Quantitative Analysis of Epigenetic Modifications in Fagopyrum Nuclei with Confocal Microscope, ImageJ, and R Studio.

Epigenetic programming plays a vital role in regulating pluripotency genes, which become activated or inactivated during the processes of dedifferentiation and differentiation during an organism's development. The analysis of epigenetic modifications has become possible through the technique of immunostaining, where specific antibodies allow the identification of a single target protein. This chapter describes a detailed protocol for the analysis of the epigenetic modifications with the use of confocal microscopy, subsequent image, and statistical analysis on the example of Fagopyrum calli with the use of nine antibodies raised against histone H3 and H4 methylation and acetylation on several lysines as well as DNA methylation.

Best practices for the execution, analysis, and data storage of plant single-cell/nucleus transcriptomics.

Single-cell and single-nucleus RNA-sequencing technologies capture the expression of plant genes at an unprecedented resolution. Therefore, these technologies are gaining traction in plant molecular and developmental biology for elucidating the transcriptional changes across cell types in a specific tissue or organ, upon treatments, in response to biotic and abiotic stresses, or between genotypes. Despite the rapidly accelerating use of these technologies, collective and standardized experimental and analytical procedures to support the acquisition of high-quality data sets are still missing. In this commentary, we discuss common challenges associated with the use of single-cell transcriptomics in plants and propose general guidelines to improve reproducibility, quality, comparability, and interpretation and to make the data readily available to the community in this fast-developing field of research.

Regulation of hair cell and stomatal size by a hair cell-specific peroxidase in the grass Brachypodium distachyon.

The leaf epidermis is the outermost cell layer forming the interface between plants and the atmosphere that must both provide a robust barrier against (a)biotic stressors and facilitate carbon dioxide uptake and leaf transpiration.1 To achieve these opposing requirements, the plant epidermis developed a wide range of specialized cell types such as stomata and hair cells. Although factors forming these individual cell types are known,2,3,4,5 it is poorly understood how their number and size are coordinated. Here, we identified a role for BdPRX76/BdPOX, a class III peroxidase, in regulating hair cell and stomatal size in the model grass Brachypodium distachyon. In bdpox mutants, prickle hair cells were smaller and stomata were longer. Because stomatal density remained unchanged, the negative correlation between stomatal size and density was disrupted in bdpox and resulted in higher stomatal conductance and lower intrinsic water-use efficiency. BdPOX was exclusively expressed in hair cells, suggesting that BdPOX cell-autonomously promotes hair cell size and indirectly restricts stomatal length. Cell-wall autofluorescence and lignin stainings indicated a role for BdPOX in the lignification or crosslinking of related phenolic compounds at the hair cell base. Ectopic expression of BdPOX in the stomatal lineage increased phenolic autofluorescence in guard cell (GC) walls and restricted stomatal elongation in bdpox. Together, we highlight a developmental interplay between hair cells and stomata that optimizes epidermal functionality. We propose that cell-type-specific changes disrupt this interplay and lead to compensatory developmental defects in other epidermal cell types.

Stem cell programming - prospects for perinatal medicine.

Recreating human cell and organ systems in vitro has tremendous potential for disease modeling, drug discovery and regenerative medicine. The aim of this short overview is to recapitulate the impressive progress that has been made in the fast-developing field of cell programming during the past years, to illuminate the advantages and limitations of the various cell programming technologies for addressing nervous system disorders and to gauge their impact for perinatal medicine.

The emergence of ecotypes in a parasitoid wasp: a case of incipient sympatric speciation in Hymenoptera?

BACKGROUND: To understand which reproductive barriers initiate speciation is a major question in evolutionary research. Despite their high species numbers and specific biology, there are only few studies on speciation in Hymenoptera. This study aims to identify very early reproductive barriers in a local, sympatric population of Nasonia vitripennis (Walker 1836), a hymenopterous parasitoid of fly pupae. We studied ecological barriers, sexual barriers, and the reduction in F1-female offspring as a postmating barrier, as well as the population structure using microsatellites. RESULTS: We found considerable inbreeding within female strains and a population structure with either three or five subpopulation clusters defined by microsatellites. In addition, there are two ecotypes, one parasitizing fly pupae in bird nests and the other on carrion. The nest ecotype is mainly formed from one of the microsatellite clusters, the two or four remaining microsatellite clusters form the carrion ecotype. There was slight sexual isolation and a reduction in F1-female offspring between inbreeding strains from the same microsatellite clusters and the same ecotypes. Strains from different microsatellite clusters are separated by a reduction in F1-female offspring. Ecotypes are separated only by ecological barriers. CONCLUSIONS: This is the first demonstration of very early reproductive barriers within a sympatric population of Hymenoptera. It demonstrates that sexual and premating barriers can precede ecological separation. This indicates the complexity of ecotype formation and highlights the general need for more studies within homogenous populations for the identification of the earliest barriers in the speciation process.

[Psychosocial Distress at Different Time Intervals after Burn Injury]. / Psychosoziale Belastungen zu verschiedenen Zeitpunkten nach Brandverletzung.

Burn injuries can result in long-term mental and physical health problems. We investigated if patients at different time periods since the burn injury differed with regard to psychosocial impairment. Patients who were treated as inpatients because of burn injury between 2006 and 2012 were asked about quality of life (Burn Specific Health Scale - Brief; BSHS-B), anxiety and depression (Hospital Anxiety and Depression Scale - Deutsche Version; HADS-D), posttraumatic stress disorder (Impact of Event Scale - Revised; IES-R), stigmatization (Perceived Stigmatization Questionnaire; PSQ) and social support (Fragebogen zur sozialen Unterstützung; F-SozU-7). The sample (146 patients) was divided into 4 groups according to the time period elapsed since the burn injury. There were no significant differences in psychosocial distress between the 4 groups. The same applied to sociodemographic and burn specific variables. 18 (12.4%) patients had the cutoff of ≥11 for anxiety and 22 (15.2%) for depression on the HADS; 16 (11.1%) patients screened positive for posttraumatic stress disorder (PTSD). No differences were found for current psycho(pharmaco)therapy and the wish for psychotherapy. The results suggest persistence of psychosocial burden over time after burn injury. Psychosocial interventions might thus be indicated even many years after burn injuries.

Validation of the German version of the Perceived Stigmatization Questionnaire/Social Comfort Questionnaire in adult burn survivors.

OBJECTIVE: To investigate the factor structure, reliability, and validity of the German version of the Perceived Stigmatization Questionnaire/Social Comfort Questionnaire (PSQ/SCQ) in burn victims. METHODS: The PSQ/SCQ was answered by 139 adult burn survivors (age M=49.69, SD=15.16 years). Factor structure was examined using a confirmatory factor analysis (CFA). Validity was investigated through correlations between the PSQ/SCQ scales and questionnaires assessing perceived social support, burn-specific health-related quality of life, symptoms of anxiety/depression, and percent of total body surface area (TBSA) burned. Additionally, the link between perceived stigmatization/social comfort and current partnership status was investigated. RESULTS: The four-factor model showed the best fit to the data with three PSQ factors (Absence of Friendly Behavior, Confused/Staring Behavior, and Hostile Behavior) and one single SCQ factor. All PSQ/SCQ scales showed good internal consistency. Higher PSQ/lower SCQ means were related to less perceived social support, less burn-specific quality of life, and more symptoms of anxiety/depression. With the exception of a positive correlation with the PSQ subscale Confused Behavior and Staring, no other significant correlations were found between the PSQ/SCQ subscales and TBSA burned. While PSQ/SCQ scores were not linked to age or gender, less perceived social stigmatization/more social comfort was reported by participants who were currently living with a partner. CONCLUSIONS: The results indicate a four-factor structure and a good validity of the PSQ/SCQ which is in line with prior research. Further studies should investigate the application of the PSQ/SCQ in individuals with appearance distinctions that are not related to burns.

Validation of the German version of the Burn Specific Health Scale-Brief (BSHS-B).

BACKGROUND: The Burn Specific Health Scale-Brief (BSHS-B) is recognized as a valid self-rating scale to evaluate quality of life after burn. AIM: To validate the translated German version of the BSHS-B. METHOD: One hundred and forty one burn survivors (65.2% men) with a mean age of 49.62 years (SD=15.16) and a mean duration after burn of 45.01 months (SD=26.18) answered the BSHS-B. Factor structure was tested by using confirmatory factor analysis, reliability (internal consistency) of the scales was determined by means of Cronbach's α. Construct validity was explored through correlations between the BSHS-B and the Short-Form 8 Health Survey (SF-8). In addition, the know-groups technique was used to determine to which degree the BSHS-B discriminates between patients with low and high burn severity based on the abbreviated burn severity index (ABSI). The Hospital Anxiety and Depression Scale (HADS) was used to examine criterion validity. RESULTS: The nine BSHS-B subscales showed good internal consistency. A second-order confirmatory factor analysis revealed the following main components: (1) Affect and Relationship, (2) Function and (3) Skin Involvement. The second-order factors were positively correlated with the SF-8 and negatively correlated with symptoms of anxiety and depression. Patients with low ABSI scored higher on all three BSHS-B domains than those with high ABSI. CONCLUSIONS: The results indicate good psychometric properties of the German BSHS-B. Further studies are needed to investigate the utility of the questionnaire in clinical routine practice, evaluation of burn management programs, and burn-specific research.