TopBP1 assembles nuclear condensates to switch on ATR signaling.

ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores.

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

Cohesin Ubiquitylation and Mobilization Facilitate Stalled Replication Fork Dynamics.

Replication fork integrity is challenged in conditions of stress and protected by the Mec1/ATR checkpoint to preserve genome stability. Still poorly understood in fork protection is the role played by the structural maintenance of chromosomes (SMC) cohesin complex. We uncovered a role for the Rsp5Bul2 ubiquitin ligase in promoting survival to replication stress by preserving stalled fork integrity. Rsp5Bul2 physically interacts with cohesin and the Mec1 kinase, thus promoting checkpoint-dependent cohesin ubiquitylation and cohesin-mediated fork protection. Ubiquitylation mediated by Rsp5Bul2 promotes cohesin mobilization from chromatin neighboring stalled forks, likely by stimulating the Cdc48/p97 ubiquitin-selective segregase, and its timely association to nascent chromatids. This Rsp5Bul2 fork protection mechanism requires the Wpl1 cohesin mobilizer as well as the function of the Eco1 acetyltransferase securing sister chromatid entrapment. Our data indicate that ubiquitylation facilitates cohesin dynamic interfacing with replication forks within a mechanism preserving stalled-fork functional architecture.

Suicidality and Personality Pathology in Adolescence: A Systematic Review.

This work presents a review of research papers examining the role of emerging personality pathology in suicidal ideation and behaviours in adolescence. Initially, 226 studies were selected in line with PRISMA guidelines, and 33 articles were finally included in this review. The data show percentages of any personality disorder diagnosis ranging from 19.5 to 22.8% in suicide attempters, while in autopsy studies, the rate of personality disorder diagnosis varied between 29.6 and 42.1%. The overwhelming majority of the studies focus on the role of borderline personality disorder (BPD) in suicidal behaviours, also highlighting its predictive role at a longitudinal level. Furthermore, the literature review shows that personality traits supposed to underlie BPD, such as affective instability, impulsivity and identity diffusion, have specific predictive links with suicidal conduct. Other personality pathology dimensions, such as aggressiveness, sadism and perfectionism that are associated with other personality disorders, namely, antisocial and narcissistic personality disorders, have also shown a significant mediating role for suicidal risk. Overall, these results seem to parallel the role of personality pathology in predicting suicide in adulthood and point to the relevance of assessing the presence of emerging patterns of personality disorders for the clinical management of suicidal risk in adolescence.

Nucleolytic processing of aberrant replication intermediates by an Exo1-Dna2-Sae2 axis counteracts fork collapse-driven chromosome instability.

Problems during DNA replication underlie genomic instability and drive malignant transformation. The DNA damage checkpoint stabilizes stalled replication forks thus counteracting aberrant fork transitions, DNA breaks and chromosomal rearrangements. We analyzed fork processing in checkpoint deficient cells by coupling psoralen crosslinking with replication intermediate two-dimensional gel analysis. This revealed a novel role for Exo1 nuclease in resecting reversed replication fork structures and counteracting the accumulation of aberrant intermediates resembling fork cleavage products. Genetic analyses demonstrated a functional interplay of Exo1 with Mus81, Dna2 and Sae2 nucleases in promoting cell survival following replication stress, suggestive of concerted nucleolytic processing of stalled forks. While Mus81 and other Structure Specific Endonucleases do not contribute to obvious collapsed fork transitions, Dna2 promotes reversed fork resection likely by facilitating Exo1 access to nascent strands. Instead, Sae2 cooperates with Exo1 in counteracting putative fork cleavage events linked to double strand breaks formation and increased gross chromosomal rearrangement rates. Our data indicate that in checkpoint deficient cells diverse nuclease activities interface to eliminate aberrant replication intermediates and prevent chromosome instability.

The Road from Pathological Narcissism to Suicidality in Adolescence: An Empirical Study.

BACKGROUND: Clinical and empirical research evidenced a complex link between pathological narcissism and the suicidal process in adulthood. Given the relevance of suicidality and the peculiar narcissistic vicissitudes of adolescence, the proposed research investigated the relationship between pathological narcissism analyzed from the multi-dimensional perspective of the Diagnostic Interview for Narcissism (DIN) and suicidal ideation conducted in a sample of Italian Adolescents. METHODS: One hundred and three Italian male and female adolescents between 12 and 18 were administered the DIN, SCIDII, CSSRS, and Kiddie-SADS with six months follow-up. RESULTS: The correlation, t-test, multiple regression analyses evidenced the association of narcissistic affective states and mood with both suicidal ideation and lethality of conduct. The increase in the dimension of grandiosity is associated with the passage to potentially highly lethal suicidal gestures. CONCLUSIONS: Suicidal ideation and conduct seem to serve the function of restoring a sense of control and self-esteem in narcissistic individuals experiencing a state of affective dysregulation. Narcissistic pathological functioning seems to play an important role in the adolescent suicidal process, quite like adulthood. Assessing an adolescent's narcissistic functioning may provide useful clinical information in understanding and managing the suicidal risk in this phase of life.

Characteristics Associated with Depression Severity in 270 Juveniles in a Major Depressive Episode.

Introduction: Severe depression is prevalent in young persons and can lead to disability and elevated suicidal risk. Objectives: To identify clinical and demographic factors associated with the severity of depression in juveniles diagnosed with a major mood disorder, as a contribution to improving clinical treatment and reducing risk of suicide. Methods: We analyzed factors associated with depression severity in 270 juveniles (aged 6-18 years) in a major depressive episode, evaluated and treated at the Bambino Gesù Children's Hospital of Rome. Depressive symptoms were rated with the revised Children's Depression Rating Scale (CDRS-R) and manic symptoms with the Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS) Mania Rating Scale (K-SADS-MRS). Bivariate comparisons were followed by multivariable linear regression modeling. Results: Depression severity was greater among females than males (55.0 vs. 47.2), with the diagnosis of a major depressive disorder (MDD) vs. bipolar disorder (BD; 53.8 vs. 49.3), and tended to increase with age (slope = 1.14). Some symptoms typical of mania were associated with greater depression severity, including mood lability, hallucinations, delusions, and irritability, whereas less likely symptoms were hyperactivity, pressured speech, grandiosity, high energy, and distractibility. Factors independently and significantly associated with greater depression severity in multivariable linear regression modeling were: MDD vs. BD diagnosis, female sex, higher anxiety ratings, mood lability, and irritability. Conclusions: Severe depression was significantly associated with female sex, the presence of some manic or psychotic symptoms, and with apparent unipolar MDD. Manic/psychotic symptoms should be assessed carefully when evaluating a juvenile depressive episode and considered in treatment planning in an effort to balance risks of antidepressants and the potential value of mood-stabilizing and antimanic agents to decrease the severity of acute episodes and reduce suicidal risk.

Manic and Depressive Symptoms in Children Diagnosed with Noonan Syndrome.

Noonan syndrome (NS) is a dominant clinically variable and genetically heterogeneous developmental disorder caused by germ-line mutations encoding components of the Ras-MAPK signaling pathway. A few studies have investigated psychopathological features occurring in individuals with NS, although they were poorly analyzed. The aim of the present work is to investigate the psychopathological features in children and adolescents with NS focusing on depressive and hypo-manic symptoms. Thirty-seven subjects with molecularly confirmed diagnosis were systematically evaluated through a psychopathological assessment. In addition, an evaluation of the cognitive level was performed. Our analyses showed a high recurrence of attention deficit and hyperactivity disorder symptoms, emotional dysregulation, irritability, and anxiety symptomatology. The mean cognitive level was on the average. The present study provides new relevant information on psychopathological features in individuals with NS. The implications for clinicians are discussed including the monitoring of mood disorders in a clinical evolution.

The Study of Motivation in the Suicidal Process: The Motivational Interview for Suicidality.

Introduction: Suicide is the outcome of a process starting with the experiences of an unbearable pain or hopelessness, passing from suicidal ideation and planning, to possible para-suicidal behaviors or actual attempts. Recent studies have evidenced the necessity to integrate approaches based on the identification of psychopathological diagnoses and other variables as possible predictors of suicidal conduct with a more clinically based approach. A clinical assessment is needed that focuses on the patients' mental state with respect to thoughts concerning death and suicide. In particular, a qualitative assessment of motivations underlying the suicidal process could represent an effective guide for clinicians engaged in the difficult field of preventing adolescents' suicidal gestures. Most instruments investigating the suicidal motivation are self-report measures, possibly resulting in a lack of sufficiently valid assessment of this area. In the present work, we present the Motivational Interview for Suicidality in Adolescence (MIS-A) aiming at identifying the motivational areas sustaining suicidal ideation and gestures in this phase of development. Materials and Methods: The identification of the different areas derives from a thorough review of the empirical literature subsequently vetted by expert clinicians who selected specific reasons behind suicidal ideation and gesture. Result: The MIS is a semi-structured clinician-report interview. The interview is composed of seven areas and 14 sub-areas, evaluated on a four-point Likert scale: illness motivated attempts area, chronic presence of internal pessimistic criticism area, sense of defeat and entrapment area, relational area, external motivated crisis area, extreme and unusual cases area, and lack of control area. Conclusions: The path followed in the creation of the MIS reflects both an empirically orientated and a clinically informed approach. Creating this MIS is the first step within a wider research project that will allow one to test the reliability of the instrument.

Analysis of Cohesin Association to Newly Replicated DNA Through Nascent Strand Binding Assay (NSBA).

Replication forks engage chromatin-bound cohesin complexes during chromosome replication. Interfacing between cohesin and replication forks influences both cohesion establishment and fork functionality. However, the mechanisms mediating this process are scarcely understood. Here we describe the nascent strand binding assay (NSBA) methodology, developed in budding yeast to discriminate the association of cohesin to either parental unreplicated or nascent DNA in the environment of replication forks. NSBA quantitatively estimates the association of a protein of interest to newly replicated DNA. For this, nascent strands are in vivo labeled with the thymine analogue bromodeoxyuridine and chromatin is immunoprecipitated to isolate a fraction enriched in DNA associated to the target protein. The abundance of nascent DNA is then assessed through BrdU immunoprecipitation followed by quantitative PCR, allowing for the parallel analysis of diverse genomic regions. While originally employed to characterize the association of cohesin to nascent sister chromatids, NSBA can be applied to study other factors dynamically associating to nascent DNA.

PCNA Deubiquitylases Control DNA Damage Bypass at Replication Forks.

DNA damage tolerance plays a key role in protecting cell viability through translesion synthesis and template switching-mediated bypass of genotoxic polymerase-blocking base lesions. Both tolerance pathways critically rely on ubiquitylation of the proliferating-cell nuclear antigen (PCNA) on lysine 164 and have been proposed to operate uncoupled from replication. We report that Ubp10 and Ubp12 ubiquitin proteases differentially cooperate in PCNA deubiquitylation, owing to distinct activities on PCNA-linked ubiquitin chains. Ubp10 and Ubp12 associate with replication forks in a fashion determined by Ubp10 dependency on lagging-strand PCNA residence, and they downregulate translesion polymerase recruitment and template switch events engaging nascent strands. These findings reveal PCNAK164 deubiquitylation as a key mechanism for the modulation of lesion bypass during replication, which might set a framework for establishing strand-differential pathway choices. We propose that damage tolerance is tempered at replication forks to limit the extension of bypass events and sustain chromosome replication rates.