bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced Hierarchical Models with Application to Estimating Health Effects of Simultaneous Exposure to Multiple Pollutants
Authors: Bobb Jennifer F. , Dominici Francesca , Peng Roger D.
Published: September 2013

Hierarchical models (HMs) have been used extensively in multisite time series studies of air pollution and health to estimate health effects of a single pollutant adjusted for other pollutants and other time varying factors. Recently, the US Environmental Protection Agency has called for research quantifying health effects of simultaneous exposure to many air pollutants. However, straightforward application of HMs in this context is challenged by the need to specify a random-effect distribution on a high dimensional vector of nuisance parameters. Here we introduce the reduced HM as a general statistical approach for analysing correlated data with many nuisance parameters. For reduced HMs we first calculate the integrated likelihood of the parameter of interest (e.g. the excess number of deaths attributed to simultaneous exposure to high levels of many pollutants), and we then specify a flexible random-effect distribution directly on this parameter. Simulation studies show that the reduced HM performs comparably with the full HM in many scenarios and even performs better in some cases, particularly when the multivariate random-effect distribution of the full HM is misspecified. Methods are applied to estimate relative risks of cardio-vascular hospital admissions associated with simultaneous exposure to elevated levels of particulate matter and ozone in 51 US counties during 1999–2005.
Fully Bayesian Hierarchical Modelling in Two Stages, with Application to Meta-Analysis
Authors: Lunn David , Barrett Jessica , Sweeting Michael , Thompson Simon
Published: September 2013

Meta-analysis is often undertaken in two stages, with each study analysed separately in stage 1 and estimates combined across studies in stage 2. The study-specific estimates are assumed to arise from normal distributions with known variances equal to their corresponding estimates. In contrast, a one-stage analysis estimates all parameters simultaneously. A Bayesian one-stage approach offers additional advantages, such as the acknowledgement of uncertainty in all parameters and greater flexibility. However, there are situations when a two-stage strategy is compelling, e.g. when study-specific analyses are complex and/or time consuming. We present a novel method for fitting the full Bayesian model in two stages, hence benefiting from its advantages while retaining the convenience and flexibility of a two-stage approach. Using Markov chain Monte Carlo methods, posteriors for the parameters of interest are derived separately for each study. These are then used as proposal distributions in a computationally efficient second stage. We illustrate these ideas on a small binomial data set; we also analyse motivating data on the growth and rupture of abdominal aortic aneurysms. The two-stage Bayesian approach closely reproduces a one-stage analysis when it can be undertaken, but can also be easily carried out when a one-stage approach is difficult or impossible.
Finite Mixture Modelling in Mass Spectrometry Analysis
Authors: Melnykov Volodymyr
Published: September 2013

Secondary ion mass spectrometry is a popular physical technique that allows learning elemental and chemical compositions of various substances. In some cases, the mass spectrum of the sample studied is not observable without that of the liquid solution in which the investigated substance must be placed. To separate the informative part of the observed mixed spectrum from the irrelevant liquid solution part, we develop an approach that is based on finite mixtures. Finite mixture modelling is a statistical technique that is capable of accounting for various shapes and patterns in data. We discuss a mixture model constructed with bell-shaped discrete component distributions that can be employed in the mass spectrometry framework for extracting the informative part of the mixed spectrum of organic objects. The results for assessing variability in the mass spectrum extracted are derived. The procedure is thoroughly illustrated on simulated data sets and applied to several real life problems that are concerned with complex organic dyes placed in a glycerol liquid matrix. The results obtained have clear chemical interpretation and suggest that the approach proposed is very promising.
Assessing the Heterogeneity of Treatment Effects via Potential Outcomes of Individual Patients
Authors: Zhang Zhiwei , Wang Chenguang , Nie Lei , Soon Guoxing
Published: September 2013

There is growing interest in understanding the heterogeneity of treatment effects (HTE), which has important implications in treatment evaluation and selection. The standard approach to assessing HTE (i.e. subgroup analyses based on known effect modifiers) is informative about the heterogeneity between subpopulations but not within. It is arguably more informative to assess HTE in terms of individual treatment effects, which can be defined by using potential outcomes. However, estimation of HTE based on potential outcomes is challenged by the lack of complete identifiability. The paper proposes methods to deal with the identifiability problem by using relevant information in baseline covariates and repeated measurements. If a set of covariates is sufficient for explaining the dependence between potential outcomes, the joint distribution of potential outcomes and hence all measures of HTE will then be identified under a conditional independence assumption. Possible violations of this assumption can be addressed by including a random effect to account for residual dependence or by specifying the conditional dependence structure directly. The methods proposed are shown to reduce effectively the uncertainty about HTE in a trial of human immunodeficiency virus.
The Use of Sample Weights in Multivariate Multilevel Models with an Application to Income Data Collected by Using a Rotating Panel Survey
Authors: Veiga Alinne , Smith Peter W. F. , Brown James J.
Published: September 2013

Longitudinal data from labour force surveys permit the investigation of income dynamics at the individual level. However, the data often originate from surveys with a complex multistage sampling scheme. In addition, the hierarchical structure of the data that is imposed by the different stages of the sampling scheme often represents the natural grouping in the population. Motivated by how income dynamics differ between the formal and informal sectors of the Brazilian economy and the data structure of the Brazilian Labour Force Survey, we extend the probability-weighted iterative generalized least squares estimation method. Our method is used to fit multivariate multilevel models to the Brazilian Labour Force Survey data where the covariance structure between occasions at the individual level is modelled. We conclude that there are significant income differentials and that incorporating the weights in the parameter estimation has some effect on the estimated coefficients and standard errors.
The Use of Accuracy Indicators to Correct for Survey Measurement Error
Authors: Da Silva Damião N. , Skinner Chris
Published: September 2013

An accuracy indicator is an observed variable which is related to the size of measurement error. Basic and extended models are introduced to represent the properties of a binary accuracy indicator. Under specified assumptions, it is shown that an accuracy indicator can identify a measurement error model. An approach to estimating a distribution function is presented together with methodology for variance estimation. The approach is applied to data on earnings from the British Household Panel Survey, where the accuracy indicator is whether or not a payslip is observed. A validation study provides justification for the modelling assumptions.
Handling Initial Conditions and Endogenous Covariates in Dynamic/Transition Models for Binary Data with Unobserved Heterogeneity
Authors: Skrondal Anders , Rabe-Hesketh Sophia
Published: September 2013

Distinguishing between longitudinal dependence due to the effects of previous responses on subsequent responses and dependence due to unobserved heterogeneity is important in many disciplines. For example, wheezing is an inflammatory reaction that may ‘remodel’ a child's airway structure and thereby affect the probability of future wheezing (state dependence). Alternatively, children could vary in their susceptibilities because of unobserved covariates such as genes (unobserved heterogeneity). For binary responses, distinguishing between state dependence and unobserved heterogeneity is typically accomplished by using dynamic/transition models that include both a lagged response and a random intercept. Naive maximum likelihood estimators can be severely inconsistent because of two kinds of endogeneity problem: lack of independence of the initial response and the random intercept (the initial conditions problem) and lack of independence of the covariates and the random intercept (the endogenous covariates problem). We clarify and unify previous work on handling these problems in the disconnected literatures of statistics and econometrics, suggest improved methods, investigate the asymptotic performance of competing methods and provide practical recommendations. The recommended methods are applied to longitudinal data on children's wheezing, where we investigate the extent of state dependence and unobserved heterogeneity and whether there is an effect of maternal smoking.
Flexible Regression Models Over River Networks
Authors: O'Donnell David , Rushworth Alastair , Bowman Adrian W. , Marian Scott E. , Hallard Mark
Published: September 2013

Many statistical models are available for spatial data but the vast majority of these assume that spatial separation can be measured by Euclidean distance. Data which are collected over river networks constitute a notable and commonly occurring exception, where distance must be measured along complex paths and, in addition, account must be taken of the relative flows of water into and out of confluences. Suitable models for this type of data have been constructed based on covariance functions. The aim of the paper is to place the focus on underlying spatial trends by adopting a regression formulation and using methods which allow smooth but flexible patterns. Specifically, kernel methods and penalized splines are investigated, with the latter proving more suitable from both computational and modelling perspectives. In addition to their use in a purely spatial setting, penalized splines also offer a convenient route to the construction of spatiotemporal models, where data are available over time as well as over space. Models which include main effects and spatiotemporal interactions, as well as seasonal terms and interactions, are constructed for data on nitrate pollution in the River Tweed. The results give valuable insight into the changes in water quality in both space and time.
Characterizing Spatial and Chronological Target Selection of Serial Offenders
Authors: Hering Amanda S. , Bair Sean
Published: September 2013

Using 31 unique crime series from two US cities, the spatial pattern that individual criminal decision makers follow is investigated. Locations within a city vary in the likelihood for crimes of a given type to occur, and this is accounted for by using kernel density estimation based on all crimes of each type. Kernel density estimation is highly influenced by the bandwidth, so an objective approach is used to select the estimate. Then, the kernel density estimate for each type of crime is incorporated in an inhomogeneous K-function that can identify significant spatial clustering and/or uniformity at various spatial scales for each crime series. We find that robbery series are more likely to exhibit uniformity than burglary series, which tend to show strong clustering. In addition, the order in which new crimes are added to a series is found to follow an interesting pattern that does not always support the theory of offender as forager in which criminals first cluster crimes and then gradually disperse them. Half of the robbery series exhibit a spatial distribution that begins dispersed and develops clusters as increasingly more events are added to the series, indicating that they often return to locations of earlier crimes after having committed crimes elsewhere.
Latent Variable Models that Account for Atypical Responses
Authors: Moustaki Irini , Knott Martin
Published: September 2013

Responses to a set of indicators, or items, or variables are often used in social sciences for measuring unobserved constructs as attitudes. Latent variable models, which are also known as factor analysis models, are used for linking the observed responses to the latent constructs. Often, some respondents provide random responses to the items. We distinguish between two response strategies: a primary response strategy that is driven by the latent variable of interest and a secondary response strategy that can be characterized as random. We propose an extended latent variable model for binary responses that models the secondary response mechanism through a latent class model implemented as an unobserved pseudoitem. We allow for the secondary response strategy that is employed by some respondents to be a function of the latent variable of interest and covariates. Not taking into account the proportion of responses generated by secondary strategies in the data can affect parameter estimates and the goodness of fit. Covariates are used to identify the demographic characteristics of those who choose a secondary response strategy and increase the precision of model estimation. We fit our proposed model to two data sets: one from a section of the 1990 Workplace Industrial Relations Survey and one from a section of the 2007 British Social Attitudes Survey.
At the mercy of others – for better or worse
Authors: Dybvik Tove Katrin , Gjengedal Eva , Lykkeslet Else
Published: September 2013

Many elderly people spend their remaining days in nursing homes and are thus affected by issues pertaining to a good life in a nursing home. The Norwegian study presented in this article aimed to gain greater knowledge and understanding of the significant factors associated with a good life in a nursing home. Qualitative in‐depth interviews with six nursing home residents aged 80 to 96 years were conducted to collect data. The aim of the analysis was to identify typical aspects related to a good life in a nursing home according to descriptions provided by nursing home residents. The findings identified both physical and psychosocial elements, and the following three main themes emerged: receiving help when the body fails is good, meaningful days despite loss and limitations, and time as a friend or foe. In conclusion, our findings indicate that it is important to understand that a good life has a deeper meaning because it challenges the existential preconditions for life.
Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope
Authors: Toyota Masatsugu , Ikeda Norifumi , Sawai‐Toyota Satoe , Kato Takehide , Gilroy Simon , Tasaka Masao , Morita Miyo Terao
Published: September 2013

The starch‐statolith hypothesis proposes that starch‐filled amyloplasts act as statoliths in plant gravisensing, moving in response to the gravity vector and signaling its direction. However, recent studies suggest that amyloplasts show continuous, complex movements in Arabidopsis shoots, contradicting the idea of a so‐called ‘static’ or ‘settled’ statolith. Here, we show that amyloplast movement underlies shoot gravisensing by using a custom‐designed centrifuge microscope in combination with analysis of gravitropic mutants. The centrifuge microscope revealed that sedimentary movements of amyloplasts under hypergravity conditions are linearly correlated with gravitropic curvature in wild‐type stems. We next analyzed the hypergravity response in the shoot gravitropism 2 (sgr2) mutant, which exhibits neither a shoot gravitropic response nor amyloplast sedimentation at 1 g. sgr2 mutants were able to sense and respond to gravity under 30 g conditions, during which the amyloplasts sedimented. These findings are consistent with amyloplast redistribution resulting from gravity‐driven movements triggering shoot gravisensing. To further support this idea, we examined two additional gravitropic mutants, phosphoglucomutase (pgm) and sgr9, which show abnormal amyloplast distribution and reduced gravitropism at 1 g. We found that the correlation between hypergravity‐induced amyloplast sedimentation and gravitropic curvature of these mutants was identical to that of wild‐type plants. These observations suggest that Arabidopsis shoots have a gravisensing mechanism that linearly converts the number of amyloplasts that settle to the ‘bottom’ of the cell into gravitropic signals. Further, the restoration of the gravitropic response by hypergravity in the gravitropic mutants that we tested indicates that these lines probably have a functional gravisensing mechanism that is not triggered at 1 g.
Mononuclear cells from a rare blood donor, after freezing under good manufacturing practice conditions, generate red blood cells that recapitulate the rare blood phenotype
Authors: Masiello Francesca , Tirelli Valentina , Sanchez Massimo , van den Akker Emile , Gabriella Girelli , Marconi Maurizio , Villa Maria Antonietta , Rebulla Paolo , Hashmi Ghazala , Whitsett Carolyn , Migliaccio Anna Rita
Published: September 2013

Background

Cultured red blood cells (cRBCs) from cord blood (CB) have been proposed as transfusion products. Whether buffy coats discarded from blood donations (adult blood [AB]) may be used to generate cRBCs for transfusion has not been investigated.

Study Design and Methods

Erythroid progenitor cell content and numbers and blood group antigen profiles of erythroblasts (ERYs) and cRBCs generated in human erythroid massive amplification (HEMA) culture by CB (n = 7) and AB (n = 33, three females, three males, one AB with rare blood antigens cryopreserved using CB protocols) were compared.

Results

Variability was observed both in progenitor cell content (twofold) and number of ERYs generated (1 log) by CB and AB in HEMA. The average progenitor cell contents of the subset of AB and CB analyzed were similar. AB generated numbers of ERYs three times lower (p < 0.01) than CB in HEMA containing fetal bovine serum but similar to CB in HEMA containing human proteins. Female AB contained two times fewer (p < 0.05) erythroid progenitor cells but generated numbers of ERYs similar to those generated by male AB. Cryopreserved AB with a rare blood group phenotype and shipped to another laboratory generated great numbers of ERYs, 90% of which matured into cRBCs. Blood group antigen expression was consistent with the donor genotype for ERYs generated both by CB and AB but concordant with that of native RBCs only for cells derived from AB.

Conclusion

Buffy coats from regular donors, including a donor with rare phenotypes stored under conditions established for CB, are not inferior to CB for the generation of cRBCs.
A Novel Prevailing Torque Threaded Fastener and Its Analysis
Authors: Ranjan B. S. C. , Vikranth H. N. , Ghosal Ashitava
Published: September 2013

We present a novel concept of a threaded fastener that is resistant to loosening under vibration. The anti-loosening feature does not use any additional element and is based on modifying the geometry of the thread in the bolt. In a normal nut and bolt combination, the axial motion of the nut or bolt is linearly related to the rotation by a constant pitch. In the proposed concept, the axial motion in the bolt is chosen to be a cubic function of the rotation, while for the nut, the axial motion remains linearly related to the rotation. This mismatch results in interference during the tightening process and additional torque required to overcome this interference gives rise to the enhanced anti-loosening property. In addition, the cubic curve is designed to ensure that the mismatch results in stresses and deformation in the elastic region of the chosen material. This ensures that the nut can be removed and reused while maintaining a repeatable anti-loosening property in the threaded fastener. A finite element analysis demonstrates the feasibility of this concept.
Comparison of Two Similar Mathematical Models for Tolerance Analysis: T-Map and Deviation Domain
Authors: Mansuy Mathieu , Giordano Max , Davidson Joseph K.
Published: September 2013

The major part of production cost of a manufacturing product is set during the design stage and especially by the tolerancing choice. Therefore, a lot of work involves trying to simulate the impact of these choices and provide an automatic optimization. For integrating this modeling in computer aided design (cad) software, the tolerancing must be modeled by a mathematical tool. Numerous models have been developed but few of them are really efficient. Two advanced models are “T-map” model developed by Joseph K. Davidson and “deviation domain” developed by Max Giordano. Despite the graphical representation of these two models seems to be similar, they have significant differences in their construction and their resolution method. These similarities and differences highlight the needs of tolerancing modeling tool in each kind of problems, especially in case of assembly with parallel links.
High current hybrid single walled carbon nanotube/graphene field emitters
Authors: Leberl Daniela , Hensel Bernhard , Kapitza Heinrich , Zeininger Heinrich , Tedde Sandro F.
Published: September 2013

Solution processed single walled carbon nanotube (CNT)/graphene hybrid samples are a low-cost alternative to chemical vapor deposition processed CNTs or graphene samples for field emission due to their fast and simple fabrication process. The electron field emission of this hybrid system was characterized regarding maximum current and long-term stability. The influence of measurement conditions such as chamber pressure, pulse-on time, and duty cycle on current–voltage characteristics was investigated. Furthermore, the anode size, anode geometry, and anode material were varied. The current density reached up to 2.9 A/cm2 when using a very small anode area of 0.8 mm2. A surface modification was observed after the emission with high current density. Very high maximum currents up to 400 mA in IV measurements and 90 mA in pulsed long-term stability measurements were achieved with thin hybrid layers of 1 μm.
Epigenetic Regulation of Mouse Sex Determination by the Histone Demethylase Jmjd1a
Authors: Kuroki Shunsuke , Matoba Shogo , Akiyoshi Mika , Matsumura Yasuko , Miyachi Hitoshi , Mise Nathan , Abe Kuniya , Ogura Atsuo , Wilhelm Dagmar , Koopman Peter , Nozaki Masami , Kanai Yoshiakira , Shinkai Yoichi , Tachibana Makoto
Published: September 2013

More Determined Sex

Although several transcription factors participate in mammalian sex determination, the contribution from specific epigenetic regulation is just being revealed.

Kuroki
et al.

(p.
1106
) show that a JmjC domain–containing protein, Jmjd1a, catalyzes H3K9 demethylation of the Y-linked sex-determining gene
Sry
in mice to enable its expression above the required threshold level. Ablation of Jmjd1a function results in mouse male-to-female sex reversal, hence not only revealing a mechanism of
Sry
regulation but also the pivotal role of epigenetic regulation in mammalian sex determination.
Direct Determination of Absolute Molecular Stereochemistry in Gas Phase by Coulomb Explosion Imaging
Authors: Pitzer Martin , Kunitski Maksim , Johnson Allan S. , Jahnke Till , Sann Hendrik , Sturm Felix , Schmidt Lothar Ph. H. , Schmidt-Böcking Horst , Dörner Reinhard , Stohner Jürgen , Kiedrowski Julia , Reggelin Michael , Marquardt Sebastian , Schießer Alexander , Berger Robert , Schöffler Markus S.
Published: September 2013

Absolute Images

Molecules are held together by a balance of charge between negative electrons and positive nuclei. When multiple electrons are expelled by laser irradiation, the remaining, mutually repulsive nuclei fly apart in a Coulomb explosion. Instead of traditional x-ray diffraction methods that require crystalline samples,

Pitzer
et al.

(p.
1096
) show that by tracking the fragment trajectories from laser-induced Coulomb explosions of relatively simple gas phase molecules, they can determine the absolute stereochemical configuration of enantiomers (mirror-image isomers).
Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways
Authors: Walker Mark C. , Thuronyi Benjamin W. , Charkoudian Louise K. , Lowry Brian , Khosla Chaitan , Chang Michelle C. Y.
Published: September 2013

Stitching in Fluoroacetate

Polyketide synthase enzymes stitch together an impressively diverse series of organic compounds from simple acetate and propionate building blocks.

Walker
et al.

(p.
1089
) now show that these biochemical pathways can be engineered to incorporate fluoroacetate—a primary product of the only known native enzymatic fluorination route—into tri- and tetraketides. In
Escherichia coli
cells, this process shows potential as a versatile means of inserting fluorine substituents into a range of complex molecules for use in pharmaceutical and agrochemical research.
Feedback on Galaxy Formation
Authors: McNamara B. R.
Published: September 2013

High-resolution radio images reveal how jets of relativistic particles can drive the dynamics of galaxy evolution.

[Also see Report by

Morganti
et al.

]
A Too-Soft Critique of India's Growth
An Uncertain Glory
India and Its Contradictions

by Jean Drèze and Amartya Sen

Princeton University Press, Princeton, NJ, 2013. 448 pp. $29.95. ISBN 9780691160795. Allen Lane (Penguin), London, 2013. £20. ISBN 9781846147616.
Authors: Robinson Andrew
Published: September 2013

Drèze and Sen consider the deprivations and inequalities that continue to afflict India and the hurdles to alleviating those.
Uncloaking the Quantum Nature of Inelastic Molecular Collisions
Authors: Casavecchia Piergiorgio , Alexander Millard H.
Published: September 2013

Experiment and theory combine to reveal quantum resonances when hydrogen molecules hit and excite oxygen molecules.

[Also see Report by

Chefdeville
et al.

]
Tony Pawson (1952–2013)
Authors: Hunter Tony
Published: September 2013

A “titan of signaling” who established principles of protein-protein interactions that influenced our understanding of cellular signal transduction.
U.N. Taps Special Labs to Investigate Syrian Attack
Authors: Enserink Martin
Published: September 2013

Special laboratories that investigate alleged chemical weapons attacks are getting a rare call to action from the United Nations after the horrifying events in Syria.
Fluorine-Adding Bacteria May Transform Natural Product Medicines
Authors: Service Robert F.
Published: September 2013

Synthetic biology now makes it possible to add fluorine atoms just where they are desired on large drug molecules, which could revolutionize medicines based on compounds harvested from living organisms.
Chimeric Derivatives of Hepatitis B Virus Core Particles Carrying Major Epitopes of the Rubella Virus E1 Glycoprotein
Authors: Skrastina Dace , Petrovskis Ivars , Petraityte Rasa , Sominskaya Irina , Ose Velta , Liekniņa Ilva , Bogans Janis , Sasnauskas Kestutis , Pumpens Paul
Published: September 2013

Three variants of the major rubella virus (RV) E1 protein virus-neutralizing epitope from position 214 to 285 were exposed on the hepatitis B virus (HBV) C-terminally truncated core (HBcΔ) in a virus-like particle (VLP) vector and were produced in
Escherichia coli
. All three chimeras demonstrated VLPs in bacterial cell lysates, but only HBcΔ-E1(245-285) demonstrated the correct VLP structure after purification. The other chimeras, HBcΔ-E1(214-285) and HBcΔ-E1(214-240), appeared after purification as non-VLP aggregates of 100 to 900 nm in diameter according to dynamic light scattering data. All three variants possessed the intrinsic antigenic activity of RV E1, since they were recognized by natural human anti-RV E1 antibodies and induced an anti-RV E1 response in mice. HBcΔ-E1(214-240) and HBcΔ-E1(245-285) can be regarded as prototypes for a putative RV vaccine because they were able to induce antibodies recognizing natural RV E1 protein in RV diagnostic kits.
Rapid and Sensitive Detection of H7N9 Avian Influenza Virus by Use of Reverse Transcription–Loop-Mediated Isothermal Amplification
Authors: Zhang Jinhai , Feng Youjun , Hu Dan , Lv Heng , Zhu Jing , Cao Min , Zheng Feng , Zhu Jin , Gong Xiufang , Hao Lina , Srinivas Swaminath , Ren Hao , Qi Zhongtian , Li Bingjun , Wang Changjun
Published: September 2013

An epidemic of human H7N9 influenza virus infection recently emerged in China whose clinical features include high mortality and which has also resulted in serious economic loss. The novel reassortant avian-origin influenza A (H7N9) virus which was the causative agent of this epidemic raised the possibility of triggering a large-scale influenza pandemic worldwide. It seemed likely that fast molecular detection assays specific for this virus would be in great demand. Here, we report a one-step reverse transcription–loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of the hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, the minimum detection limit of which was evaluated using
in vitro
RNA transcription templates. In total, 135 samples from clinical specimens (from either patients or poultry) were tested using this method in comparison with the real-time PCR recommended by the World Health Organization (WHO). Our results showed that (i) RT-LAMP-based trials can be completed in approximately 12 to 23 min and (ii) the detection limit for the H7 gene is around 10 copies per reaction, similar to that of the real-time PCR, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method. Indeed, this excellent performance of our method was also validated by the results for a series of clinical specimens. Therefore, we believe that the simple, fast, and sensitive method of RT-LAMP might be widely applied for detection of H7N9 infections and may play a role in prevention of an influenza pandemic.
Keeping Pace with Parvovirus B19 Genetic Variability: a Multiplex Genotype-Specific Quantitative PCR Assay
Authors: Bonvicini Francesca , Manaresi Elisabetta , Bua Gloria , Venturoli Simona , Gallinella Giorgio
Published: September 2013

Three genotypes have been identified within the parvovirus B19 species (B19V), and such genetic diversity may have significant implications for the development of molecular detection assays. In the present study, B19V genetic variability has been examined on a subset of genomic sequences available in the NCBI nucleotide database, and a quantitative PCR (qPCR) assay able to detect, differentiate, and quantify all viral variants has been established. The designed primers and probes have been used for the development of alternative detection formats, based on a combined use of intercalating dye and genotype-specific hydrolysis probes. The qPCR assay analytical performances have been determined on the 1st WHO International Reference Panel for Parvovirus B19 Genotypes. The developed qPCR protocols allow for the detection of genotypes 1 to 3 with equal accuracy, and with a limit of detection (LOD) of 200 IU/ml. A comparison of routine performance was carried out with respect to a previously established assay specifically validated on B19V genotype 1. For 130 clinical samples analyzed, 126 showed concordant results (31 positive and 97 negative), while 4 showed discordant results. Overall, the genotype-specific qPCR assay showed a sensitivity of 93.94% and a specificity of 97.94%, with an agreement rate of 96.92%. The proposed qPCR assay and the alternative protocols developed, each with robust performance, may allow choice with respect to operational systems and diagnostic requirements and might contribute to provide a more reliable diagnostic service and epidemiological surveillance of B19 virus.
Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity
Authors: Han Soo-Jin , Marshall Vickie , Barsov Eugene , Quiñones Octavio , Ray Alex , Labo Nazzarena , Trivett Matthew , Ott David , Renne Rolf , Whitby Denise
Published: September 2013

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645–659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii)
in vitro
Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore,
in vitro
maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies.
Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
Authors: De León Vázquez Erika , Carey Vincent J. , Kaye Kenneth M.
Published: September 2013

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N- and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N- and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.
Aluminum Hydroxide Influences Not Only the Extent but Also the Fine Specificity and Functional Activity of Antibody Responses to Tick-Borne Encephalitis Virus in Mice
Authors: Zlatkovic Juergen , Tsouchnikas Georgios , Jarmer Johanna , Koessl Christian , Stiasny Karin , Heinz Franz X.
Published: September 2013

Aluminum hydroxide is the most widely used adjuvant in human vaccines and serves as a potent enhancer of antibody production. Its stimulatory effect strongly depends on the adsorption of the antigen to the adjuvant, which may influence antigen presentation and, as a consequence, the fine specificity of antibody responses. Such variations can have functional consequences and can modulate the effectiveness of humoral immunity. Therefore, we investigated the influence of aluminum hydroxide on the fine specificity of antibody responses in a model study in mice using an inactivated purified virus particle, the flavivirus tick-borne encephalitis (TBE) virus, as an immunogen. To dissect and quantify the specificities of polyclonal antibodies in postimmunization sera, we established a platform of immunoassays using recombinant forms of the major target of neutralizing antibodies (protein E) as well as individual domains of E (DIII and the combination of DI and DII [DI+DII]). Our analyses revealed a higher proportion of neutralizing than virion binding (as detected by enzyme-linked immunosorbent assay) antibodies after immunization with aluminum hydroxide. Furthermore, the induction of antibodies to DIII, a known target of potently neutralizing antibodies, as well as their contributions to virus neutralization were significantly greater in mice immunized with adjuvant and correlated with a higher avidity of these antibodies. Thus, our data provide evidence that aluminum hydroxide can lead to functionally relevant modulations of antibody fine specificities in addition to its known overall immune enhancement effect.
Virus Factories of
Cauliflower Mosaic Virus
Are Virion Reservoirs That Engage Actively in Vector Transmission
Authors: Bak Aurélie , Gargani Daniel , Macia Jean-Luc , Malouvet Enrick , Vernerey Marie-Stéphanie , Blanc Stéphane , Drucker Martin
Published: September 2013

Cauliflower mosaic virus
(CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.
Outer Membrane Proteins Ail and OmpF of Yersinia pestis Are Involved in the Adsorption of T7-Related Bacteriophage Yep-phi
Authors: Zhao Xiangna , Cui Yujun , Yan Yanfeng , Du Zongmin , Tan Yafang , Yang Huiying , Bi Yujing , Zhang Pingping , Zhou Lei , Zhou Dongsheng , Han Yanping , Song Yajun , Wang Xiaoyi , Yang Ruifu
Published: September 2013

Yep-phi is a T7-related bacteriophage specific to
Yersinia pestis
, and it is routinely used in the identification of
Y. pestis
in China. Yep-phi infects
Y. pestis
grown at both 20°C and 37°C. It is inactive in other
Yersinia
species irrespective of the growth temperature. Based on phage adsorption, phage plaque formation, affinity chromatography, and Western blot assays, the outer membrane proteins of
Y. pestis
Ail and OmpF were identified to be involved, in addition to the rough lipopolysaccharide, in the adsorption of Yep-phi. The phage tail fiber protein specifically interacts with Ail and OmpF proteins, and residues 518N, 519N, and 523S of the phage tail fiber protein are essential for the interaction with OmpF, whereas residues 518N, 519N, 522C, and 523S are essential for the interaction with Ail. This is the first report to demonstrate that membrane-bound proteins are involved in the adsorption of a T7-related bacteriophage. The observations highlight the importance of the tail fiber protein in the evolution and function of various complex phage systems and provide insights into phage-bacterium interactions.
Phylogenetic Patterns of Human Coxsackievirus B5 Arise from Population Dynamics between Two Genogroups and Reveal Evolutionary Factors of Molecular Adaptation and Transmission
Authors: Henquell Cécile , Mirand Audrey , Richter Jan , Schuffenecker Isabelle , Böttiger Blenda , Diedrich Sabine , Terletskaia-Ladwig Elena , Christodoulou Christina , Peigue-Lafeuille Hélène , Bailly Jean-Luc
Published: September 2013

The aim of this study was to gain insights into the tempo and mode of the evolutionary processes that sustain genetic diversity in coxsackievirus B5 (CVB5) and into the interplay with virus transmission. We estimated phylodynamic patterns with a large sample of virus strains collected in Europe by Bayesian statistical methods, reconstructed the ancestral states of genealogical nodes, and tested for selection. The genealogies estimated with the structural one-dimensional gene encoding the VP1 protein and nonstructural 3CD locus allowed the precise description of lineages over time and cocirculating virus populations within the two CVB5 clades, genogroups A and B. Strong negative selection shaped the evolution of both loci, but compelling phylogenetic data suggested that immune selection pressure resulted in the emergence of the two genogroups with opposed evolutionary pathways. The genogroups also differed in the temporal occurrence of the amino acid changes. The virus strains of genogroup A were characterized by sequential acquisition of nonsynonymous changes in residues exposed at the virus 5-fold axis. The genogroup B viruses were marked by selection of three changes in a different domain (VP1 C terminus) during its early emergence. These external changes resulted in a selective sweep, which was followed by an evolutionary stasis that is still ongoing after 50 years. The inferred population history of CVB5 showed an alternation of the prevailing genogroup during meningitis epidemics across Europe and is interpreted to be a consequence of partial cross-immunity.
Change in Hepatitis B Virus Large Surface Antigen Variant Prevalence 13 Years after Implementation of a Universal Vaccination Program in China
Authors: Bian Tao , Yan Hongxia , Shen Liping , Wang Feng , Zhang Shuang , Cao Yanqiang , Zhang Shuo , Zhang Yong , Bi Shengli
Published: September 2013

A nationwide hepatitis B virus (HBV) vaccination program was implemented in China starting in 1992. To study the change in HBV variant prevalence with massive immunization, large HBV surface protein (LHBs) genes from HBV surface antigen (HBsAg)-positive sera were amplified and sequenced. The prevalences of LHBs mutants were compared between the 1992 and 2005 surveys in child and adult groups. The prevalence of “α” determinant mutants in the children increased from 6.5% in 1992 to 14.8% in 2005, where the G145R mutant occurred most frequently. In contrast, mutation frequencies showed little difference between 1992 (9.4%) and 2005 (9.9%) in adults. Moreover, compared to the 1992 survey, the child group surface (S) protein mutation frequency specifically increased (
P
= 0.005) in the 2005 survey, but the pre-S region mutation frequency did not show a significant difference (
P
> 0.05). However, the mutation frequency in the adult group increased in both the pre-S and S regions. Furthermore, the frequencies of the disease-related pre-S2 deletion and start codon mutations were significantly higher in the adult groups than in the child groups in both the 1992 and 2005 surveys (
P
< 0.01). Massive immunization enhances the HBV S protein mutation; the prevalence of LHBs mutants, particularly disease-related mutants, tends to increase with patient age.
Evolution of Broadly Cross-Reactive HIV-1-Neutralizing Activity: Therapy-Associated Decline, Positive Association with Detectable Viremia, and Partial Restoration of B-Cell Subpopulations
Authors: Ferreira Carolina B. , Merino-Mansilla Alberto , Llano Anuska , Pérez Ignacio , Crespo Isabel , Llinas Laia , Garcia Felipe , Gatell Jose M. , Yuste Eloisa , Sanchez-Merino Victor
Published: September 2013

Little is known about the stability of HIV-1 cross-neutralizing responses. Taking into account the fact that neutralization breadth has been positively associated with plasma viral load, there is no explanation for the presence of broadly neutralizing responses in a group of patients on treatment with undetectable viremia. In addition, the B-cell profile responsible for broadly cross-neutralizing responses is unknown. Here we studied the evolution of neutralizing responses and the B-cell subpopulation distribution in a group of patients with broadly cross-reactive HIV-1-neutralizing activity. We studied neutralization breadth evolution in a group of six previously identified broadly cross-neutralizing patients and six control patients during a 6-year period with a previously described minipanel of recombinant viruses from five different subtypes. B-cell subpopulation distribution during the study was also determined by multiparametric flow cytometry. Broadly cross-neutralizing activity was transient in four broad cross-neutralizers and stable, up to 4.6 years, in the other two. In four out of five broad cross-neutralizers who initiated treatment, a neutralization breadth loss occurred after viremia had been suppressed for as much as 20 months. B-cell subpopulation analyses revealed a significant increase in the frequency of naive B cells in broadly cross-reactive samples, compared with samples with less neutralization breadth (increased from 44% to 62%). We also observed a significant decrease in tissue-like and activated memory B cells (decreased from 19% to 12% and from 17% to 9%, respectively). Our data suggest that HIV-1 broadly cross-neutralizing activity is variable over time and associated with detectable viremia and partial B-cell restoration.