A Dynamic Stochastic Model for DNA Replication Initiation in Early Embryos

Background: Eukaryotic cells seem unable to monitor replication completion during normal S phase, yet must ensure a reliable replication completion time. This is an acute problem in early Xenopus embryos since DNA replication origins are located and activated stochastically, leading to the random completion problem. DNA combing, kinetic modelling and other studies using Xenopus egg extracts have suggested that potential origins are much more abundant than actual initiation events and that the time-dependent rate of initiation, I(t), markedly increases through S phase to ensure the rapid completion of unreplicated gaps and a narrow distribution of completion times. However, the molecular mechanism that underlies this increase has remained obscure.Methodology/Principal Findings: Using both previous and novel DNA combing data we have confirmed that I(t) increases through S phase but have also established that it progressively decreases before the end of S phase. To explore plausible biochemical scenarios that might explain these features, we have performed comparisons between numerical simulations and DNA combing data. Several simple models were tested: i) recycling of a limiting replication fork component from completed replicons; ii) time-dependent increase in origin efficiency; iii) time-dependent increase in availability of an initially limiting factor, e. g. by nuclear import. None of these potential mechanisms could on its own account for the data. We propose a model that combines time-dependent changes in availability of a replication factor and a fork-density dependent affinity of this factor for potential origins. This novel model quantitatively and robustly accounted for the observed changes in initiation rate and fork density.Conclusions/Significance: This work provides a refined temporal profile of replication initiation rates and a robust, dynamic model that quantitatively explains replication origin usage during early embryonic S phase. These results have significant implications for the organisation of replication origins in higher eukaryotes

Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics

Allergens induce enhanced bronchoconstriction and leukotriene production in C5 deficient mice

BACKGROUND: Previous genetic analysis has shown that a deletion in the complement component 5 gene-coding region renders mice more susceptible to allergen-induced airway hyperresponsiveness (AHR) due to reduced IL-12 production. We investigated the role of complement in a murine model of asthma-like pulmonary inflammation. METHODS: In order to evaluate the role of complement B10 mice either sufficient or deficient in C5 were studied. Both groups of mice immunized and challenged with a house dust extract (HDE) containing high levels of cockroach allergens. Airways hyper-reactivity was determined with whole-body plesthysmography. Bronchoalveolar lavage (BAL) was performed to determine pulmonary cellular recruitment and measure inflammatory mediators. Lung homogenates were assayed for mediators and plasma levels of IgE determined. Pulmonary histology was also evaluated. RESULTS: C5-deficient mice showed enhanced AHR to methylcholine challenge, 474% and 91% increase above baseline Penh in C5-deficient and C5-sufficient mice respectively, p < 0.001. IL-12 levels in the lung homogenate (LH) were only slightly reduced and BAL IL-12 was comparable in C5-sufficient and C5-deficient mice. However, C5-deficient mice had significantly higher cysteinyl-leukotriene levels in the BAL fluid, 1913 +/- 246 pg/ml in C5d and 756 +/- 232 pg/ml in C5-sufficient, p = 0.003. CONCLUSION: These data demonstrate that C5-deficient mice show enhanced AHR due to increased production of cysteinyl-leukotrienes

Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms

A short history of the 5-HT2C receptor: from the choroid plexus to depression, obesity and addiction treatment

This paper is a personal account on the discovery and characterization of the 5-HT2C receptor (first known as the 5- HT1C receptor) over 30 years ago and how it translated into a number of unsuspected features for a G protein-coupled receptor (GPCR) and a diversity of clinical applications. The 5-HT2C receptor is one of the most intriguing members of the GPCR superfamily. Initially referred to as 5-HT1CR, the 5-HT2CR was discovered while studying the pharmacological features and the distribution of [3H]mesulergine-labelled sites, primarily in the brain using radioligand binding and slice autoradiography. Mesulergine (SDZ CU-085), was, at the time, best defined as a ligand with serotonergic and dopaminergic properties. Autoradiographic studies showed remarkably strong [3H]mesulergine-labelling to the rat choroid plexus. [3H]mesulergine-labelled sites had pharmacological properties different from, at the time, known or purported 5-HT receptors. In spite of similarities with 5-HT2 binding, the new binding site was called 5-HT1C because of its very high affinity for 5-HT itself. Within the following 10 years, the 5-HT1CR (later named 5- HT2C) was extensively characterised pharmacologically, anatomically and functionally: it was one of the first 5-HT receptors to be sequenced and cloned. The 5-HT2CR is a GPCR, with a very complex gene structure. It constitutes a rarity in theGPCR family: many 5-HT2CR variants exist, especially in humans, due to RNA editing, in addition to a few 5-HT2CR splice variants. Intense research led to therapeutically active 5-HT2C receptor ligands, both antagonists (or inverse agonists) and agonists: keeping in mind that a number of antidepressants and antipsychotics are 5- HT2CR antagonists/inverse agonists. Agomelatine, a 5-HT2CR antagonist is registered for the treatment of major depression. The agonist Lorcaserin is registered for the treatment of aspects of obesity and has further potential in addiction, especially nicotine/ smoking. There is good evidence that the 5-HT2CR is involved in spinal cord injury-induced spasms of the lower limbs, which can be treated with 5-HT2CR antagonists/inverse agonists such as cyproheptadine or SB206553. The 5-HT2CR may play a role in schizophrenia and epilepsy. Vabicaserin, a 5-HT2CR agonist has been in development for the treatment of schizophrenia and obesity, but was stopped. As is common, there is potential for further indications for 5-HT2CR ligands, as suggested by a number of preclinical and/or genome-wide association studies (GWAS) on depression, suicide, sexual dysfunction, addictions and obesity. The 5-HT2CR is clearly affected by a number of established antidepressants/antipsychotics and may be one of the culprits in antipsychotic-induced weight gain

A High Through-Put Reverse Genetic Screen Identifies Two Genes Involved in Remote Memory in Mice

Previous studies have revealed that the initial stages of memory formation require several genes involved in synaptic, transcriptional and translational mechanisms. In contrast, very little is known about the molecular and cellular mechanisms underlying later stages of memory, including remote memory (i.e. 7-day memory). To identify genes required for remote memory, we screened randomly selected mouse strains harboring known mutations. In our primary reverse genetic screen, we identified 4 putative remote memory mutant strains out of a total of 54 lines analyzed. Additionally, we found 11 other mutant strains with other abnormal profiles. Secondary screens confirmed that mutations of integrin β2 (Itgβ2) and steryl-O-acyl transferase 1 (Soat1) specifically disrupted remote memory. This study identifies some of the first genes required for remote memory, and suggests that screens of targeted mutants may be an efficient strategy to identify molecular requirements for this process

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization

<p>Abstract</p> <p>Background</p> <p>Nitrogen dioxide (NO₂) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO₂is also produced endogenously in the lung during acute inflammatory responses. NO₂can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c⁺antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c⁺cells in NO₂-promoted allergic sensitization.</p> <p>Methods</p> <p>We systemically depleted CD11c⁺cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO₂followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c⁺cells from wildtype mice were studied after exposure to NO₂and ovalbumin for cellular phenotype by flow cytometry and <it>in vitro </it>cytokine production.</p> <p>Results</p> <p>Transient depletion of CD11c⁺cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c⁺cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO₂exposure. By 48 hours, CD11c⁺MHCII⁺DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c⁺CD11b^-and CD11c⁺CD11b⁺pulmonary cells exposed to NO₂<it>in vivo </it>increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647⁺CD11c⁺MHCII⁺DCs present in MLN from NO₂-exposed mice by 48 hours. Co-cultures of ova-specific CD4⁺T cells from naïve mice and CD11c⁺pulmonary cells from NO₂-exposed mice produced IL-1, IL-12p70, and IL-6 <it>in vitro </it>and augmented antigen-induced IL-5 production.</p> <p>Conclusions</p> <p>CD11c⁺cells are critical for NO₂-promoted allergic sensitization. NO₂exposure causes pulmonary CD11c⁺cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.</p

Links Between Hydrothermal Environments, Pyrophosphate, Na+, and Early Evolution

The discovery that photosynthetic bacterial membrane-bound inorganic pyrophosphatase (PPase) catalyzed light-induced phosphorylation of orthophosphate (Pi) to pyrophosphate (PPi) and the capability of PPi to drive energy requiring dark reactions supported PPi as a possible early alternative to ATP. Like the proton-pumping ATPase, the corresponding membrane-bound PPase also is a H+-pump, and like the Na+-pumping ATPase, it can be a Na+-pump, both in archaeal and bacterial membranes. We suggest that PPi and Na+ transport preceded ATP and H+ transport in association with geochemistry of the Earth at the time of the origin and early evolution of life. Life may have started in connection with early plate tectonic processes coupled to alkaline hydrothermal activity. A hydrothermal environment in which Na+ is abundant exists in sediment-starved subduction zones, like the Mariana forearc in the W Pacific Ocean. It is considered to mimic the Archean Earth. The forearc pore fluids have a pH up to 12.6, a Na+-concentration of 0.7 mol/kg seawater. PPi could have been formed during early subduction of oceanic lithosphere by dehydration of protonated orthophosphates. A key to PPi formation in these geological environments is a low local activity of water

The needs of foster children and how to satisfy them:A systematic review of the literature

Family foster care deeply influences the needs of children and how these are satisfied. To increase our knowledge of foster children’s needs and how these are conceptualized, this paper presents a systematic literature review. Sixty- four empirical articles from six databases were reviewed and categorized (inter-rater agreement K = .78) into four categories: medical, belongingness, psychological and self-actualization needs. The results give a complete overview of needs that are specific to foster children, and what can be implemented to satisfy these needs. This study shows psychological needs are studied more often compared to the other categories, which specially relates to much attention for mental health problems. Furthermore, most articles focus on how to satisfy the needs of foster children and provide no definition or concrete conceptualization of needs. Strikingly, many articles focus on children’s problems instead of their needs, and some even use these terms interchangeably. This review illustrates that future research should employ a proper conceptualization of needs, which could also initiate a shift in thinking about needs instead of problems