Regulation of eukaryotic Mcm2-7 activity

The transfer of genetic material from one cell generation to the next requires precise genome duplication. Aberrant DNA replication can lead to genomic instability and contribute to diseases arising from an unregulated cell cycle, such as cancer. Replicative DNA polymerases require a single-stranded (ssDNA) template from which to produce newly synthesized DNA. In eukaryotes, ssDNA is generated by the heterohexameric minichromosome maintenance 2 through 7 (Mcm2-7) replicative helicase that unwinds duplex DNA. Strict temporal separation of helicase loading and activation at multiple replication origins ensures once per cell cycle replication. The processes involved in activating Mcm2-7 to unwind DNA during S phase are poorly understood. Through in vivo and in vitro analyses, the current study examines the factors involved in modulating S. cerevisiae Mcm2-7 activity. Mec1, a member of the PIKK (phosphoinositide three-kinase-related kinase) family of proteins, is involved in the response to replicative stress and DNA damage. It also plays a role during an unperturbed cell cycle and is required to phosphorylate Mcm2-7 prior to helicase activation. We characterized alleles of S. cerevisiae mec1 that alter the conserved FATC domain. Mutants of Mec1 resulted in temperature sensitive growth, sensitivity to hydroxyurea and reduced kinase activity in vitro. These mutants were also less stable than wild-type Mec1 and demonstrated reduced nuclear localization. We also identified rpn3-L140P, which encodes a component of the 19S proteasomal regulatory particle of the 26S proteasome, as a suppressor of the temperature-sensitive growth caused by mec1-W2368A. As Cdt1 is required for the nuclear import and origin loading of Mcm2-7, we also sought to investigate the interaction between these two components in more detail. Using reconstituted Mcm2-7·Cdt1 complexes from bacterial-expressed proteins, we demonstrated that these complexes exhibit lower ATPase and helicase activity than Mcm2-7. We also showed that Mcm2-7 dissociates into subcomplexes, and that Mcm3, 5 and 7 bound origins in the absence of Cdt1. We propose that the reduced ATPase activity of Mcm2-7 by Cdt1 binding is induced by structural changes in the Mcm2-7 ring. We also suggest that Cdt1 helps to stabilize the Mcm2-7 hexamer. To investigate the role of phosphorylation on Mcm2-7, we utilized a phosphomimetic mutant of Mcm4 that when incorporated into Mcm2-7 can bypass the requirement for DDK. While phosphomimetic Mcm4 demonstrated slightly lower ATPase activity than the wildtype protein, phosphomimetic Mcm2-7 complexes exhibited wildtype ATPase, helicase and DNA binding activity. Taken together, our work identifies the functional role of the C-terminal residues of Mec1 and the protein’s turnover by the proteosome. Our studies also provide new insights into the factors and processes involved in the activation of Mcm2-7 to unwind DNA

A quantitative model of the initiation of DNA replication in Saccharomyces cerevisiae predicts the effects of system perturbations.

BackgroundEukaryotic cell proliferation involves DNA replication, a tightly regulated process mediated by a multitude of protein factors. In budding yeast, the initiation of replication is facilitated by the heterohexameric origin recognition complex (ORC). ORC binds to specific origins of replication and then serves as a scaffold for the recruitment of other factors such as Cdt1, Cdc6, the Mcm2-7 complex, Cdc45 and the Dbf4-Cdc7 kinase complex. While many of the mechanisms controlling these associations are well documented, mathematical models are needed to explore the network's dynamic behaviour. We have developed an ordinary differential equation-based model of the protein-protein interaction network describing replication initiation.ResultsThe model was validated against quantified levels of protein factors over a range of cell cycle timepoints. Using chromatin extracts from synchronized Saccharomyces cerevisiae cell cultures, we were able to monitor the in vivo fluctuations of several of the aforementioned proteins, with additional data obtained from the literature. The model behaviour conforms to perturbation trials previously reported in the literature, and accurately predicts the results of our own knockdown experiments. Furthermore, we successfully incorporated our replication initiation model into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes.ConclusionsThis study establishes a robust model of the processes driving DNA replication initiation. The model was validated against observed cell concentrations of the driving factors, and characterizes the interactions between factors implicated in eukaryotic DNA replication. Finally, this model can serve as a guide in efforts to generate a comprehensive model of the mammalian cell cycle in order to explore cancer-related phenotypes

Enabling virtual radio functions on software defined radio for future wireless networks

Today's wired networks have become highly flexible, thanks to the fact that an increasing number of functionalities are realized by software rather than dedicated hardware. This trend is still in its early stages for wireless networks, but it has the potential to improve the network's flexibility and resource utilization regarding both the abundant computational resources and the scarce radio spectrum resources. In this work we provide an overview of the enabling technologies for network reconfiguration, such as Network Function Virtualization, Software Defined Networking, and Software Defined Radio. We review frequently used terminology such as softwarization, virtualization, and orchestration, and how these concepts apply to wireless networks. We introduce the concept of Virtual Radio Function, and illustrate how softwarized/virtualized radio functions can be placed and initialized at runtime, allowing radio access technologies and spectrum allocation schemes to be formed dynamically. Finally we focus on embedded Software-Defined Radio as an end device, and illustrate how to realize the placement, initialization and configuration of virtual radio functions on such kind of devices

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders – especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation

Mandate-driven networking eco-system : a paradigm shift in end-to-end communications

The wireless industry is driven by key stakeholders that follow a holistic approach of "one-system-fits-all" that leads to moving network functionality of meeting stringent End-to-End (E2E) communication requirements towards the core and cloud infrastructures. This trend is limiting smaller and new players for bringing in new and novel solutions. For meeting these E2E requirements, tenants and end-users need to be active players for bringing their needs and innovations. Driving E2E communication not only in terms of quality of service (QoS) but also overall carbon footprint and spectrum efficiency from one specific community may lead to undesirable simplifications and a higher level of abstraction of other network segments may lead to sub-optimal operations. Based on this, the paper presents a paradigm shift that will enlarge the role of wireless innovation at academia, Small and Medium-sized Enterprises (SME)'s, industries and start-ups while taking into account decentralized mandate-driven intelligence in E2E communications

Improving Interpretation of the Patient-Reported Outcomes Measurement Information System (PROMIS) Physical Function Scale for Specific Tasks in Community-Dwelling Older Adults

Background and purpose: New generic patient-reported outcomes like the Patient-Reported Outcomes Measurement Information System (PROMIS) are available to physical therapists to assess physical function. However, the interpretation of the PROMIS Physical Function (PF) T-score is abstract because it references the United States average and not specific tasks. The purposes of this study were to (1) determine convergent validity of the PROMIS PF scale with physical performance tests; (2) compare predicted performance test values to normative data; and (3) identify sets of PROMIS PF items similar to performance tests that also scale in increasing difficulty and align with normative data. Methods: Community-dwelling older adults (n = 45; age = 77.1 ± 4.6 years) were recruited for this cross-sectional analysis of PROMIS PF and physical performance tests. The modified Physical Performance Test (mPPT), a multicomponent test of mostly timed items, was completed during the same session as the PROMIS PF scale. Regression analysis examined the relationship of mPPT total and component scores (walking velocity, stair ascent, and 5 times sit to stand) with the PROMIS PF scale T-scores. Normative data were compared with regression-predicted mPPT timed performance across PROMIS PF T-scores. The PROMIS PF items most similar to walking, stair ascent, or sit to stand were identified and then PROMIS PF model parameter-calibrated T-scores for these items were compared alongside normative data. Results and discussion: There were statistically significant correlations (r = 0.32-0.64) between PROMIS PF T-score and mPPT total and component scores. Regression-predicted times for walking, stair ascent, and sit-to-stand tasks (based on T-scores) aligned with published normative values for older adults. Selected PF items for stair ascent and walking scaled well to discriminate increasing difficulty; however, sit-to-stand items discriminated only lower levels of functioning. Conclusions: The PROMIS PF T-scores showed convergent validity with physical performance and aligned with published normative data. While the findings are not predictive of individual performance, they improve clinical interpretation by estimating a range of expected performance for walking, stair ascent, and sit to stand. These findings support application of T-scores in physical therapy testing, goal setting, and wellness plans of care for community-dwelling older adults

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller "high-definition" electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients. Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes. Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects. The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets

Formation energy and interaction of point defects in two-dimensional colloidal crystals

The manipulation of individual colloidal particles using optical tweezers has allowed vacancies to be created in two-dimensional (2d) colloidal crystals, with unprecedented possibility of real-time monitoring the dynamics of such defects (Nature {\bf 413}, 147 (2001)). In this Letter, we employ molecular dynamics (MD) simulations to calculate the formation energy of single defects and the binding energy between pairs of defects in a 2d colloidal crystal. In the light of our results, experimental observations of vacancies could be explained and then compared to simulation results for the interstitial defects. We see a remarkable similarity between our results for a 2d colloidal crystal and the 2d Wigner crystal (Phys. Rev. Lett. {\bf 86}, 492 (2001)). The results show that the formation energy to create a single interstitial is $12$ lower than that of the vacancy. Because the pair binding energies of the defects are strongly attractive for short distances, the ground state should correspond to bound pairs with the interstitial bound pairs being the most probable.Comment: 5 pages, 2 figure