Supporting Employment Consultants to Implement Supported and Customized Employment.

Implementing supported and customized employment in all their components is essential for supporting job seekers with disabilities to achieve their career goals. We asked 42 employment consultants in nine employment programs to respond daily to three quick questions about their employment support activities, for 6 months. Through monthly coaching, we helped the managers of these organizations engage their teams of employment consultants to interpret the data, reflect, set goals, and take action for quality improvement. Based on the positive results of this pilot, we recommend that employment consultants be provided with data-enabled feedback that supports decision making and helps them fully implement supported and customized employment as a necessary step toward improving job seekers' employment outcomes.

Prime Editing: A New Way for Genome Editing.

Precise and efficient use of genome editing tools are hampered by the introduction of DNA double-strand breaks, donor DNA templates, or homology-directed repair. A recent study expands the genome editing toolbox with the introduction of prime editing, which overcomes previous challenges and introduces insertions, deletions, and all putative 12 types of base-to-base conversions in human cells.

BABA-Induced DNA Methylome Adjustment to Intergenerational Defense Priming in Potato to Phytophthora infestans.

We provide evidence that alterations in DNA methylation patterns contribute to the regulation of stress-responsive gene expression for an intergenerational resistance of ß-aminobutyric acid (BABA)-primed potato to Phytophthora infestans. Plants exposed to BABA rapidly modified their methylation capacity toward genome-wide DNA hypermethylation. De novo induced DNA methylation (5-mC) correlated with the up-regulation of Chromomethylase 3 (CMT3), Domains rearranged methyltransferase 2 (DRM2), and Repressor of silencing 1 (ROS1) genes in potato. BABA transiently activated DNA hypermethylation in the promoter region of the R3a resistance gene triggering its downregulation in the absence of the oomycete pathogen. However, in the successive stages of priming, an excessive DNA methylation state changed into demethylation with the active involvement of potato DNA glycosylases. Interestingly, the 5-mC-mediated changes were transmitted into the next generation in the form of intergenerational stress memory. Descendants of the primed potato, which derived from tubers or seeds carrying the less methylated R3a promoter, showed a higher transcription of R3a that associated with an augmented intergenerational resistance to virulent P. infestans when compared to the inoculated progeny of unprimed plants. Furthermore, our study revealed that enhanced transcription of some SA-dependent genes (NPR1, StWRKY1, and PR1) was not directly linked with DNA methylation changes in the promoter region of these genes, but was a consequence of methylation-dependent alterations in the transcriptional network.

An exploration of the lived experiences of non-binary individuals who have presented at a gender identity clinic in the United Kingdom.

Background: Despite an increased awareness of non-binary identity in the current social landscape, the experiences and needs of this heterogeneous community are poorly understood and represented in the research literature. Evidence indicates that social exclusion is not uncommon for individuals expressing a non-binary gender identity, with reflections in the literature that this may in turn have an impact on their psychological wellbeing. Aim: As non-binary individuals are increasingly presenting at UK gender identity clinics and requesting medical interventions, the aim of this study was to better understand their experiences and needs. Method: Two focus groups were run consisting of eight service users of a National Health Service (NHS) gender identity clinic in the United Kingdom. The transcripts of these focus groups were analysed using thematic analysis. Results: Five themes were identified: Invisibility, Managing non-binary gender identity in a binary world, Individuality, Gender dysphoria and Seeking interventions. Discussion: Clinical implications are discussed, with the recommendation for an affirmative approach that offers space for the non-binary individual to articulate their desires and come to terms with their identity. This exploration must take into consideration the person's place within a social world that can be transphobic and limited in terms of potential medical interventions. Further research is needed to better understand this marginalised community.

Germination and the Early Stages of Seedling Development in Brachypodium distachyon.

Successful germination and seedling development are crucial steps in the growth of a new plant. In this study, we investigated the course of the cell cycle during germination in relation to grain hydration in the model grass Brachypodium distachyon (Brachypodium) for the first time. Flow cytometry was performed to monitor the cell cycle progression during germination and to estimate DNA content in embryo tissues. The analyses of whole zygotic embryos revealed that the relative DNA content was 2C, 4C, 8C, and 16C. Endoreplicated nuclei were detected in the scutellum and coleorhiza cells, whereas the rest of the embryo tissues only had nuclei with a 2C and 4C DNA content. This study was accompanied by a spatiotemporal profile analysis of the DNA synthetic activity in the organs of Brachypodium embryos during germination using EdU labelling. Upon imbibition, nuclear DNA replication was initiated in the radicle within 11 h and subsequently spread towards the plumule. The first EdU-labelled prophases were observed after 14 h of imbibition. Analysis of selected genes that are involved in the regulation of the cell cycle, such as those encoding cyclin-dependent kinases and cyclins, demonstrated an increase in their expression profiles.

Epigenomic diversification within the genus Lupinus.

Deciphering the various chemical modifications of both DNA and the histone compound of chromatin not only leads to a better understanding of the genome-wide organisation of epigenetic landmarks and their impact on gene expression but may also provide some insights into the evolutionary processes. Although both histone modifications and DNA methylation have been widely investigated in various plant genomes, here we present the first study for the genus Lupinus. Lupins, which are members of grain legumes (pulses), are beneficial for food security, nutrition, health and the environment. In order to gain a better understanding of the epigenetic organisation of genomes in lupins we applied the immunostaining of methylated histone H3 and DNA methylation as well as whole-genome bisulfite sequencing. We revealed variations in the patterns of chromatin modifications at the chromosomal level among three crop lupins, i.e. L. angustifolius (2n = 40), L. albus (2n = 50) and L. luteus (2n = 52), and the legume model plant Medicago truncatula (2n = 16). Different chromosomal patterns were found depending on the specific modification, e.g. H3K4me2 was localised in the terminal parts of L. angustifolius and M. truncatula chromosomes, which is in agreement with the results that have been obtained for other species. Interestingly, in L. albus and L. luteus this modification was limited to one arm in the case of all of the chromosomes in the complement. Additionally, H3K9me2 was detected in all of the analysed species except L. luteus. DNA methylation sequencing (CG, CHG and CHH contexts) of aforementioned crop but also wild lupins such as L. cosentinii (2n = 32), L. digitatus (2n = 36), L. micranthus (2n = 52) and L. pilosus (2n = 42) supported the range of interspecific diversity. The examples of epigenetic modifications illustrate the diversity of lupin genomes and could be helpful for elucidating further epigenetic changes in the evolution of the lupin genome.

Ovaries of the white worm (Enchytraeus albidus, Annelida, Clitellata) are composed of 16-celled meroistic germ-line cysts.

The paired ovaries of E. albidus are like a bunch of grapes and are composed of clearly separated units, syncytial germ cysts (clusters), which are surrounded by a thin layer of somatic cells. Each cyst maintains the connection with the ovary by an extended stalk that is composed of somatic cells. The spatial architecture of the germ-line cysts found in E. albidus is the same as in other clitellate annelids that have been studied to date. As a rule, germ cells are located at the cyst periphery and each has only one ring canal that connects it to the common and centrally located cytoplasmic mass, the cytophore. Here we present data about the F-actin and microtubular cytoskeleton and some molecular components of the germ-line cysts. We show that the ring canals have an inner rim that is enriched with microfilaments and proteins that contain phosphotyrosine. The microtubules form a loose network in the cytoplasm of the oocyte and nurse cells; moreover, some of them pass through the ring canals to the cytophore. Numerous microtubules are also located in the somatic cells. The germ-line cysts in E. albidus ovaries consist of 16 cells, which is the lowest known number of interconnected germ cells within clitellate annelids. During oogenesis, the fate of interconnected germ cells differentiates and only one cell develops as the future egg, while the other 15 become nurse cells. This differentiation means ovary meroism. The nurse cells gather cell organelles and storage material that then pass through the ring canals and cytophore moving towards the growing oocyte. At the end of oogenesis, the vitellogenic oocyte surrounds the siblings' cells together with the cytophore and engulfs their remnants into the ooplasm. No morphological or molecular markers of the apoptosis of the nurse cells were found. Moreover, the nurse cells did not undergo polyploidisation. The measured DNA level was 4C, which indicates that these cells are not highly-specialised.

Histone H3 and H4 acetylation patterns are more dynamic than those of DNA methylation in Brachypodium distachyon embryos during seed maturation and germination.

The transition of seeds from a dry to a metabolically active state requires significant changes in both the spatial and temporal patterns of gene expression, and this transcriptional reprogramming involves various modifications of the chromatin structure. There are several factors that can greatly influence the structure of chromatin, one of which is the chemical modifications of histone proteins and DNA itself. In this study, we analysed the distribution of three epigenetic markers, i.e. acetylation of histone H4 (H4K16ac) and histone H3 (H3K18ac) as well as DNA methylation (5mC) in Brachypodium distachyon embryos during the four stages of seed development-maturation, desiccation (quiescence), imbibition and germination. Our results indicate that both H4K16ac and H3K18ac are at a very high level in embryos during seed imbibition, but that the patterns of DNA methylation are considerably more stable in embryos during seed development.

DNA replication after mutagenic treatment in Hordeum vulgare.

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed.

Lead shielding efficiency from the gamma background measurements in the salt cavern of the Polkowice-Sieroszowice copper mine.

The studies of lead shielding efficiency from the gamma background measurements were performed in the salt cavern of the copper mine - a site considered for an underground laboratory. Within the energy range of 50-2700 keV, the measured gamma-ray count rates normalized to the mass of the high-purity detectors germanium crystal are 5.93 and 6.32 s-1kg-1 for the used low-background and portable spectrometers, respectively. The gamma-ray flux of 0.124 (2) cm-2s-1 connected with the natural radioisotopes was observed by the portable HPGe, including 0.026 (1) cm-2s-1 contribution of radon decay products, whereas the photon flux at the spectrum continuum was 0.18 (5) cm-2s-1.

Clinical characteristics of cutaneous lupus erythematosus.

INTRODUCTION: Lupus erythematosus (LE) shows a wide variety of clinical manifestations, skin involvement being one of the most important. AIM: To analyze the clinical presentation of cutaneous variants of lupus erythematosus in terms of skin lesion spectrum and extracutaneous involvement. MATERIAL AND METHODS: A total of 64 patients with cutaneous LE (CLE) were included. The study was based on the "Core Set Questionnaire" developed by the European Society of Cutaneous Lupus Erythematosus (EUSCLE). Clinical severity of skin lesions was evaluated with the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). All results were subjected to statistical analysis. RESULTS: Fifteen (23.4%) patients had an acute CLE (ACLE), 26 (40.6%) subacute CLE (SCLE) and 21 (32.8%) chronic CLE (CCLE). Two (3.2%) individuals only demonstrated urticarial vasculitis as a cutaneous manifestation of LE and these patients were excluded. Patients with ACLE were characterized by the earliest onset of the disease (mean age of 31.9 ±15.0 years; p < 0.001). On average, 4.8 ±1.8 criteria of systemic LE were found in the ACLE group compared to 2.7 ±1.3 criteria in SCLE and 2.5 ±1.5 criteria in CCLE (p < 0.001). The highest activity of skin lesions according to CLASI was found in the SCLE group (p = 0.002). On the other hand, the most severe skin damage was observed in CCLE (p < 0.01). CONCLUSIONS: Each variant of CLE differs significantly from the others in respect of various aspects of clinical manifestations. Due to a number of different variants of LE skin lesions, a unified classification of CLE still remains a challenge.

In situ analysis of epigenetic modifications in the chromatin of Brachypodium distachyon embryos.

Epigenetic modifications of the chromatin structure are crucial for many biological processes and act on genes during the development and germination of seeds. The spatial distribution of 3 epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 was investigated in 'matured,' 'dry,' 'imbibed" and 'germinating' embryos of a model grass, Brachypodium. Our results indicate that the patterns of epigenetic modification differ in the various types of tissues of embryos that were analyzed. Such a tissue-specific manner of these modifications may be linked to the switch of the gene expression profiles in various organs of the developing embryo.

Functional analysis of the new barley gene HvKu80 indicates that it plays a key role in double-strand DNA break repair and telomere length regulation.

Genotoxic stress causes a reduced stability of the plant genome and has a detrimental effect on plant growth and productivity. Double-strand breaks (DSBs) are the most harmful of all DNA lesions because they cause the loss of genetic information on both strands of the DNA helix. In the presented study the coding and genomic sequences of the HvKu80 gene were determined. A mutational analysis of two fragments of HvKu80 using TILLING (Targeting Induced Local Lesions IN Genomes) allowed 12 mutations to be detected, which resulted in identification of 11 alleles. Multidirectional analyses demonstrated that the HvKu80 gene is involved in the elimination of DSBs in Hordeum vulgare. The barley mutants carrying the identified ku80.c and ku80.j alleles accumulated bleomycin-induced DSBs to a much greater extent than the parent cultivar 'Sebastian'. The altered reaction of the mutants to DSB-inducing agent and the kinetics of DNA repair in these genotypes are associated with a lower expression level of the mutated gene. The study also demonstrated the significant role of the HvKu80 gene in the regulation of telomere length in barley.

Alleles of newly identified barley gene HvPARP3 exhibit changes in efficiency of DNA repair.

Genome integrity is constantly challenged by endo- and exogenous DNA-damaging factors. The influence of genotoxic agents causes an accumulation of DNA lesions, which if not repaired, become mutations that can cause various abnormalities in a cell metabolism. The main pathway of DSB repair, which is based on non-homologous recombination, is canonical non-homologous end joining (C-NHEJ). It has been shown that this mechanism is highly conserved in both Pro- and Eukaryotes. The mechanisms that underlie DSB repair through C-NHEJ have mainly been investigated in mammalian systems, and therefore our knowledge about this process is much more limited as far as plants, and crop plants in particular, are concerned. Recent studies have demonstrated that PARP3 is an important response factor to the presence of DSB in a genome. The aims of this study were to identify the sequence of the barley PARP3 gene, to perform a mutational analysis of the sequence that was identified using the TILLING (Targeting Induced Local Lesions IN Genomes) method and to phenotype the mutants that were identified through their exposure to mutagenic treatment with the DSB-inducing chemical--bleomycin. A functional analysis led to the identification of a series of parp3 alleles. The mutants were characterized using several different approaches, including quantifying the DSB and γH2AX foci, which validated the function of the HvPARP3 gene in DSB repair in barley. The potential involvement of the HvPARP3 gene in the regulation of telomere length in barley was also analyzed.

Spatial distribution of epigenetic modifications in Brachypodium distachyon embryos during seed maturation and germination.

Seed development involves a plethora of spatially and temporally synchronised genetic and epigenetic processes. Although it has been shown that epigenetic mechanisms, such as DNA methylation and chromatin remodelling, act on a large number of genes during seed development and germination, to date the global levels of histone modifications have not been studied in a tissue-specific manner in plant embryos. In this study we analysed the distribution of three epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 in 'matured', 'dry' and 'germinating' embryos of a model grass, Brachypodium distachyon (Brachypodium). Our results indicate that the abundance of these modifications differs considerably in various organs and tissues of the three types of Brachypodium embryos. Embryos from matured seeds were characterised by the highest level of H4K5ac in RAM and epithelial cells of the scutellum, whereas this modification was not observed in the coleorhiza. In this type of embryos H3K4me2 was most evident in epithelial cells of the scutellum. In 'dry' embryos H4K5ac was highest in the coleorhiza but was not present in the nuclei of the scutellum. H3K4me1 was the most elevated in the coleoptile but absent from the coleorhiza, whereas H3K4me2 was the most prominent in leaf primordia and RAM. In embryos from germinating seeds H4K5ac was the most evident in the scutellum but not present in the coleoptile, similarly H3K4me1 was the highest in the scutellum and very low in the coleoptile, while the highest level of H3K4me2 was observed in the coleoptile and the lowest in the coleorhiza. The distinct patterns of epigenetic modifications that were observed may be involved in the switch of the gene expression profiles in specific organs of the developing embryo and may be linked with the physiological changes that accompany seed desiccation, imbibition and germination.

Epigenetic chromatin modifications in barley after mutagenic treatment.

In addition to their normal developmental processes, plants have evolved complex genetic and epigenetic regulatory mechanisms to cope with various environmental stresses. It has been shown that both DNA methylation and histone modifications are involved in DNA damage response to various types of stresses. In this study, we focused on the involvement of two mutagenic agents, chemical (maleic acid hydrazide; MH) and physical (gamma rays), on the global epigenetic modifications of chromatin in barley. Our results indicate that both mutagens strongly influence the level of histone methylation and acetylation. Moreover, we found that gamma irradiation, in contrast to MH, has a more robust influence on the DNA methylation level. This is the first study that brings together mutagenic treatment along with its impact at the level of epigenetic modifications examined using the immunohistochemical method.

Tissue-specific epigenetic modifications in root apical meristem cells of Hordeum vulgare.

Epigenetic modifications of chromatin structure are essential for many biological processes, including growth and reproduction. Patterns of DNA and histone modifications have recently been widely studied in many plant species, although there is virtually no data on the spatial and temporal distribution of epigenetic markers during plant development. Accordingly, we have used immunostaining techniques to investigate epigenetic modifications in the root apical meristem of Hordeum vulgare. Histone H4 acetylation (H4K5ac), histone H3 dimethylation (H3K4me2, H3K9me2) and DNA methylation (5mC) patterns were established for various root meristem tissues. Distinct levels of those modifications were visualised in the root cap, epidermis, cortex and vascular tissues. The lateral root cap cells seem to display the highest level of H3K9me2 and 5mC. In the epidermis, the highest level of 5mC and H3K9me2 was detected in the nuclei from the boundary of the proximal meristem and the elongation zone, while the vascular tissues were characterized by the highest level of H4K5ac. Some of the modified histones were also detectable in the cytoplasm in a highly tissue-specific manner. Immunolocalisation of epigenetic modifications of chromatin carried out in this way, on longitudinal or transverse sections, provides a unique topographic context within the organ, and will provide some answers to the significant biological question of tissue differentiation processes during root development in a monocotyledon plant species.

Epigenetic modifications of nuclei differ between root meristematic tissues of Hordeum vulgare.

Recent studies on the role of epigenetic modifications during plant development emphasize the fact that both positional information and tissue specificity are essential factors that establish epigenetic marks and thus determine cell fate and differentiation processes. The root apical meristem (RAM ), which contains stem cells and generates radial patterns of tissues, is an ideal model for studying the correlation between cell position and cell-type differentiation, with particular emphasis on the patterns, global levels, and landscapes of epigenetic modifications. To date, there has been no clear evidence for differential levels of histone and DNA modification across root meristematic tissues. Our study clearly indicates that levels of modifications with potential epigenetic effects vary between RAM tissues. Of particular interest is that histone H4 acetylation in the epidermis is not simply replication-dependent and probably plays a role in epidermal cell differentiation.

Histone H3 methylation patterns in Brassica nigra, Brassica juncea, and Brassica carinata species.

Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific.

Epigenetic chromatin modifications in Brassica genomes.

Epigenetic modifications such as histone and DNA methylation are highly conserved among eukaryotes, although the nuclear patterns of these modifications vary between different species. Brassica species represent a very attractive model for analysis of epigenetic changes because of their differences in genome size, ploidy level, and the organization of heterochromatin blocks. Brassica rapa and B. oleracea are diploid species, and B. napus is an allotetraploid species that arose from the hybridization of these two diploids. We found that patterns of DNA and histone H3 methylation differ between Brassica species. The most prominent differences concern the two diploids. DNA methylation was present exclusively in the heterochromatin only in B. rapa. In B. oleracea and B. napus this modification was detected in both euchromatin and heterochromatin. A similar pattern was observed for dimethylation of lysine 9. Dimethylation of lysine 4 is a typical marker of euchromatin in Brassica species, like it is in other plant species. We conclude that the diploid species differ in patterns of analyzed epigenetic modifications and the allotetraploid B. napus has combined patterns from both diploids. Differences in patterns of DNA and histone H3 methylation between Brassica species can be attributed mainly to the genome structure and heterochromatin localization rather than ploidy level.