Complex Regulatory Circuits Research Articles

Deletion of luxI increases luminescence of Vibrio fischeri.

The regulation of bioluminescence by Vibrio fischeri is a textbook example of bacterial quorum-dependent pheromone signaling. The canonical regulatory model is that an autoinducer pheromone produced by LuxI accumulates as cells achieve a high density, and this LuxI-generated signal stimulates LuxR to activate transcription of the lux operon that underlies bioluminescence. The surprising observation that LuxI is dispensable for inducing bioluminescence forces a re-evaluation of the role of luxI. More broadly, the results underscore the potential deceptiveness of complex regulatory circuits, particularly those in which bacteria produce multiple related signaling molecules.

Abstract 5019: Comprehensive pan-cancer analysis of cfDNA methylation marks in tumors reveals complex epigenetic regulatory circuits and diagnostic biomarkers

Abstract Early detection of tumor activity in clinically asymptomatic patients has the potential to improve quality of life and prevent unnecessary exposure to toxic therapies. DNA methylation is one of the most studied, stable, and fundamental epigenetic marks and represents a prominent alteration in most cancers. Analysis of DNA methylation, particularly in liquid biopsies has been introduced in cancer diagnosis and risk stratification. Despite indisputable achievements, translation into clinical practice is lagging behind, primarily due to 1) challenges associated with the analysis of DNA methylation, 2) the fragmented nature of cell-free DNA (cfDNA), aggravated by bisulfite treatment, 3) lack of clinical validation of reported cfDNA methylation marks, and 4) the paucity of knowledge about the functional consequences of cfDNA methylation marks within tumors. Using a sample pooling strategy, complemented with robust whole genome and reduced representation methylation sequencing, we establish a pan-cancer cfDNA methylation resource encompassing 12 cancer entities with diverse clinical phenotypes and treatment modalities. We built this compendium using a robustly performing whole genome and reduced representaion enzymatic methylation sequencing of more than 150 cfDNA and tumor DNA. We complement our data by analyzing DNA methylation data from 15 cancer entities (more than 7,000 patients) within the TCGA. We developed an enzymatic digital PCR (dPCR) approach for pan-cancer but also entity-specific analysis of cfDNA methylation at single base-resolution. Using this dPCR assay, we analyzed cfDNA and tumor DNA samples from more than 400 patients and compared enzymatic digestion with bisulfite treatment. We demonstrate that this resource can allow for the identification of entity-agnostic and specific markers, and that cfDNA methylation patterns are mirrored in tumor samples. The approach can be applied for methylation profiling of yet unanalyzed entities. Finally, we demonstrate unconventional epigenetic regulation of gene expression by methylated DNA-binding transcription factors whose activities are tissue- and context-specific and dosage-dependent. This work, therefore, provides a reference resource to identify minimally invasive, entity-specific and pan-cancer markers applicable for diagnostics, surveillance and patient stratification. It also sets the stage for further investigation of tissue- and context-specific epigenetic regulation of gene expression in health and disease by methylated DNA-binding transcription factors. Citation Format: Smiths Sengkwawoh Lueong, Martin Metzenmacher, Gregor Zaun, Gina Lauren Mayer, Erik Jan Hemmer, Katharina Lückerath, Kelsey Pomykala, Balazs Hegedüs, Peter A. Horn, Marija Trajkovic-Arsic, Tibor Szarvas, Renáta Váraljai, Corinna Keup, Ingeborg Tinhofer, Stephen George, Sabine Kasimir-Bauer, Samuel Peña-Llopis, Alexander Roesch, Cornelius Kürten, Lukas Boosfeld, Kirsten Bruderek, Christopher Darr, Thomas Hilser, Viktor Grünwald, Sven Brandau, Boris Hadaschik, Hans Neubauer, Irene Irene Esposito Irene Esposito, Tanja Fehm, Csilla Oláh, Anita Csizmarik, Fabinshy Thangarajah, Laura Reetz, Ghanam Jamal, Basant Kumar Thakur, Halime Kalkavan, Alexander Schramm, Martin Schuler, Jens T. Siveke. Comprehensive pan-cancer analysis of cfDNA methylation marks in tumors reveals complex epigenetic regulatory circuits and diagnostic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5019.

Neurogenic Regulation of Cerebral Blood Flow

The brain has a well-developed vascular network, which allows it to consume up to 15% of the cardiac output with a low mass relative to the whole-body weight. Normally, the metabolic demands of the brain depend considerably on the intensity of functioning of its different departments, which requires constant regulation of the local blood flow level. On the other hand, the state of systemic hemodynamics can have a significant impact on the organ blood flow. Complex and multilevel mechanisms of the regulation of organ cerebral blood flow are aimed at minimizing the possible adverse effects of systemic hemodynamics impairment. The importance of precise and prompt regulation of cerebral blood flow is reinforced by the absence of energy reserves or substrates for its autonomous production in the nervous tissue. The main mechanisms of cerebral blood flow regulation include: myogenic regulation, influence of local humoral influences and vasoactive substances (hormones, metabolites) of systemic blood flow, changes in blood gas composition (increase or decrease in blood oxygen or carbon dioxide tension). In addition, endothelium-dependent mechanisms of regulation are distinguished. Finally, one more level of cerebral arterial tone regulation is represented by the effect of neurotransmitters released from vasomotor fibers terminals of sympathetic and parasympathetic sections of autonomic nervous system, as well as from subcortical neurons and cortical interneurons terminals. In the present review, the principles of neurogenic regulation of cerebral blood flow are considered. The neurogenic regulation of vascular tone is the most complex regulatory circuit. The autonomic innervation of cerebral vessels has significant features that distinguish it from that in most other organs of the great circulatory circle. In addition to the autonomic innervation proper, the vessels of the brain receive sensory innervation, and the small intracerebral arterioles are also innervated -directly by subcortical neurons and cortical interneurons. In this connection, a deeper understanding of the molecular mechanisms of the neurogenic regulation of cerebral blood flow may serve as a basis for the development of new methods of treatment of severe brain diseases based on neuromodulation in the long term.

Exploring the interplay between PARP1 and circRNA biogenesis and function.

PARP1 (poly-ADP-ribose polymerase 1) is a multidomain protein with a flexible and self-folding structure that allows it to interact with a wide range of biomolecules, including nucleic acids and target proteins. PARP1 interacts with its target molecules either covalently via PARylation or non-covalently through its PAR moieties induced by auto-PARylation. These diverse interactions allow PARP1 to participate in complex regulatory circuits and cellular functions. Although the most studied PARP1-mediated functions are associated with DNA repair and cellular stress response, subsequent discoveries have revealed additional biological functions. Based on these findings, PARP1 is now recognized as a major modulator of gene expression. Several discoveries show that this multifunctional protein has been intimately connected to several steps of mRNA biogenesis, from transcription initiation to mRNA splicing, polyadenylation, export, and translation of mRNA to proteins. Nevertheless, our understanding of PARP1's involvement in the biogenesis of both coding and noncoding RNA, notably circular RNA (circRNA), remains restricted. In this review, we outline the possible roles of PARP1 in circRNA biogenesis. A full examination of the regulatory roles of PARP1 in nuclear processes with an emphasis on circRNA may reveal new avenues to control dysregulation implicated in the pathogenesis of several diseases such as neurodegenerative disorders and cancers. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Processing > Splicing Regulation/Alternative Splicing.

Gene regulatory network inference in the era of single-cell multi-omics.

The interplay between chromatin, transcription factors and genes generates complex regulatory circuits that can be represented as gene regulatory networks (GRNs). The study of GRNs is useful to understand how cellular identity is established, maintained and disrupted in disease. GRNs can be inferred from experimental data - historically, bulk omics data - and/or from the literature. The advent of single-cell multi-omics technologies has led to the development of novel computational methods that leverage genomic, transcriptomic and chromatin accessibility information to infer GRNs at an unprecedented resolution. Here, we review the key principles of inferring GRNs that encompass transcription factor-gene interactions from transcriptomics and chromatin accessibility data. We focus on the comparison and classification of methods that use single-cell multimodal data. We highlight challenges in GRN inference, in particular with respect to benchmarking, and potential further developments using additional data modalities.

HSV Replication: Triggering and Repressing STING Functionality.

Herpes simplex virus (HSV) has persisted within human populations due to its ability to establish both lytic and latent infection. Given this, human hosts have evolved numerous immune responses to protect against HSV infection. Critical in this defense against HSV, the host protein stimulator of interferon genes (STING) functions as a mediator of the antiviral response by inducing interferon (IFN) as well as IFN-stimulated genes. Emerging evidence suggests that during HSV infection, dsDNA derived from either the virus or the host itself ultimately activates STING signaling. While a complex regulatory circuit is in operation, HSV has evolved several mechanisms to neutralize the STING-mediated antiviral response. Within this review, we highlight recent progress involving HSV interactions with the STING pathway, with a focus on how STING influences HSV replication and pathogenesis.

Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF–promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF–promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF–promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF–promoter binding assay, which not only complements currently available approaches for DNA–protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria–host interface.

Nlp enhances biofilm formation by Yersinia pestis biovar microtus

Biofilms formed by Yersinia pestis are able to attach to and block flea's proventriculus, which stimulates the transmission of this pathogen from fleas to mammals. In this study, we found that Nlp (YP1143) enhanced biofilm formation by Y. pestis and had regulatory effects on biofilm-associated genes at the transcriptional level. Phenotypic assays, including colony morphology assay, crystal violet staining, and Caenorhabditis elegans biofilm assay, disclosed that Nlp strongly promoted biofilm formation by Y. pestis. Further gene regulation assays showed that Nlp stimulated the expression of hmsHFRS, hmsCDE and hmsB, while had no regulatory effect on the expression of hmsT and hmsP at the transcriptional level. These findings promoted us to gain more understanding of the complex regulatory circuits controlling biofilm formation by Y. pestis.

RNase III Participates in the Adaptation to Temperature Shock and Oxidative Stress in Escherichia coli.

Bacteria thrive in ever-changing environments by quickly remodeling their transcriptome and proteome via complex regulatory circuits. Regulation occurs at multiple steps, from the transcription of genes to the post-translational modification of proteins, via both protein and RNA regulators. At the post-transcriptional level, the RNA fate is balanced through the binding of ribosomes, chaperones and ribonucleases. We aim to decipher the role of the double-stranded-RNA-specific endoribonuclease RNase III and to evaluate its biological importance in the adaptation to modifications of the environment. The inactivation of RNase III affects a large number of genes and leads to several phenotypical defects, such as reduced thermotolerance in Escherichia coli. In this study, we reveal that RNase III inactivation leads to an increased sensitivity to temperature shock and oxidative stress. We further show that RNase III is important for the induction of the heat shock sigma factor RpoH and for the expression of the superoxide dismutase SodA.

A comprehensive integrated post-GWAS analysis of Type 1 diabetes reveals enhancer-based immune dysregulation.

Type 1 diabetes (T1D) is an organ-specific autoimmune disease, whereby immune cell-mediated killing leads to loss of the insulin-producing β cells in the pancreas. Genome-wide association studies (GWAS) have identified over 200 genetic variants associated with risk for T1D. The majority of the GWAS risk variants reside in the non-coding regions of the genome, suggesting that gene regulatory changes substantially contribute to T1D. However, identification of causal regulatory variants associated with T1D risk and their affected genes is challenging due to incomplete knowledge of non-coding regulatory elements and the cellular states and processes in which they function. Here, we performed a comprehensive integrated post-GWAS analysis of T1D to identify functional regulatory variants in enhancers and their cognate target genes. Starting with 1,817 candidate T1D SNPs defined from the GWAS catalog and LDlink databases, we conducted functional annotation analysis using genomic data from various public databases. These include 1) Roadmap Epigenomics, ENCODE, and RegulomeDB for epigenome data; 2) GTEx for tissue-specific gene expression and expression quantitative trait loci data; and 3) lncRNASNP2 for long non-coding RNA data. Our results indicated a prevalent enhancer-based immune dysregulation in T1D pathogenesis. We identified 26 high-probability causal enhancer SNPs associated with T1D, and 64 predicted target genes. The majority of the target genes play major roles in antigen presentation and immune response and are regulated through complex transcriptional regulatory circuits, including those in HLA (6p21) and non-HLA (16p11.2) loci. These candidate causal enhancer SNPs are supported by strong evidence and warrant functional follow-up studies.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1−/− mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1−/− mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured Uch-l1−/− SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

Glucose-Binding of Periplasmic Protein GltB Activates GtrS-GltR Two-Component System in Pseudomonas aeruginosa.

A two-component system GtrS-GltR is required for glucose transport activity in P. aeruginosa and plays a key role during P. aeruginosa-host interactions. However, the mechanism of action of GtrS-GltR has not been definitively established. Here, we show that gltB, which encodes a periplasmic glucose binding protein, is essential for the glucose-induced activation of GtrS-GltR in P. aeruginosa. We determined that GltB is capable of binding to membrane regulatory proteins including GtrS, the sensor kinase of the GtrS-GltR TCS. We observed that alanine substitution of glucose-binding residues abolishes the ability of GltB to promote the activation of GtrS-GltR. Importantly, like the gtrS deletion mutant, gltB deletion mutant showed attenuated virulence in both Drosophila melanogaster and mouse models of infection. In addition, using CHIP-seq experiments, we showed that the promoter of gltB is the major in vivo target of GltR. Collectively, these data suggest that periplasmic binding protein GltB and GtrS-GltR TCS form a complex regulatory circuit that regulates the virulence of P. aeruginosa in response to glucose.

Evolution of Pseudomonas aeruginosa toward higher fitness under standard laboratory conditions

Identifying genetic factors that contribute to the evolution of adaptive phenotypes in pathogenic bacteria is key to understanding the establishment of infectious diseases. In this study, we performed mutation accumulation experiments to record the frequency of mutations and their effect on fitness in hypermutator strains of the environmental bacterium Pseudomonas aeruginosa in comparison to the host-niche-adapted Salmonella enterica. We demonstrate that P. aeruginosa, but not S. enterica, hypermutators evolve toward higher fitness under planktonic conditions. Adaptation to increased growth performance was accompanied by a reversible perturbing of the local genetic context of membrane and cell wall biosynthesis genes. Furthermore, we observed a fine-tuning of complex regulatory circuits involving multiple di-guanylate modulating enzymes that regulate the transition between fast growing planktonic and sessile biofilm-associated lifestyles. The redundancy and local specificity of the di-guanylate signaling pathways seem to allow a convergent shift toward increased growth performance across niche-adapted clonal P. aeruginosa lineages, which is accompanied by a pronounced heterogeneity of their motility, virulence, and biofilm phenotypes.

Root-derived trans-zeatin cytokinin protects Arabidopsis plants against photoperiod stress.

Recently, a novel type of abiotic stress caused by a prolongation of the light period-coined photoperiod stress-has been described in Arabidopsis. During the night after the prolongation of the light period, stress and cell death marker genes are induced. The next day, strongly stressed plants display a reduced photosynthetic efficiency and leaf cells eventually enter programmed cell death. The phytohormone cytokinin (CK) acts as a negative regulator of this photoperiod stress syndrome. In this study, we show that Arabidopsis wild-type plants increase the CK concentration in response to photoperiod stress. Analysis of cytokinin synthesis and transport mutants revealed that root-derived trans-zeatin (tZ)-type CKs protect against photoperiod stress. The CK signalling proteins ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 2 (AHP2), AHP3 and AHP5 and transcription factors ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), ARR10 and ARR12 are required for the protective activity of CK. Analysis of higher order B-type arr mutants suggested that a complex regulatory circuit exists in which the loss of ARR10 or ARR12 can rescue the arr2 phenotype. Together the results revealed the role of root-derived CK acting in the shoot through the two-component signalling system to protect from the negative consequences of strong photoperiod stress.

A mechanistic model for individualised treatment of anxiety disorders based on predictive neural biomarkers.

Increased amygdala responsiveness is the hallmark of fear and a characteristic across patients with anxiety disorders. The amygdala is embedded in a complex regulatory circuit. Multiple different mechanisms may elevate amygdala responsiveness and lead to the occurrence of an anxiety disorder. While top-down control by the prefrontal cortex (PFC) downregulates amygdala responses, the locus coeruleus (LC) drives up amygdala activation via noradrenergic projections. This indicates that the same fearful phenotype may result from different neural mechanisms. We propose a mechanistic model that defines three different neural biomarkers causing amygdala hyper-responsiveness in patients with anxiety disorders: (a) inherent amygdala hypersensitivity, (b) low prefrontal control and (c) high LC drive. First-line treatment for anxiety disorders is exposure-based cognitive behavioural therapy, which strengthens PFC recruitment during emotion regulation and thus targets low-prefrontal control. A treatment response rate around 50% (Loerinc et al., 2015, Clinical Psychological Reviews, 42, 72-82) might indicate heterogeneity of underlying neurobiological mechanisms among patients, presumably leading to high variation in treatment benefit. Transforming insights from cognitive neuroscience into applicable clinical heuristics to categorise patients based on their underlying biomarker may support individualised treatment selection in psychiatry. We review literature on the three anxiety-related mechanisms and present a mechanistic model that may serve as a rational for pathology-based diagnostic and biomarker-guided treatment selection in psychiatry.

Chromosome Missegregation in Single Human Oocytes Is Related to the Age and Gene Expression Profile.

The growing trend for women to postpone childbearing has resulted in a dramatic increase in the incidence of aneuploid pregnancies. Despite the importance to human reproductive health, the events precipitating female age-related meiotic errors are poorly understood. To gain new insight into the molecular basis of age-related chromosome missegregation in human oocytes, we combined the transcriptome profiles of twenty single oocytes (derived from females divided into two groups according to age <35 and ≥35 years) with their chromosome status obtained by array comparative genomic hybridization (aCGH). Furthermore, we compared the transcription profile of the single oocyte with the surrounding cumulus cells (CCs). RNA-seq data showed differences in gene expression between young and old oocytes. Dysregulated genes play a role in important biological processes such as gene transcription regulation, cytoskeleton organization, pathways related to RNA maturation and translation. The comparison of the transcription profile of the oocyte and the corresponding CCs highlighted the differential expression of genes belonging to the G protein-coupled receptor superfamily. Finally, we detected the loss of a X chromosome in two oocytes derived from women belonging to the ≥35 years age group. These aneuploidies may be caused by the detriment of REEP4, an endoplasmic reticulum protein, in women aged ≥35 years. Here we gained new insight into the complex regulatory circuit between the oocyte and the surrounding CCs and uncovered a new putative molecular basis of age-related chromosome missegregation in human oocytes.

MicroRNA Targeting Nicotinamide Adenine Dinucleotide Phosphate Oxidases in Cancer.

Significance: Reactive oxygen species (ROS) production occurs primarily in the mitochondria as a by-product of cellular metabolism. ROS are also produced by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in response to growth factors and cytokines by normal physiological signaling pathways. NADPH oxidase, a member of NADPH oxidase (NOX) family, utilizes molecular oxygen (O2) to generate ROS such as hydrogen peroxide and superoxide. Imbalance between ROS production and its elimination is known to be the major cause of various human diseases. NOX family proteins are exclusively involved in ROS production, which makes them attractive target(s) for the treatment of ROS-mediated diseases including cancer. Recent Advances: Molecules such as Keap1/nuclear factor erythroid 2-related factor 2 (Nrf2), N-methyl-d-aspartic acid (NMDA) receptors, nuclear factor-kappaB, KRAS, kallistatin, gene associated with retinoic-interferon-induced mortality-19, and deregulated metabolic pathways are involved in ROS production in association with NADPH oxidase. Critical Issues: Therapeutic strategies targeting NADPH oxidases in ROS-driven cancers are not very effective due to its complex regulatory circuit. Tumor suppressor microRNAs (miRNAs) viz. miR-34a, miR-137, miR-99a, and miR-21a-3p targeting NADPH oxidases are predominantly downregulated in ROS-driven cancers. miRNAs also regulate other cellular machineries such as Keap1/Nrf2 pathway and NMDA receptors involved in ROS production and consequently drug resistance. Here, we discuss the structure, function, and metabolic role of NADPH oxidase, NOX family protein-protein interaction, their association with other pathways, and NADPH oxidase alteration by miRNAs. Moreover, we also discuss and summarize studies on NADPH oxidase associated with various malignancies and their therapeutic implications. Future Directions: Targeting NADPH oxidases through miRNAs appears to be a promising strategy for the treatment of ROS-driven cancer.

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

An unorthodox partnership in DNA repair pathway choice.

Complex regulatory circuits determine whether DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways, a carefully balanced equilibrium of which is critical for genome stability. In this issue of EMBO Reports, Deng etal [1] report that a novel p53-suppressed long noncoding RNA (lncRNA), PRLH1, interacts with and stabilizes the E3 ubiquitin ligase RNF169 to stimulate HDR-mediated DSB repair and proliferation of p53-deficient cancer cells. These findings suggest a new regulatory principle in modulating DSB repair pathway selection that may contribute to tumorigenesis.

Regulation of Eicosanoid Pathways by MicroRNAs.

Over the last years, many microRNAs (miRNAs) have been identified that regulate the formation of bioactive lipid mediators such as prostanoids and leukotrienes. Many of these miRNAs are involved in complex regulatory circuits necessary for the fine-tuning of biological functions including inflammatory processes or cell growth. A better understanding of these networks will contribute to the development of novel therapeutic strategies for the treatment of inflammatory diseases and cancer. In this review, we provide an overview of the current knowledge of miRNA regulation in eicosanoid pathways with special focus on novel miRNA functions and regulatory circuits of leukotriene and prostaglandin biosynthesis.