bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake
Authors: Grinter Rhys , Josts Inokentijs , Zeth Kornelius , Roszak Aleksander W. , McCaughey Laura C. , Cogdell Richard J. , Milner Joel J. , Kelly Sharon M. , Byron Olwyn , Walker Daniel
Published: May 2014

The colicin‐like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram‐negative bacteria by parasitizing nutrient uptake systems. We have structurally characterized the colicin M‐like bacteriocin, pectocin M2, which is active against strains of Pectobacterium spp. This unusual bacteriocin lacks the intrinsically unstructured translocation domain that usually mediates translocation of these bacteriocins across the outer membrane, containing only a single globular ferredoxin domain connected to its cytotoxic domain by a flexible α‐helix, which allows it to adopt two distinct conformations in solution. The ferredoxin domain of pectocin M2 is homologous to plant ferredoxins and allows pectocin M2 to parasitize a system utilized by Pectobacterium to obtain iron during infection of plants. Furthermore, we identify a novel ferredoxin‐containing bacteriocin pectocin P, which possesses a cytotoxic domain homologous to lysozyme, illustrating that the ferredoxin domain acts as a generic delivery module for cytotoxic domains in Pectobacterium.
A universal DNA extraction and PCR amplification method for fungal rDNA sequence‐based identification
Authors: Romanelli A. M. , Fu J. , Herrera M. L. , Wickes B. L.
Published: May 2014

Accurate identification of fungal pathogens using a sequence‐based approach requires an extraction method that yields template DNA pure enough for polymerase chain reaction (PCR) or other types of amplification. Therefore, the objective of this study was to develop and standardise a rapid, inexpensive DNA extraction protocol applicable to the major fungal phyla, which would yield sufficient template DNA pure enough for PCR and sequencing. A total of 519 clinical and culture collection strains, comprised of both yeast and filamentous fungi, were prepared using our extraction method to determine its applicability for PCR, which targeted the ITS and D1/D2 regions in a single PCR amplicon. All templates were successfully amplified and found to yield the correct strain identification when sequenced. This protocol could be completed in approximately 30 min and utilised a combination of physical and chemical extraction methods but did not require organic solvents nor ethanol precipitation. The method reduces the number of tube manipulations and yielded suitable template DNA for PCR amplification from all phyla that were tested.
Tulipa intermedia sp. nov. (Liliaceae) from the Ferghana Depression, Uzbekistan
Authors: Tojibaev K. S. , de Groot J. J. , Naralieva N. M.
Published: May 2014

A new species, Tulipa intermedia, collected in the Ferghana Depression is described. The new species belongs to section T. sect. Kolpakowskianae Raamsd. ex Zonn. & Veldkamp. It is similar to T. scharipovii Tojibaev and T. talassica Lazkov but is distinguished by morphological characters of bulbs and flowers and by its ecology, growing only in Artemisia steppe on plains and in low foothills with gravelly slopes. From T. scharipovii, the new species differs by non‐coriaceous and strongly prolongated bulb tunics, pubescence of tunics, shorter stem, shape of outer petals and filaments, and by its pale yellow (not green) bottle‐shaped ovary. Two varieties of T. intermedia differing in the colour of tepals and filaments were observed growing together and are formally described.
Open access support groups for people experiencing personality disorders: Do group members' experiences reflect the theoretical foundations of the SUN project?
Authors: Gillard Steve , White Rachel , Miller Steve , Turner Kati
Published: May 2014

Objectives

The SUN Project is an innovative, open access support group, based in the community, for people experiencing personality disorders, developed in response to UK Department of Health policy advocating improvements in personality disorders services. The aim of this article is to critically explore where and how the theoretically informed model underpinning the SUN Project is reflected in the view and experiences of people attending the project.

Design

This article reports an in‐depth, qualitative interview‐based study employing a critical realist approach.

Methods

As part of a larger study about self‐care and mental health, in‐depth qualitative interviews were held with 38 people new to the SUN Project, and again 9 months later. Data were extracted that were relevant to core components of the project model and were subjected to thematic analysis. The critical realist approach was used to move back and forth between empirical data and theory underpinning the SUN project, providing critical insight into the model.

Results

Participant accounts were broadly concordant with core components of the SUN Project's underlying model: Open access and self‐referral; group therapeutic processes; community‐based support; service users as staff. There were some tensions between interviewee accounts and theoretical aspects of the model, notably around the challenges that group processes presented for some individuals.

Conclusions

The model underlying the SUN Project is useful in informing good practice in therapeutic, community‐based peer support groups for people experiencing personality disorders. Careful consideration should be given to a limited multi‐modal approach, providing focused one‐to‐one support for vulnerable individuals who find it hard to engage in group processes.

Practitioner points

Facilitated peer support groups based in the community may act as a powerful therapeutic resource for people experiencing personality disorders.

Promoting open access and self‐referral to support groups may increase feelings of empowerment and engagement for people experiencing personality disorders.

Some individuals experiencing personality disorders who could potentially benefit from therapeutic groups may need focused one‐to‐one support to do so.
Ceteris Paribus, I Could Have Done Otherwise
Authors: Whittle Ann
Published: May 2014

In this paper, I explore an alternative to the Principle of Alternate Possibilities for Moral Responsibility—the Ceteris Paribus Principle of Alternative Possibilities for Moral Responsibility. I consider motivations for this principle and answer some objections to it.
Why Are We Certain that We Exist?
Authors: Billon Alexandre
Published: May 2014

Descartes was certain that he was thinking and he was accordingly certain that he existed. Like Descartes, we seem to be more certain of our thoughts and our existence than of anything else. What is less clear is the reasonwhywe are thus certain. Philosophers throughout history have provided different interpretations of thecogito, disagreeing both on the kind of thoughts it characterizes and on the reasons for its cogency. According to what we may call theempiricist interpretationof thecogito, I can only claim to be certain of havingexperiences, and this certainty, as well as that of my own existence, stems from their phenomenal and subjective character. According torationalist interpretations, on the other hand, I am certain of having some self‐reflexivepropositional attitudes, and this certainty derives from their rational features. Psychiatric patients suffering from acute forms of depersonalization or of the Cotard syndrome often doubt that they think and exist, and might even believe that they don't. I argue that their study allows us to favor the empiricist interpretation of thecogito.
The relationship between organisational orientation and research and development/technology commercialisation performance
Authors: Dhewanto Wawan , Sohal Amrik S.
Published: May 2014

This paper explores organisational orientations related to research and development/technology commercialisation and their influence on technology commercialisation performance. A number of hypotheses are tested using structural equation modelling using data collected through a quantitative survey. The hypothesis that customer orientation and innovation orientation are related to technology commercialisation capability and the hypothesis that technology commercialisation capability is related to technology commercialisation performance are accepted, while the hypothesis that competitor orientation is related to technology commercialisation capability is not supported. From a theoretical perspective, the research contributes to a broader application of the resource‐based view in technology commercialisation research area, through exploring intangible resources (organisational orientations) and capability (technology commercialisation capability) that relate to technology commercialisation performance. From a practical perspective, in order to improve technology commercialisation performance, managers should monitor the speed of each commercialisation step: initiate the development of products (research); develop products (development); and launch the products to the market, so that the product arrives on the market in an appropriate time. Managers should also consider matching product customisation and market segment so that appropriate products are delivered to appropriate market segments. In addition, managers should monitor technology developments in the related industry, select appropriate technologies, acquire the technologies and integrate the technologies into their products, so that the improved product performs better than the previous product and competitors’ product.
Mobilising the elective diaspora: US–German academic exchanges since 1945
Authors: Jöns Heike , Mavroudi Elizabeth , Heffernan Michael
Published: May 2014

This article responds to recent calls by geographers for more critical, non‐essentialist and flexible conceptualisations of diaspora by developing the notion of an ‘elective diaspora’. This concept is elaborated using the case study of a specific knowledge diaspora, namely visiting researchers from the USA in the Federal Republic of Germany (FRG), who were funded by the Alexander von Humboldt Foundation, a research agency re‐established by the FRG in 1953. The analysis shows how the Humboldt programmes for transnational academic mobility and collaboration drew disproportionately on US‐based academics with biographical ties to German‐speaking central Europe, which has contributed to the proliferation of US–German knowledge networks in the post‐war period and prompts us to reconsider existing notions of diaspora in two ways. First, we emphasise the nuances of the researchers' emotional attachment to German language and culture. We point out that this cannot only be caused by biographical ties through birth and ancestry but also by other family relations, partnerships, friendships, work/living experiences, language skills and cultural knowledge. Second, we stress the elective nature of diasporic identities and belonging by emphasising that individuals can choose whether they wish to support diasporic networks of one or more communities and cultures they feel connected to. We suggest that this civic rather than ‘ethno’‐territorial understanding of diasporic networks has wider relevance for theorisations of diaspora, for studies of transnational mobility and knowledge transfer, and for university and public policies seeking to attract talent from abroad.
New Ethnographic Film in the New China
Authors: Gillette Maris Boyd
Published: May 2014

Over the past two decades, a new ethnographic cinema of China has taken shape, one element of an explosion of documentaries produced about the People's Republic. In this volume, five China ethnographers discuss making ethnographic films in China, thus addressing in part the need, long noted in anthropology, for filmmakers to write about production and postproduction of ethnographic films. In this introduction, I sketch a history of ethnographic films about China and reflect on these five new ethnographic films in light of past practices, the theoretical literature on ethnographic cinema, and the ethnography of China.
Chaiqian/Demolition: Reflections on Media Practice
Authors: Sniadecki J.P.
Published: May 2014

In 2008, I spent three weeks filming a demolition site in the center ofChengdu, the capital ofChina'sSichuan province.Icollaborated with migrant workers, site managers, and city dwellers to produce the feature‐length filmChaiqian (Demolition), which offers a sensual and open‐ended portrait of migrant labor, urban space, and ephemerality. This essay presents a set of reflections on the production process and aesthetic choices of the film, and draws on sensory ethnography, phenomenology, and the concept of cinephilia to explore the ontological differences between written ethnography and film.Iobserve that the conventional approach to media production in anthropology has tended to limit the multitude of meaning inherent in images and sound by reducing them to mere illustrations of an argument and/or cultural data. While this dominant approach has its specific uses, especially in terms of pedagogy,Iargue that equal value ought to be placed on an approach to media anthropology that not only embraces sensuality and ambiguity but also appreciates the multivalent powers peculiar to sound and image, and refrains from constraining visuality and aurality to the wholly linguistic and propositional. The essay concludes with a meditation on the value that such an approach—with its accommodation of multiple interpretations of the image—possesses for historiography and the construction of alternative archives.
MakingBored in Heaven: A Film about Ritual Sensation
Authors: Dean Cora , Dean Kenneth
Published: May 2014

This essay discusses the making of the documentary filmBored inHeaven: A Film about Ritual Sensation(2010). We first discuss our theoretical framework, then following the content and organization of the film, we provide historical and ethnographic context on the return of the gods toSoutheastChina over the past few decades. Throughout the essay, we discuss how the film was made in order to enter into and respond to realms of ritual sensation within an overall, overwhelming ritual event, collectively performed by villagers in this part ofChina. Different sections reflect on camera angles, length of shots, montage, narration, rhythm and breathing, overstimulation and spectacle, alternative temporality, shamanic possession, and cinematic form. The conclusion discusses key themes of the flow of affect within communal self‐expression and the future of local ritual.
Genetic Analysis of Dobrava–Belgrade Virus from Western Serbia – A Newly Detected Focus in the Balkan Peninsula
Authors: Stamenković G. , Nikolić V. , Blagojević J. , Bugarski‐Stanojević V. , Adnađević T. , Stanojević M. , Vujošević M.
Published: May 2014

Dobrava–Belgrade virus (DOBV) is a hantavirus species that causes the most severe form of haemorrhagic fever with renal syndrome (HFRS) in Europe. DOBV has been detected in three Apodemus rodents: A. flavicollis, A. agrarius and A. ponticus. These emerging viruses appear throughout the Balkan Peninsula including Serbia as its central part. In this study, we examined the seroprevalence, molecular epidemiology and phylogenetics of DOBV from A. flavicollis captured at six Serbian localities. Furthermore, we applied microsatellite typing of host animal genome to analyse the role of host kinship in DOBV animal transmission. The overall IgG seropositivity rate over 3 years (2008–2010) was 11.9% (22/185). All seropositive samples were subjected to RT‐PCR and DNA sequencing for S and L genome segments (pos. 291–1079 nt and 2999–3316 nt, respectively). DOBV was genetically detected in three samples from mountain Tara in western Serbia, a newly detected DOBV focus in the Balkans. No sequence data from human cases from Serbia are available for the studied period. However, collected DOBV isolates in this work phylogenetically clustered together with isolates from Serbian human cases dating from 2002, with 1.9% nucleotide divergence. We determined the level of kinship between seropositive and seronegative animal groups and found no significant difference, suggesting that horizontal virus transmission in the studied population was the same within and among the hatches. Our findings are the first genetic detection of DOBV in rodents in Serbia. We confirm wide and continuous hantavirus presence in the examined parts of the Balkans, underlying the necessity of continual monitoring of hantavirus circulation in A. flavicollis.
Industrial Gas Turbine Performance: Compressor Fouling and On-Line Washing
Authors: Igie Uyioghosa , Pilidis Pericles , Fouflias Dimitrios , Ramsden Kenneth , Laskaridis Panagiotis
Published: May 2014

Industrial gas turbines are susceptible to compressor fouling, which is the deposition and accretion of airborne particles or contaminants on the compressor blades. This paper demonstrates the blade aerodynamic effects of fouling through experimental compressor cascade tests and the accompanied engine performance degradation using turbomatch, an in-house gas turbine performance software. Similarly, on-line compressor washing is implemented taking into account typical operating conditions comparable with industry high pressure washing. The fouling study shows the changes in the individual stage maps of the compressor in this condition, the impact of degradation during part-load, influence of control variables, and the identification of key parameters to ascertain fouling levels. Applying demineralized water for 10 min, with a liquid-to-air ratio of 0.2%, the aerodynamic performance of the blade is shown to improve, however most of the cleaning effect occurred in the first 5 min. The most effectively washed part of the blade was the pressure side, in which most of the particles deposited during the accelerated fouling. The simulation of fouled and washed engine conditions indicates 30% recovery of the lost power due to washing.
Thermal Degradation and Burnishing Wear of Thin Carbon Film by Frictional Heat Generation
Authors: Lee Sungae , He Muyang , Yeo Chang-Dong
Published: May 2014

The burnishing wear of carbon films found in dynamic microdevices could be attributed to both mechanical stress and temperature rise by frictional heat generation. In this study, novel modeling and experiment were performed to investigate the burnishing wear mechanism of carbon film during high speed sliding contact. An improved thermomechanical contact model for a single asperity was extended to rough surface contact. The contact stress and surface temperature rise were examined at various contact conditions. To verify the thermal degradation of the carbon film by frictional heat flux, micro-Raman spectroscopy measurement was performed on actual burnishing failure sample.
Failure Mechanics—Part I: The Coordination Between Elasticity Theory and Failure Theory for all Isotropic Materials
Authors: Christensen Richard M.
Published: May 2014

Failure mechanics is comprised of the failure theory for homogeneous and isotropic materials along with all of its implications and applications. The present failure theory is found to have an intimate connection with elasticity behavior even though plasticity may also transpire. This becomes apparent and useful when the classical theory of elasticity is renormalized to give a simpler and more transparent (but still exact) formalism. The connection or coordination between elasticity and failure then explicitly occurs through the use of the renormalized Poisson's ratio to characterize the ductility of failure. With this unification of failure theory and elasticity theory, failure mechanics can be extended to explain other anomalous aspects of mechanical behavior and prepare it for applications.
Quantitative Analysis of the Nonlinear Displacement–Load Behavior of the Lumbar Spine
Authors: Hanlon Andrew D. , Cook Daniel J. , Yeager Matthew S. , Cheng Boyle C.
Published: May 2014

There is currently no universal model or fitting method to characterize the visco-elastic behavior of the lumbar spine observed in displacement versus load hysteresis loops. In this study, proposed methods for fitting these loops, along with the metrics obtained, were thoroughly analyzed. A spline fitting technique was shown to provide a consistent approximation of spinal kinetic behavior that can be differentiated and integrated. Using this tool, previously established metrics were analyzed using data from two separate studies evaluating different motion preservation technologies. Many of the metrics, however, provided no significant differences beyond range of motion analysis. Particular attention was paid to how different definitions of the neutral zone capture the high-flexibility region often seen in lumbar hysteresis loops. As a result, the maximum slope was introduced and shown to be well defined. This new parameter offers promise as a descriptive measurement of spinal instability in vitro and may have future implications in clinical diagnosis and treatment of spinal instability. In particular, it could help in assigning treatments to specific stabilizing effects in the lumbar spine.
Using Full Scale Experiments to Verify a Simulation Used to Analyze the Safety of Rail Vehicles During Large Earthquakes
Authors: Nishimura Kazuhiko , Terumichi Yoshiaki , Morimura Tsutomu , Adachi Masahito , Morishita Yoshitaka , Miwa Masahiro
Published: May 2014

A simulation model is used to calculate the rocking motion of a vehicle and how antiderailing guard rails work to prevent derailment when subject to large ground excitation and the results are verified through full scale testing. (1) The simple vehicle–track model should properly represent the rocking mechanism. (2) The effect of vehicle speed on the wheel/rail slide is also properly represented, thus the wheel/rail creep law model can be applied to the analysis of a running vehicle experiencing large rocking motion. (3) The action of the guard rail was equal in both the simulation and the full scale test, thus indicating that the simple antiderailing guard rail model sufficiently represents the dynamic interaction of the wheel/guard rail and the effect.
Laser Engineered Net Shaping Process for 316L/15% Nickel Coated Titanium Carbide Metal Matrix Composite
Authors: Amano R. S. , Marek S. , Schultz B. F. , Rohatgi P. K.
Published: May 2014

This paper presents the investigation of the macrostructure, microstructure, and solidification structure of a 316L/15% nickel coated TiC metal matrix composite produced by the laser engineered net shaping (LENS™) process. The focus of this work was to (1) identify the solidification structure and to estimate growth/cooling rates at the solid/liquid interface, (2) identify and quantify discontinuities in the build structure, and (3) examine the effect of solidification and thermal history on the sample microstructure to further the understanding of the LENS process. A Numerical method was also developed to examine the influence of material type and LENS™ process parameters on the forming of the specific microstructures from thermodynamics and fluid dynamics point of view. Samples of 316L stainless steel were examined, microstructures of samples were used to estimate the corresponding cooling rate, and the cooling rate was compared with the results of numerical modeling. The computational results show reasonable agreement with experimentally determined cooling rate.
A Pilot Study of a Continuum Shoulder Exoskeleton for Anatomy Adaptive Assistances
Authors: Xu Kai , Zhao Jiangran , Qiu Dong , Wang You
Published: May 2014

Many existing exoskeletons have followed a similar design concept that a rigid kinematic chain is actuated to mobilize a human wearer in spite of the intended applications. For performance-augmenting applications where an exoskeleton is usually paired with a specific wearer, the human–machine kinematic compatibility might be well maintained. However, in a clinical setting for rehabilitation where one exoskeleton is often shared by a group of patients, it will be difficult for the therapists to guarantee the on-site adjustments would accurately fit the exoskeleton to each individual patient with his/her unique anatomy. This paper proposes a continuum shoulder exoskeleton design to realize anatomy adaptive assistances (AAAs) for hemiparetic patients in a purely assistive mode where patient's limb motions are passive. The shoulder exoskeleton conforms to distinct human anatomies adaptively due to its intrinsic flexibility but still manages to deliver motion assistances in a consistent way. The design concept and the system descriptions are elaborated, including kinematics, statics, system construction, actuation, experimental validation, backbone shape identification, motion compensation, manikin trials, etc. The results suggest that it is possible to design a continuum exoskeleton to assist different patients with their limb movements, while no mechanical adjustments on the exoskeleton shall be performed.
The general low-frequency prediction for asymptotically nonlinear material functions in oscillatory shear
Authors: Bharadwaj N. Ashwin , Ewoldt Randy H.
Published: May 2014

We use a fourth-order fluid expansion to make general predictions for asymptotically nonlinear material functions in oscillatory shear, a characterization protocol sometimes known as medium-amplitude oscillatory shear. The calculation applies to any viscoelastic fluid in the terminal regime defined by the limit of Deborah number much less than one. Two viscous nonlinearities appear at third order, with shear stress scaling as  ω3, and are interrelated by a constant multiplicative factor. Two elastic nonlinearities appear at fourth order, with shear stress scaling as ω4, and are also interrelated by a constant multiplicative factor. These nonlinear measures are decoupled from the linear material functions G′ and G″ because they depend on different expansion coefficients. Experimental measurements are presented for all four asymptotic shear material functions using a linear and well-entangled homopolymer of polyisoprene. The experimental observations are consistent with both the predicted frequency scaling and the predicted interrelations in the terminal regime. Signs and magnitudes cannot be universally predicted, leaving these as free parameters that depend on the specifics of the material microstructure or constitutive model. These general results explain previous observations involving different materials and constitutive models, and provide an important reference for future experiments, analytical results, and numerical computations of these rheological fingerprints.
Improved Survival with T Cell Clonotype Stability After Anti–CTLA-4 Treatment in Cancer Patients
Authors: Cha Edward , Klinger Mark , Hou Yafei , Cummings Craig , Ribas Antoni , Faham Malek , Fong Lawrence
Published: May 2014

CTLA-4 blockade induces T cell repertoire turnover, but high-frequency clonotypes are maintained in patients with favorable outcomes.
Photoactivation of Endogenous Latent Transforming Growth Factor–β1 Directs Dental Stem Cell Differentiation for Regeneration
Authors: Arany Praveen R. , Cho Andrew , Hunt Tristan D. , Sidhu Gursimran , Shin Kyungsup , Hahm Eason , Huang George X. , Weaver James , Chen Aaron Chih-Hao , Padwa Bonnie L. , Hamblin Michael R. , Barcellos-Hoff Mary Helen , Kulkarni Ashok B. , J. Mooney David
Published: May 2014

Low-power laser–activated endogenous latent transforming growth factor–β1 (LTGF-β1) directs resident dental stem cell differentiation to promote dentin regeneration.
CD4 T Cells with Effector Memory Phenotype and Function Develop in the Sterile Environment of the Fetus
Authors: Zhang Xiaoming , Mozeleski Brian , Lemoine Sebastien , Dériaud Edith , Lim Annick , Zhivaki Dania , Azria Elie , Le Ray Camille , Roguet Gwenaelle , Launay Odile , Vanet Anne , Leclerc Claude , Lo-Man Richard
Published: May 2014

Umbilical cord blood from healthy neonates with no known infectious exposure contains T cells with an effector memory–like phenotype.
Carbapenem-Resistant Klebsiella pneumoniae Strains Exhibit Diversity in Aminoglycoside-Modifying Enzymes, Which Exert Differing Effects on Plazomicin and Other Agents
Authors: Almaghrabi Reem , Clancy Cornelius J. , Doi Yohei , Hao Binghua , Chen Liang , Shields Ryan K. , Press Ellen G. , Iovine Nicole M. , Townsend Bethany M. , Wagener Marilyn M. , Kreiswirth Barry , Nguyen M. Hong
Published: May 2014

We measured
in vitro
activity of plazomicin, a next-generation aminoglycoside, and other aminoglycosides against 50 carbapenem-resistant
Klebsiella pneumoniae
strains from two centers and correlated the results with the presence of various aminoglycoside-modifying enzymes (AMEs). Ninety-four percent of strains were sequence type 258 (ST258) clones, which exhibited 5
ompK36
genotypes; 80% and 10% of strains produced
Klebsiella pneumoniae
carbapenemase 2 (KPC-2) and KPC-3, respectively. Ninety-eight percent of strains possessed AMEs, including AAC(6′)-Ib (98%), APH(3′)-Ia (56%), AAC(3)-IV (38%), and ANT(2″)-Ia (2%). Gentamicin, tobramycin, and amikacin nonsusceptibility rates were 40, 98, and 16%, respectively. Plazomicin MICs ranged from 0.25 to 1 μg/ml. Tobramycin and plazomicin MICs correlated with gentamicin MICs (
r
= 0.75 and 0.57, respectively). Plazomicin exerted bactericidal activity against 17% (1× MIC) and 94% (4× MIC) of strains. All strains with AAC(6′)-Ib were tobramycin-resistant; 16% were nonsusceptible to amikacin. AAC(6′)-Ib combined with another AME was associated with higher gentamicin, tobramycin, and plazomicin MICs than AAC(6′)-Ib alone (
P
= 0.01, 0.0008, and 0.046, respectively). The presence of AAC(3)-IV in a strain was also associated with higher gentamicin, tobramycin, and plazomicin MICs (
P
= 0.0006,
P
< 0.0001, and
P
= 0.01, respectively). The combination of AAC(6′)-Ib and another AME, the presence of AAC(3)-IV, and the presence of APH(3′)-Ia were each associated with gentamicin resistance (
P
= 0.0002, 0.003, and 0.01, respectively). In conclusion, carbapenem-resistant
K. pneumoniae
strains (including ST258 clones) exhibit highly diverse antimicrobial resistance genotypes and phenotypes. Plazomicin may offer a treatment option against strains resistant to other aminoglycosides. The development of molecular assays that predict antimicrobial responses among carbapenem-resistant
K. pneumoniae
strains should be a research priority.
Alternative Strategies for Proof-of-Principle Studies of Antibacterial Agents
Authors: Dalhoff Axel , Weintraub Andrej , Nord Carl Erik
Published: May 2014

The proof that a new antibacterial agent is not only active
in vitro
but also effective
in vivo
under clinically relevant conditions is currently provided (i) by using appropriate nonclinical models of infection and pharmacokinetic-pharmacodynamic (PK-PD) analysis providing evidence of the likelihood of clinical efficacy and (ii) by examining the study drug in exploratory clinical trials, as well as dose and schedule finding during phase II of clinical development. This approach is both time-consuming and costly. Furthermore, PK-PD targets for any novel antibacterial agent cannot be derived from studies with experimental animals. Therefore, alternative strategies have to be identified to prove the principle that a novel antibacterial agent is active under clinically relevant conditions. This review summarizes evidence that the quantitative analysis of shifts in the viable counts of pathogens in infected patients or the evaluation of the PD effect of an investigational agent on indicator organisms of the human resident microflora or colonizers of healthy volunteers, if paralleled with PK monitoring of serum and the target site, provides an alternative to a classical proof-of-principle study in the course of a phase II study program.
Antifungal Resistance to Fluconazole and Echinocandins Is Not Emerging in Yeast Isolates Causing Fungemia in a Spanish Tertiary Care Center
Authors: Marcos-Zambrano Laura Judith , Escribano Pilar , Sánchez Carlos , Muñoz Patricia , Bouza Emilio , Guinea Jesús
Published: May 2014

Accurate knowledge of fungemia epidemiology requires identification of strains to the molecular level. Various studies have shown that the rate of resistance to fluconazole ranges from 2.5% to 9% in
Candida
spp. isolated from blood samples. However, trends in antifungal resistance have received little attention and have been studied only using CLSI M27-A3 methodology. We assessed the fungemia epidemiology in a large tertiary care institution in Madrid, Spain, by identifying isolates to the molecular level and performing antifungal susceptibility testing according to the updated breakpoints of European Committee for Antimicrobial Susceptibility Testing (EUCAST) definitive document (EDef) 7.2. We studied 613 isolates causing 598 episodes of fungemia in 544 patients admitted to our hospital (January 2007 to December 2013). Strains were identified after amplification and sequencing of the ITS1-5.8S-ITS2 region and further tested for
in vitro
susceptibility to amphotericin B, fluconazole, posaconazole, voriconazole, micafungin, and anidulafungin. Resistance was defined using EUCAST species-specific breakpoints, and epidemiological cutoff values (ECOFFs) were applied as tentative breakpoints. Most episodes were caused by
Candida albicans
(46%),
Candida parapsilosis
(28.7%),
Candida glabrata
(9.8%), and
Candida tropicalis
(8%). Molecular identification enabled us to better detect cryptic species of
Candida guilliermondii
and
C. parapsilosis
complexes and episodes of polyfungal fungemia. The overall percentage of fluconazole-resistant isolates was 5%, although it was higher in
C. glabrata
(8.6%) and non-
Candida
yeast isolates (47.4%). The rate of resistance to echinocandins was 4.4% and was mainly due to the presence of intrinsically resistant non-
Candida
species. Resistance mainly affected non-
Candida
yeasts. The rate of resistance to fluconazole and echinocandins did not change considerably during the study period.
Whole-Genome Analyses of Enterococcus faecium Isolates with Diverse Daptomycin MICs
Authors: Diaz Lorena , Tran Truc T. , Munita Jose M. , Miller William R. , Rincon Sandra , Carvajal Lina P. , Wollam Aye , Reyes Jinnethe , Panesso Diana , Rojas Natalia L. , Shamoo Yousif , Murray Barbara E. , Weinstock George M. , Arias Cesar A.
Published: May 2014

Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a “last-resort” antibiotic against vancomycin-resistant
Enterococcus faecium
(VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in
E. faecium
. Thus, in order to gain insights into the genetic bases of DAP resistance in
E. faecium
, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19
E. faecium
with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120→Ala and Trp73→Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of
liaFSR
or
yycFGHIJ
mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.
In Vitro
and
In Vivo
Assessment of Dermatophyte Acquired Resistance to Efinaconazole, a Novel Triazole Antifungal
Authors: Iwata Atsushi , Watanabe Yoko , Kumagai Naomichi , Katafuchi-Nagashima Maria , Sugiura Keita , Pillai Radhakrishnan , Tatsumi Yoshiyuki
Published: May 2014

Efinaconazole is a novel triazole antifungal drug for the topical treatment of onychomycosis, a nail infection caused mainly by dermatophytes. We assessed the potential of efinaconazole to induce resistance in dermatophytes by continuous exposure of
Trichophyton rubrum
strains to efinaconazole
in vitro
(12 passages) and in a guinea pig onychomycosis model (8 weeks). There was no evidence of efinaconazole resistance development in the tested strains under the experimental conditions used.
Different Interaction Profiles of Direct-Acting Anti-Hepatitis C Virus Agents with Human Organic Anion Transporting Polypeptides
Authors: Furihata Tomomi , Matsumoto Shogo , Fu Zhongguo , Tsubota Akihito , Sun Yuchen , Matsumoto Sayaka , Kobayashi Kaoru , Chiba Kan
Published: May 2014

Simeprevir (SMV), asunaprevir (ASV), daclatasvir (DCV), and sofosbuvir (SFV), which are newly developed direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) infection, are among the key components of anti-HCV regimens. Preclinical studies have identified inhibitory properties for some of these DAAs against organic anion transporting polypeptide 1B (OATP1B) functions. However, their details remain mostly uncharacterized. Because OATP1B1 and OATP1B3 play determinant roles in the pharmacokinetics of various drugs via their uptake into human hepatocytes, it is plausible that the inhibition of these OATP1Bs by a DAA would create a potential risk of drug-drug interaction, which has been an emerging concern in anti-HCV therapy. Accordingly, in the present study, we intended to clarify the inhibitory characteristics of newly developed DAAs toward OATP1B1 and -1B3 functions. The results of our coincubation inhibition assays have shown that all tested DAAs could inhibit OATP1B1 functions and that SMV, ASV, and DCV (to a lesser extent), but not SFV, exhibited long-lasting preincubation inhibitory effects on OATP1B1 functions. It was also found that the preincubation inhibitory effects of SMV and ASV could augment their coincubation inhibition potency. Furthermore, significant, but differential, inhibitory effects of the DAAs on the OATP1B3 function were identified. To summarize, our results clearly show that the newly developed DAAs are newly identified OATP1B1 and OATP1B3 inhibitors with distinctive interaction properties. It is believed that these inhibition profiles will provide essential information to all concerned parties with respect to the clinical significance of DAA-mediated inhibition of OATP1Bs in anti-HCV therapy.
Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors
Authors: Xu Hong-Tao , Colby-Germinario Susan P. , Oliveira Maureen , Rajotte Daniel , Bethell Richard , Wainberg Mark A.
Published: May 2014

A W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.
SAMHD1 Has Differential Impact on the Efficacies of HIV Nucleoside Reverse Transcriptase Inhibitors
Authors: Huber Andrew D. , Michailidis Eleftherios , Schultz Megan L. , Ong Yee T. , Bloch Nicolin , Puray-Chavez Maritza N. , Leslie Maxwell D. , Ji Juan , Lucas Anthony D. , Kirby Karen A. , Landau Nathaniel R. , Sarafianos Stefan G.
Published: May 2014

Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.
Giardia muris Infection in Mice Is Associated with a Protective Interleukin 17A Response and Induction of Peroxisome Proliferator-Activated Receptor Alpha
Authors: Dreesen Leentje , De Bosscher Karolien , Grit Grietje , Staels Bart , Lubberts Erik , Bauge Eric , Geldhof Peter
Published: May 2014

The protozoan parasiteGiardia duodenalis(Giardia lamblia) is one of the most commonly found intestinal pathogens in mammals, including humans. In the current study, aGiardia muris-mouse model was used to analyze cytokine transcription patterns and histological changes in intestinal tissue at different time points during infection in C57BL/6 mice. Since earlier work revealed the upregulation of peroxisome proliferator-activated receptors (PPARs) inGiardia-infected calves, a second aim was to investigate the potential activation of PPARs in the intestines of infected mice. The most important observation in all mice was a strong upregulation ofil17astarting around 1 week postinfection. The significance of interleukin 17A (IL-17A) in orchestrating a protective immune response was further demonstrated in an infection trial or experiment using IL-17 receptor A (IL-17RA) knockout (KO) mice: whereas in wild-type (WT) mice, cyst secretion dropped significantly after 3 weeks of infection, the IL-17RA KO mice were unable to clear the infection. Analysis of the intestinal response further indicated peroxisome proliferator-activated receptor alpha (PPARα) induction soon after the initial contact with the parasite, as characterized by the transcriptional upregulation ofpparaitself and several downstream target genes such aspltpandcpt1. Overall, PPARα did not seem to have any influence on the immune response againstG. muris, since PPARα KO animals expressedil-17aand could clear the infection similar to WT controls. In conclusion, this study shows for the first time the importance of IL-17 production in the clearance of aG. murisinfection together with an early induction of PPARα. The effect of the latter, however, is still unclear.
Development of an
Ex Vivo
Porcine Lung Model for Studying Growth, Virulence, and Signaling of Pseudomonas aeruginosa
Authors: Harrison Freya , Muruli Aneesha , Higgins Steven , Diggle Stephen P.
Published: May 2014

Research into chronic infection by bacterial pathogens, such as
Pseudomonas aeruginosa
, uses various
in vitro
and live host models. While these have increased our understanding of pathogen growth, virulence, and evolution, each model has certain limitations.
In vitro
models cannot recapitulate the complex spatial structure of host organs, while experiments on live hosts are limited in terms of sample size and infection duration for ethical reasons; live mammal models also require specialized facilities which are costly to run. To address this, we have developed an
ex vivo
pig lung (EVPL) model for quantifying
Pseudomonas aeruginosa
growth, quorum sensing (QS), virulence factor production, and tissue damage in an environment that mimics a chronically infected cystic fibrosis (CF) lung. In a first test of our model, we show that
lasR
mutants, which do not respond to 3-oxo-C
12
-homoserine lactone (HSL)-mediated QS, exhibit reduced virulence factor production in EVPL. We also show that
lasR
mutants grow as well as or better than a corresponding wild-type strain in EVPL.
lasR
mutants frequently and repeatedly arise during chronic CF lung infection, but the evolutionary forces governing their appearance and spread are not clear. Our data are not consistent with the hypothesis that
lasR
mutants act as social “cheats” in the lung; rather, our results support the hypothesis that
lasR
mutants are more adapted to the lung environment. More generally, this model will facilitate improved studies of microbial disease, especially studies of how cells of the same and different species interact in polymicrobial infections in a spatially structured environment.
Inflammatory Effects of Edwardsiella ictaluri Lipopolysaccharide Modifications in Catfish Gut
Authors: Santander Javier , Kilbourne Jacquelyn , Park Jie-Yeun , Martin Taylor , Loh Amanda , Diaz Ignacia , Rojas Robert , Segovia Cristopher , DeNardo Dale , Curtiss Roy
Published: May 2014

Bacterial lipopolysaccharides (LPS) are structural components of the outer membranes of Gram-negative bacteria and also are potent inducers of inflammation in mammals. Higher vertebrates are extremely sensitive to LPS, but lower vertebrates, like fish, are resistant to their systemic toxic effects. However, the effects of LPS on the fish intestinal mucosa remain unknown.Edwardsiella ictaluriis a primitive member of theEnterobacteriaceaefamily that causes enteric septicemia in channel catfish (Ictalurus punctatus).E. ictaluriinfects and colonizes deep lymphoid tissues upon oral or immersion infection. Both gut and olfactory organs are the primary sites of invasion. At the systemic level,E. ictaluripathogenesis is relatively well characterized, but our knowledge aboutE. ictaluriintestinal interaction is limited. Recently, we observed thatE. ictalurioligo-polysaccharide (O-PS) LPS mutants have differential effects on the intestinal epithelia of orally inoculated catfish. Here we evaluate the effects ofE. ictaluriO-PS LPS mutants by using a novel catfish intestinal loop model and compare it to the rabbit ileal loop model inoculated withSalmonella entericaserovar Typhimurium LPS. We found evident differences in rabbit ileal loop and catfish ileal loop responses toE. ictaluriandS. Typhimurium LPS. We determined that catfish respond toE. ictaluriLPS but not toS. Typhimurium LPS. We also determined thatE. ictaluriinhibits cytokine production and induces disruption of the intestinal fish epithelia in an O-PS-dependent fashion. TheE. ictaluriwild type and ΔwibTLPS mutant caused intestinal tissue damage and inhibited proinflammatory cytokine synthesis, in contrast toE. ictaluriΔgneand ΔugdLPS mutants. We concluded that theE. ictaluriO-PS subunits play a major role during pathogenesis, since they influence the recognition of the LPS by the intestinal mucosal immune system of the catfish. The LPS structure ofE. ictalurimutants is needed to understand the mechanism of interaction.