bluuebunny/crossref_metadata_2025 huggingface dataset:
The Virtuous Discourses of Private Communities
Authors: Kenna Therese E. , Dunn Kevin M.
Published: February 2009

This article reviews research on the contemporary marketing of private communities. Ultimately, the success of this marketing relies upon some existing or insipient consumer preferences. The urban studies literature offers at least five related sets of explanations for the emergence of desires for privatised residential communities, which we outline in this article. Yet, the research to date on the selling of private residential communities has tended to investigate specific features of private communities (e.g. demand, supply, representation and discourse) and has eschewed a more holistic encapsulation of the phenomenon. We use this article to propose a holistic approach that contemplates the ‘virtuous cycles’ that favour private community growth.
Assessment of fibrosis in chronic liver diseases
Authors: ZHOU Kun , LU Lun Gen
Published: February 2009

The assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decisions. Although liver biopsy is the current gold standard for fibrosis assessment, it has some risks and limitations, including intra‐observer and inter‐observer variation, sampling error and variability. In recent years, many studies and great interest have been dedicated to the development of non‐invasive tests to substitute a liver biopsy for fibrosis assessment and follow up. Advances in serological and radiological tests such as serum marker panels, transient elastography and their combinations can assess fibrosis accurately and reduce the need for a liver biopsy. But at present, all have failed to completely replace a liver biopsy because of their respective limitations and an imperfect gold standard used in current researches. The searching for an ideal surrogate is still in progress.
Early decline of the HCV core antigen can predict SVR in patients with HCV treated by Pegylated interferon plus ribavirin combination therapy
Authors: FUJINO Tatsuya , NAKAMUTA Makoto , AOYAGI Yoko , FUKUIZUMI Kunitaka , TAKEMOTO Ryosuke , YOSHIMOTO Tsuyoshi , MIYAHARA Toshihiko , HARADA Naohiko , SAKAI Hironori , NAKASHIMA Manabu , ENJOJI Munechika
Published: February 2009

OBJECTIVE:  Pegylated interferon (PEG‐IFN) plus ribavirin (RBV) combination therapy is now a popular treatment for patients with chronic hepatitis C; however, the reported sustained virologic response (SVR) rate remains at nearly 50% in genotype 1b infected patients. Therefore, it is of clinical benefit to be able to predict the effect of combination therapy on individual patients earlier in the treatment. We estimated the predictive serum HCV core antigen levels for SVR in the early therapeutic stage of combination therapy.

METHODS:  The HCV core antigen in patients with high‐level HCV viremia, in whom standard PEG‐IFNα2b plus RBV combination therapy had been completed, was measured at baseline and at 3, 7, 14, 28 and 84 days of treatment, and their SVR was determined at 24 weeks after treatment. Sixty genotype 1b‐ and 30 genotype 2‐infected patients were included.

RESULTS:  Thirty (50%) genotype 1b and 27 (90%) genotype 2 patients achieved a SVR. In genotype 1b patients the decline of HCV core antigen levels was statistically different between the SVR and non‐SVR groups. When we defined a separation level at 500 fmol/L, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for SVR at day 7 was 79.4%, 88.5%, 90%, 76.7%, and 83.3%, respectively. In genotype 2 patients, there was no significant difference in the HCV core antigen values between the SVR and non‐SVR groups.

CONCLUSION:  In genotype 1b patients, 500 fmol/L of HCV core antigen level at day 7 was the best predictor for therapeutic response in the early stage of treatment.
Effects of gut barrier dysfunction and NF‐κB activation on aggravating mechanism of severe acute pancreatitis
Authors: WANG Yi Lin , ZHENG Yun Jiang , ZHANG Zi Ping , SU Jing Ying , LEI Ruo Qing , TANG Yao Qing , ZHANG Sheng Dao
Published: February 2009

OBJECTIVE:   To study the effects of gut‐derived endotoxin translocation and NF‐κB activation on the aggravating mechanism of severe acute pancreatitis (SAP) and of treatment with pyrrolidine dithiocarbamate (PDTC) on rats with SAP.

METHODS:   SD rats were randomly divided into sham operation group (SO), SAP group, SAP + lipopolysaccharide(LPS) group, pyrrolidine dithiocarbamate (PDTC) treatment group and LPS group. Biochemical parameters and cytokines were examined in the serum. Multiple organs pathological slices were examined. Expression of NF‐κB mRNA in the liver tissue was detected by RT‐PCR. Activation of NF‐κB by the method of streptomycin avidin‐peroxidase (SP) and expression of NF‐κB p65 protein and its binding activity were analyzed by Western blot and electrophoretic mobidity shift assay (EMSA).

RESULTS:   Compared with sham operation group, the concentration of TNF‐α, alanine aminotransferase (ALT), and diamine oxidase (DAO) in serum significantly increased in SAP + LPS group (P < 0.05). Pathological changes were markedly observed in tissues and the expression of NF‐κB mRNA in the liver significantly increased (P < 0.05) also, the activation of NF‐κB and binding activity of NF‐κB p65 protein in the liver markedly increased (P < 0.01) in SAP + LPS group. Treatment with PDTC markedly reduced concentration of ALT, DAO and TNF‐α, and the expression of NF‐κB, and the pathologic scores, as well as significantly decreased the expression of NF‐κB p65 protein.

CONCLUSION:   The activation and overexpression of NF‐κB may participate in the aggravating mechanism of SAP. Treatment with PDTC has a protective effect on multiple organs damage in SAP.
DNA barcode divergence among species and genera of birds and fishes
Authors: WARD ROBERT D.
Published: February 2009

COI DNA barcoding is increasingly recognized as a significant new tool for the recognition and identification of animal species. Here, publicly available barcode data are compiled and analysed for birds (657 species) and fishes (1088 species). The proportion of species that cannot be barcode‐distinguished by this marker is approximately 6.4% for birds and 2.1–2.5% for fishes. At all hierarchical taxonomic levels (species, genera, family, order, class), fish show greater mean COI divergence than birds. If two samples are barcode‐identical, then for both birds and fishes, the probability that they are from the same species is 98–99%. The probability of conspecificity rapidly drops as divergence increases. At 2% COI divergence, this probability approximates to 1% for birds and 3% for fishes. The apparent difference between birds and fishes might partially reflect currently unrecognized cryptic species complexes in the latter. These probability estimates derive from pooled samples of birds and pooled samples of fishes, and will not apply in all situations. Recently evolved species complexes will have higher proportions of species that are barcode‐identical. As barcode data accumulate, more refined statistical analyses will become possible.
Evidence for shifting baseline syndrome in conservation
Authors: Papworth S.K. , Rist J. , Coad L. , Milner‐Gulland E.J.
Published: February 2009

Shifting baseline syndrome (SBS) is often referred to as a key issue for conservation, yet there is little evidence for its existence. The presence of SBS could influence the validity of participatory monitoring, local ecological knowledge, community based conservation, and conservation education. We outline two forms of SBS: (1) generational amnesia, where knowledge extinction occurs because younger generations are not aware of past biological conditions and (2) personal amnesia, where knowledge extinction occurs as individuals forget their own experience. Two conditions are essential to the identification of SBS: (1) biological change must be present in the system and (2) any perceived changes must be consistent with the biological data. If age or experience‐related differences in perception are then found, generational amnesia may be occurring. Alternately, if individuals believe current conditions also occurred in the past, personal amnesia may be occurring. Previous studies have not fully addressed these conditions, and hence cannot provide indisputable evidence for the existence of SBS. We present three case studies to examine these issues, which demonstrate both forms of SBS. Shifting baseline syndrome is no longer a cautionary tale, but instead is a real problem for those using human perceptions of change to inform conservation policy‐making or management.
Multifocal electroretinography amplitudes increase after photocoagulation in areas with increased retinal thickness and hard exudates
Authors: Lövestam‐Adrian Monica , Holm Kristina
Published: February 2009

Purpose:  This study aimed to evaluate local response on multifocal electroretinography (mfERG) and to assess retinal thickness with optical coherence tomography (OCT) after focal laser treatment in areas with retinal oedema and exudates in patients with diabetic retinopathy.

Methods:  Twelve diabetes patients (aged 60 ± 14 years, diabetes duration 16 ± 8 years) treated with focal or grid photocoagulation in areas with retinal oedema and/or exudates underwent mfERG and OCT before and 3 months after treatment. The average thickness (in μm) in any of the nine sectors (defined according to the ETDRS) treated with photocoagulation was measured. Amplitudes and implicit times were analysed in corresponding areas on the mfERG.

Results:  Mean mfERG amplitudes increased after photocoagulation (21.5 ± 8.0 nV/deg2 versus 16.8 ± 6.1 nV/deg2; p = 0.012), whereas no difference was seen in implicit times. Mean OCT values in the treated regions were lower at follow‐up (272 ± 23 μm versus 327 ± 79 μm; p = 0.013). No correlation was seen between changes in mfERG response and changes in OCT values. The decrease in retinal thickness was correlated with the number of laser spots applied (p = 0.002).

Conclusions:  Focal argon laser treatment is effective in reducing retinal thickness. In addition, treated areas tend to show improved retinal function as demonstrated by increased amplitudes on mfERG.
DY‐9760e Inhibits Endothelin‐1‐induced Cardiomyocyte Hypertrophy Through Inhibition of CaMKII and ERK Activities
Authors: Lu Ying‐Mei , Shioda Norifumi , Han Feng , Kamata Akifumi , Shirasaki Yasufumi , Qin Zheng‐Hong , Fukunaga Kohji
Published: February 2009

Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and extracellular signal‐regulated kinase (ERK) have pivotal roles in endothelin‐1 (ET‐1)‐induced cardiomyocyte hypertrophy. We here tested whether a novel CaM antagonist, DY‐9760e inhibits ET‐1‐induced hypertrophy through inhibition of CaMKII and ERK activities. We first confirmed that Ca2+ oscillation induced by ET‐1 treatment elicits transient activation of CaMKII and ERK in cultured cardiomyocytes. DY‐9760e treatment with 3 μM totally and partially inhibited the ET‐1‐induced CaMKII and ERK activation, respectively. The ET‐1‐induced ERK activation was also partially blocked by a CaMKII inhibitor, KN93. To confirm involvement of CaMKII activity in the ERK activation by ET‐1 and A23187, cultured cardiomyocytes were transfected with a constitutively active CaMKII. The transfection with the active CaMKII elicited ERK activation in cultured cardiomyocytes and cotransfection with dominant negative CaMKII eliminated its ERK activation. Consistent with inhibitory actions of DY‐9760e on the ET‐1‐induced CaMKII and ERK activation, induction of hypertrophy‐related genes including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was significantly inhibited by DY‐9760e treatment. Combination treatment with DY‐9760e and U0126, a MEK inhibitor, totally blocked the ET‐1‐induced ANP and BNP expression. DY‐9760e treatment (3 μM) significantly inhibited the ET‐1‐induced hypertrophy and combination treatment with DY‐9760e and U0126 totally blocked the ET‐1‐induced hypertrophy in cultured cardiomyocytes. These results suggest that DY‐9760e elicits antihypertrophic action on ET‐1‐induced cardiac hypertrophy through inhibition of CaMKII and ERK activation and that CaMKII activity in part mediates ET‐1‐induced ERK activation.
Resting Heart Rate Does Not Reflect the Degree of Beta‐Blockade in Subjects with Heart Failure on Chronic Beta‐Blocker Therapy
Authors: Mignatti Andrea , Sims Daniel B. , Colombo Paolo C. , Garcia Luis I. , Bijou Rachel , Deng Mario C. , Jorde Ulrich P.
Published: February 2009

We investigated the relationship between resting heart rate (HR) and two measures of beta‐1 receptor sensitivity/blockade: (1) the percentage of maximal predicted heart rate reached during exercise (%MPHR), and (2) the HR increase per unit of circulating norepinephrine (NE) or the chronotropic responsiveness index (CRI) in 28 patients with systolic CHF on chronic beta‐blocker therapy. Our results show that resting HR is not associated with HR response during exercise nor with beta‐1 receptor sensitivity to circulating NE.
Cardiac Functional Reserve is Diminished in Growth Hormone‐Deficient Adults
Authors: Moisey Robert , Orme Steve , Barker Diane , Lewis Nigel , Sharp Lisa , Clements Richard E. , Goldspink David F. , Tan Lip‐Bun
Published: February 2009

Various studies have shown that patients with severe growth hormone deficiency (GHD) have diverse changes in left ventricular (LV) size or performance but so far there is no direct indication of cardiac reserve ability to maintain the circulation during peak exercise. We tested the hypothesis that patients with severe GHD have reduced cardiac reserve function compared with healthy controls. Eighteen patients with severe GHD were studied and compared with 18 age‐, sex‐, and body mass index (BMI)‐matched healthy controls. Peak cardiac power and cardiorespiratory fitness were investigated using noninvasive hemodynamic measurements during maximal cardiopulmonary exercise testing. Compared with matched controls, the cardiac power of GHD patients during exercise to volitional exhaustion was significantly reduced by 15% (mean ± SD: 4.4 ± 1.0 watts (W) vs. 5.2 ± 1.0 W, P= 0.02), despite attaining similar aerobic exercise peaks (VO2 max, GHD: 2390 ± 822 mL/min vs. controls: 2461 ± 872 mL/min, P= 0.80) and similar peak respiratory exchange ratios. The lower peak cardiac power could not be accounted for by peripheral alterations because both groups reached similar peak exercise systemic vascular resistances. Patients with GHD also had lower cardiac chronotropic reserve (peak heart rate: 154 ± 21 bpm vs. 174 ± 11 bpm, P= 0.001) and a lower cardiac pressure‐generating capacity (systolic blood pressure [SBP] 160 ± 25 mmHg vs. 200 ± 15 mmHg, P < 0.0001). Using this robust noninvasive method of assessing functional cardiac pumping capacity we have for the first time shown that patients with severe GHD have a significantly impaired cardiac functional reserve associated with chronotropic incompetence and impaired pressure‐generating capacity.
Mapitjakuna—Shall I Go Away From Myself Towards You? Being‐with and Looking‐at Across Cultural Divides
Authors: Eickelkamp Ute
Published: February 2009

Anthropologists increasingly express links between individuals or collectivities through the discursively convenient prefix ‘‐inter’; inter‐personal, inter‐subjective, inter‐cultural, inter‐textual. However, attempts to describe the psychodynamics that effect the linkings that are integral to human social experience have not held pace. I want to suggest that a regard for the ontogenetic primary forms of encounter—looking, eating, playing—can substantiate the analysis of being a self among other selves within and across the boundaries of social worlds. Engaging a phenomenological and psychoanalytic perspective, this paper examines how We‐relationships have been variously structured by Pitjantjatjara Aborigines in encountering others at home and in Germany.
Nitric oxide (NO) does not contribute to the generation or action of adenosine during exercise hyperaemia in rat hindlimb
Authors: Ray Clare J. , Marshall Janice M.
Published: February 2009

Exercise hyperaemia is partly mediated by adenosine A2A‐receptors. Adenosine can evoke nitric oxide (NO) release via endothelial A2A‐receptors, but the role for NO in exercise hyperaemia is controversial. We have investigated the contribution of NO to hyperaemia evoked by isometric twitch contractions in its own right and in interaction with adenosine. In three groups of anaesthetized rats the effect of A2A‐receptor inhibition with ZM241385 on femoral vascular conductance (FVC) and hindlimb O2 consumption at rest and during isometric twitch contractions (4 Hz) was tested (i) after NO synthase inhibition with l‐NAME, and when FVC had been restored by infusion of (ii) an NO donor (SNAP) or (iii) cell‐permeant cGMP. Exercise hyperaemia was significantly reduced (32%) by l‐NAME and further significantly attenuated by ZM241385 (60% from control). After restoring FVC with SNAP or 8‐bromo‐cGMP, l‐NAME did not affect exercise hyperaemia, but ZM241385 still significantly reduced the hyperaemia by 25%. There was no evidence that NO limited muscle during contraction. These results indicate that NO is not required for adenosine release during contraction and that adenosine released during contraction does not depend on new synthesis of NO to produce vasodilatation. They also substantiate our general hypothesis that the mechanisms by which adenosine contributes to muscle vasodilatation during systemic hypoxia and exercise are different: we propose that, during muscle contraction, adenosine is released from skeletal muscle fibres independently of NO and acts directly on A2A‐receptors on the vascular smooth muscle to cause vasodilatation.
Astrocytes going live: advances and challenges
Authors: Nimmerjahn Axel
Published: February 2009

Astrocytes are one of the most numerous cell types in the CNS. They have emerged as sophisticated cells participating in a large and diverse variety of functions vital for normal brain development, adult physiology and pathology. Recent in vivo studies have provided exciting new insight into astrocyte physiology in the intact healthy brain. This review will summarize some of their most intriguing findings, discuss some of their implications, and look ahead at some of the challenges we face in studying astrocyte function in vivo.
β subunit‐specific modulations of BK channel function by a mutation associated with epilepsy and dyskinesia
Authors: Lee Urvi S. , Cui Jianmin
Published: February 2009

Large conductance Ca2+‐activated K+ (BK) channels modulate many physiological processes including neuronal excitability, synaptic transmission and regulation of myogenic tone. A gain‐of‐function (E/D) mutation in the pore‐forming α subunit (Slo1) of the BK channel was recently identified and is linked to human neurological diseases of coexistent generalized epilepsy and paroxysmal dyskinesia. Here we performed macroscopic current recordings to examine the effects of the E/D mutation on the gating kinetics, and voltage and Ca2+ dependence of the BK channel activation in the presence of four different β subunits (β1–4). These β subunits are expressed in a tissue‐specific pattern and modulate BK channel function differently, providing diversity and specificity for BK channels in various physiological processes. Our results show that in human (h) Slo1‐only channels, the E/D mutation increased the rate of opening and decreased the rate of closing, allowing a greater number of channels to open at more negative potentials both in the presence and absence of Ca2+ due to increased Ca2+ affinity and enhanced activation compared with the wild‐type channels. Even in the presence of β subunits, the E/D mutation exhibited these changes with the exception of β3b, where Ca2+ sensitivity changed little. However, quantitative examination of these changes shows the diversity of each β subunit and the differential modulation of these subunits by the E/D mutation. For example, in the presence of the β1 subunit the E/D mutation increased Ca2+ sensitivity less but enhanced channel activation in the absence of Ca2+ more than in hSlo1‐only channels, while in the presence of the β2 subunit the E/D mutation also altered inactivation properties. These findings suggest that depending on the distribution of the various β subunits in the brain, the E/D mutation can modulate BK channels differently to contribute to the pathophysiology of epilepsy and dyskinesia. Additionally, these results also have implications on physiological processes in tissues other than the brain where BK channels play an important role.
Field-emission properties of carbon nanotubes grown using Cu–Cr catalysts
Authors: Zhang Zhejuan , Chua Daniel H. C. , Gao Yang , Zhang Yanping , Tang Zhe , Tay Beng Kang , Feng Tao , Sun Zhuo , Chen Yiwei
Published: February 2009

In addition to Ni, Co, and Fe, the authors show that a copper (Cu) chromium (Cr) alloy can be a good catalyst for controlled growth of carbon nanotubes (CNTs). A thermal chemical vapor deposition was used at 600°C to deposit the CNTs, and a dual-target magnetron sputtering system was used to control the ratio of the Cu∕Cr alloy. The material properties of the CNTs, such as concentration, diameter, and density, are directly affected by the ratio of Cu∕Cr alloy, which subsequently affect the field-emission properties. Their results showed that Cr can be used to effectively control the catalytic effects of the Cu catalysts, where the quality of the CNTs could vary while the density could be controlled from 4×107to5×1010tubes∕cm2. The field-emission current density of the CNT film increases with Cu∕Cr ratio from 1∕6.4 to 4.6∕1 and decreases when the Cu∕Cr ratio is more than 4.8∕1 in the alloy film. When the Cu∕Cr ratio is at 4.6∕1, the average current density peaks at 5129μA∕cm2 at the applied field of 3.53V∕μm, showing an optimized field-emission property.
Plasma-surface interactions of advanced photoresists with C4F8∕Ar discharges: Plasma parameter dependencies
Authors: Engelmann S. , Bruce R. L. , Sumiya M. , Kwon T. , Phaneuf R. , Oehrlein G. S. , Andes C. , Graves D. , Nest D. , Hudson E. A.
Published: February 2009

One recurring problem in nanoscale processing is roughening of photoresist (PR) materials during plasma etch. We studied the plasma etch behavior of 248nm PR, 193nm PR, and poly methyladamantyl methacrylate while changing the source power level (400–1200W), adjusting the bias power to change the self-bias voltage Vdc (−50to−150V), and varying the pressure (10–80mTorr) and the amount of fluorocarbon gas additive to the Ar discharge (0%–10% c-C4F8 in Ar). The authors found that the PR removal is dominated by the ion energy and fluence. Surface fluorination enhanced the removal rates. Two linked mechanisms for the roughening behavior of the films during processing were identified. Changes of PR top surface roughening behavior in response to variations of bias power and pressure could be interpreted by a model of roughness formation which is dominated by a physical pattern transfer mechanism, i.e., roughness amplification through selective ion-induced transfer. When the plasma source power was varied, they observed that roughness formation was linked to the surface energy density deposited during processing. As the energy required to volatilize a volume element from the surface increased, the surface roughening rates grew proportionally. This conversion of excess energy into roughening was found to depend on the molecular structure of the polymer, with adamantyl polymers having a very high roughening constant. Additional effects on the etch behavior arise from fluorination of the samples, as quantified by x-ray photoelectron spectroscopy. High F 2s∕F 1s intensity ratios, which indicate deeper fluorination, were measured for rough surface conditions. Smaller F 2s∕F 1s ratios indicate near-surface fluorination and correspond to smoother top surfaces and feature sidewalls. Molecular compounds showed roughening behavior relative to the respective cross-linking behavior even when processed in pure Ar discharges, suggesting that the measured surface fluorination mirrors surface morphology. When plasma etching three-dimensional trenches and contact holes patterned in PR, the authors found that the sidewall roughness changed with process parameters in a fashion similar to that seen for blanket surface roughness introduction using the same etch conditions. A close correlation between the surface and sidewall roughness results was obtained, suggesting that their model of polymer surface roughening also applies to resist sidewall evolution during plasma etch.
Investigation of Bias-Temperature Instability in work-function-tuned high-k/metal-gate stacks
Authors: Kaczer B. , Veloso A. , Roussel Ph. J. , Grasser T. , Groeseneken G.
Published: February 2009

The impact of Vth-adjusting layers on high-k/metal-gate n- and pFinFET Vth stability is investigated. Additional insight is gained by monitoring ΔVth recovery transients over several decades in time. Vth-adjusting capping layers deposited directly on top of the gate dielectric degrade the Vth stability. The Vth stability improves as the capping layers are buried in the gate metal and are thus isolated from the gate dielectric. A combination of a capping layer thickness and depth in the metal gate is found that practically eliminates nFET Vth instability.
Influence of the electrode material on HfO2 metal-insulator-metal capacitors
Authors: Wenger Ch. , Lukosius M. , Müssig H.-J. , Ruhl G. , Pasko S. , Lohe Ch.
Published: February 2009

TaN and TiN are investigated as bottom electrode materials for metal-insulator-metal (MIM) capacitor applications. Atomic vapor deposited HfO2 films are used as high-k dielectric. The influence of the interfacial layer between HfO2 and the bottom electrode on the electrical performance of MIM capacitors is evaluated. The capacitance density as well as the capacitance voltage linearity of high-k MIM capacitors is affected by the electrode material. There is also an impact by TaN and TiN on leakage current density and breakdown strength of the devices.
Si
O
2
interfacial layer as the origin of the breakdown of high-k dielectrics stacks
Authors: Rafik M. , Ribes G. , Roy D. , Ghibaudd G.
Published: February 2009

This article investigates the breakdown of high-k/metal gate stacks under substrate injection regime. It is shown that it is initiated by a field driven breakdown of the interfacial layer (IL). Furthermore, because it is very sensitive to IL thickness variations, TBD optimization supposes a control of the high-k deposition process that can induce IL thickness fluctuations.
Francisella tularensisGenes Required for Inhibition of the Neutrophil Respiratory Burst and Intramacrophage Growth Identified by Random Transposon Mutagenesis of Strain LVS
Authors: Schulert Grant S. , McCaffrey Ramona L. , Buchan Blake W. , Lindemann Stephen R. , Hollenback Clayton , Jones Bradley D. , Allen Lee-Ann H.
Published: February 2009

Francisella tularensisis a facultative intracellular pathogen and the causative agent of tularemia. We have shown thatF. tularensissubspeciesholarcticastrain LVS prevents NADPH oxidase assembly and activation in human neutrophils, but how this is achieved is unclear. Herein, we used random transposon mutagenesis to identify LVS genes that affect neutrophil activation. Our initial screen identifiedcarA, carB, andpyrB, which encode the small and large subunits of carbamoylphosphate synthase and aspartate carbamoyl transferase, respectively. These strains are uracil auxotrophs, and their growth was attenuated on cysteine heart agar augmented with sheep blood (CHAB) or in modified Mueller-Hinton broth. Phagocytosis of the uracil auxotrophic mutants triggered a respiratory burst in neutrophils, and ingested bacteria were killed and fragmented in phagosomes that contained superoxide. Conversely, phagocytosis did not trigger a respiratory burst in blood monocytes or monocyte-derived macrophages (MDM), and phagosomes containing wild-type or mutant bacteria lacked NADPH oxidase subunits. Nevertheless, the viability of mutant bacteria declined in MDM, and ultrastructural analysis revealed that phagosome egress was significantly inhibited despite synthesis of the virulence factor IglC. Other aspects of infection, such as interleukin-1β (IL-1β) and IL-8 secretion, were unaffected. The cultivation ofcarA, carB, orpyrBon uracil-supplemented CHAB was sufficient to prevent neutrophil activation and intramacrophage killing and supported escape from MDM phagosomes, but intracellular growth was not restored unless uracil was added to the tissue culture medium. Finally, all mutants tested grew normally in both HepG2 and J774A.1 cells. Collectively, our data demonstrate that uracil auxotrophy has cell type-specific effects on the fate ofFrancisellabacteria.
Modulation of Toll-Like Receptor 2 (TLR2) and TLR4 Responses byAspergillus fumigatus
Authors: Chai Louis Y. A. , Kullberg Bart Jan , Vonk Alieke G. , Warris Adilia , Cambi Alessandra , Latgé Jean-Paul , Joosten Leo A. B. , van der Meer Jos W. M. , Netea Mihai G.
Published: February 2009

Toll-like receptor (TLR)-based signaling pathways in the host may be modulated by pathogens during the course of infection. We describe a novel immunomodulatory mechanism in whichAspergillus fumigatusconidia induce attenuation of TLR2- and TLR4-mediated interleukin (IL)-6 and IL-1β proinflammatory responses in human mononuclear cells with suppression of IL-1β mRNA transcription. Background TLR2 and TLR4 mRNA transcription was not influenced.A. fumigatusconidia induced TLR2 internalization and uptake into the phagosome with a resultant decrease in surface receptor expression.A. fumigatushyphae, on the other hand, selectively downregulated the TLR4-mediated response. These novel immunosuppressive effects may facilitate the invasiveness ofA. fumigatus.
Molecular Analyses of
Vibrio cholerae
O1 Clinical Strains, Including New Nontoxigenic Variants Isolated in Mexico during the Cholera Epidemic Years between 1991 and 2000
Authors: Lizárraga-Partida Marcial Leonardo , Quilici Marie-Laure
Published: February 2009

We studied the evolution of
Vibrio cholerae
O1 during the 1991 to 2000 cholera epidemic in Mexico by biochemical, serological, and molecular characterization of strains collected during this period. Strains were divided into toxigenic and nontoxigenic groups according to the presence or absence of genes encoding cholera toxin. As previously reported, we characterized two populations among toxigenic strains, which were present from the first year of the epidemic. BglI rRNA analysis revealed that these strains had ribotype profiles, denoted M5 and M6 in our study, that were identical to those previously designated Koblavi B5 or Popovic 5 and Popovic 6a or Tamayo B21a, respectively. Ribotype M5 was isolated between 1991 and 1993. This ribotype had a low level of genetic variation as detected by pulsed-field gel electrophoresis (PFGE). Ribotype M6 persisted from 1991 to 2000. However, PFGE profiles suggested that two epidemiologically unrelated strains coexisted within this single ribotype from 1995 until the end of the epidemic. We identified three new BglI ribotypes, Mx1, Mx2, and Mx3, from nontoxigenic
V. cholerae
O1 strains isolated between 1998 and 2000; one of them grouped strains positive for the toxin-coregulated pilus island. They differed from nontoxigenic clones isolated in Latin America and on the U.S. Gulf Coast and are probably autochthonous Mexican
V. cholerae
O1 variants. Most of these new variants were isolated from states surrounding the Gulf of Mexico, where the highest incidence of cholera in the country was recorded. Thus, the Mexican Gulf Coast, like the U.S. Gulf Coast, may act as an environmental reservoir of
V. cholerae
O1.
Sensitivity of PCR Targeting
Mycobacterium ulcerans
by Use of Fine-Needle Aspirates for Diagnosis of Buruli Ulcer
Authors: Phillips R. O. , Sarfo F. S. , Osei-Sarpong F. , Boateng A. , Tetteh I. , Lartey A. , Adentwe E. , Opare W. , Asiedu K. B. , Wansbrough-Jones M.
Published: February 2009

In a previous study, we reported that the sensitivity of PCR targeting the IS
2404
insertion sequence of
Mycobacterium ulcerans
was 98% when it was applied to 4-mm punch biopsy samples of Buruli lesions. Fine-needle aspiration (FNA) is a less traumatic sampling technique for nonulcerated lesions, and we have studied the sensitivity of PCR using FNA samples. Fine-needle aspirates were taken with a 21-gauge needle from 43 patients diagnosed clinically with
M. ulcerans
disease. Four-millimeter punch biopsies were obtained for microscopy, culture, and PCR targeting the IS
2404
insertion sequence. The sensitivity of PCR using samples obtained by FNA was 86% (95% confidence interval [95% CI], 72 to 94%) compared with that for PCR using punch biopsy samples. In this study, the sensitivities of culture and microscopy for punch biopsy samples were 44% (95% CI, 29 to 60%) and 26% (95% CI, 14 to 41%), respectively. This demonstrates that PCR on an FNA sample is a viable minimally invasive technique to diagnose
M. ulcerans
lesions.
One-Step, Multiplex, Real-Time PCR Assay with Molecular Beacon Probes for Simultaneous Detection, Differentiation, and Quantification of Human T-Cell Leukemia Virus Types 1, 2, and 3
Authors: Besson Guillaume , Kazanji Mirdad
Published: February 2009

A single-tube, multiplex, real-time PCR assay with molecular beacons was established in which various probes were used for the simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3 (HTLV-1, HTLV-2, and HTLV-3, respectively) and of simian T-cell leukemia virus types 1 and 3 (STLV-1 and STLV-3, respectively). The quantitative amplification of the standards with MT4 (HTLV-1) and C19 (HTLV-2) cell lines and a molecular clone of HTLV-3 was linear, with the simplex and multiplex methods having similar efficiencies. A maximum difference of 0.9 (mean, 0.4; range, 0.0 to 0.9) was found between threshold cycle values in single and multiplex reactions. The efficiency with each probe in the multiplex reaction was close to 100%, indicating strong linear amplification. The albumin gene was used to standardize the copy number. Comparable results for the detection and quantification of HTLV-1 were obtained with our new methods and with other real-time PCR methods described previously. With our new multiplex assay, however, we were able to detect and quantify HTLV-2 and -3 and STLV-1 and -3 in clinical specimens, with an excellent dynamic range of 10
6
to 10
0
copies per assay, which the other assays could not do. Thus, it will be possible to determine a wide range of HTLV types in both standard and clinical samples, with a detection of 1 to 10 HTLV copies in samples containing at least 100 cells. Furthermore, our system can provide evidence for multiple infections with the three HTLV types, with separate proviral load results. Our new method also could be used for epidemiological studies in Africa and in countries where HTLVs and STLVs are endemic.
Major
Mycobacterium tuberculosis
Lineages Associate with Patient Country of Origin
Authors: Reed Michael B. , Pichler Victoria K. , McIntosh Fiona , Mattia Alicia , Fallow Ashley , Masala Speranza , Domenech Pilar , Zwerling Alice , Thibert Louise , Menzies Dick , Schwartzman Kevin , Behr Marcel A.
Published: February 2009

Over recent years, there has been an increasing acknowledgment of the diversity that exists among
Mycobacterium tuberculosis
clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that “clustered” tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with ≥6 IS
6110
copies) or RFLP in combination with spoligotyping (for isolates with <6 IS
6110
copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS
6110
-copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.
Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin
Authors: Bok Karin , Prikhodko Victor G. , Green Kim Y. , Sosnovtsev Stanislav V.
Published: February 2009

Noroviruses (NVs) are recognized as a major cause of nonbacterial gastroenteritis in humans. Studies of the human NVs continue to be hampered by the inability to propagate them in any cell culture system. Until recently, most data concerning NV replication were derived from studies of feline calicivirus and rabbit hemorrhagic disease virus, which are cultivable members of the familyCaliciviridae. From such studies, it was proposed that caliciviruses induce apoptosis to facilitate the dissemination of viral progeny in the host. The discovery that MNV type 1 (MNV-1) grows in RAW264.7 cells provided the first cell culture system for use in studying the role of apoptosis in NV infection. We first showed that MNV-1 replication triggered apoptosis in infected RAW264.7 cells and then demonstrated that cell death was associated with activation of caspase-9 and caspase-3 through the mitochondrial pathway. This process was dependent on virus replication, since inactivated virus failed to induce signs of apoptosis. In order to better understand the apoptotic process induced by MNV-1 infection of RAW264.7 cells, we investigated the expression profiles of MNV-1-infected versus mock-infected cells. Survivin, a member of the inhibitor of apoptosis protein family, was found to be significantly downregulated in an inverse relationship with the virus genome replication. This study showed that, unlike other viruses that upregulate survivin, MNV-1 is the first virus found to downregulate the levels of survivin. We observed that MNV-1 replication in RAW264.7 cells activated caspases, resulting in apoptosis through the mitochondrial pathway, possibly as a result of downregulation of survivin.
Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability
Authors: Wang Qian , Kennedy Alan , Das Papia , McIntosh Pauline B. , Howell Steven A. , Isaacson Erin R. , Hinz Steven A. , Davy Clare , Doorbar John
Published: February 2009

The E1^E4 protein of human papillomavirus type 16 (HPV16) causes cytokeratin reorganization in the middle and upper epithelial layers and is thought to contribute to multiple facets of the virus life cycle. Although little is known as to how HPV16 E1^E4 (16E1^E4) functions are controlled following the first expression of this protein, the finding that low-risk E1^E4 proteins can be phosphorylated in vivo suggests an important role for kinases. Here, we show that 16E1^E4 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and PKC α, with CDK1/2 serine 32 and ERK threonine 57 phosphorylations representing the two primary events seen in cells in cycle. Interestingly, T57 phosphorylation was found to trigger a structural change in the 16E1^E4 protein that compacts the central fold region, leading to an increase in 16E1^E4 stability and overall abundance in the cell. When compared to wild-type 16E1^E4, a T57D phosphomimic was found to have greatly enhanced keratin-binding ability and an ability to modulate the binding of the unphosphorylated form, with keratin binding protecting the T57-phosphorylated form of 16E1^E4 from proteasomal degradation. In HPV16 genome-containing organotypic rafts, the T57-phosphorylated form was specifically detected in the intermediate cell layers, where productive infection occurs, suggesting that T57 phosphorylation may have a functional role at this stage of the viral life cycle. Interestingly, coexpression with 16E5 and ERK activation enhanced T57 phosphorylation, suggesting that E1^E4 and E5 may work together in vivo. Our data suggest a model in which the expression of 16E5 from the major E1^E4-E5 mRNA promotes T57 phosphorylation of E1^E4 and keratin binding, with dephosphorylation occurring following the switch to late poly(A) usage. Other forms of E1^E4, with alternative functional roles, may then increase in prevalence in the upper layers of the epithelium.
Proteinase 2AproIs Essential for Enterovirus Replication in Type I Interferon-Treated Cells
Authors: Morrison Juliet M. , Racaniello Vincent R.
Published: February 2009

ThePicornaviridaefamily comprises a diverse group of small RNA viruses that cause a variety of human and animal diseases. Some of these viruses are known to induce cleavage of components of the innate immune system and to inhibit steps in the interferon pathway that lead to the production of type I interferon. There has been no study of the effect of picornaviral infection on the events that occur after interferons have been produced. To determine whether members of theEnterovirusgenus can antagonize the antiviral activity of interferon-stimulated genes (ISGs), we pretreated cells with alpha interferon (IFN-α) and then infected the cells with poliovirus type 1, 2, or 3; enterovirus type 70; or human rhinovirus type 16. We found that these viruses were able to replicate in IFN-α-pretreated cells but that replication of vesicular stomatitis virus, aRhabdovirus, and encephalomyocarditis virus (EMCV), a picornavirus of theCardiovirusgenus, was completely inhibited. Although EMCV is sensitive to IFN-α, coinfection of cells with poliovirus and EMCV leads to EMCV replication in IFN-α-pretreated cells. The enteroviral 2A proteinase (2Apro) is essential for replication in cells pretreated with interferon, because amino acid changes in this protein render poliovirus sensitive to IFN-α. The addition of the poliovirus 2Aprogene to the EMCV genome allowed EMCV to replicate in IFN-α-pretreated cells. These results support an inhibitory role for 2Aproin the most downstream event in interferon signaling, the antiviral activities of ISGs.
The Human Cytomegalovirus UL112-113 Locus Can Activate the Full Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication Cycle
Authors: Wells Richard , Stensland Laurence , Vieira Jeffrey
Published: February 2009

Human cytomegalovirus (HCMV) infection of a cell containing latent Kaposi's sarcoma-associated herpesvirus (KSHV) results in the activation of KSHV lytic replication and the production of infectious virus. In this study, we examined the HCMV genes identified as having a role in the activation of HCMV early genes for their ability to activate KSHV lytic replication. It was found that the UL112-113 locus was able to activate the complete KSHV lytic cycle, while the UL122-123 locus, encoding the IE1 and IE2 proteins, known to be strong transactivators, did not.
The Mouse Cytomegalovirus Immediate-Early 1 Gene Is Not Required for Establishment of Latency or for Reactivation in the Lungs
Authors: Busche Andreas , Marquardt Anja , Bleich Andre , Ghazal Peter , Angulo Ana , Messerle Martin
Published: February 2009

The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins have been found to inhibit histone deacetylases, suggesting a role in the epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here we asked if an MCMV Δie1 mutant is able both to establish latency and to reactivate from the lungs of latently infected mice. Since the Δie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection with mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the Δie1 mutant and of wt MCMV from latency occurred with similar incidences in lung explant cultures at 4, 7, and 12 months postinfection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the Δie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for the establishment of latency or for the reactivation of MCMV.
Molecular Characterization of Infectious Clones of the Minute Virus of Canines Reveals Unique Features of Bocaviruses
Authors: Sun Yuning , Chen Aaron Yun , Cheng Fang , Guan Wuxiang , Johnson F. Brent , Qiu Jianming
Published: February 2009

Minute virus of canines (MVC) is a member of the genusBocavirusin the familyParvoviridae. We have molecularly cloned and sequenced the 5′- and 3′-terminal palindromes of MVC. The MVC genome, 5,404 nucleotides (nt) in length, shared an identity of 52.6% and 52.1% with that of human bocavirus and bovine parvovirus, respectively. It had distinct palindromic hairpins of 183 nt and 198 nt at the left-end and right-end termini of the genome, respectively. The left-end terminus was also found in two alternative orientations (flip or flop). Both termini shared extensive similarities with those of bovine parvovirus. Four full-length molecular clones constructed with different orientations of the left-end terminus proved to be infectious in Walter Reed canine cell/3873D (WRD) canine cells. Both MVC infection and transfection of the infectious clone in WRD cells revealed an identical RNA transcription profile that was similar to that of bovine parvovirus. Mutagenesis of the infectious clone demonstrated that the middle open reading frame encodes the NP1 protein. This protein, unique to the genusBocavirus, was essential for MVC DNA replication. Moreover, the phospholipase A2 motif in the VP1 unique region was also critical for MVC infection. Thus, our studies revealed important information about the genusBocavirusthat may eventually help us to clone the human bocavirus and study its pathogenesis.