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Schematic representation of the Griottes workflow: the program takes multiple data formats as input to generate a graph in a single command. The red boxes contain objects, the green boxes processes
a Confocal dorsal view of the zebrafish adult pallium. Glial fibrillary acidic protein (GFAP) in green, Proliferating cell nuclear antigen (PCNA) in magenta and Zonula occludens-1 (ZO1) in white or blue. ZO1 is highlighting the apical domain of the cells allowing the identification of their apical area. Inset: spotlight on a limited tissue area, the ZO1 membrane staining allows for the exact localization of cell membranes. Scale bar is 100 μm. b Griottes incorporates different network construction methods. The contact-based method connects cells sharing a common membrane, the Delaunay and Geometric graphs are commonly used graph-generation methods. c The graphs generated from a same set of nodes with different construction rules have different properties. For instance, the degree distribution of a Geometric graph is broader than that of the Contact and Delaunay graph. d Mean PCNA signal within the cells in the example tissue. Cells with an average intensity above 6500 (red line) are considered PCNA+, the other cells are PCNA−. e Thresholding intensity signals converts a network populated with continuous fluorescence signals to a network populated with categorical cell types. f. Representation of the example network (panel b) where node colors represent cell type. Left: the network is projected on the ZO1 signal. Right: the network is projected on the PCNA signal. This method reliably incorporates cell type information into the network representation of the tissue. g Left: connected cells can have widely varying contact surfaces. Right: this information can be encoded into the network by weighting the links between cells. Two differing cell-cell interfaces (pink lines) have different link weights in the network representation of the tissue (pink arrows). h The connection between cells can be quantified at scale: the histogram of link weights in the tissue shown in panel a
a Sections of a MSC spheroid imaged with a light-sheet microscope. The technique allows in-depth imaging of tissue structures. Scale bar is 50 μm. b 3D network representation of a MSC spheroid. Different cell types are identified based upon CD146 fluorescence measurements. c Comparison between the degree distribution of an example spheroid (panel b, bars) and the batch distribution (N = 5, red line). d Comparison between the link-length distribution of an example spheroid (bars) and the batch distribution (red line). e The network representation makes it possible to identify cells on the outer layer of the spheroid (red) from the inner cells (blue). We can “peel off” the outer layers successively, revealing the inner structure and composition of the spheroid. f Cell degree as a function of the layer number, the average degree is larger for the layers near the center of the spheroid. Blue dots show one example spheroid and red dashed line represents average over the experimental batch. g Distance between cell centers (in μm) as a function of the layer number. Blue dots show one example spheroid and red dashed line represents average over the experimental batch
a Reconstruction of the zebrafish telencephalon from a data table. Colors represent the different cell types entered in the table by the user. b Network construction with Griottes from a point-cloud using the Delaunay construction rule. c Degree distribution of cells composing the zebrafish telencephalon after the network construction using the Delaunay rule. d Composition of the zebrafish telencephalon: a vast majority of the cells are GFAP+/PCNA−. e From the network representation of the tissue we can extract clusters of any given cell type (left). Distribution of PCNA+ cluster sizes for clusters of two cells or more (right). f Percentage of cells that belong to a cluster of their cell type of size larger than 2. All GFAP+/PCNA− cells are connected and belong to the same cluster. Conversely, a majority of GFAP+/PCNA− and GFAP+/PCNA+ cells aren’t connected to any other cell of the same type
Article
  • Gustave RonteixGustave Ronteix
  • Andrey AristovAndrey Aristov
  • Valentin BonnetValentin Bonnet
  • [...]
  • Charles N. BaroudCharles N. Baroud
Background Microscopy techniques and image segmentation algorithms have improved dramatically this decade, leading to an ever increasing amount of biological images and a greater reliance on imaging to investigate biological questions. This has created a need for methods to extract the relevant information on the behaviors of cells and their interactions, while reducing the amount of computing power required to organize this information. Results This task can be performed by using a network representation in which the cells and their properties are encoded in the nodes, while the neighborhood interactions are encoded by the links. Here, we introduce Griottes, an open-source tool to build the “network twin” of 2D and 3D tissues from segmented microscopy images. We show how the library can provide a wide range of biologically relevant metrics on individual cells and their neighborhoods, with the objective of providing multi-scale biological insights. The library’s capacities are demonstrated on different image and data types. Conclusions This library is provided as an open-source tool that can be integrated into common image analysis workflows to increase their capacities.
 
Article
Background Kinesin-3 family motors drive diverse cellular processes and have significant clinical importance. The ATPase cycle is integral to the processive motility of kinesin motors to drive long-distance intracellular transport. Our previous work has demonstrated that kinesin-3 motors are fast and superprocessive with high microtubule affinity. However, chemomechanics of these motors remain poorly understood. Results We purified kinesin-3 motors using the Sf9-baculovirus expression system and demonstrated that their motility properties are on par with the motors expressed in mammalian cells. Using biochemical analysis, we show for the first time that kinesin-3 motors exhibited high ATP turnover rates, which is 1.3- to threefold higher compared to the well-studied kinesin-1 motor. Remarkably, these ATPase rates correlate to their stepping rate, suggesting a tight coupling between chemical and mechanical cycles. Intriguingly, kinesin-3 velocities (KIF1A > KIF13A > KIF13B > KIF16B) show an inverse correlation with their microtubule-binding affinities (KIF1A < KIF13A < KIF13B < KIF16B). We demonstrate that this differential microtubule-binding affinity is largely contributed by the positively charged residues in loop8 of the kinesin-3 motor domain. Furthermore, microtubule gliding and cellular expression studies displayed significant microtubule bending that is influenced by the positively charged insert in the motor domain, K-loop, a hallmark of kinesin-3 family. Conclusions Together, we propose that a fine balance between the rate of ATP hydrolysis and microtubule affinity endows kinesin-3 motors with distinct mechanical outputs. The K-loop, a positively charged insert in the loop12 of the kinesin-3 motor domain promotes microtubule bending, an interesting phenomenon often observed in cells, which requires further investigation to understand its cellular and physiological significance.
 
Article
Background Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. Results We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. Conclusion Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Graphical abstract Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.
 
Article
Background Cyanobacteria are the major prokaryotic primary producers occupying a range of aquatic habitats worldwide that differ in levels of salinity, making them a group of interest to study one of the major unresolved conundrums in aquatic microbiology which is what distinguishes a marine microbe from a freshwater one? We address this question using ecogenomics of a group of picocyanobacteria (cluster 5) that have recently evolved to inhabit geographically disparate salinity niches. Our analysis is made possible by the sequencing of 58 new genomes from freshwater representatives of this group that are presented here, representing a 6-fold increase in the available genomic data. Results Overall, freshwater strains had larger genomes (≈2.9 Mb) and %GC content (≈64%) compared to brackish (2.69 Mb and 64%) and marine (2.5 Mb and 58.5%) isolates. Genomic novelties/differences across the salinity divide highlighted acidic proteomes and specific salt adaptation pathways in marine isolates (e.g., osmolytes/compatible solutes - glycine betaine/ ggp/gpg/gmg clusters and glycerolipids glpK / glpA ), while freshwater strains possessed distinct ion/potassium channels, permeases (aquaporin Z), fatty acid desaturases, and more neutral/basic proteomes. Sulfur, nitrogen, phosphorus, carbon (photosynthesis), or stress tolerance metabolism while showing distinct genomic footprints between habitats, e.g., different types of transporters, did not obviously translate into major functionality differences between environments. Brackish microbes show a mixture of marine (salt adaptation pathways) and freshwater features, highlighting their transitional nature. Conclusions The plethora of freshwater isolates provided here, in terms of trophic status preference and genetic diversity, exemplifies their ability to colonize ecologically diverse waters across the globe. Moreover, a trend towards larger and more flexible/adaptive genomes in freshwater picocyanobacteria may hint at a wider number of ecological niches in this environment compared to the relatively homogeneous marine system.
 
Article
Background High-throughput live-cell imaging is a powerful tool to study dynamic cellular processes in single cells but creates a bottleneck at the stage of data analysis, due to the large amount of data generated and limitations of analytical pipelines. Recent progress on deep learning dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and tools spanning the complete range of image analysis are still needed. Results We present Cell-ACDC, an open-source user-friendly GUI-based framework written in Python, for segmentation, tracking and cell cycle annotations. We included state-of-the-art deep learning models for single-cell segmentation of mammalian and yeast cells alongside cell tracking methods and an intuitive, semi-automated workflow for cell cycle annotation of single cells. Using Cell-ACDC, we found that mTOR activity in hematopoietic stem cells is largely independent of cell volume. By contrast, smaller cells exhibit higher p38 activity, consistent with a role of p38 in regulation of cell size. Additionally, we show that, in S. cerevisiae , histone Htb1 concentrations decrease with replicative age. Conclusions Cell-ACDC provides a framework for the application of state-of-the-art deep learning models to the analysis of live cell imaging data without programming knowledge. Furthermore, it allows for visualization and correction of segmentation and tracking errors as well as annotation of cell cycle stages. We embedded several smart algorithms that make the correction and annotation process fast and intuitive. Finally, the open-source and modularized nature of Cell-ACDC will enable simple and fast integration of new deep learning-based and traditional methods for cell segmentation, tracking, and downstream image analysis. Source code: https://github.com/SchmollerLab/Cell_ACDC
 
Archaic linkage blocks. a Timeline for the formation of archaic haplotypes. As a rough estimate, the chimpanzee line separated from the h. sapiens line about 5 million years ago, while the Denisova h. split off around 500,000 years ago. This provides a time window of 0.5–5 million years, at which point mutations accumulated in the genome of the common ancestor of Denisova h. and H. sapien. The derived “archaic” haplotypes formed by these mutations were preserved in Denisova and serve as a reference for the recombination events rearranging these haplotypes in the human during a period of at least 500,000 years after separation. b Formation of archaic linkage blocks. NCO recombination between the derived archaic haplotype (blue), consisting of the derived alleles of these SNPs (solid circles), and the corresponding ancestral haplotype (green) created a set of haplotype variants that are still present in modern humans. In the given example the archaic linkage block consists of 6 SNPs of which SNPs 1, 3, and 6 are still in perfect linkage (block SNPs). The derived alleles of these SNPs form the core haplotype (hap), which represents the remaining fragment of the derived archaic haplotype. SNPs 2, 4, and 5 are singletons whose alleles had been shifted between haplotypes by NCO recombination. Their association to the derived haplotype is indicated by the linkage disequilibrium (D’a,hap) between their derived alleles (a) and the core haplotype (hap). c Example of an archaic linkage block. The panel shows the location of the SNPs of an archaic linkage block in reference to the intron/exon structure of the ESYT2 gene. In this visualization, the association of the derived alleles to the core haplotype (r² = 1) is indicated by the respective r²a,hap value. The horizontal line connecting the block SNPs indicates the location of the core haplotype; single vertical lines indicate the NCO rate and location of the singleton SNPs. d Haplotype composition of the linkage block. The panel displays the haplotype composition of the ESYT2 linkage block defined for the 99 individuals of the LWK cohort. The presence of derived alleles (red circles) and ancestral alleles (white circles) for each haplotype variant is indicated. Numbers indicate the absolute frequency of the haplotype in the cohort; downward and upward arrows on top of the plot respectively indicate retracting and expanding derived alleles, a double arrow indicates a neutral allele that switched the position in several haplotypes without affecting its allele frequency; horizontal lines refer to block SNPs
Correlation of the NCO recombination rate with functional genomic parameters. a Function vs. fitness. The NCO rate 1 – D’²a,hap was used as a proxy for the NCO recombination rate. The correlation of this parameter of about 1.9 million archaic SNPs is shown for function-associated GWAVA scores (left panel) and fitness-associated fitCons scores. The plot displays the average scores for about 1.0 million block SNPs (red dot) and 900,000 singleton SNPs (blue dots). The singleton SNPs were binned according to the 1 – D’2a,hap value defined for the LWK population. Dashed horizontal lines indicate the average scores of the entire set of archaic SNPs. Asterisks indicate significant differences between data point pairs (p < 0.05; Mann-Whitney U test). b Meiotic CO recombination hotspots. The solid line represents the average NCO rate (1 – D’2a,hap) plotted in reference to the distance of the alleles to meiotic CO recombination hotspots (defined by DMC1 ChIP-Seq tracks of human testis [20]). The dashed line of the second peak represents the hypothetical NCO rate in reference to a second recombination hotspot located at the average distance of about 67 kb. c Gene boundaries. The average NCO rate (1 – D’2a,hap), is plotted in reference their distance to the boundary of the closest annotated gene. Peaks in NCO rate are evident at both 5′- and 3′-boundaries (indicated by dashed vertical lines). Gene regions are marked in red, intergenic regions in blue; dashed horizontal line represents the average NCO rate of the entire set of archaic alleles. (d) Genic sub-regions. Gene regions (red) were further delineated into their sub-regions (5′UTRs, introns, splice sites, exons and 3′UTRs); intergenic regions (blue) were divided according to their distance to the boundaries both upstream and downstream of the genic region (0–5 kb, 5–50 kb, >50kb). Bars represent the average NCO rate (1 – D’²a,hap) in the respective region. Asterisks indicate significant differences between the indicated bars (p < 0.05; Mann-Whitney U test). e Open chromatin. The line chart depicts the association of the NCO recombination rate with open chromatin. The average NCO rate (1 – D’²a,hap) is plotted in reference to the distance of the alleles to the boundary of open chromatin (defined by ENCODE tracks of DNase I sensitivity). Gray bars indicate the number of archaic SNPs in the respective distance-bin
Correlation of the NCO recombination rate with epigenetic marks. a Heatmap and cumulative scores of histone and DNA marks. The impact of epigenetic marks on the NCO recombination rate was assessed by determining the average NCO rate (1 – D’²a,hap) for alleles located on tracks of histone marks (bivalent H3K27me3 & H3K4me1, H3K27me3, H3K4me1, H3K4me3, H3K4me2, H3K9ac, H3K27ac, H3K79me2, H3K36me3, H3K9me3), of 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC), as well as of open chromatin defined by DNase I sensitivity. To avoid any bias due to the genomic location of the SNPs, the data set was divided into the respective genic sub-region (intergenic, upstream > 5 kb, 5′ UTR, intronic, exonic, 3′ UTR, downstream < 5 kb). Each combination of epigenetic mark and genic sub-region were tested independently using Mann-Whitney U test to determine if the overlap with the respective mark led to a significant difference in the NCO rate (Additional file 3: Table S2). Left panel: The color code in the heat map indicates the deviation from the average the NCO rate Δ(1 - D’²a,hap) of the respective sub-region in reference to regions without any mark (ranging from − 0.1 (blue) to 0.1 (red)). “ns” indicates a non-significant association; solid horizontal line separates recombination promoting marks (green arrow) from recombination repressing marks (red arrow). Right panel: The bars represent the average increase in the NCO rate score for each of the marks across the entire genome. ChIP-Seq tracks of histone marks were compiled from ENCODE data of various tissues and cell lines while tracks of 5mC, DNase I, and 5hmC marks were derived from the embryonic stem cell H1 [1, 21]. CO recombination hotspots marked by DCM1 tracks were excluded from the analysis. b Fold change in track overlap. The fold change in the overlap with 5hmC tracks (red), H3K27me3/H3K4me3-defined bivalent regions (orange), open chromatin-related DNase I tracks (blue) and 5mC-free tracks (green) and 5mC-containing tracks (gray) in response to the increase in the average NCO rate (1 – D’²a,hap) is shown. The dots indicate the fold change for the binned NCO rate average. Only singleton SNPs are shown. Dashed lines represent linear regressions; slope coefficient (m) and r² value are indicated. Fold change for bivalency and DNase I was compiled from the ENCODE data set while 5mC and 5hmC were compiled from data provided for the H1 stem cell
Association of 5hmC and NCO recombination rates. a Association in epigenetically defined regions. NCO rate, chromatin state and DNA marks are shown for 15 epigenetic sub-regions comprising active transcription start sites (TssA), TssA flanking regions (TssAFlnk), transcribed regions (Tx), weak transcribed regions (TxWk), Tx flanking regions (TxFlnk), gene-associated enhancer (EnhG), enhancer (Enh), zinc finger nuclear factor regions (ZNF), repeats (Rpts), heterochromatin (Het), bivalent transcriptional start site (TssBiv), bivalent flanking regions (BivFlnk), bivalent enhancer (EnhBiv), polycomb repressed region (ReprPC), weak polycomb repressed region (ReprPCWk) and quiescent regions (Quies) [1]. Histone marks are shown in Additional file 1: Fig. S4. The calculations were carried out separately for each of 111 reference genomes using the entire data set of 1.9 million archaic SNPs. The boxplots indicate for each region the average NCO rate (1 − D’²a,hap), as well as the average track overlap of these SNPs with 5hmC marks, DNase I-defined, and 5mC-defined open chromatin. The NCO rate was calculated using the data from the LWK cohort, while 5mC, DNase I, and 5mC tracks were defined for the H1 stem cell line [1, 21]. The r- and p-values refer to a Spearman rank correlation carried out between these relative frequencies of the marks and the respective NCO rate data. b Correlation of NCO recombination rate with the 5hmC frequency. The plot shows the average NCO rate and corresponding track overlap of the SNP with 5hmC marks for each of the 15 epigenetically defined subregions. The line was derived by Pearson correlation, the p and r² values are indicated
Model of targeted and guided NCO recombination. a Targeted NCO recombination. 5hmC is enriched in functional regions such as UTRs and exons (compare Fig. 4b). Thus, utilization of marks like 5hmC would allow to target the NCO recombination machinery towards function-related loci, which would in turn undergo more frequent recombination than quiescent or non-functional regions. b Guided NCO recombination. 5hmC is also a product of oxidative C-demethylation and indicative of recently opened chromatin. As this process can also occur in response to environmental signals, newly formed 5hmC marks would direct the recombination machinery preferably to these sites. In this model, the environmental pressure would therefore “guide” genetic evolution in a Lamarckian sense
Article
Background Non-crossover (NCO) refers to a mechanism of homologous recombination in which short tracks of DNA are copied between homologue chromatids. The allelic changes are typically restricted to one or few SNPs, which potentially allow for the gradual adaptation and maturation of haplotypes. It is assumed to be a stochastic process but the analysis of archaic and modern human haplotypes revealed a striking variability in local NCO recombination rates. Methods NCO recombination rates of 1.9 million archaic SNPs shared with Denisovan hominids were defined by a linkage study and correlated with functional and genomic annotations as well as ChIP-Seq data from modern humans. Results We detected a strong correlation between NCO recombination rates and the function of the respective region: low NCO rates were evident in introns and quiescent intergenic regions but high rates in splice sites, exons, 5′- and 3′-UTRs, as well as CpG islands. Correlations with ChIP-Seq data from ENCODE and other public sources further identified epigenetic modifications that associated directly with these recombination events. A particularly strong association was observed for 5-hydroxymethylcytosine marks (5hmC), which were enriched in virtually all of the functional regions associated with elevated NCO rates, including CpG islands and ‘poised’ bivalent regions. Conclusion Our results suggest that 5hmC marks may guide the NCO machinery specifically towards functionally relevant regions and, as an intermediate of oxidative demethylation, may open a pathway for environmental influence by specifically targeting recently opened gene loci.
 
Article
Background ATP-dependent chromatin remodeling complexes are multi-protein machines highly conserved across eukaryotic genomes. They control sliding and displacing of the nucleosomes, modulating histone-DNA interactions and making nucleosomal DNA more accessible to specific binding proteins during replication, transcription, and DNA repair, which are processes involved in cell division. The SRCAP and p400/Tip60 chromatin remodeling complexes in humans and the related Drosophila Tip60 complex belong to the evolutionary conserved INO80 family, whose main function is promoting the exchange of canonical histone H2A with the histone variant H2A in different eukaryotic species. Some subunits of these complexes were additionally shown to relocate to the mitotic apparatus and proposed to play direct roles in cell division in human cells. However, whether this phenomenon reflects a more general function of remodeling complex components and its evolutionary conservation remains unexplored. Results We have combined cell biology, reverse genetics, and biochemical approaches to study the subcellular distribution of a number of subunits belonging to the SRCAP and p400/Tip60 complexes and assess their involvement during cell division progression in HeLa cells. Interestingly, beyond their canonical chromatin localization, the subunits under investigation accumulate at different sites of the mitotic apparatus (centrosomes, spindle, and midbody), with their depletion yielding an array of aberrant outcomes of mitosis and cytokinesis, thus causing genomic instability. Importantly, this behavior was conserved by the Drosophila melanogaster orthologs tested, despite the evolutionary divergence between fly and humans has been estimated at approximately 780 million years ago. Conclusions Overall, our results support the existence of evolutionarily conserved diverse roles of chromatin remodeling complexes, whereby subunits of the SRCAP and p400/Tip60 complexes relocate from the interphase chromatin to the mitotic apparatus, playing moonlighting functions required for proper execution of cell division.
 
Article
Background The high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function. Results We demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina. Conclusions Overall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions.
 
Article
Background Neuropsychiatric disorders, such as schizophrenia (SZ) and autism spectrum disorder (ASD), are common, multi-factorial and multi-symptomatic disorders. Ample evidence implicates oxidative stress, deficient repair of oxidative DNA lesions and DNA damage in the development of these disorders. However, it remains unclear whether insufficient DNA repair and resulting DNA damage are causally connected to their aetiopathology, or if increased levels of DNA damage observed in patient tissues merely accumulate as a consequence of cellular dysfunction. To assess a potential causal role for deficient DNA repair in the development of these disorders, we behaviourally characterized a mouse model in which CaMKIIa-Cre-driven postnatal conditional knockout (KO) of the core base-excision repair (BER) protein XRCC1 leads to accumulation of unrepaired DNA damage in the forebrain. Results CaMKIIa-Cre expression caused specific deletion of XRCC1 in the dorsal dentate gyrus (DG), CA1 and CA2 and the amygdala and led to increased DNA damage therein. While motor coordination, cognition and social behaviour remained unchanged, XRCC1 KO in the forebrain caused increased anxiety-like behaviour in males, but not females, as assessed by the light–dark box and open field tests. Conversely, in females but not males, XRCC1 KO caused an increase in learned fear-related behaviour in a cued (Pavlovian) fear conditioning test and a contextual fear extinction test. The relative density of the GABA(A) receptor alpha 5 subunit (GABRA5) was reduced in the amygdala and the dorsal CA1 in XRCC1 KO females, whereas male XRCC1 KO animals exhibited a significant reduction of GABRA5 density in the CA3. Finally, assessment of fast-spiking, parvalbumin-positive (PV) GABAergic interneurons revealed a significant increase in the density of PV+ cells in the DG of male XRCC1 KO mice, while females remained unchanged. Conclusions Our results suggest that accumulation of unrepaired DNA damage in the forebrain alters the GABAergic neurotransmitter system and causes behavioural deficits in relation to innate and learned anxiety in a sex-dependent manner. Moreover, the data uncover a previously unappreciated connection between BER deficiency, unrepaired DNA damage in the hippocampus and a sex-specific anxiety-like phenotype with implications for the aetiology and therapy of neuropsychiatric disorders.
 
Article
Background Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) acts as a transcriptional coactivator and regulates mitochondrial function. Various isoforms are generated by alternative splicing and differentially regulated promoters. In the heart, total PGC-1α deficiency knockout leads to dilatative cardiomyopathy, but knowledge on the complexity of cardiac isoform expression of PGC-1α remains sparse. Thus, this study aims to generate a reliable dataset on cardiac isoform expression pattern by long-read mRNA sequencing, followed by investigation of differential regulation of PGC-1α isoforms under metabolic and ischemic stress, using high-fat-high-sucrose-diet-induced obesity and a murine model of myocardial infarction. Results Murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery) was used for long-read mRNA sequencing, resulting in full-length transcriptomes including 58,000 mRNA isoforms with 99% sequence accuracy. Automatic bioinformatic analysis as well as manual similarity search against exonic sequences leads to identification of putative coding PGC-1α isoforms, validated by PCR and Sanger sequencing. Thereby, 12 novel transcripts generated by hitherto unknown splicing events were detected. In addition, we postulate a novel promoter with homologous and strongly conserved sequence in human heart. High-fat diet as well as ischemia/reperfusion (I/R) injury transiently reduced cardiac expression of PGC-1α isoforms, with the most pronounced effect in the infarcted area. Recovery of PGC-1α-isoform expression was even more decelerated when I/R was performed in diet-induced obese mice. Conclusions We deciphered for the first time a complete full-length transcriptome of the murine and human heart, identifying novel putative PGC-1α coding transcripts including a novel promoter. These transcripts are differentially regulated in I/R and obesity suggesting transcriptional regulation and alternative splicing that may modulate PGC-1α function in the injured and metabolically challenged heart.
 
Article
Background Chimeras are genetically mixed entities resulting from the fusion of two or more conspecifics. This phenomenon is widely distributed in nature and documented in a variety of animal and plant phyla. In corals, chimerism initiates at early ontogenic states (larvae to young spat) and results from the fusion between two or more closely settled conspecifics. When compared to genetically homogenous colonies (non-chimeras), the literature has listed ecological and evolutionary benefits for traits at the chimeric state, further positioning coral chimerism as an evolutionary rescue instrument. However, the molecular mechanisms underlying this suggestion remain unknown. Results To address this question, we developed field monitoring and multi-omics approaches to compare the responses of chimeric and non-chimeric colonies acclimated for 1 year at 10-m depth or exposed to a stressful environmental change (translocation from 10- to 2-m depth for 48h). We showed that chimerism in the stony coral Stylophora pistillata is associated with higher survival over a 1-year period. Transcriptomic analyses showed that chimeras lose transcriptomic plasticity and constitutively express at higher level (frontload) genes responsive to stress. This frontloading may prepare the colony to face at any time environmental stresses which explain its higher robustness. Conclusions These results show that chimeras are environmentally robust entities with an enhanced ability to cope with environmental stress. Results further document the potential usefulness of chimeras as a novel reef restoration tool to enhance coral adaptability to environmental change, and confirm that coral chimerism can be an evolutionary rescue instrument.
 
Inferred cleavage rates in mitochondrial RNA. A Starts and ends of original genuine RNA fragments should ‘stack’ at the same positions in the genome. B Cleavage ratio of a site calculated as the number of processed reads (those starting or ending either side of the site) as a proportion of total reads across the site (processed and unprocessed)
Relationship between genotype and inferred cleavage rate at multiple positions on the nuclear genome and mitochondrial transcriptome, respectively. Inferred cleavage positions are represented by red circles along the mitochondrial transcriptome, and inferred cleavage ratios are colour coded using three categories: yellow on known gene boundaries, blue within tRNAs, green on mRNAs within a tRNA flanked boundary and red on mRNA within a non-tRNA flanked boundary. Beta estimates, P-values, cleavage rates and genotypes displayed in boxplots originate from the CARTaGENE dataset (N = 799 for each plot)
Comparison of inferred cleavage rates in mitochondrial RNA between control and shRNA knock down data for candidate genes (N = 8 in each plot, 4 from KD and 4 from controls). A Control vs sh knock down cleavage ratio for TBRG4 at position 9219 near the MT-ATP6 to MT-CO3 gene boundary. B Control vs sh knock down cleavage ratio for RPS19 at position 9157 near the MT-ATP6 to MT-CO3 gene boundary
Article
Background The human mitochondrial genome is transcribed as long strands of RNA containing multiple genes, which require post-transcriptional cleavage and processing to release functional gene products that play vital roles in cellular energy production. Despite knowledge implicating mitochondrial post-transcriptional processes in pathologies such as cancer, cardiovascular disease and diabetes, very little is known about the way their function varies on a human population level and what drives changes in these processes to ultimately influence disease risk. Here, we develop a method to detect and quantify mitochondrial RNA cleavage events from standard RNA sequencing data and apply this approach to human whole blood data from > 1000 samples across independent cohorts. Results We detect 54 putative mitochondrial RNA cleavage sites that not only map to known gene boundaries, short RNA ends and RNA modification sites, but also occur at internal gene positions, suggesting novel mitochondrial RNA cleavage junctions. Inferred RNA cleavage rates correlate with mitochondrial-encoded gene expression across individuals, suggesting an impact on downstream processes. Furthermore, by comparing inferred cleavage rates to nuclear genetic variation and gene expression, we implicate multiple genes in modulating mitochondrial RNA cleavage (e.g. MRPP3, TBRG4 and FASTKD5), including a potentially novel role for RPS19 in influencing cleavage rates at a site near to the MTATP6-COX3 junction that we validate using shRNA knock down data. Conclusions We identify novel cleavage junctions associated with mitochondrial RNA processing, as well as genes newly implicated in these processes, and detect the potential impact of variation in cleavage rates on downstream phenotypes and disease processes. These results highlight the complexity of the mitochondrial transcriptome and point to novel mechanisms through which nuclear-encoded genes can potentially influence key mitochondrial processes.
 
Sample information and PCA of Global Panel C. a Geographic location of samples in Global Panel C. The circle color of each population on map corresponds to the dot color of PC plots in b. b M.Yunnan.West and corresponding populations with close affinities in the PCA of Global Panel C, using a total number of 17,101 SNPs in 709 individuals
Global ancestry inference and migration signals of M.Yunnan.West. aADMIXTURE result of Global Panel B at K = 6, using a total number of 28,462 SNPs in 600 samples. Population categories are labeled on the left. ACH, Achang; DAI, Dai; DEA, Deang; JIP, Jingpo; M.North, minorities in North China; M.South, minorities in South China; M.Highland highland minorities in China; MSEA, mainland Southeast Asians; ISEA, island Southeast Asians. b The model used for D statistic estimations. Positive and negative D values indicate the excess allele sharing with population 1 (pop1) labeled in red and with population 2 (pop2) labeled in blue, respectively. An absolute Z-score greater than 3 is generally accepted as a strong signal of gene flow. c Potential gene introgression of M.Yunnan.West estimated by D statistics. Different pairwise M.Yunnan.West population combinations were used as possible populations (pop1 and pop2) under the gene introgression from the assumed ancestor populations in Global Panel C. D value above and below 0 for an ancestor population is assumed to have a closer genetic affinity to pop1 and pop2, respectively. An absolute Z-score above 3 for an ancestor population is presented in the upper right and lower left, indicating possible gene introgression into pop1 labeled in red and pop2 labeled in blue, respectively
Recent effective population size and integrated demography model of M.Yunnan.West. a Recent demographic histories inferred by IBDNe, using populations from the NGS Panel. b Integrated demography model describing the population history of M.Yunnan.West. Population divergence and effective population size were estimated by the 5-population model of dadi. Detected population migrations based on the results of D statistics, dadi, and TreeMix are portrayed as bold dotted arrows
Shared adaptive signals among M.Yunnan.West. a PBS distribution of genes over the 99th percentile, using different combinations of M.Yunnan.West populations. Box plot outliers were removed. Blue boxes indicate population combinations sharing the apparent same ancestral makeup. b Shared adaptive signals with extreme significance (simulation P value less than 0.01) among M.Yunnan.West populations. Only signals shared by at least two populations are labeled. c Venn plot representing the overlaps of extremely significant shared adaptive genes in four M.Yunnan.West populations
Differential adaptive signals among M.Yunnan.West. Differential adaptive genes with extreme significance (P value less than 0.01) were scanned by PBS in a ACH, b DAI, c DEA, and d JIP. Different colors indicate that M.Yunnan.West populations were used as the second population and HAN was used as the third population in each calculation. Only signals detected as differential genes in at least two populations are labeled
Article
Background Yunnan is located in Southwest China and consists of great cultural, linguistic, and genetic diversity. However, the genomic diversity of ethnic minorities in Yunnan is largely under-investigated. To gain insights into population history and local adaptation of Yunnan minorities, we analyzed 242 whole-exome sequencing data with high coverage (~ 100–150 ×) of Yunnan minorities representing Achang, Jingpo, Dai, and Deang, who were linguistically assumed to be derived from three ancient lineages (the tri-genealogy hypothesis), i.e., Di-Qiang, Bai-Yue, and Bai-Pu. Results Yunnan minorities show considerable genetic differences. Di-Qiang populations likely migrated from the Tibetan area about 6700 years ago. Genetic divergence between Bai-Yue and Di-Qiang was estimated to be 7000 years, and that between Bai-Yue and Bai-Pu was estimated to be 5500 years. Bai-Pu is relatively isolated, but gene flow from surrounding Di-Qiang and Bai-Yue populations was also found. Furthermore, we identified genetic variants that are differentiated within Yunnan minorities possibly due to the living circumstances and habits. Notably, we found that adaptive variants related to malaria and glucose metabolism suggest the adaptation to thalassemia and G6PD deficiency resulting from malaria resistance in the Dai population. Conclusions We provided genetic evidence of the tri-genealogy hypothesis as well as new insights into the genetic history and local adaptation of the Yunnan minorities.
 
Article
Background Enhancers are cis-regulatory elements present in eukaryote genomes, which constitute indispensable determinants of gene regulation by governing the spatiotemporal and quantitative expression dynamics of target genes, and are involved in multiple life processes, for instance during development and disease states. The importance of enhancer activity has additionally been highlighted for immune responses in animals and plants; however, the dynamics of enhancer activities and molecular functions in plant innate immunity are largely unknown. Here, we investigated the involvement of distal enhancers in early innate immunity in Arabidopsis thaliana . Results A group of putative distal enhancers producing low-abundance transcripts either unidirectionally or bidirectionally are identified. We show that enhancer transcripts are dynamically modulated in plant immunity triggered by microbe-associated molecular patterns and are strongly correlated with open chromatin, low levels of methylated DNA, and increases in RNA polymerase II targeting and acetylated histone marks. Dynamic enhancer transcription is correlated with target early immune gene expression patterns. Cis motifs that are bound by immune-related transcription factors, such as WRKYs and SARD1, are highly enriched within upregulated enhancers. Moreover, a subset of core pattern-induced enhancers are upregulated by multiple patterns from diverse pathogens. The expression dynamics of putative immunity-related enhancers and the importance of WRKY binding motifs for enhancer function were also validated. Conclusions Our study demonstrates the general occurrence of enhancer transcription in plants and provides novel information on the distal regulatory landscape during early plant innate immunity, providing new insights into immune gene regulation and ultimately improving the mechanistic understanding of the plant immune system.
 
Article
Background Mitochondria have an essential role in regulating metabolism and integrate environmental and physiological signals to affect processes such as cellular bioenergetics and response to stress. In the metabolically active skeletal muscle, mitochondrial biogenesis is one important component contributing to a broad set of mitochondrial adaptations occurring in response to signals, which converge on the biogenesis transcriptional regulator peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α), and is central to the beneficial effects of exercise in skeletal muscle. We investigated the role of long non-coding RNA (lncRNA) taurine-upregulated gene 1 ( TUG1 ), which interacts with PGC-1α in regulating transcriptional responses to exercise in skeletal muscle. Results In human skeletal muscle, TUG1 gene expression was upregulated post-exercise and was also positively correlated with the increase in PGC-1α gene expression ( PPARGC1A ). Tug1 knockdown (KD) in differentiating mouse myotubes led to decreased Ppargc1a gene expression, impaired mitochondrial respiration and morphology, and enhanced myosin heavy chain slow isoform protein expression. In response to a Ca ²⁺ -mediated stimulus, Tug1 KD prevented an increase in Ppargc1a expression. RNA sequencing revealed that Tug1 KD impacted mitochondrial Ca ²⁺ transport genes and several downstream PGC-1α targets. Finally, Tug1 KD modulated the expression of ~300 genes that were upregulated in response to an in vitro model of exercise in myotubes, including genes involved in regulating myogenesis. Conclusions We found that TUG1 is upregulated in human skeletal muscle after a single session of exercise, and mechanistically, Tug1 regulates transcriptional networks associated with mitochondrial calcium handling, muscle differentiation and myogenesis. These data demonstrate that lncRNA Tug1 exerts regulation over fundamental aspects of skeletal muscle biology and response to exercise stimuli.
 
Article
Introduction Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect.” It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H ⁺ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H ⁺ equivalents from the cell. Results To test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H ⁺ -ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. Conclusions Therefore, cancer cells which increase export of H ⁺ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.
 
Article
Background Degrons are short linear motifs, bound by E3 ubiquitin ligase to target protein substrates to be degraded by the ubiquitin-proteasome system. Mutations leading to deregulation of degron functionality disrupt control of protein abundance due to mistargeting of proteins destined for degradation and often result in pathologies. Targeting degrons by small molecules also emerges as an exciting drug design strategy to upregulate the expression of specific proteins. Despite their essential function and disease targetability, reliable identification of degrons remains a conundrum. Here, we developed a deep learning-based model named Degpred that predicts general degrons directly from protein sequences. Results We showed that the BERT-based model performed well in predicting degrons singly from protein sequences. Then, we used the deep learning model Degpred to predict degrons proteome-widely. Degpred successfully captured typical degron-related sequence properties and predicted degrons beyond those from motif-based methods which use a handful of E3 motifs to match possible degrons. Furthermore, we calculated E3 motifs using predicted degrons on the substrates in our collected E3-substrate interaction dataset and constructed a regulatory network of protein degradation by assigning predicted degrons to specific E3s with calculated motifs. Critically, we experimentally verified that a predicted SPOP binding degron on CBX6 prompts CBX6 degradation and mediates the interaction with SPOP. We also showed that the protein degradation regulatory system is important in tumorigenesis by surveying degron-related mutations in TCGA. Conclusions Degpred provides an efficient tool to proteome-wide prediction of degrons and binding E3s singly from protein sequences. Degpred successfully captures typical degron-related sequence properties and predicts degrons beyond those from previously used motif-based methods, thus greatly expanding the degron landscape, which should advance the understanding of protein degradation, and allow exploration of uncharacterized alterations of proteins in diseases. To make it easier for readers to access collected and predicted datasets, we integrated these data into the website http://degron.phasep.pro/ .
 
Article
Background Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. Results We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 ( Rnf133 ); RING finger protein 148 ( Rnf148 ); RING finger protein 151 ( Rnf151 ); and Zinc finger SWIM-type containing 2 ( Zswim2 )]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148 , Rnf151 , and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. Conclusions Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.
 
Article
Background Various mammalian species emit ultrasonic vocalizations (USVs), which reflect their emotional state and mediate social interactions. USVs are usually analyzed by manual or semi-automated methodologies that categorize discrete USVs according to their structure in the frequency-time domains. This laborious analysis hinders the effective use of USVs as a readout for high-throughput analysis of behavioral changes in animals. Results Here we present a novel automated open-source tool that utilizes a different approach towards USV analysis, termed TrackUSF. To validate TrackUSF, we analyzed calls from different animal species, namely mice, rats, and bats, recorded in various settings and compared the results with a manual analysis by a trained observer. We found that TrackUSF detected the majority of USVs, with less than 1% of false-positive detections. We then employed TrackUSF to analyze social vocalizations in Shank3 -deficient rats, a rat model of autism, and revealed that these vocalizations exhibit a spectrum of deviations from appetitive calls towards aversive calls. Conclusions TrackUSF is a simple and easy-to-use system that may be used for a high-throughput comparison of ultrasonic vocalizations between groups of animals of any kind in any setting, with no prior assumptions.
 
Article
Background In many organisms, aging is characterized by a loss of mitochondrial homeostasis. Multiple factors such as respiratory metabolism, mitochondrial fusion/fission, or mitophagy have been linked to cell longevity, but the exact impact of each one on the aging process is still unclear. Results Using the deletion mutant collection of the fission yeast Schizosaccharomyces pombe , we have developed a genome-wide screening for mutants with altered chronological lifespan. We have identified four mutants associated with proteolysis at the mitochondria that exhibit opposite effects on longevity. The analysis of the respiratory activity of these mutants revealed a positive correlation between increased respiration rate and prolonged lifespan. We also found that the phenotype of the long-lived protease mutants could not be explained by impaired mitochondrial fusion/fission activities, but it was dependent on mitophagy induction. The anti-aging role of mitophagy was supported by the effect of a mutant defective in degradation of mitochondria, which shortened lifespan of the long-lived mutants. Conclusions Our characterization of the mitochondrial protease mutants demonstrates that mitophagy sustains the lifespan extension of long-lived mutants displaying a higher respiration potential.
 
Article
Background In a broad variety of species, muscle contraction is controlled at the neuromuscular junction (NMJ), the peripheral synapse composed of a motor nerve terminal, a muscle specialization, and non-myelinating terminal Schwann cells. While peripheral nerve damage leads to successful NMJ reinnervation in animal models, muscle fiber reinnervation in human patients is largely inefficient. Interestingly, some hallmarks of NMJ denervation and early reinnervation in murine species, such as fragmentation and poly-innervation, are also phenotypes of aged NMJs or even of unaltered conditions in other species, including humans. We have reasoned that rather than features of NMJ decline, such cellular responses could represent synaptic adaptations to accomplish proper functional recovery. Here, we have experimentally tackled this idea through a detailed comparative study of the short- and long-term consequences of irreversible (chronic) and reversible (partial) NMJ denervation in the convenient cranial levator auris longus muscle. Results Our findings reveal that irreversible muscle denervation results in highly fragmented postsynaptic domains and marked ectopic acetylcholine receptor clustering along with significant terminal Schwann cells sprouting and progressive detachment from the NMJ. Remarkably, even though reversible nerve damage led to complete reinnervation after 11 days, we found that more than 30% of NMJs are poly-innervated and around 65% of postsynaptic domains are fragmented even 3 months after injury, whereas synaptic transmission is fully recovered two months after nerve injury. While postsynaptic stability was irreversibly decreased after chronic denervation, this parameter was only transiently affected by partial NMJ denervation. In addition, we found that a combination of morphometric analyses and postsynaptic stability determinations allows discriminating two distinct forms of NMJ fragmentation, stable-smooth and unstable-blurred, which correlate with their regeneration potential. Conclusions Together, our data unveil that reversible nerve damage imprints a long-lasting reminiscence in the NMJ that results in the rearrangement of its cellular components. Instead of being predictive of NMJ decline, these traits may represent an efficient adaptive response for proper functional recovery. As such, these features are relevant targets to be considered in strategies aimed to restore motor function in detrimental conditions for peripheral innervation.
 
Article
Background According to Waddington’s epigenetic landscape concept, the differentiation process can be illustrated by a cell akin to a ball rolling down from the top of a hill (proliferation state) and crossing furrows before stopping in basins or “attractor states” to reach its stable differentiated state. However, it is now clear that some committed cells can retain a certain degree of plasticity and reacquire phenotypical characteristics of a more pluripotent cell state. In line with this dynamic model, we have previously shown that differentiating cells (chicken erythrocytic progenitors (T2EC)) retain for 24 h the ability to self-renew when transferred back in self-renewal conditions. Despite those intriguing and promising results, the underlying molecular state of those “reverting” cells remains unexplored. The aim of the present study was therefore to molecularly characterize the T2EC reversion process by combining advanced statistical tools to make the most of single-cell transcriptomic data. For this purpose, T2EC, initially maintained in a self-renewal medium (0H), were induced to differentiate for 24H (24H differentiating cells); then, a part of these cells was transferred back to the self-renewal medium (48H reverting cells) and the other part was maintained in the differentiation medium for another 24H (48H differentiating cells). For each time point, cell transcriptomes were generated using scRT-qPCR and scRNAseq. Results Our results showed a strong overlap between 0H and 48H reverting cells when applying dimensional reduction. Moreover, the statistical comparison of cell distributions and differential expression analysis indicated no significant differences between these two cell groups. Interestingly, gene pattern distributions highlighted that, while 48H reverting cells have gene expression pattern more similar to 0H cells, they are not completely identical, which suggest that for some genes a longer delay may be required for the cells to fully recover. Finally, sparse PLS (sparse partial least square) analysis showed that only the expression of 3 genes discriminates 48H reverting and 0H cells. Conclusions Altogether, we show that reverting cells return to an earlier molecular state almost identical to undifferentiated cells and demonstrate a previously undocumented physiological and molecular plasticity during the differentiation process, which most likely results from the dynamic behavior of the underlying molecular network.
 
There are three sex chromosome systems. Two systems exist in diploid organisms—XY (e.g. mammals), where male is the heterogametic sex (A), and ZW (e.g. birds), where female is the heterogametic sex (B). While in haploid organisms there is the UV system (e.g. some mosses), where the female gametophyte is U and the male is V (C)
(continued)
Gene loss and specialization over time on NRSCs. The rate of gene loss is expected to decrease with time since recombination cessation, as non-essential genes are lost early in this process, while essential genes can be maintained through purifying selection. In contrast, the rate of specialization may be more or less constant since it will likely be dependent on mutation accumulation
NRSC (in grey) mechanisms of action (Table 1). Direct mechanisms include (1) protein-coding gene expression in somatic tissues, while indirect mechanisms are exerted through (2) regulation of other chromosomes (via transcription factors, non-coding RNAs, heterochromatin effects or imprinting) or (3) sex hormones
Affected non-sexual phenotypes by Y chromosome in mammals
Article
Sex chromosomes are typically viewed as having originated from a pair of autosomes, and differentiated as the sex-limited chromosome (e.g. Y) has degenerated by losing most genes through cessation of recombination. While often thought that degenerated sex-limited chromosomes primarily affect traits involved in sex determination and sex cell production, accumulating evidence suggests they also influence traits not sex-limited or directly involved in reproduction. Here, we provide an overview of the effects of sex-limited chromosomes on non-reproductive traits in XY, ZW or UV sex determination systems, and discuss evolutionary processes maintaining variation at sex-limited chromosomes and molecular mechanisms affecting non-reproductive traits.
 
Article
Background Hoverflies (Diptera: Syrphidae) including Eupeodes corollae are important insects worldwide that provide dual ecosystem services including pest control and pollination. The larvae are dominant predators of aphids and can be used as biological control agents, and the adults are efficient pollinators. The different feeding habits of larvae and adults make hoverflies a valuable genetic resource for understanding the mechanisms underlying the evolution and adaptation to predation and pollination in insects. Results Here, we present a 595-Mb high-quality reference genome of the hoverfly E. corollae, which is typical of an aphid predator and a pollinator. Comparative genomic analyses of E. corollae and Coccinellidae (ladybugs, aphid predators) shed light on takeout genes (3), which are involved in circadian rhythms and feeding behavior and might regulate the feeding behavior of E. corollae in a circadian manner. Genes for sugar symporter (12) and lipid transport (7) related to energy production in E. corollae had homologs in pollinator honeybees and were absent in predatory ladybugs. A number of classical cytochrome P450 detoxification genes, mainly CYP6 subfamily members, were greatly expanded in E. corollae. Notably, comparative genomic analyses of E. corollae and other aphidophagous hoverflies highlighted three homologous trypsins (Ecor12299, Ecor12301, Ecor2966). Transcriptome analysis showed that nine trypsins, including Ecor12299, Ecor12301, and Ecor2966, are strongly expressed at the larval stage, and 10 opsin genes, which are involved in visual perception, are significantly upregulated at the adult stage of E. corollae. Conclusions The high-quality genome assembly provided new insights into the genetic basis of predation and pollination by E. corollae and is a valuable resource for advancing studies on genetic adaptations and evolution of hoverflies and other natural enemies.
 
The distribution of the 10,866 families across the 1179 representative genomes. A The distribution of 10,866 widely distributed protein families (columns) in 1179 representative genomes (rows) from Archaea. Data are clustered based on the presence (black) and absence (white) profiles (Jaccard distance, complete linkage). B Tree resulting from the hierarchical clustering of the genomes based on the distributions of protein families in A
The distribution of the 2632 families of the 19 modules discussed in this study. Each column represents a protein family and each row represents a genome. Data are clustered based on the presence (black)/absence (white) profiles but also based on the taxonomy of the genomes and the module membership. The first colored top bar (annotations) shows the families with (black)/without (white) a predicted annotation whereas the second colored top bar (modules) indicates the module of each family. The colored side bar indicates the taxonomic assignment of each genome
Schematic overview of integrin-like and TFIIH-like gene clusters identified in archaea. A Conserved gene clusters comprising archaeal integrin-like genes (fam15271 ) identified in five Asgard genomes. B Conserved gene clusters comprising archaeal TFIIH-like genes (fam18955) identified in three Theionarchaea and three Asgard genomes. A full gene synteny and genomic context of the genes neighboring the integrin-like (fam15271) and TFIIH-like (fam18955) genes is available in Additional file 1: Table S8
A list of the fourteen modules that are lineage specific but also well conserved within eleven major archaeal lineages. A family was counted as having a signal peptide if at least 25% of its protein sequences were predicted to have a signal peptide prediction according to the SignalP software [32]. A family was counted as having a transmembrane helix if more than half of its protein sequences were predicted to have a transmembrane helix according to the TMHMM software [33]. Families were considered hypothetical if they have neither PFAM (Domain of Unknown Function domains were excluded) nor KEGG annotations (see the supplementary dataset - Table S3 for the full list of hypothetical families). Finally, a family was considered to have bacterial homologs if the family matched with protein sequences of at least ten distinct bacterial genomes (see the “Methods” section). The core module 1 is included as a comparison
Article
Background Archaea play fundamental roles in the environment, for example by methane production and consumption, ammonia oxidation, protein degradation, carbon compound turnover, and sulfur compound transformations. Recent genomic analyses have profoundly reshaped our understanding of the distribution and functionalities of Archaea and their roles in eukaryotic evolution. Results Here, 1179 representative genomes were selected from 3197 archaeal genomes. The representative genomes clustered based on the content of 10,866 newly defined archaeal protein families (that will serve as a community resource) recapitulates archaeal phylogeny. We identified the co-occurring proteins that distinguish the major lineages. Those with metabolic roles were consistent with experimental data. However, two families specific to Asgard were determined to be new eukaryotic signature proteins. Overall, the blocks of lineage-specific families are dominated by proteins that lack functional predictions. Conclusions Given that these hypothetical proteins are near ubiquitous within major archaeal groups, we propose that they were important in the origin of most of the major archaeal lineages. Interestingly, although there were clearly phylum-specific co-occurring proteins, no such blocks of protein families were shared across superphyla, suggesting a burst-like origin of new lineages early in archaeal evolution.
 
Article
Background Human babesiosis, caused by parasites of the genus Babesia, is an emerging and re-emerging tick-borne disease that is mainly transmitted by tick bites and infected blood transfusion. Babesia duncani has caused majority of human babesiosis in Canada; however, limited data are available to correlate its genomic information and biological features. Results We generated a B. duncani reference genome using Oxford Nanopore Technology (ONT) and Illumina sequencing technology and uncovered its biological features and phylogenetic relationship with other Apicomplexa parasites. Phylogenetic analyses revealed that B. duncani form a clade distinct from B. microti, Babesia spp. infective to bovine and ovine species, and Theileria spp. infective to bovines. We identified the largest species-specific gene family that could be applied as diagnostic markers for this pathogen. In addition, two gene families show signals of significant expansion and several genes that present signatures of positive selection in B. duncani, suggesting their possible roles in the capability of this parasite to infect humans or tick vectors. Conclusions Using ONT sequencing and Illumina sequencing technologies, we provide the first B. duncani reference genome and confirm that B. duncani forms a phylogenetically distinct clade from other Piroplasm parasites. Comparative genomic analyses show that two gene families are significantly expanded in B. duncani and may play important roles in host cell invasion and virulence of B. duncani. Our study provides basic information for further exploring B. duncani features, such as host-parasite and tick-parasite interactions.
 
Article
Background Differences in morphology, ecology, and behavior through ontogeny can result in opposing selective pressures at different life stages. Most animals, however, transition through two or more distinct phenotypic phases, which is hypothesized to allow each life stage to adapt more freely to its ecological niche. How this applies to sensory systems, and in particular how sensory systems adapt across life stages at the molecular level, is not well understood. Here, we used whole-eye transcriptomes to investigate differences in gene expression between tadpole and juvenile southern leopard frogs (Lithobates sphenocephalus), which rely on vision in aquatic and terrestrial light environments, respectively. Because visual physiology changes with light levels, we also tested the effect of light and dark exposure. Results We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles and 5% for light/dark exposure. Analyses targeting a curated subset of visual genes revealed significant differential expression of genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments. Conclusions Overall, we identified extensive expression-level differences in the eyes of tadpoles and juveniles related to observed morphological and physiological changes through metamorphosis and corresponding adaptive shifts to improve vision in the distinct aquatic and terrestrial light environments these frogs inhabit during their life cycle. More broadly, these results suggest that decoupling of gene expression can mediate the opposing selection pressures experienced by organisms with complex life cycles that inhabit different environmental conditions throughout ontogeny.
 
In vitro formation and in vivo presence of the EntF* metabolite. a Sequences of the enterocin induction factor pro-peptide, mature quorum sensing peptide EntF, and its metabolite EntF*. b In vitro formation rate of EntF* from EntF in colon (n = 7) and feces (n = 4) homogenates. Bars represent the mean formation rate ± SEM from independent experiments. Statistically significant differences were determined by a Mann-Whitney U test with indicated p-values. c Apparent permeability coefficients (Papp) of PapRIV, EntF*, and EDF-analog in Caco-2 cells. Bars represent mean Papp values ± SEM (n = 6 independent experiments); the shaded area represents the limit of detection. d Flow chart displaying the experimental design stages, from serum sampling to peptide detection and further confirmation of EntF* presence in vivo. Different LC-MS methods: LC1-MS1, reversed-phase ultra-high-performance liquid chromatography (RP-UPLC) using triple quadrupole (TQ) in MRM mode; LC1-MS2, high-resolution quadrupole time-of-flight; LC1-MS3, high-resolution quadrupole-orbitrap; LC2-MS1, HILIC-amide UPLC using TQ in MRM mode. qPCR was performed on feces sample of mice from the same set to demonstrate the presence of EntF-encoding DNA sequences from E. faecium. e Chromatographic profiles of (1) negative serum sample, (2) positive serum sample, (3) serum sample from EntF*-treated mice. Chromatographic profiles were obtained using RP-UPLC with detection by electrospray ionization mass spectrometry (ESI-MS) using TQ in MRM mode (m/z = 865 ➔ 202.08 + 315.17). f Chromatographic profiles of (1) negative serum sample, (2) positive serum sample, (3) serum sample from EntF*-treated mice. Chromatographic profiles were obtained using HILIC amide UPLC with detection by ESI-MS using TQ in MRM mode (m/z = 865 ➔ 202.08 + 315.17). g Isotopic distribution of the double charged EntF* measured in a positive serum sample using RP-UPLC with detection by ESI-MS using quadrupole-orbitrap. h High-resolution tandem mass spectrum of EntF* with characteristic fragments, using RP-UPLC with detection by Q-TOF. i In vivo presence of EntF* in gnotobiotic mice treated with EntF-producing bacterial strains. Number of EntF DNA copies per gram of feces measured four days after treatment with placebo (300 μL BHI medium) (limit of detection: 10⁵ copies/g) (left). EntF* concentration in colon content. No EntF* was detected in the placebo group (the red line indicates the limit of detection) (middle). EntF* concentration in serum content. No EntF* was detected in the placebo group (the red line indicates the limit of detection) (right)
In vitro activity of the EntF* peptide. a The relative importance of the amino acids of EntF* on E-cadherin expression ranked in five different classes. Ranking (blue to red: increasing significance) was performed using the Fisher’s LSD p-values, which was confirmed using the Jenks natural breaks algorithm. Based on ranking, it is likely that the first, second and tenth amino acid of EntF* are the most important residues for EntF* activity. b Alignment between EntF* and the active domain of CXCL12. Black amino acids with a line indicate a match. Dark grey amino acids with two dots mark a similarity between the two residues. Light grey amino acids with one dot are not similar, but no gap is formed. c Antagonistic effects of Nef-M1 on EntF*-mediated E-cadherin downregulation in HCT-8 cells (EntF* n = 15; Nef-M1 n = 6; EntF* + Nef-M1 n = 6). Statistically significant differences were determined by a one-sided student's t test. d Antagonistic effects of EntF*1A on EntF*-mediated E-cadherin downregulation in HCT-8 cells (EntF* n = 15; EntF*1A n = 18; EntF* + EntF*1A n = 6). Statistically significant differences were determined by a one-sided Student’s t test. e Effect of EntF* on E-cadherin expression. A significant decrease in E-cadherin levels following EntF* or CXCL12 treatments was observed in HT-29 (n = 12), Caco-2 (n = 6) and HCT-8 (n = 15) cells. Statistically significant differences were determined by one-way ANOVA test. f Proposed E-cadherin-regulating pathway for EntF*. Schematic representation of the CXCR4 receptor and its signaling pathways, leading to the activation of EMT transcription factors, followed by the downregulation of E-cadherin expression (see also Additional file 5: Fig. S5)
Metastasis-inducing effect of EntF* in an orthotopic mouse model of colorectal cancer. a Schematic timeline of the experimental setup. Female Swiss nu/nu mice were orthotopically injected with 1 × 10⁶ luciferase-transfected HCT-8 cells at the age of 5 weeks. During the following 6 weeks, mice were daily i.p. injected with 10² nmol kg⁻¹ EntF* (n = 38), PBS control (vehicle, n = 17), or 0.1 mg kg⁻¹ EGF positive control (n = 18). Bioluminescent imaging was performed weekly to determine cancer progression. After 6 weeks, mice were euthanized and the cecum, liver and lungs collected. b Graph representing the average daily exposure of placebo-treated (black; n = 65) and EntF*-treated (gray; n = 14) female Swiss nu/nu mice. In the placebo group, mice with an EntF* serum concentration below the limit of detection (100 pM) were counted as zero. The daily exposure after i.p. injection of 10² nmol kg⁻¹ EntF* is 5 times higher than the naturally occurring EntF* levels. c Representative images of bioluminescence activity in vehicle, EntF*-treated, and EGF-treated mice. Mice were i.p. injected with 150 mg kg-1 luciferin and imaged after 10 min in supine position. d Tumor growth curves derived from the quantification of bioluminescence in vehicle, EntF*-treated, and EGF-treated mice. Based on linear regression slope comparison, EntF* and EGF treatment resulted in a significant increase in tumor growth compared to the vehicle. Data represent the mean relative increase in bioluminescence ± SEM. e Representative pictures of vehicle, EntF*-treated, and EGF-treated ceca at the end of the experiment. f Number of tumor nodules in untreated (vehicle), EntF*-treated, and EGF-treated ceca determined by macroscopic inspection of tissues. Data represent the mean nodule number ± SEM. Statistically significant differences were determined by a Mann-Whitney U test with indicated p-values
Effect size for bioluminescence and number of nodules in the cecum of the orthotopic mouse model after a 6-week treatment. a After a 6-week treatment, an effect size of 79% increase in bioluminescence for EntF* compared to the placebo was observed, while for the positive control EGF, a median effect size of 316% was obtained. When calculating the effect size according to Hedges’ G values, a medium to large effect was observed for both EntF* and EGF treatment groups, compared to the placebo group. b After a 6-week treatment, a 3-fold increase in the number of nodules on the cecum was observed for EntF* compared to the placebo PBS, while for the positive control EGF, a 4.5-fold increase was obtained. When calculating the effect size according to Hedges’ G values, a medium and large effect was observed for the EntF* and EGF treatment groups, respectively, compared to the placebo group [Fig. prepared in R-script: https://github.com/JoachimGoedhart/PlotsOfDifferences]
Histopathological evaluation of CRC metastasis after EntF* treatment. a Microscopic images of liver tissue sections from untreated (vehicle), EntF*-treated, and EGF-treated mice. Tissues were stained with Hematoxylin and Eosin (H&E) and imaged at different magnifications (10x upper row, 40x lower row). b Histopathological scores with statistically significant differences determined by a Mann-Whitney U test (n = 8 for PBS, n = 30 for EntF*, n = 9 for EGF) with indicated p-values. c Microscopic images of lung tissue sections from untreated (vehicle), EntF*-treated, and EGF-treated mice. Tissues were stained with H&E and imaged at different magnifications (10x upper row, 40x lower row). d Histopathological scores with statistically significant differences determined by a Mann-Whitney U test (n = 8 for PBS, n = 30 for EntF*, n = 9 for EGF) with indicated p-values
Article
Background Colorectal cancer, one of the most common malignancies worldwide, is associated with a high mortality rate, mainly caused by metastasis. Comparative metagenome-wide association analyses of healthy individuals and cancer patients suggest a role for the human intestinal microbiota in tumor progression. However, the microbial molecules involved in host-microbe communication are largely unknown, with current studies mainly focusing on short-chain fatty acids and amino acid metabolites as potential mediators. Quorum sensing peptides are not yet considered in this context since their presence in vivo and their ability to affect host cells have not been reported so far. Results Here, we show that EntF*, a metabolite of the quorum sensing peptide EntF produced by Enterococcus faecium, is naturally present in mice bloodstream. Moreover, by using an orthotopic mouse model, we show that EntF* promotes colorectal cancer metastasis in vivo, with metastatic lesions in liver and lung tissues. In vitro tests suggest that EntF* regulates E-cadherin expression and consequently the epithelial-mesenchymal transition, via the CXCR4 receptor. In addition, alanine-scanning analysis indicates that the first, second, sixth, and tenth amino acid of EntF* are critical for epithelial-mesenchymal transition and tumor metastasis. Conclusion Our work identifies a new class of molecules, quorum sensing peptides, as potential regulators of host-microbe interactions. We prove, for the first time, the presence of a selected quorum sensing peptide metabolite in a mouse model, and we demonstrate its effects on colorectal cancer metastasis. We believe that our work represents a starting point for future investigations on the role of microbiome in colorectal cancer metastasis and for the development of novel bio-therapeutics in other disease areas.
 
Comparison of variant load per gene for high-impact variants (MAF < 0.1). Each point represents a gene. Outliers are marked in orange (for higher load in participants with hearing difficulty) or blue (for higher load in participants with normal hearing). A shows all the data, including TTN and FBLN7, genes with a much higher variant count than all the others, and B shows the data without those two genes
Venn diagrams showing the overlap of the outlier gene lists when looking at only male, only female or all participants (outliers with intermediate frequency variants with high impact). The known deafness genes in the intersection (7 in the outlier genes in hearing difficulty, 3 in the outlier genes in normal hearing) are labelled
Schematic of the cochlear duct showing cell types (top left) and expression patterns based on the scRNAseq data downloaded from the gEAR database. The numbers show how many outlier genes were present in the cluster; “HD” for the number of outlier genes in hearing difficulty lists, “NH” for the number of outlier genes in normal hearing lists, and “Both” for where an outlier gene was present in a hearing difficulty list and a normal hearing list. See Table 2 for clusters and for gene names. RM=Reissner’s membrane; MC=marginal cells; IC=intermediate cells; BC=basal cells; RC=root cells; SpC = spindle cells; SGN=spiral ganglion neurons; IBC=inner border cells; IphC=inner phalangeal cells; IHC=inner hair cells; OHC=outer hair cells; HeC=Hensen cells; CC=cells of Claudius; IPC=inner pillar cells; OPC=outer pillar cells; DC=Deiters’ cells
Comparison of gene lists from recent UK Biobank GWAS on self-reported hearing. Labelled genes are those also identified in this study and are not included in the numbers for those sections. Known deafness genes are in bold. The Wells et al. [9] and Kalra et al. [8] analyses used the UK Biobank data only while the Ivarsdottir et al. analysis [43] included other populations from Iceland
Deafness gene counts in mice and humans. Brackets indicate orthologues (e.g. there are 66 mouse orthologues of the 67 human deafness genes)
Article
Background Age-related hearing loss is a common, heterogeneous disease with a strong genetic component. More than 100 loci have been reported to be involved in human hearing impairment to date, but most of the genes underlying human adult-onset hearing loss remain unknown. Most genetic studies have focussed on very rare variants (such as family studies and patient cohort screens) or very common variants (genome-wide association studies). However, the contribution of variants present in the human population at intermediate frequencies is hard to quantify using these methods, and as a result, the landscape of variation associated with adult-onset hearing loss remains largely unknown. Results Here we present a study based on exome sequencing and self-reported hearing difficulty in the UK Biobank, a large-scale biomedical database. We have carried out variant load analyses using different minor allele frequency and impact filters, and compared the resulting gene lists to a manually curated list of nearly 700 genes known to be involved in hearing in humans and/or mice. An allele frequency cutoff of 0.1, combined with a high predicted variant impact, was found to be the most effective filter setting for our analysis. We also found that separating the participants by sex produced markedly different gene lists. The gene lists obtained were investigated using gene ontology annotation, functional prioritisation and expression analysis, and this identified good candidates for further study. Conclusions Our results suggest that relatively common as well as rare variants with a high predicted impact contribute to age-related hearing impairment and that the genetic contributions to adult hearing difficulty may differ between the sexes. Our manually curated list of deafness genes is a useful resource for candidate gene prioritisation in hearing loss.
 
The venom gland of N. nigricollis contains functionally distinct clusters of related toxins. A Detected toxins can be grouped into three clusters with distinct distributions across the venom gland of N. nigricollis. The top spectrum shows the normalised across-tissue averaged spectrum in the m/z region corresponding to 3FTx—the dominant components of N. nigricollis venom (full spectrum in Additional file 1: Fig. S1). The below centroid spectra are extracted from each group clustered by probabilistic latent semantic analysis (pLSA), where distributions across the gland are displayed as contrast-optimised heatmaps on the right. The top image shows the section stained with haematoxylin and eosin (H&E) post-MSI acquisition, and the direction of venom secretion is indicated by the arrow. B Toxin distributions are correlated with functional and phylogenetic relationships, as shown by comparing the phylogenetic relationship and distributions of identified 3FTx. The displayed 3FTx phylogeny was reconstructed by maximum likelihood (ML) under the FLU+G4 model (left, bootstrap support at nodes, displayed as mid-point rooted), while their distribution across the venom gland as determined by MSI is shown as contrast-optimised heatmaps on the right. C The relationship between function, relatedness, and distribution is further supported by a significant correlation between pairwise ML distances and spatial correlations of the identified 3FTx (left; Spearman rank: P < 0.001, r = − 0.337, 95% CI = − 0.491 to − 0.163, R² = 0.1944) and significantly higher spatial correlations among toxins within compared to between each functional class (right; Mann-Whitney two-tailed: P = 0.0481 and < 0.0001 for neurotoxins and cytotoxins, respectively). Heatmap legend is shown in A, and scale bar represents 2 mm
The composition and activity of venoms spat by and milked from spitting cobras are nearly identical. A Overlaid total ion chromatograms (TICs) and isotope distributions of key toxins from spat (red) and milked (black) venoms with different distributions in the venom gland from N. nigricollis, showing that their peptide toxin composition is identical. B Comparisons of spat and milked venom total ion counts (TICs) from representatives of two independently evolved clades of ‘spitting cobras’ show that they are either identical or have only minor differences in the abundance of peptide toxins. C Non-reduced and reduced SDS-PAGE of spat (S) or post-spit milked (PS) venom suggest that the composition of high-molecular-weight protein toxins is also identical or highly similar. Abbreviations: N. pallida (N. pal.), H. haemachatus (H. hae.), N. nigricollis (N. nig.). D Anticoagulant activity of spat and milked venom on citrated bovine plasma, measured as the sample area under the curve (AUC) minus the mean (m) control AUC, averaged (m (AUC − cAUC)), across four replicates (see Additional file 3). E Cytotoxic activity of spat and milked venom measured via MTT cell viability assay and displayed as the venom concentration that resulted in a 50% reduction in cell viability (IC50) across three replicates (see Additional file 4). F Enzymatic PLA2 activity of spat and milked venom, measured kinetically and displayed as m(AUC − cAUC), across three replicates (see Additional file 5). Error bars represent the standard error of the mean (SEM) of triplicate readings. Dotted lines in F indicate the activity of positive control venom (Daboia russelii) selected for its high PLA2 activity to contextualise the findings shown here
Distinct toxin distributions across the venom glands are taxonomically widespread in snakes. A pLSA analysis of MSI spectra from the venom gland of N. subfulva. The top spectrum shows the normalised across-tissue averaged spectrum in the m/z region corresponding to 3FTx—the dominant components of N. subfulva venom. The below centroid spectra are extracted from each group clustered by pLSA, where distributions across the gland are displayed as contrast-optimised heatmaps on the right. The H&E-stained section is shown at the top with an arrow indicating the direction of venom secretion. B Distributions of peaks corresponding to toxins with known activities are shown as contrast-optimised heatmaps. Accession numbers for cytotoxins are UniProt P01448, P01473, and P01474 and for neurotoxins, UniProt P01424, P01400, and GenBank GIJM01004310.1. C pLSA analysis of MSI spectra from the venom gland of C. rhodostoma. The top spectrum shows the normalised across-tissue averaged spectrum across the full acquired m/z range. The below centroid spectra are extracted from each group clustered by pLSA, where distributions across the gland are displayed as contrast-optimised heatmaps on the right. The H&E-stained section is shown at the top with an arrow indicating the direction of venom secretion. D Distributions of major peaks corresponding to the m/z values given below each image are shown as contrast-optimised heatmaps
Article
Background Venoms are ecological innovations that have evolved numerous times, on each occasion accompanied by the co-evolution of specialised morphological and behavioural characters for venom production and delivery. The close evolutionary interdependence between these characters is exemplified by animals that control the composition of their secreted venom. This ability depends in part on the production of different toxins in different locations of the venom gland, which was recently documented in venomous snakes. Here, we test the hypothesis that the distinct spatial distributions of toxins in snake venom glands are an adaptation that enables the secretion of venoms with distinct ecological functions. Results We show that the main defensive and predatory peptide toxins are produced in distinct regions of the venom glands of the black-necked spitting cobra (Naja nigricollis), but these distributions likely reflect developmental effects. Indeed, we detected no significant differences in venom collected via defensive ‘spitting’ or predatory ‘biting’ events from the same specimens representing multiple lineages of spitting cobra. We also found the same spatial distribution of toxins in a non-spitting cobra and show that heterogeneous toxin distribution is a feature shared with a viper with primarily predatory venom. Conclusions Our findings suggest that heterogeneous distributions of toxins are not an adaptation to controlling venom composition in snakes. Instead, it likely reflects physiological constraints on toxin production by the venom glands, opening avenues for future research on the mechanisms of functional differentiation of populations of protein-secreting cells within adaptive contexts.
 
Article
Background Vertebrates develop their peripheral nervous system (PNS) from transient unique embryonic structures, the neural crest, and the ectodermal placodes that are located at the border of the forming central nervous system. By contrast, in the invertebrate chordates, amphioxus and ascidians, a large part of the PNS originates at the opposite of the embryo, in the ventral ectoderm. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify a neurogenic territory within which glutamatergic epidermal sensory neuron formation is controlled by the Notch pathway. Given these similarities and the phylogenetic relationships within chordates, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates. Results In order to get insights into the molecular control of ventral PNS formation and to test the hypothesis of their homology and potential contribution to the emergence of vertebrate PNS, we undertook a close comparison of ventral PNS formation in the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum. Using timed RNA-seq series, we identified novel markers of the ventral PNS during different phases of its development in both species. By extensively determining the expression of paralogous and orthologous genes, we observed that only a minority of genes have a shared expression in the ventral PNS. However, a large fraction of ventral PNS orthologous genes are expressed in the dorsally forming PNS of vertebrates. Conclusions Our work has significantly increased the molecular characterization of ventral PNS formation in invertebrate chordates. The low observed conservation of gene expression in the ventral PNS suggests that the amphioxus and ascidian ventral PNS are either not homologous, or alternatively extensive drift has occurred in their regulatory mechanisms following a long period (600 My) of separate evolution and accelerated evolution in the ascidian lineage. The homology to genes expressed in the dorsally forming PNS of vertebrates suggests that ancestral sensory neurons gene networks have been redeployed in vertebrates.
 
VE-1 regulates light-dependent transcription and the accumulation of carotenoids. a Effect of light intensity on the accumulation of carotenoids. Mycelia of the wild-type and Δve-1 mutant strains were grown for 2 days in the dark at 22 °C and illuminated for 2 min prior to incubation for 24 h in the dark at 8 °C. The plot shows the average and standard error for six measurements for each light intensity. b Light-dependent transcription and photoadaptation. Wild-type and Δve-1 mutant strains were grown for 2 days at 22 °C in the dark and then exposed to light during the times indicated prior to RNA purification and quantification by RT-PCR. The plots show the average and standard error of the mean of the relative mRNA accumulation in three independent experiments. The results from each PCR for each gene were normalized to the corresponding PCR for tub-2 to correct for sampling errors. Then, the results were normalized to those obtained with the wild type after exposure to 30 min of light. c Light-dependent phosphorylation of the photoreceptor WC-1. Mycelial samples of the wild-type and the Δve-1 mutant strains were grown for 2 days at 22 °C in the dark and then exposed to light during the times indicated. Total protein extracts were separated by SDS-PAGE, and hybridized with an antibody specific for WC-1. Two hundred micrograms of proteins was loaded per lane. Additional bands are due to the transient light-dependent WC-1 phosphorylation. As loading control, we used a Coomassie staining of each protein sample. d Subcellular localization of VE-1. Mycelial samples of the ve-1FLAG strain were grown for 2 days at 30 °C in the dark, light, or grown in the dark and exposed to light during 30 min. Total protein samples (T), or samples enriched in cytoplasmic (C) or nuclear (N) proteins were separated by SDS-PAGE, and hybridized with antibodies specific for FLAG or histone H3. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample
Light regulates the accumulation of VE-1 during conidiation. a VE-1 regulates the growth of aerial hyphae. Aerial hyphae of the wild-type, Δve-1, Δwc-1, and Δwc-1 Δve-1 strains after 3 days of growth in minimal agar at 30 °C in dark or light. The length of aerial hyphae growing up from vegetative mycelia in two representative cultures is indicated by arrows. b Length of aerial hyphae in each strain after growth at 30 °C in dark or light. Average and standard error of the mean in 3–5 independent experiments. c Accumulation of VE-1 during conidiation. Upper part. Protein samples from cultures kept in the dark or light were obtained from mycelia growing vegetatively in liquid media (M), or as supporting vegetative mycelia after 15 h (M15) or 24 h (M24) of transfer from liquid media to the surface of an agar plate with minimal media to induce conidiation. Conidiating aerial hyphae were collected after 15 h (A15) or 24 h (A24) of the induction of conidiation. We used the ve-1FLAG and the Δwc-1 ve-1FLAG strains. Bottom part. Proteins were separated by SDS-PAGE, and hybridized with an antibody specific for FLAG. One hundred micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. d VE-1 accumulates in the nuclear fraction during conidiation. Upper part. We collected samples of the ve-1FLAG strain grown in the light from vegetative mycelia (M), supporting vegetative mycelia (M15) or (M24), and aerial hyphae (A15) or (A24). Bottom part. Total protein samples (T), or samples enriched in cytoplasmic (C) or nuclear (N) proteins were separated by SDS-PAGE, and hybridized with antibodies specific for FLAG or histone H3. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample
The degradation of VE-1 by the proteasome is regulated by light. a Regulation by light of VE-1 degradation. Cultures of vegetative mycelia of the ve-1FLAG and the Δwc-1 ve-1FLAG strains were grown at 30 °C in liquid media for 24 h in the dark, light or exposed to 30 min of light, then cycloheximide was added to the cultures, and samples were collected at different times. Proteins were separated by SDS-PAGE, and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Ponceau staining of each protein sample. Each hybridization was quantified using as reference the amount of VE-1 detected at time point 0 (before addition of cycloheximide). The plot shows the average and standard error of four independent experiments. b The proteasome inhibitor thiolutin prevents the degradation of VE-1. Cultures of vegetative mycelia of the ve-1FLAG strain were grown at 30 °C in liquid media and exposed to 30 min of light, then cycloheximide and/or thiolutin were added to the cultures, and samples were removed at different times. Cultures without any additional chemical added were used as controls. Proteins were separated by SDS-PAGE, and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. The hybridization was quantified using as reference the amount of VE-1 detected at time point 0 (before addition of cycloheximide, thiolutin or both). The plot shows the average and standard error of three independent experiments. c FWD-1 and the CSN participate in the degradation of VE-1. Cultures of vegetative mycelia of the Δcsn-1 ve-1FLAG, Δcsn-5 ve-1FLAG, and Δfwd-1 ve-1FLAG strains were grown at 30 °C in liquid media for 24 h in the dark, light, or exposed to 30 min of light, then cycloheximide was added to the cultures, and samples were removed at different times. Proteins were separated by SDS-PAGE and hybridized with an antibody specific for FLAG. Seventy micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. Each hybridization was quantified using as reference the amount of VE-1 detected at time point 0 (before addition of cycloheximide). The plot shows the average and standard error of 2–3 independent experiments
Light regulates the accumulation of VE-1 in aerial hyphae by preventing its degradation. a The degradation of VE-1 in aerial hyphae requires FWD-1. Protein samples from cultures of the Δfwd-1 ve-1FLAG strain kept in the dark or light were obtained from mycelia growing vegetatively in liquid media (M), or as supporting vegetative mycelia after 48 h (M48) of transfer from liquid media to the surface of an agar plate with minimal media to induce conidiation. Conidiating aerial hyphae were collected after 48 h (A48) of the induction of conidiation. Proteins were separated by SDS-PAGE and hybridized with antibodies specific for FLAG. One hundred micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. b The degradation of VE-1 in conidia requires FWD-1 and an active CSN. Protein samples were isolated from conidia of the ve-1FLAG, Δcsn-5 ve-1FLAG, and Δfwd-1 ve-1FLAG strains isolated from cultures kept in the dark or light. Proteins were separated by SDS-PAGE and hybridized with antibodies specific for FLAG. Fifty micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. c Light promotes the accumulation of VE-1 during conidiation. Protein samples from cultures of the ve-1FLAG strain kept in the dark or light were obtained from mycelia growing vegetatively in liquid media (M), or as supporting vegetative mycelia after 15 h (M15) of transfer from liquid media to the surface of an agar plate with minimal media to induce conidiation. Conidiating aerial hyphae were collected after 15 h (A15) of the induction of conidiation. For the dark to light transfer or light to dark transfer, the M15 or A15 samples were incubated in light or dark, respectively, for the indicated times after 15 h of induction of conidiation in dark or light, respectively, and collected for protein purification. Proteins were separated by SDS-PAGE and hybridized with an antibody specific for FLAG. One hundred micrograms of proteins was loaded per lane. As loading control, we used a Coomassie staining of each protein sample. d A model for the regulation of VE-1 degradation during conidiation and light. VE-1 accumulates in the cytoplasm and nuclei of vegetative hyphae following transcription and translation. VE-1 is degraded by the proteasome in a process that requires interaction with the SCFFWD−1 and ubiquitylation (symbolized by stars) and is regulated by the CSN and light. During the development of aerial hyphae VE-1 is highly degraded but light reduces VE-1 degradation and promotes its translocation to the nucleus for gene regulation
Article
Background Fungi use light as an environmental signal to regulate developmental transitions that are key aspects of their biological cycles and that are also relevant for their dispersal and infectivity as plant or animal pathogens. In addition, light regulates the accumulation of photoprotective pigments, like carotenoids, and other secondary metabolites. Most fungal light responses occur after changes in gene transcription and we describe here a novel effect of light in the regulation of degradation of VE-1, a key component of the velvet complex, in the model fungus Neurospora crassa. The velvet complex is a fungal-specific protein complex that coordinates fungal development, secondary metabolism, and light regulation by interacting with other regulators and photoreceptors and modifying gene expression. Results We have characterized the role of VE-1 during conidiation in N. crassa. In vegetative mycelia, VE-1 is localized in the cytoplasm and nuclei and is required for light-dependent transcription but does not interact with the photoreceptor and transcription factor WC-1. VE-1 is more stable in light than in darkness during asexual development (conidiation). We have shown that this light effect requires the blue-light photoreceptor WC-1. We have characterized the role of the proteasome, the COP9 signalosome (CSN), and the adaptor component of cullin-RING ubiquitin ligases, FWD-1, in the degradation of VE-1. Conclusions We propose that this new effect of light allows the fungal cell to adapt quickly to changes in light exposure by promoting the accumulation of VE-1 for the regulation of genes that participate in the biosynthesis of photoprotective pigments.
 
Sequence-scaled linear domain organization of Pks13. a Overall view. b Structural coverage. PDB codes and residues resolved in the structures/boundaries of the crystallized constructs are indicated. For mKS and mACP2, which were modelled based on homology, percentages of sequence identity (SI) are given. c The different molecular entities produced within the current study. Colour code of the domains is as follows: ACP, red; KS, green; AT, yellow; TE, blue; LD, cyan. Interdomain linkers are in grey and the Mtb Pks13 insertion (Ins) is in dark grey
SDS-PAGE of purified Mas, PpsA and Pks13 full-length proteins and Pks13 fragments. Molecular weight ladder is on the first lane
SAXS analysis of Pks13, Mas and PpsA. a Experimental scattering curves measured at 50 mM NaCl (except otherwise stated) and for the proteins without the phosphopantetheine arm (except otherwise stated). The logarithm of intensity is displayed as a function of the momentum transfer Q. The normalized curve has been displaced downwards by 1 or 2 logarithmic units for clarity, except when precised that they are superposed. The fits obtained from modelling (AllosMod-FoXS or MultiFoXS analysis) are in dark. b Normalized Kratky plots, c, d p(r) distance distribution computed from the experimental scattering patterns. Samples are given in the following order from top to bottom: superposed Pks13(S1533A) monomer (medium-purple) and dimer of C16-Pks13(S1533A) (dark purple), Mas (purple), PpsA (lavender, 500 mM NaCl), superposed Pks13 and holo-Pks13 (i.e. without and with Ppant arms: purple and pink, respectively), fACP1‑KS‑AT (grey), fKS-AT (light green), fKS (dark green), fAT (yellow, 300 mM NaCl), AT52 (light orange, 300 mM NaCl), fACP2-TE (red) and fTE (dark blue)
Ab initio models of Pks13 and Pks13 fragments, C16-Pks13(S1533A) dimer, Mas and PpsA. Envelopes were calculated using the GASBOR program except otherwise stated: Pks13 (average envelope, pink) and Pks13 fragments (most probable envelopes: fACP1-KS-AT, grey; fKS-AT, green; fKS, dark green; AT52, yellow; fACP2-TE, red; fTE, blue), C16-Pks13(S1533A) dimer (average DAMMIN envelope, dark purple), Mas (average envelope, purple), PpsA (average envelope, lavender). Images were prepared using the software Chimera (http://www.rbvi.ucsf.edu/chimera)
Hybrid modelling of Pks13. a With the most probable population superposed on the envelope. The Pks13 insertion is circled in dark grey. b With the three populations constituting the minimal ensemble, independently fitted within the envelope. c With the three populations aligned via ACP1-KS-AT. Equivalent residues known to interact with the C16 substrate analogue in the complex between AcpP and the ketosynthase FabF (E. coli) and residues interacting with CoA of the α-chain in the AT of Pks13 are highlighted in red on the corresponding KS and AT domains. Colour code: ACPs, red; KS, green; AT, yellow; TE, dark blue; linker regions, light grey; Pks13 insertion, dark grey
Article
Background Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure‐function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis , Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. Results We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. Conclusions The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.
 
Article
Background The establishment of tissue architecture requires coordination between distinct processes including basement membrane assembly, cell adhesion, and polarity; however, the underlying mechanisms remain poorly understood. The actin cytoskeleton is ideally situated to orchestrate tissue morphogenesis due to its roles in mechanical, structural, and regulatory processes. However, the function of many pivotal actin-binding proteins in mammalian development is poorly understood. Results Here, we identify a crucial role for anillin (ANLN), an actin-binding protein, in orchestrating epidermal morphogenesis. In utero RNAi-mediated silencing of Anln in mouse embryos disrupted epidermal architecture marked by adhesion, polarity, and basement membrane defects. Unexpectedly, these defects cannot explain the profoundly perturbed epidermis of Anln-depleted embryos. Indeed, even before these defects emerge, Anln-depleted epidermis exhibits abnormalities in mitotic rounding and its associated processes: chromosome segregation, spindle orientation, and mitotic progression, though not in cytokinesis that was disrupted only in Anln-depleted cultured keratinocytes. We further show that ANLN localizes to the cell cortex during mitotic rounding, where it regulates the distribution of active RhoA and the levels, activity, and structural organization of the cortical actomyosin proteins. Conclusions Our results demonstrate that ANLN is a major regulator of epidermal morphogenesis and identify a novel role for ANLN in mitotic rounding, a near-universal process that governs cell shape, fate, and tissue morphogenesis.
 
Article
Background Escherichia coli ( E. coli ) has been one of the most studied model organisms in the history of life sciences. Initially thought just to be commensal bacteria, E. coli has shown wide phenotypic diversity including pathogenic isolates with great relevance to public health. Though pangenome analysis has been attempted several times, there is no systematic functional characterization of the E. coli subgroups according to the gene profile. Results Systematically scanning for optimal parametrization, we have built the E. coli pangenome from 1324 complete genomes. The pangenome size is estimated to be ~25,000 gene families (GFs). Whereas the core genome diminishes as more genomes are added, the softcore genome (≥95% of strains) is stable with ~3000 GFs regardless of the total number of genomes. Apparently, the softcore genome (with a 92% or 95% generation threshold) can define the genome of a bacterial species listing the critically relevant, evolutionarily most conserved or important classes of GFs. Unsupervised clustering of common E. coli sequence types using the presence/absence GF matrix reveals distinct characteristics of E. coli phylogroups B1, B2, and E. We highlight the bi-lineage nature of B1, the variation of the secretion and of the iron acquisition systems in ST11 (E), and the incorporation of a highly conserved prophage into the genome of ST131 (B2). The tail structure of the prophage is evolutionarily related to R2-pyocin (a tailocin) from Pseudomonas aeruginosa PAO1. We hypothesize that this molecular machinery is highly likely to play an important role in protecting its own colonies; thus, contributing towards the rapid rise of pandemic E. coli ST131. Conclusions This study has explored the optimized pangenome development in E. coli . We provide complete GF lists and the pangenome matrix as supplementary data for further studies. We identified biological characteristics of different E. coli subtypes, specifically for phylogroups B1, B2, and E. We found an operon-like genome region coding for a tailocin specific for ST131 strains. The latter is a potential killer weapon providing pandemic E. coli ST131 with an advantage in inter-bacterial competition and, suggestively, explains their dominance as human pathogen among E. coli strains.
 
Article
Background Circadian rhythms are important for all aspects of biology; virtually every aspect of biological function varies according to time of day. Although this is well known, variation across the day is also often ignored in the design and reporting of research. For this review, we analyzed the top 50 cited papers across 10 major domains of the biological sciences in the calendar year 2015. We repeated this analysis for the year 2019, hypothesizing that the awarding of a Nobel Prize in 2017 for achievements in the field of circadian biology would highlight the importance of circadian rhythms for scientists across many disciplines, and improve time-of-day reporting. Results Our analyses of these 1000 empirical papers, however, revealed that most failed to include sufficient temporal details when describing experimental methods and that few systematic differences in time-of-day reporting existed between 2015 and 2019. Overall, only 6.1% of reports included time-of-day information about experimental measures and manipulations sufficient to permit replication. Conclusions Circadian rhythms are a defining feature of biological systems, and knowing when in the circadian day these systems are evaluated is fundamentally important information. Failing to account for time of day hampers reproducibility across laboratories, complicates interpretation of results, and reduces the value of data based predominantly on nocturnal animals when extrapolating to diurnal humans.
 
Article
Background: DNA methylation is involved in the epigenetic regulation of gene expression during developmental processes and is primarily established by the DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B). DNMT3A is one of the most frequently mutated genes in clonal hematopoiesis and leukemia, indicating that it plays a crucial role for hematopoietic differentiation. However, the functional relevance of Dnmt3a for hematopoietic differentiation and hematological malignancies has mostly been analyzed in mice, with the specific role for human hematopoiesis remaining elusive. In this study, we therefore investigated if DNMT3A is essential for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs). Results: We generated iPSC lines with knockout of either exon 2, 19, or 23 and analyzed the impact of different DNMT3A exon knockouts on directed differentiation toward mesenchymal and hematopoietic lineages. Exon 19-/- and 23-/- lines displayed an almost entire absence of de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation efficiency was only slightly reduced in exon 19-/- and rather increased in exon 23-/- lines, while there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A-/- iHPCs recapitulate some DNA methylation patterns of acute myeloid leukemia (AML) with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed growth advantage of exon 23-/- iHPCs in a syngeneic competitive differentiation assay. Conclusions: Our results demonstrate that iPSCs with homozygous knockout of different exons of DNMT3A remain capable of mesenchymal and hematopoietic differentiation-and exon 23-/- iHPCs even gained growth advantage-despite loss of almost the entire de novo DNA methylation. Partial recapitulation of DNA methylation patterns of AML with DNMT3A mutations by our DNMT3A knockout iHPCs indicates that our model system can help to elucidate mechanisms of clonal hematopoiesis.
 
of the analysis pipeline used to identify novel orthogroups
Biological functions of novel orthogroups based on Saccharomyces cerevisiae genes. Each colored box, coded by the phylogenetic group, is a summary of the extant biological processes that are associated with past evolutionary transitions. The number at each node represents the total number of novel orthogroups for that clade. Numbers in parentheses indicate the number of species studied in each phylum. The phylogenetic hypothesis of fungal phyla interrelationships is as per references in the text.
Mitochondria-localized novel orthogroups. A The percentage of mitochondria-localized proteins among novel proteins (Novel) is significantly higher than those among all proteins (All). B Proteins mapped on the phylogeny represent mitochondria-localized novel proteins identified by the analysis pipeline in Fig. 1. The cartoon in the upper right panel shows subcellular location and the dashed rectangle in the lower right panel shows proteins involved in major mitochondrial complexes
Protein-protein interaction network of novel proteins. Protein names corresponding to the S. cerevisiae equivalents of 139 novel proteins were uploaded to the STRING database. Only networks including at least three proteins are shown
Prokaryote-derived horizontal gene transfers. A Phylogenetic relationship of LEU4 from selected species using IQ-TREE2. Only bootstrap values >90% are shown. As, Ascomycota; Ba, Basidiomycota. B Phylogenetic relationship of dihydroorotate dehydrogenases from selected species using IQ-TREE2. Mu, Mucoromycota; Zo, Zoopagomycota; Ch, Chytridiomycota; Bl, Blastocladiomycota. C Distribution of dihydroorotate dehydrogenase in fungi identified by our pipeline (see Additional file 6: Table S5)
Article
Background Fungi exhibit astonishing diversity with multiple major phenotypic transitions over the kingdom’s evolutionary history. As part of this process, fungi developed hyphae, adapted to land environments (terrestrialization), and innovated their sexual structures. These changes also helped fungi establish ecological relationships with other organisms (animals and plants), but the genomic basis of these changes remains largely unknown. Results By systematically analyzing 304 genomes from all major fungal groups, together with a broad range of eukaryotic outgroups, we have identified 188 novel orthogroups associated with major changes during the evolution of fungi. Functional annotations suggest that many of these orthogroups were involved in the formation of key trait innovations in extant fungi and are functionally connected. These innovations include components for cell wall formation, functioning of the spindle pole body, polarisome formation, hyphal growth, and mating group signaling. Innovation of mitochondria-localized proteins occurred widely during fungal transitions, indicating their previously unrecognized importance. We also find that prokaryote-derived horizontal gene transfer provided a small source of evolutionary novelty with such genes involved in key metabolic pathways. Conclusions The overall picture is one of a relatively small number of novel genes appearing at major evolutionary transitions in the phylogeny of fungi, with most arising de novo and horizontal gene transfer providing only a small additional source of evolutionary novelty. Our findings contribute to an increasingly detailed portrait of the gene families that define fungal phyla and underpin core features of extant fungi.
 
Article
Background Zika virus (ZIKV) and dengue virus (DENV) cause microcephaly and dengue hemorrhagic fever, respectively, leading to severe problems. No effective antiviral agents are approved against infections of these flaviviruses, calling for the need to develop potent therapeutics. We previously identified gossypol as an effective inhibitor against ZIKV and DENV infections, but this compound is toxic and not suitable for in vivo treatment. Results In this study, we showed that gossypol derivative ST087010 exhibited potent and broad-spectrum in vitro inhibitory activity against infections of at least ten ZIKV strains isolated from different hosts, time periods, and countries, as well as DENV-1-4 serotypes, and significantly reduced cytotoxicity compared to gossypol. It presented broad-spectrum in vivo protective efficacy, protecting ZIKV-infected Ifnar1−/− mice from lethal challenge, with increased survival and reduced weight loss. Ifnar1−/− mice treated with this gossypol derivative decreased viral titers in various tissues, including the brain and testis, after infection with ZIKV at different human isolates. Moreover, ST087010 potently blocked ZIKV vertical transmission in pregnant Ifnar1−/− mice, preventing ZIKV-caused fetal death, and it was safe for pregnant mice and their pups. It also protected DENV-2-challenged Ifnar1−/− mice against viral replication by reducing the viral titers in the brain, kidney, heart, and sera. Conclusions Overall, our data indicate the potential for further development of this gossypol derivative as an effective and safe broad-spectrum therapeutic agent to treat ZIKV and DENV diseases.
 
Article
Background Animal locomotion requires dynamic interactions between neural circuits, the body (typically muscles), and surrounding environments. While the neural circuitry of movement has been intensively studied, how these outputs are integrated with body mechanics (neuromechanics) is less clear, in part due to the lack of understanding of the biomechanical properties of animal bodies. Here, we propose an integrated neuromechanical model of movement based on physical measurements by taking Drosophila larvae as a model of soft-bodied animals. Results We first characterized the kinematics of forward crawling in Drosophila larvae at a segmental and whole-body level. We then characterized the biomechanical parameters of fly larvae, namely the contraction forces generated by neural activity, and passive elastic and viscosity of the larval body using a stress-relaxation test. We established a mathematical neuromechanical model based on the physical measurements described above, obtaining seven kinematic values characterizing crawling locomotion. By optimizing the parameters in the neural circuit, our neuromechanical model succeeded in quantitatively reproducing the kinematics of larval locomotion that were obtained experimentally. This model could reproduce the observation of optogenetic studies reported previously. The model predicted that peristaltic locomotion could be exhibited in a low-friction condition. Analysis of floating larvae provided results consistent with this prediction. Furthermore, the model predicted a significant contribution of intersegmental connections in the central nervous system, which contrasts with a previous study. This hypothesis allowed us to make a testable prediction for the variability in intersegmental connection in sister species of the genus Drosophila. Conclusions We generated a neurochemical model based on physical measurement to provide a new foundation to study locomotion in soft-bodied animals and soft robot engineering.
 
Article
Background As the most abundant epigenetic modification of eukaryotic mRNA, N6-methyladenosine (m6A) modification has been shown to play a role in mammalian nervous system development and function by regulating mRNA synthesis and degeneration. However, the role of m6A modification in retinal photoreceptors remains unknown. Results We generated the first retina-specific Mettl14-knockout mouse models using the Rho-Cre and HRGP-Cre lines and investigated the functions of Mettl14 in retinal rod and cone photoreceptors. Our data showed that loss of Mettl14 in rod cells causes a weakened scotopic photoresponse and rod degeneration. Further study revealed the ectopic accumulation of multiple outer segment (OS) proteins in the inner segment (IS). Deficiency of Mettl14 in cone cells led to the mislocalization of cone opsin proteins and the progressive death of cone cells. Moreover, Mettl14 depletion resulted in drastic decreases in METTL3/WTAP levels and reduced m6A methylation levels. Mechanistically, transcriptomic analyses in combination with MeRIP-seq illustrated that m6A depletion via inactivation of Mettl14 resulted in reduced expression levels of multiple phototransduction- and cilium-associated genes, which subsequently led to compromised ciliogenesis and impaired synthesis and transport of OS-residing proteins in rod cells. Conclusions Our data demonstrate that Mettl14 plays an important role in regulating phototransduction and ciliogenesis events and is essential for photoreceptor function and survival, highlighting the importance of m6A modification in visual function.
 
Variation screening of four wild species and evolutionary analysis of peach genome. a SNPs, small indels, SVs, and CNVs in P. mira, P. davidiana, P. kansuensis, and P. ferganensis compared to P. persica. b Core and dispensable gene families of four wild peaches and P. persica. c Gene Ontology annotation of genes specific in each species. d Estimation of divergence times of 15 species and identification of gene family expansions and contractions. Numbers on the nodes represent the divergence times from present (million years ago, Mya). MCRA, most recent common ancestor. e Distribution of Ks (synonymous mutation rate) values of orthologous genes between six genomes of the Prunus species and strawberry
The evolution of P. davidiana. a Phylogenetic tree of 126 peach accessions which included P. persica and its wild relative species. b Principal component analysis (PCA) of above accessions. c Stone steak morphology of P. dulcis, P. mira, P. davidiana, P. kansuensis, and P. ferganensis. d Percent of P. davidiana-specific contigs covered by reads from different Prunus species. e Regional collinearity between P. davidiana with P. dulcis or with P. mira genomes. The detailed location of these three regions were at Chr. 1: 4.79 Mb, Chr. 1: 5.08 Mb, and Chr. 2: 7.50 Mb in P. davidiana genome. Green and blue boxes indicate the positive and negative direction of genes. “P” and “A” letters showed the gene was presence and absence in P. davidiana genome
Identification of nematode resistance genes in P. kansuensis. a Bulked segregation analysis to locate the nematode resistance gene using a backcross population. b The Indels in the promoter and mRNA regions of one NBS-LRR genes on Chr. 2. “Ins” and “Del” indicate an insertion and deletion, respectively. And the number after the variation type indicates the length (bp) of Indels. c Expression of Prupe.2G053600 in two accessions (“Hong Gen Gan Su Tao 1#” and “Bailey”) inoculated with nematode. e Functional validation of Prupe.2G053600 through analysis of transgenic tomato plants expressing Prupe.2G053600 under nematode treatment. e Promoter activity assay. Promoters with different lengths were fused to the GUS gene in plasmid pBI101. GUS was dyed, and its activity was measured using protein extracts of tobacco. pBI101 and pBI121 indicate negative control and positive control, respectively
Selective regions associated with high-altitude adaptation in P. mira. a–c Domestication signals in accessions originating in high-altitude region compared to those in low-altitude. The signals were defined by the top 5% of πratio (a), Tajima’s D (b), and FST values (c). d Distribution of expression of genes induced by low temperature and UV of P. mira. Grey dots indicate the background genes and red dots indicate selective genes associated with high-altitude adaptation. e Detailed π ratio and FST values in the genome region of the candidate gene, Pmi02g3025 (pointed by the dashed line), which was substantially induced by low temperature. f Detailed Tajima’s D in the genome region of the candidate gene Pmi02g3025. gA. thaliana plants expressing Pmi02g3025 gene (OE) and the control (WT) treated with low temperature. h Genotypes (K indicates G/T) of a variation (Chr. 2: 28,612,439 bp) located at the promoter of Pmi02g3025 in accessions from different altitude regions
Article
Background Peach (Prunus persica) is an economically important stone fruit crop in Rosaceae and widely cultivated in temperate and subtropical regions, emerging as an excellent material to study the interaction between plant and environment. During its genus, there are four wild species of peach, all living in harsh environments. For example, one of the wild species, P. mira, originates from the Qinghai-Tibet Plateau (QTP) and exhibits strong cold/ultraviolet ray environmental adaptations. Although remarkable progresses in the gene discovery of fruit quality-related traits in peach using previous assembled genome were obtained, genomic basis of the response of these wild species to different geographical environments remains unclear. Results To uncover key genes regulating adaptability in different species and analyze the role of genetic variations in resistance formation, we performed de novo genome assembling of four wild relatives of peach (P. persica), P. mira, P. davidiana, P. kansuensis, and P. ferganensis and resequenced 175 peach varieties. The phylogenetic tree showed that the divergence time of P. mira and other wild relatives of peach was 11.5 million years ago, which was consistent with the drastic crustal movement of QTP. Abundant genetic variations were identified in four wild species when compared to P. persica, and the results showed that plant-pathogen interaction pathways were enriched in genes containing small insertions and deletions and copy number variations in all four wild relatives of peach. Then, the data were used to identify new genes and variations regulating resistance. For example, presence/absence variations which result from a hybridization event that occurred between P. mira and P. dulcis enhanced the resistance of their putative hybrid, P. davidiana. Using bulked segregant analysis, we located the nematode resistance locus of P. kansuensis in chromosome 2. Within the mapping region, a deletion in the promoter of one NBS-LRR gene was found to involve the resistance by regulating gene expression. Furthermore, combined with RNA-seq and selective sweeps analysis, we proposed that a deletion in the promoter of one CBF gene was essential for high-altitude adaptation of P. mira through increasing its resistance to low temperature. Conclusions In general, the reference genomes assembled in the study facilitate our understanding of resistance mechanism of perennial fruit crops, and provide valuable resources for future breeding and improvement.
 
Article
Background Gene expression programs are intimately linked to the interplay of active cis regulatory elements mediated by chromatin contacts and associated RNAs. Genome-wide association studies (GWAS) have identified many variants in these regulatory elements that can contribute to phenotypic diversity. However, the functional interpretation of these variants remains nontrivial due to the lack of chromatin contact information or limited contact resolution. Furthermore, the distribution and role of chromatin-associated RNAs in gene expression and chromatin conformation remain poorly understood. To address this, we first present a comprehensive interaction map of nuclear dynamics of 3D chromatin-chromatin interactions (H3K27ac BL-HiChIP) and RNA-chromatin interactions (GRID-seq) to reveal genomic variants that contribute to complex skeletal muscle traits. Results In a genome-wide scan, we provide systematic fine mapping and gene prioritization from GWAS leading signals that underlie phenotypic variability of growth rate, meat quality, and carcass performance. A set of candidate functional variants and 54 target genes previously not detected were identified, with 71% of these candidate functional variants choosing to skip over their nearest gene to regulate the target gene in a long-range manner. The effects of three functional variants regulating KLF6 (related to days to 100 kg), MXRA8 (related to lean meat percentage), and TAF11 (related to loin muscle depth) were observed in two pig populations. Moreover, we find that this multi-omics interaction map consists of functional communities that are enriched in specific biological functions, and GWAS target genes can serve as core genes for exploring peripheral trait-relevant genes. Conclusions Our results provide a valuable resource of candidate functional variants for complex skeletal muscle-related traits and establish an integrated approach to complement existing 3D genomics by exploiting RNA-chromatin and chromatin-chromatin interactions for future association studies.
 
Visual appearance of patch types maintained by Azteca ants and quantified N2 fixation activity. a An initial patch (IP), here inside a cross-sectioned internode, is built up by a foundress queen scratching off parenchyma. Patch material of established bA. alfari and cA. constructor colonies (established patches (EP)) is consisting of plant material, dead nestmates, fungi, and nematodes. Patch samples and parenchyma from the inner walls of uninhabited plant internodes were incubated for 72 h in an artificial ¹⁵N2:O2 atmosphere. d The ¹⁵N atom percent excess (APE) [%] depicted on the y-axis shows significant N2 fixation in patches, but not in the plant parenchyma (Pa). e The amount of fixed N per mg sample (dry weight) depicted on the y-axis shows variations, but insignificant differences between Azteca species (alf, A. alfari; con, A. constructor; xan, A. xanthochroa) within patch types (IP, EP). Sample sizes are given in brackets, and significant differences are indicated by asterisks (***P < 0.001). Scale bars, 1 cm (a–c)
Diazotrophic community composition in Azteca ant-associated patch samples. Taxonomic assignments of OTUs are summarized on the order level based on BLASTP analysis. On the x-axis, every bar represents the diazotrophic community per ant colony, grouped by patch type (initial patch, established patch) and ant species (Azteca alfari, A. constructor, A. xanthochroa), whereas the y-axis shows the relative read abundance of taxonomic orders
Distance-based redundancy analysis showing the variation of diazotrophic communities on OTU level a due to patch types, b within established patches due to individual ant colonies, and c within established patches due to ant species. Patch types are represented by shapes (IPs as triangles, EPs as circles). In b, multiple circles of the same color represent patch samples taken from different positions within the same plant
Relative abundance of phylogenetic nifH clusters based on classification and regression trees (CART) analysis. On the x-axis, every bar represents the diazotrophic community of an individual ant colony, grouped by patch type (initial patch, established patch) and ant species (Azteca alfari, A. constructor, A. xanthochroa), whereas the y-axis shows the relative read abundance
High heterogeneity of the nifH-transcribing community in patches of established Azteca alfari and A. constructor colonies at the time of sampling. Taxonomic assignments of OTUs are summarized on the order level based on BLASTP analysis. On the x-axis, every bar represents the diazotrophic community (DNA, bottom) and the actively transcribing community (RNA, middle) of one EP sample, grouped by ant species. The y-axis shows the relative read abundance of taxonomic orders. Triangles denote samples in which the most abundant transcribing order is not the most abundant one on the diazotrophic community level. The top graph shows the corresponding ¹⁵N atom percent excess (APE) after samples were incubated with ¹⁵N2. The dashed line denotes the median APE of all measured EP samples
Article
Background Symbiotic ant-plant associations, in which ants live on plants, feed on plant-provided food, and protect host trees against threats, are ubiquitous across the tropics, with the Azteca-Cecropia associations being amongst the most widespread interactions in the Neotropics. Upon colonization of Cecropia’s hollow internodes, Azteca queens form small patches with plant parenchyma, which are then used as waste piles when the colony grows. Patches—found in many ant-plant mutualisms—are present throughout the colony life cycle and may supplement larval food. Despite their initial nitrogen (N)-poor substrate, patches in Cecropia accommodate fungi, nematodes, and bacteria. In this study, we investigated the atmospheric N2 fixation as an N source in patches of early and established ant colonies. Results Via ¹⁵N2 tracer assays, N2 fixation was frequently detected in all investigated patch types formed by three Azteca ant species. Quantified fixation rates were similar in early and established ant colonies and higher than in various tropical habitats. Based on amplicon sequencing, the identified microbial functional guild—the diazotrophs—harboring and transcribing the dinitrogenase reductase (nifH) gene was highly diverse and heterogeneous across Azteca colonies. The community composition differed between early and established ant colonies and partly between the ant species. Conclusions Our data show that N2 fixation can result in reasonable amounts of N in ant colonies, which might not only enable bacterial, fungal, and nematode growth in the patch ecosystems but according to our calculations can even support the growth of ant populations. The diverse and heterogeneous diazotrophic community implies a functional redundancy, which could provide the ant-plant-patch system with a higher resilience towards changing environmental conditions. Hence, we propose that N2 fixation represents a previously unknown potential to overcome N limitations in arboreal ant colonies.
 
Article
Background β-1,4-endoglucanase (EG) is one of the three types of cellulases used in cellulose saccharification during lignocellulosic biofuel/biomaterial production. GsCelA is an EG secreted by the thermophilic bacterium Geobacillus sp. 70PC53 isolated from rice straw compost in southern Taiwan. This enzyme belongs to glycoside hydrolase family 5 (GH5) with a TIM-barrel structure common among all members of this family. GsCelA exhibits excellent lignocellulolytic activity and thermostability. In the course of investigating the regulation of this enzyme, it was fortuitously discovered that GsCelA undergoes a novel self-truncation/activation process that appears to be common among GH5 enzymes. Results Three diverse Gram-positive bacterial GH5 EGs, but not a GH12 EG, undergo an unexpected self-truncation process by removing a part of their C-terminal region. This unique process has been studied in detail with GsCelA. The purified recombinant GsCelA was capable of removing a 53-amino-acid peptide from the C-terminus. Natural or engineered GsCelA truncated variants, with up to 60-amino-acid deletion from the C-terminus, exhibited higher specific activity and thermostability than the full-length enzyme. Interestingly, the C-terminal part that is removed in this self-truncation process is capable of binding to cellulosic substrates of EGs. The protein truncation, which is pH and temperature dependent, occurred between amino acids 315 and 316, but removal of these two amino acids did not stop the process. Furthermore, mutations of E142A and E231A, which are essential for EG activity, did not affect the protein self-truncation process. Conversely, two single amino acid substitution mutations affected the self-truncation activity without much impact on EG activities. In Geobacillus sp. 70PC53, the full-length GsCelA was first synthesized in the cell but progressively transformed into the truncated form and eventually secreted. The GsCelA self-truncation was not affected by standard protease inhibitors, but could be suppressed by EDTA and EGTA and enhanced by certain divalent ions, such as Ca ²⁺ , Mg ²⁺ , and Cu ²⁺ . Conclusions This study reveals novel insights into the strategy of Gram-positive bacteria for directing their GH5 EGs to the substrate, and then releasing the catalytic part for enhanced activity via a spontaneous self-truncation process.
 
Article
Background The mammalian X and Y chromosomes originated from a pair of ordinary autosomes. Over the past ~180 million years, the X and Y have become highly differentiated and now only recombine with each other within a short pseudoautosomal region. While the X chromosome broadly preserved its gene content, the Y chromosome lost ~92% of the genes it once shared with the X chromosome. PRSSLY is a Y-linked gene identified in only a few mammalian species that was thought to be acquired, not ancestral. However, PRSSLY’s presence in widely divergent species—bull and mouse—led us to further investigate its evolutionary history. Results We discovered that PRSSLY is broadly conserved across eutherians and has ancient origins. PRSSLY homologs are found in syntenic regions on the X chromosome in marsupials and on autosomes in more distant animals, including lizards, indicating that PRSSLY was present on the ancestral autosomes but was lost from the X and retained on the Y in eutherian mammals. We found that across eutheria, PRSSLY’s expression is testis-specific, and, in mouse, it is most robustly expressed in post-meiotic germ cells. The closest paralog to PRSSLY is the autosomal gene PRSS55, which is expressed exclusively in testes, involved in sperm differentiation and migration, and essential for male fertility in mice. Outside of eutheria, in species where PRSSLY orthologs are not Y-linked, we find expression in a broader range of somatic tissues, suggesting that PRSSLY has adopted a germ-cell-specific function in eutherians. Finally, we generated Prssly mutant mice and found that they are fully fertile but produce offspring with a modest female-biased sex ratio compared to controls. Conclusions PRSSLY appears to be the first example of a gene that derives from the mammalian ancestral sex chromosomes that was lost from the X and retained on the Y. Although the function of PRSSLY remains to be determined, it may influence the sex ratio by promoting the survival or propagation of Y-bearing sperm.
 
Transcriptional regulation of the young genes. A A violin plot showing the expression of old and young genes. The expression of genes in transcript per million (TPM) was log10-transformed before plotting. The statistical significance of the difference was calculated using the Mann-Whitney test. B A line plot showing normalized coverage of FAIRE-seq reads around the transcription start site (TSS) of old (PS1) and young (PS19) genes. C A bar plot was drawn to show the enrichment of FAIRE-seq peaks at either the TSS-proximal (up to 1.5 kb upstream of TSS), TSS-distal (> − 1.5 kb from TSS), or both (FAIRE-seq peak present at TSS-proximal as well as TSS-distal intergenic regions) of old and young genes. Enrichment was higher than background (expected) and was calculated as the total percentage of genes present in the old or young gene categories. D An area proportional Venn diagram showing the overlap of FAIRE-seq-identified open chromatin regions and STARR-seq-identified enhancers. E A bar plot was drawn to show enrichment of enhancers at the TSS-proximal (up to 1.5 kb upstream of TSS) and TSS-distal (> − 1.5 kb from TSS) regions of old and young genes. The enrichment was seen over the background (expected), calculated as the total percentage of enhancers present in the old or young gene categories. F A box plot showing the association of STARR-seq-identified enhancers with the age of genes. Enhancer is characterized as TSS-distal if it is present > 1.5 kb upstream of the TSS, whereas TSS-proximal enhancers are located within 1.5 kb upstream of TSS. If an enhancer is present at any part of the gene body, then it is characterized as a gene body enhancer. The age of the nearest gene is plotted at the Y-axis, where phylostratum 1 denotes the oldest and phylostratum 19 denotes the youngest class of genes
Abiotic stress re-models the chromatin architecture of young genes. A line plot showing the normalized coverage of FAIRE-seq reads around the TSS (± 2000 bp) of young genes in control and stress subject rice seedlings
Nonsense-mediated RNA decay (NMD) post-transcriptionally regulates the abundance of young genes. A The accumulation of NMD features in old and young genes is presented as a bar plot. B A box plot representing the half-life (log10-transformed) of the old and young genes. C The relative change in expression of old and young genes due to UPF1 deficiency. D The association of old and young genes with the ribosome is shown as a box plot. E A box plot depicting the association of old and young genes with monosome and polysome either in pad4 or UPF1-null (upf1 pad4)