bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated Surface Defect Detection Using High-Density Data
Authors: Wells Lee J. , Shafae Mohammed S. , Camelio Jaime A.
Published: December 2015

State-of-the-art measurement technologies, such as 3D laser scanners, provide new opportunities for knowledge discovery and development of quality control (QC) strategies for complex manufacturing systems. These technologies can rapidly provide millions of data points to represent a manufactured part's surface. The resulting high-density (HD) datasets have a great potential to be used for inspecting parts for surface and feature abnormalities. The current use of these datasets for part inspection can be divided into two main categories: (1) extracting feature parameters, which does not complement the nature of these datasets as it wastes valuable data and (2) an ad hoc inspection process, where a visual representation of the data is manually analyzed, which tends to suffer from slow, inefficient, and inconsistent inspection results. To overcome these deficiencies, this paper proposes an adaptive generalized likelihood ratio (AGLR) technique to automate the surface defect inspection process using HD data. This paper presents the performance results of the proposed AGLR approach with respect to the probability of detecting varying size and magnitude defects in addition to the probability of false alarms. In addition, a formal approach for designing an optimal AGLR inspection system is proposed. Finally, simulation results are presented and analyzed to showcase the performance gains of the AGLR approach versus a more traditional generalized likelihood ratio (GLR) approach.
Actuation Configurations of Bionic Hands for a Better Anthropomorphism Index
Authors: Tavakoli Mahmoud , Enes Baptiste , Marques Lino , Feix Thomas
Published: December 2015

In this study, we analyze different actuation configurations for bionic hands in order to improve their level of anthropomorphism. We used a previously developed benchmark, the anthropomorphism index (AI), for 15 different actuation configurations of hands from one to five actuators. By comparing the AI of these configurations, we obtained important conclusions regarding the actuation strategy of the anthropomorphic hands with a limited number of actuators. Results show that the actuation configuration is very important for increasing the level of anthropomorphism of the hands. It is shown that with an appropriate actuation configuration, a configuration with lower number of actuators can result in a higher AI than a configuration with higher number of actuators. We also showed the best actuation configurations for each category of 1–5 actuators. Results can be used as a guideline for development of hands with high anthropomorphism in terms of grasping postures.
Trade Space Exploration of Magnetically Actuated Origami Mechanisms
Authors: Bowen Landen , Springsteen Kara , Frecker Mary , Simpson Timothy
Published: December 2015

Self-folding origami has the potential to be utilized in novel areas such as self-assembling robots and shape-morphing structures. Important decisions in the development of such applications include the choice of active material and its placement on the origami model. With proper active material placement, the error between the actual and target shapes can be minimized along with cost, weight, and input energy requirements. A method for creating magnetically actuated dynamic models and experimentally verifying their results is briefly reviewed, after which the joint stiffness and magnetic material approximations used in the dynamic model are discussed in more detail. Through the incorporation of dynamic models of magnetically actuated origami mechanisms into the Applied Research Laboratory's trade space visualizer (atsv), the trade spaces of self-folding dynamic models of the waterbomb base and Shafer's frog tongue are explored. Finally, a design tradeoff is investigated between target shape approximation error and the placement of magnetic material needed to reach a target shape. These two examples demonstrate the potential use of this process as a design tool for other self-folding origami mechanisms.
Miniaturized Terrestrial Walking Robot Using PVDF/PVP/PSSA Based Ionic Polymer–Metal Composite Actuator
Authors: Tien Nguyen Kim , Young Ko Seong , Park Jong-Oh , Park Sukho
Published: December 2015

This paper presents a design and fabrication of millimeter scale walking robot using ionic polymer–metal composite (IPMC) actuator as the robot's leg for walking in terrestrial environment. A small scale of new IPMC actuator based on poly-vinylidene fluoride (PVDF)/polyvinyl pyrrolidone (PVP)/polystyrene sulfuric acid (PSSA) blend membrane was fabricated and employed in this study to sustain and drive the walking robot with sufficient force and displacement. The PVDF/PVP/PSSA based IPMC actuator with a polymer mixture ratio of 15/30/55 shows improved performances than Nafion based IPMC actuator. To enhance a traction force of the walking robot and to increase the life time of IPMC actuators, the IPMC strips are covered with a thin PDMS (polydimethylsiloxane) layer. A miniaturized terrestrial walking robot (size: 18 × 11 × 12 mm, weight: 1.3 g) with a light weight robot's body which can support 2-, 4-, or 6-IPMC-leg models was designed and implemented the walking motion on the ground at the maximum speed of 0.58 mm/s.
Variable Motion/Force Transmissibility of a Metamorphic Parallel Mechanism With Reconfigurable 3T and 3R Motion
Authors: Gan Dongming , Dai Jian S. , Dias Jorge , Seneviratne Lakmal D.
Published: December 2015

This paper presents a metamorphic parallel mechanism (MPM) which can switch its motion between pure translation (3T) and pure rotation (3R). This feature stems from a reconfigurable Hooke (rT) joint of which one of the rotation axes can be altered freely. More than that, based on the reconfiguration of the rT joint, workspace of both 3T and 3R motion can be tunable, and the rotation center of the 3R motion can be controlled along a line perpendicular to the base plane. Kinematics analysis is presented based on the geometric constraints of the parallel mechanism covering both 3T and 3R motion. Following this, screw theory based motion/force transmission equations are obtained, and their characteristics are investigated and linked to the singularity analysis using Jacobian matrix. Motion/force transmission indices can be used to optimize basic design parameters of the MPM. This provides reference of this mechanism for potential applications requiring 3T and 3R motion.
A Survey of Analysis, Modeling, and Diagnostics of Diesel Fuel Injection Systems
Authors: Krogerus Tomi R. , Hyvönen Mika P. , Huhtala Kalevi J.
Published: December 2015

Diesel engines are widely used due to their high reliability, high thermal efficiency, fuel availability, and low consumption. They are used to generate power, e.g., in passenger cars, ships, power plants, marine offshore platforms, and mining and construction machines. The engine is at heart of these applications, so keeping it in good working condition is vital. Recent technical and computational advances and environmental legislation have stimulated the development of more efficient and robust techniques for the diagnostics of diesel engines. The emphasis is on the diagnostics of faults under development and the causes of engine failure or reduced efficiency. Diesel engine fuel injection plays an important role in the development of the combustion in the engine cylinder. Arguably, the most influential component of the diesel engine is the fuel injection equipment; even minor faults can cause a major loss of efficiency of the combustion and an increase in engine emissions and noise. With increased sophistication (e.g., higher injection pressures) being required to meet continuously improving noise, exhaust smoke, and gaseous emission regulations, fuel injection equipment is becoming even more susceptible to failure. The injection systems have been shown to be the largest contributing factor in diesel engine failures. Extracting the health information of components in the fuel injection system is a very demanding task. Besides the very time-consuming nature of experimental investigations, direct measurements are also limited to selected observation points. Diesel engine faults normally do not occur in a short timeframe. The modeling of typical engine faults, particularly combustion related faults, in a controlled manner is thus vital for the development of diesel engine diagnostics and fault detection. Simulation models based on physical grounds can enlarge the number of studied variables and also obtain a better understanding of localized phenomena that affect the overall behavior of the system. This paper presents a survey of the analysis, modeling, and diagnostics of diesel fuel injection systems. Typical diesel fuel injection systems and their common faults are presented. The most relevant state of the art research articles on analysis and modeling of fluid injection systems as well as diagnostics techniques and measured signals describing the behavior of the system are reviewed and the results and findings are discussed. The increasing demand and effect of legislation related to diagnostics, especially on-board diagnostics (OBD), are discussed with reference to the future progress of this field.
C60-SIMS imaging of nanoparticles within mammalian cells
Authors: Bloom Anna N. , Tian Hua , Winograd Nicholas
Published: December 2015

To achieve successful drug delivery via nanoparticles the interactions between the nanoparticle and the chemistry of the surrounding biological environment is of central importance. A thorough understanding of these interactions is necessary in order to better elucidate information regarding drug pathways and mechanisms of action in treatment protocols. As such, it is important to identify the location of the nanoparticle, the state of its functionalization, as well as any changes in the cellular environment. The use of cluster secondary ion mass spectrometry (SIMS) using C60+ primary ions makes simultaneous acquisition of this information possible. Here, SIMS has been successfully used to chemically image gold nanoparticles (AuNPs) within a model, single cell system involving macrophage-like RAW 264.7 cells. The macrophage-like properties of this cell line make it extremely well-suited for cell-uptake studies. Both AuNPs and two pharmaceutical compounds, amiodarone and elacridar, were successfully imaged within a cellular system using cluster SIMS. To verify that SIMS can also be used to detect functionalization and nanoparticles simultaneously, fluorophore-functionalized AuNPs were studied as a model system. The fluorescent characteristics of these functionalized nanoparticles enabled the visual confirmation of the presence and location of the particles within the cell.
Characterizing perceptual properties of a binaurally modulated stimulus
Authors: Reed Darrin K. , van de Par Steven
Published: December 2015

The majority of everyday listening situations involve a complex mixture of multiple sound sources. Assuming a spectro-temporally sparse target signal, e.g., speech, the binaural cues in these complex listening environments do not simply result from a single sound source, but are often rapidly switching between the most dominant sources at any given moment. To investigate the perception of rapidly switching interaural time difference (ITD) cues, a noise stimulus that periodically alternates between two different values of ITD was created. This stimulus appears to evoke a purely binaural percept of modulation, which is the focus of the studies presented here. Results indicate that listeners can reliably discriminate this ITD-switching stimulus from a stimulus composed of stationary ITD cues. Frequency discrimination of this ITD-switching stimulus was tested in a separate experiment, which showed that listeners are generally able to discriminate a 50% change in modulation frequency for reference rates below 16 Hz. The final study investigated if modulation masking exists between the monaural and binaural auditory pathways. Although a statistically significant increase in modulation detection thresholds was observed when both types of modulations were presented together, the increase was relatively small, indicating only a weak interaction.
California stem cell agency plots a race to the clinic
Authors: Servick Kelly
Published: December 2015

CIRM lays out endgame and prospects for new money.
Additive manufacturing of polymer-derived ceramics
Authors: Eckel Zak C. , Zhou Chaoyin , Martin John H. , Jacobsen Alan J. , Carter William B. , Schaedler Tobias A.
Published: December 2015

Printing ceramics into complex shapes

Some materials, such as thermoplastics and metals, are naturally suited to being 3D printed because the individual particles can be fused together by applying heat. In contrast, ceramics do not fuse together the same way. Eckel
et al.
developed a way to pattern specific preceramic monomers using either 3D printing or stereolithography into complex, curved, and porous shapes. Upon heating, they observed almost no shrinkage, and the formed parts showed exceptional thermal stability.

Science
, this issue p.
58
Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production
Authors: Sakimoto Kelsey K. , Wong Andrew Barnabas , Yang Peidong
Published: December 2015

Using light in the darkness

Solid-state devices can efficiently capture solar energy to produce chemicals and fuels from carbon dioxide. Yet biology has already developed a high-specificity, low-cost system to do just that through photosynthesis. Sakimoto
et al.
developed a biological-inorganic hybrid that combines the best of both worlds (see the Perspective by Müller). They precipitated semiconductor nanoparticles on the surface of a nonphotosynthetic bacterium to serve as a light harvester. The captured energy sustained cellular metabolism, producing acetic acid: a natural waste product of respiration.

Science
, this issue p.
74
; see also p.
34
Seeing mTORC1 specificity
Authors: Buel Gwen R. , Blenis John
Published: December 2015

Structural information reveals how a multiprotein complex responds to amino acid abundance

[Also see Research Articles by

Wolfson
et al.

and

Aylett
et al.

and

Saxton
et al.

]
New members of the family
Authors: McNutt Marcia
Published: December 2015

Some publishers add new journal titles more often than I buy new shoes. The American Association for the Advancement of Science (AAAS), the publisher of
Science
, certainly cannot be accused of that, having grown to only four journal titles in 136 years. Yet many members of the scientific community have encouraged AAAS to expand its repertoire, arguing that the
Science
family of journals is high quality, well curated and edited, and worth supporting because they enable the mission of a nonprofit scientific society that is governed by scientists working to further the best interests of the scientific community. It is thus with some pride that I describe the launch in 2016 of two new journals:
Science Robotics and Science Immunology
.
Triton Hodge Test: Improved Protocol for Modified Hodge Test for Enhanced Detection of NDM and Other Carbapenemase Producers
Authors: Pasteran Fernando , Gonzalez Lisandro J. , Albornoz Ezequiel , Bahr Guillermo , Vila Alejandro J. , Corso Alejandra
Published: December 2015

Accurate detection of carbapenemase-producing Gram-negative bacilli is of utmost importance for the control of nosocomial spread and the initiation of appropriate antimicrobial therapy. The modified Hodge test (MHT), a carbapenem inactivation assay, has shown poor sensitivity in detecting the worldwide spread of New Delhi metallo-β-lactamase (NDM). Recent studies demonstrated that NDM is a lipoprotein anchored to the outer membrane in Gram-negative bacteria, unlike all other known carbapenemases. Here we report that membrane anchoring of β-lactamases precludes detection of carbapenemase activity by the MHT. We also show that this limitation can be overcome by the addition of Triton X-100 during the test, which allows detection of NDM. We propose an improved version of the assay, called the Triton Hodge test (THT), which allows detection of membrane-bound carbapenemases with the addition of this nonionic surfactant. This test was challenged with a panel of 185 clinical isolates (145 carrying known carbapenemase-encoding genes and 40 carbapenemase nonproducers). The THT displayed test sensitivity of >90% against NDM-producing clinical isolates, while improving performance against other carbapenemases. Ertapenem provided the highest sensitivity (97 to 100%, depending on the type of carbapenemase), followed by meropenem (92.5 to 100%). Test specificity was not affected by the addition of Triton (87.5% and 92.5% with ertapenem and meropenem, respectively). This simple inexpensive test confers a large improvement to the sensitivity of the MHT for the detection of NDM and other carbapenemases.
Rapid and Accurate Determination of Lipopolysaccharide O-Antigen Types in Klebsiella pneumoniae with a Novel PCR-Based O-Genotyping Method
Authors: Fang Chi-Tai , Shih Yun-Jui , Cheong Cheng-Man , Yi Wen-Ching
Published: December 2015

Klebsiella pneumoniae
, a Gram-negative bacillus that causes life-threatening infections in both hospitalized patients and ambulatory persons, can be classified into nine lipopolysaccharide (LPS) O-antigen serotypes. The O-antigen type has important clinical and epidemiological significance. However,
K. pneumoniae
O serotyping is cumbersome, and the reagents are not commercially available. To overcome the limitations of conventional serotyping methods, we aimed to create a rapid and accurate PCR method for
K. pneumoniae
O genotyping. We sequenced the genetic determinants of LPS O antigen from serotypes O1, O2a, O2ac, O3, O4, O5, O8, O9, and O12. We established a two-step genotyping scheme, based on the two genomic regions associated with O-antigen biosynthesis. The first set of PCR primers, which detects alleles at the
wzm-wzt
loci of the
wb
gene cluster, distinguishes between O1/O2, O3, O4, O5, O8, O9, and O12. The second set of PCR primers, which detects alleles at the
wbbY
region, further differentiates between O1, O2a, and O2ac. We verified the specificity of O genotyping against the O-serotype reference strains. We then tested the sensitivity and specificity of O genotyping in
K. pneumoniae
, using the 56 K-serotype reference strains with known O serotypes determined by an inhibition enzyme-linked immunosorbent assay (iELISA). There is a very good correlation between the O genotypes and classical O serotypes. Three discrepancies were observed and resolved by nucleotide sequencing—all in favor of O genotyping. The PCR-based O genotyping, which can be easily performed in clinical and research microbiology laboratories, is a rapid and accurate method for determining the LPS O-antigen types of
K. pneumoniae
isolates.
Automated Scoring of Chromogenic Media for Detection of Methicillin-Resistant Staphylococcus aureus by Use of WASPLab Image Analysis Software
Authors: Faron Matthew L. , Buchan Blake W. , Vismara Chiara , Lacchini Carla , Bielli Alessandra , Gesu Giovanni , Liebregts Theo , van Bree Anita , Jansz Arjan , Soucy Genevieve , Korver John , Ledeboer Nathan A.
Published: December 2015

Recently, systems have been developed to create total laboratory automation for clinical microbiology. These systems allow for the automation of specimen processing, specimen incubation, and imaging of bacterial growth. In this study, we used the WASPLab to validate software that discriminates and segregates positive and negative chromogenic methicillin-resistant
Staphylococcus aureus
(MRSA) plates by recognition of pigmented colonies. A total of 57,690 swabs submitted for MRSA screening were enrolled in the study. Four sites enrolled specimens following their standard of care. Chromogenic agar used at these sites included MRSASelect (Bio-Rad Laboratories, Redmond, WA), chromID MRSA (bioMérieux, Marcy l'Etoile, France), and CHROMagar MRSA (BD Diagnostics, Sparks, MD). Specimens were plated and incubated using the WASPLab. The digital camera took images at 0 and 16 to 24 h and the WASPLab software determined the presence of positive colonies based on a hue, saturation, and value (HSV) score. If the HSV score fell within a defined threshold, the plate was called positive. The performance of the digital analysis was compared to manual reading. Overall, the digital software had a sensitivity of 100% and a specificity of 90.7% with the specificity ranging between 90.0 and 96.0 across all sites. The results were similar using the three different agars with a sensitivity of 100% and specificity ranging between 90.7 and 92.4%. These data demonstrate that automated digital analysis can be used to accurately sort positive from negative chromogenic agar cultures regardless of the pigmentation produced.
Plasmalyte: No Longer a Culprit in Causing False-Positive Galactomannan Test Results
Authors: Spriet Isabel , Lagrou Katrien , Maertens Johan , Willems Ludo , Wilmer Alexander , Wauters Joost
Published: December 2015

False-positive galactomannan (GM) results have been reported in patients treated with gluconate-containing solutions, such as Plasmalyte. The GM optical density index was tested on 33 distinct batches of Plasmalyte and was found to be negative in all of the batches, confirming that Plasmalyte is no longer a cause of false-positive GM results.
Preclinical Assessment of a Fully Automated Multiplex PCR Panel for Detection of Central Nervous System Pathogens
Authors: Hanson K. E. , Slechta E. S. , Killpack J. A. , Heyrend C. , Lunt T. , Daly J. A , Hemmert A. C. , Blaschke A. J.
Published: December 2015

We evaluated a multiplexed PCR panel for the detection of 16 bacterial, viral, and fungal pathogens in cerebrospinal fluid. Panel results were compared to routine testing, and discrepancies were resolved by additional nucleic acid amplification tests or sequencing. Overall, the positive and negative agreements across methods were 92.9% and 91.9%, respectively.
Ultrafast Assessment of the Presence of a High-Risk Mycobacterium tuberculosis Strain in a Population
Authors: Pérez-Lago Laura , Herranz Marta , Comas Iñaki , Ruiz-Serrano María Jesús , López Roa Paula , Bouza Emilio , García-de-Viedma Darío
Published: December 2015

A persistent 8-year infection by a Beijing
Mycobacterium tuberculosis
strain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission—despite opportunities—and challenged some of the advantages associated with Beijing strains.
In Vitro
Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an
In Vivo
Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus
Authors: Ferris Ryan A. , McCue Patrick M. , Borlee Grace I. , Loncar Kristen D. , Hennet Margo L. , Borlee Bradley R.
Published: December 2015

In this study, we evaluated the ability of the equine clinical treatments
N
-acetylcysteine, EDTA, and hydrogen peroxide to disrupt
in vitro
biofilms and kill equine reproductive pathogens (
Escherichia coli
,
Pseudomonas aeruginosa
, or
Klebsiella pneumoniae
) isolated from clinical cases.
N
-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of
E. coli
isolates. The CFU of recoverable
P. aeruginosa
and
K. pneumoniae
isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of
E. coli
isolates,
K. pneumoniae
isolates were observed to have a reduction in CFU, and minimal effects were observed for
P. aeruginosa
isolates. Chelating agents (EDTA formulations) reduced
E. coli
CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of
P. aeruginosa
and
K. pneumoniae
within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. An
in vivo
equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine
P. aeruginosa
isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent “biofilm-like” matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilm
in vitro
, and
P. aeruginosa
is capable of producing biofilm-like material
in vivo
.
Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia
Authors: Sasso Milène , Chastang-Dumas Elsa , Bastide Sophie , Alonso Sandrine , Lechiche Catherine , Bourgeois Nathalie , Lachaud Laurence
Published: December 2015

Pneumonia due to
Pneumocystis jirovecii
(PCP) is a frequent infection among HIV-positive or other immunocompromised patients. In the past several years, PCR on pulmonary samples has become an essential element for the laboratory diagnosis of PCP. Nevertheless, very few comparative studies of available PCR assays have been published. In this work, we evaluated the concordance between four real-time PCR assays, including three commercial kits, AmpliSens, MycAssay, and Bio-Evolution PCR, and an in-house PCR (J. Fillaux et al. 2008, J Microbiol Methods 75:258–261, doi:
http://dx.doi.org/10.1016/j.mimet.2008.06.009
), on 148 pulmonary samples. The results showed concordance rates ranging from 81.6% to 96.6% (kappa, 0.64 to 0.93). Concordance was excellent between three assays: the in-house assay, AmpliSens, and the MycAssay PCR (kappa, >0.8). The performances of these PCR assays were also evaluated according to the classification of the probability of PCP (proven, probable, possible, or no final diagnosis of PCP) based on clinical and radiological signs as well as on the direct examination of bronchoalveolar lavage samples. In the proven PCP category,
Pneumocystis jirovecii
DNA was detected with all four assays. In the probable PCP category, the in-house PCR, AmpliSens, and the MycAssay PCR were positive for all samples, while the Bio-Evolution PCR failed to detect
Pneumocystis jirovecii
DNA in two samples. In the possible PCP category, the percentage of positive samples according to PCR varied from 54.5% to 86.4%. Detection of colonized patients is discussed. Finally, among the four evaluated PCR assays, one was not suitable for colonization detection but showed good performance in the proven and probable PCP groups. For the three other assays, performances were excellent and allowed detection of a very low fungal burden.
The BioFilm Ring Test: a Rapid Method for Routine Analysis of Pseudomonas aeruginosa Biofilm Formation Kinetics
Authors: Olivares Elodie , Badel-Berchoux Stéphanie , Provot Christian , Jaulhac Benoît , Prévost Gilles , Bernardi Thierry , Jehl François
Published: December 2015

Currently, few techniques are available for the evaluation of bacterial biofilm adhesion. These detection tools generally require time for culture and/or arduous handling steps. In this work, the BioFilm Ring Test (BRT), a new technology, was used to estimate the biofilm formation kinetics of 25 strains of
Pseudomonas aeruginosa
, isolated from the sputum of cystic fibrosis (CF) patients. The principle of the new assay is based on the mobility measurement of magnetic microbeads mixed with a bacterial suspension in a polystyrene microplate. If free to move under the magnetic action, particles gather to a visible central spot in the well bottom. Therefore, the absence of spot formation in the plate reflects the bead immobilization by a biofilm in formation. The BRT device allowed us to classify the bacterial strains into three general adhesion profiles. Group 1 consists of bacteria, which are able to form a solid biofilm in <2 h. Group 2 comprises the strains that progressively set up a biofilm during 24 h. Lastly, group 3 includes the strains that stay in a planktonic form. The grouping of our strains did not differ according to culture conditions, i.e., the use of different sets of beads or culture media. The BRT is shown to be an informative tool for the characterization of biofilm-forming bacteria. Various application perspectives may be investigated for this device, such as the addition of antibiotics to the bacterial suspension to select which would have the ability to inhibit the biofilm formation.
Chemotherapy-Induced Intestinal Mucosal Barrier Damage: a Cause of Falsely Elevated Serum 1,3-Beta-
d
-Glucan Levels?
Authors: Prattes Juergen , Raggam Reinhard B. , Vanstraelen Kim , Rabensteiner Jasmin , Hoegenauer Christoph , Krause Robert , Prüller Florian , Wölfler Albert , Spriet Isabel , Hoenigl Martin
Published: December 2015

Blood citrulline and intestinal fatty acid binding protein were determined as biomarkers for intestinal mucositis. Biomarker levels were correlated with corresponding serum 1,3-beta-
d
-glucan levels in 56 samples obtained from 33 cases with underlying hematological malignancies receiving induction chemotherapy. No correlation between biomarkers of intestinal mucositis and BDG levels was observed. (This study has been registered at ClinicalTrials.gov under registration no. NCT01576653.)
Polyprotein-Driven Formation of Two Interdependent Sets of Complexes Supporting Hepatitis C Virus Genome Replication
Authors: Gomes Rafael G. B. , Isken Olaf , Tautz Norbert , McLauchlan John , McCormick Christopher J.
Published: December 2015

Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from open reading frame 2 (ORF2) and a second duplicate nonstructural (NS) polyprotein from ORF1. Using these constructs and other methods, we have assessed the polyprotein requirements for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings: those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A
cis
-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue: one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N), and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, an S1977P-mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two interdependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation.

IMPORTANCE
Positive-strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about the functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing subgenomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly, as it enables improved composite polyprotein complex formation compared to traditional
trans
-complementation systems. Finally, the differential behavior of distinct NS3 helicase knockout mutations hints that certain conformations of this enzyme might be particularly deleterious for replication.
Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins
Authors: Go Eden P. , Cupo Albert , Ringe Rajesh , Pugach Pavel , Moore John P. , Desaire Heather
Published: December 2015

We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505
env
gene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds.

IMPORTANCE
It is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.
Functional Genomics Reveals Linkers Critical for Influenza Virus Polymerase
Authors: Wang Lulan , Wu Aiping , Wang Yao E. , Quanquin Natalie , Li Chunfeng , Wang Jingfeng , Chen Hsiang-Wen , Liu Suyang , Liu Ping , Zhang Hong , Qin F. Xiao-Feng , Jiang Taijiao , Cheng Genhong
Published: December 2015

Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits but also have revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five-amino-acid insertions in the linker were nonviable. Our model further suggests that the PA linker plays an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis.

IMPORTANCE
The RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2, and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five-amino-acid insertions in individual linkers were nonviable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We further identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.
Plasmacytoid Dendritic Cells Require Direct Infection To Sustain the Pulmonary Influenza A Virus-Specific CD8 T Cell Response
Authors: Hemann Emily A. , Sjaastad Louisa E. , Langlois Ryan A. , Legge Kevin L.
Published: December 2015

Following influenza A virus (IAV) infection, development of a robust IAV-specific CD8 T cell response is required for clearance of primary infection and enhances memory protection. Following IAV infection, plasmacytoid dendritic cells (pDC) or CD8α
+
DC regulate pulmonary effector CD8 T cell responses within the lung. Without this DC-T cell interaction, insufficient effector CD8 T cells are maintained in the lungs, leading to enhanced morbidity and mortality. Previous studies have demonstrated that pDC are capable of classical presentation or cross-presentation of IAV antigens and could potentially regulate IAV-specific CD8 T cell responses through either mechanism. Our results demonstrate that pDC from the lungs of donor mice infected with an IAV that is not able to replicate in hematopoietic cells (142t-IAV), unlike donor pDC isolated from the lungs of control infected mice, are not able to rescue the host IAV-specific CD8 T cell response from apoptosis. This indicates that pDC must utilize the direct presentation pathway for this rescue. This inability of pDC from 142t-IAV donors to rescue the IAV-specific CD8 T cell response is not due to differences in the overall ability of 142t-IAV to replicate within the lungs or generate defective viral genomes or to differences in levels of costimulatory molecules required for this interaction. We further demonstrate that bypassing the antigen presentation pathway by coating the 142t-IAV pDC with IAV peptide epitopes restores their ability to rescue the IAV-specific CD8 T cell response.

IMPORTANCE
IAV continues to be a global health burden, infecting 5 to 20% of the global population annually. Continued investigation into the mechanisms that mediate protective immune responses against IAV is important to improving current vaccination and antiviral strategies antagonistic toward IAV. Our findings presented herein demonstrate a key requirement for pDC promotion of effector CD8 T cell survival: that rather than utilizing cross-presentation, pDC must be infected and utilize the endogenous pathway for presentation of antigens to CD8 T cells during
in vivo
IAV infections. This suggests that targeting presentation via the endogenous pathway in pDC could be important for the development of unique antiviral cellular therapies.
Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40
Authors: Wijesinghe Kaveesha J. , Stahelin Robert V.
Published: December 2015

Marburg virus (MARV), which belongs to the virus family
Filoviridae
, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated the
in vitro
and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface.

IMPORTANCE
Marburg virus (MARV) is a lipid-enveloped filamentous virus from the family
Filoviridae
. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane.
In vitro
and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV.
Functional Comparison of Molluscum Contagiosum Virus vFLIP MC159 with Murine Cytomegalovirus M36/vICA and M45/vIRA Proteins
Authors: Hüttmann Julia , Krause Eva , Schommartz Tim , Brune Wolfram
Published: December 2015

Molluscum contagiosum virus (MCV) gene
MC159
encodes a viral FLICE inhibitory protein (vFLIP) that inhibits caspase-8-mediated apoptosis. The MC159 protein was also reported to inhibit programmed necrosis (necroptosis) and modulate NF-κB activation by interacting with RIP1 and NEMO. The importance of MC159 during MCV infection has remained unknown, as there is no system for propagation and genetic manipulation of this virus. Here we investigated the functions of MC159 during viral infection using murine cytomegalovirus (MCMV) as a surrogate virus.
MC159
was inserted into the MCMV genome, replacing
M36
or
M45
, two MCMV genes with functions similar to those reported for
MC159
.
M36
encodes a viral inhibitor of caspase-8-induced apoptosis (vICA) and
M45
a viral inhibitor of RIP activation (vIRA), which inhibits RIP1/RIP3-mediated necroptosis. The M45 protein also blocks NF-κB activation by interacting with NEMO. When expressed by MCMV, MC159 blocked tumor necrosis factor alpha (TNF-α)-induced apoptosis of infected cells and partially restored MCMV replication in macrophages. However, MC159 did not fully replace M45, as it did not inhibit necroptosis in murine cells, but it reduced TNF-α-induced necroptosis in MCMV-infected human HT-29 cells. MC159 also differed from M45 in its effect on NF-κB. While MCMV-encoded M45 blocked NF-κB activation by TNF-α and interleukin-1β (IL-1β), MC159 inhibited TNF-α- but not IL-1β-induced NF-κB activation in infected mouse fibroblasts. These results indicate that the spectrum of MC159's functions differs depending on cell type and expression system and that a cell culture system for the propagation of MCV is needed to determine the biological relevance of presumed viral gene functions.

IMPORTANCE
MCV is a human-pathogenic poxvirus that cannot be propagated in cell culture or laboratory animals. Therefore, MCV gene products have been studied predominantly in cells expressing individual viral genes. In this study, we analyzed the function of the MCV gene
MC159
by expressing it from a different virus and comparing its functions to those of two well-characterized MCMV genes. In this system,
MC159
displayed some but not all of the previously described functions, suggesting that the functions of a viral gene depend on the conditions under which it is expressed. Until a cell culture system for the analysis of MCV becomes available, it might be necessary to analyze MCV genes in several different systems to extrapolate their biological importance.
Determination of Sequences Required for Human Endogenous Retrovirus K Transduction and Its Recognition by Foreign Retroviral Virions
Authors: Erlwein Otto , Sweeney Nathan P. , de Leon Raffaele , Wills Gillian , Robinson Mark J. , McClure Myra O.
Published: December 2015

Sequences necessary for transduction of human endogenous retrovirus (HERV)-K
con
, a consensus of the HERV-K(HML-2) family, were analyzed and found to reside in the leader/
gag
region. They act in an orientation-dependent way and consist of at least two sites working together. Having defined these sequences, we exploited this information to produce a simple system to investigate to what extent virions of HERV-K
con
, murine leukemia virus, and HIV-1 have the ability to transduce each other's genomes, leading to potential contamination of gene therapy vectors.
Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses
Authors: Carolan Louise A. , Rockman Steve , Borg Kathryn , Guarnaccia Teagan , Reading Patrick , Mosse Jennifer , Kelso Anne , Barr Ian , Laurie Karen L.
Published: December 2015

The burden of infection with seasonal influenza viruses is significant. Each year is typically characterized by the dominance of one (sub)type or lineage of influenza A or B virus, respectively. The incidence of disease varies annually, and while this may be attributed to a particular virus strain or subtype, the impacts of prior immunity, population differences, and variations in clinical assessment are also important. To improve our understanding of the impacts of seasonal influenza viruses, we directly compared clinical symptoms, virus shedding, and expression of cytokines, chemokines, and immune mediators in the upper respiratory tract (URT) of ferrets infected with contemporary A(H1N1)pdm09, A(H3N2), or influenza B virus. Gene expression in the lower respiratory tract (LRT) was also assessed. Clinical symptoms were minimal. Overall cytokine/chemokine profiles in the URT were consistent in pattern and magnitude between animals infected with influenza A and B viruses, and peak expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p40, alpha interferon (IFN-α), IFN-β, and tumor necrosis factor alpha (TNF-α) mRNAs correlated with peak levels of viral shedding. MCP1 and IFN-γ were expressed after the virus peak. Granzymes A and B and IL-10 reached peak expression as the virus was cleared and seroconversion was detected. Cytokine/chemokine gene expression in the LRT following A(H1N1)pdm09 virus infection reflected the observations seen for the URT but was delayed 2 or 3 days, as was virus replication. These data indicate that disease severities and localized immune responses following infection with seasonal influenza A and B viruses are similar, suggesting that other factors are likely to modulate the incidence and impact of seasonal influenza.

IMPORTANCE
Both influenza A and B viruses cocirculate in the human population, and annual influenza seasons are typically dominated by an influenza A virus subtype or an influenza B virus lineage. Surveillance data indicate that the burden of disease is higher in some seasons, yet it is unclear whether this is due to specific virus strains or to other factors, such as cross-reactive immunity or clinical definitions of influenza. We directly compared disease severities and localized inflammatory responses to different seasonal influenza virus strains, including the 2009 pandemic strain, in healthy naive ferrets. We found that the disease severities and the cytokine and chemokine responses were similar irrespective of the seasonal strain or the location of the infection in the respiratory tract. This suggests that factors other than the immune response to a particular virus (sub)type contribute to the variable impact of influenza virus infection in a population.