High-throughput genotyping of high-homology mutant mouse strains by next-generation sequencing.

Genotyping of knockout alleles in mice is commonly performed by end-point PCR or gene-specific/universal cassette qPCR. Both have advantages and limitations in terms of assay design and interpretation of results. As an alternative method for high-throughput genotyping, we investigated next generation sequencing (NGS) of PCR amplicons, with a focus on CRISPR-mediated exon deletions where antibiotic selection markers are not present. By multiplexing the wild type and mutant-specific PCR reactions, the genotype can be called by the relative sequence counts of each product. The system is highly scalable and can be applied to a variety of different allele types, including those produced by the International Mouse Phenotyping Consortium and associated projects. One potential challenge with any assay design is locating unique areas of the genome, especially when working with gene families or regions of high homology. These can result in misleading or ambiguous genotypes for either qPCR or end-point assays. Here, we show that genotyping by NGS can negate these issues by simple, automated filtering of undesired sequences. Analysis and genotype calls can also be fully automated, using FASTQ or FASTA input files and an in-house Perl script and SQL database.

Poison Parasite Counter: Turning Duplicitous Mass Communications Into Self-Negating Memory-Retrieval Cues.

Disinformation in politics, advertising, and mass communications has proliferated in recent years. Few counterargumentation strategies have proven effective at undermining a deceptive message over time. This article introduces the Poison Parasite Counter (PPC), a cognitive-science-based strategy for durably countering deceptive communications. The PPC involves inserting a strong (poisonous) counter-message, just once, into a close replica of a deceptive rival's original communication. In parasitic fashion, the original communication then "hosts" the counter-message, which is recalled on each reexposure to the original communication. The strategy harnesses associative memory to turn the original communication into a retrieval cue for a negating counter-message. Seven experiments (N = 3,679 adults) show that the PPC lastingly undermines a duplicitous rival's original communication, influencing judgments of communicator honesty and favorability as well as real political donations.

Genetic diagnosis of developmental disorders in the DDD study: a scalable analysis of genome-wide research data.

BACKGROUND: Human genome sequencing has transformed our understanding of genomic variation and its relevance to health and disease, and is now starting to enter clinical practice for the diagnosis of rare diseases. The question of whether and how some categories of genomic findings should be shared with individual research participants is currently a topic of international debate, and development of robust analytical workflows to identify and communicate clinically relevant variants is paramount. METHODS: The Deciphering Developmental Disorders (DDD) study has developed a UK-wide patient recruitment network involving over 180 clinicians across all 24 regional genetics services, and has performed genome-wide microarray and whole exome sequencing on children with undiagnosed developmental disorders and their parents. After data analysis, pertinent genomic variants were returned to individual research participants via their local clinical genetics team. FINDINGS: Around 80,000 genomic variants were identified from exome sequencing and microarray analysis in each individual, of which on average 400 were rare and predicted to be protein altering. By focusing only on de novo and segregating variants in known developmental disorder genes, we achieved a diagnostic yield of 27% among 1133 previously investigated yet undiagnosed children with developmental disorders, whilst minimising incidental findings. In families with developmentally normal parents, whole exome sequencing of the child and both parents resulted in a 10-fold reduction in the number of potential causal variants that needed clinical evaluation compared to sequencing only the child. Most diagnostic variants identified in known genes were novel and not present in current databases of known disease variation. INTERPRETATION: Implementation of a robust translational genomics workflow is achievable within a large-scale rare disease research study to allow feedback of potentially diagnostic findings to clinicians and research participants. Systematic recording of relevant clinical data, curation of a gene-phenotype knowledge base, and development of clinical decision support software are needed in addition to automated exclusion of almost all variants, which is crucial for scalable prioritisation and review of possible diagnostic variants. However, the resource requirements of development and maintenance of a clinical reporting system within a research setting are substantial. FUNDING: Health Innovation Challenge Fund, a parallel funding partnership between the Wellcome Trust and the UK Department of Health.

The synthesis and analysis of lignin-bound Hibbert ketone structures in technical lignins.

Understanding the structure of technical lignins resulting from acid-catalysed treatment of lignocellulosic biomass is important for their future applications. Here we report an investigation into the fate of lignin under acidic aqueous organosolv conditions. In particular we examine in detail the formation and reactivity of non-native Hibbert ketone structures found in isolated organosolv lignins from both Douglas fir and beech woods. Through the use of model compounds combined with HSQC, HMBC and HSQC-TOCSY NMR experiments we demonstrate that, depending on the lignin source, both S and G lignin-bound Hibbert ketone units can be present. We also show that these units can serve as a source of novel mono-aromatic compounds following an additional lignin depolymerisation reaction.

Suspicion About Suspicion Probes: Ways Forward.

Suspicion probes are the traditional tool employed to assess the extent to which participants suspect intentional misdirection or deception within the research context. A primary reason psychologists use deception in research settings is to prevent participants from altering their behavior in light of knowing what is being studied, which could undermine internal validity as well as threaten the generalizability of findings to the real world (i.e., external validity). The present article elucidates a number of challenges with suspicion probes. A definition and framework for conceptualizing the construct of suspicion in research settings are proposed. Following a literature review, an analysis of existing evidence, and new data on the prevalence of using and reporting suspicion probes, we conclude that suspicion is a likely problem in research practice. We provide a decision guide to help researchers navigate the numerous choices involved in addressing potential suspicion and call for a combination of (a) renewed research leading to empirically supported tools and best practices and (b) systemic changes to editorial policies, funding practices, professional standards, and research training that would increase rigor and focus on this aspect of research methodology.

CYP2E1 overexpression protects COS-7 cancer cells against ferroptosis.

Ferroptosis is a recently described form of regulated cell death initiated by the iron-mediated one-electron reduction of lipid hydroperoxides (LOOH). Cytochrome P450 2E1 (CYP2E1) induction, a consequence of genetic polymorphisms or/and gene induction by xenobiotics, may promote ferroptosis by contributing to the cellular pool of LOOH. However, CYP2E1 induction also increases the transcription of anti-ferroptotic genes that regulate the activity of glutathione peroxidase 4 (GPX4), the main ferroptosis inhibitor. Based on the above, we hypothesize that the impact of CYP2E1 induction on ferroptosis depends on the balance between pro- and anti-ferroptotic pathways triggered by CYP2E1. To test our hypothesis, ferroptosis was induced with class 2 inducers (RSL-3 or ML-162) in mammalian COS-7 cancer cells that don't express CYP2E1 (Mock cells), and in cells engineered to express human CYP2E1 (WT cells), and the impact on viability, lipid peroxidation and GPX4 was assessed. CYP2E1 overexpression protected COS-7 cancer cells against ferroptosis, evidenced by an increase in the IC50 and a decrease in lipid ROS in WT versus Mock cells after exposure to class 2 inducers. CYP2E1 overexpression produced an 80% increase in the levels of the GPX4 substrate glutathione (GSH). Increasing GSH in Mock cells protected cells against ferroptosis by ML-162. Depleting GSH, or inhibiting Nrf2 in WT cells reverted the protective effect mediated by CYP2E1, causing a decrease in the IC50 and an increase in lipid ROS after exposure to ML-162. These results show that CYP2E1 overexpression protects COS-7 cancer cells against ferroptosis, an effect probably mediated by Nrf2-dependent GSH induction.

Benign peripheral nerve tumors: treatment algorithm and reconstructive options.

Peripheral nerve tumors are mostly benign; however, their excision can result in profound deficits. Nerve reconstruction strategies offer techniques to minimize morbidity. In this prospective study, 20 consecutive patients with benign peripheral nerve tumors were treated using a single-stage surgical paradigm between 2003 and 2007. Nerve fascicles were microdissected off of the tumor; any fascicle that gave origin to the tumor (was inseparable from tumor) was reconstructed using nerve conduits. Patients were followed from 6 to 24 months. All patients had neuropathic pain before tumor excision; only 1 patient had pain persist postoperatively. Seventeen patients had complete functional recovery after nerve reconstruction. No perioperative complications occurred. Benign peripheral nerve tumors require highly specialized surgical care. Tumor excision with immediate nerve reconstruction, for fascicles inseparable from the tumor, optimizes outcomes. Nerve reconstruction with available conduits or allografts should be attempted to restore anatomic integrity to any killed fascicles, thereby minimizing possible deficits.

Preparation and Reactivity of Biomass-Derived Dihydro-Dioxins.

The depolymerization of the biopolymer lignin can give pure aromatic monomers but selective catalytic approaches remain scarce. Here, an approach was rerouted to deliver an unusual phenolic monomer. This monomer's instability proved challenging, but a degradation study identified strategies to overcome this. Heterocycles and a useful synthetic intermediate were prepared. The range of aromatics available from the ß-O-4 unit in lignin was extended.

Driving Under the Influence (of Language).

We present a unified framework which supports grounding natural-language semantics in robotic driving. This framework supports acquisition (learning grounded meanings of nouns and prepositions from human sentential annotation of robotic driving paths), generation (using such acquired meanings to generate sentential description of new robotic driving paths), and comprehension (using such acquired meanings to support automated driving to accomplish navigational goals specified in natural language). We evaluate the performance of these three tasks by having independent human judges rate the semantic fidelity of the sentences associated with paths. Overall, machine performance is 74.9%, while the performance of human annotators is 83.8%.

Unravelling the enigma of ligninOX: can the oxidation of lignin be controlled?

As societal challenges go, the development of efficient biorefineries as a means of reducing our dependence on petroleum refineries is high on the list. One of the core strengths of the petroleum refinery is its ability to produce a huge range of different products using all of the components of the starting material. In contrast, the target of using all the biopolymers present in lignocellulosic biomass is far from realised. Even though our ability to use the carbohydrate-based components has advanced, our plans for lignin lag behind (with the notable exception of vanillin production). One approach to lignin usage is its controlled depolymerisation. This study focuses on an increasingly popular approach to this challenge which involves highly selective lignin oxidation to give a material often referred to as ligninOX. But what do we mean by ligninOX? In this study we show that it is possible to form many different types of ligninOX depending on the oxidation conditions that are used. We show that variations in the levels of processing of the ß-O-4, the ß-ß and a third linkage occur. Through use of this information, we can form a well-defined ligninOX from six different hardwood lignins. This process is reproducible and can be carried out on a large scale. With a source of well-defined ligninOX in hand, we show that it can be converted to simple aromatic monomers and that any remaining ligninOX is sufficiently soluble for further processing to be carried out.

Imaging multidrug resistance with 4-[18F]fluoropaclitaxel.

Multidrug resistance (MDR) is a cause of treatment failure in many cancer patients. MDR refers to a phenotype whereby a tumor is resistant to a large number of natural chemotherapeutic drugs. Having prior knowledge of the presence of such resistance would decrease morbidity from unsuccessful therapy and allow for the selection of individuals who may benefit from the coadministration of MDR-inhibiting drugs. The Tc-99m-labeled single-photon-emitting radiotracers sestamibi and tetrofosmin have shown some predictive value. However, positron-emitting radiotracers, which allow for dynamic quantitative imaging, hold promise for a more accurate and specific identification of MDRtumors.MDR-expressing tumors are resistant to paclitaxel, which is commonly used as a chemotherapeutic agent. 4-[18F]Fluoropaclitaxel (FPAC) is a PET-radiolabeled analogue of paclitaxel. Preclinical studies have shown the uptake of FPAC to be inversely proportional to tumor MDR expression. FPAC PET imaging in normal volunteers shows biodistribution to be similar to that in nonhuman primates. Imaging in a breast cancer patient showed FPAC localization in a primary tumor that responded to chemotherapy, while failure to localize in mediastinal disease corresponded with only partial response.FPAC PET imaging shows promise for the noninvasive pretreatment identification of MDR-expressing tumors. While much additional work is needed, this work represents a step toward image-guided personalized medicine.

Fractionation and DOSY NMR as Analytical Tools: From Model Polymers to a Technical Lignin.

One key challenge hindering the valorization of lignin is its structural complexity. Artificial lignin-like materials provide a stepping stone between the simplicity of model compounds and the complexity of lignin. Here, we report an optimized synthesis of an all-G ß-O-4 polymer 1 designed to model softwood lignin. After acetylation, the polymer Ac-1(V) was fractionated using a protocol that involved only volatile organic solvents, which left no insoluble residue. Using diffusion ordered spectroscopy NMR in combination with gel permeation chromatography, it was revealed that this fractionated material behaved like a flexible linear polymer in solution (average α > 0.5). Acetylated kraft lignin was subsequently processed using the same fractionation protocol. By comparison with the model polymer, we propose that the acetylated kraft lignin is composed of two classes of materials that exhibit contrasting physical properties. One is comparable to the acetylated all-G ß-O-4 polymer Ac-1, and the second has a significantly different macromolecular structure.

Finding function: evaluation methods for functional genomic data.

BACKGROUND: Accurate evaluation of the quality of genomic or proteomic data and computational methods is vital to our ability to use them for formulating novel biological hypotheses and directing further experiments. There is currently no standard approach to evaluation in functional genomics. Our analysis of existing approaches shows that they are inconsistent and contain substantial functional biases that render the resulting evaluations misleading both quantitatively and qualitatively. These problems make it essentially impossible to compare computational methods or large-scale experimental datasets and also result in conclusions that generalize poorly in most biological applications. RESULTS: We reveal issues with current evaluation methods here and suggest new approaches to evaluation that facilitate accurate and representative characterization of genomic methods and data. Specifically, we describe a functional genomics gold standard based on curation by expert biologists and demonstrate its use as an effective means of evaluation of genomic approaches. Our evaluation framework and gold standard are freely available to the community through our website. CONCLUSION: Proper methods for evaluating genomic data and computational approaches will determine how much we, as a community, are able to learn from the wealth of available data. We propose one possible solution to this problem here but emphasize that this topic warrants broader community discussion.

Saying What You're Looking For: Linguistics Meets Video Search.

We present an approach to searching large video corpora for clips which depict a natural-language query in the form of a sentence. Compositional semantics is used to encode subtle meaning differences lost in other approaches, such as the difference between two sentences which have identical words but entirely different meaning: The person rode the horse versus The horse rode the person. Given a sentential query and a natural-language parser, we produce a score indicating how well a video clip depicts that sentence for each clip in a corpus and return a ranked list of clips. Two fundamental problems are addressed simultaneously: detecting and tracking objects, and recognizing whether those tracks depict the query. Because both tracking and object detection are unreliable, our approach uses the sentential query to focus the tracker on the relevant participants and ensures that the resulting tracks are described by the sentential query. While most earlier work was limited to single-word queries which correspond to either verbs or nouns, we search for complex queries which contain multiple phrases, such as prepositional phrases, and modifiers, such as adverbs. We demonstrate this approach by searching for 2,627 naturally elicited sentential queries in 10 Hollywood movies.

A new system for comparative functional genomics of Saccharomyces yeasts.

Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.

Predicting human nocturnal nonvisual responses to monochromatic and polychromatic light with a melanopsin photosensitivity function.

The short-wavelength (blue) light sensitivity of human circadian, neurobehavioral, neuroendocrine, and neurophysiological responses is attributed to melanopsin. Whether melanopsin is the sole factor in determining the efficacy of a polychromatic light source in driving nonvisual responses, however, remains to be established. Monochromatic (λ(max) 437, 479, and 532 nm administered singly and in combination with 479 nm light) and polychromatic (color temperature: 4000 K and 17000 K) light stimuli were photon matched for their predicted ability to stimulate melanopsin, and their capacity to affect nocturnal melatonin levels, auditory reaction time, and subjective alertness and mood was assessed. Young, healthy male participants aged 18-35 yrs (23.6 ± 3.6 yrs [mean ± SD]; n=12) participated in 12 overnight sessions that included an individually timed 30-min nocturnal light stimulus on the rising limb of the melatonin profile. At regular intervals before, during, and after the light stimulus, subjective mood and alertness were verbally assessed, blood samples were taken for analysis of plasma melatonin levels, and an auditory reaction time task (psychomotor vigilance task; PVT) was performed. Proc GLM (general linear model) repeated-measures ANOVA (analysis of variance) revealed significantly lower melatonin suppression with the polychromatic light conditions (4000 and 17000 K) compared to the "melanopsin photon-matched" monochromatic light conditions (p< .05). In contrast, subjective alertness was significantly lower under the 479 nm monochromatic light condition compared to the 437 and 532 nm monochromatic and both polychromatic light conditions. The alerting responses more reflected the total photon content of the light stimulus. The demonstration that the melatonin suppression response to polychromatic light was significantly lower than predicted by the melanopsin photosensitivity function suggests this function is not the sole consideration when trying to predict the efficacy of broadband lighting. The different spectral sensitivity of subjective alertness and melatonin suppression responses may imply a differential involvement of the cone photopigments. An analysis of the photon densities in specific wavelength bands for the polychromatic lights used in this and the authors' previous study suggests the spectral composition of a polychromatic light source, and particularly the very short-wavelength content, may be critical in determining response magnitude for the neuroendocrine and neurobehavioral effects of nocturnal light.

Exploring the human genome with functional maps.

Human genomic data of many types are readily available, but the complexity and scale of human molecular biology make it difficult to integrate this body of data, understand it from a systems level, and apply it to the study of specific pathways or genetic disorders. An investigator could best explore a particular protein, pathway, or disease if given a functional map summarizing the data and interactions most relevant to his or her area of interest. Using a regularized Bayesian integration system, we provide maps of functional activity and interaction networks in over 200 areas of human cellular biology, each including information from approximately 30,000 genome-scale experiments pertaining to approximately 25,000 human genes. Key to these analyses is the ability to efficiently summarize this large data collection from a variety of biologically informative perspectives: prediction of protein function and functional modules, cross-talk among biological processes, and association of novel genes and pathways with known genetic disorders. In addition to providing maps of each of these areas, we also identify biological processes active in each data set. Experimental investigation of five specific genes, AP3B1, ATP6AP1, BLOC1S1, LAMP2, and RAB11A, has confirmed novel roles for these proteins in the proper initiation of macroautophagy in amino acid-starved human fibroblasts. Our functional maps can be explored using HEFalMp (Human Experimental/Functional Mapper), a web interface allowing interactive visualization and investigation of this large body of information.

Confirming preferences or collecting data? Information search strategies and romantic partner selection.

This article investigates two kinds of information search strategies in the context of selecting romantic partners. Confirmatory searching occurs when people ask for more information about a romantic partner in order to validate or confirm their assessment. Balanced searches are characterized by a search for risk information for partners rated as attractive and for attractiveness information about partners rated as risky in order to attain a more complete evaluation. A factorial survey computer program randomly constructed five types of partner descriptions and college-age respondents evaluated nine descriptions in terms of both health risk and romantic attractiveness outcomes. The results show little evidence of balanced search strategies: for all vignette types the respondents searched for attractiveness information. Regression analysis of the search outcomes showed no difference between males and females in the desire for attractiveness or risk information, the amount of additional information desired, or the proportion of descriptions for which more information was desired. However, an attractive physical appearance did increase the amount of additional information desired and the proportion of vignettes for which more information was desired. The results were generally inconsistent with a balanced search hypothesis; a better characterization of the respondents' strategy might be "confirmatory bias."

Evaluating the risk and attractiveness of romantic partners when confronted with contradictory cues.

Research shows that people engage in "risky" sex with "safe" partners and in "safer" sex with "riskier" partners. How is the determination of "risky" or "safe" status made? Factorial survey methodology was used to randomly construct descriptions of romantic partners based on attractive and/or risky characteristics. Respondents evaluated 20 descriptions for attractiveness, health risk, likelihood of going on a date, likelihood of unprotected sex, and likelihood of STD/HIV infection. Respondents were most attracted to and perceived the least risk from attractive descriptions and were least attracted to and perceived the most risk from the risky descriptions. The differences between the "conflicting information" descriptions are attributable to a primacy effect: descriptions that began with attractiveness information but end with risk information were evaluated more positively than those that began with risk and ended with attractive information.