EdU induces DNA damage response and cell death in mESC in culture

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2′-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.     

S-phase progression in mammalian cells: modelling the influence of nuclear organization

The control of DNA replication is of fundamental importance as cell proliferation demands that identical copies of the genetic material are passed to the two daughter cells that form during mitosis. These genetic copies are generated in the preceding S phase, where the entire DNA complement of the mother cell must be copied exactly once. As part of this process, it is known that different regions of mammalian genomes are replicated at specific times of a temporally defined replication programme. The key feature of this programme is that active genes in euchromatin are replicated before inactive ones in heterochromatin. This separation of S phase into periods where different classes of chromatin are duplicated is important in maintaining changes in gene expression that define individual cell types. Recent attempts to understand the structure of the S-phase timing programme have focused on the use of genome-wide strategies that inevitably use DNA isolated from large cell populations for analysis. However, this approach provides a composite view of events that occur within a population without knowledge of the cell-to-cell variability across the population. In this review, we attempt to combine information generated using genome-wide and single cell strategies in order to develop a coherent molecular understanding of S-phase progression. During this integration, we have explored how available information can be introduced into a modelling environment that best describes S-phase progression in mammalian cells.     

Perception,understanding, and association between psychological stress and skin aging: Quantitative surveys of Asian women aged 18–34 years,dermatologists, and psychologists in China and Japan

Background

Skin aging involves a variety of structural and functional changes, under the influence of various factors. Preaging skin is a relatively new concept describing self-perceived signs of skin aging that appear in the early 20s–30s and may be triggered by psychological stress. However, it is unclear how young women and healthcare professionals (HCP) understand the association between stress and skin aging.

Aims

We sought to explore the perceptions of stress-related skin aging among young women and HCPs.

Methods

We performed online surveys of 403 young women (18–34 years), 60 dermatologists, and 60 psychologists residing in major cities in China and Japan. Questions covered skin signs, understanding/perceptions of stress–aging connection, and demographics. Young women also completed DASS-21 to assess their stress level, which was dichotomized as normal or mild–extremely severe.

Results

The stress level was normal in 52.6% or mild–extremely severe in 47.4% of young women. Greater proportions of women in the mild–extremely severe stress group reported skin manifestations associated with preaging, the top three being “rough skin” (39.3% vs. 24.1%), “slow metabolic rate” (28.8% vs. 14.2%), and “dull skin” (43.5% vs. 29.2%). The top three skin manifestations showing the strongest perceived associations with stress were: “dark eye circles,” “slow metabolic rate,” and “dull skin” (among young women); “acne,” “dry skin,” and “skin rash” (among HCPs).

Conclusions

Young women frequently report high levels of psychological stress and signs of skin aging. Perceptions of stress–skin aging association differ between young women and HCPs.     

Non-convulsive status epilepticus in elderly individuals: report of four representative cases

OBJECTIVE: the purpose of this article is to describe the clinical and electroencephalographic features of four elderly patients diagnosed as having non-convulsive status epilepticus. METHODS: four females ranging in age from 74 to 81 years were admitted to our hospital because of confusion and altered mental state. We recognised four distinctive entities: i) Absence status in a patient with pre-existing idiopathic generalised epilepsy; ii) De novo absence status of late onset precipitated by benzodiazepine withdrawal; iii) Complex partial status epilepticus in a patient with a focal brain lesion; iv) Subtle generalised status epilepticus in a comatose subject representing the final phase of convulsive status epilepticus. CONCLUSIONS: the identification of non-convulsive status epilepticus may be particularly arduous in elderly subjects and, therefore, a high level of suspicion is essential to obtain an early diagnosis. An urgent electroencephalogram is considered as the method of choice in the diagnostic evaluation of non-convulsive status epilepticus. Finally, non-convulsive status epilepticus should be included among the causes of coma in older individuals.     

Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress

Both Myc and Ras oncogenes impact cellular metabolism, deregulate redox homeostasis and trigger DNA replication stress (RS) that compromises genomic integrity. However, how are such oncogene‐induced effects evoked and temporally related, to what extent are these kinetic parameters shared by Myc and Ras, and how are these cellular changes linked with oncogene‐induced cellular senescence in different cell context(s) remain poorly understood. Here, we addressed the above‐mentioned open questions by multifaceted comparative analyses of human cellular models with inducible expression of c‐Myc and H‐RasV12 (Ras), two commonly deregulated oncoproteins operating in a functionally connected signaling network. Our study of DNA replication parameters using the DNA fiber approach and time‐course assessment of perturbations in glycolytic flux, oxygen consumption and production of reactive oxygen species (ROS) revealed the following results. First, overabundance of nuclear Myc triggered RS promptly, already after one day of Myc induction, causing slow replication fork progression and fork asymmetry, even before any metabolic changes occurred. In contrast, Ras overexpression initially induced a burst of cell proliferation and increased the speed of replication fork progression. However, after several days of induction Ras caused bioenergetic metabolic changes that correlated with slower DNA replication fork progression and the ensuing cell cycle arrest, gradually leading to senescence. Second, the observed oncogene‐induced RS and metabolic alterations were cell‐type/context dependent, as shown by comparative analyses of normal human BJ fibroblasts versus U2‐OS sarcoma cells. Third, the energy metabolic reprogramming triggered by Ras was more robust compared to impact of Myc. Fourth, the detected oncogene‐induced oxidative stress was due to ROS (superoxide) of non‐mitochondrial origin and mitochondrial OXPHOS was reduced (Crabtree effect). Overall, our study provides novel insights into oncogene‐evoked metabolic reprogramming, replication and oxidative stress, with implications for mechanisms of tumorigenesis and potential targeting of oncogene addiction.     

The Intersection of Social Media Sites and Narrative Therapy in Treating Substance Use in Urban African American Adolescents

Social media has become prominent in the lives of many adolescents. This article addresses the intersection of social media sites and narrative therapy in treating substance use in urban African American adolescents. Risk factors of substance use in urban African American adolescents will be discussed, along with highlighting the importance of addressing issues around race, gender, and class in therapy, through a sociocultural lens. A case vignette is presented to demonstrate how narrative therapy techniques can be used to address therapy goals, specifically utilizing Facebook as an intervention tool to make progress in treatment.