Drosophila Genome Research Articles

Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner

Proximity-dependent biotin labelling revealed undescribed participants of the ecdysone response in Drosophila. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. The EcR/Usp heterodimer was found to collaborate with nuclear pore subunits, chromatin remodelers, and architectural proteins. Many proteins identified through proximity-dependent labelling with EcR/Usp were described previously as functional components of an ecdysone response, corroborating the potency of this labelling method. A link to ecdysone response was confirmed for some newly discovered regulators by immunoprecipitation of prepupal nuclear extract with anti-EcR antibodies and functional experiments in Drosophila S2 cells. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. CP190 was found to co-immunoprecipitate with the EcR subunit of EcR/Usp in a 20E-independent manner. ChIP-Seq experiments revealed only partial overlapping between CP190 and EcR bound sites in the Drosophila genome and complete absence of CP190 binding at 20E-dependent enhancers. Analysis of Hi-C data demonstrated an existence of remote interactions between 20E-dependent enhancers and CP190 sites which suggests formation of a protein complex between EcR/Usp and CP190 through the space. Our results support the previous concept that CP190 has a role in stabilization of specific chromatin loops for proper activation of transcription of genes regulated by 20E hormone.

Ancient evolutionary signals of protein-coding sequences allow the discovery of new genes in the Drosophila melanogaster genome

BackgroundThe current growth in DNA sequencing techniques makes of genome annotation a crucial task in the genomic era. Traditional gene finders focus on protein-coding sequences, but they are far from being exhaustive. The number of this kind of genes continuously increases due to new experimental data and development of improved bioinformatics algorithms.ResultsIn this context, AnABlast represents a novel in silico strategy, based on the accumulation of short evolutionary signals identified by protein sequence alignments of low score. This strategy potentially highlights protein-coding regions in genomic sequences regardless of traditional homology or translation signatures. Here, we analyze the evolutionary information that the accumulation of these short signals encloses. Using the Drosophila melanogaster genome, we stablish optimal parameters for the accurate gene prediction with AnABlast and show that this new strategy significantly contributes to add genes, exons and pseudogenes regions, yet to be discovered in both already annotated and new genomes.ConclusionsAnABlast can be freely used to analyze genomic regions of whole genomes where it contributes to complete the previous annotation.

Ab Initio Construction and Evolutionary Analysis of Protein-Coding Gene Families with Partially Homologous Relationships: Closely Related Drosophila Genomes as a Case Study.

How have genes evolved within a well-known genome phylogeny? Many protein-coding genes should have evolved as a whole at the gene level, and some should have evolved partly through fragments at the subgene level. To comprehensively explore such complex homologous relationships and better understand gene family evolution, here, with de novo-identified modules, the subgene units which could consecutively cover proteins within a set of closely related species, we applied a new phylogeny-based approach that considers evolutionary models with partial homology to classify all protein-coding genes in nine Drosophila genomes. Compared with two other popular methods for gene family construction, our approach improved practical gene family classifications with a more reasonable view of homology and provided a much more complete landscape of gene family evolution at the gene and subgene levels. In the case study, we found that most expanded gene families might have evolved mainly through module rearrangements rather than gene duplications and mainly generated single-module genes through partial gene duplication, suggesting that there might be pervasive subgene rearrangement in the evolution of protein-coding gene families. The use of a phylogeny-based approach with partial homology to classify and analyze protein-coding gene families may provide us with a more comprehensive landscape depicting how genes evolve within a well-known genome phylogeny.

Genomic features of the fall armyworm (Spodoptera frugiperda) (J.E. Smith) yield insights into its defense system and flight capability

AbstractThe fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) is an agriculturally important insect pest and causes significant economic loss due to its resistance to insecticide and high dispersal ability (i.e. long‐distance flyer). We performed comparative genomics analysis based on the recently released chromosome‐scale genome of the FAW to investigate the genetic mechanisms of FAWs defense system and flight ability. The gene families encoding receptors for bitter or toxic substances and detoxification enzymes, such as cytochrome P450, carboxylesterase and glutathione‐S‐transferase, were expanded in FAW, enabling its ability to detect and detoxify many plant secondary compounds. The chromosomal locations of five detoxification‐related genes (i.e. ABC, CCE, GST, P450, and UGT) were analyzed, which revealed the density of these genes across the FAW genome. Several genes like Mio, Sik2, CRTC, and Lpin were positively selected in FAW, which were associated with energy metabolism, lipid metabolism, and/or muscle development, and thus possibly contributed to the high flight ability of FAW. We also analyzed the homologs to antimicrobial genes reported in the Drosophila genome, and identified 2 attacins, 6 cecropins, 3 gloverins, and 2 moricins in FAW. The findings of this study will broadly expand our knowledge on the biology of the devastating FAW, and contribute to the development of new pest management strategies for the control of major agricultural pests like FAW.

Genomic organization of Polycomb Response Elements and its functional implication in Drosophila and other insects

The epigenetic memory is an essential aspect of multicellular organisms to maintain several cell types and their gene expression pattern. This complex process uses a number of protein factors and specific DNA elements within the developmental cues to achieve this. The protein factors involved in the process are the Polycomb group (PcG) members, and, accordingly, the DNA sequences that interact with these proteins are called Polycomb Response Elements (PREs). Since the PcG proteins are highly conserved among higher eukaryotes, including insects, and function at thousands of sites in the genomes, it is expected that PREs mayalso be present across the genome.However, the studies on PREs in insect species, other thanDrosophila, is currently lacking.We took a bioinformatics approach to develop an inclusive PRE prediction tool, 'PRE Mapper', to address this need. By applying this tool on the Drosophila melanogaster genome, we predicted greater than 20,000 PREs.When comparedwith the available PRE prediction methods, this tool shows far better performance by correctly identifying the in vivo binding sites of PcG proteins, identified by genome-scale ChIP experiments. Further analysis of the predicted PREs shows their cohabitation with chromatin domain boundary elements at several places in the Drosophila genome, possibly defining a composite epigenetic module.We analysed 10 insect genomes in this context and find several conserved features in PREs across the insect species with some variations in their occurrence frequency. These analyses leading to the identification of PREin insect genomes contribute to our understanding of epigenetic mechanisms in these organisms.

A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila.

In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. Here, we show that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset ofneurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. Our study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female.

Induction of reporter genes expression by ionizing radiation and toxins influence on the Drosophila melanogaster genome

In the human environment, there are many factors that can cause genotoxic and mutagenic effects and one of them is ionizing radiation. Radioactive substances like radon, thorium, radium and their decay products during decomposition are able to emit not only highly penetrating gamma radiation, but also form large heavy alpha-particles. Alpha-particles have a low penetrating power, but high ionization energy, about 10 times greater than gamma-rays. Consequently, we researched genotoxic effects of alpha-radiation using a test-system with reporter genes on the Drosophila melanogaster. In nature, the main source of alpha-particles is colorless radioactive gas radon, its isotopes and daughter decay products. It is technically difficult to create sufficiently intense and directed beams of α-radiation on the long-lived radon isotope – 222Rn. Therefore, for experiments was chosen the 238Pu (Plutonium) isotope, which has the necessary α-line at decay equal to 5.5 MeV. The genotoxic activity of alpha-particles was tested on Drosophila melanogaster with genotype containing genes of luminous proteins. Working of the genetic construction based on the GAL4-UAS system, often used in research to determine gene expression. Genotype also included GADD45 protein gene and GFP gene (green fluorescent protein). This genes combination allows to observe glow of Drosophila organs and tissues after stress factor impact. The sensitivity of construction was checked by chemical mutagen cisplatin, which led to the larvae death at high concentrations, and caused glow of organs at low concentrations. The larvae were exposed to alpha-radiation for 20 hours. Preparations were analyzed on a light microscope with a blue filter and glow of the larval organs was observed. The glow intensity was depending from radiation source. In other words, higher radiation dose caused brighter glow, which correlates with synthesis of luminous protein level. This proves that alpha-radiation from radon and its decay daughter products has a genetic effect. Keywords: alpha radiation, radon, Drosophila, GFP, genotoxicity.

Comparative Analysis of the Structure of Three piRNA Clusters in the Drosophila melanogaster Genome

Here we attempt to reconstruct the sequence of events that led to the formation of three regulatory piRNA clusters, namely, 20A, 38C and flamenco in the Drosophila melanogaster genome. Both the 38C and flamenco clusters include inverted sequences, which potentially form double-stranded RNA hairpins. We present evidence in favor of the well-known hypothesis of piRNA clusters as "transposon traps". According to this model, the presence of the only copy of the transposon in the genome indicates that its expression is suppressed by an RNA-interference mechanism immediately after the mobile element enters the piRNA cluster. We also discuss high the structural variability of piRNAs in Drosophila clusters and cases of horizontal transmobile elements between related species.

The Effect of the Human MeCP2 gene on Drosophila melanogaster behavior and p53 inhibition as a model for Rett Syndrome

The purpose of this study was to explore the effect of introducing the human methyl CPG binding protein 2 gene (MeCP2) into the Drosophila melanogaster genome, examine how it would affect protein levels of p53, and test how inhibiting p53 with pifithrin-alpha would affect behavioral and physical patterns in fruit flies. One goal was to observe if the symptoms of Rett Syndrome, a neurodegenerative disease in humans, are reflected in Drosophila melanogaster. This was achieved by differentiating the behavior and physical aspects of wild-type flies from flies expressing the full-length MeCP2 gene and the mutated MeCP2 gene (R106W). Differences in eye structure, climbing (the use of locomotor skills), geotaxis, as well as differences in the flies’ abilities to fly were analyzed. In addition, after these series of tests, the MeCP2 protein and the p53 protein, endogenous to Drosophila, were extracted and their levels were compared in each of the populations of flies. Then, after the Drosophila consumed pifithrin-alpha hydrobromide, their behavior, physical appearance, and protein levels were again monitored. After conducting these experiments, the effects of full-length and mutant MeCP2 on Drosophila melanogaster’s psychomotor skills were analyzed, along with their effects on how the treatment of pifithrin-alpha affected the level of MeCP2 and p53 in the various populations of flies. In conclusion, some of the Rett Syndrome symptoms were recapitulated in Drosophila, and a subset of those were partially ameliorated by the introduction of pifithrin-alpha.

Building a pipeline to solicit expert knowledge from the community to aid gene summary curation.

Brief summaries describing the function of each gene’s product(s) are of great value to the research community, especially when interpreting genome-wide studies that reveal changes to hundreds of genes. However, manually writing such summaries, even for a single species, is a daunting task; for example, the Drosophila melanogaster genome contains almost 14 000 protein-coding genes. One solution is to use computational methods to generate summaries, but this often fails to capture the key functions or express them eloquently. Here, we describe how we solicited help from the research community to generate manually written summaries of D. melanogaster gene function. Based on the data within the FlyBase database, we developed a computational pipeline to identify researchers who have worked extensively on each gene. We e-mailed these researchers to ask them to draft a brief summary of the main function(s) of the gene’s product, which we edited for consistency to produce a ‘gene snapshot’. This approach yielded 1800 gene snapshot submissions within a 3-month period. We discuss the general utility of this strategy for other databases that capture data from the research literature.Database URL: https://flybase.org/

3D genome organisation in Drosophila.

Ever since Thomas Hunt Morgan's discovery of the chromosomal basis of inheritance by using Drosophila melanogaster as a model organism, the fruit fly has remained an essential model system in studies of genome biology, including chromatin organisation. Very much as in vertebrates, in Drosophila, the genome is organised in territories, compartments and topologically associating domains (TADs). However, these domains might be formed through a slightly different mechanism than in vertebrates due to the presence of a large and potentially redundant set of insulator proteins and the minor role of dCTCF in TAD boundary formation. Here, we review the different levels of chromatin organisation in Drosophila and discuss mechanisms and factors that might be involved in TAD formation. The dynamics of TADs and enhancer-promoter interactions in the context of transcription are covered in the light of currently conflicting results. Finally, we illustrate the value of polymer modelling approaches to infer the principles governing the three-dimensional organisation of the Drosophila genome.

Identification of novel genes associated with longevity in Drosophila melanogaster - a computational approach.

Despite a growing number of studies on longevity in Drosophila, genetic factors influencing lifespan are still poorly understood. In this paper we propose a conceptually new approach for the identification of novel longevity-associated genes and potential target genes for SNPs in non-coding regions by utilizing the knowledge of co-location of various loci, governed by the three-dimensional architecture of the Drosophila genome. Firstly, we created networks between genes/genomic regions harboring SNPs deemed to be significant in two longevity GWAS summary statistics datasets using intra- and inter-chromosomal interaction frequencies (Hi-C data) as a measure of co-location. These networks were further extended to include regions strongly interacting with previously selected regions. Using various network measures, literature search and additional bioinformatics resources, we investigated the plausibility of genes found to have genuine association with longevity. Several of the newly identified genes were common between the two GWAS datasets and these possessed human orthologs. We also found that the proportion of non-coding SNPs in borders between topologically associated domains is significantly higher than expected by chance. Assuming co-location, we investigated potential target genes for non-coding SNPs. This approach therefore offers a stepping stone to identification of novel genes and SNP targets linked to human longevity.

Position Effects Influence Transvection in Drosophila melanogaster.

Transvection is an epigenetic phenomenon wherein regulatory elements communicate between different chromosomes in trans, and is thereby dependent upon the three-dimensional organization of the genome. Transvection is best understood in Drosophila, where homologous chromosomes are closely paired in most somatic nuclei, although similar phenomena have been observed in other species. Previous data have supported that the Drosophila genome is generally permissive to enhancer action in trans, a form of transvection where an enhancer on one homolog activates gene expression from a promoter on a paired homolog. However, the capacity of different genomic positions to influence the quantitative output of transvection has yet to be addressed. To investigate this question, we employed a transgenic system that assesses and compares enhancer action in cis and in trans at defined chromosomal locations. Using the strong synthetic eye-specific enhancer GMR, we show that loci supporting strong cis-expression tend to support robust enhancer action in trans, whereas locations with weaker cis-expression show reduced transvection in a fluorescent reporter assay. Our subsequent analysis is consistent with a model wherein the chromatin state of the transgenic insertion site is a primary determinant of the degree to which enhancer action in trans will be supported, whereas other factors such as locus-specific variation in somatic homolog pairing are of less importance in influencing position effects on transvection.

BEN-solo factors partition active chromatin to ensure proper gene activation in Drosophila

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units.

Deducing the Role of Virus Genome-Derived PIWI-Associated RNAs in the Mosquito-Arbovirus Arms Race.

The P-element-induced wimpy testis (PIWI)-associated RNA (piRNA) pathway is known for its role in the protection of genome integrity in the germline of Drosophila melanogaster by silencing transposable elements. The piRNAs that target transposons originate from piRNA clusters in transposon-rich regions of the Drosophila genome and are processed by three PIWI family proteins. In Aedes aegypti and Aedes albopictus mosquitoes, which are two of the most important vectors of arthropod-borne viruses (arboviruses), the number of PIWI family genes has expanded and some are expressed in somatic, as well as germline, tissues. These discoveries have led to active research to explore the possible expanded functional roles of the piRNA pathway in vector mosquitoes. Virus genome-derived piRNAs (which will be referred to as (virus name) vpiRNAs) have been demonstrated in Aedes spp. cultured cells and mosquitoes after infection by arthropod-borne alpha-, bunya-, and flaviviruses. However, although Culex quinquefasciatus also is an important arbovirus vector and has an expansion of PIWI family genes, vpiRNAs have seldom been documented in this genus after arbovirus infection. Generation of complementary DNA (cDNA) fragments from RNA genomes of alpha-, bunya-, and flaviviruses (viral-derived cDNAs, vDNAs) has been demonstrated in cultured Aedes spp. cells and mosquitoes, and endogenous viral elements (EVEs), cDNA fragments of non-retroviral RNA virus genomes, are found more abundantly in genomes of Ae. aegypti and Ae. albopictus than other vector mosquitoes. These observations have led to speculation that vDNAs are integrated into vector genomes to form EVEs, which serve as templates for the transcription of antiviral vpiRNA precursors. However, no EVEs derived from alphavirus genomes have been demonstrated in genomes of any vector mosquito. In addition, although EVEs have been shown to be a source of piRNAs, the preponderance of EVEs described in Aedes spp. vectors are more closely related to the genomes of persistently infecting insect-specific viruses than to acutely infecting arboviruses. Furthermore, the signature patterns of the “ping-pong” amplification cycle that maintains transposon-targeting piRNAs in Drosophila are also evident in alphavirus and bunyavirus vpiRNAs, but not in vpiRNAs of flaviviruses. These divergent observations have rendered deciphering the mechanism(s) of biogenesis and potential role of vpiRNAs in the mosquito–arbovirus arms race difficult, and the focus of this review will be to assemble major findings regarding vpiRNAs and antiviral immunity in the important arbovirus vectors from Aedes and Culex genera.

CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.

Construction of a genome-wide transgenic UAS-cDNA/ORF library in Drosophila based on the binary GAL4/UAS system has been severely hampered by technical difficulties, although genome-wide cDNA or ORF resources of Drosophila, human, and mouse have been publicly available for more than a decade. Here, we developed a new method named CRISPR-based modular assembly (CRISPRmass) for the high-throughput construction of a genome-wide UAS-cDNA/ORF library from publicly available cDNA/ORF resources. Through cleavage of shared vector sequences of cDNA/ORF plasmids by CRISPR/Cas9 and subsequent insertion of UAS modules by Gibson assembly, the procedure of construction of such a library by CRISPRmass is standardized as massively parallel two-step test tube reactions before bacterial transformation. Using CRISPRmass, we generated 5551 UAS-cDNA/ORF constructs covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection, and among them, 5518 were generated within 3 mo by three people. Our results show that CRISPRmass allows modulization, simplicity, efficiency, and adaptability in the generation of a genome-wide UAS-cDNA/ORF plasmid library by using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various genome-wide libraries in general and is an alternative to Gateway technology in high-throughput plasmid library editing. Furthermore, the more than 5500 UAS-cDNA/ORF plasmids of Drosophila genes serve as a powerful resource for gain-of-function (GOF) screening in cultured cells and for generation of a transgenic UAS-cDNA/ORF library in Drosophila.

Odor-Specific Deactivation Defects in a Drosophila Odorant-Binding Protein Mutant.

Insect odorant-binding proteins (OBPs) are a large, diverse group of low-molecular weight proteins secreted into the fluid bathing olfactory and gustatory neuron dendrites. The best-characterized OBP, LUSH (OBP76a) enhances pheromone sensitivity enabling detection of physiological levels of the male-specific pheromone, 11-cis vaccenyl acetate. The role of the other OBPs encoded in the Drosophila genome is largely unknown. Here, using clustered regularly interspaced short palindromic repeats/Cas9, we generated and characterized the loss-of-function phenotype for two genes encoding homologous OBPs, OS-E (OBP83b) and OS-F (OBP83a). Instead of activation defects, these extracellular proteins are required for normal deactivation of odorant responses to a subset of odorants. Remarkably, odorants detected by the same odorant receptor are differentially affected by the loss of the OBPs, revealing an odorant-specific role in deactivation kinetics. In stark contrast to lush mutants, the OS-E/F mutants have normal activation kinetics to the affected odorants, even at low stimulus concentrations, suggesting that these OBPs are not competing for these ligands with the odorant receptors. We also show that OS-E and OS-F are functionally redundant as either is sufficient to revert the mutant phenotype in transgenic rescue experiments. These findings expand our understanding of the roles of OBPs to include the deactivation of odorant responses.

Structure of a P element transposase-DNA complex reveals unusual DNA structures and GTP-DNA contacts.

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.

Structural variants exhibit widespread allelic heterogeneity and shape variation in complex traits

It has been hypothesized that individually-rare hidden structural variants (SVs) could account for a significant fraction of variation in complex traits. Here we identified more than 20,000 euchromatic SVs from 14 Drosophila melanogaster genome assemblies, of which ~40% are invisible to high specificity short-read genotyping approaches. SVs are common, with 31.5% of diploid individuals harboring a SV in genes larger than 5kb, and 24% harboring multiple SVs in genes larger than 10kb. SV minor allele frequencies are rarer than amino acid polymorphisms, suggesting that SVs are more deleterious. We show that a number of functionally important genes harbor previously hidden structural variants likely to affect complex phenotypes. Furthermore, SVs are overrepresented in candidate genes associated with quantitative trait loci mapped using the Drosophila Synthetic Population Resource. We conclude that SVs are ubiquitous, frequently constitute a heterogeneous allelic series, and can act as rare alleles of large effect.

Highly structured homolog pairing reflects functional organization of the Drosophila genome

Trans-homolog interactions have been studied extensively in Drosophila, where homologs are paired in somatic cells and transvection is prevalent. Nevertheless, the detailed structure of pairing and its functional impact have not been thoroughly investigated. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, showing that homologs pair with varying precision genome-wide, in addition to establishing trans-homolog domains and compartments. We also elucidate the structure of pairing with unprecedented detail, observing significant variation across the genome and revealing at least two forms of pairing: tight pairing, spanning contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional implication genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing, including the disruption of some interaction peaks.