bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrating Research into design foundations Curriculum: Learning and Testing Theory
Authors: Martinson Barbara E.
Published: October 2008

Issue

Design students can integrate theoretical knowledge from foundation–level design courses into real–world applications through basic research exercises.

Goal

Through the development of research questions, students gain experience in problem seeking, basic research methods, ond analytical skills. They learn first–hand that the seemingly abstract concepts taught in foundation classes can be tested and new knowledge gained.

Application

Introducing students to basic theory testing through the use of hypothesis development and application of basic research methods provides a link between design concepts and how those concepts function in the designed environment. Students utilize the course readings not as passive learners but as active challengers of concepts. They extend the concept from the visual to the cognitive thus enhancing integration of the knowledge and experience.

Description

Students collaboratively develop research questions based on course readings. They discuss the merits of each question and come to agreement on a question and suitable hypothesis. The class then establishes a method for testing the hypothesis and carries out the research. The collected data are analyzed, and the class works together to draw conclusions.

Conclusion

Students increase their ability to read critically and learn to develop meaningful research questions research questions. Active involvement in examining applications of design theory provides students with a greater understanding of the design concept being tested and helps them to make the cognitive link between theory and practice.
Orbital Motion of Electrically Charged Spheres in Microgravity
Authors: Banerjee Shubho , Andring Kevin , Campbell Desmond , Janeski John , Keedy Daniel , Quinn Sean , Hoffmeister Brent
Published: October 2008

The similar mathematical forms of Coulomb's law and Newton's law of gravitation suggest that two uniformly charged spheres should be able to orbit each other just as two uniform spheres of mass are known to do. In this paper we describe an experiment that we performed to demonstrate such an orbit. This is the first published account of a successful orbit using electrostatic forces.
The Smallest Tweezers in the World
Authors: Lewalle Alexandre
Published: October 2008

A pair of fine tweezers and a steady hand may well be enough to pick up a grain of sand, but what would you use to hold something hundreds of times smaller still, the size of only one micron? The answer is to use a device that is not mechanical in nature but that relies instead on the tiny forces that light exerts on small particles: “optical tweezers.” In recent years, this technique has become central to nanotechnology for the manipulation of small particles, even individual molecules. It is also an ideal illustration of how classroom physics is applied to cutting-edge research, combining concepts such as the vector nature of momentum and force, Newton's laws, optics, the wave-particle duality of light, and thermodynamics. The physics behind optical tweezers has many layers of complexity, but it can be reduced to a basic principle: the conservation of momentum. This paper guides the reader through a much simplified demonstration of this “tweezing effect” using a question-answer approach, leaving the reader with the choice to treat each step as a problem exercise.
Soda Pop Fizz-ics
Authors: Coffey Tonya
Published: October 2008

This paper is a brief summary of an “open-ended” physics experiment that gave my students a glimpse of what real physics research is like. They found it to be both fun and challenging. The subject of the experiment is the well-known Diet Coke and Mentos reaction.1 A more detailed description of the experimental results has been published elsewhere.2
The Cornstarch Flamethrower
Authors: Concannon Tom
Published: October 2008

Doing physics “magic shows” for the general public or for local area schools is usually an integral part of any physics department's outreach program. These demonstration shows should not only teach fundamental physics principles with “standard” demonstrations (like the rocket cart) but should also include the “wow!” types of demonstrations for maximum audience impact. Presenters quickly discover the audience's thirst for spectacular demonstrations; if too few “wow!” demonstrations are performed, the presenter can lose the audience's attention, or, worse, the audience may get bored and leave. Some of the more exciting demonstrations include, for example, the Van de Graaff generator, a large Tesla coil, the bed of nails, liquid nitrogen experiments, imploding gas cans, breaking a glass container with sound waves, and igniting cornstarch powder. The focus of this paper is a suggested improvement to this last demonstration, to enhance the “dust explosion” effect for demonstration show purposes. At the end, I also describe a small-scale version for use in classrooms, where the physics and chemistry of dust explosions can be explored in more detail than possible in a demonstration show atmosphere.
Graphs as a Problem-Solving Tool in 1-D Kinematics
Authors: Desbien Dwain M.
Published: October 2008

In this age of the microcomputer-based lab (MBL), students are quite accustomed to looking at graphs of position, velocity, and acceleration versus time.1 A number of textbooks argue convincingly that the slope of the velocity graph gives the acceleration, the area under the velocity graph yields the displacement, and the area under the acceleration gives the change in velocity.2 I also use dimensional analysis to help make the arguments. While students are very often encouraged to sketch graphs of motion to help build a better understanding,3 too little emphasis is placed on the fact that such graphs can be used to actually solve problems. This paper shows two examples of how to use sketches of velocity and acceleration graphs to solve 1-D motion problems.
Fourier Analysis of Musical Intervals
Authors: LoPresto Michael C.
Published: October 2008

Use of a microphone attached to a computer to capture musical sounds and software to display their waveforms and harmonic spectra has become somewhat commonplace.1 A recent article in The Physics Teacher aptly demonstrated the use of MacScope2 in just such a manner as a way to teach Fourier analysis.3 A logical continuation of this project is to use MacScope not just to analyze the Fourier composition of musical tones but also musical intervals.
Podcasting a Physics Lecture
Authors: McDonald James E. R.
Published: October 2008

The technology of podcasting, or creating audio or video files that can be subscribed to over the Internet, has grown in popularity over the past few years. Many educators have already begun realizing the potential of delivering such customized content, but most efforts have focused on lecture-style humanities courses or multimedia arts courses. At the University of Hartford, I have made audio recordings of physics lectures available as podcasts for four semesters. Despite the fact that physics is math-intensive and inherently visual, the students have found the podcasts to be very useful.
An Equilibrium Board to Demonstrate Both Torques and Translational Forces
Authors: Kinsler Mark , Kinzel Evelyn
Published: October 2008

We have devised a simple and inexpensive laboratory exercise that improves upon the traditional “force table” by treating the torques and translational forces imparted to an object with equal importance. The result is an intuitively satisfying illustration of all the conditions required for equilibrium, accompanied by a rigorous exercise in the resolution of vectors and the calculation of torques.
A Simple Demonstration for the Static Ladder Problem
Authors: Belloni Mario
Published: October 2008

In the teaching of statics, the ladder problem is a standard two-dimensional exercise. If students can understand how to set up this problem, they can solve almost any other two-dimensional problem. Solving this or any other statics problem requires identifying the forces (such that ΣF = 0) and where they act, and determining the length of the moment arm and the angle between the moment arm and the force (such that Στ = 0). In teaching the ladder problem we begin with a simple diagram of the leaning ladder and ask the students to identify the forces on the ladder and where they act. Students will readily identify three forces: the ladder's weight (mg), the normal force of the ground on the ladder, N, and the force of the wall on the ladder, Fwall, as shown in Fig. 1.
MC Hammer's Contribution to Physics
Authors: Roberts Wade
Published: October 2008

Column Editor's note: You never know what will be the “hook” that will capture the imagination of students and get them excited about learning. Wade Roberts, who has been the physics teacher at Centennial High School in Franklin, TN, since 1998, shares a technique he uses to help students relate to the ideas being presented. Use your imagination to envision the fun his students seem to have as they learn about vector resolution.
Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy
Authors: Keller Philipp J. , Schmidt Annette D. , Wittbrodt Joachim , Stelzer Ernst H.K.
Published: October 2008

A long-standing goal of biology is to map the behavior of all cells during vertebrate embryogenesis. We developed digital scanned laser light sheet fluorescence microscopy and recorded nuclei localization and movement in entire wild-type and mutant zebrafish embryos over the first 24 hours of development. Multiview in vivo imaging at 1.5 billion voxels per minute provides “digital embryos,” that is, comprehensive databases of cell positions, divisions, and migratory tracks. Our analysis of global cell division patterns reveals a maternally defined initial morphodynamic symmetry break, which identifies the embryonic body axis. We further derive a model of germ layer formation and show that the mesendoderm forms from one-third of the embryo's cells in a single event. Our digital embryos, with 55 million nucleus entries, are provided as a resource.
Uropathogens and Host Characteristics
Authors: Tabibian James H. , Gornbein Jeffrey , Heidari Arash , Dien Sarah L. , Lau Valerie H. , Chahal Puneet , Churchill Bernard M. , Haake David A.
Published: October 2008

Uropathogens other than
Escherichia coli
occur with greater frequency in patients with risk factors for urinary tract infection (UTI). However, little is known about associations between uropathogen species and host characteristics. Three hundred nineteen urine specimens containing a balanced distribution of uropathogen species were selected from inpatients and outpatients at a university hospital clinical microbiology laboratory. Information on host characteristics was retrospectively collected by chart review. Differences in the frequencies of host characteristics in UTI groups, as defined by the causative uropathogen, were compared by chi-square/Fisher analysis. Multivariate classification and regression tree analysis were used to identify host characteristic subsets that distinguish among uropathogen groups. In this exploratory study, several uropathogen species were found to be strongly linked to host characteristics relevant to UTI. Patients with
Pseudomonas aeruginosa
UTIs were more likely to have undergone urinary tract procedures (43% versus 15% overall), to have a neurogenic bladder (29% versus 12% overall), to have received recent antibiotic therapy (52% versus 24% overall), and to be male (76% versus 28% overall). Patients with
Proteus mirabilis
UTIs were more likely to have a foreign body in the lower urinary tract (48% versus 30% overall). The classification tree identified males over the age of 27 years who had undergone a prior urinary tract procedure as belonging to a host characteristic profile associated with
P. aeruginosa
UTI: 38% of patients with
P. aeruginosa
UTIs fit this profile. These data may be useful for planning future targeted prophylaxis studies.
Comparison of Gen-Probe Transcription-Mediated Amplification, Abbott PCR, and Roche PCR Assays for Detection of Wild-Type and Mutant Plasmid Strains of
Chlamydia trachomatis
in Sweden
Authors: Møller Jens Kjølseth , Pedersen Lisbeth Nørum , Persson Kenneth
Published: October 2008

The clinical performance of two nucleic acid amplification assays targeting the cryptic plasmid and two assays targeting rRNA molecules in
Chlamydia trachomatis
was examined. First-catch urine samples from Malmoe, Sweden, were tested for
C. trachomatis
with the Abbott real-time PCR assay m2000 and an in-house PCR for the new variant strain of
C. trachomatis
with a deletion in the cryptic plasmid. Aliquots of the urine samples were sent to Aarhus, Denmark, and further examined with the Roche COBAS Amplicor CT (RCA) PCR, the Gen-Probe Aptima Combo 2 assay (AC2) targeting the
C. trachomatis
23S rRNA, and the Aptima
C. trachomatis
assay (ACT) targeting the 16S rRNA molecule. A positive prevalence of 9% (163/1,808 urine samples examined) was detected according to the combined reference standard. The clinical sensitivity and specificity of the four assays were as follows: for ACT, 100% (163/163) and 99.9% (1,643/1,645), respectively; for AC2, 100% (163/163) and 99.6% (1,640/1,645); for m2000, 68.7% (112/163) and 99.9% (1,644/1,645); for RCA, 63.8% (104/163) and 99.9% (1,643/1,645). The two Gen-Probe assays detected all mutant strains characterized by the in-house PCR as having the deletion in the cryptic plasmid, whereas the Roche and the Abbott PCRs targeting the plasmid were both unable to detect the plasmid mutant. The difference in clinical sensitivity between the plasmid PCR assays m2000 and RCA, on the one hand, and the rRNA assays AC2 and ACT, on the other, could be attributed almost exclusively to the presence of the plasmid mutant in about one-quarter of the
Chlamydia
-positive samples examined.
Molecular Epidemiological Identification of
Neisseria gonorrhoeae
Clonal Clusters with Distinct Susceptibility Profiles Associated with Specific Groups at High Risk of Contracting Human Immunodeficiency Virus and Syphilis
Authors: Wong Wing-Wai , Huang Chung-Ter , Li Lan-Hui , Chiang Chien-Chou , Chen Bor-Dong , Li Shu-Ying
Published: October 2008

From April 2006 to August 2007, a total of 146
Neisseria gonorrhoeae
isolates collected from 139 male patients in Taipei, Taiwan, were analyzed by
N. gonorrhoeae
multiantigen sequence typing (NG-MAST) and antibiotic susceptibility testing. The resistance rates of all isolates to ciprofloxacin, cefpodoxime, and cefixime were 76.7 (112/146), 21.2 (31/146), and 16.4% (24/146), respectively. NG-MAST identified 71 sequence types (STs), of which 21 STs contained 2 to 21 isolates. The isolates that belonged to the three major ST clusters typically were from patients who had specific epidemiological characteristics (such as sexual orientation and human immunodeficiency virus status). The major ST clones exhibited distinct resistance profiles and are associated with specific groups at high risk of human immunodeficiency virus and syphilis infections.
Mycobacterium tuberculosis
Population Structures Differ Significantly on Two Indonesian Islands
Authors: Parwati Ida , van Crevel Reinout , Sudiro Mirawati , Alisjahbana Bachti , Pakasi Trevino , Kremer Kristin , van der Zanden Adri , van Soolingen Dick
Published: October 2008

Comparison of
Mycobacterium tuberculosis
genotype distributions in different areas might help to find determinants of the emergence of certain genotypes, such as the Beijing family. In this study,
M. tuberculosis
isolates originating from patients from two Indonesian islands were genotyped, and possible associations with patients' characteristics and drug resistance were explored. A high degree of genetic diversity was observed among the
M. tuberculosis
strains, and a significant difference was found in the geographical distribution of genotype families. The predominant Beijing genotype family was isolated from 268 of 813 patients from West Java (33.0%) versus 12 of 84 patients from Timor (14.3%) (
P
= 0.002). Family F (East African-Indian) (33.3%) and family D (Latin American and Mediterranean) (20.0%) were more prevalent in Timor. No significant associations were found between genotype families and age, vaccination with
Mycobacterium bovis
BCG, previous treatment, disease localization, or drug resistance. Possible explanations for the differences in the geographical distribution of the
M. tuberculosis
genotypes are discussed.
Absence of CTX-M Enzymes but High Prevalence of Clones, Including Clone ST131, among Fecal
Escherichia coli
Isolates from Healthy Subjects Living in the Area of Paris, France
Authors: Leflon-Guibout Véronique , Blanco Jorge , Amaqdouf Karim , Mora Azucena , Guize Louis , Nicolas-Chanoine Marie-Hélène
Published: October 2008

Quinolone-resistant and CTX-M-15-producing
Escherichia coli
isolates belonging to clone ST131 have been reported in the community. This study was designed to identify these
E. coli
isolates in the stools of 332 independent healthy subjects living in the area of Paris, France. Stools were plated on media without antibiotics, in order to obtain the dominant (Dm) fecal
E. coli
strain, and with nalidixic acid (NAL) and cefotaxime. Quinolone susceptibility, phylogenetic groups, and molecular profiles, including multilocus sequence types (ST), were determined for all NAL-resistant (NAL-R) isolates. Groups were also determined for the Dm strains from participants with NAL-R isolates and from a subgroup without NAL-R isolates. All B2 isolates were typed; pulsed-field gel electrophoresis was performed for the ST131 isolates, and the results were compared with those for intercontinental clone ST131. Two participants (0.6%) had extended-spectrum β-lactamase-producing (SHV-2, TEM-52) fecal
E. coli
isolates, and 51 (15%) had NAL-R isolates; 51% of NAL-R isolates belonged to phylogenetic group A, 31% to group D, 16% to group B2, and 2% to group B1. The Dm strain was NAL-R in 3.3% of the 332 subjects. Forty-nine percent of the NAL-R isolates belonged to clones: ST10 and ST606 for group A isolates, ST117 and ST393 for group D isolates. Of all B2 isolates studied from 100 subjects (8 NAL-R strains; 19 NAL-susceptible dominant strains), 52% belonged to three clones: ST131 (
n
= 7), ST95 (
n
= 4), and ST141 (
n
= 3). This is the first study to show the presence of fecal
E. coli
isolates of clone ST131 in 7% of independent healthy subjects not colonized by CTX-M-15-producing isolates.
Transmission Classification Model To Determine Place and Time of Infection of Tuberculosis Cases in an Urban Area
Authors: de Vries G. , Baars H. W. M. , Šebek M. M. G. G. , van Hest N. A. H. , Richardus J. H.
Published: October 2008

We conducted a population-based study in the Rotterdam region of The Netherlands to determine the place and time of infection of tuberculosis (TB) cases using conventional epidemiological and genotyping information. In particular, we focused on the extent of misclassification if genotyping was not combined with epidemiological information. Cases were divided into those with a unique mycobacterial DNA fingerprint, a clustering fingerprint, and an unknown fingerprint. We developed transmission classification trees for each category to determine whether patients were infected in a foreign country or recently (≤2 years) or remotely (>2 years) infected in The Netherlands. Of all TB cases during the 12-year study period, 38% were infected in a foreign country, 36% resulted from recent transmission in The Netherlands, and 18% resulted from remote infection in The Netherlands, while in the remaining cases (9%) either the time or place of infection could not be determined. The conventional epidemiological data suggested that at least 29% of clustered cases were not part of recent chains of transmission. Cases with unknown fingerprints, almost all culture negative, relatively frequently had confirmed epidemiological links with a recent pulmonary TB case in The Netherlands and were more often identified by contact tracing. Our findings highlight the idea that genotyping should be combined with conventional epidemiological investigation to establish the place and time of infection of TB cases as accurately as possible. A standardized way of classifying TB into recently, remotely, and foreign-acquired disease provides indicators for surveillance and TB control program performance that can be used to decide on interventions and allocation of resources.
Identification of Minimal Predictors of Avian Pathogenic
Escherichia coli
Virulence for Use as a Rapid Diagnostic Tool
Authors: Johnson Timothy J. , Wannemuehler Yvonne , Doetkott Curt , Johnson Sara J. , Rosenberger Sandra C. , Nolan Lisa K.
Published: October 2008

To identify traits that predict avian pathogenic
Escherichia coli
(APEC) virulence, 124 avian
E. coli
isolates of known pathogenicity and serogroup were subjected to virulence genotyping and phylogenetic typing. The results were analyzed by multiple-correspondence analysis. From this analysis, five genes carried by plasmids were identified as being the most significantly associated with highly pathogenic APEC strains:
iutA
,
hlyF
,
iss
,
iroN
, and
ompT
. A multiplex PCR panel targeting these five genes was used to screen a collection of 994 avian
E. coli
isolates. APEC isolates were clearly distinguished from the avian fecal
E. coli
isolates by their possession of these genes, suggesting that this pentaplex panel has diagnostic applications and underscoring the close association between avian
E. coli
virulence and the possession of ColV plasmids. Also, the sharp demarcation between APEC isolates and avian fecal
E. coli
isolates in their plasmid-associated virulence gene content suggests that APEC isolates are well equipped for a pathogenic lifestyle, which is contrary to the widely held belief that most APEC isolates are opportunistic pathogens. Regardless, APEC isolates remain an important problem for poultry producers and a potential concern for public health professionals, as growing evidence suggests a possible role for APEC in human disease. Thus, the pentaplex panel described here may be useful in detecting APEC-like strains occurring in poultry production, along the food chain, and in human disease. This panel may be helpful toward clarifying potential roles of APEC in human disease, ascertaining the source of APEC in animal outbreaks, and identifying effective targets of avian colibacillosis control.
Aichi Virus, Norovirus, Astrovirus, Enterovirus, and Rotavirus Involved in Clinical Cases from a French Oyster-Related Gastroenteritis Outbreak
Authors: Le Guyader Françoise S. , Le Saux Jean-Claude , Ambert-Balay Katia , Krol Joanna , Serais Ophelie , Parnaudeau Sylvain , Giraudon Hélène , Delmas Gilles , Pommepuy Monique , Pothier Pierre , Atmar Robert L.
Published: October 2008

Following a flooding event close to a shellfish production lagoon, 205 cases of gastroenteritis were linked to oyster consumption. Twelve stool samples from different individuals were collected. Analysis showed that eight samples were positive for multiple enteric viruses, and one stool sample had seven different enteric viruses. Analysis of shellfish implicated in the outbreak allowed detection of the same diversity of enteric viruses, with some viral genomic sequences being identical to those obtained from stool sample analysis. Shellfish were contaminated by as many as five different enteric viruses. For the first time in Europe, Aichi virus was identified in oyster samples. Shellfish samples collected over 3 weeks following the outbreak showed a progressive decline in the level of virus contamination as measured by the virus diversity detected and by quantitative reverse transcription-PCR.
Association of Tuberculous Endometritis with Infertility and Other Gynecological Complaints of Women in India
Authors: Kumar P. , Shah N. P. , Singhal A. , Chauhan D. S. , Katoch V. M. , Mittal S. , Kumar S. , Singh M. K. , Gupta S. Datta , Prasad H. K.
Published: October 2008

Endometrial biopsy samples derived from 393 patients with assorted gynecological complaints were investigated for mycobacterial infection. By employment of four different techniques, mycobacterial pathogens were detected irrespective of the nature/type of clinical complaint.
Mycobacterium tuberculosis
was the predominant pathogen detected among the samples investigated.
Hepatitis and Human Bocavirus Primary Infection in a Child with T-Cell Deficiency
Authors: Kainulainen Leena , Waris Matti , Söderlund-Venermo Maria , Allander Tobias , Hedman Klaus , Ruuskanen Olli
Published: October 2008

Human bocavirus (HBoV) is one of the recently identified respiratory viruses. The clinical features of HBoV infection have not yet been fully elucidated. We report an invasive HBoV infection and hepatitis in a boy with cartilage-hair hypoplasia and severe T-cell immunodeficiency.
Comparison of Classical Serotyping and PremiTest Assay for Routine Identification of Common
Salmonella enterica
Serovars
Authors: Wattiau Pierre , Van Hessche Mieke , Schlicker Christine , Vander Veken Heidi , Imberechts Hein
Published: October 2008

The commercial PremiTest
Salmonella
kit uses a multiplexed DNA typing test aimed at identifying common serovars of
Salmonella enterica
. It was used in assays over a 9-month period in the Belgian reference laboratory that performs the routine identification of
Salmonella
strains of animal origin. A blind analysis of 754 strains was conducted in parallel by classical serotyping and the PremiTest assay. Full results were available for 685 strains (90.8%) by serotyping, while the remaining 69 strains were found to be nontypeable due to either a lack of surface antigen expression or autoagglutination properties. When the PremiTest assay (version 4.2) was performed with crude bacterial extracts, it identified 658 strains (87.3%), including most strains found to be nontypeable by serotyping. In contrast, it gave no, wrong, dual, or noninterpretable results for 96 strains, for which 23 were caused by assay failures. When purified DNA instead of crude extracts were tested, the number of strains successfully identified to the serovar level increased to 714 (94.7%), while all assay failures were cleared. Our conclusion is that, in its actual development stage, the application of the investigated kit to purified DNA samples offers a valuable alternative to classical serotyping for laboratories performing the routine identification of
Salmonella
strains belonging to commonly encountered serovars and isolated from a given geographical area, assuming that the system has been validated beforehand with a significant number of strains originating from that particular area.
DNA-Level Diversity and Relatedness of
Helicobacter pylori
Strains in Shantytown Families in Peru and Transmission in a Developing-Country Setting
Authors: Herrera Phabiola M. , Mendez Melissa , Velapatiõ Billie , Santivaẽz Livia , Balqui Jacqueline , Finger S. Alison , Sherman Jonathan , Zimic Mirko , Cabrera Lilia , Watanabe Jose , Rodríguez Carlos , Gilman Robert H. , Berg Douglas E.
Published: October 2008

The efficiency of transmission of a pathogen within families compared with that between unrelated persons can affect both the strategies needed to control or eradicate infection and how the pathogen evolves. In industrialized countries, most cases of transmission of the gastric pathogen
Helicobacter pylori
seems to be from mother to child. An alternative model, potentially applicable among the very poor in developing countries, where infection is more common and the sanitary infrastructure is often deficient, invokes frequent transmission among unrelated persons, often via environmental sources. In the present study, we compared the genotypes of
H. pylori
from members of shantytown households in Peru to better understand the transmission of
H. pylori
in developing-country settings.
H. pylori
cultures and/or DNAs were obtained with informed consent by the string test (a minimally invasive alternative to endoscopy) from at least one child and one parent from each of 62 families. The random amplified polymorphic DNA fingerprints of 57 of 81 (70%) child-mother strain pairs did not match, nor did the diagnostic gene sequences (>1% DNA sequence difference), independent of the child's age (range, 1 to 39 years). Most strains from siblings or other paired family members were also unrelated. These results suggest that
H. pylori
infections are often community acquired in the society studied. Transmission between unrelated persons should facilitate the formation of novel recombinant genotypes by interstrain DNA transfer and selection for genotypes that are well suited for individual hosts. It also implies that the effective prevention of
H. pylori
infection and associated gastroduodenal disease will require anti-
H. pylori
measures to be applied communitywide.
Neuroadapted Yellow Fever Virus Strain 17D: a Charged Locus in Domain III of the E Protein Governs Heparin Binding Activity and Neuroinvasiveness in the SCID Mouse Model
Authors: Nickells Janice , Cannella Maria , Droll Deborah A. , Liang Yan , Wold William S. M. , Chambers Thomas J.
Published: October 2008

A molecular clone of yellow fever virus (YFV) strain 17D was used to identify critical determinants of mouse neuroinvasiveness previously localized to domain III of the neuroadapted SPYF-MN virus envelope protein. Three candidate virulence substitutions (305F→V, 326K→E, and 380R→T) were individually evaluated for their roles in this phenotype in a SCID mouse model. The virus containing a glutamic acid residue at position 326 of the envelope protein (326E) caused rapidly lethal encephalitis, with a mortality rate and average survival time resembling those of the parental SPYF-MN virus. Determinants at positions 380 (380T) and 305 (305V) did not independently affect neuroinvasiveness. Testing a panel of viruses with various amino acid substitutions at position 326 revealed that attenuation of neuroinvasiveness required a positively charged residue (lysine or arginine) at this position. Molecular-modeling studies suggest that residues 326 and 380 contribute to charge clusters on the lateral surface of domain III that constitute putative heparin binding sites, as confirmed by studies of heparin inhibition of plaque formation. The neuroinvasiveness of YFVs in the SCID model correlated inversely with sensitivity to heparin. These findings establish that residue 326 in domain III of the E protein is a critical determinant of YFV neuroinvasiveness in the SCID mouse model. Together with modeling of domain III from virulent YFV strains, the data suggest that heparin binding activity involving lysine at position 326 may be a modulator of YFV virulence phenotypes.
Lack of Heparan Sulfate Expression in B-Cell Lines: Implications for Kaposi's Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus 68 Infections
Authors: Jarousse Nadine , Chandran Bala , Coscoy Laurent
Published: October 2008

Kaposi's sarcoma-associated herpesvirus (KSHV) and its murine homolog, murine gammaherpesvirus 68 (MHV68), are lymphotropic viruses that establish latent infection in their host. Surprisingly, while B cells are the main viral reservoir in vivo, B-cell lines are poorly permissive to infection by either MHV68 or KSHV. Here, we report that most B-cell lines express very little to no cell surface heparan sulfate (HS), a glycosaminoglycan that is essential for infection by these viruses. We found that Ext1, a key enzyme in the biosynthesis of HS, was expressed at a low level in these cells. Transfection of B-cell lines with Ext1 restored high HS expression at the cell surface. Overexpression of Ext1 in murine A20 and M12 B-cell lines increased MHV68 surface binding and enhanced the efficiency of infection. Finally, although it was not sufficient to allow efficient infection, the expression of HS on BJAB cells promoted KSHV binding at the cell surface. Thus, our results indicate that MHV68 and KSHV cycles are blocked in B-cell lines at the binding step due to a lack of surface HS.
Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells
Authors: Sinigalia Elisa , Alvisi Gualtiero , Mercorelli Beatrice , Coen Donald M. , Pari Gregory S. , Jans David A. , Ripalti Alessandro , Palù Giorgio , Loregian Arianna
Published: October 2008

The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV
ori
Lyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy.
Passive Immunization Reduces Murine Cytomegalovirus-Induced Brain Pathology in Newborn Mice
Authors: Cekinović Đurđica , Golemac Mijo , Pugel Ester Pernjak , Tomac Jelena , Čičin-Šain Luka , Slavuljica Irena , Bradford Russell , Misch Sonja , Winkler Thomas H. , Mach Michael , Britt William J. , Jonjić Stipan
Published: October 2008

Human cytomegalovirus (HCMV) is the most frequent cause of congenital viral infections in humans and frequently leads to long-term central nervous system (CNS) abnormalities that include learning disabilities, microcephaly, and hearing loss. The pathogenesis of the CNS infection has not been fully elucidated and may arise as a result of direct damage of CMV-infected neurons or indirectly secondary to inflammatory response to infection. We used a recently established model of mouse CMV (MCMV) infection in newborn mice to analyze the contribution of humoral immunity to virus clearance from the brain. In brains of MCMV-infected newborn mice treated with immune serum, the titer of infectious virus was reduced below detection limit, whereas in the brains of mice receiving control (nonimmune) serum significant amounts of virus were recovered. Moreover, histopathological and immunohistological analyses revealed significantly less CNS inflammation in mice treated with immune serum. Treatment with MCMV-specific monoclonal antibodies also resulted in the reduction of virus titer in the brain. Recipients of control serum or irrelevant antibodies had more viral foci, marked mononuclear cell infiltrates, and prominent glial nodules in their brains than mice treated with immune serum or MCMV-specific antibodies. In conclusion, our data indicate that virus-specific antibodies have a protective role in the development of CNS pathology in MCMV-infected newborn mice, suggesting that antiviral antibodies may be an important component of protective immunological responses during CMV infection of the developing CNS.
Topology and Membrane Anchoring of the Coronavirus Replication Complex: Not All Hydrophobic Domains of nsp3 and nsp6 Are Membrane Spanning
Authors: Oostra Monique , Hagemeijer Marne C. , van Gent Michiel , Bekker Cornelis P. J. , te Lintelo Eddie G. , Rottier Peter J. M. , de Haan Cornelis A. M.
Published: October 2008

Coronaviruses express two very large replicase polyproteins, the 16 autoproteolytic cleavage products of which collectively form the membrane-anchored replication complexes. How these structures are assembled is still largely unknown, but it is likely that the membrane-spanning members of these nonstructural proteins (nsps) are responsible for the induction of the double-membrane vesicles and for anchoring the replication complexes to these membranes. For 3 of the 16 coronavirus nsps—nsp3, nsp4, and nsp6—multiple transmembrane domains are predicted. Previously we showed that, consistent with predictions, nsp4 occurs in membranes with both of its termini exposed in the cytoplasm (M. Oostra et al., J. Virol. 81:12323-12336, 2007). Strikingly, however, for both nsp3 and nsp6, predictions based on a multiple alignment of 27 coronavirus genome sequences indicate an uneven number of transmembrane domains. As a consequence, the proteinase domains present in nsp3 and nsp5 would be separated from their target sequences by the lipid bilayer. To look into this incongruity, we studied the membrane disposition of nsp3 and nsp6 of the severe acute respiratory syndrome coronavirus and murine hepatitis virus by analyzing tagged forms of the proteins expressed in cultured cells. Contrary to the predictions, in both viruses, both proteins had their amino terminus, as well as their carboxy terminus, exposed in the cytoplasm. We established that two of the three hydrophobic domains in nsp3 and six of the seven in nsp6 are membrane spanning. Subsequently, we verified that in nsp4, all four hydrophobic domains span the lipid bilayer. The occurrence of conserved non-membrane-spanning hydrophobic domains in nsp3 and nsp6 suggests an important function for these domains in coronavirus replication.
Memory CD4+T-Cell-Mediated Protection from Lethal Coronavirus Encephalomyelitis
Authors: Savarin Carine , Bergmann Cornelia C. , Hinton David R. , Ransohoff Richard M. , Stohlman Stephen A.
Published: October 2008

The antiviral role of CD4+T cells in virus-induced pathologies of the central nervous system (CNS) has not been explored extensively. Control of neurotropic mouse hepatitis virus (JHMV) requires the collaboration of CD4+and CD8+T cells, with CD8+T cells providing direct perforin and gamma interferon (IFN-γ)-mediated antiviral activity. To distinguish bystander from direct antiviral contributions of CD4+T cells in virus clearance and pathology, memory CD4+T cells purified from wild type (wt), perforin-deficient (PKO), and IFN-γ-deficient (GKO) immune donors were transferred to immunodeficient SCID mice prior to CNS challenge. All three donor CD4+T-cell populations controlled CNS virus replication at 8 days postinfection, indicating IFN-γ- and perforin-independent antiviral function. Recipients of GKO CD4+T cells succumbed more rapidly to fatal disease than untreated control infected mice. In contrast, wt and PKO donor CD4+T cells cleared infectious virus to undetectable levels and protected from fatal disease. Recipients of all CD4+T-cell populations exhibited demyelination. However, it was more severe in wt CD4+T-cell recipients. These data support a role of CD4+T cells in virus clearance and demyelination. Despite substantial IFN-γ-independent antiviral activity, IFN-γ was crucial in providing protection from death. IFN-γ reduced neutrophil accumulation and directed macrophages to white matter but did not ameliorate myelin loss.
The Zinc RING Finger of Bovine Herpesvirus 1-Encoded bICP0 Protein Is Crucial for Viral Replication and Virulence
Authors: Saira Kazima , Chowdhury Shafiqul , Gaudreault Natasha , da Silva Leticia , Henderson Gail , Doster Alan , Jones Clinton
Published: October 2008

Bovine herpesvirus 1 (BHV-1) infected cell protein 0 (bICP0) stimulates productive infection, in part by activating viral gene expression. The C
3
HC
4
zinc RING finger of bICP0 is crucial for activating viral transcription and productive infection. In this study, we used a bacterial artificial chromosome containing a wild-type (wt) virulent BHV-1 strain to generate a single amino acid mutation in the C
3
HC
4
zinc RING finger of bICP0. This virus (the 51g mutant) contains a cysteine-to-glycine mutation (51st amino acid) in the C
3
HC
4
zinc RING finger of bICP0. A plasmid expressing the 51g mutant protein did not transactivate viral promoter activity as efficiently as wt bICP0. The 51g mutant virus expressed higher levels of the bICP0 protein than did the 51g rescued virus (51gR) but yielded reduced virus titers following infection of permissive bovine cells. The 51g mutant virus, but not the 51gR virus, grew poorly in bovine cells pretreated with imiquimod to stimulate interferon production. During acute infection of calves, levels of infectious virus were 2 to 3 logs lower in ocular or nasal swabs with 51g than with 51gR. Calves latently infected with the 51g mutant did not reactivate from latency because virus shedding did not occur in ocular or nasal cavities. As expected, calves latently infected with 51gR reactivated from latency following dexamethasone treatment. These studies demonstrate that mutation of a single well-conserved cysteine residue in the C
3
HC
4
zinc RING finger of bICP0 has dramatic effects on the growth properties of BHV-1.
Complexes of Human Papillomavirus Type 16 E6 Proteins Form Pseudo-Death-Inducing Signaling Complex Structures during Tumor Necrosis Factor-Mediated Apoptosis
Authors: Filippova Maria , Filippov Valery A. , Kagoda Mercy , Garnett Theodore , Fodor Nadya , Duerksen-Hughes Penelope J.
Published: October 2008

High-risk strains of human papillomavirus (HPV) such as HPV type 16 (HPV16) and HPV18 are causative agents of most human cervical carcinomas. E6, one of the oncogenes encoded by HPV16, possesses a number of biological and transforming functions. We have previously shown that the binding of E6 to host apoptotic proteins such as tumor necrosis factor (TNF) R1, the adaptor protein FADD, and procaspase 8 results in a significant modification of the normal flow of apoptotic events. For example, E6 can bind to and accelerate the degradation of FADD. In addition, full-length E6 binds to the TNF R1 death domain and can also bind to and accelerate the degradation of procaspase 8. In contrast, the binding of small splice isoforms known as E6* results in the stabilization of procaspase 8. In this report, we propose a model for the ability of HPV16 E6 to both sensitize and protect cells from TNF as well as to protect cells from Fas. We demonstrate that both the level of E6 expression and the ratio between full-length E6 and E6* are important factors in the modification of the host extrinsic apoptotic pathways and show that at high levels of E6 expression, the further sensitization of U2OS, NOK, and Ca Ski cells to TNF-mediated apoptosis is most likely due to the formation of a pseudo-death-inducing signaling complex structure that includes complexes of E6 proteins.
Accelerated Prion Replication in, but Prolonged Survival Times of, Prion-Infected CXCR3−/−Mice
Authors: Riemer Constanze , Schultz Julia , Burwinkel Michael , Schwarz Anja , Mok Simon W. F. , Gültner Sandra , Bamme Theresa , Norley Stephen , van Landeghem Frank , Lu Bao , Gerard Craig , Baier Michael
Published: October 2008

Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3−/−) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3−/−mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrPScand 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3−/−compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrPScin disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrPSc.
RNA-Binding Protein hnRNP D Modulates Internal Ribosome Entry Site-Dependent Translation of Hepatitis C Virus RNA
Authors: Paek Ki Young , Kim Chon Saeng , Park Sung Mi , Kim Jong Heon , Jang Sung Key
Published: October 2008

Hepatitis C virus (HCV) is one of the major causative agents of virus-related hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. Translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of the genome. Here, we report that a cellular protein, hnRNP D, interacts with the 5′ border of HCV IRES (stem-loop II) and promotes translation of HCV mRNA. Overexpression of hnRNP D in mammalian cells enhances HCV IRES-dependent translation, whereas knockdown of hnRNP D with small interfering RNAs (siRNAs) inhibits translation. In addition, sequestration of hnRNP D with an interacting DNA oligomer inhibits the translation of HCV mRNA in an in vitro system. Ribosome profiling experiments reveal that HCV RNA is redistributed from heavy to light polysome fractions upon suppression of the hnRNP D level using specific siRNA. These results collectively suggest that hnRNP D plays an important role in the translation of HCV mRNA through interactions with the IRES. Moreover, knockdown of hnRNP D with siRNA significantly hampers infection by HCV. A potential role of hnRNP D in HCV proliferation is discussed.
Functional Pseudotyping of Human Immunodeficiency Virus Type 1 Vectors by Western Equine Encephalitis Virus Envelope Glycoprotein
Authors: Poluri Ananthalakshmi , Ainsworth Rebecca , Weaver Scott C. , Sutton Richard E.
Published: October 2008

We investigated the ability of western equine encephalitis virus envelope glycoproteins (WEEV GP) to pseudotype lentiviral vectors. The titers of WEEV GP-pseudotyped human immunodeficiency virus type 1 (HIV) ranged as high as 8.0 × 10
4
IU/ml on permissive cells. Sera from WEEV-infected mice specifically neutralized these pseudotypes; cell transduction was also sensitive to changes in pH. The host range of the pseudotyped particles in vitro was somewhat limited, which is atypical for most alphaviruses. HIV vectors pseudotyped by WEEV GP may be a useful tool for characterizing WEEV cell binding and entry and screening for small-molecule inhibitors.
Vaccinia Virus A26 and A27 Proteins Form a Stable Complex Tethered to Mature Virions by Association with the A17 Transmembrane Protein
Authors: Howard Amanda R. , Senkevich Tatiana G. , Moss Bernard
Published: October 2008

During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.