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The nematode Caenorhabditis elegans is a simple model host for studying the interaction between bacterial pathogens and the metazoan innate immune system. In the last two decades, much focus has been given to studying intestinal infection in the worm by the clinical strain Pseudomonas aeruginosa PA14. Powerful genetic and molecular tools in both species facilitate the identification and analysis of bacterial virulence factors as well as host defense factors.However, findings from these studies are confounded by the use of the genetically, metabolically, and physiologically divergent E. coli OP50 as a food source and as the non-virulent control. Here we report the use of P. aeruginosa z11 strain as a preferable control for PA14 infection studies. We demonstrate that many aspects of worm behavior, health-span, longevity, food attraction, brood size, lifespan, and pathogen avoidance and survival are not affected in z11. We show that the use of z11 as a control for transcriptomics analysis can increase the discovery power. In particular, we identified three novel pathogenesis-related genes in C. elegans. Our findings accentuate the importance of choosing an appropriate control environment for host-pathogen studies.

In the last decade, microfluidic methods have proven to be powerful tools for Caenorhabditis elegans research, offering advanced manipulation of worms and precise control of experimental conditions. The advantages of microfluidic chips include their capability of immobilization, automated sorting, and longitudinal measurement, and more. In this review, we focus on control components that are widely used in the design of microfluidic devices, and discuss their functions and working principles that enable advanced manipulation on a chip. Understanding these components will ease the onboarding of researchers inexperienced with microfluidics and help them bring the power of microfluidics to new applications.

Recently, applications of mathematical and computational models to biological processes have helped investigators to systematically interpret data, test hypotheses built on experimental data, generate new hypotheses, and guide the design of new experiments, protocols, and synthetic biological systems. Availability of diverse quantitative data is a prerequisite for successful mathematical modeling. The ability to acquire high-quality quantitative data for a broad range of biological processes and perform precise perturbation makes C. elegans an ideal model system for such studies. In this primer, we examine the general procedure of modeling biological systems and demonstrate this process using the heat-shock response in C. elegans as a case study. Our goal is to facilitate the initial discussion between worm biologists and their potential collaborators from quantitative disciplines.

In the last decade, microfluidic methods have proven to be powerful tools for Caenorhabditis elegans research, offering advanced manipulation of worms and precise control of experimental conditions. The advantages of microfluidic chips include their capability of immobilization, automated sorting, and longitudinal measurement, and more. In this review, we focus on control components that are widely used in the design of microfluidic devices, and discuss their functions and working principles that enable advanced manipulation on a chip. Understanding these components will ease the onboarding of researchers inexperienced with microfluidics and help them bring the power of microfluidics to new applications.

Significance Unlike their eukaryotic counterparts, bacterial cells are composed of a single compartment. This allows many rapidly diffusing macromolecules, such as proteins and mRNAs, to be evenly distributed in the cell. Important exceptions are proteins embedded in the cell membrane, which transport material and information across the membrane. Often these proteins attach to the membrane before their translation is complete, anchoring their mRNAs to the vicinity of the membrane. This coupling between translation and localization suggests that the dynamics of translation may shape the spatial organization. In this paper, we use a canonical model of nonequilibrium statistical physics to characterize this connection and show how tunable kinetic properties allow the cell to regulate the spatial organization of both mRNAs and proteins., Unlike most macromolecules that are homogeneously distributed in the bacterial cell, mRNAs that encode inner-membrane proteins can be concentrated near the inner membrane. Cotranslational insertion of the nascent peptide into the membrane brings the translating ribosome and the mRNA close to the membrane. This suggests that kinetic properties of translation can determine the spatial organization of these mRNAs and proteins, which can be modulated through posttranscriptional regulation. Here we use a simple stochastic model of translation to characterize the effect of mRNA properties on the dynamics and statistics of its spatial distribution. We show that a combination of the rate of translation initiation, the availability of secretory apparatuses, and the composition of the coding region determines the abundance of mRNAs near the membrane, as well as their residence time. We propose that the spatiotemporal dynamics of mRNAs can give rise to protein clusters on the membrane and determine their size distribution.

Significance Animals have evolved mechanisms for gathering information from the environment to make apt decisions. An important example is the regulation of feeding, where animals weigh nutritional value with the associated energetic costs. Many species feed in short bursts interspersed with pauses, yet the function and advantages of this strategy are unknown. We suggest that these bursts are used both to collect food and to gather information. Using quantifiable feeding behavior from Caenorhabditis elegans , we define a model that formalizes this decision process. The model shows that burst feeding is advantageous when information about the expected food is noisy. This conceptualization of feeding behavior suggests optimal feeding strategies in response to conflicting inputs and to fluctuating environments.

The heat shock response is the organized molecular response to stressors which disrupt proteostasis, potentially leading to protein misfolding and aggregation. While the regulation of the heat shock response is well-studied in single cells, its coordination at the cell, tissue, and systemic levels of a multicellular organism is poorly understood. To probe the interplay between systemic and cell-autonomous responses, we studied the upregulation of HSP-16.2, a molecular chaperone induced throughout the intestine ofCaenorhabditis elegansfollowing a heat shock, by taking longitudinal measurements in a microfluidic environment. Based on the dynamics of HSP-16.2 accumulation, we showed that a combination of heat shock temperature and duration define the intensity of stress inflicted on the worm and identified two regimes of low and high intensity stress. Modeling the underlying regulatory dynamics implicated the saturation of heat shock protein mRNA production in defining these two regimes and emphasized the importance of time separation between transcription and translation in establishing these dynamics. By applying a heat shock and measuring the response in separate parts of the animals, we implicated thermosensory neurons in accelerating the response and transducing information within the animal. We discuss possible implications of the systemic and cell level aspects and how they coordinate to facilitate the organismal response.

H3K9me3 (histone H3 modified with tri-methylation at lysine 9) is a hallmark of transcriptional silencing and heterochromatin. However, its global effects on the genome, including euchromatin, are less well understood. Here we develop Formaldehyde-Assisted Identification of Regulatory Elements (FAIRE) forC. elegansto examine the chromatin configuration of mutants that lack virtually all H3K9me3, while leaving H3K9me1 and H3K9me2 intact. We find that nucleosomes are mildly disrupted, and levels of H3K9me2 and H3K27me3 rise in mutant embryos. In addition to these expected changes, the most dramatic change occurs in euchromatin: many regions encompassing transcription start sites (TSSs) gain an average of two nucleosomes in mutants. The affected regions normally lack H3K9me3, revealing a locus non-autonomous role for H3K9me3. Affected TSSs are associated with genes that are active in epithelia and muscles, and implicated in development, locomotion, morphogenesis and transcription. Mutant embryos develop normally under ideal laboratory conditions but die when challenged, with defects in morphogenesis and development. Our findings reveal that H3K9me3 protects transcription start sites within euchromatin from nucleosome deposition. These results may be relevant to mammals, where diseases that disrupt the nuclear lamina and heterochromatin can alter epithelial and muscle gene expression.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

Multifunctional pioneers Proteins surround cellular DNA to silence gene expression. Early in development, proteins such as pioneer transcription factors facilitate the opening of this silenced chromatin structure. Hsu et al. describe an additional role for the PHA-4 pioneer transcription factor in nematode worms. PHA-4 recruited RNA polymerase II to target promoters before transcriptional onset, and this activity preceded its chromatin-opening duty. The multifunctional role identified for PHA-4 may be shared by other pioneer factors. Science , this issue p. 1372

Exposure to high temperatures has an adverse effect on cellular processes and results in activation of the cellular heat shock response (HSR), a highly conserved program of inducible genes to maintain protein homeostasis1. The insulin/IGF-1 signaling (IIS) pathway, which has diverse roles from metabolism to stress response and longevity, is activated as part of the HSR2–4. Recent evidence suggest that the IIS pathway is able to affect proteostasis non-autonomously5,6, yet it is not known if it is activated autonomously in stressed cells or systemically as part of an organismic program. In Caenorhabditis elegans, the single forkhead box O (FOXO) homologue DAF-16 functions as the major target of the IIS pathway7 and, together with the heat-shock factor HSF-1, induce the expression of small heat shock proteins in response to heat shock8–10,3. Here we use a novel microfluidic device that allows precise control of the spatiotemporal temperature profile to show that cellular activation of DAF-16 integrates local temperature sensation with systemic signals. We demonstrate that DAF-16 activation in head sensory neurons is essential for DAF-16 activation in other tissues, but show that no known thermosensory neuron is individually required. Our findings demonstrate that systemic and cell-autonomous aspects of stress response act together to facilitate a coordinated cellular response at the organismic level.

Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

Animals integrate physiological and environmental signals to modulate their food uptake. The nematode C. elegans, whose food uptake consists of pumping bacteria from the environment into the gut, provides excellent opportunities for discovering principles of conserved regulatory mechanisms. Here we show that worms implement a graded feeding response to the concentration of environmental bacteria by modulating a commitment to bursts of fast pumping. Using long-term, high-resolution, longitudinal recordings of feeding dynamics under defined conditions, we find that the frequency and duration of pumping bursts increase and the duration of long pauses diminishes in environments richer in bacteria. The bioamine serotonin is required for food-dependent induction of bursts as well as for maintaining their high rate of pumping through two distinct mechanisms. We identify the differential roles of distinct families of serotonin receptors in this process and propose that regulation of bursts is a conserved mechanism of behaviour and motor control., Regulating food intake is an important physiological mechanism. Here, the authors use a custom microfluidic device to investigate feeding dynamics in C. elegans, and identify roles of serotonergic neurons in regulating bursts of feeding in response to food availability.

Small regulatory RNAs (sRNAs) in bacteria regulate many important cellular activities under normal conditions and in response to stress. Many sRNAs bind to the mRNA targets at or near the 5' untranslated region (UTR) resulting in translation inhibition and accelerated degradation. Often the sRNA-binding site is adjacent to or overlapping with the ribosomal binding site (RBS), suggesting a possible interplay between sRNA and ribosome binding. Here we combine quantitative experiments with mathematical modeling to reveal novel features of the interaction between small RNAs and the translation machinery at the 5'UTR of a target mRNA. By measuring the response of a library of reporter targets with varied RBSs, we find that increasing translation rate can lead to increased repression. Quantitative analysis of these data suggests a recruitment model, where bound ribosomes facilitate binding of the sRNA. We experimentally verified predictions of this model for the cell-to-cell variability of target expression. Our findings offer a framework for understanding sRNA silencing in the context of bacterial physiology.

Two decades into the genomics era the question of mapping sequence to function has evolved from identifying functional elements to characterizing their quantitative properties including, in particular, their specificity and efficiency. Here, we use a large-scale approach to establish a quantitative map between the sequence of a bacterial regulatory RNA and its efficiency in modulating the expression of its targets. Our approach generalizes the sort-seq method, introduced recently to analyze promoter sequences, in order to accurately quantify the efficiency of a large library of sequence variants. We focus on two small RNAs (sRNAs) in E. coli, DsrA and RyhB, and their regulation of both repressed and activated targets. In addition to precisely identifying functional elements in the sRNAs, our data establish quantitative relationships between structural and energetic features of the sRNAs and their regulatory activity, and characterize a large set of direct and indirect interactions between nucleotides. A core of these interactions supports a model where specificity can be enhanced by a rigid molecular structure. Both sRNAs exhibit a modular design with limited cross-interactions, dividing the requirements for structural stability and target binding among modules.

Animal response to changes in environmental cues is a complex dynamical process that occurs at diverse molecular and cellular levels. To gain a quantitative understanding of such processes, it is desirable to observe many individuals, subjected to repeatable and well defined environmental cues over long time periods. Here we present WormSpa, a microfluidic system where worms are individually confined in optimized chambers. We show that worms in WormSpa are neither stressed nor starved, and in particular exhibit pumping and egg-laying behaviors equivalent to those of freely behaving worms. We demonstrate the applicability of WormSpa for studying stress response and physiological processes. WormSpa is simple to make and easy to operate, and its design is modular, making it straightforward to incorporate available microfluidic technologies. We expect that WormSpa would open novel avenues of research, hitherto impossible or impractical.

Small animals such as the roundworm C. elegans are excellent models for studying bacterial infection and host response, as well as for genetic and chemical screens. A key methodology is the killing assay, in which the number of surviving animals is tracked as a function of the time post infection. This is a labor-intensive procedure, prone to human error and subjective choices, and often involves undesired perturbation to the animals and their environment. In addition, the survival of animals is just one aspect of a multi-dimensional complex biological process. Here we report a microfluidic-based approach for performing killing assays in worms, compatible with standard assays performed on solid media. In addition to providing accurate and reproducible survival curves at a considerably reduced labor, this approach allows acquisition of a multitude of quantitative data with minimal undesired perturbations. These measurements are obtained automatically at a worm-by-worm resolution using a custom image processing workflow. The proposed approach is simple, scalable, and extendable, and is significantly more economical than standard manual protocols.

Background The nematode Caenorhabditis elegans is widely used for studying small neural circuits underlying behavior. In particular, the rhythmic feeding motions collectively termed pharyngeal pumping are regulated by a nearly autonomous network of 20 neurons of 14 types. Despite much progress achieved through laser ablation, genetics, electrophysiology, and optogenetics, key questions regarding the regulation of pumping remain open. New method We describe the implementation and application of a scalable automated method for measuring pumping in controlled environments. Our implementation is affordable and flexible: key hardware and software elements can be modified to accommodate different requirements. Results We demonstrate prolonged measurements under controlled conditions and the resulting high quality data. We show the scalability of our method, enabling high throughput, and its suitability for maintaining static and dynamic conditions. When food availability was oscillated, pumping rates were low as compared to steady conditions and pumping activity was not reliably modulated in response to changes in food concentration. Comparison with existing method The prevailing method for measuring rates of pumping relies on scoring by visual inspection of short recordings. Our automated method compares well with manual scoring. It enables detailed statistical characterization under experimental conditions not previously accessible and minimizes unintentional bias. Conclusions Our approach adds a powerful tool for studying pharyngeal pumping. It enhances the experimental versatility of assaying genetic and pharmacological manipulations and the ability to characterize the resulting behavior. Both the experimental setup and the analysis can be readily adapted to additional challenging motion detection problems.

Animals integrate physiological and environmental signals to modulate their food uptake. Failure to regulate feeding may have devastating results, including obesity and diabetes, underscoring the importance of understanding its underlying mechanisms. The nematodeC. elegans, whose food uptake consists of pumping bacteria from the environment into the gut, provides excellent opportunities for discovering principles of conserved regulatory mechanisms. Here we show that worms implement a graded feeding response to the concentration of environmental bacteria by modulating a commitment to bursts of fast pumping. Using long-term, high-resolution, longitudinal recordings of feeding dynamics under defined conditions, we find that the frequency and duration of pumping bursts increase and the duration of long pauses diminishes in environments richer in bacteria. The bioamine serotonin, a known feeding regulator in metazoa, is required for food-dependent induction of bursts as well as for maintaining their high rate of pumping through two distinct mechanisms. Following this phenotype quantitatively we identify the essential serotonergic neurons and the differential roles of distinct families of serotonin receptors. We propose that regulation of bursts is a conserved mechanism of behavior and motor control.

Background Fast responses can provide a competitive advantage when resources are inhomogeneously distributed. The nematode Caenorhabditis elegans was shown to modulate locomotion on a lawn of bacterial food in serotonin (5-HT)-dependent manners. However, potential roles for serotonergic signaling in responding to food discovery are poorly understood. Results We found that 5-HT signaling in C. elegans facilitates efficient exploitation in complex environments by mediating a rapid response upon encountering food. Genetic or cellular manipulations leading to deficient serotonergic signaling resulted in gradual responses and defective exploitation of a patchy foraging landscape. Physiological imaging revealed that the NSM serotonergic neurons responded acutely upon encounter with newly discovered food and were key to rapid responses. In contrast, the onset of responses of ADF serotonergic neurons preceded the physical encounter with the food. The serotonin-gated chloride channel MOD-1 and the ortholog of mammalian 5-HT1 metabotropic serotonin receptors SER-4 acted in synergy to accelerate decision-making. The relevance of responding rapidly was demonstrated in patchy environments, where the absence of 5-HT signaling was detrimental to exploitation. Conclusions Our results implicate 5-HT in a novel form of decision-making, demonstrate its fitness consequences, suggest that NSM and ADF act in concert to modulate locomotion in complex environments, and identify the synergistic action of a channel and a metabotropic receptor in accelerating C. elegans decision-making. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0232-y) contains supplementary material, which is available to authorized users.

Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. Here we demonstrate that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals.

During the last decade small regulatory RNA (srRNA) emerged as central players in the regulation of gene expression in all kingdoms of life. Multiple pathways for srRNA biogenesis and diverse mechanisms of gene regulation may indicate that srRNA regulation evolved independently multiple times. However, small RNA pathways share numerous properties, including the ability of a single srRNA to regulate multiple targets. Some of the mechanisms of gene regulation by srRNAs have significant effect on the abundance of free srRNAs that are ready to interact with new targets. This results in indirect interactions among seemingly unrelated genes, as well as in a crosstalk between different srRNA pathways. Here we briefly review and compare the major srRNA pathways, and argue that the impact of srRNA is always at the system level. We demonstrate how a simple mathematical model can ease the discussion of governing principles. To demonstrate these points we review a few examples from bacteria and animals.

Caenorhabditis elegans is particularly suitable for obtaining quantitative data about behavior, neuronal activity, gene expression, ecological interactions, quantitative traits, and much more. To exploit the full potential of these data one seeks to interpret them within quantitative models. Using two examples from the C. elegans literature we briefly explore several types of modeling approaches relevant to worm biology, and show how they might be used to interpret data, formulate testable hypotheses, and suggest new experiments. We emphasize that the choice of modeling approach is strongly dependent on the questions of interest and the type of available knowledge.

Caenorhabditis elegans is particularly suitable for obtaining quantitative data about behavior, neuronal activity, gene expression, ecological interactions, quantitative traits, and much more. To exploit the full potential of these data one seeks to interpret them within quantitative models. Using two examples from the C. elegans literature we briefly explore several types of modeling approaches relevant to worm biology, and show how they might be used to interpret data, formulate testable hypotheses, and suggest new experiments. We emphasize that the choice of modeling approach is strongly dependent on the questions of interest and the type of available knowledge.

Biological homeostasis invokes modulatory responses aimed at stabilizing internal conditions. Using tunable photo- and mechano-stimulation, we identified two distinct categories of homeostatic responses during the sleep-like state of Caenorhabditis elegans (lethargus). In the presence of weak or no stimuli, extended motion caused a subsequent extension of quiescence. The neuropeptide Y receptor homolog, NPR-1, and an inhibitory neuropeptide known to activate it, FLP-18, were required for this process. In the presence of strong stimuli, the correlations between motion and quiescence were disrupted for several minutes but homeostasis manifested as an overall elevation of the time spent in quiescence. This response to strong stimuli required the function of the DAF-16/FOXO transcription factor in neurons, but not that of NPR-1. Conversely, response to weak stimuli did not require the function of DAF-16/FOXO. These findings suggest that routine homeostatic stabilization of sleep may be distinct from homeostatic compensation following a strong disturbance. DOI: http://dx.doi.org/10.7554/eLife.04380.001, eLife digest The regenerative properties of sleep are required by all animals, with even the simplest animal, the nematode Caenorhabditis elegans, displaying a sleep-like state called lethargus. During development, nematodes must pass through four larval stages en route to adulthood, and the end of each stage is preceded by a period of lethargus lasting 2 to 3 hr. Human sleep is divided into distinct stages that recur in a prescribed order throughout the night. Nematodes, on the other hand, simply experience alternating periods of activity and stillness as they sleep. Nevertheless, in both species, any disruptions to sleep automatically lead to adjustments of the rest of the sleep cycle to compensate for the disturbance and to ensure that the organism gets an adequate amount of sleep overall. To date, it has been assumed that a single mechanism is responsible for adjusting the sleep cycle after any disturbance, regardless of its severity. However, Nagy, Tramm, Sanders et al. now show that this is not the case in C. elegans. Sleeping nematodes that were lightly disturbed by exposing them to light or to vibrations—causing them to briefly increase their activity levels—compensated for the disturbance by lengthening their next inactive period. By contrast, worms that were vigorously agitated by stronger vibrations showed a different response: the alternating pattern of stillness and activity was disrupted for several minutes, followed by an overall increase in the length of time spent in the stillness phase. Experiments using genetically modified worms revealed that these two responses involve distinct molecular pathways. A signaling molecule called neuropeptide Y affects the response to minor sleep disruptions, whereas a transcription factor called DAF-16/FOXO is involved in the corresponding role after major disruptions. Given that neuropeptide Y has already been implicated in sleep regulation in humans and flies, it is not implausible that similar mechanisms may occur in response to disturbances of our own sleep. DOI: http://dx.doi.org/10.7554/eLife.04380.002

Post-transcriptional regulation by miRNAs is a widespread and highly conserved phenomenon in metazoans, with several hundreds to thousands of conserved binding sites for each miRNA, and up to two thirds of all genes under miRNA regulation. At the same time, the effect of miRNA regulation on mRNA and protein levels is usually quite modest and associated phenotypes are often weak or subtle. This has given rise to the notion that the highly interconnected miRNA regulatory network exerts its function less through any individual link and more via collective effects that lead to a functional interdependence of network links. We present a Bayesian framework to quantify conservation of miRNA target sites using vertebrate whole-genome alignments. The increased statistical power of our phylogenetic model allows detection of evolutionary correlation in the conservation patterns of site pairs. Such correlations could result from collective functions in the regulatory network. For instance, co-conservation of target site pairs supports a selective benefit of combinatorial regulation by multiple miRNAs. We find that some miRNA families are under pronounced co-targeting constraints, indicating a high connectivity in the regulatory network, while others appear to function in a more isolated way. By analyzing coordinated targeting of different curated gene sets, we observe distinct evolutionary signatures for protein complexes and signaling pathways that could reflect differences in control strategies. Our method is easily scalable to analyze upcoming larger data sets, and readily adaptable to detect high-level selective constraints between other genomic loci. We thus provide a proof-of-principle method to understand regulatory networks from an evolutionary perspective., Author Summary Sequence conservation patterns can be used to assess the functional importance of a genomic locus, e.g. a binding site of a regulatory factor. If one locus is functionally coupled with another, they do not evolve independently and their conservation patterns are correlated. We used evolutionary correlations to study the vertebrate miRNA regulatory network, which is densely connected and shows rapid evolutionary rewiring. Using a Bayesian framework, we identify topological features of this network that are under weak but significant selection, such as combinatorial regulation of one gene by multiple miRNAs, or the coordinated targeting of protein complexes and signaling pathways. Our method is easily scalable to include more genomes as they are being sequenced and can be used to study correlations between other kinds of loci.

Small regulatory RNAs are central players in the regulation of many cellular processes across all kingdoms of life. Experiments in mouse and human have shown that a typical small RNA may regulate the expression of many different genes, suggesting that small RNAs act as global regulators. It is noted though that most targets respond only weakly to the presence of the small RNA. At the same time, evidence in bacteria and animals suggest that the phenotypes associated with small RNA mutants are only due to a few of their targets. Here we assume that targets regulated by a small RNA to control function is in fact small, and propose that the role of the many other weak targets is to confer robustness to the regulation of these few principal targets. Through mathematical modeling we show that auxiliary targets may significantly buffer both number and kinetic fluctuations of the principal targets, with only minor slowdown in the kinetics of response. Analysis of genomic data suggests that auxiliary targets experience a nonspecific evolutionary pressure, playing a role at the system level. Our work is of importance for studies on small RNA functions, and impacts on the understanding of small RNA evolution.

Correlations between two variables of a high-dimensional system can be indicative of an underlying interaction, but can also result from indirect effects. Inverse Ising inference is a method to distinguish one from the other. Essentially, the parameters of the least constrained statistical model are learned from the observed correlations such that direct interactions can be separated from indirect correlations. Among many other applications, this approach has been helpful for protein structure prediction, because residues which interact in the 3D structure often show correlated substitutions in a multiple sequence alignment. In this context, samples used for inference are not independent but share an evolutionary history on a phylogenetic tree. Here, we discuss the effects of correlations between samples on global inference. Such correlations could arise due to phylogeny but also via other slow dynamical processes. We present a simple analytical model to address the resulting inference biases, and develop an exact method accounting for background correlations in alignment data by combining phylogenetic modeling with an adaptive cluster expansion algorithm. We find that popular reweighting schemes are only marginally effective at removing phylogenetic bias, suggest a rescaling strategy that yields better results, and provide evidence that our conclusions carry over to the frequently used mean-field approach to the inverse Ising problem., Comment: 18 pages, 6 figures; accepted at New J Phys

We present a coupled two-way clustering approach to gene microarray data analysis. The main idea is to identify subsets of the genes and samples, such that when one of these is used to cluster the other, stable and significant partitions emerge. The search for such subsets is a computationally complex task. We present an algorithm, based on iterative clustering, that performs such a search. This analysis is especially suitable for gene microarray data, where the contributions of a variety of biological mechanisms to the gene expression levels are entangled in a large body of experimental data. The method was applied to two gene microarray data sets, on colon cancer and leukemia. By identifying relevant subsets of the data and focusing on them we were able to discover partitions and correlations that were masked and hidden when the full dataset was used in the analysis. Some of these partitions have clear biological interpretation; others can serve to identify possible directions for future research.

We introduce a method for validation of results obtained by clustering analysis of data. The method is based on resampling the available data. A figure of merit that measures the stability of clustering solutions against resampling is introduced. Clusters that are stable against resampling give rise to local maxima of this figure of merit. This is presented first for a one-dimensional data set, for which an analytic approximation for the figure of merit is derived and compared with numerical measurements. Next, the applicability of the method is demonstrated for higher-dimensional data, including gene microarray expression data.

High-density DNA arrays, used to monitor gene expression at a genomic scale, have produced vast amounts of information which require the development of efficient computational methods to analyze them. The important first step is to extract the fundamental patterns of gene expression inherent in the data. This paper describes the application of a novel clustering algorithm, Super-Paramagnetic Clustering (SPC) to analysis of gene expression profiles that were generated recently during a study of the yeast cell cycle. SPC was used to organize genes into biologically relevant clusters that are suggestive for their co-regulation. Some of the advantages of SPC are its robustness against noise and initialization, a clear signature of cluster formation and splitting, and an unsupervised self-organized determination of the number of clusters at each resolution. Our analysis revealed interesting correlated behavior of several groups of genes which has not been previously identified.

Background Sort-seq is an effective approach for simultaneous activity measurements in a large-scale library, combining flow cytometry, deep sequencing, and statistical inference. Such assays enable the characterization of functional landscapes at unprecedented scale for a wide-reaching array of biological molecules and functionalities in vivo. Applications of sort-seq range from footprinting to establishing quantitative models of biological systems and rational design of synthetic genetic elements. Nearly as diverse are implementations of this technique, reflecting key design choices with extensive impact on the scope and accuracy the results. Yet how to make these choices remains unclear. Here we investigate the effects of alternative sort-seq designs and inference methods on the information output using mathematical formulation and simulations. Results We identify key intrinsic properties of any system of interest with practical implications for sort-seq assays, depending on the experimental goals. The fluorescence range and cell-to-cell variability specify the number of sorted populations needed for quantitative measurements that are precise and unbiased. These factors also indicate cases where an enrichment-based approach that uses a single sorted population can offer satisfactory results. These predications of our model are corroborated using re-analysis of published data. We explore implications of these results for quantitative modeling and library design. Conclusions Sort-seq assays can be streamlined by reducing the number of sorted populations, saving considerable resources. Simple preliminary experiments can guide optimal experiment design, minimizing cost while maintaining the maximal information output and avoiding latent biases. These insights can facilitate future applications of this highly adaptable technique. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2533-5) contains supplementary material, which is available to authorized users.