Chronic endoplasmic reticulum stress in myotonic dystrophy type 2 promotes autoimmunity via mitochondrial DNA release.

Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases.

Proverb comprehension in schizophrenia: A comprehensive review and meta-analysis.

BACKGROUND: Examination of proverb comprehension has a long tradition in clinical diagnostics of individuals with schizophrenia (iSCZ). Deficits in the comprehension are considered common. Interpretations of proverbs are traditionally measured by their degree of abstraction and concreteness ('literalness'), but iSCZ's responses may also be illogical or 'bizarre'. Experimental research on proverb comprehension starts in the 1940s. Since then, the specificity of proverb tests has often been questioned, but has never been the subject of a meta-analysis. The aim of this meta-analysis is to include all experimental research, including historical studies, that meets quality criteria and compares the responses to proverbs in iSCZ with those in healthy controls (HC) or clinical controls (CC). METHODS: PubMed, Web of Science, and PsycInfo databases were searched. After coding 121 articles, 27 (median publication year 1982) were included and multi-level meta-analyses performed. Moderator analyses were performed on response format (multiple-choice vs. verbal responses), proverb test, scoring method, language, acute vs. chronic stage of iSCZ, time of publication, clinical vs. healthy control group, age, IQ/education, and gender. Publication bias was assessed using funnel plots, trim and fill method and Egger's test. RESULTS: The search identified 27 eligible studies for inclusion. Studies were published between 1956 and 2020 and predominantly older than 30 years (median: 1982). The Gorham Proverbs Test was the most established test and predominantly conducted in English. CC mostly consisted of depressive disorders. Pooled estimates yielded statistically significant less abstract (g = -1.00; 95%CI, -1.34 to -1.67), more concrete (g = 0.69; 95%CI, 0.35-1.03), and more bizarre (g = 1.08; 95%CI, 0.74-1.41) responses in iSCZ compared to controls. The type of control group moderated all three effects, with greater differences of iSCZ compared to HC than to CC in abstraction and bizarreness, and no significant group difference between iSCZ and CC in concreteness. Meta-regressions indicated IQ/education and age as possible sources of variability in abstraction and bizarreness. CONCLUSIONS: While lower abstraction and higher bizarreness seems a characteristic of iSCZ, the diagnostic specificity of a concrete response was astonishingly low. The lack of a unified definition for concretism and limited consideration of cultural diversity contributed to these complex findings. Future research should focus on exploring the qualitative aspects of proverb comprehension and the association between symptomatology types and misinterpretations to improve diagnostic accuracy.

The cyto-linker and scaffolding protein "plectin" mis-localization leads to softening of cancer cells.

Discovering tools to prevent cancer progression requires understanding the fundamental differences between normal and cancer cells. More than a decade ago, atomic force microscopy (AFM) revealed cancer cells' softer body compared to their healthy counterparts. Here, we investigated the mechanism underlying the softening of cancerous cells in comparison with their healthy counterparts based on AFM high resolution stiffness tomography and 3D confocal microscopy. We showed microtubules (MTs) network in invasive ductal carcinoma cell cytoskeleton is basally located and segmented for around 400 nm from the cell periphery. Additionally, the cytoskeleton scaffolding protein plectin exhibits a mis-localization from the cytoplasm to the surface of cells in the carcinoma which justifies the dissociation of the MT network from the cell's cortex. Furthermore, the assessment of MTs' persistence length using a worm-like-chain (WLC) model in high resolution AFM images showed lower persistence length of the single MTs in ductal carcinoma compared to that in the normal state. Overall, these tuned mechanics support the invasive cells to ascertain more flexibility under compressive forces in small deformations. These data provide new insights into the structural origins of cancer aids in progression.

Irony detection in patients with borderline personality disorder: an experimental study examining schizotypal traits, response biases and empathy.

BACKGROUND: In verbal irony we often convey meanings that oppose the literal words. To look behind these words, we need to integrate perspectives of ourselves, others, and their beliefs about us. Although patients with borderline personality disorder (BPD) experience problems in social cognition and schizotypal symptoms, research on irony comprehension mainly focused on the schizophrenic spectrum. Accounting for possible negative biases in BPD, the current study examined the detection of praising and critical irony in a text messaging interface. METHODS: The cross-sectional study included 30 patients and 30 matched controls, who completed measures of cognitive and affective empathy (Interpersonal Reactivity Index, IRI), schizotypal (Schizotypal Personality Questionnaire; SPQ), and borderline symptoms (Borderline Symptom List; BSL-23) and the irony detection task. The irony task contained critical and praising remarks embedded in text messages. Asking for literality (ironic vs. literal) and intention ratings (critical to praising) of the stimuli, it allowed to analyze the sensitivity of literality detection as well as implicit and explicit response biases in a signal detection framework. RESULTS: Borderline symptoms explained lower sensitivity for the detection of literal and ironic statements across groups. Whereas HC showed a negativity bias when implicitly asked about the literalness of the statement, patients with BPD perceived praising utterances as less praising when explicitly asked about their perceived intention. Neither empathy nor schizotypy explained outcomes beyond borderline symptoms. CONCLUSIONS: This was the first study to show lower detection of verbal irony in patients with BPD. While patients were less biased when asked about the literality of a statement, they perceived praising remarks as less positive on explicit measurements. The results highlight the importance of congruent, transparent communication in promoting epistemic trust in individuals with BPD.

SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA.

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

Chromatin Ubiquitination Guides DNA Double Strand Break Signaling and Repair.

Chromatin is the context for all DNA-based molecular processes taking place in the cell nucleus. The initial chromatin structure at the site of the DNA damage determines both, lesion generation and subsequent activation of the DNA damage response (DDR) pathway. In turn, proceeding DDR changes the chromatin at the damaged site and across large fractions of the genome. Ubiquitination, besides phosphorylation and methylation, was characterized as an important chromatin post-translational modification (PTM) occurring at the DNA damage site and persisting during the duration of the DDR. Ubiquitination appears to function as a highly versatile "signal-response" network involving several types of players performing various functions. Here we discuss how ubiquitin modifiers fine-tune the DNA damage recognition and response and how the interaction with other chromatin modifications ensures cell survival.

The Chromatin Architectural Protein CTCF Is Critical for Cell Survival upon Irradiation-Induced DNA Damage.

CTCF is a nuclear protein initially discovered for its role in enhancer-promoter insulation. It has been shown to play a role in genome architecture and in fact, its DNA binding sites are enriched at the borders of chromatin domains. Recently, we showed that depletion of CTCF impairs the DNA damage response to ionizing radiation. To investigate the relationship between chromatin domains and DNA damage repair, we present here clonogenic survival assays in different cell lines upon CTCF knockdown and ionizing irradiation. The application of a wide range of ionizing irradiation doses (0-10 Gy) allowed us to investigate the survival response through a biophysical model that accounts for the double-strand breaks' probability distribution onto chromatin domains. We demonstrate that the radiosensitivity of different cell lines is increased upon lowering the amount of the architectural protein. Our model shows that the deficiency in the DNA repair ability is related to the changes in the size of chromatin domains that occur when different amounts of CTCF are present in the nucleus.

Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction.

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture.

FUS-dependent liquid-liquid phase separation is important for DNA repair initiation.

RNA-binding proteins (RBPs) are emerging as important effectors of the cellular DNA damage response (DDR). The RBP FUS is implicated in RNA metabolism and DNA repair, and it undergoes reversible liquid-liquid phase separation (LLPS) in vitro. Here, we demonstrate that FUS-dependent LLPS is necessary for the initiation of the DDR. Using laser microirradiation in FUS-knockout cells, we show that FUS is required for the recruitment to DNA damage sites of the DDR factors KU80, NBS1, and 53BP1 and of SFPQ, another RBP implicated in the DDR. The relocation of KU80, NBS1, and SFPQ is similarly impaired by LLPS inhibitors, or LLPS-deficient FUS variants. We also show that LLPS is necessary for efficient γH2AX foci formation. Finally, using superresolution structured illumination microscopy, we demonstrate that the absence of FUS impairs the proper arrangement of γH2AX nanofoci into higher-order clusters. These findings demonstrate the early requirement for FUS-dependent LLPS in the activation of the DDR and the proper assembly of DSB repair complexes.

Trauma exposure therapy in a pregnant woman suffering from complex posttraumatic stress disorder after childhood sexual abuse: risk or benefit?

Background: Mental disorders during pregnancy are common and affect the health of mother and child. Despite a relatively high prevalence rate, treatment options have not been investigated systematically. Particularly symptoms of posttraumatic stress disorder (PTSD) may increase significantly during the course of pregnancy. However, proper guidelines for psychotherapeutic treatment of PTSD during pregnancy do not exist. Objective: In this article, we aimed at discussing the effects of untreated PTSD on pregnancy and postpartum mother-child bonding as well as exposure therapy during pregnancy. Method: To do so, we present the case of a pregnant woman with complex PTSD following childhood sexual abuse. At the time of hospitalization, the patient was pregnant in the second trimester and reported intrusive re-experiencing of the traumatic events, nightmares, anxiety and helplessness as well as an impairing level of irritability during social situations. After a careful discussion of the case within our department and at the annual conference of the German Association of Psychiatry, Psychotherapy and Psychosomatics, we decided to treat the patient with dialectical behavior therapy for PTSD (DBT-PTSD) including exposure therapy under the regular observation of a gynecologist. Psychometric measurements (Davidson Trauma Scale (DTS) and Borderline Symptom- List-23 (BSL-23) were used to observe the course of treatment regarding common PTSD-symptoms and disturbances in self-organization (DSO). Results: The intensity of intrusions and hyperarousal increased from the date of admission, reached the maximum when exposure started and decreased below baseline-level at the end of treatment. Avoidance behavior continually decreased from the beginning until the end of therapy. Decreased BSL-23 values show major improvements regarding DSO. To our knowledge, the course of pregnancy was not affected by treatment-induced psychological and physical symptoms.Conclusions: DBT- PTSD is a potential treatment option for patients suffering from PTSD during pregnancy. Yet, further (epigenetic) research in this field is urgently needed.

 Antecedentes: Los trastornos mentales durante el embarazo son frecuentes y afectan la salud de la madre y el niño. A pesar de las relativamente altas tasas de prevalencia, las opciones de tratamiento aún no han sido investigadas sistemáticamente. Particularmente los síntomas de trastorno de estrés postraumático (TEPT) pueden aumentar significativamente durante el embarazo. Sin embargo, no existen guías adecuadas de tratamientos psicoterapéuticos para el TEPT durante el embarazo.Objetivo: En este artículo, nuestro objetivo fue discutir los efectos del TEPT no tratado en el embarazo y el vínculo madre-hijo postparto, así como la terapia de exposición durante el embarazo.Método: Para ello, presentamos el caso de una mujer embarazada con TEPT complejo por un abuso sexual en su infancia. Al momento de la hospitalización, la paciente estaba cursando su segundo trimestre de embarazo y reportaba re-experimentación intrusiva de los eventos traumáticos, pesadillas, ansiedad y desesperanza así como tambien empeoramiento de los niveles de irritabilidad en situaciones sociales. Tras una discusión cuidadosa del caso en nuestro departamento y en la reunión anual de la Asociación Alemana de Psiquiatría, Psicoterapia y Psicosomática, decidimos tratar a la paciente con terapia dialéctica conductual para TEPT (DBT-TEPT) incluida la terapia de exposición bajo observación regular de un ginecólogo. Se usaron medidas psicométricas (la Escala de Trauma de Davidson (DTS) y la Lista de síntomas Borderline-23 (BSL-23) para observar el curso del tratamiento con respecto a los síntomas comunes de TEPT y las alteraciones en la auto-organización (DSO). Resultados: La intensidad de las intrusiones e hiperalerta aumentaron desde el ingreso, alcanzando un máximo cuando la exposición comenzó y disminuyeron bajo el nivel basal al final del tratamiento. La conducta evitativa disminuyó continuamente desde el inicio hasta el final de la terapia. Los valores disminuidos del BSL-23 muestran una mejoría importante en relación a la DSO. Hasta donde, el curso del embarazo no se afectó por los síntomas psicológicos y físicos inducidos por el tratamiento. Conclusiones: La DBT-TEPT es una opción de tratamiento potencial para pacientes que sufren de TEPT durante el embarazo. Sin embargo, se necesitan con urgencia más investigaciones (epigenéticas) en este campo.

Characterisation of the novel spontaneously immortalized and invasively growing human skin keratinocyte line HaSKpw.

We here present the spontaneously immortalised cell line, HaSKpw, as a novel model for the multistep process of skin carcinogenesis. HaSKpw cells were established from the epidermis of normal human adult skin that, without crisis, are now growing unrestricted and feeder-independent. At passage 22, clonal populations were established and clone7 (HaSKpwC7) was further compared to the also spontaneously immortalized HaCaT cells. As important differences, the HaSKpw cells express wild-type p53, remain pseudodiploid, and show a unique chromosomal profile with numerous complex aberrations involving chromosome 20. In addition, HaSKpw cells overexpress a pattern of genes and miRNAs such as KRT34, LOX, S100A9, miR21, and miR155; all pointing to a tumorigenic status. In concordance, HaSKpw cells exhibit reduced desmosomal contacts that provide them with increased motility and a highly migratory/invasive phenotype as demonstrated in scratch- and Boyden chamber assays. In 3D organotypic cultures, both HaCaT and HaSKpw cells form disorganized epithelia but only the HaSKpw cells show tumorcell-like invasive growth. Together, HaSKpwC7 and HaCaT cells represent two spontaneous (non-genetically engineered) "premalignant" keratinocyte lines from adult human skin that display different stages of the multistep process of skin carcinogenesis and thus represent unique models for analysing skin cancer development and progression.

Familiarity, empathy and comprehension of metaphors in patients with borderline personality disorder.

Research on figurative language has a long tradition in psychiatry, as it is employed in psychotherapy and its (mis)comprehension plays a substantial role in differential diagnostics of schizophrenic spectrum disorders. Although often associated with empathy and mentalization, it has never been addressed in borderline personality disorder (BPD). Therefore, this study investigated metaphor comprehension and its relationship to cognitive and affective empathy in 20 patients with BPD and 20 matched healthy controls who completed a metaphor task comprising conventional metaphors (CM), novel metaphors (NM), meaningless stimuli (MS), and a rating scale of familiarity, a factor known to influence performance. For cognitive and affective empathy, the interpersonal reactivity index was applied. At first patients with BPD seemed to have significantly more problems in comprehending CM, but not NM or MS, and were less familiar with CM. When familiarity with the stimulus was controlled, this difference disappeared. As for empathy, only fantasy was positively related to familiar CM beyond borderline symptoms. Results indicate that the comprehension of novel metaphorical meaning is preserved in patients with BPD.

Processive DNA synthesis is associated with localized decompaction of constitutive heterochromatin at the sites of DNA replication and repair.

Constitutive heterochromatin is considered as a functionally inert genome compartment, important for its architecture and stability. How such stable structure is maintained is not well understood. Here, we apply four different visualization schemes to label it and investigate its dynamics during DNA replication and repair. We show that replisomes assemble over the heterochromatin in a temporally ordered manner. Furthermore, heterochromatin undergoes transient decompaction locally at the active sites of DNA synthesis. Using selective laser microirradiation conditions that lead to damage repaired via processive DNA synthesis, we measured similarly local decompaction of heterochromatin. In both cases, we could not observe large-scale movement of heterochromatin to the domain surface. Instead, the processive DNA synthesis machinery assembled at the replication/repair sites. Altogether, our data are compatible with a progression of DNA replication/repair along the chromatin in a dynamic mode with localized and transient decompaction that does not globally remodels the whole heterochromatin compartment.

A New Test for Irony Detection: The Influence of Schizotypal, Borderline, and Autistic Personality Traits.

Irony has repeatedly been suggested as a language based social cognition task. It has been argued to show specific variances in psychiatric disorders and healthy adults with certain personality traits. Above that, irony comprehension is based on a complex interplay of the informational context, the relationship of the conversational partners, and the personality of the recipient. The present study developed a video-based German language test for a systematic examination of irony detection accuracy (Tuerony). The test includes (i) a stereotypical conversation partner (doctor, actor) in (ii) different perspectives (direct interaction, neutral observer) and (iii) a bilateral chat history on a conventional messenger service interface with ironic criticism, ironic praise, literal criticism, and literal praise. Based on the continuous approach of psychiatric symptoms, schizotypal, borderline, and autistic personality traits were associated with irony detection accuracy in a healthy sample. Given the often reported role of mentalization in irony detection, these associations were also investigated. First, a broad variance of irony comprehension in our healthy sample could be shown. Second, schizotypal and borderline, but not autistic traits were significantly negatively associated with irony detection accuracy. Finally, in the current healthy sample, neither variation of the conversational context nor mentalization characteristics were significantly associated with performance beyond personality traits. The current results therefore highlight two aspects for future research in irony comprehension: the importance of ecological valid tests and the role of the individual personality of the recipient.

DNA replication dynamics of vole genome and its epigenetic regulation.

BACKGROUND: The genome of some vole rodents exhibit large blocks of heterochromatin coupled to their sex chromosomes. The DNA composition and transcriptional activity of these heterochromatin blocks have been studied, but little is known about their DNA replication dynamics and epigenetic composition. RESULTS: Here, we show prominent epigenetic marks of the heterochromatic blocks in the giant sex chromosomes of female Microtus cabrerae cells. While the X chromosomes are hypoacetylated and cytosine hypomethylated, they are either enriched for macroH2A and H3K27me3 typical for facultative heterochromatin or for H3K9me3 and HP1 beta typical for constitutive heterochromatin. Using pulse-chase replication labeling and time-lapse microscopy, we found that the heterochromatic block enriched for macroH2A/H3K27me3 of the X chromosome is replicated during mid-S-phase, prior to the heterochromatic block enriched for H3K9me3/HP1 beta, which is replicated during late S-phase. To test whether histone acetylation level regulates its replication dynamics, we induced either global hyperacetylation by pharmacological inhibition or by targeting a histone acetyltransferase to the heterochromatic region of the X chromosomes. Our data reveal that histone acetylation level affects DNA replication dynamics of the sex chromosomes' heterochromatin and leads to a global reduction in replication fork rate genome wide. CONCLUSIONS: In conclusion, we mapped major epigenetic modifications controlling the structure of the sex chromosome-associated heterochromatin and demonstrated the occurrence of differences in the molecular mechanisms controlling the replication timing of the heterochromatic blocks at the sex chromosomes in female Microtus cabrerae cells. Furthermore, we highlighted a conserved role of histone acetylation level on replication dynamics across mammalian species.

DNA replication and repair kinetics of Alu, LINE-1 and satellite III genomic repetitive elements.

BACKGROUND: Preservation of genome integrity by complete, error-free DNA duplication prior to cell division and by correct DNA damage repair is paramount for the development and maintenance of an organism. This holds true not only for protein-encoding genes, but also it applies to repetitive DNA elements, which make up more than half of the human genome. Here, we focused on the replication and repair kinetics of interspersed and tandem repetitive DNA elements. RESULTS: We integrated genomic population level data with a single cell immunofluorescence in situ hybridization approach to simultaneously label replication/repair and repetitive DNA elements. We found that: (1) the euchromatic Alu element was replicated during early S-phase; (2) LINE-1, which is associated with AT-rich genomic regions, was replicated throughout S-phase, with the majority being replicated according to their particular histone marks; (3) satellite III, which constitutes pericentromeric heterochromatin, was replicated exclusively during the mid-to-late S-phase. As for the DNA double-strand break repair process, we observed that Alu elements followed the global genome repair kinetics, while LINE-1 elements repaired at a slower rate. Finally, satellite III repeats were repaired at later time points. CONCLUSIONS: We conclude that the histone modifications in the specific repeat element predominantly determine its replication and repair timing. Thus, Alu elements, which are characterized by euchromatic chromatin features, are repaired and replicated the earliest, followed by LINE-1 elements, including more variegated eu/heterochromatic features and, lastly, satellite tandem repeats, which are homogeneously characterized by heterochromatic features and extend over megabase-long genomic regions. Altogether, this work reemphasizes the need for complementary approaches to achieve an integrated and comprehensive investigation of genomic processes.

Enhancing Psychosis-Spectrum Nosology Through an International Data Sharing Initiative.

The latent structure of schizotypy and psychosis-spectrum symptoms remains poorly understood. Furthermore, molecular genetic substrates are poorly defined, largely due to the substantial resources required to collect rich phenotypic data across diverse populations. Sample sizes of phenotypic studies are often insufficient for advanced structural equation modeling approaches. In the last 50 years, efforts in both psychiatry and psychological science have moved toward (1) a dimensional model of psychopathology (eg, the current Hierarchical Taxonomy of Psychopathology [HiTOP] initiative), (2) an integration of methods and measures across traits and units of analysis (eg, the RDoC initiative), and (3) powerful, impactful study designs maximizing sample size to detect subtle genomic variation relating to complex traits (the Psychiatric Genomics Consortium [PGC]). These movements are important to the future study of the psychosis spectrum, and to resolving heterogeneity with respect to instrument and population. The International Consortium of Schizotypy Research is composed of over 40 laboratories in 12 countries, and to date, members have compiled a body of schizotypy- and psychosis-related phenotype data from more than 30000 individuals. It has become apparent that compiling data into a protected, relational database and crowdsourcing analytic and data science expertise will result in significant enhancement of current research on the structure and biological substrates of the psychosis spectrum. The authors present a data-sharing infrastructure similar to that of the PGC, and a resource-sharing infrastructure similar to that of HiTOP. This report details the rationale and benefits of the phenotypic data collective and presents an open invitation for participation.

Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks.

The replication of the genome is a highly organized process, both spatially and temporally. Although a lot is known on the composition of the basic replication machinery, how its activity is regulated is mostly unknown. Several chromatin properties have been proposed as regulators, but a potential role of the nuclear DNA position remains unclear. We made use of the prominent structure and well-defined heterochromatic landscape of mouse pericentric chromosome domains as a well-studied example of late replicating constitutive heterochromatin. We established a method to manipulate its nuclear position and evaluated the effect on replication timing, DNA compaction and epigenetic composition. Using time-lapse microscopy, we observed that constitutive heterochromatin, known to replicate during late S-phase, was replicated in mid S-phase when repositioned to the nuclear periphery. Out-of-schedule replication resulted in deficient post-replicative maintenance of chromatin modifications, namely silencing marks. We propose that repositioned constitutive heterochromatin was activated in trans according to the domino model of origin firing by nearby (mid S) firing origins. In summary, our data provide, on the one hand, a novel approach to manipulate nuclear DNA position and, on the other hand, establish nuclear DNA position as a novel mechanism regulating DNA replication timing and epigenetic maintenance.

Single Cell Gel Electrophoresis for the Detection of Genomic Ribonucleotides.

Single cell gel electrophoresis or comet assay enables the quantification of DNA damage such as single-strand or double-strand breaks on a single cell level. Here, we describe a variant of this method for the detection of ribonucleotides embedded in genomic DNA. Briefly, cells are embedded in agarose on a microscopic slide, lysed under high salt and alkaline conditions and then subjected to in situ treatment with E. coli RNase HII which nicks 5' to a ribonucleotide within the context of a DNA duplex thereby converting genomic ribonucleotides into strand breaks. After unwinding of genomic DNA using a highly alkaline buffer, electrophoresis under mild alkaline conditions is performed resulting in formation of comets due to migration of fragmented DNA toward the anode. Following SYBR Gold staining comets can be visualized by fluorescence microscopy. In this setting, the length and the intensity of comets formed reflect the level of genomic ribonucleotides present in a given cell.

Super-Resolution Microscopy Techniques and Their Potential for Applications in Radiation Biophysics.

Fluorescence microscopy is an essential tool for imaging tagged biological structures. Due to the wave nature of light, the resolution of a conventional fluorescence microscope is limited laterally to about 200 nm and axially to about 600 nm, which is often referred to as the Abbe limit. This hampers the observation of important biological structures and dynamics in the nano-scaled range ~10 nm to ~100 nm. Consequentially, various methods have been developed circumventing this limit of resolution. Super-resolution microscopy comprises several of those methods employing physical and/or chemical properties, such as optical/instrumental modifications and specific labeling of samples. In this article, we will give a brief insight into a variety of selected optical microscopy methods reaching super-resolution beyond the Abbe limit. We will survey three different concepts in connection to biological applications in radiation research without making a claim to be complete.