can be selectively switched off or on by targeting the peptide to the desired gene target. It was also suggested that by combining an appropriate zinc finger peptide with other effector or functional domains, e.g. from nucleases or integrases to form chimaeric proteins, genomes could be modified or manipulated. The first example of the power of the method was published in 1994 when a three-finger protein was constructed to block the expression of a human oncogene transformed into a mouse cell line. The same paper also described how a reporter gene was activated by targeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that, by combining zinc fingers with other effector or functional domains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could be manipulated or modified. Several applications of such engineered ZFPs are described here, including some of therapeutic importance, and also their adaptation for breeding improved crop plants.