bluuebunny/crossref_metadata_2025 huggingface dataset:
A velocity map imaging study of gold-rare gas complexes: Au–Ar, Au–Kr, and Au–Xe
Authors: Hopkins W. Scott , Woodham Alex P. , Plowright Richard J. , Wright Timothy G. , Mackenzie Stuart R.
Published: June 2010

The ultraviolet photodissociation dynamics of the gold-rare gas atom van der Waals complexes (Au–RG, RG=Ar, Kr, and Xe) have been studied by velocity map imaging. Photofragmentation of Au–Ar and Au–Kr at several wavelengths permits extrapolation to zero of the total kinetic energy release (TKER) spectra as monitored in the Au(P23/2∘[5d106p]) fragment channel, facilitating the determination of ground state dissociation energies of D0″(Au–Ar)=149±13 cm−1 and D0″(Au–Kr)=240±19 cm−1, respectively. In the same spectral region, transitions to vibrational levels of an Ω′=1/2 state of the Au–Xe complex result in predissociation to the lower Au(P21/2∘[5d106p])+Xe(S10[5p6]) fragment channel for which TKER extrapolation yields a value of D0″(Au–Xe)=636±27 cm−1. Asymmetric line shapes for transitions to the v′=14 level of this state indicate coupling to the Au(P23/2∘[5d106p])+Xe(S10[5p6]) continuum, which allows us to refine this value to D0″(Au–Xe)=607±5 cm−1. The dissociation dynamics of this vibrational level have been studied at the level of individual isotopologues by fitting the observed excitation spectra to Fano profiles. These fits reveal a remarkable variation in the predissociation dynamics for different Au–Xe isotopologues. For Au–Ar and Au–Xe, the determined ground state dissociation energies are in good agreement with recent theoretical calculations; the agreement of the Au–Kr value with theory is less satisfactory.
Quantum Monte Carlo calculated potential energy curve for the helium dimer
Authors: Wu Xuebin , Hu Xianru , Dai Yunchuan , Du Chenlei , Chu Shibin , Hu Leibo , Deng Jianbo , Feng Yuanping
Published: June 2010

We report on the results of both the diffusion quantum Monte Carlo (DMC) and reptation quantum Monte Carlo (RMC) methods on the potential energy curve of the helium dimer. We show that it is possible to obtain a highly accurate description of the helium dimer. An improved stochastic reconfiguration technique is employed to optimize the many-body wave function, which is the starting point for highly accurate simulations based on the DMC and RMC methods. We find that the results of these methods are in excellent agreement with the best theoretical results at short range, especially the recently developed RMC method, yield particularly accurate results with reduced statistical error, which gives very excellent agreement across the whole potential curve. For the equilibrium internuclear distance of 5.6 bohrs, the calculated total energy with RMC method is −5.807 483 599±0.000 000 016 hartree and the corresponding well depth is −11.003±0.005 K.
Synthesis, anisotropy, and superconducting properties of LiFeAs single crystal
Authors: Song Yoo Jang , Ghim Jin Soo , Min Byeong Hun , Kwon Yong Seung , Jung Myung Hwa , Rhyee Jong-Soo
Published: June 2010

A LiFeAs single crystal with Tconset∼19.7 K was grown in a sealed tungsten crucible using the Bridgeman method. The electrical resistivity experiments revealed a ratio of room temperature to residual resistivity of approximately 46 and 18 for the in-plane and out-of plane directions, respectively. The estimated anisotropic resistivity, γρ=ρc/ρab, was approximately 3.3 at Tconset. The upper critical fields had large Hc2∥ab and Hc2∥c values of 83.4 T and 72.5 T, respectively, and an anisotropy ratio is γH=Hc2∥ab/Hc2∥c∼1.15. The high upper critical field value and small anisotropy highlight the potential use of LiFeAs in a variety of applications. The calculated critical current density (Jc) from the M-H loop is approximately 103 A/cm2
Molecular packing and chemical association in liquid water simulated using ab initio hybrid Monte Carlo and different exchange-correlation functionals
Authors: Weber Valéry , Merchant Safir , Dixit Purushottam D. , Asthagiri D.
Published: June 2010

In the free energy of hydration of a solute, the chemical contribution is given by the free energy required to expel water molecules from the coordination sphere and the packing contribution is given by the free energy required to create the solute-free coordination sphere (the observation volume) in bulk water. With the simple point charge/extended (SPC/E) water model as a reference, we examine the chemical and packing contributions in the free energy of water simulated using different electron density functionals. The density is fixed at a value corresponding to that for SPC/E water at a pressure of 1 bar. The chemical contribution shows that water simulated at 300 K with BLYP is somewhat more tightly bound than water simulated at 300 K with revised PBE (revPBE) functional or at 350 K with the BLYP and BLYP-D functionals. The packing contribution for various radii of the observation volume is studied. In the size range where the distribution of water molecules in the observation volume is expected to be Gaussian, the packing contribution is expected to scale with the volume of the observation sphere. Water simulated at 300 K with the revPBE and at 350 K with BLYP-D or BLYP conforms to this expectation, but the results suggest an earlier onset of system size effects in the BLYP 350 K and revPBE 300 K systems than that observed for either BLYP-D 350 K or SPC/E. The implication of this observation for constant pressure simulations is indicated. For water simulated at 300 K with BLYP, in the size range where Gaussian distribution of occupation is expected, we instead find non-Gaussian behavior, and the packing contribution scales with surface area of the observation volume, suggesting the presence of heterogeneities in the system.
Tunable thin film bulk acoustic wave resonators with improved Q-factor
Authors: Vorobiev A. , Gevorgian S.
Published: June 2010

The tunable solidly mounted Ba0.25Sr0.75TiO3 (BSTO) thin film bulk acoustic wave resonators (TFBARs) with improved Q-factor are fabricated and characterized. The BSTO films are grown by magnetron sputtering at temperature 600 °C and extremely low sputter gas pressure 2 mTorr using on-axis configuration. The measured TFBARs Q-factor is more than 250 and mechanical Q-factor is more than 350 at 5 GHz resonance frequency. The improvement in the Q-factor is associated with reduction in the BSTO film grain misorientation. The latter is responsible for generation of shear waves leaking through the Bragg reflector and corresponding acoustic loss.
A synthesis route of gold nanoparticles without using a reducing agent
Authors: Vargas-Hernandez C. , Mariscal M. M. , Esparza R. , Yacaman M. J.
Published: June 2010

In the present work we show that synthesis of gold nanoparticles (NPs) could be performed by microwave-assisted technique without the need of adding any reducing agent. Only water and the gold salt precursor are necessary to generate the NPs under the influence of microwaves. The produced NPs have been characterized by state-of-art microscopy techniques, like high resolution transmission electron microscopy, scanning electron microscopy, and energy-dispersive x-ray. Theoretical calculations have been performed to support the experimental findings. It is expected that the present work opens routes for synthesis of NPs using green, fast, and safe methods.
Kinetics of Ta ions penetration into porous low-k dielectrics under bias-temperature stress
Authors: He Ming , Ou Ya , Wang Pei-I , Lu Toh-Ming
Published: June 2010

It is known that Ta, a popular diffusion barrier material, can itself penetrate into low-k dielectrics under bias-temperature stress. In this work, we derived a model which directly correlates the diffusivity of Ta ions to the rate of flatband voltage shift (FBS) of the Ta/methyl silsesquixane (MSQ)/Si capacitors. From our experimentally measured constant FBS rate, the Ta diffusivity and activation energy were determined. It appears that an increase in the porosity of MSQ film enhances the Ta diffusivity but does not affect the associated activation energy. This suggests the Ta ion diffusion is mainly through interconnected pore surfaces.
Current transport and barrier height evaluation in Ni/InAlN/GaN Schottky diodes
Authors: Donoval D. , Chvála A. , Šramatý R. , Kováč J. , Carlin J.-F. , Grandjean N. , Pozzovivo G. , Kuzmík J. , Pogany D. , Strasser G. , Kordoš P.
Published: June 2010

The current-voltage characteristics of the Ni/InAlN/GaN Schottky diodes were measured at various temperatures in the range of 300–700 K. The experimental data were analyzed considering different current-transport mechanisms. From the analysis it follows that the tunneling current, which might be due to a multistep tunneling along dislocations, is the dominant component at all temperatures in the samples investigated. The barrier height of the Ni/InAlN/GaN Schottky diodes, evaluated from the thermionic emission current, shows a slightly negative temperature coefficient and its value at 300 K is ≥1.46 eV. This is significantly higher barrier height than reported before (<1 eV). This discrepancy follows from an incorrect evaluation using the intercept and slope of a measured characteristic without separation of the individual current components.
Roles of interfacial TiOxN1−x layer and TiN electrode on bipolar resistive switching in TiN/TiO2/TiN frameworks
Authors: Kwak June Sik , Do Young Ho , Bae Yoon Cheol , Im Hyun Sik , Yoo Jong Hee , Sung Min Gyu , Hwang Yun Taek , Hong Jin Pyo
Published: June 2010

Reversible counter-clockwise and clockwise resistive switching in a TiN/TiO2/TiN structure was studied by different polarities of bias voltage. The nature of the bipolar switching phenomenon is related to the creation and annihilation of filament paths caused by redox reactions at locally confined interfaces between the TiO2 layer and TiN electrode. The analysis of electron energy loss spectroscopy (EELS) confirmed the formation of interfacial TiOxN1−x layer between the TiO2 and TiN bottom electrode. The TiOxN1−x layer reduces current levels of ON and OFF states by partially blocking oxygen ion drift to the TiN bottom electrode.
Improvement of memory performance by high temperature annealing of the Al2O3 blocking layer in a charge-trap type flash memory device
Authors: Park Jong Kyung , Park Youngmin , Lim Sung Kyu , Oh Jae Sub , Joo Moon Sig , Hong Kwon , Cho Byung Jin
Published: June 2010

The effect of postdeposition annealing (PDA) of the Al2O3 blocking layer in a charge-trap type memory device is investigated. Significant improvements are achieved by high temperature PDA at 1100 °C, achieving faster operation speed, good charge retention, and a wide program/erase window. Experimental evidence shows that the underlying mechanism is not the changes in the band gap of the crystallized Al2O3 but is due to the higher trap density in the Si3N4 trapping layer at a deeper energy level by the intermixing between Al2O3 and Si3N4. The reduced trapping efficiency of the annealed Al2O3 also helps improve the retention property.
Electronic structures of fully fluorinated and semifluorinated zinc oxide sheets
Authors: Wang Yanli , Ding Yi , Ni Jun , Shi Siqi , Li Chaorong , Shi Jianjun
Published: June 2010

Using first-principles calculations, we investigate the structural, electronic, and magnetic properties of fully fluorinated and semifluorinated ZnO sheets. We find the fully fluorinated ZnO sheet prefers a twist-chair conformation, which is a nonmagnetic semiconductor. While for the semifluorinated sheet, the F-center conformation is the most stable and exhibits half-metallic behavior. The half-metallicity is attributed to the decrease in the charge transfer from Zn to O atoms after fluorination, which leads to the partial occupancy of the O 2p orbitals and spin-polarization in the semifluorinated sheet. Our studies demonstrate that fluorination is an efficient route to tune the electronic structures of ZnO nanostructures.
Pinning of recombination-enhanced dislocation motion in 4H–SiC: Role of Cu and EH1 complex
Authors: Chen Bin , Matsuhata Hirofumi , Sekiguchi Takashi , Ohyanagi Takasumi , Kinoshita Akimasa , Okumura Hajime
Published: June 2010

We report on the pinning of recombination-enhanced dislocation motion in 4H–SiC by the implantation of Cu. The Cu was found to be preferentially gettered at basal plane dislocations (BPDs). Both EH1 and Z1/2 center were detected in 4H–SiC by cathodoluminescence. It was noticed that the EH1 has high luminescence intensity at the central part of the BPDs, while the Z1/2 does not. The complex of Cu and EH1 is regarded to be the cause for the pinning effect. The possible reason for the pinning is discussed.
Theory of electroluminescence intensity and insights into recombination in thin film solar cells
Authors: Brown Gregory , Faifer Vladimir , Cardozo Ben , Bykov Eugene , Contreras Miguel
Published: June 2010

Equations describing the electroluminescence (EL) intensity as a function of material properties are derived for thin film solar cells and experimentally validated using Cu(In,Ga)Se2 solar cells. EL intensity at constant voltage is controlled by the electronic properties of the neutral bulk even when the diode current is controlled by recombination in the space charge region. Using a combination of techniques, it is found that recombination in the quasineutral bulk does not correlate with recombination in the space charge region. Differences between EL measurements on thin film cells and crystalline silicon cells are discussed including the effects of secondary barriers.
Element 117 yields long-lived decay products
Authors: Miller Johanna
Published: June 2010

Two isotopes of the newly discovered element decay to give nine more previously unobserved nuclei.
Effect of gypsum on establishment, persistence and productivity of lucerne and annual pasture legumes on two grey Vertosols in southern New South Wales
Authors: Dear B. S. , Peoples M. B. , Hayes R. C. , Swan A. D. , Chan K. Y. , Oates A. A. , Morris S. G. , Orchard B. A.
Published: June 2010

Changes in pasture yield and botanical composition due to gypsum application were examined on Vertosols at two locations of differing soil sodicity, Grogan and Morangarell, in southern New South Wales. Two pasture treatments were examined. One was an annual pasture comprised of 3 annual legumes (2 subterranean clover Trifolium subterraneum L. cultivars, Clare and Riverina, and balansa clover T. michelianum Savi cv. Paradana), while the second treatment consisted of lucerne (Medicago sativa L.) cv. Aquarius sown in a mixture with the same annual legumes.
Gypsum had no effect on the establishment or persistence of lucerne at either site. Gypsum increased the number of subterranean clover seedlings present in autumn in annual swards at the more sodic Grogan site in each of the 4 years, but provided no difference when the clover was in a mixture with lucerne. Annual legume seed yields in annual-only swards increased with gypsum by up to 58% at Grogan and 38% at Morangarell. Seed yields of both cultivars of subterranean clover declined as a proportion of the total annual legume seed bank when lucerne was included in the mixture, in contrast to balansa clover (at Grogan) and the naturalised annual legumes, burr medic (M. polymorpha L.) and woolly clover (T. tomentosum L.), which all increased in relative seed yield in the presence of lucerne.
Total pasture production at the Grogan site increased with gypsum by up to 15% per annum in annual swards and 36% in lucerne swards depending on the season. Yield responses to gypsum by the lucerne component were observed in 10 of the 13 seasonal yield measurements taken at Grogan. However, total pasture yield and seasonal yields were unaffected by both gypsum and pasture type at the less sodic Morangarell site. It was concluded that sowing a diverse mixture of annual legumes or polycultures was conducive to maintaining productive pastures on these spatially variable soils.
Lucerne dried the soil profile (0.15–1.15 m) more than annual pastures at both sites. The combination of gypsum and lucerne enhanced water extraction at depth (0.6–1.15 m) at the Grogan site increasing the size of the dry soil buffer whereas gypsum increased soil water at depth (>0.6 m) under annual swards.
Activating WASP mutations associated with X-linked neutropenia result in enhanced actin polymerization, altered cytoskeletal responses, and genomic instability in lymphocytes
Authors: Westerberg Lisa S. , Meelu Parool , Baptista Marisa , Eston Michelle A. , Adamovich David A. , Cotta-de-Almeida Vinicius , Seed Brian , Rosen Michael K. , Vandenberghe Peter , Thrasher Adrian J. , Klein Christoph , Alt Frederick W. , Snapper Scott B.
Published: June 2010

X-linked neutropenia (XLN) is caused by activating mutations in the Wiskott-Aldrich syndrome protein (WASP) that result in aberrant autoinhibition. Although patients with XLN appear to have only defects in myeloid lineages, we hypothesized that activating mutations of WASP are likely to affect the immune system more broadly. We generated mouse models to assess the role of activating WASP mutations associated with XLN (XLN-WASP) in lymphocytes. XLN-WASP is expressed stably in B and T cells and induces a marked increase in polymerized actin. XLN-WASP–expressing B and T cells migrate toward chemokines but fail to adhere normally. In marked contrast to WASP-deficient cells, XLN-WASP–expressing T cells proliferate normally in response to cell-surface receptor activation. However, XLN-WASP–expressing B cells fail to proliferate and secrete lower amounts of antibodies. Moreover, XLN-WASP expression in lymphocytes results in modestly increased apoptosis associated with increased genomic instability. These data indicate that there are unique requirements for the presence and activation status of WASP in B and T cells and that WASP-activating mutations interfere with lymphocyte cell survival and genomic stability.
Some hematopoietic stem cells are more equal than others
Authors: Hock Hanno
Published: June 2010

Hematopoietic stem cells (HSCs) save lives in routine clinical practice every day, as they are the key element in transplantation-based therapies for hematologic malignancies. The success of clinical stem cell transplantation critically relies on the ability of stem cells to reconstitute the hematopoietic system for many decades after the administration of the powerful chemotherapy and/or irradiation that is required to eradicate malignant cells, but also irreversibly ablates patients’ own blood forming capacity. Surprisingly, despite enormous efforts and continuous progress in the field, our understanding of the basic biology of HSCs is still rather incomplete. Several recent studies substantially refine our understanding of the cells at the very top of the hematopoietic hierarchy, and suggest that we may need to revise the criteria we typically use to identify and define HSCs.
Found in translation: the human equivalent of mouse CD8+ dendritic cells
Authors: Villadangos Jose A. , Shortman Ken
Published: June 2010

The murine dendritic cell network comprises multiple subsets with distinct functions, but few of their human counterparts have been described. New data now reveals the likely human equivalent of the mouse DC subset specialized in cross-presentation.
Autoantibodies against cardiac troponin I in patients with congestive heart failure
Authors: Düngen Hans‐Dirk , Platzeck Maria , Vollert Jörn , Searle Julia , Müller Christian , Reiche Janett , Mehrhof Felix , Müller Reinhold , Möckel Martin
Published: June 2010

Aims

In this randomized, double‐blind clinical study, we investigated the relationship between autoantibodies against cardiac troponin I (cTnI) and disease severity in elderly congestive heart failure (CHF) patients before and after titration of beta‐blocker therapy.

Methods and results

Anti‐cTnI, cTnI, and N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) levels in blood from 138 patients (73 ± 5.6 years, 48% male) with CHF in American Heart Association stages A–C were measured at baseline and after titration of beta‐blockers to maximal tolerated dose. Median follow‐up period was 85 days. Anti‐cTnI was measured using an experimental assay (Abbott Diagnostics) and is expressed as a relative value unit (RVU) in relation to the mean value of a low‐level control sample. Anti‐cTnI values in CHF patients were also compared with measurements taken in a normal reference population of 300 healthy individuals (50% male). Cardiac troponin I and NT‐proBNP levels were measured using routine assays (Architect® Abbott Diagnostics and Roche Diagnostics®). Median anti‐cTnI was 0.53 and 0.56 RVU in healthy and CHF subjects, respectively (P = n.s.), and increased significantly to 0.67 RVU (P < 0.001) after beta‐blocker titration. Mean cTnI values were 0.021 µg/L at baseline and fell significantly to 0.0046 µg/L at follow‐up (P < 0.001). Median NT‐proBNP values were 352 ng/L at baseline and 414 ng/L after titration (P = n.s.). In contrast to NT‐proBNP and cTnI, anti‐cTnI was not associated with the severity of CHF at baseline or follow‐up.

Conclusion

Levels of anti‐cTnI tend to increase in elderly patients with CHF following titration of beta‐blocker therapy but do not correlate with disease severity. Anti‐cTnI is not a useful biomarker for heart failure diagnosis, prognosis, or monitoring. In contrast, levels of cTnI decreased following therapy and did correlate with disease severity.
Modeling T-cell acute lymphoblastic leukemia induced by the SCL and LMO1 oncogenes
Authors: Tremblay Mathieu , Tremblay Cédric S. , Herblot Sabine , Aplan Peter D. , Hébert Josée , Perreault Claude , Hoang Trang
Published: June 2010

Deciphering molecular events required for full transformation of normal cells into cancer cells remains a challenge. In T-cell acute lymphoblastic leukemia (T-ALL), the genes encoding the TAL1/SCL and LMO1/2 transcription factors are recurring targets of chromosomal translocations, whereas NOTCH1 is activated in >50% of samples. Here we show that the SCL and LMO1 oncogenes collaborate to expand primitive thymocyte progenitors and inhibit later stages of differentiation. Together with pre-T-cell antigen receptor (pre-TCR) signaling, these oncogenes provide a favorable context for the acquisition of activating Notch1 mutations and the emergence of self-renewing leukemia-initiating cells in T-ALL. All tumor cells harness identical and specific Notch1 mutations and Tcrβ clonal signature, indicative of clonal dominance and concurring with the observation that Notch1 gain of function confers a selective advantage to SCL-LMO1 transgenic thymocytes. Accordingly, a hyperactive Notch1 allele accelerates leukemia onset induced by SCL-LMO1 and bypasses the requirement for pre-TCR signaling. Finally, the time to leukemia induced by the three transgenes corresponds to the time required for clonal expansion from a single leukemic stem cell, suggesting that SCL, LMO1, and Notch1 gain of function, together with an active pre-TCR, might represent the minimum set of complementing events for the transformation of susceptible thymocytes.
Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly
Authors: Lydeard John R. , Lipkin-Moore Zachary , Sheu Yi-Jun , Stillman Bruce , Burgers Peter M. , Haber James E.
Published: June 2010

Break-induced replication (BIR) is an efficient homologous recombination (HR) pathway employed to repair a DNA double-strand break (DSB) when homology is restricted to one end. All three major replicative DNA polymerases are required for BIR, including the otherwise nonessential Pol32 subunit. Here we show that BIR requires the replicative DNA helicase (Cdc45, the GINS, and Mcm2–7 proteins) as well as Cdt1. In contrast, both subunits of origin recognition complex (ORC) and Cdc6, which are required to create a prereplication complex (pre-RC), are dispensable. The Cdc7 kinase, required for both initiation of DNA replication and post-replication repair (PRR), is also required for BIR. Ubiquitination and sumoylation of the DNA processivity clamp PCNA play modest roles; in contrast, PCNA alleles that suppress pol32Δ's cold sensitivity fail to suppress its role in BIR, and are by themselves dominant inhibitors of BIR. These results suggest that origin-independent BIR involves cross-talk between normal DNA replication factors and PRR.
Generation of Retroviral Particles for the Spleen Necrosis Virus (SNV)-Based Vector System and Their Use in Transduction of Various Cell Types: Figure 1.
Authors: Parveen Zahida , Mukhtar Muhammad , Pomerantz Roger J.
Published: June 2010

INTRODUCTION

Genetically engineered retroviruses are widely used for gene delivery into human cells. A number of investigators have studied spleen necrosis virus (SNV) as a vehicle for gene delivery. Vectors developed from SNV and its closely associated avian reticuloendotheliosis virus strain A (REV-A) can be used for gene transfer into a variety of cells, including primary hematopoietic cells and human brain and post-mitotic neuronal cells that are difficult to transduce with other vector systems. SNV-based vector systems have the advantage of being quite safe, because wild-type SNV is unable to infect human cells and has less preference for integration into transcriptionally active sites or genes. However, the generation of retroviral vectors requires cotransfection of more than one plasmid into a packaging cell line, which is a tedious process. The development of stable packaging cell lines expressing envelope (Env) proteins and the structural proteins Gag-Pol will enhance mass production of retroviral vectors for future gene therapy experiments both in vitro and in vivo. This protocol describes the generation of retroviral particles for the SNV-based vector system. These particles can then be used for transduction of various cell types; as an example, a technique for transduction of post-mitotic neurons is also presented.
Production of SV40-Derived Vectors
Authors: Strayer David S. , Mitchell Christine , Maier Dawn A. , Nichols Carmen N.
Published: June 2010

INTRODUCTION

Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Here we describe a protocol for production and purification of these vectors. These procedures require only packaging cells (e.g., COS-7) and circular vector genome DNA. Amplification involves repeated infection of packaging cells with vector produced by transfection. Cotransfection is not required in any step. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients and resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. These approaches are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. These vectors are best applied to long-term expression of proteins normally encoded by mammalian cells or by viruses that infect mammalian cells, or of untranslated RNAs (e.g., RNA interference). The preparative approaches described facilitate application of these vectors and allow almost any laboratory to exploit their strengths for diverse gene delivery applications.
Titering Replication-Defective rSV40 Vectors
Authors: Strayer David S. , Mitchell Christine , Maier Dawn A. , Nichols Carmen N.
Published: June 2010

INTRODUCTION

Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Techniques used to produce, purify, and quantitate these vectors are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients. Resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. Viruses are titered by quantitative polymerase chain reaction (qPCR), described here. qPCR measures the number of rSV40 genomes in purified viral stocks using primers specific for the rSV40, coupled with SYBR Green detection of PCR products. Sample purity is assessed using qPCR via melt (dissociation) curve analysis. The only specialized equipment necessary is a quantitative real-time PCR machine.
Purification of RNA Using TRIzol (TRI Reagent)
Authors: Rio Donald C. , Ares Manuel , Hannon Gregory J. , Nilsen Timothy W.
Published: June 2010

INTRODUCTION

TRIzol solubilization and extraction is a relatively recently developed general method for deproteinizing RNA. This method is particularly advantageous in situations where cells or tissues are enriched for endogenous RNases or when separation of cytoplasmic RNA from nuclear RNA is impractical. TRIzol (or TRI Reagent) is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously solubilizes biological material and denatures protein. After solubilization, the addition of chloroform causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase. Therefore, RNA, DNA, and protein can be purified from a single sample (hence, the name TRIzol). TRIzol extraction is also an effective method for isolating small RNAs, such as microRNAs, piwi-associated RNAs, or endogeneous, small interfering RNAs. However, TRIzol is expensive and RNA pellets can be difficult to resuspend. Thus, the use of TRIzol is not recommend when regular phenol extraction is practical.
Ethanol Precipitation of RNA and the Use of Carriers
Authors: Rio Donald C. , Ares Manuel , Hannon Gregory J. , Nilsen Timothy W.
Published: June 2010

INTRODUCTION

Recovery of deproteinized RNA almost always is performed via precipitation with ethanol. If the RNA is free of contaminants (i.e., it is well extracted) as determined by spectrophotometry, it will resuspend easily and be ready for any subsequent manipulation. Ethanol precipitation is quite useful because it provides quantitative recovery of any-sized RNA, from several kilobases to 20 nucleotides (nt). Major considerations in this protocol are RNA concentration, pellet identification, and resuspension.
Polyacrylamide Gel Electrophoresis of RNA: Figure 1.
Authors: Rio Donald C. , Ares Manuel , Hannon Gregory J. , Nilsen Timothy W.
Published: June 2010

INTRODUCTION

Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. This technique is generally applicable for RNA detection, quantification, purification by size, and quality assessment. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to ≤20 nucleotides (nt). In certain circumstances, e.g., resolving different conformers of RNAs or RNA-protein complexes, native gels are appropriate. The only disadvantage to acrylamide gels is that they are not suitable for analyzing large RNAs (≥600 nt); for such applications, agarose gels are preferred. This protocol describes how to prepare, load, and run polyacrylamide gels for RNA analysis.
Nondenaturing Agarose Gel Electrophoresis of RNA
Authors: Rio Donald C. , Ares Manuel , Hannon Gregory J. , Nilsen Timothy W.
Published: June 2010

INTRODUCTION

Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. There are two common types of gel: polyacrylamide and agarose. For most applications involving RNAs of ≤600 nucleotides, denaturing acrylamide gels are most appropriate. In contrast, agarose gels are generally used to analyze RNAs of ≥600 nucleotides, and are especially useful for analysis of mRNAs (e.g., by Northern blotting). RNA analysis on agarose gels is essentially identical to DNA analysis (except that the gel boxes used must be dedicated to RNA work or to other ribonuclease-free work). Here we describe the use of straightforward Tris borate, EDTA (TBE) gels for routine analysis. These gels are appropriate for determining the quantity and integrity of RNA before using it for other applications. This procedure should not be used to determine size with accuracy, because the RNA will not remain in its extended state throughout the run.
In Ovo Live Imaging of Avian Embryos: Figure 1.
Authors: Kulesa Paul M. , Bailey Caleb M. , Cooper Cameron , Fraser Scott E.
Published: June 2010

INTRODUCTION

Vertebrate development is best studied in an intact embryo model, but a robust interface between time-lapse microscopy and in vivo embryo health and maintenance can be difficult to achieve in model systems that rely on external factors for life support. This protocol presents a system for in ovo culture and time-lapse imaging of fluorescently labeled cells within living avian embryos, using a Teflon membrane that is oxygen-permeable and liquid-impermeable. The protocol describes the Teflon membrane assembly (the assembly size can be changed to fit smaller eggs, such as those of the quail), its interface with the egg window, and the use of an upright microscope and heated chamber. The use of the system is demonstrated in chick embryos by following individual fluorescently labeled neural crest cells, a multipotent stem cell-like population that differentiates into a wide range of derivatives and travels extensively throughout the embryo. By combining in ovo culture with confocal or two-photon four-dimensional time-lapse imaging, embryo health can be maintained for up to 5 d, and neural crest cell behaviors can be visualized for long periods of time (~36 h). This technique has been adapted to study somitogenesis.
Multi-Position Photoactivation and Multi-Time Acquisition for Large-Scale Cell Tracing in Avian Embryos
Authors: Steen Joseph , Morrison Jason A. , Kulesa Paul M.
Published: June 2010

INTRODUCTION

Vertebrate development is a complex orchestration of cell and tissue movements. Tracing individual cell positions can rapidly become a large-scale problem because cell numbers often grow exponentially in the early embryo. A typical approach consists of fluorescently marking small numbers of cells within a large number of embryos, followed by comprehensive three-dimensional static or time-lapse imaging to map cell positions. However, for large-scale cell tracing, such as during organogenesis, the time, effort, and expense of this approach can be limiting. The multi-position acquisition method can be used to capture more than one location on a microscope stage and allow for multi-specimen imaging. When combined with photoactivation cell labeling, a tool for selective cell marking using laser excitation, multi-position imaging offers a powerful tool for rapid data acquisition. This protocol describes the technique and demonstrates its use to map cell movements in the chick spinal cord, using slice culture explants. The details of multiple slice culture preparation, multi-position photoactivation, and multi-time acquisition are described. These events are coordinated by setting up blocks of microscope instructions that execute sequentially. This method significantly decreases the time, effort, microscopy, and embryo costs by a factor of the number of specimens imaged per session, typically six.
Purification of the Mitotic Checkpoint Complex (MCC) and the Anaphase Promoting Complex/Cyclosome (APC/C) from HeLa Cells
Authors: Sudakin Valery
Published: June 2010

INTRODUCTION

In this protocol, active anaphase promoting complex/cyclosome (APC/C) and its inhibitor, mitotic checkpoint complex (MCC), are purified from HeLa cell extracts. The two complexes are first separated by gel filtration and ion exchange chromatography. The MCC is further purified by an additional gel filtration step. The APC/C activity and the ability of MCC to inhibit the APC/C are assayed at every stage of the purification procedure by in vitro ubiquitination assays. The composition and distribution of the complexes are monitored by Western blot analysis.
Assembling Webs of Support: Child Domestic Workers in India
Authors: Wasiuzzaman Shaziah , Wells Karen
Published: June 2010

This paper uses ethnographic and qualitative interview data with Muslim child domestic workers, their families and employers to investigate the social ties between young workers and their employers. Our analysis shows that working‐class families use children’s domestic work with middle‐class families as part of a web of resources to protect them from economic shocks and to enable them to afford to meet the cost of social obligations. We show that in this particular context, a town in Uttar Pradesh in north India, hiring domestic workers locks employers into relations of social obligation with their employees and their families. We conclude that these webs of support are enabled precisely because the domestic workers are children and not adults; their status as children makes it possible for the labour contract to be mystified and reconfigured as a social relationship.
Combining Work and School: The Dynamics of Girls’ Involvement in Agricultural Work in Andhra Pradesh, India
Authors: Morrow Virginia , Vennam Uma
Published: June 2010

Child labour in India has long been the focus of research, policy concern and intervention. This article presents an analysis of children’s involvement in agricultural work, particularly cottonseed production, drawing on evidence gathered for Young Lives in 2007 and 2008. In parts of Andhra Pradesh, children work in cotton fields for two or three months of the year, with marked gender and age differentiation. In the mid‐1990s, there was reportedly a cultural as well as an economic basis for children’s work in cottonseed pollination, when it was believed that pre‐pubescent girls were preferred, as they were considered ‘pure’. However, the reasons for preferring children are now largely financial and practical. The article focuses on accounts from two girls who highlighted the importance of this form of work in their everyday lives and its consequences for their schooling.
Barriers to research utilisation by staff nurses in a university hospital
Authors: Kocaman Gulseren , Seren Seyda , Lash Ayhan A , Kurt Seyyare , Bengu Nergiz , Yurumezoglu Havva A
Published: June 2010

Aim.  To identify self‐reported perceptions of the barriers to research utilisation by Turkish staff nurses working in a university hospital and to ascertain whether involvement in research‐related activities influenced their perception of the barriers.

Background.  Research utilisation in nursing has been a prominent concern for the last 30 years. While investigators in several countries have identified nurses’ perception of barriers to research utilisation, this is the first study to investigate such barriers in Turkey.

Design.  This is a descriptive, cross sectional study with a sample of 329 staff nurses working in a university hospital. Two instruments were used; a demographics questionnaire that investigated research activities and the Barriers Scale to measure perceptions. An additional item, ‘studies are published in English’ was added to the scale.

Results.  The lack of time to implement new ideas was the most frequently, 84·5%, perceived barrier. The second most frequently perceived barrier was difficulty in reading English. The top six barriers were all related to the setting. Additionally, research‐active nurses found insufficient time less of a barrier but unawareness of research findings and being isolated from knowledgeable colleagues as greater barriers to research utilisation.

Conclusions.  Turkish nurses’ perception of barriers is similar to the findings in other countries, i.e., that the top six barriers were related to setting. Language barrier compares similarly with studies in other countries that investigated this variable.

Relevance to clinical practice.  The gap between theory and practice in nursing can be minimised by identifying strategies to increase research utilisation in clinical settings. Such strategies can also improve patient care outcomes.
Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis
Authors: Shcherbakov Dmitri , Akbergenov Rashid , Matt Tanja , Sander Peter , Andersson Dan I. , Böttger Erik C.
Published: June 2010

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.
Gain‐of‐function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor, Aer
Authors: Campbell Asharie J. , Watts Kylie J. , Johnson Mark S. , Taylor Barry L.
Published: June 2010

The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD‐containing PAS domain. Here, we randomly mutagenized the region encoding residues 14–119 of the PAS domain and found 72 aerotaxis‐defective mutants, 24 of which were gain‐of‐function, signal‐on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS–FAD domain in NifL from Azotobacter vinlandii. Signal‐on lesions clustered in the FAD binding pocket, the β‐scaffolding and in the N‐cap loop. We suggest that the signal‐on lesions mimic the ‘signal‐on’ state of the PAS domain, and therefore may be markers for the signal‐in and signal‐out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the β‐scaffolding and the N‐cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS β‐scaffold and residue Q248C in the HAMP AS‐2 helix.
Metabolic shutdown in Escherichia coli cells lacking the outer membrane channel TolC
Authors: Dhamdhere Girija , Zgurskaya Helen I.
Published: June 2010

The outer membrane channel TolC is a key component of multidrug efflux and type I secretion transporters in Escherichia coli. Mutational inactivation of TolC renders cells highly susceptible to antibiotics and leads to defects in secretion of protein toxins. Despite impairment of various transport functions, no growth defects were reported in cells lacking TolC. Unexpectedly, we found that the loss of TolC notably impairs cell division and growth in minimal glucose medium. The TolC‐dependent phenotype was further exacerbated by the loss of ygiB and ygiC genes expressed in the same operon as tolC and their homologues yjfM and yjfC located elsewhere on the chromosome. Our results show that this growth deficiency is caused by depletion of the critical metabolite NAD+ and high NADH/NAD+ ratios. The increased amounts of PspA and decreased rates of NADH oxidation in ΔtolC membranes indicated stress on the membrane and dissipation of a proton motive force. We conclude that inactivation of TolC triggers metabolic shutdown in E. coli cells grown in minimal glucose medium. The ΔtolC phenotype is partially rescued by YgiBC and YjfMC, which have parallel functions independent from TolC.
UVA1 phototherapy: a review of mechanism and therapeutic application
Authors: York Nathan R. , Jacobe Heidi T.
Published: June 2010

Ultraviolet radiation (UVR) phototherapy has been associated with both deleterious and beneficial effects to patients with both localized and systemic skin disorders. Phototherapy is advantageous in diseases of the epidermis and dermis, as it provides the most direct approach minimizing systemic side effects. Most recently, ultraviolet A1 (UVA1) phototherapy has emerged as a specific UVR phototherapeutic mechanism. It has shown to be therapeutic in a number of sclerosing skin conditions and other dermatitides, in many cases proving to be more effective than other phototherapy modalities. Treatment advantages of UVA1 phototherapy include the ability to penetrate into the deep layers of the skin to affect changes on disease‐causing T cells, as well as activation of endothelial cells to promote neovascularization. UVA1 therapy also has been shown to be relatively free of side effects associated with other phototherapy regimens, including erythema and cellular transformation. These properties make UVA1 phototherapy an important treatment option for many debilitating skin conditions.