Chk1 requirement for high global rates of replication fork progression during normal vertebrate S phase

Chk1 protein kinase maintains replication fork stability in metazoan cells in response to DNA damage and DNA replication inhibitors. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which Chk1 maintains global replication fork rates during normal vertebrate S phase. We report that replication fork rates in Chk1¿/¿ chicken DT40 cells are on average half of those observed with wild-type cells. Similar results were observed if Chk1 was inhibited or depleted in wild-type DT40 cells or HeLa cells by incubation with Chk1 inhibitor or small interfering RNA. In addition, reduced rates of fork extension were observed with permeabilized Chk1¿/¿ cells in vitro. The requirement for Chk1 for high fork rates during normal S phase was not to suppress promiscuous homologous recombination at replication forks, because inhibition of Chk1 similarly slowed fork progression in XRCC3¿/¿ DT40 cells. Rather, we observed an increased number of replication fibers in Chk1¿/¿ cells in which the nascent strand is single-stranded, supporting the idea that slow global fork rates in unperturbed Chk1¿/¿ cells are associated with the accumulation of aberrant replication fork structure

KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage

The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation

Counterion Condensation on Spheres in the Salt-free Limit

A highly-charged spherical colloid in a salt-free environment exerts such a powerful attraction on its counterions that a certain fraction condenses onto the surface of a particle. The degree of condensation depends on the curvature of the surface. So, for instance, condensation is triggered on a highly-charged sphere only if the radius exceeds a certain critical radius \collrad^{*}. \collrad^{*} is expected to be a simple function of the volume fraction of particles. To test these predictions, we prepare spherical particles which contain a covalently-bound ionic liquid, which is engineered to dissociate efficiently in a low-dielectric medium. By varying the proportion of ionic liquid to monomer we synthesise nonpolar dispersions of highly-charged spheres which contain essentially no free co-ions. The only ions in the system are counterions generated by the dissociation of surface-bound groups. We study the electrophoretic mobility of this salt-free system as a function of the colloid volume fraction, the particle radius, and the bare charge density and find evidence for extensive counterion condensation. At low electric fields, we observe excellent agreement with Poisson-Boltzmann predictions for counterion condensation on spheres. At high electric fields however, where ion advection is dominant, the electrophoretic mobility is enhanced significantly which we attribute to hydrodynamic stripping of the condensed layer of counterions from the surface of the particle.Comment: 13 pages, 9 figures and two table

Akt: A Double-Edged Sword in Cell Proliferation and Genome Stability

The Akt family of serine/threonine protein kinases are key regulators of multiple aspects of cell behaviour, including proliferation, survival, metabolism, and tumorigenesis. Growth-factor-activated Akt signalling promotes progression through normal, unperturbed cell cycles by acting on diverse downstream factors involved in controlling the G1/S and G2/M transitions. Remarkably, several recent studies have also implicated Akt in modulating DNA damage responses and genome stability. High Akt activity can suppress ATR/Chk1 signalling and homologous recombination repair (HRR) via direct phosphorylation of Chk1 or TopBP1 or, indirectly, by inhibiting recruitment of double-strand break (DSB) resection factors, such as RPA, Brca1, and Rad51, to sites of damage. Loss of checkpoint and/or HRR proficiency is therefore a potential cause of genomic instability in tumor cells with high Akt. Conversely, Akt is activated by DNA double-strand breaks (DSBs) in a DNA-PK- or ATM/ATR-dependent manner and in some circumstances can contribute to radioresistance by stimulating DNA repair by nonhomologous end joining (NHEJ). Akt therefore modifies both the response to and repair of genotoxic damage in complex ways that are likely to have important consequences for the therapy of tumors with deregulation of the PI3K-Akt-PTEN pathway

Vasilii Belov and the Russian idea

Vasilii Ivanovich Belov (1932-2012) was one of the most wellknown and outspoken of the ‘village writers’ who came to prominence in the Soviet Union in the 1960s, and in the age of ‘glasnost’ and post-Soviet times attracted much attention and even vilification for his nationalist and apparently xenophobic views. In his documentary account of village life and customs published in the journal Nash sovremennik between 1979 and 1982, and subsequently in illustrated book form, Belov provides in great detail and with much personal warmth the history of the northern Russian village, its people, customs and way of life as it has developed over the centuries, showing a place seemingly outside of time and untouched by historical developments. Lad: ocherki o narodnoi estetike may yet prove to be his most enduring contribution to post-Soviet reflections on ‘the Russian idea’, and Russia’s relationship with Europe

A Qualitative Evaluation of Mental Health Clinic Staff Perceptions of Barriers and Facilitators to Treating Tobacco Use

Introduction: Veterans with mental health disorders smoke at high rates, but encounter low rates of tobacco treatment. We sought to understand barriers and facilitators to treating tobacco use in VA mental health clinics. Methods: This qualitative study was part of a trial evaluating a telephone care coordination program for smokers using mental health services at six VA facilities. We conducted semi-structured interviews with 14 staff: 12 mental health clinic staff working at the parent study\u27s intervention sites (n = 6 psychiatrists, three psychologists, two social workers, one NP), as well as one psychiatrist and one psychologist on the VA\u27s national tobacco advisory committee. Interviews were transcribed and inductively coded to identify themes. Results: Five barriers themes emerged: (1) competing priorities, (2) patient challenges/resistance, (3) complex staffing/challenging cross-discipline coordination, (4) mixed perceptions about whether tobacco is a mental health care responsibility, and (5) limited staff training/comfort in treating tobacco. Five facilitators themes emerged: (1) reminding mental health staff about tobacco, (2) staff belief in the importance of addressing tobacco, (3) designating a cessation medication prescriber, (4) linking tobacco to mental health outcomes and norms, and (5) limiting mental health staff burden. Conclusions: VA mental health staff struggle with knowing that tobacco use is important, but they face competing priorities, encounter patient resistance, are conflicted on their role in addressing tobacco, and lack tobacco training. They suggested strategies at multiple levels that would help overcome those barriers that can be used to design interventions that improve tobacco treatment delivery for mental health patients. Implications: This study builds upon the existing literature on the high rates of smoking, but low rates of treatment, in people with mental health diagnoses. This study is one of the few qualitative evaluations of mental health clinic staff perceptions of barriers and facilitators to treating tobacco. The study results provide a multi-level framework for developing strategies to improve the implementation of tobacco treatment programs in mental health clinics

Analysis of nucleation using mean first-passage time data from molecular dynamics simulation

We introduce a method for the analysis of nucleation using mean first-passage time (MFPT) statistics obtained by molecular dynamics simulation. The method is based on the Becker-Döring model for the dynamics of a nucleation-mediated phase change and rigorously accounts for the system size dependence of first-passage statistics. It is thus suitable for the analysis of systems in which the separation between time scales for nucleation and growth is small, due to either a small free energy barrier or a large system size. The method is made computationally practical by an approximation of the first-passage time distribution based on its cumulant expansion. Using this approximation, the MFPT of the model can be fit to data from molecular dynamics simulation in order to estimate valuable kinetic parameters, including the free energy barrier, critical nucleus size, and monomer attachment pre-factor, as well as the steady-state rates of nucleation and growth. The method is demonstrated using a case study on nucleation of n-eicosane crystals from the melt. For this system, we found that the observed distribution of first-passage times do not follow an exponential distribution at short times, rendering it incompatible with the assumptions made by some other methods. Using our method, the observed distribution of first-passage times was accurately described, and reasonable estimates for the kinetic parameters and steady-state rates of nucleation and growth were obtained

Debunking Lesbian Bed Death:Using Coarsened Exact Matching to Compare Sexual Practices and Satisfaction of Lesbian and Heterosexual Women

The current study examined the prevalence and correlates of over 50 sexual practices in a national survey of heterosexual and lesbian women in relationships. Coarsened exact matching was used to create comparable samples of heterosexual (n = 2510) and lesbian (n = 283) women on six demographic factors, including relationship length. Heterosexual and lesbian women were equally likely to be sexually satisfied (66% heterosexual women vs. 68% lesbian women). Compared to heterosexuals, lesbians were more likely to report having sex 0–1 times per month (11% vs. 23%) and were less likely to report having sex greater than once per month (89% vs. 77%). Among women who had been in relationships for longer than 5 years, heterosexual women were less likely than lesbian women to report having sex 0–1 times per month (15%; 42%). This steeper drop in sexual frequency among lesbian women than heterosexual women has pejoratively been labeled lesbian bed death. Rather than accept the label “lesbian bed death” as characterizing these sexual relationships, we turn our attention to what we call lesbian bed intimacies: the myriad ways that lesbian women incorporate behaviors promoting emotional connection, romance, and mood setting, as well as relying on a wide variety of specific sexual acts (e.g., use of sex toys) and sexual communication. Compared to heterosexual women, lesbian women were more likely to usually to always receive oral sex during sex in the past month (28%; 47%) and to use sex toys in the past year (40%; 62%). In their last sexual encounter, lesbian women were more likely to say “I love you” (67%; 80%), have sex longer than 30 min (48%; 72%), and engage in gentle kissing (80%; 92%). These intimacies likely help explain why sexual satisfaction was similar in these groups despite notable differences in sexual frequency.</p

Debunking Lesbian Bed Death: Using Coarsened Exact Matching to Compare Sexual Practices and Satisfaction of Lesbian and Heterosexual Women

The current study examined the prevalence and correlates of over 50 sexual practices in a national survey of heterosexual and lesbian women in relationships. Coarsened exact matching was used to create comparable samples of heterosexual (n = 2510) and lesbian (n = 283) women on six demographic factors, including relationship length. Heterosexual and lesbian women were equally likely to be sexually satisfied (66% heterosexual women vs. 68% lesbian women). Compared to heterosexuals, lesbians were more likely to report having sex 0–1 times per month (11% vs. 23%) and were less likely to report having sex greater than once per month (89% vs. 77%). Among women who had been in relationships for longer than 5 years, heterosexual women were less likely than lesbian women to report having sex 0–1 times per month (15%; 42%). This steeper drop in sexual frequency among lesbian women than heterosexual women has pejoratively been labeled lesbian bed death. Rather than accept the label “lesbian bed death” as characterizing these sexual relationships, we turn our attention to what we call lesbian bed intimacies: the myriad ways that lesbian women incorporate behaviors promoting emotional connection, romance, and mood setting, as well as relying on a wide variety of specific sexual acts (e.g., use of sex toys) and sexual communication. Compared to heterosexual women, lesbian women were more likely to usually to always receive oral sex during sex in the past month (28%; 47%) and to use sex toys in the past year (40%; 62%). In their last sexual encounter, lesbian women were more likely to say “I love you” (67%; 80%), have sex longer than 30 min (48%; 72%), and engage in gentle kissing (80%; 92%). These intimacies likely help explain why sexual satisfaction was similar in these groups despite notable differences in sexual frequency

Effects of memory load and distraction on performance and event-related slow potentials in a visuospatial working memory task

Brain electrical activity related to working memory was recorded at 15 scalp electrodes during a visuospatial delayed response task. Participants (N = 18) touched the remembered position of a target on a computer screen after either a 1 or 8 sec delay. These memory trials were compared to sensory trials in which the target remained present throughout the delay and response periods. Distracter stimuli identical to the target were briefly presented during the delay on 30% of trials. Responses were less accurate in memory than sensory trials, especially after the long delay. During the delay slow potentials developed that were significantly more negative in memory than sensory trials. The difference between memory and sensory trials was greater at anterior than posterior electrodes. On trials with distracters, the slow potentials generated by memory trials showed further enhancement of negativity whereas there were minimal effects on accuracy of performance. The results provide evidence that engagement of visuospatial working memory generates slow wave negativity with a timing and distribution consistent with frontal activation. Enhanced brain activity associated with working memory is required to maintain performance in the presence of distraction. © 1997 by the Massachusetts Institute of Technolog