A rewiring of DNA replication mediated by MRE11 exonuclease underlies primed-to-naive cell de-differentiation.

Mouse embryonic stem cells (mESCs) in the primed pluripotency state, which resembles the post-implantation epiblast, can be de-differentiated in culture to a naive state that resembles the pre-implantation inner cell mass. We report that primed-to-naive mESC transition entails a significant slowdown of DNA replication forks and the compensatory activation of dormant origins. Using isolation of proteins on nascent DNA coupled to mass spectrometry, we identify key changes in replisome composition that are responsible for these effects. Naive mESC forks are enriched in MRE11 nuclease and other DNA repair proteins. MRE11 is recruited to newly synthesized DNA in response to transcription-replication conflicts, and its inhibition or genetic downregulation in naive mESCs is sufficient to restore the fork rate of primed cells. Transcriptomic analyses indicate that MRE11 exonuclease activity is required for the complete primed-to-naive mESC transition, demonstrating a direct link between DNA replication dynamics and the mESC de-differentiation process.

A protocol to study three-dimensional genome structure in individual mutant preimplantation mouse embryos.

Hi-C studies the three-dimensional structure of the genome by detecting genome-wide chromatin regions that are in spatial proximity within the nucleus. We developed single-blastocyst Hi-C in mutant mouse embryos to genotype them on sequence. We describe steps for embryo fixation and nuclei permeabilization, after which chromatin is digested and re-ligated having incorporated a biotin-labeled nucleotide at the ligation junction. After cross-link reversal, we then detail purification of immobilized chimeric DNA ligations, library generation, sequencing, and genome-wide analysis of interactions. For complete details on the use and execution of this protocol, please refer to Andreu et al. (2022).1.

Different NIPBL requirements of cohesin-STAG1 and cohesin-STAG2.

Cohesin organizes the genome through the formation of chromatin loops. NIPBL activates cohesin's ATPase and is essential for loop extrusion, but its requirement for cohesin loading is unclear. Here we have examined the effect of reducing NIPBL levels on the behavior of the two cohesin variants carrying STAG1 or STAG2 by combining a flow cytometry assay to measure chromatin-bound cohesin with analyses of its genome-wide distribution and genome contacts. We show that NIPBL depletion results in increased cohesin-STAG1 on chromatin that further accumulates at CTCF positions while cohesin-STAG2 diminishes genome-wide. Our data are consistent with a model in which NIPBL may not be required for chromatin association of cohesin but it is for loop extrusion, which in turn facilitates stabilization of cohesin-STAG2 at CTCF positions after being loaded elsewhere. In contrast, cohesin-STAG1 binds chromatin and becomes stabilized at CTCF sites even under low NIPBL levels, but genome folding is severely impaired.

Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin.

BACKGROUND: The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood. RESULTS: The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast. CONCLUSION: Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.

Essential Roles of Cohesin STAG2 in Mouse Embryonic Development and Adult Tissue Homeostasis.

Cohesin mediates sister chromatid cohesion and 3D genome folding. Two versions of the complex carrying STAG1 or STAG2 coexist in somatic vertebrate cells. STAG2 is commonly mutated in cancer, and germline mutations have been identified in cohesinopathy patients. To better understand the underlying pathogenic mechanisms, we report the consequences of Stag2 ablation in mice. STAG2 is largely dispensable in adults, and its tissue-wide inactivation does not lead to tumors but reduces fitness and affects both hematopoiesis and intestinal homeostasis. STAG2 is also dispensable for murine embryonic fibroblasts in vitro. In contrast, Stag2-null embryos die by mid-gestation and show global developmental delay and defective heart morphogenesis, most prominently in structures derived from secondary heart field progenitors. Both decreased proliferation and altered transcription of tissue-specific genes contribute to these defects. Our results provide compelling evidence on cell- and tissue-specific roles of different cohesin complexes and how their dysfunction contributes to disease.

Specific Contributions of Cohesin-SA1 and Cohesin-SA2 to TADs and Polycomb Domains in Embryonic Stem Cells.

Cohesin exists in two variants carrying either STAG/SA1 or SA2. Here we have addressed their specific contributions to the unique spatial organization of the mouse embryonic stem cell genome, which ensures super-enhancer-dependent transcription of pluripotency factors and repression of lineage-specification genes within Polycomb domains. We find that cohesin-SA2 facilitates Polycomb domain compaction through Polycomb repressing complex 1 (PRC1) recruitment and promotes the establishment of long-range interaction networks between distant Polycomb-bound promoters that are important for gene repression. Cohesin-SA1, in contrast, disrupts these networks, while preserving topologically associating domain (TAD) borders. The diverse effects of both complexes on genome topology may reflect two modes of action of cohesin. One, likely involving loop extrusion, establishes overall genome arrangement in TADs together with CTCF and prevents excessive segregation of same-class compartment regions. The other is required for organization of local transcriptional hubs such as Polycomb domains and super-enhancers, which define cell identity.

Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization.

Two variant cohesin complexes containing SMC1, SMC3, RAD21 and either SA1 (also known as STAG1) or SA2 (also known as STAG2) are present in all cell types. We report here their genomic distribution and specific contributions to genome organization in human cells. Although both variants are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-containing cohesin complexes (hereafter referred to as cohesin-SA2) localize to enhancers lacking CTCF, are linked to tissue-specific transcription and cannot be replaced by the SA1-containing cohesin complex (cohesin-SA1) when SA2 is absent, a condition that has been observed in several tumors. Downregulation of each of these variants has different consequences for gene expression and genome architecture. Our results suggest that cohesin-SA1 preferentially contributes to the stabilization of topologically associating domain boundaries together with CTCF, whereas cohesin-SA2 promotes cell-type-specific contacts between enhancers and promoters independently of CTCF. Loss of cohesin-SA2 rewires local chromatin contacts and alters gene expression. These findings provide insights into how cohesin mediates chromosome folding and establish a novel framework to address the consequences of mutations in cohesin genes in cancer.

Conservation Status of the Family Orchidaceae in Spain Based on European, National, and Regional Catalogues of Protected Species.

This report reviews the European, National, and Regional catalogues of protected species, focusing specifically on the Orchidaceae family to determine which species seem to be well-protected and where they are protected. Moreover, this examination highlights which species appear to be underprotected and therefore need to be included in some catalogues of protection or be catalogued under some category of protection. The national and regional catalogues that should be implemented are shown, as well as what species should be included within them. This report should be a helpful guideline for environmental policies about orchid's conservation in Spain, at least at the regional and national level. Around 76% of the Spanish orchid flora are listed with any figure of protection or included in any red list, either nationally (about 12-17%) or regionally (72%).

Polymeric imidazolium ionic liquids as valuable stationary phases in gas chromatography: chemical synthesis and full characterization.

Seven new functionalized polymerizable ionic liquids were chemically prepared, and later applied for the preparation of polymeric stationary phases in gas chromatography. These coated GC columns, which exhibited good thermal stabilities (240-300°C) and very high efficiencies (3120-4200 plates/m), have been characterized using the Abraham solvation parameter model. The chromatographic behavior of these polymeric IL columns has been deeply studied observing excellent selectivities in the separation of many organic substances such as alkanes, ketones, alcohols, amines or esters in mixtures of polar and non polar solvents or fragrances. Remarkably, the challenging separation of xylene isomers has been possible using a bis(trifluoromethylsulfonyl)amide based imidazolium IL coated column as a gas chromatography stationary phase.

Characterization of hexacationic imidazolium ionic liquids as effective and highly stable gas chromatography stationary phases.

Polycationic ionic liquids (ILs) are an attractive class of ILs with great potential applicability as gas chromatography stationary phases. A family of hexacationic imidazolium ILs derived from the cycloalkanol family was chemically first prepared in a straightforward manner and then applied for analytical separation purposes. Four tuneable engineering vectors, namely cation ring size structure, anion nature, spatial disposition of cycloalkanol substituents and O-substitution, were considered as experimental parameters for the design of the desired ionic liquids. A total number of five new phases based on a common benzene core respectively exhibited column efficiencies around to 2500 plates/m, broad operating temperature ranges and also, even more importantly, good thermal stabilities (bleeding temperature between 260 and 365°C), finding variations in the selectivity and analytes elution orders depending on the IL structures. Their solvation characteristics were evaluated using the Abraham solvation parameter model, establishing clear correlations between their cation structure and retention capability with respect to certain analytes. The study of relationships between the ILs structure and solvation parameters gives us an idea of the IL stationary phase to be used for specific separations.

Experimental design applied to the analysis of volatile compounds in apple juice by headspace solid-phase microextraction.

A simple and fast method based on solid-phase microextraction (SPME) followed by fast gas chromatography (Fast GC) has been developed for the analysis of volatile compounds in Asturian apple juices employed in the cider production. Three different fiber coatings have been checked (PDMS, PDMS-DVB and CAR-PDMS) and PDMS-DVB has been presented to be the most suitable one. Experimental design has been employed in the optimization of extraction factors and robustness assessment. The use of Fast GC allowed the separation of 14 compounds (esters, aldehydes and alcohols) in approximately 4 min, clearly reducing the analysis time when compared to conventional GC. Good linearity, recoveries and repeatability of the solid-phase microextraction were obtained with r(2) values, recoveries and relative standard deviations ranging from 0.9822 to 0.9998, 83.2 to 109.8% and 0.5 to 11.7%, respectively, using standard solution.

Evaluation of new ionic liquids as high stability selective stationary phases in gas chromatography.

Two ionic liquids (ILs), namely (S,S)-1-butyl-3-(2'-hydroxy-cyclohexyl)-3H-imidazol-1-ium tetrafluoroborate and (S,S)-1-butyl-3-(2'-acetyl-cyclohexyl)-3H-imidazol-1-ium tetrafluoroborate have been employed as stationary phases in capillary gas chromatography. These new phases exhibit a column efficiency of 1,600 and 2,100 plates m(-1) for IL 1 and IL 2, respectively, a wide operating temperature range and good thermal stability (bleeding temperature of 250 °C for IL 1 and 160 °C for IL 2). Inverse gas chromatography (GC) analyses were used to study the solvation properties of these ILs through a linear solvation energy model. The application of these ILs as new GC stationary phases was studied. These stationary phases exhibited unique selectivity for many organic substances, such as alkanes, ketones, esters, and aromatic compounds. The efficient separation of several mixtures containing compounds of different polarities and the good separation of fatty acid methyl esters (FAMEs) and cis/trans isomers indicate that these ILs may be applicable as a new type of GC stationary phases.

A novel method for the determination of total 1,3-octanediols in apple juice via 1,3-dioxanes by solid-phase microextraction and high-speed gas chromatography.

In this work, a novel, simple and fast method based on solid-phase microextraction (SPME) followed by high-speed gas chromatography (HSGC) was developed for the analysis of total 1,3-octanediols in apple juices by means of derivatization reaction to volatile 1,3-dioxanes. The derivatization reaction, SPME conditions, glycosidically bound fraction and 1,3-nonanediol as a surrogate standard were studied. The formation of 1,3-dioxanes from 1,3-diols was confirmed by GC-MS. The method was validated obtaining a regression coefficient (r(2)) of 0.9996, precisions between 0.3 and 9.8%, extraction recoveries in the range 94.7-112.2% and LOD of 2.9 microg l(-1). Experimental design has been employed in the optimization of extraction factors and robustness assessment. The method was applied to the analysis of 21 Asturian apple varieties finding a double reciprocal relationship between the concentrations of saturated and unsaturated 1,3-octanediol.

Comparison of several models for calculating ozone stomatal fluxes on a Mediterranean wheat cultivar (Triticum durum Desf. cv. Camacho).

Ozone stomatal fluxes were modeled for a 3-year period following different approaches for a commercial variety of durum wheat (Triticum durum Desf. cv. Camacho) at the phenological stage of anthesis. All models performed in the same range, although not all of them afforded equally significant results. Nevertheless, all of them suggest that stomatal conductance would account for the main percentage of ozone deposition fluxes. A new modeling approach was tested, based on a 3-D architectural model of the wheat canopy, and fairly accurate results were obtained. Plant species-specific measurements, as well as measurements of stomatal conductance and environmental parameters, were required. The method proposed for calculating ozone stomatal fluxes (FO(3_3-D)) from experimental gs data and modeling them as a function of certain environmental parameters in conjunction with the use of the YPLANT model seems to be adequate, providing realistic estimates of the canopy FO(3_3-D), integrating and not neglecting the contribution of the lower leaves with respect to the flag leaf, although a further development of this model is needed.

Dyspnea, ventilatory pattern, and changes in dynamic hyperinflation related to the intensity of constant work rate exercise in COPD.

STUDY OBJECTIVE: We undertook the present study to investigate the perception of dyspnea (with respect to changes in end-inspiratory and end-expiratory lung volumes), during four different levels of high-intensity constant work rate exercise (CWRE) in patients with severe COPD. DESIGN: Crossover descriptive study with consecutively recruited subjects. SETTING: Tertiary university hospital. PATIENTS: Twenty-seven subjects with severe COPD (mean [+/- SD] age, 65 +/- 5 years of age; mean FEV1, 43 +/- 8% predicted; and mean inspiratory capacity [IC]; 74 +/- 14% predicted). MEASUREMENTS AND RESULTS: Subjects randomly performed four high-intensity CWRE tests (conducted at 65%, 75%, 85%, and 95% of their symptom-limited peak work rate). Dyspnea, leg fatigue, and IC were determined every 2 min during exercise with breath-by-breath gas exchange and ventilatory measurements. There was a good correlation between the resting IC percent predicted and the oxygen uptake (V(O2)) peak (r = 0.64 to 0.69 between the IC percent predicted and V(O2) peak at the four work rates). There were significant differences (p < 0.01) in mean respiratory rate (33 +/- 6, 35 +/- 6, 37 +/- 6, and 38 +/- 6 min), peak dyspnea score (5.9 +/- 1.3, 6.3 +/- 1.4, 6.8 +/- 1.2, and 6.9 +/- 1.6), minute ventilation (45.0 +/- 8.7, 43.8 +/- 7.7, 43.1 +/- 8.7, and 42.8 +/- 8.0 L/min), leg fatigue (4.8 +/- 1.3, 5.1 +/- 1.3, 5.7 +/- 1.4, and 5.8 +/- 1.4), and end-tidal carbon dioxide partial pressure (4.41 +/- 0.36, 4.53 +/- 0.33, 4.66 +/- 0.31, and 4.76 +/- 0.24 kPa), respectively, for tests conducted at 65%, 75%, 85%, and 95% of their symptom-limited peak work rate, and in mean end-expiratory lung volume ([EELV] 4.55 +/- 0.44, 4.69 +/- 0.43, and 4.79 +/- 0.43 L), respectively, for tests conducted at 65%, 75%, and 85% of their symptom-limited peak work rate. In multivariable analysis, the factors that were independently correlated with dyspnea (p < 0.05) were EELV, peak inspiratory flow, and leg fatigue/discomfort. CONCLUSION: In COPD subjects with flow limitation at rest, the perception of dyspnea increased nonlinearly with the magnitude of high-intensity CWRE in association with a faster respiratory pattern and an increase in EELV. At the highest work rates, it appeared that a reduction in tidal volume and ventilation peak may have limited the tolerance to exercise.