bluuebunny/crossref_metadata_2025 huggingface dataset:
ULTRASONOGRAPHIC APPEARANCE OF THE LOWER URINARY TRACT IN FIFTEEN NORMAL HORSES
Authors: DIAZ OLGA SECO , SMITH GARY , REEF VIRGINIA B.
Published: October 2007

The purpose of this study was to describe the normal ultrasonographic appearance of the caudal portion of the equine ureters, pelvic urethra, and urinary bladder and to assess ureteral contractility. Fifteen horses with no evidence of urinary tract disease (five females, five intact males and five geldings) were studied. The lower urinary tract was evaluated ultrasonographically using a transrectal approach. The normal ultrasonographic appearance and anatomic location of structures of the lower urinary tract were evaluated and anatomic relationships described. Both ureters were identified dorsal to the bladder and at their openings into the bladder. Their relationship and different appearance from the male deferent ducts/ampullae and vesicular glands was noted. Ultrasonographic measurements of the diameter of the ureters and urethra were obtained and the ureteral, urethral, and urinary bladder wall thickness recorded. The normal parameters established in this study will be useful during sonographic examination of horses with suspected urinary tract disease. Transrectal ultrasonographic examination is valuable in assessing the presence and frequency of ureteral contractions as well as in detecting structural abnormalities of the lower urinary tract. The use of high‐frequency transrectal ultrasound transducers allows for excellent visualization of the structures of the equine lower urinary tract.
ASSOCIATION BETWEEN MALIGNANCY AND SONOGRAPHIC HETEROGENEITY IN CANINE AND FELINE ABDOMINAL LYMPH NODES
Authors: KINNS JENNIFER , MAI WILFRIED
Published: October 2007

Abdominal lymph node enlargement is frequently noted sonographically. Certain sonographic features can be used to suggest whether lymphadenopathy is more likely benign or malignant. Specific changes in size, shape, echogenicity, and Doppler flow patterns have an association with malignancy. In this retrospective case–control study, the association between abdominal lymph node heterogeneity and malignancy was evaluated. Twenty‐three canine and 18 feline patients with ultrasonographically heterogeneous abdominal nodes were evaluated for presence of benign or malignant lymphadenopathy. Controls were animals with lymph node enlargement of uniform echogeneity. Twenty‐one (91%) of heterogeneous canine lymph nodes were malignant, and there was a significant association between heterogeneity and malignancy in canine abdominal lymph nodes (P=0.024). Seven (63%) heterogeneous feline lymph nodes were malignant, with no significant association between heterogeneity and malignancy (P=0.537).
COMPARISON OF DOPPLER‐DERIVED AORTIC VELOCITIES OBTAINED FROM VARIOUS TRANSDUCER SITES IN HEALTHY DOGS AND CATS
Authors: RIESEN SABINE C. , DOHERR MARCUS G. , LOMBARD CHRISTOPHE W.
Published: October 2007

In normal dogs and dogs with subaortic stenosis, it is known that the subcostal transducer site provides higher left ventricular ejection velocities than does the left apical site. We hypothesized that aortic flow velocities could also be obtained from the right parasternal long‐axis view, optimized for the placement of the Doppler cursor as parallel as possible into the aortic root. In 15 healthy dogs and 13 healthy cats, high‐pulsed repetition frequency Doppler flow velocity measurements in the proximal aorta were performed using two‐dimensional echocardiographic guidance. The mean [±standard error of the mean (SEM)] peak aortic flow velocities in healthy dogs were as follows: subcostal site 1.46±0.05 m/s; apical site 1.12±0.06 m/s; right parasternal long‐axis site 1.09±0.05 m/s. In healthy cats, the following peak aortic flow velocities were observed: apical site 0.87±0.03 m/s; right parasternal long‐axis site 0.87±0.03 m/s. Aortic flow velocities obtained from the subcostal site were significantly higher in healthy dogs than those obtained from the left apical and right parasternal long‐axis site (P<0.001). There was no statistical difference between the peak aortic flow velocities obtained from right parasternal long‐axis and left apical transducer position in all groups. We conclude therefore that right parasternal long‐axis and left apical‐derived aortic flow velocities are similar and may be used interchangeably in healthy dogs and cats.
PERIRENAL EFFUSION IN DOGS AND CATS WITH ACUTE RENAL FAILURE
Authors: HOLLOWAY ANDREW , O'BRIEN ROBERT
Published: October 2007

Perirenal fluid accumulation has been described as an ultrasonographic feature of urine leakage, hemorrhage, abscessation, or neoplasia. The purpose of this retrospective study was to report perirenal effusion as an additional ultrasonographic finding in canine and feline patients with acute renal failure. The causes of acute renal failure in 18 patients included nephrotoxicity (4), leptospirosis (3), ureteral obstruction (2), renal lymphoma (2), ureteronephrolithiasis (2), prostatic urethral obstruction (1) and interstitial nephritis and ureteritis (1). An underlying cause was not identified in three patients. The sonographic finding of perirenal fluid was bilateral in 15 patients. Unilateral perirenal fluid was identified ipsilateral to the site of ureteric obstruction in two patients. Large effusions extended into the caudal retroperitoneal space. Additional sonographic findings suggestive of renal parenchymal disease included mild (5), moderate (5) or severe (2) pyelectasia, increased renal echogenicity (11), increased (9) or decreased renal size (2) and ureteral and/or renal calculi (3). There did not appear to be an association between the volume of perirenal fluid and the severity of renal dysfunction. All patients with large effusions underwent euthanasia. Perirenal fluid developing in acute renal failure is thought to be an ultrafiltrate associated with tubular back‐leak into the renal interstitium that overwhelms lymphatic drainage within the perirenal and retroperitoneal connective tissues although obstruction to urine flow may also play a role. Localized perirenal retroperitoneal free fluid may be a useful ultrasonographic feature to assist with the characterization of, and determination of prognosis in, patients with suspected renal disease.
EFFECTS OF MEASUREMENT OF PLASMA ACTIVITY INPUT ON NORMALIZATION OF GLOMERULAR FILTRATION RATE TO PLASMA VOLUME IN DOGS
Authors: KAMPA NARUEPON , LORD PETER , MARIPUU ENN , HOPPE ASTRID
Published: October 2007

Glomerular filtration rate (GFR) normalized to body fluid volumes to adjust for differing body size and conformation is more physiologically correct than a relationship with body weight (BW). GFR can be normalized to plasma volume by a renographic method that uses the Rutland–Patlak plot with plasma activity and kidney activity inputs. A plasma time–activity curve is obtained from a region of interest (ROI) of the left ventricle (LV), the size of which is in theory not critical. The aims of the study were to evaluate the effect of different LV ROI sizes, the effect of extravascular activity in the thorax over the LV ROI, and different time intervals for the semilogarithmic LV plot. Seventy‐two scintigrams were used, with three different‐sized automatic and a manual LV ROI, all with and without subtracting extravascular activity, and with LV curve time intervals of 30–120 s and 60–240 s. GFR/plasma volume was not affected by LV ROI sizes but significantly affected by extravascular activity subtraction and different time intervals. Subtracting extravascular activity from the LV ROI did not improve precision, but increased variability caused by different LV ROI sizes and time intervals chosen for the LV plot. The ROI for measuring extravascular activity apparently contained a considerable and variable intravascular component, which when subtracted, created noisy and unreliable LV curves. Manual LV ROI, without extravascular subtraction, and a time interval for LV input between 1 and 4 min are recommended as they gave the least variability determined by statistical analysis. With these methods, normal individual GFR/plasma volume in normal beagle dogs was 29.2±6.5 ml/min/l.
Original Articles: Epidemiology and Neurobiology of Female Sexual Dysfunction
Authors: Clayton Anita H.
Published: October 2007

Introduction

Although several conceptual frameworks for female sexual dysfunction (FSD) have been advanced, there still is considerable disagreement over what constitutes a normal vs. abnormal response. Sexual dysfunction is a disturbance in sexual functioning involving one or multiple phases of the sexual response cycle or pain associated with sexual activity, while a sexual disorder includes both dysfunction and marked distress.

Aim

Review the literature regarding the epidemiology and neurobiology of FSD.

Methods

Review of the literature.

Results

While a wide range of epidemiologic studies has been published, it is still difficult to determine the scope of FSD and sexual disorders in the general population. It is becoming clear that good sexual health is associated with good physical and mental health as well as compatible relationships with one's sexual partner. Central nervous system (CNS) control of the sexual response is a relatively new area of scientific exploration.

Conclusions

We are improving our understanding of the contributions of the CNS neuroendocrine and neurotransmitter systems that modulate sexual behavior.
Clinical Application of CNS-Acting Agents in FSD
Authors: Perelman Michael A.
Published: October 2007

Introduction

Human sexual response involves a complex sequencing of interrelated mind/body processes. Few treatment options exist that address the complex multilayered etiological determinants of female sexual dysfunction (FSD).

Aim

Review and evaluate the clinical application of centrally acting agents for the treatment of FSD, with particular emphasis on recently released data on bremelanotide.

Methods

Review of literature on the treatment of FSD.

Results

Approaches to FSD treatment have ranged from psychological counseling through a variety of medical interventions along the physiological pathways of sexual function and dysfunction. Despite a great deal of research, especially into hormonal and vasoactive substances, few therapeutics have broad consistent applicability and have withstood scientific scrutiny. Recent clinical trials have investigated the potential role of agents which act on the central nervous system for the treatment of FSD.

Conclusions

While the recent data supporting the therapeutic use of centrally acting agents as a monotherapy for FSD are cautiously encouraging, there would seem to be tremendous promise for these centrally acting compounds to be integrated with treatment approaches that utilize other pathways in a multilayered, individualized approach to care.
Difficulties Experienced by Families With Disabled Children
Authors: Sen Esine , Yurtsever Sabire
Published: October 2007

PURPOSE. This study was designed to determine the difficulties experienced by families with disabled children.

DESIGN AND METHOD. This descriptive research study was carried out in one public and two private rehabilitation centers. A questionnaire was used for data collection.

RESULTS. Families did not have enough knowledge about their child's condition. Mothers felt severe sadness, and they indicated that after having a disabled child, their social life, working life, and family relationships were all affected. Families also have financial problems.

PRACTICE IMPLICATIONS. Parents with a disabled child need support from professionals. Nurses have a vital role in providing support for these families.
‘Driving people crazy’: A geography of boy racers in Christchurch, New Zealand
Authors: Falconer Ryan , Kingham Simon
Published: October 2007

Abstract:  So‐called ‘boy racers’ form a subculture, which is subject to ongoing public scrutiny and media attention, usually negative. This study aimed to deconstruct the term ‘boy racer’ and to explore the subculture in detail, within the context of Christchurch, New Zealand, using a phenomenological ethnographic approach. This included conducting semistructured interviews and observing the activities of participants. Results show the subculture has a relatively diverse membership and a range of associated behaviours, activities and attitudes. Moreover, performances within the subculture are linked to place, and are seen by other members of the community as threats to normative boundaries.
Sporting narratives and globalization: Making links between the All Black tours of 1905 and 2005
Authors: Lewis Nicolas , Winder Gordon
Published: October 2007

Abstract:  In this paper, we use commodity chain ideas to compare the All Blacks rugby tour of Great Britain and Ireland in 2005 with the formative 1905 tour, and to relate their cultural economy to their political economy. We argue that ‘globalization’ has become imagined and institutionalized in new ways that more thoroughly exploit rugby's production values. We highlight the value‐adding potential of a 100‐year‐old tension between a colonial/imperial and a national identity. Our point is that fresh insights can be drawn from bringing cultural economy sensitivities to political economy approaches and concerns with identity and consumption to those of value and production.
Vegetation Change and Soil Nutrient Distribution along an Oasis‐Desert Transitional Zone in Northwestern China
Authors: Chen Bao‐Ming , Wang Gen‐Xuan , Cheng Dong‐Liang , Deng Jian‐Min , Peng Shao‐Lin , An Fu‐Bo
Published: October 2007

Many studies have focused on soil nutrient heterogeneity and islands of fertility in arid ecosystems. However, few have been conducted on an oasis‐desert transitional zone where there is a vegetation pattern changing from shrubs to annual herbs. The goal of the present study was to understand vegetation and soil nutrient heterogenity along an oasis‐desert transitional zone in northwestern China. Three replicated sampling belts were selected at 200 m intervals along the transitional zone. Twenty‐one quadrats (10 × 10 m) at 50 m intervals were located along each sampling belt. The vegetation cover was estimated through the quadrats, where both the soil under the canopy and the open soil were sampled simultaneously. The dominated shrub was Haloxylon ammodendron in the areas close to the oasis and Nitraria tangutorum dominated the areas close to the desert. In general, along the transitional zone the vegetation cover decreased within 660 m, increased above 660 m and decreased again above 1 020 m (close to the desert). The soil nutrients (organic matter, total N, NO3− and NH4+) showed significant differences along the zone. The soil nutrients except the soil NH4+ under the canopy were higher than those in open soil, confirming “islands of fertility” or nutrient enrichment. Only a slight downward trend of the level of “islands of fertility” for soil organic matter appeared in the area within 900 m. Soil organic matter both under canopy and in interspace showed a positive correlation with the total vegetation cover, however, there was no significant correlation between the other soil nutrients and the total vegetation cover. We also analyzed the relationship between the shrubs and annuals and the soil nutrients along the zone. Similarly, there was no significant correlation between them, except soil organic matter with the annuals. The results implied that annual plants played an important role in soil nutrient enrichment in arid ecosystem.
Mass Production of Intergeneric Chromosomal Translocations through Pollen Irradiation of Triticum durum‐Haynaldia villosa Amphiploid
Authors: Bie Tong‐De , Cao Ya‐Ping , Chen Pei‐Du
Published: October 2007

Haynaldia villosa possesses a lot of important agronomic traits and has been a powerful gene resource for wheat improvement. However, only several wheat–H. villosa translocation lines have been reported so far. In this study, we attempted to develop an efficient method for inducing wheat–H. villosa chromosomal translocations. Triticum durum‐Haynaldia villosa amphiploid pollen treated with 1 200 rad 60Co‐γ‐rays was pollinated to Triticum aestivum cv. ‘Chinese Spring’. Ninety‐eight intergeneric translocated chromosomes between T. durum and H. villosa were detected by genomic in situ hybridization in 44 of 61 M1 plants, indicating a translocation occurrence frequency of 72.1%; much higher than ever reported. There were 26, 62 and 10 translocated chromosomes involving whole arm translocations, terminal translocations, and intercarlary translocations, respectively. Of the total 108 breakage‐fusion events, 79 involved interstitial regions and 29 involved centric regions. The ratio of small segment terminal translocations (W·W‐V) was much higher than that of large segment terminal translocations (W‐V·V). All of the M1 plants were self‐sterile, and their backcross progeny was all obtained with ‘Chinese Spring’ as pollen donors. Transmission analysis showed that most of the translocations were transmittable. This study provides a new strategy for rapid mass production of wheat‐alien chromosomal translocations, especially terminal translocations that will be more significant for wheat improvement.
Use of Sera from Humans and Dolphins with Lacaziosis and Sera from Experimentally Infected Mice for Western Blot Analyses of
Lacazia loboi
Antigens
Authors: Mendoza Leonel , Belone Andréa F. F. , Vilela Raquel , Rehtanz Manuela , Bossart Gregory D. , Reif John S. , Fair Patricia A. , Durden Wendy N. , St. Leger Judy , Travassos Luiz R. , Rosa Patricia S.
Published: October 2007

Antibodies in the sera of patients with lacaziosis recognized an ∼193-kDa antigen and other
Lacazia loboi
antigens.
Paracoccidioides brasiliensis
gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in
L. loboi
extracts. This study is the first to examine the humoral response to
L. loboi
antigens by using multiple host sera.
Serologic Evidence for Reactivation of Cryptococcosis in Solid-Organ Transplant Recipients
Authors: Saha D. C. , Goldman D. L. , Shao X. , Casadevall A. , Husain S. , Limaye A. P. , Lyon M. , Somani J. , Pursell K. , Pruett T. L. , Singh N.
Published: October 2007

Cryptococcosis is a significant infection with a high mortality in solid-organ transplant recipients. Nonetheless, the pathogenesis of this disease is poorly understood. It has been hypothesized that cryptococcosis may result from either primary infection or reactivation of a latent infection. Sera were obtained from transplant recipients prior to transplantation and at the time they developed cryptococcosis. Control sera were obtained before and after transplant from patients who did not develop cryptococcosis. Sera were tested for antibodies against
Cryptococcus neoformans
by using an immunoblot assay. Antibody responses were also compared with those observed in sera from rats with experimental pulmonary cryptococcosis. In all, 52% of the transplant recipients who developed cryptococcosis exhibited serologic evidence of cryptococcal infection before transplantation. These patients developed cryptococcosis significantly earlier after transplant than patients without preexisting reactivity did (5.6 ± 3.4 months compared to 40.6 ± 63.8 months, respectively [
P
= 0.0011]). The results from our study suggest that a substantial proportion of transplant-associated cryptococcosis cases result from the reactivation of a latent infection. These findings also highlight the potential utility of serologic studies in identifying patients at risk for the development of cryptococcosis after transplantation.
Western Blot Assay Using Recombinant p26 Antigen for Detection of Equine Infectious Anemia Virus-Specific Antibodies
Authors: Alvarez I. , Gutierrez G. , Ostlund E. , Barrandeguy M. , Trono K.
Published: October 2007

We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.
Evaluation of a Novel PCR-Based Assay for Detection and Identification of
Chlamydia trachomatis
Serovars in Cervical Specimens
Authors: Quint Koen , Porras Carolina , Safaeian Mahboobeh , González Paula , Hildesheim Allan , Quint Wim , van Doorn Leen-Jan , Silva Sandra , Melchers Willem , Schiffman Mark , Rodríguez Ana Cecilia , Wacholder Sholom , Freer Enrique , Cortes Bernal , Herrero Rolando
Published: October 2007

The aims of this study were to compare a novel PCR-based
Chlamydia trachomatis
detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available
C. trachomatis
detection Hybrid Capture 2 (HC2) assay and to investigate the
C. trachomatis
serovar distribution among young women in a rural Costa Rican study population. A total of 5,828 sexually active women participating in a community-based trial in Costa Rica were tested for
C. trachomatis
by HC2. A sample of 1,229 specimens consisting of 100% HC2
C. trachomatis
-positive specimens (
n
= 827) and a random sample of 8% HC2
C. trachomatis
-negative specimens (
n
= 402) were tested with the Ct-DT assay. Agreement between the two assays was determined by the unweighted kappa statistic. Discrepant specimens were tested with a second commercially available test (COBAS TaqMan). The Ct-DT-positive specimens were further analyzed with the Ct-DT genotyping step to investigate the distribution of 14 different
C. trachomatis
serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3). After accounting for the sampling fraction selected for Ct-DT testing, crude agreement with the HC2 assay was 98% and the kappa was 0.92 (95% confidence interval [CI], 0.89 to 0.97). The 33 discordant samples that were further analyzed with the COBAS TaqMan test showed better agreement with the Ct-DT assay (31/33,
P
< 0.001). Among the 806 Ct-DT-positive samples, serovar E was the most common serovar (31%), followed by serovars F and D (both 21%) and serovar I (15%). In conclusion, the novel Ct-DT assay permits reliable detection and identification of
C. trachomatis
serovars.
Novel Light-Upon-Extension Real-Time PCR Assays for Detection and Quantification of Genogroup I and II Noroviruses in Clinical Specimens
Authors: Nordgren Johan , Bucardo Filemón , Dienus Olaf , Svensson Lennart , Lindgren Per-Eric
Published: October 2007

Norovirus is now recognized as the leading cause of nonbacterial acute gastroenteritis in adults, causing numerous outbreaks worldwide. We have developed two novel light-upon-extension (LUX) real-time PCR assays for detection and quantification of norovirus genogroups I and II. The LUX system uses a fluorophore attached to one primer having a self-quenching hairpin structure, making it cost-effective and specific. The assays were evaluated against clinical stool specimens (
n
= 103) from Sweden and Nicaragua and compared to established methods. The norovirus assay detected more positive stool specimens (47/103) than conventional PCR (39/103) and corresponded to a TaqMan real-time PCR, with the exception of one specimen. Furthermore, the assays correctly identified all (
n
= 11) coded control specimens in a reference panel containing various genogroups and genotypes. Both LUX real-time PCR assays had a wide dynamic range, detecting from ≤10
1
to 10
7
genes per reaction, resulting in a theoretical lower limit of ≤∼20 000 viruses per gram of stool. No cross-reactivity was noticed with specimens containing other enteric viruses, and by using melting curve analysis we could differentiate between norovirus genogroups I and II.
Comparison between Quantitative Nucleic Acid Sequence-Based Amplification, Real-Time Reverse Transcriptase PCR, and Real-Time PCR for Quantification ofLeishmaniaParasites
Authors: van der Meide Wendy , Guerra Jorge , Schoone Gerard , Farenhorst Marit , Coelho Leíla , Faber William , Peekel Inge , Schallig Henk
Published: October 2007

DNA or RNA amplification methods for detection ofLeishmaniaparasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification ofLeishmaniaparasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higherr2values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification ofLeishmaniaparasites, since it was highly sensitive and reproducible and the procedure was relatively fast.
Molecular Epidemiology of
Escherichia coli
Producing Extended-Spectrum β-Lactamases Isolated in Rome, Italy
Authors: Carattoli Alessandra , García-Fernández Aurora , Varesi Paola , Fortini Daniela , Gerardi Serena , Penni Adriano , Mancini Carlo , Giordano Alessandra
Published: October 2007

Escherichia coli
strains producing extended-spectrum β-lactamases (ESBLs) are a major problem in many different hospitals worldwide, causing outbreaks as well as sporadic infections. The prevalence of
Escherichia coli
ESBL producers was analyzed in a surveillance study performed on the population attending the Policlinico Umberto I, the largest university hospital in Rome, Italy. We also investigated genotypes, pathogenicity islands, and plasmids in the ESBL-positive
E. coli
isolates as further markers that are useful in describing the epidemiology of the infections. In this survey, 163 nonreplicate isolates of
Escherichia coli
were isolated from patients from 86 different wards, and 28 were confirmed as ESBL producers. A high prevalence (26/28) of CTX-M-15 producers was observed within the bacterial population circulating in this hospital, and the dissemination of this genetic trait was associated with the spread of related strains; however, these do not have the characteristics of a single epidemic clone spreading. The dissemination was also linked to horizontal transfer among the prevalent
E. coli
genotypes of multireplicon plasmids showing FIA, FIB, and FII replicons in various combinations, which are well adapted to the
E. coli
species. The analysis of related bacteria suggests a probable interpatient transmission occurring in several wards, causing small outbreaks.
Evaluation of the Vitek 2 System with a Variety of
Staphylococcus
Species
Authors: Delmas Julien , Chacornac Jean Paul , Robin Frédéric , Giammarinaro Philippe , Talon Régine , Bonnet Richard
Published: October 2007

The Vitek 2 gram-positive (GP) card was compared with an oligonucleotide array approach for the identification of 190
Staphylococcus
strains, including 35 species, isolated from clinical and environmental specimens. The GP card provided a rapid and reliable identification of most species, whatever their origin.
Mycobacterium bovis
BCG Vertebral Osteomyelitis after Intravesical BCG Therapy, Diagnosed by PCR-Based Genomic Deletion Analysis
Authors: Nikaido Takuya , Ishibashi Kei , Otani Koji , Yabuki Shoji , Konno Shinichi , Mori Shuichi , Ohashi Kazutaka , Ishida Takashi , Nakano Michiko , Yamaguchi Osamu , Suzutani Tatsuo , Kikuchi Shinichi
Published: October 2007

We report a case of
Mycobacterium bovis
BCG vertebral osteomyelitis 1.8 years after intravesical BCG therapy for bladder cancer. We differentiated BCG from other
Mycobacterium tuberculosis
complex members by PCR analysis of deletion regions and started an appropriate chemotherapy regimen resulting in the remission of symptoms within 1 month.
Influence of Type of Culture Medium on Characterization ofMycobacterium aviumsubsp.paratuberculosisSubtypes
Authors: Cernicchiaro N. , Wells S. J. , Janagama H. , Sreevatsan S.
Published: October 2007

We evaluated the effects of culture and/or enrichment methods on the selection ofMycobacterium aviumsubsp.paratuberculosissubtypes.M. aviumsubsp.paratuberculosisisolates from bovine fecal samples processed using a centrifugation protocol were investigated in both liquid (MGIT ParaTb tubes) and solid (Herrold's egg yolk medium) culture media. For this evaluation,M. aviumsubsp.paratuberculosissubtyping was based on the sequence variation in two of the most discriminatory short sequence repeat loci, i.e., mononucleotide G and trinucleotide GGT, in isolates from liquid and solid cultures. This study identified the existence of one major predominating fingerprint (>13G-5GGT) in bovine fecal samples, regardless of the type of medium used for isolation. Matched-pair analysis of subtypes showed that 69% of samples presented unique subtypes in the two culture media used, while 31% shared the same G-GGT allele. Furthermore, the liquid culture method appeared to select for a more genotypically diverse subtype population than the solid culture method. The variety of subtypes observed between liquid and solid cultures obtained from the same fecal samples suggests that the culture method could provide a “microbiological” bias and lead to a discrepancy in the growth ofM. aviumsubsp.paratuberculosisstrains. In conclusion, this study identified that these two types of culture media determined differential growth ofM. aviumsubsp.paratuberculosisstrains and that this should be considered in evaluating detection capabilities of diagnostic tests or interpreting data from molecular epidemiological studies performed using conventional solid fecal culture or automated liquid medium culture methods.
Lodderomyces elongisporus
Masquerading as
Candida parapsilosis
as a Cause of Bloodstream Infections
Authors: Lockhart Shawn R. , Messer Shawn A. , Pfaller Michael A. , Diekema Daniel J.
Published: October 2007

Ten yeast bloodstream isolates identified as
Candida parapsilosis
by conventional methods grew as turquoise blue colonies on Chromagar media. Subsequent sequence analysis showed that these isolates were the species
Lodderomyces elongisporus
. To our knowledge, this is the first published report of
L. elongisporus
as a cause of human disease.
The Antiapoptotic Herpes Simplex Virus Glycoprotein J Localizes to Multiple Cellular Organelles and Induces Reactive Oxygen Species Formation
Authors: Aubert Martine , Chen Zheng , Lang Robin , Dang Chung H. , Fowler Carla , Sloan Derek D. , Jerome Keith R.
Published: October 2007

The
Us5
gene of herpes simplex virus (HSV) encodes glycoprotein J (gJ). The only previously reported function of gJ was its ability to inhibit apoptosis. However, the mechanism by which gJ prevents apoptosis is not understood, and it is not known whether gJ mediates additional cellular effects. In this study, we evaluated the expression, localization, and cellular effects of
Us5
/gJ.
Us5
was first expressed 4 h after infection. gJ was detectable at 6 h and was expressed in glycosylated and unglycosylated forms.
Us5
was regulated as a late gene, with partial dependency on DNA replication for expression.
Us5
expression was delayed in the absence of ICP22; furthermore, expression of
Us5
in
trans
protected cells from apoptosis induced by an HSV mutant with deletion of ICP27, suggesting that the antiapoptotic effects of ICP22 and ICP27 are mediated in part through effects on gJ expression. Within HSV-infected or
Us5
-transfected cells, gJ was distributed widely, especially to the endoplasmic reticulum,
trans
-Golgi network, and early endosomes. gJ interacted with F
o
F
1
ATP synthase subunit 6 by a yeast two-hybrid screen and had strong antiapoptotic effects, which were mediated by protein rather than mRNA. Antiapoptotic activity required the extracellular and transmembrane domains of gJ, but not the intracellular domain. Consistent with inhibition of F
o
F
1
ATP synthase function,
Us5
was required for HSV-induced reactive oxygen species (ROS) formation, and gJ was sufficient to induce ROS in
Us5
-transfected cells. Thus, HSV gJ is a multifunctional protein, modulating other cellular processes in addition to inhibition of apoptosis.
The Tyrosine Kinase sf-Stk and Its Downstream Signals Are Required for Maintenance of Friend Spleen Focus-Forming Virus-Induced Fibroblast Transformation
Authors: Jelacic Tanya M. , Thompson Delores , Hanson Charlotte , Cmarik Joan L. , Nishigaki Kazuo , Ruscetti Sandra
Published: October 2007

Infection of erythroid progenitor cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia and eventually to erythroleukemia in susceptible strains of mice. The viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), activating both and inducing Epo-independent proliferation. Recently, we discovered that coexpression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. In the current study, we demonstrate that sf-Stk and its downstream effectors are critical to this transformation. Unlike SFFV-derived erythroleukemia cells, which depend on PU.1 expression for maintenance of the transformed phenotype, SFFV gp55-sf-Stk-transformed fibroblasts are negative for PU.1. Underscoring the importance of sf-Stk to fibroblast transformation, knockdown of sf-Stk abolished the ability of these cells to form anchorage-independent colonies. Like SFFV-infected erythroid cells, SFFV gp55-sf-Stk-transformed fibroblasts express high levels of phosphorylated MEK, ERK, phosphatidylinositol 3-kinase (PI3K), Gab1/2, Akt, Jun kinase (JNK), and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 mitogen-activated protein kinase (MAPK) stress response is suppressed in the transformed fibroblasts. Inhibition of either JNK or the PI3K pathway decreases both monolayer proliferation and anchorage-independent growth of the transformed fibroblasts as does the putative kinase inhibitor luteolin, but inhibition of p38 MAPK has no effect. Our results indicate that sf-Stk is a molecular endpoint of transformation that could be targeted directly or with agents against its downstream effectors.
Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate
Authors: Oh Jangsuk , Chang Kevin W. , Wierzchoslawski Rafal , Alvord W. Gregory , Hughes Stephen H.
Published: October 2007

The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3′ end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5′ end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5′ end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo.
Akt Plays a Critical Role in Replication of Nonsegmented Negative-Stranded RNA Viruses
Authors: Sun Minghao , Fuentes Sandra M. , Timani Khalid , Sun Dengyun , Murphy Chris , Lin Yuan , August Avery , Teng Michael N. , He Biao
Published: October 2007

The order
Mononegavirales
(comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs.
Mutational Analyses of Packaging Signals in Influenza Virus PA, PB1, and PB2 Genomic RNA Segments
Authors: Liang Yuhong , Huang Taoying , Ly Hinh , Parslow Tristram G. , Liang Yuying
Published: October 2007

The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal
cis
-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3′ and 5′ termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5′ sites in the PA or PB1 coding regions but extended across the entire (80-base) 5′ coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.
Coupling Genetics and Proteomics To Identify Aphid Proteins Associated with Vector-Specific Transmission of Polerovirus (
Luteoviridae
)
Authors: Yang Xiaolong , Thannhauser T. W. , Burrows Mary , Cox-Foster Diana , Gildow Fred E. , Gray Stewart M.
Published: October 2007

Cereal yellow dwarf virus
-RPV (CYDV-RPV) is transmitted specifically by the aphids
Rhopalosiphum padi
and
Schizaphis graminum
in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F
2
progeny from crosses of vector and nonvector genotypes of
S
.
graminum
showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F
2
genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of
S
.
graminum
to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.
Human Astrovirus Coat Protein Inhibits Serum Complement Activation via C1, the First Component of the Classical Pathway
Authors: Bonaparte Rheba S. , Hair Pamela S. , Banthia Deepa , Marshall Dawn M. , Cunnion Kenji M. , Krishna Neel K.
Published: October 2007

Human astroviruses (HAstVs) belong to a family of nonenveloped, icosahedral RNA viruses that cause noninflammatory gastroenteritis, predominantly in infants. Eight HAstV serotypes have been identified, with a worldwide distribution. While the HAstVs represent a significant public health concern, very little is known about the pathogenesis of and host immune response to these viruses. Here we demonstrate that HAstV type 1 (HAstV-1) virions, specifically the viral coat protein (CP), suppress the complement system, a fundamental component of the innate immune response in vertebrates. HAstV-1 virions and purified CP both suppress hemolytic complement activity. Hemolytic assays utilizing sera depleted of individual complement factors as well as adding back purified factors demonstrated that HAstV CP suppresses classical pathway activation at the first component, C1. HAstV-1 CP bound the A chain of C1q and inhibited serum complement activation, resulting in decreased C4b, iC3b, and terminal C5b-9 formation. Inhibition of complement activation was also demonstrated for HAstV serotypes 2 to 4, suggesting that this phenomenon is a general feature of these human pathogens. Since complement is a major contributor to the initiation and amplification of inflammation, the observed CP-mediated inhibition of complement activity may contribute to the lack of inflammation associated with astrovirus-induced gastroenteritis. Although diverse mechanisms of inhibition of complement activation have been described for many enveloped animal viruses, this is the first report of a nonenveloped icosahedral virus CP inhibiting classical pathway activation at C1.
Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis
Authors: Zhang Ping , Jacobs Bertram L. , Samuel Charles E.
Published: October 2007

The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (ΔE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ΔE3L virus was increased by nearly 2 log
10
in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ΔE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ΔE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the α subunit of protein synthesis initiation factor 2 (eIF-2α) was elevated severalfold in ΔE3L-infected PKR-sufficient, but not PKR-deficient, cells. WT virus did not significantly increase PKR or eIF-2α phosphorylation in either PKR-sufficient or -deficient cells, both of which supported efficient WT viral protein production. Finally, apoptosis induced by infection of PKR-sufficient HeLa cells with ΔE3L virus was blocked by a caspase antagonist, but mutant virus growth was not rescued, suggesting that translation inhibition rather than apoptosis activation is a principal factor limiting virus growth.
Effect of Cell Polarization on Hepatitis C Virus Entry
Authors: Mee Christopher J. , Grove Joe , Harris Helen J. , Hu Ke , Balfe Peter , McKeating Jane A.
Published: October 2007

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.