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 specie

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

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.NC

Perinatal hospice care in the opinion of nurses and midwives

Objectives: Perinatal medicine is a relatively new, dynamically developing branch of medicine. Its main purpose is takingcare of a woman in the pre-conception period, pregnancy and delivery, as well as taking care of a newborn baby.The main aim of the study was to assess the state of knowledge and opinion on hospice perinatal care of professionallyactive nurses and midwives.Material and methods: An original and anonymous questionnaire containing 30 questions was used for the study. 572 nursesand midwives from the Silesian Voivodeship took part in the study. The obtained data were analyzed.Results: Only 31.6% of respondents defined the level of their knowledge of pregnancy and neonatal care as high. 12.8%of respondents were able to indicate the definition of perinatal care and accurately determine its goals. The women participatingin the study were in favor of enclosing the information about not attempting resuscitation (DNAR) in medicalrecord of children with incurable disease diagnosed in fetal life (99.3%).Conclusions: The study showed deficits in practical and theoretical knowledge of nurses and midwives in the area ofhospice perinatal care. Lack of proper preparation is also one of the most frequently mentioned difficulties in taking careof a child and family with poor prognosis

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

Defining the Cell Wall, Cell Cycle and Chromatin Landmarks in the Responses of Brachypodium distachyon to Salinity

Excess salinity is a major stress that limits crop yields. Here, we used the model grass Brachypodium distachyon (Brachypodium) reference line Bd21 in order to define the key molecular events in the responses to salt during germination. Salt was applied either throughout the germination period (“salt stress”) or only after root emergence (“salt shock”). Germination was affected at 100 mM and root elongation at 75 mM NaCl. The expression of arabinogalactan proteins (AGPs), FLA1, FLA10, FLA11, AGP20 and AGP26, which regulate cell wall expansion (especially FLA11), were mostly induced by the “salt stress” but to a lesser extent by “salt shock”. Cytological assessment using two AGP epitopes, JIM8 and JIM13 indicated that “salt stress” increases the fluorescence signals in rhizodermal and exodermal cell wall. Cell division was suppressed at >75 mM NaCl. The cell cycle genes (CDKB1, CDKB2, CYCA3, CYCB1, WEE1) were induced by “salt stress” in a concentration-dependent manner but not CDKA, CYCA and CYCLIN-D4-1-RELATED. Under “salt shock”, the cell cycle genes were optimally expressed at 100mMNaCl. These changes were consistent with the cell cycle arrest, possibly at the G1 phase. The salt-induced genomic damage was linked with the oxidative events via an increased glutathione accumulation. Histone acetylation and methylation and DNA methylation were visualized by immunofluorescence. Histone H4 acetylation at lysine 5 increased strongly whereas DNA methylation decreased with the application of salt. Taken together, we suggest that salt-induced oxidative stress causes genomic damage but that it also has epigenetic effects, which might modulate the cell cycle and AGP expression gene. Based on these landmarks, we aim to encourage functional genomics studies on the responses of Brachypodium to salt

Brachypodium distachyon - a model plant to study grass genome structure, dynamics and evolution

Plenary Session: Present status of cell, tissue and organ in vitro cultur

Ultrastructural visualization of 3D chromatin folding using volume electron microscopy and DNA in situ hybridization.

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states

Zagrożenia zdrowotne wśród dzieci i młodzieży. T. 1

Praca recenzowana / Peer-reviewed pape

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030