Comparing the Hierarchy of Keywords in On-Line News Portals

Hierarchical organization is prevalent in networks representing a wide range of systems in nature and society. An important example is given by the tag hierarchies extracted from large on-line data repositories such as scientific publication archives, file sharing portals, blogs, on-line news portals, etc. The tagging of the stored objects with informative keywords in such repositories has become very common, and in most cases the tags on a given item are free words chosen by the authors independently. Therefore, the relations among keywords appearing in an on-line data repository are unknown in general. However, in most cases the topics and concepts described by these keywords are forming a latent hierarchy, with the more general topics and categories at the top, and more specialized ones at the bottom. There are several algorithms available for deducing this hierarchy from the statistical features of the keywords. In the present work we apply a recent, co-occurrence-based tag hierarchy extraction method to sets of keywords obtained from four different on-line news portals. The resulting hierarchies show substantial differences not just in the topics rendered as important (being at the top of the hierarchy) or of less interest (categorized low in the hierarchy), but also in the underlying network structure. This reveals discrepancies between the plausible keyword association frameworks in the studied news portals.     

Efficient enumeration of phylogenetically informative substrings.

We study the problem of enumerating substrings that are common amongst genomes that share evolutionary descent. For example, one might want to enumerate all identical (therefore conserved) substrings that are shared between all mammals and not found in non-mammals. Such collection of substrings may be used to identify conserved subsequences or to construct sets of identifying substrings for branches of a phylogenetic tree. For two disjoint sets of genomes on a phylogenetic tree, a substring is called a tag if it is found in all of the genomes of one set and none of the genomes of the other set. We present a near-linear time algorithm that finds all tags in a given phylogeny; and a sublinear space algorithm (at the expense of running time) that is more suited for very large data sets. Under a stochastic model of evolution, we show that a simple process of tag-generation essentially captures all possible ways of generating tags. We use this insight to develop a faster tag discovery algorithm with a small chance of error. However, since tags are not guaranteed to exist in a given data set, we generalize the notion of a tag from a single substring to a set of substrings. We present a linear programming-based approach for finding approximate generalized tag sets. Finally, we use our tag enumeration algorithm to analyze a phylogeny containing 57 whole microbial genomes. We find tags for all nodes in the phylogeny except the root for which we find generalized tag sets.     

Challenges and opportunities in the purification of recombinant tagged proteins

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.     

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background

Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.

Methodology/Principal Findings

Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.

Conclusions and Significance

The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.     

Toward Trash That Thinks: Product Tags for Environmental Management

In this article, we explore several options for linking information technology to materials and products through the use of bar codes and radio-frequency identification (RFID) tags, and the implications for product life-cycle management. We also describe tests with existing and modified tags, both on and inside products, as would be needed for environmental management applications.
Bar codes are cheap and have an existing infrastructure; RFID tags are more expensive and less widespread, but they can be read without a line of sight between the tag and the reader. Bar codes and RFID tags carrying basic product information could link to different databases for a range of applications. Product tags could increase recycling efficiency by automating the sorting of recyclables or by linking to product dismantling instructions during the recycling process. Product tags could provide incentives for good waste management, through Universal Product Code (UPC) bar-code recycling coupons or through RFID tag automatic recycling credits for curbside collection programs. Measures to encourage the development of these types of applications include moves toward competitive, market-based waste management systems, the encouragement of experimental systems, and coordination between manufacturers and waste management industries.     

PRODUCTION OF SMALL ANTIBACTERIAL PEPTIDES USING SILKWORM–BACULOVIRUS PROTEIN EXPRESSION SYSTEM

The recombinant proteins with strong antimicrobial activity are known to be very difficult to express using bacterial expression system. Here, human β-defensin (DEFB) 1, DEFB2, and DEFB3 were successfully produced using a silkworm–baculovirus protein expression system. We have generated four baculoviruses for each DEFB protein to compare the effect of different peptide tags in secretion into silkworm larval hemolymph. Interestingly, the best performing peptide tags for the secretion were different among DEFBs: C-terminal GST-H8 tag for DEFB1, N-terminal H8 tag for DEFB2, and C-terminal H8 tag for DEFB3, respectively. In addition, the colony count assay demonstrated that the recombinant DEFB2 s showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, and Paenibacillus thiaminolyticus.     

Development of an automated method of detecting stereotyped feeding events in multisensor data from tagged rorqual whales

The introduction of animal‐borne, multisensor tags has opened up many opportunities for ecological research, making previously inaccessible species and behaviors observable. The advancement of tag technology and the increasingly widespread use of bio‐logging tags are leading to large volumes of sometimes extremely detailed data. With the increasing quantity and duration of tag deployments, a set of tools needs to be developed to aid in facilitating and standardizing the analysis of movement sensor data. Here, we developed an observation‐based decision tree method to detect feeding events in data from multisensor movement tags attached to fin whales (Balaenoptera physalus). Fin whales exhibit an energetically costly and kinematically complex foraging behavior called lunge feeding, an intermittent ram filtration mechanism. Using this automated system, we identified feeding lunges in 19 fin whales tagged with multisensor tags, during a total of over 100 h of continuously sampled data. Using movement sensor and hydrophone data, the automated lunge detector correctly identified an average of 92.8% of all lunges, with a false‐positive rate of 9.5%. The strong performance of our automated feeding detector demonstrates an effective, straightforward method of activity identification in animal‐borne movement tag data. Our method employs a detection algorithm that utilizes a hierarchy of simple thresholds based on knowledge of observed features of feeding behavior, a technique that is readily modifiable to fit a variety of species and behaviors. Using automated methods to detect behavioral events in tag records will significantly decrease data analysis time and aid in standardizing analysis methods, crucial objectives with the rapidly increasing quantity and variety of on‐animal tag data. Furthermore, our results have implications for next‐generation tag design, especially long‐term tags that can be outfitted with on‐board processing algorithms that automatically detect kinematic events and transmit ethograms via acoustic or satellite telemetry.     

Lanthanoid tagging via an unnatural amino acid for protein structure characterization

Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.     

Hierarchy measure for complex networks

Nature, technology and society are full of complexity arising from the intricate web of the interactions among the units of the related systems (e.g., proteins, computers, people). Consequently, one of the most successful recent approaches to capturing the fundamental features of the structure and dynamics of complex systems has been the investigation of the networks associated with the above units (nodes) together with their relations (edges). Most complex systems have an inherently hierarchical organization and, correspondingly, the networks behind them also exhibit hierarchical features. Indeed, several papers have been devoted to describing this essential aspect of networks, however, without resulting in a widely accepted, converging concept concerning the quantitative characterization of the level of their hierarchy. Here we develop an approach and propose a quantity (measure) which is simple enough to be widely applicable, reveals a number of universal features of the organization of real-world networks and, as we demonstrate, is capable of capturing the essential features of the structure and the degree of hierarchy in a complex network. The measure we introduce is based on a generalization of the m-reach centrality, which we first extend to directed/partially directed graphs. Then, we define the global reaching centrality (GRC), which is the difference between the maximum and the average value of the generalized reach centralities over the network. We investigate the behavior of the GRC considering both a synthetic model with an adjustable level of hierarchy and real networks. Results for real networks show that our hierarchy measure is related to the controllability of the given system. We also propose a visualization procedure for large complex networks that can be used to obtain an overall qualitative picture about the nature of their hierarchical structure.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Comparison of affinity tags for protein purification

Affinity tags are highly efficient tools for purifying proteins from crude extracts. To facilitate the selection of affinity tags for purification projects, we have compared the efficiency of eight elutable affinity tags to purify proteins from Escherichia coli, yeast, Drosophila, and HeLa extracts. Our results show that the HIS, CBP, CYD (covalent yet dissociable NorpD peptide), Strep II, FLAG, HPC (heavy chain of protein C) peptide tags, and the GST and MBP protein fusion tag systems differ substantially in purity, yield, and cost. We find that the HIS tag provides good yields of tagged protein from inexpensive, high capacity resins but with only moderate purity from E. coli extracts and relatively poor purification from yeast, Drosophila, and HeLa extracts. The CBP tag produced moderate purity protein from E. coli, yeast, and Drosophila extracts, but better purity from HeLa extracts. Epitope-based tags such as FLAG and HPC produced the highest purity protein for all extracts but require expensive, low capacity resin. Our results suggest that the Strep II tag may provide an acceptable compromise of excellent purification with good yields at a moderate cost.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Evaluation of tag entanglement as a factor in harmonic radar studies of insect dispersal

The observation of insects and other small organisms entangled in the habitat after the addition of vertical or trailing electronic tags to their body has generated concerns on the suitability of harmonic radars to track the dispersal of insects. This study compared the walking behavior of adult Colorado potato beetle (Leptinotarsa decemlineata (Say) Chrysomelidae), plum curculio (Conotrachelus nenuphar (Herbst) Curculionidae), and western corn rootworm (Diabrotica virgifera virgifera (LeConte) Chrysomelidae) with and without vertical and or trailing tags in field plots or arenas. The frequency of the larger Colorado potato beetles crossing bare ground or grassy plots was unaffected by the presence of an 8 cm trailing harmonic radar tag. However, plum curculios and western corn rootworms, were either unable to walk with a 4 cm trailing tag (plum curculio) or displayed a reduced ability to successfully cross a bare ground arena. Our results revealed the significant impact of vegetation on successful insect dispersal, whether tagged or not. The vertical movement of these insects on stems, stalks, and tubes was also unaffected by the presence of vertical tags. Trailing tags had a significant negative effect on the vertical movement of the western corn rootworm. Results show that harmonic radar technology is a suitable method for studying the walking paths of the three insects with appropriate tag type and size. The nuisance factor generated by appropriately sized tags was small relative to that of vegetation.     

Rapid purification and crystal structure analysis of a small protein carrying two terminal affinity tags

Small peptide tags are often fused to proteins to allow their affinity purification in high-throughput structure analysis schemes. To assess the compatibility of small peptide tags with protein crystallization and to examine if the tags alter the three-dimensional structure, the N-terminus of the chicken alpha-spectrin SH3 domain was labeled with a His6 tag and the C-terminus with a StrepII tag. The resulting protein, His6-SH3-StrepII, consists of 83 amino-acid residues, 23 of which originate from the tags. His6-SH3-StrepII is readily purified by dual affinity chromatography, has very similar biophysical characteristics as the untagged protein domain and crystallizes readily from a number of sparse-matrix screen conditions. The crystal structure analysis at 2.3 A resolution proves native-like structure of His6-SH3-StrepII and shows the entire His6 tag and part of the StrepII tag to be disordered in the crystal. Obviously, the fused affinity tags did not interfere with crystallization and structure analysis and did not change the protein structure. From the extreme case of His6-SH3-StrepII, where affinity tags represent 27% of the total fusion protein mass, we extrapolate that protein constructs with N- and C-terminal peptide tags may lend themselves to biophysical and structural investigations in high-throughput regimes.