Herding of sheep by dogs is a powerful example of one individual causing many unwilling individuals to move in the same direction. Similar phenomena are central to crowd control, cleaning the environment and other engineering problems. Despite single dogs solving this 'shepherding problem' every day, it remains unknown which algorithm they employ or whether a general algorithm exists for shepherding. Here, we demonstrate such an algorithm, based on adaptive switching between collecting the agents when they are too dispersed and driving them once they are aggregated. Our algorithm reproduces key features of empirical data collected from sheep-dog interactions and suggests new ways in which robots can be designed to influence movements of living and artificial agents.
Hox and other homeobox-containing genes encode critical transcriptional regulators of animal development. Although these genes are well known for their roles in the body axis and appendage development, little is known regarding the mechanisms by which these factors influence chromatin landscapes. Chromatin structure can have a profound influence on gene expression during animal body formation. However, when applied to developing embryos, conventional chromatin analysis of genes and cis-regulatory modules (CRMs) typically lacks the required cell type-specific resolution due to the heterogeneous nature of animal bodies. Here we present a strategy to analyze both the composition and conformation of in vivo-tagged CRM sequences in a cell type-specific manner, using as a system Drosophila embryos. We term this method cgChIP (cell- and gene-specific Chromatin Immunoprecipitation) by which we access and analyze regulatory chromatin in specific cell types. cgChIP is an in vivo method designed to analyze genetic elements derived from limited cell populations. cgChIP can be used for both the analysis of chromatin structure (e.g., long-distance interactions between DNA elements) and the composition of histones and histone modifications and the occupancy of transcription factors and chromatin modifiers. This method was applied to the Hox target gene Distalless (Dll), which encodes for a homeodomain-containing transcription factor critical for the formation of appendages in Drosophila. However, cgChIP can be applied in diverse animal models to better dissect CRM-dependent gene regulation and body formation in developing animals.
The closely related members of the Hox family of homeodomain transcription factors have similar DNA-binding preferences as monomers, yet carry out distinct functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA-binding specificities. To test this hypothesis we developed a new experimental and computational platform, termed SELEX-seq, to characterize DNA-binding specificities of Hox-based multiprotein complexes. We found that complex formation with the same cofactor reveals latent specificities that are not observed for monomeric Hox factors. The findings from this in vitro platform are consistent with in vivo data, and the "latent specificity" concept serves as a precedent for how the specificities of similar transcription factors might be distinguished in vivo. Importantly, the SELEX-seq platform is flexible and can be used to determine the relative affinities to any DNA sequence for any transcription factor or multiprotein complex.
There is increasing evidence that animal groups can maintain coordinated behaviour and make collective decisions based on simple interaction rules. Effective collective action may be further facilitated by individual variation within groups, particularly through leader-follower polymorphisms. Recent studies have suggested that individual-level personality traits influence the degree to which individuals use social information, are attracted to conspecifics, or act as leaders/followers. However, evidence is equivocal and largely limited to laboratory studies. We use an automated data-collection system to conduct an experiment testing the relationship between personality and collective decision-making in the wild. First, we report that foraging flocks of great tits (Parus major) show strikingly synchronous behaviour. A predictive model of collective decision-making replicates patterns well, suggesting simple interaction rules are sufficient to explain the observed social behaviour. Second, within groups, individuals with more reactive personalities behave more collectively, moving to within-flock areas of higher density. By contrast, proactive individuals tend to move to and feed at spatial periphery of flocks. Finally, comparing alternative simulations of flocking with empirical data, we demonstrate that variation in personality promotes within-patch movement while maintaining group cohesion. Our results illustrate the importance of incorporating individual variability in models of social behaviour.
Hippo signaling limits organ growth by inhibiting the transcriptional coactivator Yorkie. Despite the key role of Yorkie in both normal and oncogenic growth, the mechanism by which it activates transcription has not been defined. We report that Yorkie binding to chromatin correlates with histone H3K4 methylation and is sufficient to locally increase it. We show that Yorkie can recruit a histone methyltransferase complex through binding between WW domains of Yorkie and PPxY sequence motifs of NcoA6, a subunit of the Trithorax-related (Trr) methyltransferase complex. Cell culture and in vivo assays establish that this recruitment of NcoA6 contributes to Yorkie's ability to activate transcription. Mammalian NcoA6, a subunit of Trr-homologous methyltransferase complexes, can similarly interact with Yorkie's mammalian homolog YAP. Our results implicate direct recruitment of a histone methyltransferase complex as central to transcriptional activation by Yorkie, linking the control of cell proliferation by Hippo signaling to chromatin modification.
Obesity is often associated with higher hospital costs because of longer length of stay (LOS) but this has not been well studied in the kidney transplant population. Therefore, we used national data to compare LOS in select groups of morbidly obese and normal weight recipients after kidney transplant.
Associations in mixed-species foraging groups are common in animals, yet have rarely been explored in the context of collective behaviour. Despite many investigations into the social and ecological conditions under which individuals should form groups, we still know little about the specific behavioural rules that individuals adopt in these contexts, or whether these can be generalized to heterospecifics. Here, we studied collective behaviour in flocks in a community of five species of woodland passerine birds. We adopted an automated data collection protocol, involving visits by RFID-tagged birds to feeding stations equipped with antennae, over two winters, recording 91?576 feeding events by 1904 individuals. We demonstrated highly synchronized feeding behaviour within patches, with birds moving towards areas of the patch with the largest proportion of the flock. Using a model of collective decision making, we then explored the underlying decision rule birds may be using when foraging in mixed-species flocks. The model tested whether birds used a different decision rule for conspecifics and heterospecifics, and whether the rules used by individuals of different species varied. We found that species differed in their response to the distribution of conspecifics and heterospecifics across foraging patches. However, simulating decisions using the different rules, which reproduced our data well, suggested that the outcome of using different decision rules by each species resulted in qualitatively similar overall patterns of movement. It is possible that the decision rules each species uses may be adjusted to variation in mean species abundance in order for individuals to maintain the same overall flock-level response. This is likely to be important for maintaining coordinated behaviour across species, and to result in quick and adaptive flock responses to food resources that are patchily distributed in space and time.
Walking behavior is context-dependent, resulting from the integration of internal and external influences by specialized motor and pre-motor centers. Neuronal programs must be sufficiently flexible to the locomotive challenges inherent in different environments. Although insect studies have contributed substantially to the identification of the components and rules that determine locomotion, we still lack an understanding of how multi-jointed walking insects respond to changes in walking orientation and direction and strength of the gravitational force. In order to answer these questions we measured with high temporal and spatial resolution the kinematic properties of untethered Drosophila during inverted and vertical walking. In addition, we also examined the kinematic responses to increases in gravitational load. We find that animals are capable of shifting their step, spatial and inter-leg parameters in order to cope with more challenging walking conditions. For example, flies walking in an inverted orientation decreased the duration of their swing phase leading to increased contact with the substrate and, as a result, greater stability. We also find that when flies carry additional weight, thereby increasing their gravitational load, some changes in step parameters vary over time, providing evidence for adaptation. However, above a threshold that is between 1 and 2 times their body weight flies display locomotion parameters that suggest they are no longer capable of walking in a coordinated manner. Finally, we find that functional chordotonal organs are required for flies to cope with additional weight, as animals deficient in these proprioceptors display increased sensitivity to load bearing as well as other locomotive defects.
The approach presented in this article represents a generalizable and adaptable methodology for identifying complex interactions in educational systems and for investigating how manipulation of these systems may affect educational outcomes of interest. Multilayer Minimum Spanning Tree and Monte-Carlo methods are used. A virtual Sandbox University is created in order to facilitate effective identification of successful and stable initiatives within higher education, which can affect students' credits and student retention - something that has been lacking up until now. The results highlight the importance of teacher feedback and teacher-student rapport, which is congruent with current educational findings, illustrating the methodology's potential to provide a new basis for further empirical studies of issues in higher education from a complex systems perspective.
Over the past decades many countries have experienced rapid changes in their economies, their democratic institutions and the values of their citizens. Comprehensive data measuring these changes across very different countries has recently become openly available. Between country similarities suggest common underlying dynamics in how countries develop in terms of economy, democracy and cultural values. We apply a novel Bayesian dynamical systems approach to identify the model which best captures the complex, mainly non-linear dynamics that underlie these changes. We show that the level of Human Development Index (HDI) in a country drives first democracy and then higher emancipation of citizens. This change occurs once the countries pass a certain threshold in HDI. The data also suggests that there is a limit to the growth of wealth, set by higher emancipation. Having reached a high level of democracy and emancipation, societies tend towards equilibrium that does not support further economic growth. Our findings give strong empirical evidence against a popular political science theory, known as the Human Development Sequence. Contrary to this theory, we find that implementation of human-rights and democratisation precede increases in emancipative values.
Data arising from social systems is often highly complex, involving non-linear relationships between the macro-level variables that characterize these systems. We present a method for analyzing this type of longitudinal or panel data using differential equations. We identify the best non-linear functions that capture interactions between variables, employing Bayes factor to decide how many interaction terms should be included in the model. This method punishes overly complicated models and identifies models with the most explanatory power. We illustrate our approach on the classic example of relating democracy and economic growth, identifying non-linear relationships between these two variables. We show how multiple variables and variable lags can be accounted for and provide a toolbox in R to implement our approach.
Observations of the flight paths of pigeons navigating from familiar locations have shown that these birds are able to learn and subsequently follow habitual routes home. It has been suggested that navigation along these routes is based on the recognition of memorized visual landmarks. Previous research has identified the effect of landmarks on flight path structure, and thus the locations of potentially salient sites. Pigeons have also been observed to be particularly attracted to strong linear features in the landscape, such as roads and rivers. However, a more general understanding of the specific characteristics of the landscape that facilitate route learning has remained out of reach. In this study, we identify landscape complexity as a key predictor of the fidelity to the habitual route, and thus conclude that pigeons form route memories most strongly in regions where the landscape complexity is neither too great nor too low. Our results imply that pigeons process their visual environment on a characteristic spatial scale while navigating and can explain the different degrees of success in reproducing route learning in different geographical locations.
Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.
We present OnTheFly (http://bhapp.c2b2.columbia.edu/OnTheFly/index.php), a database comprising a systematic collection of transcription factors (TFs) of Drosophila melanogaster and their DNA-binding sites. TFs predicted in the Drosophila melanogaster genome are annotated and classified and their structures, obtained via experiment or homology models, are provided. All known preferred TF DNA-binding sites obtained from the B1H, DNase I and SELEX methodologies are presented. DNA shape parameters predicted for these sites are obtained from a high throughput server or from crystal structures of protein-DNA complexes where available. An important feature of the database is that all DNA-binding domains and their binding sites are fully annotated in a eukaryote using structural criteria and evolutionary homology. OnTheFly thus provides a comprehensive view of TFs and their binding sites that will be a valuable resource for deciphering non-coding regulatory DNA.
The Yorkie/Yap transcriptional coactivator is a well-known regulator of cellular proliferation in both invertebrates and mammals. As a coactivator, Yorkie (Yki) lacks a DNA binding domain and must partner with sequence-specific DNA binding proteins in the nucleus to regulate gene expression; in Drosophila, the developmental regulators Scalloped (Sd) and Homothorax (Hth) are two such partners. To determine the range of target genes regulated by these three transcription factors, we performed genome-wide chromatin immunoprecipitation experiments for each factor in both the wing and eye-antenna imaginal discs. Strong, tissue-specific binding patterns are observed for Sd and Hth, while Yki binding is remarkably similar across both tissues. Binding events common to the eye and wing are also present for Sd and Hth; these are associated with genes regulating cell proliferation and "housekeeping" functions, and account for the majority of Yki binding. In contrast, tissue-specific binding events for Sd and Hth significantly overlap enhancers that are active in the given tissue, are enriched in Sd and Hth DNA binding sites, respectively, and are associated with genes that are consistent with each factors previously established tissue-specific functions. Tissue-specific binding events are also significantly associated with Polycomb targeted chromatin domains. To provide mechanistic insights into tissue-specific regulation, we identify and characterize eye and wing enhancers of the Yki-targeted bantam microRNA gene and demonstrate that they are dependent on direct binding by Hth and Sd, respectively. Overall these results suggest that both Sd and Hth use distinct strategies - one shared between tissues and associated with Yki, the other tissue-specific, generally Yki-independent and associated with developmental patterning - to regulate distinct gene sets during development.
"Collective intelligence" and "wisdom of crowds" refer to situations in which groups achieve more accurate perception and better decisions than solitary agents. Whether groups outperform individuals should depend on the kind of task and its difficulty, but the nature of this relationship remains unknown. Here we show that colonies of Temnothorax ants outperform individuals for a difficult perception task but that individuals do better than groups when the task is easy. Subjects were required to choose the better of two nest sites as the quality difference was varied. For small differences, colonies were more likely than isolated ants to choose the better site, but this relationship was reversed for large differences. We explain these results using a mathematical model, which shows that positive feedback between group members effectively integrates information and sharpens the discrimination of fine differences. When the task is easier the same positive feedback can lock the colony into a suboptimal choice. These results suggest the conditions under which crowds do or do not become wise.
Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.
Adult Drosophila walk using six multi-jointed legs, each controlled by ?50 leg motoneurons (MNs). Although MNs have stereotyped morphologies, little is known about how they are specified. Here, we describe the function of Hox genes and homothorax (hth), which encodes a Hox co-factor, in Drosophila leg MN development. Removing either Hox or Hth function from a single neuroblast (NB) lineage results in MN apoptosis. A single Hox gene, Antennapedia (Antp), is primarily responsible for MN survival in all three thoracic segments. When cell death is blocked, partially penetrant axon branching errors are observed in Hox mutant MNs. When single MNs are mutant, errors in both dendritic and axon arborizations are observed. Our data also suggest that Antp levels in post-mitotic MNs are important for specifying their identities. Thus, in addition to being essential for survival, Hox and hth are required to specify accurate MN morphologies in a level-dependent manner.
Inference of interaction rules of animals moving in groups usually relies on an analysis of large scale system behaviour. Models are tuned through repeated simulation until they match the observed behaviour. More recent work has used the fine scale motions of animals to validate and fit the rules of interaction of animals in groups. Here, we use a Bayesian methodology to compare a variety of models to the collective motion of glass prawns (Paratya australiensis). We show that these exhibit a stereotypical phase transition, whereby an increase in density leads to the onset of collective motion in one direction. We fit models to this data, which range from: a mean-field model where all prawns interact globally; to a spatial Markovian model where prawns are self-propelled particles influenced only by the current positions and directions of their neighbours; up to non-Markovian models where prawns have memory of previous interactions, integrating their experiences over time when deciding to change behaviour. We show that the mean-field model fits the large scale behaviour of the system, but does not capture the observed locality of interactions. Traditional self-propelled particle models fail to capture the fine scale dynamics of the system. The most sophisticated model, the non-Markovian model, provides a good match to the data at both the fine scale and in terms of reproducing global dynamics, while maintaining a biologically plausible perceptual range. We conclude that prawns movements are influenced by not just the current direction of nearby conspecifics, but also those encountered in the recent past. Given the simplicity of prawns as a study system our research suggests that self-propelled particle models of collective motion should, if they are to be realistic at multiple biological scales, include memory of previous interactions and other non-Markovian effects.
The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkies association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkies transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.
Coordinated walking in vertebrates and multi-legged in vertebrates such as Drosophila melanogaster requires a complex neural network coupled to sensory feedback. An understanding of this network will benefit from systems such as Drosophila that have the ability to genetically manipulate neural activities. However, the flys small size makes it challenging to analyze walking in this system. In order to overcome this limitation, we developed an optical method coupled with high-speed imaging that allows the tracking and quantification of gait parameters in freely walking flies with high temporal and spatial resolution. Using this method, we present a comprehensive description of many locomotion parameters, such as gait, tarsal positioning, and intersegmental and left-right coordination for wild type fruit flies. Surprisingly, we find that inactivation of sensory neurons in the flys legs, to block proprioceptive feedback, led to deficient step precision, but interleg coordination and the ability to execute a tripod gait were unaffected.DOI:http://dx.doi.org/10.7554/eLife.00231.001.
The study of social identity and crowd psychology looks at how and why individual people change their behaviour in response to others. Within a group, a new behaviour can emerge first in a few individuals before it spreads rapidly to all other members. A number of mathematical models have been hypothesized to describe these social contagion phenomena, but these models remain largely untested against empirical data. We used Bayesian model selection to test between various hypotheses about the spread of a simple social behaviour, applause after an academic presentation. Individuals probability of starting clapping increased in proportion to the number of other audience members already infected by this social contagion, regardless of their spatial proximity. The cessation of applause is similarly socially mediated, but is to a lesser degree controlled by the reluctance of individuals to clap too many times. We also found consistent differences between individuals in their willingness to start and stop clapping. The social contagion model arising from our analysis predicts that the time the audience spends clapping can vary considerably, even in the absence of any differences in the quality of the presentations they have heard.
Homeotic selector (Hox) proteins often bind DNA cooperatively with cofactors such as Extradenticle (Exd) and Homothorax (Hth) to achieve functional specificity in vivo. Previous studies identified the Hox YPWM motif as an important Exd interaction motif. Using a comparative approach, we characterize the contribution of this and additional conserved sequence motifs to the regulation of specific target genes for three Drosophila Hox proteins. We find that Sex combs reduced (Scr) uses a simple interaction mechanism, where a single tryptophan-containing motif is necessary for Exd-dependent DNA-binding and in vivo functions. Abdominal-A (AbdA) is more complex, using multiple conserved motifs in a context-dependent manner. Lastly, Ultrabithorax (Ubx) is the most flexible, in that it uses multiple conserved motifs that function in parallel to regulate target genes in vivo. We propose that using different binding mechanisms with the same cofactor may be one strategy to achieve functional specificity in vivo.
Hox transcription factors exhibit an evolutionarily conserved functional hierarchy, termed phenotypic suppression, in which the activity of posterior Hox proteins dominates over more anterior Hox proteins. Using directly regulated Hox targeted reporter genes in Drosophila, we show that posterior Hox proteins suppress the activities of anterior ones by competing for cofactor-dependent DNA binding. Furthermore, we map a motif in the posterior Hox protein Abdominal-A (AbdA) that is required for phenotypic suppression and facilitates cooperative DNA binding with the Hox cofactor Extradenticle (Exd). Together, these results suggest that Hox-specific motifs endow posterior Hox proteins with the ability to dominate over more anterior ones via a cofactor-dependent DNA-binding mechanism.
Collective motion, where large numbers of individuals move synchronously together, is achieved when individuals adopt interaction rules that determine how they respond to their neighbors movements and positions. These rules determine how group-living animals move, make decisions, and transmit information between individuals. Nonetheless, few studies have explicitly determined these interaction rules in moving groups, and very little is known about the interaction rules of fish. Here, we identify three key rules for the social interactions of mosquitofish (Gambusia holbrooki): (i) Attraction forces are important in maintaining group cohesion, while we find only weak evidence that fish align with their neighbors orientation; (ii) repulsion is mediated principally by changes in speed; (iii) although the positions and directions of all shoal members are highly correlated, individuals only respond to their single nearest neighbor. The last two of these rules are different from the classical models of collective animal motion, raising new questions about how fish and other animals self-organize on the move.
DNA shape variation and the associated variation in minor groove electrostatic potential are widely exploited by proteins for DNA recognition. Here we show that the hydroxyl radical cleavage pattern is a quantitative measure of DNA backbone solvent accessibility, minor groove width, and minor groove electrostatic potential, at single nucleotide resolution. We introduce maps of DNA shape and electrostatic potential as tools for understanding how proteins recognize binding sites in a genome. These maps reveal periodic structural signals in yeast and Drosophila genomic DNA sequences that are associated with positioned nucleosomes.
Annual GP appraisal provides a standardised system to identify poorly performing or underperforming doctors as well as development needs. Mentoring is widely used for enhancing personal and professional development and might help address these needs. However, doubts have been expressed in the literature about the strength of the supporting evidence. The aim of this pilot study was to explore the possible benefits of using a specified action learning approach to mentoring through a prospective study design.
Members of transcription factor families typically have similar DNA binding specificities yet execute unique functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA binding specificities. To test this hypothesis, we developed an experimental and computational platform, SELEX-seq, that can be used to determine the relative affinities to any DNA sequence for any transcription factor complex. Applying this method to all eight Drosophila Hox proteins, we show that they obtain novel recognition properties when they bind DNA with the dimeric cofactor Extradenticle-Homothorax (Exd). Exd-Hox specificities group into three main classes that obey Hox gene collinearity rules and DNA structure predictions suggest that anterior and posterior Hox proteins prefer DNA sequences with distinct minor groove topographies. Together, these data suggest that emergent DNA recognition properties revealed by interactions with cofactors contribute to transcription factor specificities in vivo.
The emergence of similar collective patterns from different self-propelled particle models of animal groups points to a restricted set of "universal" classes for these patterns. While universality is interesting, it is often the fine details of animal interactions that are of biological importance. Universality thus presents a challenge to inferring such interactions from macroscopic group dynamics since these can be consistent with many underlying interaction models. We present a Bayesian framework for learning animal interaction rules from fine scale recordings of animal movements in swarms. We apply these techniques to the inverse problem of inferring interaction rules from simulation models, showing that parameters can often be inferred from a small number of observations. Our methodology allows us to quantify our confidence in parameter fitting. For example, we show that attraction and alignment terms can be reliably estimated when animals are milling in a torus shape, while interaction radius cannot be reliably measured in such a situation. We assess the importance of rate of data collection and show how to test different models, such as topological and metric neighbourhood models. Taken together our results both inform the design of experiments on animal interactions and suggest how these data should be best analysed.
The proximodistal (PD) axis of the Drosophila leg is thought to be established by the combined gradients of two secreted morphogens, Wingless (Wg) and Decapentaplegic (Dpp). According to this model, high [Wg+Dpp] activates Distalless (Dll) and represses dachshund (dac) in the distal cells of the leg disc, while intermediate [Wg+Dpp] activates dac in medial tissue. To test this model we identified and characterized a dac cis-regulatory element (dac RE) that recapitulates dacs medial expression domain during leg development. Counter to the gradient model, we find that Wg and Dpp do not act in a graded manner to activate RE. Instead, dac RE is activated directly by Dll and repressed distally by a combination of factors, including the homeodomain protein Bar. Thus, medial leg fates are established via a regulatory cascade in which Wg+Dpp activate Dll and then Dll directly activates dac, with Wg+Dpp as less critical, permissive inputs.
The Hox genes are responsible for generating morphological diversity along the anterior-posterior axis during animal development. The Drosophila Hox gene Ultrabithorax (Ubx), for example, is required for specifying the identity of the third thoracic (T3) segment of the adult, which includes the dorsal haltere, an appendage required for flight, and the ventral T3 leg. Ubx mutants show homeotic transformations of the T3 leg towards the identity of the T2 leg and the haltere towards the wing. All Hox genes, including Ubx, encode homeodomain containing transcription factors, raising the question of what target genes Ubx regulates to generate these adult structures. To address this question, we carried out whole genome ChIP-chip studies to identify all of the Ubx bound regions in the haltere and T3 leg imaginal discs, which are the precursors to these adult structures. In addition, we used ChIP-chip to identify the sites bound by the Hox cofactor, Homothorax (Hth). In contrast to previous ChIP-chip studies carried out in Drosophila embryos, these binding studies reveal that there is a remarkable amount of tissue- and transcription factor-specific binding. Analyses of the putative target genes bound and regulated by these factors suggest that Ubx regulates many downstream transcription factors and developmental pathways in the haltere and T3 leg. Finally, we discovered additional DNA sequence motifs that in some cases are specific for individual data sets, arguing that Ubx and/or Hth work together with many regionally expressed transcription factors to execute their functions. Together, these data provide the first whole-genome analysis of the binding sites and target genes regulated by Ubx to specify the morphologies of the adult T3 segment of the fly.
How social-living animals make collective decisions is currently the subject of intense scientific interest, with increasing focus on the role of individual variation within the group. Previously, we demonstrated that during paired flight in homing pigeons, a fully transitive leadership hierarchy emerges as birds are forced to choose between their own and their partners habitual routes. This stable hierarchy suggests a role for individual differences mediating leadership decisions within homing pigeon pairs. What these differences are, however, has remained elusive. Using novel quantitative techniques to analyse habitual route structure, we show here that leadership can be predicted from prior route-following fidelity. Birds that are more faithful to their own route when homing alone are more likely to emerge as leaders when homing socially. We discuss how this fidelity may relate to the leadership phenomenon, and propose that leadership may emerge from the interplay between individual route confidence and the dynamics of paired flight.
Pigeons home along idiosyncratic habitual routes from familiar locations. It has been suggested that memorized visual landmarks underpin this route learning. However, the inability to experimentally alter the landscape on large scales has hindered the discovery of the particular features to which birds attend. Here, we present a method for objectively classifying the most informative regions of animal paths. We apply this method to flight trajectories from homing pigeons to identify probable locations of salient visual landmarks. We construct and apply a Gaussian process model of flight trajectory generation for pigeons trained to home from specific release sites. The model shows increasing predictive power as the birds become familiar with the sites, mirroring the animals learning process. We subsequently find that the most informative elements of the flight trajectories coincide with landscape features that have previously been suggested as important components of the homing task.
Hox proteins frequently select and regulate their specific target genes with the help of cofactors like Extradenticle (Exd) and Homothorax (Hth). For the Drosophila Hox protein Sex combs reduced (Scr), Exd has been shown to position a normally unstructured portion of Scr so that two basic amino acid side chains can insert into the minor groove of an Scr-specific DNA-binding site. Here we provide evidence that another Drosophila Hox protein, Deformed (Dfd), uses a very similar mechanism to achieve specificity in vivo, thus generalizing this mechanism. Furthermore, we show that subtle differences in the way Dfd and Scr recognize their specific binding sites, in conjunction with non-DNA-binding domains, influence whether the target gene is transcriptionally activated or repressed. These results suggest that the interaction between these DNA-binding proteins and the DNA-binding site determines the architecture of the Hox-cofactor-DNA ternary complex, which in turn determines whether the complex recruits coactivators or corepressors.
The distribution of secreted morphogens in a developing organ determines its form by instructing size, shape and pattern. Pentagone has been identified as a secreted factor that controls the distribution of the morphogen Decapentaplegic (Dpp) in the Drosophila melanogaster wing.
The radically distinct morphologies of arthropod and tetrapod legs argue that these appendages do not share a common evolutionary origin. Yet, despite dramatic differences in morphology, it has been known for some time that transcription factors encoded by the Distalless (Dll)/Dlx gene family play a critical role in the development of both structures. Here we show that a second transcription factor family encoded by the Sp8 gene family, previously implicated in vertebrate limb development, also plays an early and fundamental role in arthropod leg development. By simultaneously removing the function of two Sp8 orthologs, buttonhead (btd) and Sp1, during Drosophila embryogenesis, we find that adult leg development is completely abolished. Remarkably, in the absence of these factors, transformations from ventral to dorsal appendage identities are observed, suggesting that adult dorsal fates become derepressed when ventral fates are eliminated. Further, we show that Sp1 plays a much more important role in ventral appendage specification than btd and that Sp1 lies genetically upstream of Dll. In addition to these selector-like gene functions, Sp1 and btd are also required during larval stages for the growth of the leg. Vertebrate Sp8 can rescue many of the functions of the Drosophila genes, arguing that these activities have been conserved, despite more than 500 million years of independent evolution. These observations suggest that an ancient Sp8/Dlx gene cassette was used in an early metazoan for primitive limb-like outgrowths and that this cassette was co-opted multiple times for appendage formation in multiple animal phyla.
Specific interactions between proteins and DNA are fundamental to many biological processes. In this review, we provide a revised view of protein-DNA interactions that emphasizes the importance of the three-dimensional structures of both macromolecules. We divide protein-DNA interactions into two categories: those when the protein recognizes the unique chemical signatures of the DNA bases (base readout) and those when the protein recognizes a sequence-dependent DNA shape (shape readout). We further divide base readout into those interactions that occur in the major groove from those that occur in the minor groove. Analogously, the readout of the DNA shape is subdivided into global shape recognition (for example, when the DNA helix exhibits an overall bend) and local shape recognition (for example, when a base pair step is kinked or a region of the minor groove is narrow). Based on the >1500 structures of protein-DNA complexes now available in the Protein Data Bank, we argue that individual DNA-binding proteins combine multiple readout mechanisms to achieve DNA-binding specificity. Specificity that distinguishes between families frequently involves base readout in the major groove, whereas shape readout is often exploited for higher resolution specificity, to distinguish between members within the same DNA-binding protein family.
Proteins rely on a variety of readout mechanisms to preferentially bind specific DNA sequences. The nucleosome offers a prominent example of a shape readout mechanism where arginines insert into narrow minor groove regions that face the histone core. Here we compare DNA shape and arginine recognition of three nucleosome core particle structures, expanding on our previous study by characterizing two additional structures, one with a different protein sequence and one with a different DNA sequence. The electrostatic potential in the minor groove is shown to be largely independent of the underlying sequence but is, however, dominated by groove geometry. Our results extend and generalize our previous observation that the interaction of arginines with narrow minor grooves plays an important role in stabilizing the deformed DNA in the nucleosome.
Many registration scenarios involve aligning more than just two images. These image sets-called ensembles-are conventionally registered by choosing one image as a template, and every other image is registered to it. This pairwise approach is problematic because results depend on which image is chosen as the template. The issue is particularly acute for multisensor ensembles because different sensors create images with different features. Also, pairwise methods use only a fraction of the available data at a time. In this paper, we propose a maximum-likelihood clustering method that registers all the images in a multisensor ensemble simultaneously. Experiments involving rigid-body and affine transformations show that the clustering method is more robust and accurate than competing pairwise registration methods. Moreover, the clustering results can be used to form a rudimentary segmentation of the image ensemble.
The accurate control of cell proliferation and survival is critical for animal development. The Hippo tumor suppressor pathway regulates both of these parameters by controlling the nuclear availability of the transcriptional coactivator Yorkie (Yki), which regulates downstream target genes together with Scalloped (Sd), a DNA-binding protein. Here we provide evidence that Yki can also regulate target genes in conjunction with Homothorax (Hth) and Teashirt (Tsh), two DNA-binding transcription factors expressed in the uncommitted progenitor cells of the Drosophila eye imaginal disc. Clonal analyses demonstrate that Hth and Tsh promote cell proliferation and protect eye progenitor cells from apoptosis. Genetic epistasis experiments suggest that Hth and Tsh execute these functions with Yki, in part by up-regulating the microRNA bantam. A physical interaction between Hth and Yki can be detected in cell culture, and we show that Hth and Yki are bound to a DNA sequence approximately 14 kb upstream of the bantam hairpin in eye imaginal disc cells, arguing that this regulation is direct. These data suggest that the Hippo pathway uses different DNA-binding transcription factors depending on the cellular context. In the eye disc, Hth and Tsh provide spatial information to this pathway, promoting cell proliferation and survival in the progenitor domain.
Hox proteins are well known for executing highly specific functions in vivo, but our understanding of the molecular mechanisms underlying gene regulation by these fascinating proteins has lagged behind. The premise of this review is that an understanding of gene regulation-by any transcription factor-requires the dissection of the cis-regulatory elements that they act upon. With this goal in mind, we review the concepts and ideas regarding gene regulation by Hox proteins and apply them to a curated list of directly regulated Hox cis-regulatory elements that have been validated in the literature. Our analysis of the Hox-binding sites within these elements suggests several emerging generalizations. We distinguish between Hox cofactors, proteins that bind DNA cooperatively with Hox proteins and thereby help with DNA-binding site selection, and Hox collaborators, proteins that bind in parallel to Hox-targeted cis-regulatory elements and dictate the sign and strength of gene regulation. Finally, we summarize insights that come from examining five X-ray crystal structures of Hox-cofactor-DNA complexes. Together, these analyses reveal an enormous amount of flexibility into how Hox proteins function to regulate gene expression, perhaps providing an explanation for why these factors have been central players in the evolution of morphological diversity in the animal kingdom.
The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanism: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analysing the three-dimensional structures of protein-DNA complexes, here we show that the binding of arginine residues to narrow minor grooves is a widely used mode for protein-DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a prominent example of this effect. Minor-groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings indicate that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA-binding specificity.
For gene products that must be present in cells at defined concentrations, expression levels must be tightly controlled to ensure robustness against environmental, genetic, and developmental noise. By studying the regulation of the concentration-sensitive Drosophila melanogaster Hox gene Ultrabithorax (Ubx), we found that Ubx enhancer activities respond to both increases in Ubx levels and genetic background. Large, transient increases in Ubx levels are capable of silencing all enhancer input into Ubx transcription, resulting in the complete silencing of this gene. Small increases in Ubx levels, brought about by duplications of the Ubx locus, cause sporadic silencing of subsets of Ubx enhancers. Ubx enhancer silencing can also be induced by outcrossing laboratory stocks to D. melanogaster strains established from wild flies from around the world. These results suggest that enhancer activities are not rigidly determined, but instead are sensitive to genetic background. Together, these findings suggest that enhancer silencing may be used to maintain gene product levels within the correct range in response to natural genetic variation.
Locomotion in adult Drosophila depends on motor neurons that target a set of multifibered muscles in the appendages. Here, we describe the development of motor neurons in adult Drosophila, focusing on those that target the legs. Leg motor neurons are born from at least 11 neuroblast lineages, but two lineages generate the majority of these cells. Using genetic single-cell labeling methods, we analyze the birth order, muscle targeting, and dendritic arbors for most of the leg motor neurons. Our results reveal that each leg motor neuron is born at a characteristic time of development, from a specific lineage, and has a stereotyped dendritic architecture. Motor axons that target a particular leg segment or muscle have similar dendritic arbors but can derive from different lineages. Thus, although Drosophila uses a lineage-based method to generate leg motor neurons, individual lineages are not dedicated to generate neurons that target a single leg segment or muscle type.
Limb development requires the elaboration of a proximodistal (PD) axis, which forms orthogonally to previously defined dorsoventral (DV) and anteroposterior (AP) axes. In arthropods, the PD axis of the adult leg is subdivided into two broad domains, a proximal coxopodite and a distal telopodite. We show that the progressive subdivision of the PD axis into these two domains occurs during embryogenesis and is reflected in the cis-regulatory architecture of the Distalless (Dll) gene. Early Dll expression, governed by the Dll304 enhancer, is in cells that can give rise to both domains of the leg as well as to the entire dorsal (wing) appendage. A few hours after Dll304 is activated, the activity of this enhancer fades, and two later-acting enhancers assume control over Dll expression. The LT enhancer is expressed in cells that will give rise to the entire telopodite, and only the telopodite. By contrast, cells that activate the DKO enhancer will give rise to a leg-associated larval sensory structure known as the Keilins organ (KO). Cells that activate neither LT nor DKO, but had activated Dll304, will give rise to the coxopodite. In addition, we describe the trans-acting signals controlling the LT and DKO enhancers, and show, surprisingly, that the coxopodite progenitors begin to proliferate approximately 24 hours earlier than the telopodite progenitors. Together, these findings provide a complete and high-resolution fate map of the Drosophila appendage primordia, linking the primary domains to specific cis-regulatory elements in Dll.
The discovery of the sulfonamide herbicides, which inhibit the enzyme acetolactate synthase (ALS), has resulted in many investigations to exploit their herbicidal activity. One area which proved particularly productive was the N-aryltriazolo[1,5-c]pyrimidine sulfonamides, providing three commercial herbicides, cloransulam-methyl, diclosulam and florasulam. Additional structure-activity investigations by reversing the sulfonamide linkage resulted in the discovery of triazolopyrimidine sulfonamides with cereal crop selectivity and high levels of grass and broadleaf weed control. Research efforts to exploit these high levels of weed activity ultimately led to the discovery of penoxsulam, a new herbicide developed for grass, sedge and broadleaf weed control in rice. Synthetic efforts and structure-activity relationships leading to the discovery of penoxsulam will be discussed.
Collective animal behaviour is the study of how interactions between individuals produce group level patterns, and why these interactions have evolved. This study has proved itself uniquely interdisciplinary, involving physicists, mathematicians, engineers as well as biologists. Almost all experimental work in this area is related directly or indirectly to mathematical models, with regular movement back and forth between models, experimental data and statistical fitting. In this paper, we describe how the modelling cycle works in the study of collective animal behaviour. We classify studies as addressing questions at different levels or linking different levels, i.e. as local, local to global, global to local or global. We also describe three distinct approaches-theory-driven, data-driven and model selection-to these questions. We show, with reference to our own research on species across different taxa, how we move between these different levels of description and how these various approaches can be applied to link levels together.
Over 6,000 fragments from the genome of Drosophila melanogaster were analyzed for their ability to drive expression of GAL4 reporter genes in the third-instar larval imaginal discs. About 1,200 reporter genes drove expression in the eye, antenna, leg, wing, haltere, or genital imaginal discs. The patterns ranged from large regions to individual cells. About 75% of the active fragments drove expression in multiple discs; 20% were expressed in ventral, but not dorsal, discs (legs, genital, and antenna), whereas ?23% were expressed in dorsal but not ventral discs (wing, haltere, and eye). Several patterns, for example, within the leg chordotonal organ, appeared a surprisingly large number of times. Unbiased searches for DNA sequence motifs suggest candidate transcription factors that may regulate enhancers with shared activities. Together, these expression patterns provide a valuable resource to the community and offer a broad overview of how transcriptional regulatory information is distributed in the Drosophila genome.
Regulation of gene expression in eukaryotes is an extremely complex process. In this review, we break down several critical steps, emphasizing new data and techniques that have expanded current gene regulatory models. We begin at the level of DNA sequence where cis-regulatory modules (CRMs) provide important regulatory information in the form of transcription factor (TF) binding sites. In this respect, CRMs function as instructional platforms for the assembly of gene regulatory complexes. We discuss multiple mechanisms controlling complex assembly, including cooperative DNA binding, combinatorial codes, and CRM architecture. The second section of this review places CRM assembly in the context of nucleosomes and condensed chromatin. We discuss how DNA accessibility and histone modifications contribute to TF function. Lastly, new advances in chromosomal mapping techniques have provided increased understanding of intra- and interchromosomal interactions. We discuss how these topological maps influence gene regulatory models.
Mitochondrial structure and function are highly dynamic, but the potential roles for cell signaling pathways in influencing these properties are not fully understood. Reduced mitochondrial function has been shown to cause cell cycle arrest, and a direct role of signaling pathways in controlling mitochondrial function during development and disease is an active area of investigation. Here, we show that the conserved Yorkie/YAP signaling pathway implicated in the control of organ size also functions in the regulation of mitochondria in Drosophila as well as human cells. In Drosophila, activation of Yorkie causes direct transcriptional up-regulation of genes that regulate mitochondrial fusion, such as opa1-like (opa1) and mitochondria assembly regulatory factor (Marf), and results in fused mitochondria with dramatic reduction in reactive oxygen species (ROS) levels. When mitochondrial fusion is genetically attenuated, the Yorkie-induced cell proliferation and tissue overgrowth are significantly suppressed. The function of Yorkie is conserved across evolution, as activation of YAP2 in human cell lines causes increased mitochondrial fusion. Thus, mitochondrial fusion is an essential and direct target of the Yorkie/YAP pathway in the regulation of organ size control during development and could play a similar role in the genesis of cancer.
The evolutionary divergence of sexual signals is often important during the formation of new animal species, but our understanding of the origin of signal diversity is limited [1, 2]. Sensory drive, the optimization of communication signal efficiency through matching to the local environment, has been highlighted as a potential promoter of diversification and speciation . The swordtail characin (Corynopoma riisei) is a tropical fish in which males display a flag-like ornament that elicits female foraging behavior during courtship. We show that the shape of the male ornament covaries with female diet across natural populations. More specifically, natural populations in which the female diet is more dominated by ants exhibit male ornaments more similar to the shape of an ant. Feeding experiments confirm that females habituated to a diet of ants prefer to bite at male ornaments from populations with a diet more dominated by ants. Our results show that the male ornament functions as a "fishing lure" that is diversifying in shape to match local variation in female search images employed during foraging. This direct link between variation in female feeding ecology and the evolutionary diversification of male sexual ornaments suggests that sensory drive may be a common engine of signal divergence.
Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1 (Fujioka and Jaynes, 2012). Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. We localized cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function.
We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunoprecipitate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. We show that the local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5 regulatory region is in close proximity to the Dll promoter. In Dll-nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type-specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and nonexpressing cell types, respectively.
Animal appendages require a proximodistal (PD) axis, which forms orthogonally from the two main body axes, anteroposterior and dorsoventral. In this review, we discuss recent advances that begin to provide insights into the molecular mechanisms controlling PD axis formation in the Drosophila leg. In this case, two morphogens, Wingless (Wg) and Decapentaplegic (Dpp), initiate a genetic cascade that, together with growth of the leg imaginal disc, establishes the PD axis. The analysis of cis-regulatory modules (CRMs) that control the expression of genes at different positions along the PD axis has been particularly valuable in dissecting this complex process. From these experiments, it appears that only one concentration of Wg and Dpp are required to initiate PD axis formation by inducing the expression of Distal-less (Dll), a homeodomain-encoding gene that is required for leg development. Once Dll is turned on, it activates the medially expressed gene dachshund (dac). Cross-regulation between Dll and dac, together with cell proliferation in the growing leg imaginal disc, results in the formation of a rudimentary PD axis. Wg and Dpp also initiate the expression of ligands for the EGFR pathway, which in turn induces the expression of a series of target genes that pattern the distal-most portion of the leg.
Inference of interaction rules of animals moving in groups usually relies on an analysis of large scale system behaviour. Models are tuned through repeated simulation until they match the observed behaviour. More recent work has used the fine scale motions of animals to validate and fit the rules of interaction of animals in groups. Here, we use a Bayesian methodology to compare a variety of models to the collective motion of glass prawns (Paratya australiensis). We show that these exhibit a stereotypical phase transition, whereby an increase in density leads to the onset of collective motion in one direction. We fit models to this data, which range from: a mean-field model where all prawns interact globally; to a spatial Markovian model where prawns are self-propelled particles influenced only by the current positions and directions of their neighbours; up to non-Markovian models where prawns have memory of previous interactions, integrating their experiences over time when deciding to change behaviour. We show that the mean-field model fits the large scale behaviour of the system, but does not capture fine scale rules of interaction, which are primarily mediated by physical contact. Conversely, the Markovian self-propelled particle model captures the fine scale rules of interaction but fails to reproduce global dynamics. The most sophisticated model, the non-Markovian model, provides a good match to the data at both the fine scale and in terms of reproducing global dynamics. We conclude that prawns movements are influenced by not just the current direction of nearby conspecifics, but also those encountered in the recent past. Given the simplicity of prawns as a study system our research suggests that self-propelled particle models of collective motion should, if they are to be realistic at multiple biological scales, include memory of previous interactions and other non-Markovian effects.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.