bluuebunny/crossref_metadata_2025 huggingface dataset:
A code for transcription initiation in mammalian genomes
Authors: Frith Martin C. , Valen Eivind , Krogh Anders , Hayashizaki Yoshihide , Carninci Piero , Sandelin Albin
Published: November 2007

Genome-wide detection of transcription start sites (TSSs) has revealed that RNA Polymerase II transcription initiates at millions of positions in mammalian genomes. Most core promoters do not have a single TSS, but an array of closely located TSSs with different rates of initiation. As a rule, genes have more than one such core promoter; however, defining the boundaries between core promoters is not trivial. These discoveries prompt a re-evaluation of our models for transcription initiation. We describe a new framework for understanding the organization of transcription initiation. We show that initiation events are clustered on the chromosomes at multiple scales—clusters within clusters—indicating multiple regulatory processes. Within the smallest of such clusters, which can be interpreted as core promoters, the local DNA sequence predicts the relative transcription start usage of each nucleotide with a remarkable 91% accuracy, implying the existence of a DNA code that determines TSS selection. Conversely, the total expression strength of such clusters is only partially determined by the local DNA sequence. Thus, the overall control of transcription can be understood as a combination of large- and small-scale effects; the selection of transcription start sites is largely governed by the local DNA sequence, whereas the transcriptional activity of a locus is regulated at a different level; it is affected by distal features or events such as enhancers and chromatin remodeling.
Features of 5′-splice-site efficiency derived from disease-causing mutations and comparative genomics
Authors: Roca Xavier , Olson Andrew J. , Rao Atmakuri R. , Enerly Espen , Kristensen Vessela N. , Børresen-Dale Anne-Lise , Andresen Brage S. , Krainer Adrian R. , Sachidanandam Ravi
Published: November 2007

Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5′-splice-site (5′ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies between 5′ss nucleotides as a conserved feature of the entire set of 5′ss. These dependencies are also conserved in human–mouse pairs of orthologous 5′ss. Many disease-associated 5′ss mutations disrupt these dependencies, as can some human SNPs that appear to alter splicing. The consistency of the evidence signifies the relevance of this approach and suggests that 5′ss SNPs play a role in complex diseases.
Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA
Authors: Korshunova Yulia , Maloney Rebecca K. , Lakey Nathan , Citek Robert W. , Bacher Blaire , Budiman Arief , Ordway Jared M. , McCombie W. Richard , Leon Jorge , Jeddeloh Jeffrey A. , McPherson John D.
Published: November 2007

Cytosine-methylation changes are stable and thought to be among the earliest events in tumorigenesis. Theoretically, DNA carrying tumor-specifying methylation patterns escape the tumors and may be found circulating in the sera from cancer patients, thus providing the basis for development of noninvasive clinical tests for early cancer detection. Indeed, using methylation-specific PCR-based techniques, several groups reported the detection of tumor-associated methylated DNA in the sera from cancer patients with varying clinical success. However, by design, such analytical approaches allow assessment of the presence of molecules with only one methylation pattern, leaving the bigger picture unexplored. The limited knowledge about circulating DNA methylation patterns hinders the efficient development of clinical methylation tests and testing platforms. Here, we report the results of a comprehensive methylation pattern analysis from breast cancer clinical tissues and sera obtained using massively parallel bisulphite pyrosequencing. The four loci studied were recently discovered by our group, and demonstrated to be powerful epigenetic biomarkers of breast cancer. The detailed analysis of more than 700,000 DNA fragments derived from more than 50 individuals (cancer and cancer-free) revealed an unappreciated complexity of genomic cytosine-methylation patterns in both tissue derived and circulating DNAs. Both tumor and cancer-free tissues (as well as sera) contained molecules with nearly every conceivable cytosine-methylation pattern at each locus. Tumor samples displayed more variation in methylation level than normal samples. Importantly, by establishing the methylation landscape within circulating DNA, this study has better defined the development challenges facing DNA methylation-based cancer-detection tests.
The genome-wide determinants of human and chimpanzee microsatellite evolution
Authors: Kelkar Yogeshwar D. , Tyekucheva Svitlana , Chiaromonte Francesca , Makova Kateryna D.
Published: November 2007

Mutation rates of microsatellites vary greatly among loci. The causes of this heterogeneity remain largely enigmatic yet are crucial for understanding numerous human neurological diseases and genetic instability in cancer. In this first genome-wide study, the relative contributions of intrinsic features and regional genomic factors to the variation in mutability among orthologous human–chimpanzee microsatellites are investigated with resampling and regression techniques. As a result, we uncover the intricacies of microsatellite mutagenesis as follows. First, intrinsic features (repeat number, length, and motif size), which all influence the probability and rate of slippage, are the strongest predictors of mutability. Second, mutability increases nonuniformly with length, suggesting that processes additional to slippage, such as faulty repair, contribute to mutations. Third, mutability varies among microsatellites with different motif composition likely due to dissimilarities in secondary DNA structure formed by their slippage intermediates. Fourth, mutability of mononucleotide microsatellites is impacted by their location on sex chromosomes vs. autosomes and inside vs. outside of Alu repeats, the former confirming the importance of replication and the latter suggesting a role for gene conversion. Fifth, transcription status and location in a particular isochore do not influence microsatellite mutability. Sixth, compared with intrinsic features, regional genomic factors have only minor effects. Finally, our regression models explain ∼90% of variation in microsatellite mutability and can generate useful predictions for the studies of human diseases, forensics, and conservation genetics.
Structural and Functional Analysis of SGT1 Reveals That Its Interaction with HSP90 Is Required for the Accumulation of Rx, an R Protein Involved in Plant Immunity
Authors: Botër Marta , Amigues Béatrice , Peart Jack , Breuer Christian , Kadota Yasuhiro , Casais Catarina , Moore Geoffrey , Kleanthous Colin , Ochsenbein Francoise , Shirasu Ken , Guerois Raphaël
Published: November 2007

SGT1 (for suppressor of G2 allele of skp1) and RAR1 (for required for Mla12 resistance) are highly conserved eukaryotic proteins that interact with the molecular chaperone HSP90 (for heat shock protein90). In plants, SGT1, RAR1, and HSP90 are essential for disease resistance triggered by a number of resistance (R) proteins. Here, we present structural and functional characterization of plant SGT1 proteins. Random mutagenesis of Arabidopsis thaliana SGT1b revealed that its CS (for CHORD-SGT1) and SGS (for SGT1 specific) domains are essential for disease resistance. NMR-based interaction surface mapping and mutational analyses of the CS domain showed that the CHORD II domain of RAR1 and the N-terminal domain of HSP90 interact with opposite sides of the CS domain. Functional analysis of the CS mutations indicated that the interaction between SGT1 and HSP90 is required for the accumulation of Rx, a potato (Solanum tuberosum) R protein. Biochemical reconstitution experiments suggest that RAR1 may function to enhance the SGT1–HSP90 interaction by promoting ternary complex formation.
Arabidopsis MALE STERILITY1 Encodes a PHD-Type Transcription Factor and Regulates Pollen and Tapetum Development
Authors: Ito Takuya , Nagata Noriko , Yoshiba Yoshu , Ohme-Takagi Masaru , Ma Hong , Shinozaki Kazuo
Published: November 2007

The Arabidopsis thaliana MALE  STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper–like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper–like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants.
MALE STERILITY1 Is Required for Tapetal Development and Pollen Wall Biosynthesis
Authors: Yang Caiyun , Vizcay-Barrena Gema , Conner Katie , Wilson Zoe A.
Published: November 2007

The Arabidopsis  thaliana MALE STERILITY1 (MS1) gene is critical for viable pollen formation and has homology to the PHD-finger class of transcription factors; however, its role in pollen development has not been fully defined. We show that MS1 transcription appears to be autoregulated by the wild-type MS1 transcript or protein. Using a functional green fluorescent protein (GFP) fusion to analyze the temporal and spatial expression of MS1, we demonstrate that the MS1:GFP protein is nuclear localized within the tapetum and is expressed in a developmentally regulated manner between late tetraspore and microspore release, then rapidly breaks down, probably by ubiquitin-dependent proteolysis. Absence of MS1 expression results in changes in tapetal secretion and exine structure. Microarray analysis has shown that 260 (228 downregulated and 32 upreglated) genes have altered expression in young ms1 buds. These genes are primarily associated with pollen wall and coat formation; however, a number of transcription factors and Cys proteases have also been identified as the putative primary regulatory targets of MS1. Ectopic expression of MS1 alters transcriptional regulation of vegetative gene expression, resulting in stunted plants with increased levels of branching, partially fertile flowers and an apparent increase in wall material on mature pollen. MS1 therefore plays a critical role in the induction of pollen wall and pollen coat materials in the tapetum and, ultimately, the production of viable pollen.
Nurses perceptions of older patients integrity in long‐term institutions
Authors: Teeri Sari , Välimäki Maritta , Katajisto Jouko , Leino‐Kilpi Helena
Published: November 2007

Aim:  This article reports on a study into nurses’ perceptions of how the integrity of older patients is maintained in long‐term care institutions in Finland.

Background:  Patient integrity is an important aspect of ethical decision‐making in nursing care and respect for integrity is central to good care. Problems may occur in the maintenance of older patients’ integrity because of reduced communication skills and decision‐making authority. Data collection was by means of a purpose‐designed structured questionnaire in a sample of 222 nurses from four purposively selected long‐term institutions in 2004. The response rate was 74%. The questionnaire consisted of a background data sheet and integrity items in three categories: psychological, physical and social integrity.

Results:  The nurses gave the highest ratings for the maintenance of physical integrity, particularly for respectful and gentle touching. Nonetheless, according to nurses patients were often tied to their bed or chair. The second highest ratings were give to the maintenance of social integrity. Most nurses felt that the patients had good contact with the outside world, mainly family members. On the other hand loneliness was a more common problem for older people in institutions. The maintenance of psychological integrity received the lowest rating. Satisfaction with the job and with the quality of service provided correlated positively with the nurses’ views on the maintenance of patient integrity.

Conclusion:  Nurses take the view that patient integrity is maintained reasonably well in long‐term institutions. There are, however, some problematic areas that require special attention, particularly in the maintenance of psychological integrity. Future research needs to look at how patients and their relatives view the situation and to explore different training delivery options that can help raise the ethical quality of nursing care.
Spatial autocorrelation in plant communities: vegetation texture versus species composition
Authors: Lawrence Lodge Rachael H. E. , Anderson Barbara J. , de Groot Adrian , Bill Amber , McQueen Amelia A. M. , Steel John B. , Mistral Maia , Mason Norman W. H. , Bastow Wilson J.
Published: November 2007

Vegetation texture, describing plant communities by the range of characters present in them, can describe functional variation more effectively than using the identities of the species. We ask whether spatial trends, as seen in spatial autocorrelation (SAc), are stronger in vegetation texture. We also ask in what environment SAc is stronger, whether local or over longer distances, and with what measure of distance.

Roadside vegetation was sampled across an orographic region of the South Island, New Zealand, from a high‐rainfall zone up to just above treeline (the “Wet” side) and then down into a rain‐shadow area (the “Dry” side). Species composition was recorded in quadrats placed in 30 clusters. Five functional plant characters were measured on all species encountered: characters that have been reported in the literature to correlate with light‐capture, heat budget and nutrient strategy. SAc was calculated using a saturation‐response type of relation in species composition, and also in texture using the five functional characters.

There was significant species‐composition SAc in almost all comparisons, but the maximum percentage of variation explained was 25%. The fit was almost always higher using local, short‐distance, comparisons than using comparisons over a whole side (Wet or Dry). On average dissimilarity had reached 90% of its asymptote after ca 250 m. It is concluded that the SAc was mainly due to local factors. SAc relations almost always predicted the presence/absence of species more accurately than their abundance. Distances along the road generally gave a very slightly better fit to species composition than surface distances, suggesting that there might be dispersal limitation. On the Wet side, elevation in overall comparisons gave a still better fit, implying environmental control too. The strength of SAc in texture was strongest on the Dry side, and using overall comparisons, abundance weighting and surface distances, but in other cases texture SAc was weaker than SAc in species composition.

It is concluded that the saturation‐response formula used here has theoretical advantages over previous approaches. The SAc in this vegetation seems to be caused by a combination of dispersal limitation, broad environmental trends and especially local environmental effects such as disturbance. SAc in texture may have been weaker overall on the Wet side than on the Dry because the main environmental difference across the former is in frost, and characters correlated with frost resistance have not yet been included in texture analyses.
Comment on "Roadless Space of the Conterminous United States"
Authors: Girvetz Evan H. , Jaeger Jochen A. G. , Thorne James H.
Published: November 2007

Watts
et al
. (Reports, 6 May 2007, p. 736) introduced a metric of landscape pattern called roadless volume (RV). However, as with most previous metrics, RV does not explicitly address ecological processes. We demonstrate that RV can produce results inconsistent with the notion of landscape connectivity and contend that more ecologically relevant metrics are available.
Social Comparison Affects Reward-Related Brain Activity in the Human Ventral Striatum
Authors: Fliessbach K. , Weber B. , Trautner P. , Dohmen T. , Sunde U. , Elger C. E. , Falk A.
Published: November 2007

Whether social comparison affects individual well-being is of central importance for understanding behavior in any social environment. Traditional economic theories focus on the role of absolute rewards, whereas behavioral evidence suggests that social comparisons influence well-being and decisions. We investigated the impact of social comparisons on reward-related brain activity using functional magnetic resonance imaging (fMRI). While being scanned in two adjacent MRI scanners, pairs of subjects had to simultaneously perform a simple estimation task that entailed monetary rewards for correct answers. We show that a variation in the comparison subject's payment affects blood oxygenation level–dependent responses in the ventral striatum. Our results provide neurophysiological evidence for the importance of social comparison on reward processing in the human brain.
Transposase-Derived Transcription Factors Regulate Light Signaling in
Arabidopsis
Authors: Lin Rongcheng , Ding Lei , Casola Claudio , Ripoll Daniel R. , Feschotte Cédric , Wang Haiyang
Published: November 2007

Plants use light to optimize growth and development. The photoreceptor phytochrome A (phyA) mediates various far-red light–induced responses. We show that
Arabidopsis FHY3
and
FAR1
, which encode two proteins related to
Mutator
-like transposases, act together to modulate phyA signaling by directly activating the transcription of
FHY1
and
FHL
, whose products are essential for light-induced phyA nuclear accumulation and subsequent light responses. FHY3 and FAR1 have separable DNA binding and transcriptional activation domains that are highly conserved in
Mutator
-like transposases. Further, expression of
FHY3
and
FAR1
is negatively regulated by phyA signaling. We propose that FHY3 and FAR1 represent transcription factors that have been co-opted from an ancient
Mutator
-like transposase(s) to modulate phyA-signaling homeostasis in higher plants.
Requirement of Inositol Pyrophosphates for Full Exocytotic Capacity in Pancreatic β Cells
Authors: Illies Christopher , Gromada Jesper , Fiume Roberta , Leibiger Barbara , Yu Jia , Juhl Kirstine , Yang Shao-Nian , Barma Deb K. , Falck John R. , Saiardi Adolfo , Barker Christopher J. , Berggren Per-Olof
Published: November 2007

Inositol pyrophosphates are recognized components of cellular processes that regulate vesicle trafficking, telomere length, and apoptosis. We observed that pancreatic β cells maintain high basal concentrations of the pyrophosphate diphosphoinositol pentakisphosphate (Ins
P
7
or IP
7
). Inositol hexakisphosphate kinases (IP6Ks) that can generate IP
7
were overexpressed. This overexpression stimulated exocytosis of insulin-containing granules from the readily releasable pool. Exogenously applied IP
7
dose-dependently enhanced exocytosis at physiological concentrations. We determined that IP6K1 and IP6K2 were present in β cells. RNA silencing of IP6K1, but not IP6K2, inhibited exocytosis, which suggests that IP6K1 is the critical endogenous kinase. Maintenance of high concentrations of IP
7
in the pancreatic β cell may enhance the immediate exocytotic capacity and consequently allow rapid adjustment of insulin secretion in response to increased demand.
Superconducting Pair Correlations in an Amorphous Insulating Nanohoneycomb Film
Authors: Stewart M. D. , Yin Aijun , Xu J. M. , Valles James M.
Published: November 2007

The Cooper pairing mechanism that binds single electrons to form pairs in metals allows electrons to circumvent the exclusion principle and condense into a single superconducting or zero-resistance state. We present results from an amorphous bismuth film system patterned with a nanohoneycomb array of holes, which undergoes a thickness-tuned insulator-superconductor transition. The insulating films exhibit activated resistances and magnetoresistance oscillations dictated by the superconducting flux quantum
h
/2
e
. This 2
e
period is direct evidence indicating that Cooper pairing is also responsible for electrically insulating behavior.
Emission of Coherent THz Radiation from Superconductors
Authors: Ozyuzer L. , Koshelev A. E. , Kurter C. , Gopalsami N. , Li Q. , Tachiki M. , Kadowaki K. , Yamamoto T. , Minami H. , Yamaguchi H. , Tachiki T. , Gray K. E. , Kwok W.-K. , Welp U.
Published: November 2007

Compact solid-state sources of terahertz (THz) radiation are being sought for sensing, imaging, and spectroscopy applications across the physical and biological sciences. We demonstrate that coherent continuous-wave THz radiation of sizable power can be extracted from intrinsic Josephson junctions in the layered high-temperature superconductor Bi
2
Sr
2
CaCu
2
O
8
. In analogy to a laser cavity, the excitation of an electromagnetic cavity resonance inside the sample generates a macroscopic coherent state in which a large number of junctions are synchronized to oscillate in phase. The emission power is found to increase as the square of the number of junctions reaching values of 0.5 microwatt at frequencies up to 0.85 THz, and persists up to ∼50 kelvin. These results should stimulate the development of superconducting compact sources of THz radiation.
Neutralizing Activity of Vaccine-Induced Antibodies to TwoBacillus anthracisToxin Components, Lethal Factor and Edema Factor
Authors: Taft Sarah C. , Weiss Alison A.
Published: November 2007

Anthrax vaccine adsorbed (AVA; BioThrax), the current FDA-licensed human anthrax vaccine, contains various amounts of the three anthrax toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF). While antibody to PA is sufficient to mediate protection against anthrax in animal models, it is not known if antibodies to LF or EF contribute to protection in humans. Toxin-neutralizing activity was evaluated in sera from AVA-vaccinated volunteers, all of whom had antibody responses to LF and EF, as well as PA. The contribution of antibodies to LF and EF was assessed using mouse macrophage J774A.1 cells by examining neutralization of LF-induced lysis using alamarBlue reduction and neutralization of EF-induced cyclic AMP increases by enzyme-linked immunosorbent assay. Antibody responses to LF and EF were low compared to those to PA, and the amount of LF or EF in the assay could exceed the amount of antibodies to LF or EF. Higher titers were seen for most individuals when the LF or EF concentration was limiting compared to when LF or EF was in excess, initially suggesting that antibody to LF or EF augmented protection. However, depletion of LF and EF antibodies in sera did not result in a significant decrease in toxin neutralization. Overall, this study suggests that AVA-induced LF and EF antibodies do not significantly contribute to anthrax toxin neutralization in humans and that antibodies to PA are sufficient to neutralize toxin activity.
Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus
Authors: Konishi Eiji , Kitai Yoko , Kondo Takashi
Published: November 2007

Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.
Development of Real-Time Multiplex Nucleic Acid Sequence-Based Amplification for Detection of
Mycoplasma pneumoniae, Chlamydophila pneumoniae
, and
Legionella
spp. in Respiratory Specimens
Authors: Loens K. , Beck T. , Ursi D. , Overdijk M. , Sillekens P. , Goossens H. , Ieven M.
Published: November 2007

Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect
Mycoplasma pneumoniae, Chlamydophila pneumoniae
, and
Legionella
spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the
M. pneumoniae, C. pneumoniae
, and
Legionella pneumophila
16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of
M. pneumoniae
-,
C. pneumoniae
-, and
L. pneumophila
-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of
M. pneumoniae, C. pneumoniae
, and
Legionella
spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.
Chlamydia trachomatis
Serovars among Strains Isolated from Members of Rural Indigenous Communities and Urban Populations in Australia
Authors: Bandea Claudiu I. , Debattista Joseph , Joseph Kahaliah , Igietseme Joseph , Timms Peter , Black Carolyn M.
Published: November 2007

We genotyped
Chlamydia trachomatis
strains from 45 women or men living in either a rural indigenous community or in urban heterosexual communities. We found six different
C. trachomatis
serovars: E (
n
= 22; 48.9%), F (
n
= 10; 22.2%), J/Ja (
n
= 5; 11.1%), D/Da (
n
= 4; 8.9%), G (
n
= 3; 6.7%), and K (
n
= 1; 2.2%). The distribution of
C. trachomatis
serovars among members of the indigenous rural and the urban Australian communities appears similar to that in other Western countries.
Novel Varicella-Zoster Virus Glycoprotein E Gene Mutations Associated with Genotypes A and D
Authors: Schmidt-Chanasit Jonas , Bleymehl Karoline , Schäd Susanne G. , Gross Gerd , Ulrich Rainer G. , Doerr Hans Wilhelm
Published: November 2007

Here, we describe the association of certain varicella-zoster virus (VZV) genotypes with unique glycoprotein E (gE) gene mutations. Within 45 analyzed VZV wild-type strains of genotypes A and D, five novel gE mutations were discovered. A statistically significant (
P
< 0.0001) association of certain gE mutations with VZV genotype D was found.
Rapid Preclinical Detection of Sheeppox Virus by a Real-Time PCR Assay
Authors: Balinsky C. A. , Delhon G. , Smoliga G. , Prarat M. , French R. A. , Geary S. J. , Rock D. L. , Rodriguez L. L.
Published: November 2007

Sheeppox virus
(SPPV) is a member of the
Capripoxvirus
(CaPV) genus of the
Poxviridae
family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples.
Optochin Resistance among
Streptococcus pneumoniae
Strains Colonizing Healthy Children in Portugal
Authors: Nunes Sónia , Sá-Leão Raquel , de Lencastre Hermínia
Published: November 2007

Two percent of 1,973 pneumococcus strains isolated from carriers since 2001 in Portugal were found to be optochin resistant. These strains belonged to eight serotypes (and some were nontypeable), and they had diverse genetic backgrounds. Novel optochin-resistant lineages were detected over time, suggesting that there was a continuous, although sporadic, emergence of optochin resistance.
Hsp40 Facilitates Nuclear Import of the Human Immunodeficiency Virus Type 2 Vpx-Mediated Preintegration Complex
Authors: Cheng Xiaogang , Belshan Michael , Ratner Lee
Published: November 2007

Human immunodeficiency virus type 2 (HIV-2) Vpx is required for nuclear translocation of the viral preintegration complex (PIC) in quiescent cells. In order to decipher the mechanism of action of Vpx, a cDNA library was screened with the yeast two-hybrid assay, resulting in the identification of heat shock protein 40, Hsp40/DnaJB6, as a Vpx-interactive protein. Interaction with Vpx was confirmed by glutathione
S
-transferase (GST) pull-down and coimmunoprecipitation assays. Overexpression of Hsp40/DnaJB6 enhanced Vpx nuclear import, whereas overexpression of a nuclear localization mutant of Hsp40/DnaJB6 (H31Q) or down-regulation of Hsp40/DnaJB6 by small interfering RNA (siRNA) reduced the nuclear import of Vpx. Hsp40/DnaJB6 competed with the Pr55
Gag
precursor protein for the binding of Vpx and incorporation into virus-like particles. Overexpression of Hsp40/DnaJB6 promoted viral PIC nuclear import, whereas siRNA down-regulation of Hsp40/DnaJB6 inhibited PIC nuclear import. These results demonstrate a role for Hsp40/DnaJB6 in the regulation of HIV-2 PIC nuclear transport.
Intracellular Targeting of a Hordeiviral Membrane-Spanning Movement Protein: Sequence Requirements and Involvement of an Unconventional Mechanism
Authors: Schepetilnikov Mikhail V. , Solovyev Andrey G. , Gorshkova Elena N. , Schiemann Joachim , Prokhnevsky Alexey I. , Dolja Valerian V. , Morozov Sergey Y.
Published: November 2007

The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of
Poa semilatent virus
. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of
Nicotiana benthamiana
leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.
Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles
Authors: Graff Judith , Zhou Yi-Hua , Torian Udana , Nguyen Hanh , St. Claire Marisa , Yu Claro , Purcell Robert H. , Emerson Suzanne U.
Published: November 2007

Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.
Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1
Authors: Upla Paula , Marjomäki Varpu , Nissinen Liisa , Nylund Camilla , Waris Matti , Hyypiä Timo , Heino Jyrki
Published: November 2007

Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.
Human Papillomavirus E1 Helicase Interacts with the WD Repeat Protein p80 To Promote Maintenance of the Viral Genome in Keratinocytes
Authors: Côté-Martin Alexandra , Moody Cary , Fradet-Turcotte Amélie , D'Abramo Claudia M. , Lehoux Michaël , Joubert Simon , Poirier Guy G. , Coulombe Benoit , Laimins Laimonis A. , Archambault Jacques
Published: November 2007

Due to the limited coding capacity of their small genomes, human papillomaviruses (HPV) rely extensively on host factors for the completion of their life cycles. Accordingly, most HPV proteins, including the replicative helicase E1, engage in multiple protein interactions. The fact that conserved regions of E1 have not yet been ascribed a function prompted us to use tandem affinity protein purification (TAP) coupled to mass spectrometry to identify novel targets of this helicase. This method led to the discovery of a novel interaction between the N-terminal 40 amino acids of HPV type 11 (HPV11) E1 and the cellular WD repeat protein p80 (WDR48). We found that interaction with p80 is conserved among E1 proteins from anogenital HPV but not among cutaneous or animal types. Colocalization studies showed that E1 can redistribute p80 from the cytoplasm to the nucleus in a manner that is dependent on the E1 nuclear localization signal. Three amino acid substitutions in E1 proteins from HPV11 and -31 were identified that abrogate binding to p80 and its relocalization to the nucleus. In HPV31 E1, these substitutions reduced but did not completely abolish transient viral DNA replication. HPV31 genomes encoding two of the mutant E1 proteins were not maintained as episomes in immortalized primary keratinocytes, whereas one encoding the third mutant protein was maintained at a very low copy number. These findings suggest that the interaction of E1 with p80 is required for efficient maintenance of the viral episome in undifferentiated keratinocytes.
Phase I Trial of a CD8+T-Cell Peptide Epitope-Based Vaccine for Infectious Mononucleosis
Authors: Elliott Suzanne L. , Suhrbier Andreas , Miles John J. , Lawrence Greg , Pye Stephanie J. , Le Thuy T. , Rosenstengel Andrew , Nguyen Tam , Allworth Anthony , Burrows Scott R. , Cox John , Pye David , Moss Denis J. , Bharadwaj Mandvi
Published: November 2007

A single blind, randomized, placebo-controlled, single-center phase I clinical trial of a CD8+T-cell peptide epitope vaccine against infectious mononucleosis was conducted with 14 HLA B*0801-positive, Epstein-Barr virus (EBV)-seronegative adults. The vaccine comprised the HLA B*0801-restricted peptide epitope FLRGRAYGL and tetanus toxoid formulated in a water-in-oil adjuvant, Montanide ISA 720. FLRGRAYGL-specific responses were detected in 8/9 peptide-vaccine recipients and 0/4 placebo vaccine recipients by gamma interferon enzyme-linked immunospot assay and/or limiting-dilution analysis. The same T-cell receptor Vβ CDR3 sequence that is found in FLRGRAYGL-specific T cells from most EBV-seropositive individuals could also be detected in the peripheral blood of vaccine recipients. The vaccine was well tolerated, with the main side effect being mild to moderate injection site reactions. After a 2- to 12-year follow-up, 1/2 placebo vaccinees who acquired EBV developed infectious mononucleosis, whereas 4/4 vaccinees who acquired EBV after completing peptide vaccination seroconverted asymptomatically. Single-epitope vaccination did not predispose individuals to disease, nor did it significantly influence development of a normal repertoire of EBV-specific CD8+T-cell responses following seroconversion.
Induction of Paclitaxel Resistance by the Kaposi's Sarcoma-Associated Herpesvirus Latent Protein LANA2
Authors: Muñoz-Fontela C. , Marcos-Villar L. , Hernandez F. , Gallego P. , Rodriguez E. , Arroyo J. , Gao S.-J. , Avila J. , Rivas C.
Published: November 2007

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent of both KS and primary effusion lymphoma (PEL). Although treatment with paclitaxel has significant antitumor activity in KS, drug resistance represents a major obstacle for improving the overall response and survival of PEL patients. The transcriptional pattern of KSHV is cell/tissue specific, as revealed by the fact that the viral latent protein LANA2 is detected exclusively in B cells. This paper focuses on the mechanism of paclitaxel resistance observed in PEL cells. Here we show that LANA2 protein modulates microtubule dynamics through its direct binding to polymerized microtubules, preventing microtubule stabilization induced by paclitaxel. This is the first demonstration of paclitaxel resistance induced by a viral protein and suggests a link between the expression of LANA2 and the resistance of PEL cells to paclitaxel.
Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II
Authors: Chang Jinhong , Nie Xingcao , Chang Ho Eun , Han Ziying , Taylor John
Published: November 2007

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.
Bovine Viral Diarrhea Virus Core Is an Intrinsically Disordered Protein That Binds RNA
Authors: Murray Catherine L. , Marcotrigiano Joseph , Rice Charles M.
Published: November 2007

Pestiviruses, including bovine viral diarrhea virus (BVDV), are important animal pathogens and close relatives of hepatitis C virus. Pestivirus particles are composed of an RNA genome, a host-derived lipid envelope, and four virion-encoded structural proteins, core (C), E
rns
, E1, and E2. Core is a small, highly basic polypeptide that is processed by three enzymatic cleavages before its incorporation into virions. Little is known about its biological properties or its role in virion assembly and structure. We have purified BVDV core protein and characterized it biochemically. We have determined that the processed form of core lacks significant secondary structure and is instead intrinsically disordered. Consistent with its highly basic sequence, we observed that core binds to RNA, although with low affinity and little discernible specificity. We found that BVDV core protein was able to functionally replace the nonspecific RNA binding and condensing region of an unrelated viral capsid protein. Together these results suggest that the in vitro properties of core may reflect its mechanism of action in RNA packaging and virion morphogenesis.
Immune Serum Produced by DNA Vaccination Protects Hamsters against Lethal Respiratory Challenge with Andes Virus
Authors: Hooper Jay W. , Ferro Anthony M. , Wahl-Jensen Victoria
Published: November 2007

Hantavirus pulmonary syndrome (HPS) is a highly pathogenic disease (40% case fatality rate) carried by rodents chronically infected with certain viruses within the genus
Hantavirus
of the family
Bunyaviridae
. The primary mode of transmission to humans is thought to be inhalation of excreta from infected rodents; however, ingestion of contaminated material and rodent bites are also possible modes of transmission. Person-to-person transmission of HPS caused by one species of hantavirus, Andes virus (ANDV), has been reported. Previously, we reported that ANDV injected intramuscularly causes a disease in Syrian hamsters that closely resembles HPS in humans. Here we tested whether ANDV was lethal in hamsters when it was administered by routes that more accurately model the most common routes of human infection, i.e., the subcutaneous, intranasal, and intragastric routes. We discovered that ANDV was lethal by all three routes. Remarkably, even at very low doses, ANDV was highly pathogenic when it was introduced by the mucosal routes (50% lethal dose [LD
50
], ∼100 PFU). We performed passive transfer experiments to test the capacity of neutralizing antibodies to protect against lethal intranasal challenge. The neutralizing antibodies used in these experiments were produced in rabbits vaccinated by electroporation with a previously described ANDV M gene-based DNA vaccine, pWRG/AND-M. Hamsters that were administered immune serum on days −1 and +5 relative to challenge were protected against intranasal challenge (21 LD
50
). These findings demonstrate the utility of using the ANDV hamster model to study transmission across mucosal barriers and provide evidence that neutralizing antibodies produced by DNA vaccine technology can be used to protect against challenge by the respiratory route.
Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations
Authors: Segura María Mercedes , Garnier Alain , Di Falco Marcos Rafael , Whissell Gavin , Meneses-Acosta Angélica , Arcand Normand , Kamen Amine
Published: November 2007

The Moloney murine leukemia virus (MMLV) belongs to the
Retroviridae
family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin β1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed.