Exploiting Overlapping Advantages of <i>in vitro</i> and <i>in cellulo</i> Selection Systems to Isolate a Novel High-affinity cJun Antagonist

We have combined two peptide library-screening systems, exploiting the benefits offered by both to select novel antagonistic agents of cJun. CIS display is an <i>in vitro</i> cell-free system that allows very large libraries (≤10¹⁴) to be interrogated. However, affinity-based screening conditions can poorly reflect those relevant to therapeutic application, particularly for difficult intracellular targets, and can lead to false positives. In contrast, an <i>in cellulo</i> screening system such as the Protein-fragment Complementation Assay (PCA) selects peptides with high target affinity while additionally profiling for target specificity, protease resistance, solubility, and lack of toxicity in a more relevant context. A disadvantage is the necessity to transform cells, limiting library sizes that can be screened to ≤10⁶. However, by combining both cell-free and cell-based systems, we isolated a peptide (CPW) from a ∼10¹⁰ member library, which forms a highly stable interaction with cJun (<i>T</i>_m = 63 °C, <i>K</i>_d = 750 nM, Δ<i>G</i> = −8.2 kcal/mol) using the oncogenic transcriptional regulator Activator Protein-1 (AP-1) as our exemplar target. In contrast, CIS display alone selected a peptide with low affinity for cJun (<i>T</i>_m = 34 °C, <i>K</i>_d = 25 μM, Δ<i>G</i> = −6.2 kcal/mol), highlighting the benefit of CIS → PCA. Furthermore, increased library size with CIS → PCA vs PCA alone allows the freedom to introduce noncanonical options, such as interfacial aromatics, and solvent exposed options that may allow the molecule to explore alternative structures and interact with greater affinity and efficacy with the target. CIS → PCA therefore offers significant potential as a peptide-library screening platform by synergistically combining the relative attributes of both assays to generate therapeutically interesting compounds that may otherwise not be identified

Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing

We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity

Athena: Advanced air launched space booster

The infrastructure for routine, reliable, and inexpensive access of space is a goal that has been actively pursued over the past 50 years, but has yet not been realized. Current launch systems utilize ground launching facilities which require the booster vehicle to plow up through the dense lower atmosphere before reaching space. An air launched system on the other hand has the advantage of being launched from a carrier aircraft above this dense portion of the atmosphere and hence can be smaller and lighter compared to its ground based counterpart. The goal of last year's Aerospace Engineering Course 483 (AE 483) was to design a 227,272 kg (500,000 lb.) air launched space booster which would beat the customer's launch cost on existing launch vehicles by at least 50 percent. While the cost analysis conducted by the class showed that this goal could be met, the cost and size of the carrier aircraft make it appear dubious that any private company would be willing to invest in such a project. To avoid this potential pitfall, this year's AE 483 class was to design as large an air launched space booster as possible which can be launched from an existing or modification to an existing aircraft. An initial estimate of the weight of the booster is 136,363 kg (300,000 lb.) to 159,091 kg (350,000 lb.)

Developmental and computational perspectives on infant social cognition

Adults effortlessly and automatically infer complex pat- terns of goals, beliefs, and other mental states as the causes of others’ actions. Yet before the last decade little was known about the developmental origins of these abilities in early infancy. Our understanding of infant social cognition has now improved dramatically: even preverbal infants appear to perceive goals, preferences (Kushnir, Xu, & Wellman, in press), and even beliefs from sparse observations of inten- tional agents’ behavior. Furthermore, they use these infer- ences to predict others’ behavior in novel contexts and to make social evaluations (Hamlin, Wynn, & Bloom, 2007). Keywords: Social cognition; Cognitive Development; Computational Modeling; Theory of Min

ProxiMAX randomisation:a new technology for non-degenerate saturation mutagenesis of contiguous codons

Back in 2003, we published ‘MAX’ randomisation, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomisation saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an alpha-helix, as in zinc finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple, contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomisation, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomisation uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialised chemistry, reagents nor equipment and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in pre-defined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomisation is particularly relevant to antibody engineering

Associations of occupational standing with musculoskeletal symptoms: A systematic review with meta-analysis

Objective Given the high exposure to occupational standing in specific occupations, and recent initiatives to encourage intermittent standing among white-collar workers, a better understanding of the potential health consequences of occupational standing is required. We aimed to review and quantify the epidemiological evidence on associations of occupational standing with musculoskeletal symptoms. Design A systematic review was performed. Data from included articles were extracted and described, and meta-analyses conducted when data were sufficiently homogeneous. Data sources Electronic databases were systematically searched. Eligibility criteria Peer-reviewed articles on occupational standing and musculoskeletal symptoms from epidemiological studies were identified. Results Of the 11 750 articles screened, 50 articles reporting 49 studies were included (45 cross-sectional and 5 longitudinal; n=88 158 participants) describing the associations of occupational standing with musculoskeletal symptoms, including low-back (39 articles), lower extremity (14 articles) and upper extremity (18 articles) symptoms. In the meta-analysis, 'substantial' (&gt;4 hours/workday) occupational standing was associated with the occurrence of low-back symptoms (pooled OR (95% CI) 1.31 (1.10 to 1.56)). Evidence on lower and upper extremity symptoms was too heterogeneous for meta-analyses. The majority of included studies reported statistically significant detrimental associations of occupational standing with lower extremity, but not with upper extremity symptoms. Conclusions The evidence suggests that substantial occupational standing is associated with the occurrence of low-back and (inconclusively) lower extremity symptoms, but there may not be such an association with upper extremity symptoms. However, these conclusions are tentative as only limited evidence was found from high-quality, longitudinal studies with fully adjusted models using objective measures of standing

Genome-enabled insights into the biology of thrips as crop pests

Background The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. Results We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. Conclusions Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species

Milepost GCC: Machine Learning Enabled Self-tuning Compiler

International audienceTuning compiler optimizations for rapidly evolving hardwaremakes porting and extending an optimizing compiler for each new platform extremely challenging. Iterative optimization is a popular approach to adapting programs to a new architecture automatically using feedback-directed compilation. However, the large number of evaluations required for each program has prevented iterative compilation from widespread take-up in production compilers. Machine learning has been proposed to tune optimizations across programs systematically but is currently limited to a few transformations, long training phases and critically lacks publicly released, stable tools. Our approach is to develop a modular, extensible, self-tuning optimization infrastructure to automatically learn the best optimizations across multiple programs and architectures based on the correlation between program features, run-time behavior and optimizations. In this paper we describeMilepostGCC, the first publicly-available open-source machine learning-based compiler. It consists of an Interactive Compilation Interface (ICI) and plugins to extract program features and exchange optimization data with the cTuning.org open public repository. It automatically adapts the internal optimization heuristic at function-level granularity to improve execution time, code size and compilation time of a new program on a given architecture. Part of the MILEPOST technology together with low-level ICI-inspired plugin framework is now included in the mainline GCC.We developed machine learning plugins based on probabilistic and transductive approaches to predict good combinations of optimizations. Our preliminary experimental results show that it is possible to automatically reduce the execution time of individual MiBench programs, some by more than a factor of 2, while also improving compilation time and code size. On average we are able to reduce the execution time of the MiBench benchmark suite by 11% for the ARC reconfigurable processor.We also present a realistic multi-objective optimization scenario for Berkeley DB library using Milepost GCC and improve execution time by approximately 17%, while reducing compilatio

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac