bluuebunny/crossref_metadata_2025 huggingface dataset:
Financial Restructuring in Fresh‐Start Chapter 11 Reorganizations
Authors: Heron Randall A. , Lie Erik , Rodgers Kimberly J.
Published: November 2009

We find that firms substantially reduce their debt burden in “fresh‐start” Chapter 11 reorganizations, yet they emerge with higher debt ratios than what is typical in their respective industries. While cross‐sectional regressions reveal that post‐reorganization debt ratios are more in line with the predictions of the static trade‐off theory, they also reveal that pre‐reorganization debt ratios affect post‐reorganization debt ratios. Collectively, these results suggest that impediments in Chapter 11 prevent firms from completely resetting their capital structures. We also find that firms that reported positive operating income leading up to Chapter 11 emerge faster, suggesting that it is quicker to remedy strictly financial distress than economic distress.
Determinants of the Size and Composition of US Corporate Boards: 1935‐2000
Authors: Lehn Kenneth M. , Patro Sukesh , Zhao Mengxin
Published: November 2009

We examine the determinants of the size and composition of corporate boards for a sample of 82 US companies that survived during the period 1935‐2000. Our hypotheses lead to predictions that firm size, growth opportunities, merger activity, and geographical expansion are important determinants of these board characteristics. We find empirical evidence that the four variables are significant determinants of the size and/or composition of boards. After controlling for these determinants of board characteristics, we find no robust relation between firm performance and either board size or composition.
A Multistage Model of Loans and the Role of Relationships
Authors: Chakravarty Sugato , Yilmazer Tansel
Published: November 2009

We develop a multistage model of the loan granting process to understand the contradictory findings of the existing literature on bank‐borrower relationships, credit availability, and loan rates. Upon estimating our model with the 1993, 1998, and 2003 versions of the Survey of Small Business Finances data set, we find that relationships matter in a borrower's decision whether to apply for a loan and in the loan approval/rejection decision by the financial institution. However, the effect of relationships on loan rates depends on the prevailing economic climate. While firms with preexisting relationships obtain credit at lower rates during periods of economic expansion, loan rates are not negatively correlated with preexisting relationships during periods of economic recession.
Information Diffusion among International Fund Managers: Multicountry Evidence
Authors: Parwada Jerry T. , Yang Joey W.
Published: November 2009

Investors seeking exposure to global equity markets commonly buy international mutual funds managed by locally based fund managers. How competitive is this form of intermediated investing? We investigate whether international equity fund managers mimic each other's portfolio holdings and analyze the performance implications of these actions. Managers based in the same country have more stocks held in common than those of their peers in other countries. Correlated trading among domestic fund managers contributes significantly to this pattern. Cross‐border managers' portfolio holdings and trades are also relevant to the actions of domestic managers. Stock selection strategies based on mimicry and differentiation both deliver short‐term superior performance. Mimicked sales occur while prices are rising.
Investment Timing for Dynamic Business Expansion
Authors: Blazenko George W. , Pavlov Andrey D.
Published: November 2009

We investigate the timing of business expansion. With an indefinite sequence of growth opportunities that have constant returns to scale, current investment neither displaces nor impairs future returns. In a dynamic setting with expansion restricted to a fraction of firm size, the endogenously determined cost of capital uniformly exceeds the value maximizing return threshold for expansion. Taking this into account, a manager accelerates investment to facilitate larger and more valuable future investments when earnings stochastically improve. This result is the opposite of deferral that the investment literature recommends due to irreversibility. This means that the managerial application of the cost of capital as an expansion hurdle rate is improperly conservative.
Are Interlocked Directors Effective Monitors?
Authors: Devos Erik , Prevost Andrew , Puthenpurackal John
Published: November 2009

This paper examines whether the presence of interlocked directors on a board is associated with weak governance. For a sample of 3,566 firm‐years spanning 2001 to 2003, we find that firms with lower industry‐adjusted firm performance are more likely to have interlocked directors. We document that shareholders react negatively to the formation of director interlocks and find that the presence of interlocked directors is associated with lower than optimal pay‐performance sensitivity of CEO incentive compensation and reduced sensitivity of CEO turnover to firm performance. Collectively, our results suggest that the presence of interlocked directors is indicative of weak governance.
An Empirical Comparison of Option‐Pricing Models in Hedging Exotic Options
Authors: An Yunbi , Suo Wulin
Published: November 2009

This paper examines the empirical performance of various option‐pricing models in hedging exotic options, such as barrier options and compound options. A practical and relevant testing approach is adopted to capture the essence of model risk in option pricing and hedging. Our results indicate that the exotic feature of the option under consideration has a great impact on the relative performance of different option‐pricing models. In addition, for any given model, the more “exotic” the option, the poorer the hedging effectiveness.
The Telling Trades of Mutual Funds
Authors: Nicolosi Gina
Published: November 2009

Under the assumption that mutual funds trade at quarter commencement, some funds exhibit and exploit persistent stock selection talent; that is, the stocks purchased consistently outperform the stocks sold, and the higher turnover of these funds indicates that managers are capitalizing on their forecasting abilities. However, any evidence of sustained stock selection skill disappears when alternate trade‐timing assumptions are considered, suggesting that some skilled managers are electing to trade earlier than previously assumed. Overall, the results question the appropriateness of the quarter‐end trading assumption and the validity of existing studies that employ it.
Enhanced charge storage characteristics of silicon nanocrystals fabricated by electron-beam coevaporation of Si and SiOx(x=1 or 2)
Authors: Chen Chen , Jia Rui , Li Weilong , Li Haofeng , Ye Tianchun , Liu Xinyu , Liu Ming , Kasai Seiya , Tamotsu Hashizume , Wu Nanjian
Published: November 2009

In this article, a simple and flexible electron-beam coevaporation (EBCE) technique has been reported of fabrication of the silicon nanocrystals (Si NCs) and their application to the nonvolatile memory. For EBCE, the Si and SiOx(x=1 or 2) were used as source materials. Transmission electron microscopy images and Raman spectra measurement verified the formation of the Si NCs. The average size and area density of the Si NCs can be adjusted by increasing the Si:O weight ratio in source material, which has a great impact on the crystalline volume fraction of the deposited film and on the charge storage characteristics of the Si NCs. A memory window as large as 6.6 V under ±8 V sweep voltage was observed for the metal-oxide-semiconductor capacitor structure with the embedded Si NCs.
Patterning decomposable polynorbornene with electron beam lithography to create nanochannels
Authors: Devlin Nicole R. , Brown Devin K. , Kohl Paul A.
Published: December 2009

Unity® 4671E sacrificial material is a decomposable negative tone polymer sensitive to ultraviolet radiation. In this study, it is shown that Unity® 4671E can also be patterned by electron beam lithography. Nanochannels with a width of 65nm and a pitch of 200nm have been fabricated. The developed Unity® 4671E patterns can thermally decompose and the products can permeate through the encapsulating material leaving nanocavities. This direct write electron-beam process has fewer processing steps than other published fabrication methods.
Retention-failure mechanism of TaN∕CuxO∕Cu resistive memory with good data retention capability
Authors: Wan H. J. , Zhou P. , Ye L. , Lin Y. Y. , Wu J. G. , Wu H. , Chi M. H.
Published: November 2009

Data retention characteristics and a failure mechanism of TaN∕CuxO∕Cu resistive memory device are investigated by a temperature-accelerated test method. Data retention capability at 85°C is sufficiently longer than 10years by using two different methods: simple extrapolation and Arrhenius equation. The high resistance state fails to low resistance state and low resistance state fails to high resistance state at the elevated temperature. It is attributed that different retention-failure mechanisms are responsible for high resistance state and low resistance state, respectively. A filament/charge trapped combined model is presented to clarify the retention-failure mechanism.
Efficacy of Cetuximab in the Treatment of Ménétrier’s Disease
Authors: Fiske William H. , Tanksley Jarred , Nam Ki Taek , Goldenring James R. , Slebos Robbert J. C. , Liebler Daniel C. , Abtahi Amir M. , La Fleur Bonnie , Ayers Gregory D. , Lind Christopher D. , Washington Mary K. , Coffey Robert J.
Published: November 2009

An antibody to the epidermal growth factor receptor successfully treats nine patients with a disease in which the lining of the stomach abnormally proliferates.
In Situ Regulation of DC Subsets and T Cells Mediates Tumor Regression in Mice
Authors: Ali Omar A. , Emerich Dwaine , Dranoff Glenn , Mooney David J.
Published: November 2009

An implanted copolymer matrix that incorporates inflammatory cytokines, an immune danger signal, and tumor antigens elicits an immune response network that can eradicate established tumors in mice.
Use of Forensic Methods Under Exigent Circumstances Without Full Validation
Authors: Schutzer Steven E. , Keim Paul , Czerwinski Ken , Budowle Bruce
Published: November 2009

Terrorist attacks and other emergency circumstances, such as infectious outbreaks, might require the use of forensic assays that have not been fully validated to identify the perpetrator(s) rapidly to stop further harm.
Ménétrier’s Disease Therapy: Rebooting Mucosal Signaling
Authors: Nalle Sam C. , Turner Jerrold R.
Published: November 2009

EGFR neutralization provides hope of sustained remission in Ménétrier’s disease patients.
Too Much of a Good Thing
Authors: Khidr Lily
Published: November 2009

Receptors gone wild may be linked to type 2 diabetes.
Deep Sequencing
Authors: Khidr Lily
Published: November 2009

Genes that cause rare disorders are uncovered by deep resequencing of protein-coding regions.
Catching Cancer Early
Authors: McDonald Heather B.
Published: November 2009

Islands of expanded clonal cells may be biomarkers of cancer progression in ulcerative colitis.
Evaluation of Two Immunoblot Assays and a Western Blot Assay for the Detection of Antisyphilis Immunoglobulin G Antibodies
Authors: Welch Ryan J. , Litwin Christine M.
Published: November 2009

In the present study, two immunoglobulin G (IgG) immunoblot assays and one IgG Western blot assay were compared to the rapid plasma reagin test (RPR), the fluorescent treponemal antibody absorption test (FTA-ABS), and the
Treponema pallidum
particle agglutination assay (TP-PA). The agreement levels of the Viramed, Virotech, and MarDx assays were 97.0%, 96.4%, and 99.4%, and the agreements of samples inconclusive by FTA-ABS and resolved by TP-PA were 91.7%, 83.3%, and 69.4%, respectively.
Long-Term Observation of Subunit Vaccine F1-rV270 against
Y
ersinia pestis
in Mice
Authors: Wang Zuyun , Zhou Lei , Qi Zhizhen , Zhang Qingwen , Dai Ruixia , Yang Yonghai , Cui Baizhong , Wang Hu , Yang Ruifu , Wang Xiaoyi
Published: November 2009

Long-term protection and antibody response for the subunit vaccine F1-rV270 were determined by using the mouse model. Antibodies to F1 and rV270 were still detectable over a period of 518 days. The complete protection against lethal challenge of
Yersinia pestis
could be achieved up to day 518 after primary immunization.
Validation and Clinical Application of a Molecular Method for Identification of
Histoplasma capsulatum
in Human Specimens in Colombia, South America
Authors: Muñoz Cesar , Gómez Beatriz L. , Tobón Angela , Arango Karen , Restrepo Angela , Correa Margarita M. , Muskus Carlos , Cano Luz Elena , González Angel
Published: November 2009

The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the
H
istoplasma
capsulatum
-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (
n
= 20), patients suspected of having respiratory disease with negative fungal cultures (
n
= 29), and patients with other proven infections (
n
= 60). Additionally, a sizable collection of DNA from cultures of
H
.
capsulatum
and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented ≥98% identity with
H.
capsulatum
. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of
H.
capsulatum
DNA is a useful test in areas where mycosis caused by this organism is endemic.
First Report of Sudden Death Due to Myocarditis Caused by Adenovirus Serotype 3
Authors: Treacy Ann , Carr Michael J. , Dunford Linda , Palacios Gustavo , Cannon Gemma A. , O'Grady Anthony , Moran Julie , Hassan Jaythoon , Loy Aisling , Connell Jeff , Devaney Deirdre , Kelehan Peter , Hall William W.
Published: November 2009

Myocarditis is a rare cause of sudden death in childhood. We describe the sudden death of a child from viral myocarditis, which we demonstrate was likely caused by an uncontrolled inflammatory response to a disseminated adenovirus serotype 3 infection originating in the tonsil.
Comparison of the AF146527 and B1 Repeated Elements, Two Real-Time PCR Targets Used for Detection of
Toxoplasma gondii
Authors: Wahab Tara , Edvinsson Benjamin , Palm Daniel , Lindh Johan
Published: November 2009

Previous studies have reported the increased sensitivity of PCR targeting AF146527 over that of PCR targeting the B1 gene for diagnosis of toxoplasmosis. The present study suggests that the AF146527 element was absent in 4.8% of human
Toxoplasma gondii
-positive samples tested. The data argue that the
B1
gene may be the preferred diagnostic target.
Nocardia pseudobrasiliensis
as an Emerging Cause of Opportunistic Infection after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Lebeaux David , Lanternier Fanny , Degand Nicolas , Catherinot Emilie , Podglajen Isabelle , Rubio Marie-Thérèse , Suarez Felipe , Lecuit Marc , Mainardi Jean-Luc , Lortholary Olivier
Published: November 2009

We report the case of a 55-year-old man who exhibited a nodular pneumonia 4 months after an allogeneic hematopoietic stem cell transplantation. Culture of the bronchoalveolar lavage fluid revealed
Nocardia pseudobrasiliensis
. This recently described carbapenem-resistant species should be included in the differential diagnosis of fungal infection in this setting.
Incidence, Diversity, and Molecular Epidemiology of Sapoviruses in Swine across Europe
Authors: Reuter Gábor , Zimšek-Mijovski Janet , Poljšak-Prijatelj Mateja , Di Bartolo Ilaria , Ruggeri Franco Maria , Kantala Tuija , Maunula Leena , Kiss István , Kecskeméti Sándor , Halaihel Nabil , Buesa Javier , Johnsen Christina , Hjulsager Charlotte K. , Larsen Lars E. , Koopmans Marion , Böttiger Blenda
Published: November 2009

Porcine sapovirus is an enteric calicivirus in domestic pigs that belongs to the familyCaliciviridae. Some porcine sapoviruses are genetically related to human caliciviruses, which has raised public health concerns over animal reservoirs and the potential cross-species transmission of sapoviruses. We report on the incidence, genetic diversity, and molecular epidemiology of sapoviruses detected in domestic pigs in a comprehensive study conducted in six European countries (Denmark, Finland, Hungary, Italy, Slovenia, and Spain) between 2004 and 2007. A total of 1,050 swine fecal samples from 88 pig farms were collected and tested by reverse transcription-PCR for sapoviruses, and positive findings were confirmed by sequencing. Sapoviruses were detected in 80 (7.6%) samples collected on 39 (44.3%) farms and in every country. The highest prevalence was seen among piglets aged 2 to 8 weeks, and there was no significant difference in the proportion of sapovirus-positive findings for healthy animals and animals with diarrhea in Spain and Denmark (the only countries where both healthy animals and animals with diarrhea were tested). On the basis of the sequence of the RNA polymerase region, highly heterogeneous populations of viruses representing six different genogroups (genogroups III, VI, VII, and VIII, including potential new genogroups IX and X) were identified, with a predominance of genogroup GIII (50.6%). Genogroup VIII, found in five of the six countries, had the highest degree of homology (up to 66% at the amino acid level) to human sapovirus strains. Sapoviruses are commonly circulating and endemic agents in swine herds throughout Europe. Highly heterogeneous and potential new genogroups of sapoviruses were found in pigs; however, no “human-like” sapoviruses were detected.
Characterization of Baseline Methicillin-Resistant
Staphylococcus aureus
Isolates Recovered from Phase IV Clinical Trial for Linezolid
Authors: Mendes Rodrigo E. , Sader Helio S. , Deshpande Lalitagauri M. , Diep Binh An , Chambers Henry F. , Jones Ronald N.
Published: November 2009

A total of 565 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates were collected mostly from Europe and the Americas (2004 to 2007) during a phase IV clinical trial comparing linezolid with vancomycin for the treatment of complicated skin and skin structure infections proven to be due to MRSA. The isolates were tested for their susceptibilities by the broth microdilution method, they were tested for inducible clindamycin resistance by the D-test, and they were screened for heterogeneous resistance to vancomycin (heterogeneously vancomycin-intermediate
S. aureus
[hVISA]) by the Etest macromethod. The isolates were evaluated for the MRSA genotype by pulsed-field gel electrophoresis, staphylococcal protein A (
spa
) typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome
mec
(SCC
mec
) typing. All isolates were inhibited by 4 μg/ml of linezolid (MIC
50
and MIC
90
, 2 and 4 μg/ml, respectively). The vast majority of isolates (92.4%) were resistant to erythromycin, and high clindamycin resistance rates were observed (28.5% constitutive and 16.3% inducible). Only 1.0% of the isolates were hVISA. Isolates from the United States were predominantly USA300 sequence type 8 (ST8)-SCC
mec
type IV (78.5%), followed by a lower prevalence of USA100 ST5-SCC
mec
type II isolates (14.2%). Strains belonging to the ST5 lineage were widely distributed in Portugal, South American countries, and Mexico. MRSA strains belonging to ST8-SCC
mec
type IV predominated in Russia (80.0%) and also emerged in Venezuela and Colombia. The epidemic MRSA type 15 clone predominated in the United Kingdom (55.6%) and Spain (100%). In addition, a new MLST profile (ST1071) was observed in South Africa. This study demonstrated the presence of major clones in particular regions (ST8 in the United States, ST5 in Latin America and Portugal, ST22 in Spain and the United Kingdom); however, emerging clones were identified, suggesting that the epidemiology of MRSA continues to evolve.
Prevalence of Hepatitis E Virus in Chinese Blood Donors
Authors: Guo Qing-Shun , Yan Qiang , Xiong Jun-Hui , Ge Sheng-Xiang , Shih J. Wai-Kuo , Ng Mun-Hon , Zhang Jun , Xia Ning-Shao
Published: November 2009

A point prevalence study of hepatitis E virus (HEV) in Chinese blood donors was conducted, and the prevalences of antibodies against HEV immunoglobulin G (IgG) and IgM among Chinese blood donors were 32.60% and 0.94%, respectively. HEV viremia was 0.07%.
Production of the Subtilase AB
5
Cytotoxin by Shiga Toxin-Negative
Escherichia coli
Authors: Tozzoli Rosangela , Caprioli Alfredo , Cappannella Stefano , Michelacci Valeria , Marziano Maria Luisa , Morabito Stefano
Published: November 2009

The subtilase cytotoxin (SubAB) is an AB
5
toxin described in certain Shiga toxin (Stx)-producing
Escherichia coli
(STEC) strains that usually lack the locus for enterocyte effacement (LEE). We report for the first time the production of SubAB by two Stx-negative
E. coli
strains, isolated from unrelated cases of childhood diarrhea. The characterization of the SubAB-coding genes showed a 90% nucleotide sequence similarity with that of the prototype
subAB
, located on the virulence plasmid of the STEC O113 strain 98NK2 (pO113). In both strains,
subAB
was physically associated with
tia
, an invasion genetic determinant of enterotoxigenic
E. coli
. The strains were negative for the
saa
gene, encoding an adhesin located on pO113 and present in many of the SubAB-positive strains described so far. PCR screening of 61 STEC and 100 Stx-negative
E. coli
strains in our collection revealed the presence of
subAB
in five LEE-negative STEC strains but not in the Stx-negative strains.
subAB
was contiguous to
tia
in three of the positive strains, which were all negative for
saa
. These results indicate that SubAB production is not restricted to STEC and suggest that a
subAB
-
tia
putative pathogenicity island is involved in the dissemination of
subAB
genes, as an alternative to plasmid pO113.
Molecular Characteristics of Occult Hepatitis B Virus from Blood Donors in Southeast China
Authors: Yuan Quan , Ou Shan-Hai , Chen Chang-Rong , Ge Sheng-Xiang , Pei Bin , Chen Qing-Rui , Yan Qiang , Lin Yong-Cai , Ni Hong-Ying , Huang Cheng-Hao , Yeo Anthony E. T. , Shih James W. K. , Zhang Jun , Xia Ning-Shao
Published: November 2009

The characteristics of 30 carriers with occult hepatitis B virus (HBV) infection (OBI) were compared with those of 30 individuals diagnosed as being HBV carriers at the time of blood donation, 60 asymptomatic carriers, and 60 chronic hepatitis patients. The prevalence of genotype C was significantly higher in carriers with OBIs than in any other HBsAg-positive (HBsAg
+
) group (
P
< 0.001). Specific amino acid substitutions in the regions from amino acids 117 to 121 and amino acids 144 to 147 located in the major hydrophilic region of the S gene were associated with carriers with OBIs (
P
< 0.01 for carriers with OBIs versus HBsAg
+
donors, carriers with OBIs versus HBsAg
+
asymptomatic carriers, and carriers with OBIs versus HBsAg
+
chronic hepatitis patients). G145R was the major variation in the HBV isolates responsible for local occult HBV infections.
Multicenter Study Evaluating the Role of Enterococci in Secondary Bacterial Peritonitis
Authors: Cercenado Emilia , Torroba Luis , Cantón Rafael , Martínez-Martínez Luis , Chaves Fernando , García-Rodríguez Jose Angel , Lopez-Garcia Carmen , Aguilar Lorenzo , García-Rey César , García-Escribano Nuria , Bouza Emilio
Published: November 2009

A 1-year prospective multicenter study was performed to explore the significance of the presence of enterococci in cultures of peritoneal fluid from patients with secondary bacterial peritonitis in seven Spanish hospitals. The clinical records of patients with positive peritoneal fluid cultures were reviewed and distributed into cases (patients with cultures yielding enterococci) and controls (patients with cultures not yielding enterococci). Of a total of 158 records, 38 (24.1%) were cases and 120 (75.9%) were controls. The percentages or the scores (cases versus controls) for the variables included in the multivariate analysis were as follows: age of >50 years, 89.5% versus 68.3%; malignancy, 39.5% versus 18.3%; chronic obstructive pulmonary disease (COPD), 15.8% versus 4.2%; postoperative peritonitis, 55.3% versus 30.1%; nosocomial onset, 57.9% versus 34.2%; a higher Charlson comorbidity index, 3.29 ± 3.38 versus 1.84 ± 2.31; APACHE II score, 10.71 ± 4.37 versus 8.76 ± 5.49; ultimately or rapidly fatal disease, 63.2% versus 34.8%; need for surgical reintervention, 36.1% versus 15.1%; and admission to an intensive care unit, 45.9% versus 30.8%. In the multivariate analysis, enterococci were associated only with postoperative peritonitis (
P
= 0.009; odds ratio [OR] = 5.0; 95% confidence interval [CI] = 1.49 to 16.80), a higher Charlson comorbidity index (
P
= 0.002; OR = 1.30; 95% CI = 1.11 to 1.54), and COPD (
P
= 0.046; OR = 6.50; 95% CI = 1.04 to 40.73). The results of this study showed that enterococci were associated with comorbidity. An association with mortality could not be demonstrated.
Enhanced Detection of Enteroviruses in Clinical Samples by Reverse Transcription-PCR Using Complementary Locked Primer Technology
Authors: Hong JiYoung , Kang Byunghak , Kim Ahyoun , Hwang Seoyoun , Lee Sunwha , Kim Jonghyen , Lee Hyun-Young , Kang Sang-Hyeon , Cheon Doo-Sung
Published: November 2009

To increase detection sensitivity, we modified primers using complementary locked primer (CLP) technology. The sensitivity of the reverse transcription-PCR (RT-PCR) with CLP-modified primers was 10- to 100-fold higher than that of RT-PCR without these primers. CLP-modified primers can increase sensitivity, providing a widely accessible method for molecular diagnosis.
Occurrence of Mixed Populations of Influenza A Viruses That Can Be Maintained through Transmission in a Single Host and Potential for Reassortment
Authors: Furuse Yuki , Suzuki Akira , Kishi Makiko , Nukiwa Nao , Shimizu Midori , Sawayama Rumi , Fuji Naoko , Oshitani Hitoshi
Published: November 2009

Reassortment, which is the rearrangement of viral gene segments in a host cell infected with two different viruses, is an important mechanism for the evolution of influenza viruses. Mixed infections with multiple virus types could lead to reassortment. To better understand the occurrence of quasispecies in a single host, we investigated mixed infections in individual isolates of seasonal influenza A viruses using amantadine sensitivity as a marker. We cultured viruses with amantadine and performed sequencing, restriction fragment length polymorphism analysis, cloning, and quantitative PCR to detect mixed populations. Culturing with amantadine showed evidence of a high number of mixed populations, while the other assays could hardly detect mixed populations. The existence of quasispecies in each isolate was common. However, the proportion of these, which can be less than 1%, is too low to be detected by conventional methods. Such mixed populations in which reassortment occurs may have a significant role in the evolution of viruses.
Correlation of Bacterial Colonization Status between Mother and Child: the Generation R Study
Authors: Lebon Ankie , Moll Henriëtte A. , Tavakol Mehri , van Wamel Willem J. , Jaddoe Vincent W. V. , Hofman Albert , Verbrugh Henri A. , van Belkum Alex
Published: November 2009

Determinants of bacterial colonization in children have been described. In the Generation R Study, a population-based cohort study, we determined whether the colonization statuses of mothers and children are correlated. Such a correlation was observed for
Staphylococcus aureus
and
Haemophilus influenzae
. Direct transmission, genetic susceptibility and/or unidentified environmental factors may play a role here.
High-Throughput Pooling and Real-Time PCR-Based Strategy for Malaria Detection
Authors: Taylor Steve M. , Juliano Jonathan J. , Trottman Paul A. , Griffin Jennifer B. , Landis Sarah H. , Kitsa Paluku , Tshefu Antoinette K. , Meshnick Steven R.
Published: November 2009

Molecular assays can provide critical information for malaria diagnosis, speciation, and drug resistance, but their cost and resource requirements limit their application to clinical malaria studies. This study describes the application of a resource-conserving testing algorithm employing sample pooling for real-time PCR assays for malaria in a cohort of 182 pregnant women in Kinshasa. A total of 1,268 peripheral blood samples were collected during the study. Using a real-time PCR assay that detects all
Plasmodium
species, microscopy-positive samples were amplified individually; the microscopy-negative samples were amplified after pooling the genomic DNA (gDNA) of four samples prior to testing. Of 176 microscopy-positive samples, 74 were positive by the real-time PCR assay; the 1,092 microscopy-negative samples were initially amplified in 293 pools, and subsequently, 35 samples were real-time PCR positive (3%). With the real-time PCR result as the referent standard, microscopy was 67.9% sensitive (95% confidence interval [CI], 58.3% to 76.5%) and 91.2% specific (95% CI, 89.4% to 92.8%) for malaria. In total, we detected 109 parasitemias by real-time PCR and, by pooling samples, obviated over 50% of reactions and halved the cost of testing. Our study highlights both substantial discordance between malaria diagnostics and the utility and parsimony of employing a sample pooling strategy for molecular diagnostics in clinical and epidemiologic malaria studies.
Mycobacterium tuberculosis
Spoligotypes in Monterrey, Mexico
Authors: Molina-Torres Carmen A. , Moreno-Torres Elisa , Ocampo-Candiani Jorge , Rendon Adrian , Blackwood Kym , Kremer Kristin , Rastogi Nalin , Welsh Oliverio , Vera-Cabrera Lucio
Published: November 2009

Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180
Mycobacterium tuberculosis
clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS
6110
-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (
n
= 8), SIT92 (
n
= 3), SIT70 (
n
= 2), and SIT3038 (
n
= 1). When the molecular detection of mutations in the
katG
and
rpoB
genes were analyzed in these isolates with low copy numbers of IS
6110
, only two isolates shared the same IS
6110
, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of
M. tuberculosis
isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.
Role of AKT Kinase in Measles Virus Replication
Authors: Carsillo Mary , Kim Dhohyung , Niewiesk Stefan
Published: November 2009

Many RNA and DNA viruses activate serine-threonine kinase AKT to increase virus replication. In contrast, measles virus (MV) infection leads to downregulation of AKT. This is thought to be beneficial for the virus because it correlates with immune suppression. To determine whether this is a sacrifice for the virus, we used a recombinant virus and transfected cells expressing constitutively active AKT and evaluated its effect on virus replication.
In vitro
, overexpression of AKT did not influence virus replication but did affect (cell-type dependent) virus release.
In vivo
, the recombinant virus did not abrogate inhibition of proliferation of spleen cells from MV-infected cotton rats.
Immunization with Live Attenuated Influenza Viruses That Express Altered NS1 Proteins Results in Potent and Protective Memory CD8
+
T-Cell Responses
Authors: Mueller Scott N. , Langley William A. , Carnero Elena , García-Sastre Adolfo , Ahmed Rafi
Published: November 2009

The generation of vaccines that induce long-lived protective immunity against influenza virus infections remains a challenging goal. Ideally, vaccines should elicit effective humoral and cellular immunity to protect an individual from infection or disease. Cross-reactive T- and B-cell responses that are elicited by live virus infections may provide such broad protection. Optimal induction of T-cell responses involves the action of type I interferons (IFN-I). Influenza virus expressed nonstructural protein 1 (NS1) functions as an inhibitor of IFN-I and promotes viral growth. We wanted to examine the priming of CD8
+
T-cell responses to influenza virus in the absence of this inhibition of IFN-I production. We generated recombinant mouse-adapted influenza A/PR/8/34 viruses with NS1 truncations and/or deletions that also express the gp33-41 epitope from lymphocytic choriomeningitis virus. Intranasal infection of mice with the attenuated viruses primed long-lived T- and B-cell responses despite significantly reduced viral replication in the lungs compared to wild-type virus. Antigen-specific CD8
+
T cells expanded upon rechallenge and generated increased protective memory T-cell populations after boosting. These results show that live attenuated influenza viruses expressing truncated NS1 proteins can prime protective immunity and may have implications for the design of novel modified live influenza virus vaccines.
Hemagglutinin-Pseudotyped Green Fluorescent Protein-Expressing Influenza Viruses for the Detection of Influenza Virus Neutralizing Antibodies
Authors: Martínez-Sobrido Luis , Cadagan Richard , Steel John , Basler Christopher F. , Palese Peter , Moran Thomas M. , García-Sastre Adolfo
Published: November 2009

Influenza virus is a highly contagious virus that causes yearly epidemics and occasional pandemics of great consequence. Influenza virus neutralizing antibodies (NAbs) are promising prophylactic and therapeutic reagents. Detection of NAbs in serum samples is critical to evaluate the prevalence and spread of new virus strains. Here we describe the development of a simple, sensitive, specific, and safe screening assay for the rapid detection of NAbs against highly pathogenic influenza viruses under biosafety level 2 (BSL-2) conditions. This assay is based on the use of influenza viruses in which the hemagglutinin (HA) gene is replaced by a gene expressing green fluorescent protein (GFP). These GFP-expressing influenza viruses replicate to high titers in HA-expressing cell lines, but in non-HA-expressing cells, their replication is restricted to a single cycle.
Nonpathogenesis of Simian Immunodeficiency Virus Infection Is Associated with Reduced Inflammation and Recruitment of Plasmacytoid Dendritic Cells to Lymph Nodes, Not to Lack of an Interferon Type I Response, during the Acute Phase
Authors: Campillo-Gimenez Laure , Laforge Mireille , Fay Michèle , Brussel Audrey , Cumont Marie-Christine , Monceaux Valérie , Diop Ousmane , Lévy Yves , Hurtrel Bruno , Zaunders John , Corbeil Jacques , Elbim Carole , Estaquier Jérôme
Published: November 2009

Divergent Toll-like receptor 7 (TLR7) and TLR9 signaling has been proposed to distinguish pathogenic from nonpathogenic simian immunodeficiency virus infection in primate models. We demonstrate here that increased expression of type I interferon in pathogenic rhesus macaques compared to nonpathogenic African green monkeys was associated with the recruitment of plasmacytoid dendritic cells in the lymph nodes and the presence of an inflammatory environment early after infection, instead of a difference in the TLR7/9 response.
A Single Amino Acid Difference in Human APOBEC3H Variants Determines HIV-1 Vif Sensitivity
Authors: Zhen Anjie , Wang Tao , Zhao Ke , Xiong Yong , Yu Xiao-Fang
Published: November 2009

Several variants of APOBEC3H (A3H) have been identified in different human populations. Certain variants of this protein are particularly potent inhibitors of retrotransposons and retroviruses, including HIV-1. However, it is not clear whether HIV-1 Vif can recognize and suppress the antiviral activity of A3H variants, as it does with other APOBEC3 proteins. We now report that A3H_Haplotype II (HapII), a potent inhibitor of HIV-1 in the absence of Vif, can indeed be degraded by HIV-1 Vif. Vif-induced degradation of A3H_HapII was blocked by the proteasome inhibitor MG132 and a Cullin5 (Cul5) dominant negative mutant. In addition, Vif mutants that were incapable of assembly with the host E3 ligase complex factors Cul5, ElonginB, and ElonginC were also defective for A3H_HapII suppression. Although we found that Vif hijacks the same E3 ligase to degrade A3H_HapII as it does to inactivate APOBEC3G (A3G) and APOBEC3F (A3F), more Vif motifs were involved in A3H_HapII inactivation than in either A3G or A3F suppression. In contrast to A3H_HapII, A3H_Haplotype I (HapI), which differs in only three amino acids from A3H_HapII, was resistant to HIV-1 Vif-mediated degradation. We also found that residue 121 was critical for determining A3H sensitivity and binding to HIV-1 Vif.
Viral Oncolysis That Targets Raf-1 Signaling Control of Nuclear Transport
Authors: Riolobos Laura , Valle Noelia , Hernando Eva , Maroto Beatriz , Kann Michael , Almendral José M.
Published: November 2009

The central role of Raf protein kinase isoforms in human cancer demands specific anti-Raf therapeutic inhibitors. Parvoviruses are currently used in experimental cancer therapy due to their natural oncotropism and lytic life cycle. In searching for mechanisms underlying parvovirus oncolysis, we found that trimers of the major structural protein (VP) of the parvovirus minute virus of mice (MVM), which have to be imported into the nucleus for capsid assembly, undergo phosphorylation by the Raf-1 kinase. Purified Raf-1 phosphorylated the capsid subunits
in vitro
to the two-dimensional pattern found in natural MVM infections. VP trimers isolated from mammalian cells translocated into the nucleus of digitonin-permeabilized human cells. In contrast, VP trimers isolated from insect cells, which are devoid of Raf-1, were neither phosphorylated nor imported into the mammalian nucleus. However, the coexpression of a constitutively active Raf-1 kinase in insect cells restored VP trimer phosphorylation and nuclear transport competence. In MVM-infected normal and transformed cells, Raf-1 inhibition resulted in cytoplasmic retention of capsid proteins, preventing their nuclear assembly and progeny virus maturation. The level of Raf-1 activity in cancer cells was consistent with the extent of VP specific phosphorylation and with the permissiveness to MVM infection. Thus, Raf-1 control of nuclear translocation of MVM capsid assembly intermediates provides a novel target for viral oncolysis. MVM may reinforce specific therapies against frequent human cancers with deregulated Raf signaling.
Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry
Authors: Hernaez Bruno , Alonso Covadonga
Published: November 2009

African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na
+
/H
+
ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.
Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags
Authors: Teterina Natalya L. , Levenson Eric A. , Ehrenfeld Ellie
Published: November 2009

The 2A proteins of the
Picornaviridae
enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in
cis
and in several cellular proteins in
trans
. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of
Discosoma
sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in
trans
and supported translation and replication of viral RNA, both
in vitro
and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.
The Alpha/Beta Interferon Receptor Provides Protection against Influenza Virus Replication but Is Dispensable for Inflammatory Response Signaling
Authors: Goodman Alan G. , Zeng Hui , Proll Sean C. , Peng Xinxia , Cillóniz Cristian , Carter Victoria S. , Korth Marcus J. , Tumpey Terrence M. , Katze Michael G.
Published: November 2009

The innate immune response provides the first line of defense against foreign pathogens by responding to molecules that are a signature of a pathogenic infection. Certain RNA viruses, such as influenza virus, produce double-stranded RNA as an intermediate during the replication life cycle, which activates pathogen recognition receptors capable of inducing interferon production. By engaging interferon receptors, interferon activates the JAK-STAT pathway and results in the positive feedback of interferon production, amplifying the response to viral infection. To examine how deficiencies in interferon signaling affect the cellular response to infection, we performed influenza virus infections of mouse embryonic fibroblasts lacking the alpha/beta interferon receptor, the gamma interferon receptor, or both. In the absence of the alpha/beta interferon receptor, we observed increased viral replication but decreased activation of PKR, Stat1, and NF-κB; the presence or absence of the gamma interferon receptor did not exhibit discernible differences in these readouts. Analysis of gene expression profiles showed that while cells lacking the alpha/beta interferon receptor exhibited decreased levels of transcription of antiviral genes, genes related to inflammatory and apoptotic responses were transcribed to levels similar to those of cells containing the receptor. These results indicate that while the alpha/beta interferon receptor is needed to curb viral replication, it is dispensable for the induction of certain inflammatory and apoptotic genes. We have identified potential pathways, via interferon regulatory factor 3 (IRF3) activation or
Hoxa13
,
Polr2a
,
Nr4a1
, or
Ing1
induction, that contribute to this redundancy. This study illustrates another way in which the host has evolved to establish several overlapping mechanisms to respond to viral infections.
Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons
Authors: Österlund Pamela , Pirhonen Jaana , Ikonen Niina , Rönkkö Esa , Strengell Mari , Mäkelä Sanna M. , Broman Mia , Hamming Ole J. , Hartmann Rune , Ziegler Thedi , Julkunen Ilkka
Published: November 2009

In less than 3 months after the first cases of swine origin 2009 influenza A (H1N1) virus infections were reported from Mexico, WHO declared a pandemic. The pandemic virus is antigenically distinct from seasonal influenza viruses, and the majority of human population lacks immunity against this virus. We have studied the activation of innate immune responses in pandemic virus-infected human monocyte-derived dendritic cells (DC) and macrophages. Pandemic A/Finland/553/2009 virus, representing a typical North American/European lineage virus, replicated very well in these cells. The pandemic virus, as well as the seasonal A/Brisbane/59/07 (H1N1) and A/New Caledonia/20/99 (H1N1) viruses, induced type I (alpha/beta interferon [IFN-α/β]) and type III (IFN-λ1 to -λ3) IFN, CXCL10, and tumor necrosis factor alpha (TNF-α) gene expression weakly in DCs. Mouse-adapted A/WSN/33 (H1N1) and human A/Udorn/72 (H3N2) viruses, instead, induced efficiently the expression of antiviral and proinflammatory genes. Both IFN-α and IFN-β inhibited the replication of the pandemic (H1N1) virus. The potential of IFN-λ3 to inhibit viral replication was lower than that of type I IFNs. However, the pandemic virus was more sensitive to the antiviral IFN-λ3 than the seasonal A/Brisbane/59/07 (H1N1) virus. The present study demonstrates that the novel pandemic (H1N1) influenza A virus can readily replicate in human primary DCs and macrophages and efficiently avoid the activation of innate antiviral responses. It is, however, highly sensitive to the antiviral actions of IFNs, which may provide us an additional means to treat severe cases of infection especially if significant drug resistance emerges.