BACKGROUND: Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions. RESULTS: By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9 kb and another within 13-16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp (base pair) and a second arm at 13,447-13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. CONCLUSIONS: Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

BACKGROUND: Horizontal gene transfer (HGT) is an evolutionary mechanism of adaptive importance, which has been deeply studied in wine S. cerevisiae strains, where those acquired genes conferred improved traits related to both transport and metabolism of the nutrients present in the grape must. However, little is known about HGT events that occurred in wild Saccharomyces yeasts and how they determine their phenotypes. RESULTS: Through a comparative genomic approach among Saccharomyces species, we detected a subtelomeric segment present in the S. uvarum, S. kudriavzevii, and S. eubayanus species, belonging to the first species to diverge in the Saccharomyces genus, but absent in the other Saccharomyces species. The segment contains three genes, two of which were characterized, named DGD1 and DGD2. DGD1 encodes dialkylglicine decarboxylase, whose specific substrate is the non-proteinogenic amino acid 2-aminoisobutyric acid (AIB), a rare amino acid present in some antimicrobial peptides of fungal origin. DGD2 encodes putative zinc finger transcription factor, which is essential to induce the AIB-dependent expression of DGD1. Phylogenetic analysis showed that DGD1 and DGD2 are closely related to two adjacent genes present in Zygosaccharomyces. CONCLUSIONS: The presented results show evidence of an early HGT event conferring new traits to the ancestor of the Saccharomyces genus that could be lost in the evolutionary more recent Saccharomyces species, perhaps due to loss of function during the colonization of new habitats.

BACKGROUND: Through the evolution of novel wing structures, bats (Order Chiroptera) became the only mammalian group to achieve powered flight. This achievement preceded the massive adaptive radiation of bats into diverse ecological niches. We investigate some of the developmental processes that underlie the origin and subsequent diversification of one of the novel membranes of the bat wing: the plagiopatagium, which connects the fore- and hind limb in all bat species. RESULTS: Our results suggest that the plagiopatagium initially arises through novel outgrowths from the body flank that subsequently merge with the limbs to generate the wing airfoil. Our findings further suggest that this merging process, which is highly conserved across bats, occurs through modulation of the programs controlling the development of the periderm of the epidermal epithelium. Finally, our results suggest that the shape of the plagiopatagium begins to diversify in bats only after this merging has occurred. CONCLUSIONS: This study demonstrates how focusing on the evolution of cellular processes can inform an understanding of the developmental factors shaping the evolution of novel, highly adaptive structures.

BACKGROUND: Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae, and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets, and pharmacological manipulation of an upstream activator. RESULTS: We find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this time point. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We propose that Al1 expression in future white scales might be related to an observed downregulation of the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3-hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling. CONCLUSIONS: We propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis.

BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

BACKGROUND: Non-invasive recordings of gross neural activity in humans often show responses to omitted stimuli in steady trains of identical stimuli. This has been taken as evidence for the neural coding of prediction or prediction error. However, evidence for such omission responses from invasive recordings of cellular-scale responses in animal models is scarce. Here, we sought to characterise omission responses using extracellular recordings in the auditory cortex of anaesthetised rats. We profiled omission responses across local field potentials (LFP), analogue multiunit activity (AMUA), and single/multi-unit spiking activity, using stimuli that were fixed-rate trains of acoustic noise bursts where 5% of bursts were randomly omitted. RESULTS: Significant omission responses were observed in LFP and AMUA signals, but not in spiking activity. These omission responses had a lower amplitude and longer latency than burst-evoked sensory responses, and omission response amplitude increased as a function of the number of preceding bursts. CONCLUSIONS: Together, our findings show that omission responses are most robustly observed in LFP and AMUA signals (relative to spiking activity). This has implications for models of cortical processing that require many neurons to encode prediction errors in their spike output.

BACKGROUND: Common seadragons (Phyllopteryx taeniolatus, Syngnathidae) are an emblem of the diverse endemic fauna of Australia's southern rocky reefs, the newly recognized "Great Southern Reef." A lack of assessments spanning this global biodiversity hotspot in its entirety is currently hampering an understanding of the factors that have contributed to its diversity. The common seadragon has a wide range across Australia's entire temperate south and includes a geogenetic break over a former land bridge, which has called its status as a single species into question. As a popular aquarium display that sells for high prices, common seadragons are also vulnerable to illegal capture. RESULTS: Here, we provide range-wide nuclear sequences (986 variable Ultraconserved Elements) for 198 individuals and mitochondrial genomes for 140 individuals to assess species status, identify genetic units and their diversity, and trace the source of two poached individuals. Using published data of the other two seadragon species, we found that lineages of common seadragons have diverged relatively recently (< 0.63 Ma). Within common seadragons, we found pronounced genetic structure, falling into three major groups in the western, central, and eastern parts of the range. While populations across the Bassian Isthmus were divergent, there is also evidence for secondary contact since the passage opened. We found a strong cline of genetic diversity from the range center tapering symmetrically towards the range peripheries. Based on their genetic similarities, the poached individuals were inferred to have originated from around Albany in southwestern Australia. CONCLUSIONS: We conclude that common seadragons constitute a single species with strong geographic structure but coherence through gene flow. The low genetic diversity on the east and west coasts is concerning given that these areas are projected to face fast climate change. Our results suggest that in addition to their life history, geological events and demographic expansions have all played a role in shaping populations in the temperate south. These insights are an important step towards understanding the historical determinants of the diversity of species endemic to the Great Southern Reef.

BACKGROUND: Circular RNA (circRNA) molecules, generated through non-canonical back-splicing of exon-exon junctions, have recently been implicated in diverse biological functions including transcriptional regulation and modulation of protein interactions. CircRNAs are emerging as a key component of the complex neural transcriptome implicated in brain development. However, the specific expression patterns and functions of circRNAs in human neuronal differentiation have not been explored. RESULTS: Using total RNA sequencing analysis, we identified expressed circRNAs during the differentiation of human neuroepithelial stem (NES) cells into developing neurons and discovered that many circRNAs originated from host genes associated with synaptic function. Interestingly, when assessing population data, exons giving rise to circRNAs in our dataset had a higher frequency of genetic variants. Additionally, screening for RNA-binding protein sites identified enrichment of Splicing Factor Proline and Glutamine Rich (SFPQ) motifs in increased circRNAs, several of which were reduced by SFPQ knockdown and enriched in SFPQ ribonucleoprotein complexes. CONCLUSIONS: Our study provides an in-depth characterisation of circRNAs in a human neuronal differentiation model and highlights SFPQ as both a regulator and binding partner of circRNAs elevated during neuronal maturation.

As organisms evolve, the effects of mutations change as a result of epistatic interactions with other mutations accumulated along the line of descent. This can lead to shifts in adaptability or robustness that ultimately shape subsequent evolution. Here, we review recent advances in measuring, modeling, and predicting epistasis along evolutionary trajectories, both in microbial cells and single proteins. We focus on simple patterns of global epistasis that emerge in this data, in which the effects of mutations can be predicted by a small number of variables. The emergence of these patterns offers promise for efforts to model epistasis and predict evolution.

BACKGROUND: The axonemal microtubules of primary cilium undergo a conserved protein posttranslational modification (PTM) - polyglutamylation. This reversible procedure is processed by tubulin tyrosine ligase-like polyglutamylases to form secondary polyglutamate side chains, which are metabolized by the 6-member cytosolic carboxypeptidase (CCP) family. Although polyglutamylation modifying enzymes have been linked to ciliary architecture and motility, it was unknown whether they also play a role in ciliogenesis. RESULTS: In this study, we found that CCP5 expression is transiently downregulated upon the initiation of ciliogenesis, but recovered after cilia are formed. Overexpression of CCP5 inhibited ciliogenesis, suggesting that a transient downregulation of CCP5 expression is required for ciliation initiation. Interestingly, the inhibitory effect of CCP5 on ciliogenesis does not rely on its enzyme activity. Among other 3 CCP members tested, only CCP6 can similarly suppress ciliogenesis. Using CoIP-MS analysis, we identified a protein that potentially interacts with CCP - CP110, a known negative regulator of ciliogenesis, whose degradation at the distal end of mother centriole permits cilia assembly. We found that both CCP5 and CCP6 can modulate CP110 level. Particularly, CCP5 interacts with CP110 through its N-terminus. Loss of CCP5 or CCP6 led to the disappearance of CP110 at the mother centriole and abnormally increased ciliation in cycling RPE-1 cells. Co-depletion of CCP5 and CCP6 synergized this abnormal ciliation, suggesting their partially overlapped function in suppressing cilia formation in cycling cells. In contrast, co-depletion of the two enzymes did not further increase the length of cilia, although CCP5 and CCP6 differentially regulate polyglutamate side-chain length of ciliary axoneme and both contribute to limiting cilia length, suggesting that they may share a common pathway in cilia length control. Through inducing the overexpression of CCP5 or CCP6 at different stages of ciliogenesis, we further demonstrated that CCP5 or CCP6 inhibited cilia formation before ciliogenesis, while shortened the length of cilia after cilia formation. CONCLUSION: These findings reveal the dual role of CCP5 and CCP6. In addition to regulating cilia length, they also retain CP110 level to suppress cilia formation in cycling cells, pointing to a novel regulatory mechanism for ciliogenesis mediated by demodifying enzymes of a conserved ciliary PTM, polyglutamylation.

BACKGROUND: Changes in gene expression levels during brain development are thought to have played an important role in the evolution of human cognition. With the advent of high-throughput sequencing technologies, changes in brain developmental expression patterns, as well as human-specific brain gene expression, have been characterized. However, interpreting the origin of evolutionarily advanced cognition in human brains requires a deeper understanding of the regulation of gene expression, including the epigenomic context, along the primate genome. Here, we used chromatin immunoprecipitation sequencing (ChIP-seq) to measure the genome-wide profiles of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), both of which are associated with transcriptional activation in the prefrontal cortex of humans, chimpanzees, and rhesus macaques. RESULTS: We found a discrete functional association, in which H3K4me3HP gain was significantly associated with myelination assembly and signaling transmission, while H3K4me3HP loss played a vital role in synaptic activity. Moreover, H3K27acHP gain was enriched in interneuron and oligodendrocyte markers, and H3K27acHP loss was enriched in CA1 pyramidal neuron markers. Using strand-specific RNA sequencing (ssRNA-seq), we first demonstrated that approximately 7 and 2% of human-specific expressed genes were epigenetically marked by H3K4me3HP and H3K27acHP, respectively, providing robust support for causal involvement of histones in gene expression. We also revealed the co-activation role of epigenetic modification and transcription factors in human-specific transcriptome evolution. Mechanistically, histone-modifying enzymes at least partially contribute to an epigenetic disturbance among primates, especially for the H3K27ac epigenomic marker. In line with this, peaks enriched in the macaque lineage were found to be driven by upregulated acetyl enzymes. CONCLUSIONS: Our results comprehensively elucidated a causal species-specific gene-histone-enzyme landscape in the prefrontal cortex and highlighted the regulatory interaction that drove transcriptional activation.

BACKGROUND: Pseudozyma flocculosa is a highly efficient biocontrol agent (BCA) of powdery mildews whose mode of action remains elusive. It is known to secrete unique effectors during its interaction with powdery mildews but effectors have never been shown to be part of the arsenal of a BCA. Here, we characterize the role of the effector Pf2826 released by Pseudozyma flocculosa during its tripartite interaction with barley and the pathogen fungus Blumeria graminis f. sp. hordei. RESULTS: We utilized CRISPR-Cas9-based genome editing and confirmed that secreted P. flocculosa effector Pf2826 is required for full biocontrol activity. We monitored the localization of the effector Pf2826 with C-terminal mCherry tag and found it localized around the haustoria and on powdery mildew spores. His-tagged Pf2826 recombinant protein was expressed, purified, and used as bait in a pull-down assay from total proteins extracted during the tripartite interaction. Potential interactors were identified by LC-MS/MS analysis after removing unspecific interactions found in the negative controls. A two-way yeast two-hybrid assay validated that Pf2826 interacted with barley pathogenesis-related (PR) proteins HvPR1a and chitinase and with an effector protein from powdery mildew. CONCLUSIONS: In contrast to the usual modes of action of competition, parasitism, and antibiosis ascribed to BCAs, this study shows that effector pf2826 plays a vital role in the biocontrol activity of P. flocculosa by interacting with plant PR proteins and a powdery mildew effector, altering the host-pathogen interaction.

BACKGROUND: The regulation of glycolysis and autophagy during feeding and metamorphosis in holometabolous insects is a complex process that is not yet fully understood. Insulin regulates glycolysis during the larval feeding stage, allowing the insects to grow and live. However, during metamorphosis, 20-hydroxyecdysone (20E) takes over and regulates programmed cell death (PCD) in larval tissues, leading to degradation and ultimately enabling the insects to transform into adults. The precise mechanism through which these seemingly contradictory processes are coordinated remains unclear and requires further research. To understand the coordination of glycolysis and autophagy during development, we focused our investigation on the role of 20E and insulin in the regulation of phosphoglycerate kinase 1 (PGK1). We examined the glycolytic substrates and products, PGK1 glycolytic activity, and the posttranslational modification of PGK1 during the development of Helicoverpa armigera from feeding to metamorphosis. RESULTS: Our findings suggest that the coordination of glycolysis and autophagy during holometabolous insect development is regulated by a balance between 20E and insulin signaling pathways. Glycolysis and PGK1 expression levels were decreased during metamorphosis under the regulation of 20E. Insulin promoted glycolysis and cell proliferation via PGK1 phosphorylation, while 20E dephosphorylated PGK1 via phosphatase and tensin homolog (PTEN) to repress glycolysis. The phosphorylation of PGK1 at Y194 by insulin and its subsequent promotion of glycolysis and cell proliferation were important for tissue growth and differentiation during the feeding stage. However, during metamorphosis, the acetylation of PGK1 by 20E was key in initiating PCD. Knockdown of phosphorylated PGK1 by RNA interference (RNAi) at the feeding stage led to glycolysis suppression and small pupae. Insulin via histone deacetylase 3 (HDAC3) deacetylated PGK1, whereas 20E via acetyltransferase arrest-defective protein 1 (ARD1) induced PGK1 acetylation at K386 to stimulate PCD. Knockdown of acetylated-PGK1 by RNAi at the metamorphic stages led to PCD repression and delayed pupation. CONCLUSIONS: The posttranslational modification of PGK1 determines its functions in cell proliferation and PCD. Insulin and 20E counteractively regulate PGK1 phosphorylation and acetylation to give it dual functions in cell proliferation and PCD.

BACKGROUND: Post mortem human brain tissue is an essential resource to study cell types, connectivity as well as subcellular structures down to the molecular setup of the central nervous system especially with respect to the plethora of brain diseases. A key method is immunostaining with fluorescent dyes, which allows high-resolution imaging in three dimensions of multiple structures simultaneously. Although there are large collections of formalin-fixed brains, research is often limited because several conditions arise that complicate the use of human brain tissue for high-resolution fluorescence microscopy. RESULTS: In this study, we developed a clearing approach for immunofluorescence-based analysis of perfusion- and immersion-fixed post mortem human brain tissue, termed human Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / In situ hybridization-compatible Tissue-hYdrogel (hCLARITY). hCLARITY is optimized for specificity by reducing off-target labeling and yields very sensitive stainings in human brain sections allowing for super-resolution microscopy with unprecedented imaging of pre- and postsynaptic compartments. Moreover, hallmarks of Alzheimer's disease were preserved with hCLARITY, and importantly classical 3,3'-diaminobenzidine (DAB) or Nissl stainings are compatible with this protocol. hCLARITY is very versatile as demonstrated by the use of more than 30 well performing antibodies and allows for de- and subsequent re-staining of the same tissue section, which is important for multi-labeling approaches, e.g., in super-resolution microscopy. CONCLUSIONS: Taken together, hCLARITY enables research of the human brain with high sensitivity and down to sub-diffraction resolution. It therefore has enormous potential for the investigation of local morphological changes, e.g., in neurodegenerative diseases.

BACKGROUND: A fraction of all genomes is composed of transposable elements (TEs) whose mobility needs to be carefully controlled. In gonads, TE activity is repressed by PIWI-interacting RNAs (piRNAs), a class of small RNAs synthesized by heterochromatic loci enriched in TE fragments, called piRNA clusters. Maintenance of active piRNA clusters across generations is secured by maternal piRNA inheritance providing the memory for TE repression. On rare occasions, genomes encounter horizontal transfer (HT) of new TEs with no piRNA targeting them, threatening the host genome integrity. Naïve genomes can eventually start to produce new piRNAs against these genomic invaders, but the timing of their emergence remains elusive. RESULTS: Using a set of TE-derived transgenes inserted in different germline piRNA clusters and functional assays, we have modeled a TE HT in Drosophila melanogaster. We have found that the complete co-option of these transgenes by a germline piRNA cluster can occur within four generations associated with the production of new piRNAs all along the transgenes and the germline silencing of piRNA sensors. Synthesis of new transgenic TE piRNAs is linked to piRNA cluster transcription dependent on Moonshiner and heterochromatin mark deposition that propagates more efficiently on short sequences. Moreover, we found that sequences located within piRNA clusters can have different piRNA profiles and can influence transcript accumulation of nearby sequences. CONCLUSIONS: Our study reveals that genetic and epigenetic properties, such as transcription, piRNA profiles, heterochromatin, and conversion efficiency along piRNA clusters, could be heterogeneous depending on the sequences that compose them. These findings suggest that the capacity of transcriptional signal erasure induced by the chromatin complex specific of the piRNA cluster can be incomplete through the piRNA cluster loci. Finally, these results have revealed an unexpected level of complexity that highlights a new magnitude of piRNA cluster plasticity fundamental for the maintenance of genome integrity.

BACKGROUND: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. RESULTS: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. CONCLUSIONS: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

BACKGROUND: The factors that maintain phenotypic and genetic variation within a population have received long-term attention in evolutionary biology. Here the genetic basis and evolution of the geographically widespread variation in twig trichome color (from red to white) in a shrub Melastoma normale was investigated using Pool-seq and evolutionary analyses. RESULTS: The results show that the twig trichome coloration is under selection in different light environments and that a 6-kb region containing an R2R3 MYB transcription factor gene is the major region of divergence between the extreme red and white morphs. This gene has two highly divergent groups of alleles, one of which likely originated from introgression from another species in this genus and has risen to high frequency (> 0.6) within each of the three populations under investigation. In contrast, polymorphisms in other regions of the genome show no sign of differentiation between the two morphs, suggesting that genomic patterns of diversity have been shaped by homogenizing gene flow. Population genetics analysis reveals signals of balancing selection acting on this gene, and it is suggested that spatially varying selection is the most likely mechanism of balancing selection in this case. CONCLUSIONS: This study demonstrate that polymorphisms on a single transcription factor gene largely confer the twig trichome color variation in M. normale, while also explaining how adaptive divergence can occur and be maintained in the face of gene flow.

BACKGROUND: Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon. RESULTS: Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies-including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5' intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450. CONCLUSIONS: These findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.

BACKGROUND: Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. RESULTS: We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. CONCLUSIONS: Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

BACKGROUND: The Western mosquitofish, Gambusia affinis, is a model for sex chromosome organization and evolution of female heterogamety. We previously identified a G. affinis female-specific marker, orthologous to the aminomethyl transferase (amt) gene of the related platyfish (Xiphophorus maculatus). Here, we have analyzed the structure and differentiation of the G. affinis W-chromosome, using a cytogenomics and bioinformatics approach. RESULTS: The long arm of the G. affinis W-chromosome (Wq) is highly enriched in dispersed repetitive sequences, but neither heterochromatic nor epigenetically silenced by hypermethylation. In line with this, Wq sequences are highly transcribed, including an active nucleolus organizing region (NOR). Female-specific SNPs and evolutionary young transposable elements were highly enriched and dispersed along the W-chromosome long arm, suggesting constrained recombination. Wq copy number expanded elements also include female-specific transcribed sequences from the amt locus with homology to TE. Collectively, the G. affinis W-chromosome is actively differentiating by sex-specific copy number expansion of transcribed TE-related elements, but not (yet) by extensive sequence divergence or gene decay. CONCLUSIONS: The G. affinis W-chromosome exhibits characteristic genomic properties of an evolutionary young sex chromosome. Strikingly, the observed sex-specific changes in the genomic landscape are confined to the W long arm, which is separated from the rest of the W-chromosome by a neocentromere acquired during sex chromosome evolution and may thus have become functionally insulated. In contrast, W short arm sequences were apparently shielded from repeat-driven differentiation, retained Z-chromosome like genomic features, and may have preserved pseudo-autosomal properties.

BACKGROUND: Anthocyanins, a class of specialized metabolites that are ubiquitous among plant species, have attracted a great deal of attention from plant biologists due to their chemical diversity. They confer purple, pink, and blue colors that attract pollinators, protect plants from ultraviolet (UV) radiation, and scavenge reactive oxygen species (ROS) to facilitate plant survival during abiotic stress. In a previous study, we identified Beauty Mark (BM) in Gossypium barbadense as an activator of the anthocyanin biosynthesis pathway; this gene also directly led to the formation of a pollinator-attracting purple spot. RESULTS: Here, we found that a single nucleotide polymorphism (SNP) (C/T) within the BM coding sequence was responsible for variations in this trait. Transient expression assays of BM from G. barbadense and G. hirsutum in Nicotiana benthamiana using luciferase reporter gene also suggested that SNPs in the coding sequence could be responsible for the absent beauty mark phenotype observed in G. hirsutum. We next demonstrated that the beauty mark and UV floral patterns are associated phenotypes and that UV exposure resulted in increased ROS generation in floral tissues; BM thus contributed to ROS scavenging in G. barbadense and wild cotton plants with flowers containing the beauty mark. Furthermore, a nucleotide diversity analysis and Tajima's D Test suggested that there have been strong selective sweeps in the GhBM locus during G. hirsutum domestication. CONCLUSIONS: Taken together, these results suggest that cotton species differ in their approaches to absorbing or reflecting UV light and thus exhibit variations in floral anthocyanin biosynthesis to scavenge reactive ROS; furthermore, these traits are related to the geographic distribution of cotton species.

Finding the optimal balance between survival and reproduction is a central puzzle in life-history theory. The terminal investment hypothesis predicts that when individuals encounter a survival threat that compromises future reproductive potential, they will increase immediate reproductive investment to maximise fitness. Despite decades of research on the terminal investment hypothesis, findings remain mixed. We examined the terminal investment hypothesis with a meta-analysis of studies that measured reproductive investment of multicellular iteroparous animals after a non-lethal immune challenge. We had two main aims. The first was to investigate whether individuals, on average, increase reproductive investment in response to an immune threat, as predicted by the terminal investment hypothesis. We also examined whether such responses vary adaptively on factors associated with the amount of reproductive opportunities left (residual reproductive value) in the individuals, as predicted by the terminal investment hypothesis. The second was to provide a quantitative test of a novel prediction based on the dynamic threshold model: that an immune threat increases between-individual variance in reproductive investment. Our results provided some support for our hypotheses. Older individuals, who are expected to have lower residual reproductive values, showed stronger mean terminal investment response than younger individuals. In terms of variance, individuals showed a divergence in responses, leading to an increase in variance. This increase in variance was especially amplified in longer-living species, which was consistent with our prediction that individuals in longer-living species should respond with greater individual variation due to increased phenotypic plasticity. We find little statistical evidence of publication bias. Together, our results highlight the need for a more nuanced view on the terminal investment hypothesis and a greater focus on the factors that drive individual responses.

BACKGROUND: Hybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events. RESULTS: We found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids. CONCLUSIONS: Our results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events-as expected by mitotic repair of rare chromosomal breaks-but rather of acute episodes involving many LOH events in a short period of time.

BACKGROUND: Gene duplication events are critical for the evolution of new gene functions. Aristaless is a major regulator of distinct developmental processes. It is most known for its role during appendage development across animals. However, more recently other distinct biological functions have been described for this gene and its duplicates. Butterflies and moths have two copies of aristaless, aristaless1 (al1) and aristaless2 (al2), as a result of a gene duplication event. Previous work in Heliconius has shown that both copies appear to have novel functions related to wing color patterning. Here we expand our knowledge of the expression profiles associated with both ancestral and novel functions of Al1 across embryogenesis and wing pigmentation. Furthermore, we characterize Al2 expression, providing a comparative framework between gene copies within the same species, allowing us to understand the origin of new functions following gene duplication. RESULTS: Our work shows that the expression of both Al1 and Al2 is associated with the ancestral function of sensory appendage (leg, mouth, spines, and eyes) development in embryos. Interestingly, Al1 exhibits higher expression earlier in embryogenesis while the highest levels of Al2 expression are shifted to later stages of embryonic development. Furthermore, Al1 localization appears extranuclear while Al2 co-localizes tightly with nuclei earlier, and then also expands outside the nucleus later in development. Cellular expression of Al1 and Al2 in pupal wings is broadly consistent with patterns observed during embryogenesis. We also describe, for the first time, how Al1 localization appears to correlate with zones of anterior/posterior elongation of the body during embryonic growth, showcasing a possible new function related to Aristaless' previously described role in appendage extension. CONCLUSIONS: Overall, our data suggest that while both gene copies play a role in embryogenesis and wing pigmentation, the duplicates have diverged temporally and mechanistically across those functions. Our study helps clarify principles behind sub-functionalization and gene expression evolution associated with developmental functions following gene duplication events.