Automatic Understanding of Image and Video Advertisements

There is more to images than their objective physical content: for example, advertisements are created to persuade a viewer to take a certain action. We propose the novel problem of automatic advertisement understanding. To enable research on this problem, we create two datasets: an image dataset of 64,832 image ads, and a video dataset of 3,477 ads. Our data contains rich annotations encompassing the topic and sentiment of the ads, questions and answers describing what actions the viewer is prompted to take and the reasoning that the ad presents to persuade the viewer ("What should I do according to this ad, and why should I do it?"), and symbolic references ads make (e.g. a dove symbolizes peace). We also analyze the most common persuasive strategies ads use, and the capabilities that computer vision systems should have to understand these strategies. We present baseline classification results for several prediction tasks, including automatically answering questions about the messages of the ads.Comment: To appear in CVPR 2017; data available on http://cs.pitt.edu/~kovashka/ad

High-throughput expression and purification of human solute carriers for structural and biochemical studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening

Adapting nanobody scaffolds as protein chaperones for effective crystallization and good protein behaviour in cryo-EM

In the past three decades, structural biology has developed tremendously with the establishment of the X-ray crystallography and cryo-EM techniques. In order to satisfy the needs of those two techniques, methods have been developed to engineer the proteins of interest. One of the popular methods is binder-assisted structure solution. Among the various kinds of protein binders, nanobodies are one of the most frequently used tool in both X-ray crystallography and cryo-EM. Nanobodies are a small single-domain antibody (VHH) of around 15 kDa, and consist of the complementarity determining regions (CDRs) and the scaffold (non-CDRs). The residues in the nanobody scaffold are relatively constant and the CDRs determine the specificity for the target protein. Until now, there have been hundreds of successful cases of nanobody-assisted structure solution, including both soluble proteins and membrane proteins. However, in crystallography, there has never been reports about the success rate of nanobodies as a crystal chaperone, and the nanobody scaffolds have never been optimized for crystallization purposes. Moreover, in cryo-EM, there was no convenient imaging scaffolds to enable structure solution of small proteins. In this study, I set out to address those two problems. A few target:nanobody pairs were first selected and evaluated the success rate of crystallization. It turned out that only one pair (MAGEB1:nanobody) successfully crystalized out of the 17 pairs. In order to further demonstrate that the nanobody scaffold was optimizable, I selected one of the failed pairs (RECQL5:nanobody_G0-001) and carried out surface mutagenesis on the nanobody, which successfully led to a 2.8 Å structure. This demonstrated that the nanobody scaffold had large potential to be optimized for crystallization purposes. Thorough bioinformatics research was then carried out on nanboody-nanobody crystal contacts, in order to find the most suitable surface of the nanobody scaffold for engineering. After the unsupervised density based clustering (DBSCAN), the largest class of nanobody-nanobody crystal contacts was discovered. This class of interfaces was named as ‘Nb-α’. This finding provided guidance on where to start with the scaffold engineering. Extensive engineering was carried out around the Nb-α following bioinformatics research. 5 iterations of mutagenesis were performed to make the RECQL5:nanobody complex consistently well-diffracting, which identified 4 key mutations along with the truncation of the C-terminal tail. Those modifications led to the robust dimerization and tetramerization of nanobodies in the crystal lattice, giving the name ‘Gluebody’ to the optimized scaffold. However, the Gluebody scaffold did not show significant improvement for other target proteins, and therefore it did not solve all problems in crystallization. The tetrameric arrangement of the Gluebodies had two rigid interfaces perpendicular to each other, which if in solution did not dissociate, would make the tetrameric-Gluebody an imaging scaffold with D2 symmetry for cryo-EM. Initial trials of disulfide linked Gluebodies were taken on two target proteins, which led to the 2D classification results as good as the target protein alone when the thesis was written. The work is still on-going to invent a new tool for cryo-EM structure solution. In conclusion, the discovery process of Gluebodies is the first systematic study on the nanobody scaffold optimization for crystallization purposes. For the first time, the naïve nanobody scaffold is demonstrated to be optimizable for crystallization purposes, and the Gluebody mutations are designed based on bioinformatics research, which are demonstrated to improve the crystal reliability and diversity of packing forms of RECQL5. Meanwhile, the tetrameric-Gluebody assembly is discovered, based on which a symmetric imaging scaffold for cryo-EM could be invented. On the other hand, the Gluebody mutations could not solve all problems in crystallization as it did not show significant assistance for other target proteins. The combination of other approaches would be necessary depending on the target protein

Real-Time Energy Management Strategy for Fuel Cell Vehicles Based on DP and Rule Extraction

Energy management strategy (EMS), as a core technology in fuel cell vehicles (FCVs), profoundly influences the lifespan of fuel cells and the economy of the vehicle. Aiming at the problem of the EMS of FCVs based on a global optimization algorithm not being applicable in real-time, a rule extraction-based EMS is proposed for fuel cell commercial vehicles. Based on the results of the dynamic programming (DP) algorithm in the CLTC-C cycle, the deep learning approach is employed to extract output power rules for fuel cell, leading to the establishment of a rule library. Using this library, a real-time applicable rule-based EMS is designed. The simulated driving platform is built in a CARLA, SUMO, and MATLAB/Simulink joint simulation environment. Simulation results indicate that the proposed strategy yields savings ranging from 3.64% to 8.96% in total costs when compared to the state machine-based strategy

Structural basis for activity switching in polymerases determining the fate of let-7 pre-miRNAs

Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3′ end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3′ oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer

Table_6_Comparative genomics reveals new insights into the evolution of the IncA and IncC family of plasmids.XLSX

Incompatibility groups IncA and IncC plasmids are of great concern due to their ability to disseminate antibiotic resistance in bacteria via conjugative transfer. A deep understanding of their genomic structures and evolutionary characteristics is of great significance for improving our knowledge about its multidrug-resistance evolution and dissemination. However, current knowledge of their backbone structure, features of core functional modules and the characteristics of variable regions is based on a few plasmids, which highlights the need for a comprehensive systematic study. The present study thoroughly compared and analysed 678 IncA and IncC plasmid genomes. We found that their core functional genes were occasionally deficient and sometimes existed as multiple functional copies/multiple families, which resulted in much diversity. The phylogeny of 13 core functional genes corresponded well to the plasmid subtypes. The conjugative transfer system gained diverse complexity and exhibited many previously unnoticed types with multiple combinations. The insertion of mobile genetic elements (MGEs) in plasmids varied between types and was present in 4 insertion spots in different types of plasmids with certain types of transposons, integrons and insertion sequences. The impact of gene duplication, deletion, the insertion of MGEs, genome rearrangement and recombination resulted in the complex dynamic variable backbone of IncA and IncC plasmids. And IncA and IncC plasmids were more complex than their closest relative SXT/R391 integrative conjugative elements (ICEs), which included nearly all of the diversity of SXT/R391 in key systems. Our work demonstrated a global and systematic view of the IncA and IncC plasmids and provides many new insights into their genome evolution.</p

Table_5_Comparative genomics reveals new insights into the evolution of the IncA and IncC family of plasmids.XLSX

Incompatibility groups IncA and IncC plasmids are of great concern due to their ability to disseminate antibiotic resistance in bacteria via conjugative transfer. A deep understanding of their genomic structures and evolutionary characteristics is of great significance for improving our knowledge about its multidrug-resistance evolution and dissemination. However, current knowledge of their backbone structure, features of core functional modules and the characteristics of variable regions is based on a few plasmids, which highlights the need for a comprehensive systematic study. The present study thoroughly compared and analysed 678 IncA and IncC plasmid genomes. We found that their core functional genes were occasionally deficient and sometimes existed as multiple functional copies/multiple families, which resulted in much diversity. The phylogeny of 13 core functional genes corresponded well to the plasmid subtypes. The conjugative transfer system gained diverse complexity and exhibited many previously unnoticed types with multiple combinations. The insertion of mobile genetic elements (MGEs) in plasmids varied between types and was present in 4 insertion spots in different types of plasmids with certain types of transposons, integrons and insertion sequences. The impact of gene duplication, deletion, the insertion of MGEs, genome rearrangement and recombination resulted in the complex dynamic variable backbone of IncA and IncC plasmids. And IncA and IncC plasmids were more complex than their closest relative SXT/R391 integrative conjugative elements (ICEs), which included nearly all of the diversity of SXT/R391 in key systems. Our work demonstrated a global and systematic view of the IncA and IncC plasmids and provides many new insights into their genome evolution.</p

Image_5_Comparative genomics reveals new insights into the evolution of the IncA and IncC family of plasmids.TIF

Incompatibility groups IncA and IncC plasmids are of great concern due to their ability to disseminate antibiotic resistance in bacteria via conjugative transfer. A deep understanding of their genomic structures and evolutionary characteristics is of great significance for improving our knowledge about its multidrug-resistance evolution and dissemination. However, current knowledge of their backbone structure, features of core functional modules and the characteristics of variable regions is based on a few plasmids, which highlights the need for a comprehensive systematic study. The present study thoroughly compared and analysed 678 IncA and IncC plasmid genomes. We found that their core functional genes were occasionally deficient and sometimes existed as multiple functional copies/multiple families, which resulted in much diversity. The phylogeny of 13 core functional genes corresponded well to the plasmid subtypes. The conjugative transfer system gained diverse complexity and exhibited many previously unnoticed types with multiple combinations. The insertion of mobile genetic elements (MGEs) in plasmids varied between types and was present in 4 insertion spots in different types of plasmids with certain types of transposons, integrons and insertion sequences. The impact of gene duplication, deletion, the insertion of MGEs, genome rearrangement and recombination resulted in the complex dynamic variable backbone of IncA and IncC plasmids. And IncA and IncC plasmids were more complex than their closest relative SXT/R391 integrative conjugative elements (ICEs), which included nearly all of the diversity of SXT/R391 in key systems. Our work demonstrated a global and systematic view of the IncA and IncC plasmids and provides many new insights into their genome evolution.</p

Image_1_Comparative genomics reveals new insights into the evolution of the IncA and IncC family of plasmids.TIFF

Incompatibility groups IncA and IncC plasmids are of great concern due to their ability to disseminate antibiotic resistance in bacteria via conjugative transfer. A deep understanding of their genomic structures and evolutionary characteristics is of great significance for improving our knowledge about its multidrug-resistance evolution and dissemination. However, current knowledge of their backbone structure, features of core functional modules and the characteristics of variable regions is based on a few plasmids, which highlights the need for a comprehensive systematic study. The present study thoroughly compared and analysed 678 IncA and IncC plasmid genomes. We found that their core functional genes were occasionally deficient and sometimes existed as multiple functional copies/multiple families, which resulted in much diversity. The phylogeny of 13 core functional genes corresponded well to the plasmid subtypes. The conjugative transfer system gained diverse complexity and exhibited many previously unnoticed types with multiple combinations. The insertion of mobile genetic elements (MGEs) in plasmids varied between types and was present in 4 insertion spots in different types of plasmids with certain types of transposons, integrons and insertion sequences. The impact of gene duplication, deletion, the insertion of MGEs, genome rearrangement and recombination resulted in the complex dynamic variable backbone of IncA and IncC plasmids. And IncA and IncC plasmids were more complex than their closest relative SXT/R391 integrative conjugative elements (ICEs), which included nearly all of the diversity of SXT/R391 in key systems. Our work demonstrated a global and systematic view of the IncA and IncC plasmids and provides many new insights into their genome evolution.</p