bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstructing solid state nanopore shape from electrical measurements
Authors: Liebes Yael , Drozdov Maria , Avital Yotam Y. , Kauffmann Yaron , Rapaport Hanna , Kaplan Wayne D. , Ashkenasy Nurit
Published: December 2010

The dependence of nanopore biosensor conductance signal on the nanopore shape makes it important to decipher the latter with high precision. We show here that the three dimensional shape of a nanopore, extracted from electron microscopy analysis, allows for modeling the conductance of the nanopore over a wide range of ionic strengths. Furthermore, we demonstrate that the dependence of the nanopore conductance on ionic strength can be used to accurately extract the nanopore shape, eliminating the need for lengthy electron microscopy analysis. The suggested methodology can be used to monitor changes in the nanopore shape and evaluate them during electrical characterization.
Light out-coupling enhancement of organic light-emitting devices with microlens array
Authors: Yang J. P. , Bao Q. Y. , Xu Z. Q. , Li Y. Q. , Tang J. X. , Shen S.
Published: December 2010

Light out-coupling efficiency of organic light-emitting devices from high-index glass substrate into air is enhanced by attaching ordered microlens arrays, which are fabricated by a roll-to-roll mold transfer process. The dependence of microlens geometries on light extraction is analyzed experimentally and theoretically. An increase of 60% in the light out-coupling with an optimized elliptical microlens array is achieved over a conventional device without affecting the electroluminescent spectrum.
Response to “Comment on ‘Nonlinear gyrokinetic theory with polarization drift’” [Phys. Plasmas 17, 124701 (2010)]
Authors: Wang Lu , Hahm T. S.
Published: December 2010

We agree that there is no contradiction between Wang and Hahm [Phys. Plasmas 17, 082304 (2010)] and Sosenko et al. [Phys. Scr. 64, 264 (2001)]. However, by explicitly evaluating the change in phase-volume due to the electrostatic potential [T. S. Hahm, Phys. Plasmas 3, 4658 (1996)], Wang and Hahm [Phys. Plasmas 17, 082304 (2010)] has demonstrated that the polarization density remains the same even when the polarization drift explicitly appears in the gyrocenter equations of motion, and has derived an energy invariant in general toroidal geometry.
Electrical control of fine-structure splitting in self-assembled quantum dots for entangled photon pair creation
Authors: Mar J. D. , Xu X. L. , Sandhu J. S. , Irvine A. C. , Hopkinson M. , Williams D. A.
Published: December 2010

The fine-structure splitting δ1 of the bright exciton doublet in self-assembled InGaAs/GaAs quantum dots is measured as a function of the applied in-plane electric field using time-resolved Kerr rotation. We observe a significant reduction of δ1 with increasing electric field, suggesting a symmetrizing of the electron and hole wave functions. For sufficiently high fields, it is shown that δ1 can be made less than the quantum dot homogeneous linewidth γ, which is calculated using an appropriate model. This may open the way for the electrical control of polarization-entangled photon pair creation through the radiative biexciton cascade.
Resonant waveguide grating imager for live cell sensing
Authors: Ferrie Ann M. , Wu Qi , Fang Ye
Published: December 2010

We report on a resonant waveguide grating imager for high throughput screening using live cells. This imager can generate a snapshot image of all biosensors in a 384-well microtiter plate with a time resolution of ∼3 s and a spatial resolution of 80 μm. This imager is well tolerant to variability in plate configurations and cell confluency. The resonant wavelength and its shifts induced by cell responses at each pixel correlate well with cell confluency. Data filtration protocol can be used to improve assay quality for partially confluent cells.
Some memories of Chandra
Authors: Wald Robert M.
Published: December 2010

Five noted scientists, all close colleagues and friends of Subrahmanyan Chandrasekhar, share thoughts and memories of the man whose centennial we celebrate.
At work in the trenches of science diplomacy
Authors: Kramer David
Published: December 2010

The US Agency for International Development has its first science adviser in 19 years.
Chandra: A biographical portrait
Authors: Wali Kameshwar C.
Published: December 2010

The complexities of three countries—India, England, and the US—helped produce a scientist of rare stature and greatness.
Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes
Authors: Vujičić-Žagar Andreja , Pijning Tjaard , Kralj Slavko , López Cesar A. , Eeuwema Wieger , Dijkhuizen Lubbert , Dijkstra Bauke W.
Published: December 2010

Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from
Lactobacillus reuteri
180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like (
β
/
α
)
8
-barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a “U”-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product.
The interaction of human natural killer cells with either unpolarized or polarized macrophages results in different functional outcomes
Authors: Bellora Francesca , Castriconi Roberta , Dondero Alessandra , Reggiardo Giorgio , Moretta Lorenzo , Mantovani Alberto , Moretta Alessandro , Bottino Cristina
Published: December 2010

The cross-talk among cells of the innate immunity can greatly affect both innate and adaptive responses. Here we analyzed the molecular interactions between human natural killer (NK) cells and autologous macrophages. Activated NK cells killed M0 and M2, whereas M1 macrophages were more resistant to lysis because of their higher expression of HLA class I molecules. Following exposure to LPS or bacillus Calmette–Guérin, M0 and M2, but not polarized (endotoxin tolerant) M1 macrophages, induced strong activation of resting NK cells. The expression of CD69 and CD25 activation markers and the acquisition of cytotoxicity against tumor cells and immature dendritic cells required soluble factors being mostly contact independent. On the contrary, IFN-γ production was contact dependent and required the interaction of DNAM-1 and 2B4 (on NK) with their ligands on macrophages as well as IL-18. IL-18 was involved also in the acquisition of CCR7 by NK cells. Interestingly, M0 and M2 cells expressed a membrane-bound form of IL-18, which was released in small amounts after LPS treatment. Our data indicate that, upon interaction with M0 macrophages exposed to microbial products, NK cells may amplify classical type 1 immune responses. In addition, M1-polarizing stimuli can rescue M2 macrophages from their immunomodulatory state and shape their functional behavior toward NK stimulatory capability.
Benefits, status and effectiveness of Continuous Professional Development for teachers in England
Authors: Opfer V. Darleen , Pedder David
Published: December 2010

Reported here is one part of the nationally representative, Schools and Continuing Professional Development in England – State of the Nation research study which was funded by the Training and Development Agency for Schools (TDA). This paper sets out to understand how teachers and school leaders in England perceive the benefits and effectiveness of CPD activity. The investigation of benefits and effectiveness of CPD presented in the paper focuses on the various impacts that could result from participation, the forms and features of activities, as well as the conditions that make effectiveness more or less likely to occur.
Access to Continuous Professional Development by teachers in England
Authors: Opfer V. Darleen , Pedder David
Published: December 2010

This paper sets out to investigate issues of access to continuous professional development faced by teachers in England. Specifically, the paper attempts to understand the types of CPD activities that are offered to teachers, the types of activities in which teachers participated and whether a disconnect occurs between activities offered and those needed by teachers. Further, the paper also explores the barriers to access to high quality CPD that exist for teachers at both the individual and school levels. Data for the analyses presented were collected as part of the nationally representative, Schools and Continuing Professional Development in England – State of the Nation research study which was funded by the Training and Development Agency for Schools (TDA).
Improved Intertask Coordination after Extensive Dual-Task Practice
Authors: Liepelt Roman , Strobach Tilo , Frensch Peter , Schubert Torsten
Published: December 2010

This study examines whether an improved intertask coordination skill is acquired during extensive dual-task training and whether it can be transferred to a new dual-task situation. Participants practised a visual–manual task and an auditory–vocal task. These tasks were trained in two groups matched in dual-task performance measures before practice: a single-task practice group and a hybrid practice group (including single-task and dual-task practice). After practice, the single-task practice group was transferred to the same dual-task situation as that for the hybrid practice group (Experiment 1), both groups were transferred to a dual-task situation with a new visual task (Experiment 2), and both groups were transferred to a dual-task situation with a new auditory task matched in task difficulty (Experiment 3). The results show a dual-task performance advantage in the hybrid practice group over the single-task practice group in the practised dual-task situation (Experiment 1), the manipulated visual-task situation (Experiment 2), and the manipulated auditory-task situation (Experiment 3). In all experiments, the dual-task performance advantage was consistently found for the auditory task only. These findings suggest that extended dual-task practice improves the skill to coordinate two tasks, which may be defined as an accelerated switching operation between both tasks. This skill is relatively robust against changes of the component visual and auditory tasks. We discuss how the finding of task coordination could be integrated in present models of dual-task research.
Individualized Catheter Surveillance among Neonates: A Prospective, 8-Year, Single-Center Experience
Authors: Zingg Walter , Posfay-Barbe Klara M. , Pfister Riccardo E. , Touveneau Sylvie , Pittet Didier
Published: December 2010

Objective.

To monitor trends in central line-associated bloodstream infections and clinical sepsis (CLABICS) among neonates and to determine risk factors for infection, especially dwell time.

Design.

Prospective, single-center cohort study conducted from 2001 through 2008.

Setting.

University-affiliated tertiary care center.

Methods.

Individualized surveillance of catheter use and CLABICS episodes was conducted. Data were obtained via regular on-site visits made 3 times a week. Trends over time were estimated by Poisson regression, and risk factor analysis was conducted using a Cox proportional hazards model and logistic regression.

Results.

In all, 1,124 neonates were exposed to 2,210 central lines for a total of 12,746 catheter-days and 11,467 catheter-days at risk. The median duration of catheter use was 8 (interquartile range, 5–11) days for peripherally inserted central catheters (PICCs) and 4 (interquartile range, 2-6) days for umbilical catheters; 102 CLABICS episodes were detected. The median time to infection was 7 days. Incidence densities were 8.5 CLABICS episodes per 1,000 catheter-days at risk and 8.0 CLABICS episodes per 1,000 catheter-days. The highest rates were identified among neonates weighing 750 g or lower (14.9 CLABICS episodes per 1,000 catheter days at risk) and for PICCs (13.2 CLABICS episodes per 1,000 catheter days at risk). Catheter dwell time was associated with CLABICS for all umbilical catheters (odds ratio [OR], 1.2 per day of use [95% confidence interval {CI}, 1.1–1.3] P< .001) and for PICCs for up to 7 days (OR, 1.2 [95% CI, 1.1–1.4]; P = .041), but not thereafter (OR, 1.0 [95% CI, 0.9–1.1]; P = .90).

Conclusion.

Catheter dwell time is a risk factor for CLABICS during the first 7 days, irrespective of catheter type. After 7 days, PICCs are less likely to become infected.
Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments
Authors: Lorente-Rodríguez Andrés , Barlowe Charles
Published: December 2010

The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.
The Zds proteins control entry into mitosis and target protein phosphatase 2A to the Cdc25 phosphatase
Authors: Wicky Sidonie , Tjandra Hendri , Schieltz David , Yates John , Kellogg Douglas R.
Published: December 2010

The Wee1 kinase restrains entry into mitosis by phosphorylating and inhibiting cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing Cdk1 inhibitory phosphorylation. Experiments in diverse systems have established that Wee1 and Cdc25 are regulated by protein phosphatase 2A (PP2A), but a full understanding of the function and regulation of PP2A in entry into mitosis has remained elusive. In budding yeast, entry into mitosis is controlled by a specific form of PP2A that is associated with the Cdc55 regulatory subunit (PP2ACdc55). We show here that related proteins called Zds1 and Zds2 form a tight stoichiometric complex with PP2ACdc55and target its activity to Cdc25 but not to Wee1. Conditional inactivation of the Zds proteins revealed that their function is required primarily at entry into mitosis. In addition, Zds1 undergoes cell cycle–dependent changes in phosphorylation. Together, these observations define a role for the Zds proteins in controlling specific functions of PP2ACdc55and suggest that upstream signals that regulate PP2ACdc55may play an important role in controlling entry into mitosis.
TRAF2 phosphorylation promotes NF-κB–dependent gene expression and inhibits oxidative stress-induced cell death
Authors: Zhang Laiqun , Blackwell Ken , Altaeva Aliya , Shi Zhaohui , Habelhah Hasem
Published: December 2010

Tumor necrosis factor α (TNF-α) receptor–associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α–induced cell death but promotes oxidative stress–induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α–induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.
H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo
Authors: Ejlassi-Lassallette Aïda , Mocquard Eloïse , Arnaud Marie-Claire , Thiriet Christophe
Published: December 2010

While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.
State reduction for network intervention in probabilistic Boolean networks
Authors: Qian Xiaoning , Ghaffari Noushin , Ivanov Ivan , Dougherty Edward R.
Published: December 2010

Motivation: A key goal of studying biological systems is to design therapeutic intervention strategies. Probabilistic Boolean networks (PBNs) constitute a mathematical model which enables modeling, predicting and intervening in their long-run behavior using Markov chain theory. The long-run dynamics of a PBN, as represented by its steady-state distribution (SSD), can guide the design of effective intervention strategies for the modeled systems. A major obstacle for its application is the large state space of the underlying Markov chain, which poses a serious computational challenge. Hence, it is critical to reduce the model complexity of PBNs for practical applications.

Results: We propose a strategy to reduce the state space of the underlying Markov chain of a PBN based on a criterion that the reduction least distorts the proportional change of stationary masses for critical states, for instance, the network attractors. In comparison to previous reduction methods, we reduce the state space directly, without deleting genes. We then derive stationary control policies on the reduced network that can be naturally induced back to the original network. Computational experiments study the effects of the reduction on model complexity and the performance of designed control policies which is measured by the shift of stationary mass away from undesirable states, those associated with undesirable phenotypes. We consider randomly generated networks as well as a 17-gene gastrointestinal cancer network, which, if not reduced, has a 217 × 217 transition probability matrix. Such a dimension is too large for direct application of many previously proposed PBN intervention strategies.

Contact:  xqian@cse.usf.edu

Supplementary information:  Supplementary information are available at Bioinformatics online.
Identifying functional miRNA–mRNA regulatory modules with correspondence latent dirichlet allocation
Authors: Liu Bing , Liu Lin , Tsykin Anna , Goodall Gregory J. , Green Jeffrey E. , Zhu Min , Kim Chang Hee , Li Jiuyong
Published: December 2010

Motivation: MicroRNAs (miRNAs) are small non-coding RNAs that cause mRNA degradation and translational inhibition. They are important regulators of development and cellular homeostasis through their control of diverse processes. Recently, great efforts have been made to elucidate their regulatory mechanism, but the functions of most miRNAs and their precise regulatory mechanisms remain elusive. With more and more matched expression profiles of miRNAs and mRNAs having been made available, it is of great interest to utilize both expression profiles to discover the functional regulatory networks of miRNAs and their target mRNAs for potential biological processes that they may participate in.

Results: We present a probabilistic graphical model to discover functional miRNA regulatory modules at potential biological levels by integrating heterogeneous datasets, including expression profiles of miRNAs and mRNAs, with or without the prior target binding information. We applied this model to a mouse mammary dataset. It effectively captured several biological process specific modules involving miRNAs and their target mRNAs. Furthermore, without using prior target binding information, the identified miRNAs and mRNAs in each module show a large proportion of overlap with predicted miRNA target relationships, suggesting that expression profiles are crucial for both target identification and discovery of regulatory modules.

Contact:  bing.liu@unisa.edu.au; jiuyong.li@unisa.edu.au

Supplementary information:  Supplementary data are available at Bioinformatics online.
Interactive microbial genome visualization with GView
Authors: Petkau Aaron , Stuart-Edwards Matthew , Stothard Paul , Van Domselaar Gary
Published: December 2010

Summary: GView is a Java application for viewing and examining prokaryotic genomes in a circular or linear context. It accepts standard sequence file formats and an optional style specification file to generate customizable, publication quality genome maps in bitmap and scalable vector graphics formats. GView features an interactive pan-and-zoom interface, a command-line interface for incorporation in genome analysis pipelines, and a public Application Programming Interface for incorporation in other Java applications.

Availability: GView is a freely available application licensed under the GNU Public License. The application, source code, documentation, file specifications, tutorials and image galleries are available at http://gview.ca

Contact:  gary.van.domselaar@phac-aspc.gc.ca
GLay: community structure analysis of biological networks
Authors: Su Gang , Kuchinsky Allan , Morris John H. , States David J. , Meng Fan
Published: December 2010

Summary: GLay provides Cytoscape users an assorted collection of versatile community structure algorithms and graph layout functions for network clustering and structured visualization. High performance is achieved by dynamically linking highly optimized C functions to the Cytoscape JAVA program, which makes GLay especially suitable for decomposition, display and exploratory analysis of large biological networks.

Availability:  http://brainarray.mbni.med.umich.edu/glay/

Contact:  sugang@umich.edu
Improving the quality of protein similarity network clustering algorithms using the network edge weight distribution
Authors: Apeltsin Leonard , Morris John H. , Babbitt Patricia C. , Ferrin Thomas E.
Published: December 2010

Motivation: Clustering protein sequence data into functionally specific families is a difficult but important problem in biological research. One useful approach for tackling this problem involves representing the sequence dataset as a protein similarity network, and afterwards clustering the network using advanced graph analysis techniques. Although a multitude of such network clustering algorithms have been developed over the past few years, comparing algorithms is often difficult because performance is affected by the specifics of network construction. We investigate an important aspect of network construction used in analyzing protein superfamilies and present a heuristic approach for improving the performance of several algorithms.

Results: We analyzed how the performance of network clustering algorithms relates to thresholding the network prior to clustering. Our results, over four different datasets, show how for each input dataset there exists an optimal threshold range over which an algorithm generates its most accurate clustering output. Our results further show how the optimal threshold range correlates with the shape of the edge weight distribution for the input similarity network. We used this correlation to develop an automated threshold selection heuristic in order to most optimally filter a similarity network prior to clustering. This heuristic allows researchers to process their protein datasets with runtime efficient network clustering algorithms without sacrificing the clustering accuracy of the final results.

Availability: Python code for implementing the automated threshold selection heuristic, together with the datasets used in our analysis, are available at http://www.rbvi.ucsf.edu/Research/cytoscape/threshold_scripts.zip.

Contact:  tef@cgl.ucsf.edu

Supplementary information:  Supplementary data are available at Bioinformatics online.
Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae
Authors: Munding Elizabeth M. , Igel A. Haller , Shiue Lily , Dorighi Kristel M. , Treviño Lisa R. , Ares Manuel
Published: December 2010

Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p; however, other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination and for activation of Ndt80p, the activator of the transcriptional network required for subsequent steps in the program. Thus, the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This study reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs.
Alternative cell death mechanisms in development and beyond
Authors: Yuan Junying , Kroemer Guido
Published: December 2010

A canonical regulatory pathway involving the members of the Bcl-2 and caspase families has been established to regulate developmental apoptosis in nematodes and flies. However, mutant mice that have major deficiencies in this apoptosis pathway show only relatively minor developmental defects. Recent revelations indicate that multiple mechanisms are involved in regulating cell death during mammalian development, tissue homeostasis, and pathological cell loss. Here, we critically evaluate the evidence demonstrating the existence of alternative cell death mechanisms, including apoptosis of lower organisms in the absence of canonical apoptosis mediators, autophagic cell death, necroptosis, elimination by shedding, keratinocyte death by cornification, and cell–cell cannibalism by entosis. The physiological relevance of alternative cell death mechanisms as primary and backup mechanisms is discussed.
RNAs actively cycle on the Sm-like protein Hfq
Authors: Fender Aurélie , Elf Johan , Hampel Kornelia , Zimmermann Bastian , Wagner E. Gerhart H.
Published: December 2010

Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar Kd values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1- 2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentration-driven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA–Hfq complexes are ≈1 min. We propose that competitor RNA associates transiently with RNA–Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the “strong binding–high turnover” paradox and permits efficient use of the Hfq pool.
Microinjection of Xenopus Embryos
Authors: Sive Hazel L. , Grainger Robert M. , Harland Richard M.
Published: December 2010

INTRODUCTION

This article describes the factors that should be considered when microinjecting Xenopus oocytes. Forward planning is particularly important for embryo injection because the frogs lay eggs only for a limited time, and once the eggs are fertilized there is a very short period during which the embryos are suitable for injection.
Imaging Neuropeptide Release in the Drosophila Neuromuscular Junction (NMJ)
Authors: Levitan Edwin S. , Shakiryanova Dinara
Published: December 2010

INTRODUCTION

Electrophysiological studies of synaptic function cannot directly reveal the internal workings of the nerve terminal and do not robustly report release of neuropeptides and neurotrophins. These limitations can now be overcome with the presynaptic expression of green fluorescent protein (GFP) indicators of vesicle motion, release, and signaling. This protocol describes how to image single wavelength and ratiometric fluorescence resonance energy transfer (FRET)-based GFP indicators with fluorescence microscopy in living synaptic boutons of the Drosophila neuromuscular junction (NMJ). The steps for setting up the imaging equipment for epifluorescence microscopy are given, followed by special considerations for preparing the larval NMJ for peptide release studies.
Creating Zinc Finger Nucleases Using a Modular-Assembly Approach
Authors: Porteus Matthew
Published: December 2010

INTRODUCTION

Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but has not been available in other systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousandfold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This protocol describes how to use a polymerase chain reaction (PCR) technique to assemble new three-finger proteins and how to create a new ZFN.
Testing a Three-Finger Zinc Finger Nuclease Using a GFP Reporter System
Authors: Porteus Matthew
Published: December 2010

INTRODUCTION

Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but it has not been available in other systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousandfold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This protocol describes how to test newly designed ZFNs using a cell-based green fluorescent protein (GFP) reporter assay to determine if they are active in a mammalian cell-culture-based system.
Calibration of the Injection Volume for Microinjection of Xenopus Oocytes and Embryos
Authors: Sive Hazel L. , Grainger Robert M. , Harland Richard M.
Published: December 2010

INTRODUCTION

Microinjection of Xenopus oocytes or embryos with messenger RNA (mRNA) or DNA is a powerful technique for studying development. Before microinjection can be performed, the injection volume must be calibrated carefully. This protocol describes the calibration procedure for a pressure injector.
Imaging Cell Movements in Egg-Cylinder Stage Mouse Embryos
Authors: Srinivas Shankar
Published: December 2010

INTRODUCTION

Cell movements in the pregastrulation egg cylinder mouse embryo play an important role in patterning. The stereotypic movement of the anterior visceral endoderm converts a proximal-distal axis to an anteroposterior axis by properly positioning the primitive streak. The epiblast at this stage is also characterized by a great deal of cell mixing, about which very little is known. Visualizing such cell movements can help us understand their role in embryonic development. This protocol describes a method to isolate and culture the egg cylinder-stage mouse embryo, as well as an approach for time-lapse imaging of embryos cultured in vivo.
Acquisition of 4D DIC Microscopic Data to Determine Cell Contacts in Caenorhabditis elegans Embryos
Authors: Walston Timothy , Hardin Jeff
Published: December 2010

INTRODUCTION

The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. Acquisition of stacks of images throughout the thickness of the embryo over time is a crucial method for identifying the positions and contacts between cells. Such four-dimensional (4D) microscopy is a routine tool in laboratories that study early C. elegans development. Differential interference contrast (DIC) microscopy is the focus here because of its broad availability, common use for C. elegans imaging, and wide applicability to microscopic analysis of embryos of other organisms. This protocol describes the use of a custom script within μManager’s Beanshell scripting language. The script is helpful for reducing the number of shutter open/close events during 4D acquisition.
Analysis of 4D DIC Microscopic Data to Determine Cell Contacts in Caenorhabditis elegans Embryos
Authors: Walston Timothy , Hardin Jeff
Published: December 2010

INTRODUCTION

The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. Identification of cell contacts is important for understanding how cells within the embryo can be polarized by their neighbors. This protocol describes a technique for identifying cell membranes and potential contacts between different blastomeres in the embryo and for following those contacts through development. This protocol involves manual segmentation of the membrane of each blastomere from four-dimensional (4D) differential interference contrast (DIC) data sets. Although this technique is described for use with C. elegans embryos, it will work with any 4D DIC data set. The recent development of a pleckstrin homology (PH)-domain tagged::GFP expressed in C. elegans embryos simplifies this analysis; however, many organisms lack appropriate green fluorescent protein (GFP) transgenes. The use of the GFP transgene improves on laser-mediated techniques for labeling embryonic cell membranes with fluorescent dye. Such methods require that a laser be used to carefully permeabilize the eggshell for entry of the dye into the embryo. The technique described in this protocol requires only easily obtainable 4D DIC data sets.
Fluorescence Perturbation Techniques to Study Mobility and Molecular Dynamics of Proteins in Live Cells: FRAP, Photoactivation, Photoconversion, and FLIP
Authors: Bancaud Aurélien , Huet Sébastien , Rabut Gwénaël , Ellenberg Jan
Published: December 2010

INTRODUCTION

The technique of fluorescence recovery after photobleaching (FRAP) was introduced in the mid-1970s to study the diffusion of biomolecules in living cells. For several years, it was used mainly by a small number of biophysicists who had developed their own photobleaching systems. Since the mid-1990s, FRAP has gained increasing popularity because of the conjunction of two factors: First, photobleaching techniques are easily implemented on confocal laser-scanning microscopes (CLSMs), and so FRAP has become available to anyone who has access to such equipment. Second, the advent of green fluorescent protein (GFP) has allowed easy fluorescent tagging of proteins and their observation in living cells. Thanks both to the versatility of modern CLSMs, which allow control of laser intensity at any point of the image, and to the development of new fluorescent probes, additional photoperturbation techniques have emerged during the last few years. After the photoperturbation event, one observes and then analyzes how the fluorescence distribution relaxes toward the steady state. Because the photochemical perturbation of suitable fluorophores is essentially irreversible, changes of fluorescence intensity in the perturbed and unperturbed regions are due to the exchange of tagged molecules between those regions. This article first discusses the materials required for performing FRAP experiments on a CLSM and the software for data analysis. It then describes general considerations on how to perform FRAP experiments as well as the necessary controls. Finally, different possible ways to analyze the data are presented.
Creating Zinc Finger Nucleases to Manipulate the Genome in a Site-Specific Manner Using a Modular-Assembly Approach
Authors: Porteus Matthew
Published: December 2010

INTRODUCTION

Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but it has not been available in other genetic systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousandfold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This article describes how to identify potential targets for ZFN cutting. It also focuses on how to assemble ZFNs to recognize target sequences of the form 5′-GNNGNNGNN-3′. It is likely that there will be improvements in making the individual finger modules. High-throughput methods that combine both selection and assembly are being developed, which will broaden the number of sites that can be targeted, optimize the overall structure of the ZFNs, and increase the possible experimental and therapeutic uses for ZFNs.
Immunosuppressive therapies in the management of immune‐mediated marrow failures in adults: where we stand and where we are going
Authors: Risitano Antonio M.
Published: December 2010

Immunosuppression is a key treatment strategy for aplastic anaemia (AA) and the related immune‐mediated bone marrow failure syndromes (BMFS). For the last 20 years the standard immunosuppressive regimen for AA patients has been anti‐thymocyte globulin (ATG) plus ciclosporin A (CyA), which results in response rates ranging between 50% and 70%, and even higher overall survival. However, primary and secondary failures after immunosuppressive therapy remain frequent, and to date all attempts aiming to overcome this problem have been unfruitful. This article reviews the state of the art of current immunosuppressive therapies for AA, focusing on open questions linked to standard immunosuppressive treatment, and on experimental immunosuppressive strategies which could lead to future improvement of current treatments. Specific immunosuppressive strategies employed for other BMFS, such as lineage‐restricted marrow failures, myelodysplastic syndromes and large granular lymphocyte leukaemia‐associated cytopenias, are also briefly discussed.
Factors associated with treatment restriction orders and hospice in older nursing home residents
Authors: Lu Chu‐Yun , Johantgen Meg
Published: December 2010

Aim.  The purpose of the study is to examine factors associated with do‐not‐resuscitate orders, do‐not‐hospitalise orders and hospice care in older nursing home residents at admission.

Background.  Although hospice care is viewed as the ‘gold standard,’ geographic availability and financial reimbursement limits its use. Treatment restriction orders may represent alternative approaches to defining wishes for end‐of‐life care.

Design.  A descriptive correlational study design was employed to examine the use of four care directives and hospice in newly admitted older people NH residents using Maryland Minimum Data Set 2·0 and the On‐Line Survey Certification and Reporting in 2000. Analyses reflected 10 023 unduplicated admission records from 77 NHs.

Results.  The prevalence of do‐not‐resuscitate and do‐not‐hospitalise orders at admission was 28 and 3·4%, respectively. A very small percentage of residents received hospice care on admission (1·7%). Appropriately, health‐related characteristics had a strong influence on use of do‐not‐resuscitate orders, do‐not‐hospitalise orders and hospice care. However, identified predictors were varied among do‐not‐resuscitate orders, do‐not‐hospitalise orders and hospice care. Moreover, multivariate logistical modelling found that non‐Medicare insurance significantly influenced the likelihood of do‐not‐resuscitate orders, do‐not‐hospitalise orders and hospice uses; White race increased the likelihood of having a do‐not‐resuscitate and do‐not‐hospitalise order. Treatment restriction orders were associated with an increased of likelihood of hospice use.

Conclusions.  As policy and reimbursement barriers to hospice use are likely to persist, treatment restriction orders should be used to focus communication with residents, families and providers, with the ultimate goal of more widespread implementation of hospice care principles.

Relevance to clinical practice.  White race was consistently associated with increasing the likelihood of having do‐not‐resuscitate and do‐not‐hospitalise orders, supporting the importance of cultural sensitivity in advanced care planning. With the association between do‐not‐hospitalise orders and hospice use, treatment restriction orders should be used as potential triggers to prompting end‐of‐life care.
Swedish mothers’ experience of continuous Kangaroo Mother Care
Authors: Blomqvist Ylva Thernström , Nyqvist Kerstin Hedberg
Published: December 2010

Aims.  To characterise the first infants receiving continuous Kangaroo Mother Care from birth to discharge in a Swedish neonatal intensive care unit and to investigate their mothers’ experiences of this model of care.

Background.  Admission of a newborn infant to a neonatal intensive care unit commonly implies separation of the new mother from her infant. Kangaroo Mother Care is a model of neonatal care which supports the parental role as primary care‐giver and contributes to minimising the separation between the infant and parents.

Design.  A retrospective survey design.

Method.  A purposive sample consisting of 23 mother–infant pairs. Relevant infant data were obtained from their medical records. A questionnaire with questions about the infant’s care and regarding Kangaroo Mother Care was designed for this study.

Results.  The infants were born at a gestational age of 31–41 weeks, birth weight ranging from 1715–3700 g. The mothers of these moderately preterm and ill newborn infants showed good acceptance of the idea of providing their infants with continuous Kangaroo Mother Care during their stay at the neonatal intensive care unit. The mothers’ evaluations of this method were predominantly positive. Negative comments concerned lack of information about practical application of the method, and some mothers perceived their infants’ care during the night as exhausting. No mother would have preferred not to perform continuous Kangaroo Mother Care or to terminate Kangaroo Mother Care earlier than they did.

Conclusions.  These mothers accepted this model of care very well, provided that they received the help and support they required.

Relevance to clinical practice.  Mothers whose infants are admitted to an neonatal intensive care units in settings similar to the study setting should be offered opportunities to be present and provide Kangaroo Mother Care for their infants, to the extent that they are able and willing to do so and as permitted by the infant’s medical condition and care.