Chromosome Research

During meiosis, homologous chromosomes pair to form synaptonemal complexes (SCs) and exchange genetic material through a process known as meiotic recombination. First, programmed DNA double-strand breaks form, followed by the assembly of recombination foci on SCs. These foci mark the sites of recombination intermediates and future crossovers. Distributions of recombination foci along SCs have been studied in many eukaryotes, revealing the interplay between recombination patterns and genome evolution. However, in fish, data on recombination patterns are scarce, and, for the majority of groups, completely absent. Here, we measure the positions of MLH1 foci in 3,504 SCs from 219 male meiotic cells of an African annual killifish Nothobranchius virgatus, a representative of a genus with remarkable karyotype and genome diversity, and present a detailed statistical analysis of its recombination patterns. We found that, in contrast to the several other fish species characterised to date, recombination in N. virgatus occurs across almost entire chromosome arms, excluding (peri)centromeres and telomeres. In the longest SCs, we observed a proximal and a distal peak of the recombination focus frequency and explained the peaks by chromosome pairing dynamics. We also revealed the typical positions of focus pairs, demonstrated interference between foci, with the minimal interfocus distance of 4 m, and described regions of the total recombination suppression near centromeres and telomeres. In sum, our study provides a detailed analysis of recombination patterns in a killifish with a fully acrocentric karyotype and contributes to cytogenomic and statistical methodology for future exploration of meiotic recombination patterns.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Bdelloid rotifers are known to survive desiccation and high doses of ionizing radiation. This extreme resistance is notably due to their capacity to cope with numerous DNA double-strand breaks (DSBs). Genes encoding key components of the non-homologous end joining (NHEJ) DNA repair pathway are strongly upregulated in the bdelloid rotifer Adineta vaga following exposure to ionizing radiation. Considering the notably high doses tolerated by these organisms, their capacity to efficiently restore genome integrity is particularly striking. Although NHEJ is generally regarded as less accurate than homologous recombination (HR), the absence of major genomic rearrangements in the descendants of irradiated rotifers suggests that DNA repair occurs with high fidelity. Terwagne et al. recently reported a delayed repair in germline nuclei, occurring during oocyte development when homologous chromosomes pair, thereby enabling template-based repair through HR. In this study, we established an in situ hybridization approach on A. vaga cryosections to investigate the spatial and temporal expression of key actors involved in NHEJ, HR, and Base excision repair (BER) pathways in somatic and germline tissues. We show that NHEJ (KU80) and BER-related genes (PARPs) as well as A. vaga Ligase E (putatively involved in DNA repair) are expressed early after radiation exposure in the somatic syncytium. In contrast, HR-related genes (Rad51: two paralogs, Rad54), as well as PCNA (involved in DNA replication, NER, BER, HR) are expressed later in maturing oocytes, indicating the activation of a delayed homologous recombination repair pathway in germline nuclei. Nurse cells, which express genes associated with both HR and NHEJ pathways, may rely on both mechanisms for their own DNA repair while also supplying mRNAs to the maturing oocyte. Our results provide new evidence for a differential regulation of DNA DSB repair pathways between soma and germline in bdelloids, with NHEJ predominating in somatic tissues and HR in the germline of A. vaga. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/722046v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3b1f3borg.highwire.dtl.DTLVardef@17f5eb5org.highwire.dtl.DTLVardef@122ef14org.highwire.dtl.DTLVardef@7e4413_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure:C_FLOATNO Summary of in situ hybridization results: genes coding for actors of NHEJ are expressed in the somatic nuclei and in the nurse nuclei of Adineta vaga individuals 2.5 hours post X-rays radiation, while genes coding for HR actors and PCNA (involved in multiple pathways including DNA replication and DNA repair: NER, BER, MR, HR) are expressed in the nurse nuclei 2.5 hours post radiation, and later in the maturing oocyte during oogenesis and in the laid eggs. Genes coding for actors highly expressed post-radiation, involved in the BER pathway appear to be only expressed in the somatic syncytium 2.5 hours post radiation, as well as the gene coding for the Ligase E, likely involved in DNA repair. C_FIG

The field of genetics of bird migration advances, driven by exponential refinements of sequencing and tracking technologies. In willow warblers (Phylloscopus trochilus), a complex repeat-rich region named MARB (Migration Associated Repeat Block) has recently been found to correlate with the routes taken by individual birds from Europe to their African wintering grounds. However, the genomic location of this region remains unknown. Here, we characterized MARB using a combination of approaches to understand how it evolved. We describe the region using long-read genome assemblies of two willow warbler subspecies (P. t. trochilus and P. t. acredula), two related species, the common chiffchaff (P. collybita) and the greenish warbler (P. trochiloides), and whole genome sequencing data from 76 willow warblers. Finally, we applied karyotyping and fluorescent in situ hybridization techniques on willow warbler spermatocytes to cytogenetically locate MARB. Due to the many repeats, we cannot order scaffolds in silico, but probe hybridization on the karyotype shows that MARB constitutes a single locus (~27.5 Mb) spanning most of the 11th largest chromosome in the willow warbler genome. Interestingly, the MARB regions of all species share several characteristics such as relatively high GC content (50%), a high density of specific repeat families and notably, more than 800 olfactory receptor sequences. Regions homologous to MARB may exist in several migrant bird genomes, though currently unassembled due to their complexity. Resolving these in species with similar migratory polymorphisms to willow warblers will be essential to determine whether MARB influences migratory behaviour across species.

Microexons are highly conserved fragments of exons ranging from 3 to 51 nucleotides (nts), representing a precise but poorly understood layer of post-transcriptional regulation outside the central nervous system. While their role in neuronal development is well documented, their behavior in peripheral tissues remains largely uncharacterized. In this study, we utilized VAST-TOOLS to perform a comprehensive meta-analysis of alternative microexon splicing across independent transcriptomic datasets spanning hepatic, pulmonary, renal, and colonic tissues. By comparing the diseased and wild-type (WT) profiles, we identified a robust set of differentially spliced microexons (DSMs) unique to each disease. Our findings suggest that microexon dysregulation in the liver, lung, kidney and colon may not be a primary driver of specific diseases, but rather a signature of a broader collapse in cellular splicing homeostasis. We propose that this phenomenon of differential splicing, particularly within critical hub proteins, fundamentally compromises protein interaction networks, thereby priming the cell for the diverse phenotypic failures observed across chronic disease states.

Abstract/SummaryGross chromosomal rearrangements are a hallmark of many diseases and cancers. The study of their biogenesis and the mechanisms underlying their formation is greatly facilitated by the availability of genetic reporter assays in model organisms. We present here a novel GCR assay developed in fission yeast, a highly relevant model for understanding genome instability related to human biology. The reporter employs canavanine counter-selection to detect GCRs within a chromosomal context. Using this assay, we identified natural hotspots for GCRs, including inverted long terminal repeats (IR-LTRs). Structural analysis of GCR events showed that IR-LTR-induced GCRs mainly result in either terminal deletions with adjacent inverted duplications or repair via long-range break-induced replication (BIR). Deleting IR-LTRs reduces the GCR rate and reveals another hotspot driven by BIR between homeologous aldo/keto reductase genes on opposite arms of chromosome I. This is the first evidence that BIR can occur in S. pombe on long tracks reaching up to 600 kb. Besides highlighting genome rearrangement hotspots, the assay also identifies regulators of genome instability in fission yeast. Loss of Nup132, a component of the nuclear pore complex, increases IR-LTRs-induced GCRs, while the budding yeast homolog Nup133 has no effect on the stability of a structurally similar IR. In contrast, disrupting djc9, which encodes a conserved histone H3-H4 binding protein, decreases GCR rates. Overall, this sensitive GCR assay enables the identification of factors that control spontaneous and fragile motif-induced chromosomal instability, including those conserved in humans but lost through evolution in other organisms.

Understanding cis-regulatory elements (CREs) at the single cell level is fundamental to deciphering transcriptional changes during development, cell differentiation, and homeostasis. Recent studies have shown that arbitrary peak-calling thresholds complicate data interpretation and cross-study comparisons. Furthermore, due to the inherent sparsity of single-nuclei ATAC-seq (snATAC-seq) data, distinguishing between truly inaccessible regions and technical dropouts remains challenging. Our analysis of snATAC-seq experiments performed in a well-established cell line suggests that the dichotomy between accessible (open) or inaccessible (close) CREs is misleading. Thousands of accessible regions are present in a very small fraction of cells of the population but they are repeatedly identified, suggesting that they have a low accessibility or are only transiently accessible. However, depending on the detection threshold selected they could be considered as either genuine CREs or noise. To resolve this inconsistency, we propose a model where chromatin accessibility is treated as a continuum, defined by a probability of accessibility (Pa) for each accessible region across cell types and conditions. Through computational simulations, we demonstrate that snATAC-seq results can be explained by a simple "balls into bins" probability model, offering a theoretical framework for calculating Pa distributions from any snATAC-seq dataset. Furthermore, we examine how Pa distributions shift following activation of the TGF{beta} signaling pathway. This probabilistic approach removes the reliance on arbitrary thresholds and supports a more quantitative, and dynamic understanding of accessible regions function.

Centromeric chromatin is defined by the presence of the histone H3 variant CENP-A, which forms a specialized nucleosome required for kinetochore assembly. Compared to canonical H3 nucleosomes, CENP-A nucleosomes exhibit an open DNA conformation that leaves an additional 13 base pairs of DNA accessible at the entry and exit sites. While the CENP-A N-helix has previously been implicated in promoting this enhanced DNA breathing, the contributions of the intrinsically disordered N-terminal tail and adjacent latch regions of CENP-A in nucleosome conformation remain unknown. The intrinsically disordered N-terminal regions of histone H3 are known to facilitate interactions with DNA to stabilize overall nucleosome conformation. Here, we systematically tested the contribution of each N-terminal histone region to maintaining H3 histones by utilizing a combination of MNase digestion assays and coarse-grained molecular dynamics simulations of H3/CENP-A chimera histone nucleosomes containing targeted swaps of the tail, latch, and N-helix regions. Removal or substitution of individual H3 with CENP-A N-terminal regions increased DNA accessibility and nucleosome unwrapping. While any single CENP-A N-terminal region was sufficient to open the canonical nucleosomal DNA conformation, replacement of any single CENP-A N-terminal region with its H3 counterpart was insufficient to restore the wrapped DNA conformation characteristic of canonical H3 nucleosomes. Instead, progressive incorporation of multiple H3-derived regions produced increasingly closed DNA conformations, demonstrating that the H3 tail, latch, and N-helices act cooperatively to stabilize wrapped nucleosomal DNA. Taken together, these findings demonstrate that the more restricted DNA breathing of canonical nucleosomes arises from coordinated contributions across multiple N-terminal regions and suggest that the multi-region redundancy in the conformational flexibility of the centromeric nucleosome could emphasize the importance of retaining flexibility in the centromeric nucleosome, even upon post-translational modification and binding to structural proteins. SIGNIFICANCEThe centromere is marked by nucleosomes containing CENP-A, which adopt a more open and accessible DNA conformation than canonical nucleosomes. However, the molecular determinants underlying this difference remain unclear. Previous structural investigations of the centromeric nucleosome have placed less emphasis on the intrinsically disordered N-terminal regions of CENP-A. Here, we systematically dissect the contributions of the N-terminal tail, latch, and N-helix via MNase digestion assays and molecular dynamics simulations on nucleosomes containing H3/CENP-A chimeras. We demonstrate that no individual H3-derived region is sufficient to impart a closed conformation to the nucleosomal DNA. Instead, multiple regions act together to stabilize DNA, revealing that nucleosome conformation is controlled by concerted histone-DNA interactions.

Biological sequences are known to be not random. Thus, the comparison of in silico restriction fragment distributions of random and biological sequences may be an indicator of this non-randomness. Our analyses show that for most of the tested combinations of restriction enzyme and genome sequence the fragments per Megabase of the biological sequence deviate at least more then 10% from the corresponding random sequence. This deviation goes into both directions, i.e. clearly increased values are as common as clearly decreased values. Although there is no species- or restriction-enzyme-specific effect, a clear impact of the GC content both of the restriction site and of the genome sequence can be seen. In contrast to the random sequences, the genome sequences show distinct peaks in their fragment length distributions, hinting to repetitive elements such as transposons.

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

Citrullination is a charge-modifying post-translational modification whereby proteinogenic arginine is converted to the non-coded amino acid citrulline by calcium-activated protein arginine deiminases (PADs; EC 3.5.3.15). The five known PAD enzymes in humans (PADs 1, 2, 3, 4, and 6) are differentially expressed and have distinct targets, including histones. While some PAD histone citrullination sites are known, a comprehensive investigation of all histone tail arginines targeted by catalytically active PADs 1-4 is lacking. Here, we sought to identify PAD citrullination sites in histone tails, both within histone peptides and in reconstituted nucleosomes. Toward this objective, we utilized a real-time 1H-15N NMR spectroscopy-based assay. By monitoring both arginine and citrulline backbone amide peak intensities over time, we identified sites of citrullination in 15N-labeled histone tails within peptides and reconstituted nucleosome core particles. We found that PADs 1, 2, and 4 citrullinate all directly observable histone tail arginines to varying degrees. This is distinct from PAD3, which only moderately citrullinates H2A and H4 arginine residues and does not modify H3 tail arginines. Together, these data suggest a level of histone arginine specificity by each PAD. Furthermore, histone tail citrullination is altered within nucleosomes compared to isolated peptides, which we interpret to reflect changes in conformation and accessibility. We speculate that citrullination increases nucleosomal histone tail dynamics, with implications for crosstalk between sites of histone citrullination and other important sites of regulation by PTMs (including lysines) within and between tails.

Sarcomeres, the basic repeating unit of striated muscle, are joined together by crosslinked actin filaments found at the boundaries of muscle sarcomeres, termed Z-discs. Z-discs play a key role in cardiac signalling and disease, however, the arrangement and function of many of the proteins present in the Z-disc remain to be understood. Here, we determined the organisation of 3 key proteins, ZASP, [a]-Actinin-2 and the Z1Z2 epitope of titin, located within the Z-disc. We fluorescently labelled these proteins in cardiac myofibrils using Adhirons specific to each protein and used interferometric photoactivated localization microscopy (iPALM) to obtain the 3D position of these proteins to a high precision (<10nm in x,y,z). We then used PERPL (Pattern Extraction from Relative Positions of Localisations) to analyse patterns in the relative positions of the proteins and reveal their underlying organisation. This analysis revealed that ZASP and [a]-Actinin-2 have a similar repeating organisation, but that the organisation of Z1Z2 is different.

Calpains constitute an ancient, extensive family of calcium-dependent cysteine proteases found in some bacteria and most eukaryotes. They are involved in a wide variety of developmental and cellular processes and are implicated in major human diseases, yet it remains to be seen if they have a common core function explaining their widespread and varied presence across taxa. Beyond their core CysPc catalytic domain, calpains contain diverse domain combinations and can be either cytosolic or membrane bound. Here we hypothesize a general role for both cytosolic and transmembrane calpains in cellular cytokinesis through positional anchoring and organization of microtubules (MTs). We propose that during plant cell division, the singular transmembrane calpain DEK1 localizes and organizes the array of cortical MTs from the microtubule organizing center (MTOC) to establish the location of the preprophase band and/or the site of cell plate formation according to the positional activation of DEK1 proteins in the nuclear membrane. Similarly, during cell division in animals, their calpains may be involved in setting the point of membrane invagination via their association with membrane-bound proteins. This proposition adds to the current picture of animal MTOC/centrosome function and suggests how a calcium peak during the initial cytokinetic furrowing might be transmitted. We discuss this novel mechanistic model for calpain activity in the context of data from the animal and plant literature, as well as of our novel discovery here of calpain sequences in both brown and red algal genomes. Finally, we speculate that the ancestral role of calpains in early eukaryotes, before the split into the major eukaryotic supergroups, may have been to facilitate the formation and function of MT arrays in flagella and cilia. From this origin, calpains may have developed new functions in eukaryote cell division processes by anchoring centrosomes/MTOC to set the cell division orientations that are especially important for complex multicellularity.

Target enrichment methods have provided unprecedented advances in phylogenomics. Targeting hundreds of conserved regions has proven to be a good tradeoff between cost and efficiency, while being useful for museomics and diversified non-model clades. Unfortunately, current methods used for identifying such regions involve high degrees of conservation within targeted elements, usually pushing researchers to rely on flanking data with little guarantee for homology. With a growing number of high quality genomes available throughout the Tree of Life emerges new opportunities to improve marker selection. In this study, we introduce GABBI, a new method for designing target capture probes by taking advantage of genome alignments, avoiding the selection of a single reference genome that can cause notable biases. We compare GABBI-derived markers to the most commonly used probe design method, PHYLUCE, at two taxonomic scales, the weevil superfamily Curculionoidea and the tribe Pachyrhynchini. At both taxonomic scales, results show that our new method allows identifying more variable loci that prove to be more phylogenetically resolutive than the PHYLUCE-derived ones. Doing so, we provide the first probe set specifically designed for weevils, targeting a wide set of 4,255 shared homologous regions, encouraging future research on systematics and macroevolution of one of the most diverse and economically important groups of insects. By providing GABBI as an automated and open-access pipeline, we hope to open new probe design opportunities to other taxonomic groups that face similar phylogenetic obstacles.

Forest trees pose numerous potential challenges to phylogenomic inference. Their large effective population sizes and relatively long generation times lead to deep allele coalescence and consequently incomplete lineage sorting (ILS), which biases inferences of divergence times toward older ages and introduces gene tree discordance. Deep phylogenetic divergences, reaching back into the Paleocene, introduce reference-mapping biases. Introgression--the movement of genes between lineages--may result in different phylogenies being inferred depending on which individuals are included in analysis, even if the plurality of the genome favors the divergence history unaffected by introgression. These factors influence phylogenetic inference across the Tree of Life but are particularly prevalent in forest trees. Oaks (Quercus) are notable for all three influences. In addition, our knowledge of the oak phylogeny is currently based strongly on restriction site associated DNA sequencing (RADseq) datasets published over the past decade, which may introduce additional sources of uncertainty. In this chapter, we analyze a 322-species RADseq dataset and genome resequencing data from across the genus to address sources of uncertainty in our understanding of the global oak phylogeny, which we hope will serve as a model for other research groups working on comparable woody plant groups.

Few studies assessed the performance of population-based phasing combined with parental genotypes to infer recombination on whole genome sequence (WGS) data. In this study, our objective was to evaluate whether Shapeit2 duoHMM, a Hidden Markov Model using parental genotypes, infers recombination events reliably on WGS and with narrower intervals than SNP arrays. We based our analysis on the overlap between recombination events inferred by Merlin on SNP genotypes and Shapeit2 on WGS and SNP genotypes. We used a sample of 61 extended families from the GeneSTAR study with TopMED freeze 8 WGS on 580 sequenced subjects (60% of sample). Shapeit2 was run with a window size of 500 kilobases and 200 states on WGS. To mimic a SNP array, we extracted genotypes of 355,112 autosomal markers on the Illumina OmniExpress array. The number of recombination events per meiosis inferred by Shapeit2 on the WGS data (36.8) was aligned with the expected numbers over autosomes (35.7), although Merlin overestimated this number (115.0). 73% of Shapeit2 recombination events on WGS were detected by Merlin, a proportion rising to 91% when restricting to events also inferred by Shapeit2 on OmniExpress genotypes. Furthermore, Shapeit2 recombination intervals were narrower on WGS than OmniExpress genotypes (median of 4,530 bp vs. 49,458 bp). This suggests that Shapeit2 on WGS is a reliable and accurate method for inferring recombination events.

Rapid and specific diagnosis of viral and bacterial infections is a significant challenge in medicine and veterinary science, especially in the case of epidemically dangerous pathogens. The African swine fever virus (ASFV), for example, causes annual outbreaks among livestock, resulting in significant economic losses for farmers. DNA aptamers have been identified as a promising tool for point-of-care diagnostics, being highly specific to the target and stable ambient temperatures during storage. In this study, we describe the selection of DNA aptamers targeting the p54 viral protein using a single-round selection process. These aptamers were able to bind both to recombinant protein and inactivated ASFV viral particles. Analysis of the newly generated aptamers revealed a dependence of affinity and thermal stability on Ni2+ content, which was a dopant in the selection process. In some cases, the affinity increased 100 times, and melting temperature increased by 30{degrees}C. We have identify two novel DNA motifs that bound 2-3 Ni2+ or Zn2+ ions.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Computational workshops are common in evolutionary biology and are used to share discipline-specific tools and skills with researchers. Despite the perceived importance of these workshops, there is no common set of criteria for workshop success, and there are few peer-reviewed studies investigating the efficacy of workshops or assessing the value of particular instructional techniques in this context. Here, we focused on one key element of a successful workshop: its ability to increase participants motivation to use the methods and tools presented during the workshop. We analyzed the goals, perceptions, and future plans of research practitioners engaging in a workshop on phylogenetic methods of historical biogeography using pre- and post-workshop surveys. Overall, the workshop was successful at motivating participants, and survey responses provided insights into participants perceptions of different activities, including "participatory live coding". Apart from this case study, we aim to highlight the importance of developing a common set of workshop goals in collaboration with other workshop stakeholders and the need for specialized, validated tools for assessing the efficacy of computational workshops for researchers.

This investigation aimed at compiling all phylogenetic lineages within and around genus Cyanoboletus. The evolutionary inference obtained from the nuclear ribosomal genes internal transcribed spacer region (ITS) suggests that part of the species currently classified in Cyanoboletus belong in lineages separate from the genus, thus suggesting a narrower boundary that includes only the species that develop a strong staining reaction to touch and to air exposure of the context. The separate lineages are the monotypic Cupreoboletus genus and a few species that do not develop such reaction, which are part of a clade together with genera Cacaoporus and Acyanoboletus, thus broadening the concept of Cacaoporus to encompass all of them. The emerging 3C perspective of Cupreoboletus, Cacaoporus and Cyanoboletus offers a remarkably consistent morphological diagnosis, overcoming the problems of a too broad concept for Cyanoboletus. This work reveals that Boletus neotropicus, B. novae-zelandiae and B. sensibilis belong respectively in Cyanoboletus, Cacaoporus and Lanmaoa, and by studying multigene alignment concatenates it identifies lineages that probably represent undescribed species: at least four in Cacaoporus and at least five in Cyanoboletus. Diagnostic tables and dichotomic keys are presented by geographic region. The present work also includes a study of the phylogenetic position of Neoboletus flavosanguineus, a species once classified in Cyanoboletus. The complexity of assigning species epithets in some lineages is addressed, namely for the boundaries between Cacaoporus instabilis and Ca. fagaceophilus as well as the diversity under the names Cyanoboletus sinopulverulentus and Cy. pulverulentus. The overall picture of evolutionary lineages sets a framework for the choice of reference data that can provide, in future phylogenetic studies that involve the 3C, a balanced and efficient coverage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/724631v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@7f618corg.highwire.dtl.DTLVardef@dd6a14org.highwire.dtl.DTLVardef@5f7399org.highwire.dtl.DTLVardef@9e7443_HPS_FORMAT_FIGEXP M_FIG C_FIG