bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of Three-Dimensional Microstructure Model for Discontinuously Reinforced Composite by Modified Random Sequential Absorption Method
Authors: Zhou Jiming , Qi Lehua , Gokhale Arun M.
Published: December 2015

Computer simulation of mechanical behavior of discontinuously reinforced composites containing randomly oriented short-fibers/whiskers presents an attractive opportunity for reduction of the number of experiments and resources required for microstructure design of such advanced materials. It is desirable to perform such simulations using microstructure model that accounts for randomness in angular orientations and locations of the short fibers/whiskers. In this contribution, a methodology is presented for efficient simulation of the required microstructural model through modification of well-known random sequential adsorption (RSA) algorithm for microstructure simulation through its application to the microstructure of Mg–alloy matrix composite containing randomly oriented short carbon fibers. The modified RSA algorithm enhances accuracy and efficiency of the complex geometric details of the randomly oriented short-fiber reinforced composite microstructure. Simulated microstructural model of composite is implemented in abaqus to simulate the mechanical response of the Mg–matrix composite containing randomly oriented short carbon fibers. The generated complex microstructure model in abaqus code is sliced into thin slices for reducing computing resources. The simulated results from multiple sliced models were averaged to approximate the result for the full volume element. The simulated mechanical response by use of multiple sliced models is validated via comparison with the experimental data.
Simplified Finite Element Models to Study the Dry Collapse of Straight and Curved Flexible Pipes
Authors: Neto Alfredo Gay , Martins Clóvis de Arruda , Malta Eduardo Ribeiro , Tanaka Rafael Loureiro , Godinho Carlos Alberto Ferreira
Published: December 2015

Dry collapse is one of the possible failure modes of flexible pipes. It refers to the situation in which no damage occurs in the flexible pipe external sheath. In this scenario, all layers of the pipe withstand the external pressure loading in a deep-water application. Such a situation is addressed in this work, which proposes some simplified modeling techniques to represent straight and curved flexible pipes subjected to external pressure, undergoing dry collapse during simulation procedure. The results of the proposed models are compared to other reference results, from a fully three-dimensional (3D) finite element model. Good agreement has been got, even with the proposed simplifications with a large reduction in computational cost when compared to full 3D model.
Mistuned Forced Response Predictions of an Embedded Rotor in a Multistage Compressor
Authors: Besem Fanny M. , Kielb Robert E. , Galpin Paul , Zori Laith , Key Nicole L.
Published: December 2015

This paper covers a comprehensive forced response analysis conducted on a multistage compressor and compared with the largest forced response experimental data set ever obtained in the field. The steady-state aerodynamic performance and stator wake predictions compare well with the experimental data, although losses are underestimated. Coupled and uncoupled unsteady simulations are conducted on the stator–rotor configuration. It is shown that the use of a decoupled method for forced response cannot yield accurate results for cases with strong inter-row interactions. The individual and combined contributions of the upstream and downstream stators are also assessed. The downstream stator is found to have a tremendous impact on the forced response predictions due to the constructive interactions of the two stator rows. Finally, predicted mistuned blade amplitudes are compared to mistuned experimental data. The average amplitudes match the experiments very well, while the maximum response amplitude is underestimated.
Roughness Effects on Flow and Heat Transfer for Additively Manufactured Channels
Authors: Stimpson Curtis K. , Snyder Jacob C. , Thole Karen A. , Mongillo Dominic
Published: December 2015

Recent technological advances in the field of additive manufacturing (AM), particularly with direct metal laser sintering (DMLS), have increased the potential for building gas turbine components with AM. Using the DMLS for turbine components broadens the design space and allows for increasingly small and complex geometries to be fabricated with little increase in time or cost. Challenges arise when attempting to evaluate the advantages of the DMLS for specific applications, particularly because of how little is known regarding the effects of surface roughness. This paper presents pressure drop and heat transfer results of flow through small, as produced channels that have been manufactured using the DMLS in an effort to better understand roughness. Ten different coupons made with the DMLS all having multiple rectangular channels were evaluated in this study. Measurements were collected at various flow conditions and reduced to a friction factor and a Nusselt number. Results showed significant augmentation of these parameters compared to smooth channels, particularly with the friction factor for minichannels with small hydraulic diameters. However, augmentation of Nusselt number did not increase proportionally with the augmentation of the friction factor.
Three-Dimensional Reconstruction of Blood Vessels of the Human Retina by Fractal Interpolation
Authors: Guedri Hichem , Malek Jihen , Belmabrouk Hafedh
Published: December 2015

In this work, data from two-dimensional (2D) images of the human retina were taken as a case study. First, the characteristic data points had been removed using the Douglas–Peucker (DP) method, and subsequently, more data points were added using random fractal interpolation approach, to reconstruct a three-dimensional (3D) model of the blood vessel. By visualizing the result, we can see that all the small blood vessels in the human retina are more visible and detailed. This algorithm of 3D reconstruction has the advantage of being fast with calculation time less than 40 s and also can reduce the 3D image storage level on a disk with a reduction ratio between 78% and 96.65%.
Efficient Energy Harvesting Using Piezoelectric Compliant Mechanisms: Theory and Experiment
Authors: Ma Xiaokun , Wilson Andrew , Rahn Christopher D. , Trolier-McKinstry Susan
Published: December 2015

Piezoelectric energy harvesters typically perform poorly in the low frequency, low amplitude, and intermittent excitation environment of human movement. In this paper, a piezoelectric compliant mechanism (PCM) energy harvester is designed that consists of a polyvinylidene diflouoride (PVDF) unimorph clamped at the base and attached to a compliant mechanism at the tip. The compliant mechanism has two flexures that amplify the tip displacement to produce large motion of a proof mass and a low frequency first mode with an efficient (nearly quadratic) shape. The compliant mechanism is fabricated as a separate, relatively rigid frame with flexure hinges, simplifying the fabrication process, and surrounding and protecting the piezoelectric unimorph. The bridge structure of the PCM also self-limits the response to large amplitude impacts, improving the device robustness. Experiments show that the compliant hinge stiffness can be carefully tuned to approach the theoretical high power output and mode shape efficiency.
Numerical Tracking of Limit Points for Direct Parametric Analysis in Nonlinear Rotordynamics
Authors: Xie Lihan , Baguet Sébastien , Prabel Benoit , Dufour Régis
Published: December 2015

A frequency-domain approach for direct parametric analysis of limit points (LPs) of nonlinear dynamical systems is presented in this paper. Instead of computing responses curves for several values of a given system parameter, the direct tracking of LPs is performed. The whole numerical procedure is based on the harmonic balance method (HBM) and can be decomposed in three distinct steps. First, a response curve is calculated by HBM combined with a continuation technique until an LP is detected. Then this starting LP is used to initialize the direct tracking of LPs which is based on the combination of a so-called extended system and a continuation technique. With only one computation, a complete branch of LPs is obtained, which provides the stability boundary with respect to system parameters such as nonlinearity or excitation level. Several numerical examples demonstrate the capabilities and the performance of the proposed method.
Semi-Exact Natural Frequencies for Kirchhoff–Love Plates Using Wave-Based Phase Closure
Authors: Leamy Michael J.
Published: December 2015

This paper presents semi-exact, closed-form algebraic expressions for the natural frequencies of Kirchhoff–Love plates by analyzing plane waves, their edge reflections, and their phase closure. The semi-exact nature is such that the analysis exactly satisfies plate boundary conditions along each edge when taken in isolation, but not fully when combined, and thus is approximate near a corner. As frequency increases, the expressions become increasingly more accurate. For clamped square plates, closed-form expressions are reported in algebraic form for the first time. These expressions are developed by tracing the path of plane waves as they reflect from edges while accounting for phase changes over a total trip. This change includes phase addition/subtraction due to edge reflections. A natural frequency is identified as a frequency in which three phase changes (in the plate's horizontal, vertical, and path directions) each sum to an integer multiple of 2π, enforcing phase closure along each direction. A solution of the subsequent equations is found in closed form, for multiple boundary conditions, such that highly convenient algebraic expressions result for the plate natural frequencies. The expressions are exact for the case of all sides simply supported, while for other boundary conditions, the expressions are semi-exact. For the practically important and difficult case of a fully clamped plate, the expressions for a square plate yield the first 20 nondimensional natural frequencies to within 0.06% of their exact values.
An Aerodynamic Parameter for Low-Pressure Turbine Flutter
Authors: Barbarossa Fernando , Parry Anthony B. , Green Jeffrey S. , di Mare Luca
Published: December 2015

This paper presents a study on low-pressure (LP) turbine bending flutter. The study is performed using a semi-analytical model, which is validated against experimental and computational fluid dynamics (CFD) data. The validation highlights the ability of even the simplest models to represent accurately the flutter behavior of the LP turbine assemblies. A parametric study is then performed into the effect of modifications of the blade camber line on the stability of bending modes. Variations in maximum camber position, leading edge and trailing edge metal angles, and stagger are considered. The modifications are applied to a family of aerodynamically well designed aerofoils and to a family of aerodynamically poorly designed aerofoils. Trends relating damping to the parameters describing the camber line modifications are identified. These trends are found to apply to both families of aerofoils. Furthermore, it is found that the behavior of all aerofoils studied can be characterized by their behavior at a specific flow angle and by a simple algebraic relation based on true incidence. This behavior also seems to be universal.
Unsteady Measurements of Periodic Effects in a Transonic Compressor With Casing Treatments
Authors: Brandstetter Christoph , Wartzek Fabian , Werner Jan , Schiffer Heinz-Peter , Heinichen Frank
Published: December 2015

Application of nonaxisymmetric casing treatments (CTs) can extend the operating range of a transonic compressor significantly. Recent CT designs have proven successful at achieving operating range extension without efficiency loss under design conditions. Two different CT designs were investigated on a high-speed one and a half stage test rig using extensive instrumentation. The stage setup is representative of the front stage of a modern high-pressure compressor. Results of particle image velocimetry (PIV) measurements taken in the blade tip region underneath the CT show a significantly modified flow structure compared to the smooth casing reference case. Blockage zone, secondary flow, and shock structures are affected by the CT, especially in highly throttled operating conditions. The stall inception process of the system with axial slots shows unexpected behavior, with modal activities that are not observed without CT. These activities are resolved using unsteady wall pressure (WP) and hot wire measurements.
Increased Red Blood Cell Stiffness Increases Pulmonary Vascular Resistance and Pulmonary Arterial Pressure
Authors: Schreier David A. , Forouzan Omid , Hacker Timothy A. , Sheehan John , Chesler Naomi
Published: December 2015

Patients with sickle cell anemia (SCD) and pulmonary hypertension (PH) have a significantly increased risk of sudden death compared to patients with SCD alone. Sickled red blood cells (RBCs) are stiffer, more dense, more frequently undergo hemolysis, and have a sixfold shorter lifespan compared to normal RBCs. Here, we sought to investigate the impact of increased RBC stiffness, independent of other SCD-related biological and mechanical RBC abnormalities, on the hemodynamic changes that ultimately cause PH and increase mortality in SCD. To do so, pulmonary vascular impedance (PVZ) measures were recorded in control C57BL6 mice before and after ∼50 μl of blood (Hct = 45%) was extracted and replaced with an equal volume of blood containing either untreated RBCs or RBCs chemically stiffened with glutaraldehyde (Hct = 45%). Chemically stiffened RBCs increased mean pulmonary artery pressure (mPAP) (13.5 ± 0.6 mmHg at baseline to 23.2 ± 0.7 mmHg after the third injection), pulmonary vascular resistance (PVR) (1.23 ± 0.11 mmHg*min/ml at baseline to 2.24 ± 0.14 mmHg*min/ml after the third injection), and wave reflections (0.31 ± 0.02 at baseline to 0.43 ± 0.03 after the third injection). Chemically stiffened RBCs also decreased cardiac output, but did not change hematocrit, blood viscosity, pulmonary arterial compliance, or heart rate. The main finding of this study is that increased RBC stiffness alone affects pulmonary pulsatile hemodynamics, which suggests that RBC stiffness plays an important role in the development of PH in patients with SCD.
Uncertainty Quantification of Large-Eddy Spray Simulations
Authors: Van Dam Noah , Rutland Chris
Published: December 2015

Two uncertainty quantification (UQ) techniques, latin-hypercube sampling (LHS) and polynomial chaos expansion (PCE), have been used in an initial UQ study to calculate the effect of boundary condition uncertainty on Large-eddy spray simulations. Liquid and vapor penetration as well as multidimensional liquid and vapor data were used as response variables. The Morris one-at-a-time (MOAT) screening method was used to identify the most important boundary conditions. The LHS and PCE methods both predict the same level of variability in the response variables, which was much larger than the corresponding experimental uncertainty. Nested grids were used in conjunction with the PCE method to examine the effects of subsets of boundary condition variables. Numerical modeling parameters had a much larger effect on the resulting spray predictions; the uncertainty in spray penetration or multidimensional spray contours from physically derived boundary conditions was close to the uncertainty of the measurements.
Probabilistic Pressurized Thermal Shock Analysis for a Reactor Pressure Vessel Considering Plume Cooling Effect
Authors: Qian Guian , González-Albuixech V. F. , Niffenegger Markus , Sharabi Medhat
Published: December 2015

The inner surface of a reactor pressure vessel (RPV) is assumed to be subjected to pressurized thermal shocks (PTSs) caused by the injection of emergency cooling water. The downstream is not homogeneous but typically in a plume shape coming from the inlet nozzles. In this paper, both deterministic and probabilistic methods are used to assess the integrity of a model RPV subjected to PTS. The favor code is used to calculate the probabilities for crack initiation and failure of the RPV considering crack distributions based on cracks observed in the Shoreham and PVRUF RPVs. The study shows that peak KI of the cracks inside the plume increases about 33% compared with that outside. The conditional probability inside the plume is more than eight orders of magnitude higher than outside the plume. In order to be conservative, it is necessary to consider the plume effect in the integrity assessment.
Assessment of the Cyclic Behavior of Structural Components Using Novel Approaches
Authors: Panagiotou K. D. , Spiliopoulos K. V.
Published: December 2015

To extend the life of a structure, or a component, which is subjected to cyclic thermomechanical loading history, one has to provide safety margins against excessive inelastic deformations that may lead either to low-cycle fatigue or to ratcheting. Direct methods constitute a convenient tool toward this direction. Two direct methods that have been named residual stress decomposition method (RSDM) and residual stress decomposition method for shakedown (RSDM-S) have recently appeared in the literature. The first method may predict any cyclic elastoplastic state for a given cyclic loading history. The second method RSDM-S that is based upon RSDM is suggested for the shakedown analysis of structures. Both methods may be directly implemented in any finite-element (FE) code. An elastic perfectly plastic material with a von Mises yield surface has been assumed. In this work, through their application to structures that are used as benchmarks in the literature, both methods, applied together, prove their efficiency and capacity to determine shakedown boundaries and reveal unsafe conditions.
Development of Inverse Analysis Method of Heat Conduction and Thermal Stress for Elbow—Part II
Authors: Hojo Kiminobu , Ochi Mayumi , Ioka Seiji , Kubo Shiro
Published: December 2015

An inverse heat conduction analysis method for piping elbow was developed to estimate the temperature and stress distribution on the inner surface by measuring the outer surface temperature. In the paper, the accuracy for the thermal stress calculation using the inverse heat conduction analysis method was confirmed by comparing with the reference results from normal FE heat conduction and thermal stress analyses. In the case of the measured-basis fluid temperature input from a high temperature–pressure test, the inverse analysis method estimated the maximum stress change by 7% conservative comparing with the normal FE analyses.
Kinematic Analysis of Congruent Multilayer Tessellations
Authors: Klett Yves , Middendorf Peter
Published: December 2015

Rigidly foldable origami tessellations exhibit interesting kinematic properties. Several tessellation types (most prominently Miura-ori) have shown potential for technical usage in aerospace and general lightweight construction. In addition to static (e.g., as core structures for sandwich components) and single-layer kinematic (e.g., deployable) applications, new possibilities arise from the combination of several layers of tessellations with congruent kinematics. This paper presents an analytical description of the kinematics of multilayered, or stacked, globally plane tessellations which retain rigid/isometric foldability by congruent, compatible movement.
Study of the electrical and chemical properties of the multistep deposited and two-step (ultraviolet ozone cum rapid thermal) annealed HfO2 gate stack
Authors: Yew Kwang Sing , Ang Diing Shenp , Tang Lei Jun , Pan Jisheng
Published: December 2015

The authors show that the TiN/HfO2/SiOx gate stack, formed via multistep deposition cum two-step anneal [comprising a room-temperature ultraviolet ozone (RTUVO) anneal and a subsequent rapid thermal anneal (RTA) at 420 °C], exhibits more superior electrical characteristics as compared to the gate stacks formed via multistep deposition cum single-step anneal (either RTUVO anneal or 420 °C RTA). The former exhibits more than an order of magnitude smaller gate current density, a 14-fold increase in the time-to-breakdown, and reduced positive oxide trapped charge as compared to the latter. The enhanced performance and reliability are attributed to the improved formation of Hf–O bonds in HfO2, resulting from the efficient incorporation of oxygen atoms facilitated by the thermal activation of the absorbed ozone. The findings provide insights into the improvement mechanism by the two-step anneal method for high-k last integration scheme.
New Verapamil Analogs Inhibit Intracellular Mycobacteria without Affecting the Functions of Mycobacterium-Specific T Cells
Authors: Abate Getahun , Ruminiski Peter G. , Kumar Malkeet , Singh Kawaljit , Hamzabegovic Fahreta , Hoft Daniel F. , Eickhoff Christopher S. , Selimovic Asmir , Campbell Mary , Chibale Kelly
Published: December 2015

There is a growing interest in repurposing mycobacterial efflux pump inhibitors, such as verapamil, for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and
Mycobacterium tuberculosis
-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and gamma interferon (IFN-γ). Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow-derived macrophages from ATG5
flox/flox
(control) and ATG5
flox/flox
Lyz-Cre mice. Our results showed that despite the well-recognized effects of verapamil on calcium channels and autophagy, its action on intracellular
M. tuberculosis
does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on
M. tuberculosis
-specific T cells were assessed using flow cytometry following the stimulation of peripheral blood mononuclear cells from TB-skin-test-positive donors with
M. tuberculosis
whole-cell lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of
M. tuberculosis
-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular
Mycobacterium bovis
BCG, and one of the three analogs (KSV21) inhibited intracellular
M. tuberculosis
replication at concentrations that did not inhibit
M. tuberculosis
-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular
M. tuberculosis
. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs.
Abalone Hemocyanin Blocks the Entry of Herpes Simplex Virus 1 into Cells: a Potential New Antiviral Strategy
Authors: Talaei Zanjani Negar , Miranda-Saksena Monica , Valtchev Peter , Diefenbach Russell J. , Hueston Linda , Diefenbach Eve , Sairi Fareed , Gomes Vincent G. , Cunningham Anthony L. , Dehghani Fariba
Published: December 2015

A marine-derived compound, abalone hemocyanin, from
Haliotis rubra
was shown to have a unique mechanism of antiviral activity against herpes simplex virus 1 (HSV-1) infections.
In vitro
assays demonstrated the dose-dependent and inhibitory effect of purified hemocyanin against HSV-1 infection in Vero cells with a 50% effective dose (ED
50
) of 40 to 50 nM and no significant toxicity. In addition, hemocyanin specifically inhibited viral attachment and entry by binding selectively to the viral surface glycoproteins gD, gB, and gC, probably by mimicking their receptors. However, hemocyanin had no effect on postentry events and did not block infection by binding to cellular receptors for HSV. By the use of different mutants of gD and gB and a competitive heparin binding assay, both protein charge and conformation were shown to be the driving forces of the interaction between hemocyanin and viral glycoproteins. These findings also suggested that hemocyanin may have different motifs for binding to each of the viral glycoproteins B and D. The dimer subunit of hemocyanin with a 10-fold-smaller molecular mass exhibited similar binding to viral surface glycoproteins, showing that the observed inhibition did not require the entire multimer. Therefore, a small hemocyanin analogue could serve as a new antiviral candidate for HSV infections.
Characterization of Antifungal Activity and Nail Penetration of ME1111, a New Antifungal Agent for Topical Treatment of Onychomycosis
Authors: Tabata Yuji , Takei-Masuda Naomi , Kubota Natsuki , Takahata Sho , Ohyama Makoto , Kaneda Kaori , Iida Maiko , Maebashi Kazunori
Published: December 2015

Fungal nail infection (onychomycosis) is a prevalent disease in many areas of the world, with a high incidence approaching 23%. Available antifungals to treat the disease suffer from a number of disadvantages, necessitating the discovery of new efficacious and safe antifungals. Here, we evaluate the
in vitro
antifungal activity and nail penetration ability of ME1111, a novel antifungal agent, along with comparator drugs, including ciclopirox, amorolfine, terbinafine, and itraconazole. ME1111 showed potent antifungal activity against
Trichophyton rubrum
and
Trichophyton mentagrophytes
(the major etiologic agents of onychomycosis) strains isolated in Japan and reference fungal strains with an MIC range of 0.12 to 0.5 mg/liter and an MIC
50
and MIC
90
of 0.5 mg/liter for both. Importantly, none of the tested isolates showed an elevated ME1111 MIC. Moreover, the antifungal activity of ME1111 was minimally affected by 5% wool keratin powder in comparison to the other antifungals tested. The ME1111 solution was able to penetrate human nails and inhibit fungal growth in a dose-dependent manner according to the TurChub assay. In contrast, 8% ciclopirox and 5% amorolfine nail lacquers showed no activity under the same conditions. ME1111 demonstrated approximately 60-fold-greater selectivity in inhibition of
Trichophyto
n spp. than of human cell lines. Our findings demonstrate that ME1111 possesses potent antidermatophyte activity, maintains this activity in the presence of keratin, and possesses excellent human nail permeability. These results suggest that ME1111 is a promising topical medication for the treatment of onychomycosis and therefore warrants further clinical evaluation.
Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11
Authors: Yang Mai Lee , Uhrig John , Vu Kiem , Singapuri Anil , Dennis Michael , Gelli Angie , Thompson George R.
Published: December 2015

Cryptococcus gattii
isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in
C. gattii
is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (
mdr1
Δ) and another strain lacking Pdr11 (
pdr11
Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37°C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 μg/ml for the wild-type isolate to <0.03 μg/ml for the
pdr11
Δ strain and reverted to 32 μg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in
C. gattii
. Genomic and expression differences between resistant and susceptible
C. gattii
clinical isolates should be assessed further in order to identify other potential mechanisms of resistance.
Next-Generation Sequencing and Comparative Analysis of Sequential Outbreaks Caused by Multidrug-Resistant Acinetobacter baumannii at a Large Academic Burn Center
Authors: Kanamori Hajime , Parobek Christian M. , Weber David J. , van Duin David , Rutala William A. , Cairns Bruce A. , Juliano Jonathan J.
Published: December 2015

Next-generation sequencing (NGS) analysis has emerged as a promising molecular epidemiological method for investigating health care-associated outbreaks. Here, we used NGS to investigate a 3-year outbreak of multidrug-resistant
Acinetobacter baumannii
(MDRAB) at a large academic burn center. A reference genome from the index case was generated using
de novo
assembly of PacBio reads. Forty-six MDRAB isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and sequenced using an Illumina platform. After mapping to the index case reference genome, four samples were excluded due to low coverage, leaving 42 samples for further analysis. Multilocus sequence types (MLST) and the presence of acquired resistance genes were also determined from the sequencing data. A transmission network was inferred from genomic and epidemiological data using a Bayesian framework. Based on single-nucleotide variant (SNV) differences, this MDRAB outbreak represented three sequential outbreaks caused by distinct clones. The first and second outbreaks were caused by sequence type 2 (ST2), while the third outbreak was caused by ST79. For the second outbreak, the MLST and PFGE results were discordant. However, NGS-based SNV typing detected a recombination event and consequently enabled a more accurate phylogenetic analysis. The distribution of resistance genes varied among the three outbreaks. The first- and second-outbreak strains possessed a
bla
OXA-23-like
group, while the third-outbreak strains harbored a
bla
OXA-40-like
group. NGS-based analysis demonstrated the superior resolution of outbreak transmission networks for MDRAB and provided insight into the mechanisms of strain diversification between sequential outbreaks through recombination.
Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae
Authors: Ioannou Petros , Andrianaki Aggeliki , Akoumianaki Tonia , Kyrmizi Irene , Albert Nathaniel , Perlin David , Samonis George , Kontoyiannis Dimitrios P. , Chamilos Georgios
Published: December 2015

The modest
in vitro
activity of echinocandins against
Aspergillus
implies that host-related factors augment the action of these antifungal agents
in vivo
. We found that, in contrast to the other antifungal agents (voriconazole, amphotericin B) tested, caspofungin exhibited a profound increase in activity against various
Aspergillus
species under conditions of cell culture growth, as evidenced by a ≥4-fold decrease in minimum effective concentrations (MECs) (
P
= 0. 0005). Importantly, the enhanced activity of caspofungin against
Aspergillus
spp. under cell culture conditions was strictly dependent on serum albumin and was not observed with the other two echinocandins, micafungin and anidulafungin. Of interest, fluorescently labeled albumin bound preferentially on the surface of germinating
Aspergillus
hyphae, and this interaction was further enhanced upon treatment with caspofungin. In addition, supplementation of cell culture medium with albumin resulted in a significant, 5-fold increase in association of fluorescently labeled caspofungin with
Aspergillus
hyphae (
P
< 0.0001). Collectively, we found a novel synergistic interaction between albumin and caspofungin, with albumin acting as a potential carrier molecule to facilitate antifungal drug delivery to
Aspergillus
hyphae.
Is a New Standard Needed for Diffusion Methods for
In Vitro
Susceptibility Testing of Fosfomycin against Pseudomonas aeruginosa?
Authors: Díez-Aguilar María , Martínez-García Laura , Cantón Rafael , Morosini María Isabel
Published: December 2015

We analyzed fosfomycin susceptibility results in
Pseudomonas aeruginosa
clinical isolates obtained by MIC gradient strips and disk diffusion methods using two different inocula, 10
8
and 10
6
CFU/ml, and compared them to the agar dilution reference method. Essential and categorical agreements were 93.6% and 95%, respectively, for the 10
6
CFU/ml alternative inoculum, and they were 67.6% and 78.2%, respectively, for the standard inoculum (10
8
CFU/ml). The use of the 10
6
CFU/ml inoculum improves the agreement values and inhibition zone readings.
Searching for the Optimal Predictor of Ciprofloxacin Resistance in Klebsiella pneumoniae by Using
In Vitro
Dynamic Models
Authors: Strukova Elena N. , Portnoy Yury A. , Romanov Andrey V. , Edelstein Mikhail V. , Zinner Stephen H. , Firsov Alexander A.
Published: December 2015

There is growing evidence of applicability of the hypothesis of the mutant selection window (MSW), i.e., the range between the MIC and the mutant prevention concentration (MPC), within which the enrichment of resistant mutants is most probable. However, it is not clear if MPC-based pharmacokinetic variables are preferable to the respective MIC-based variables as interstrain predictors of resistance. To examine the predictive power of the ratios of the area under the curve (AUC
24
) to the MPC and to the MIC, the selection of ciprofloxacin-resistant mutants of three
Klebsiella pneumoniae
strains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC
24
/MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. Resistant
K. pneumoniae
mutants were intensively enriched at an AUC
24
/MIC ratio of 60 to 360 h (AUC
24
/MPC ratio from 2.5 to 15 h) but not at the lower or higher AUC
24
/MIC and AUC
24
/MPC ratios, in accordance with the MSW hypothesis. AUC
24
/MPC and AUC
24
/MIC relationships with areas under the time courses of ciprofloxacin-resistant
K. pneumoniae
(AUBC
M
) were bell shaped. These relationships predict highly variable “antimutant” AUC
24
/MPC ratios (20 to 290 h) compared to AUC
24
/MIC ratios (1,310 to 2,610 h). These findings suggest that the potential of the AUC
24
/MPC ratio as an interstrain predictor of
K. pneumoniae
resistance is lower than that of the AUC
24
/MIC ratio.
Characterization of Polymyxin B Biodistribution and Disposition in an Animal Model
Authors: Manchandani Pooja , Zhou Jian , Ledesma Kimberly R. , Truong Luan D. , Chow Diana S.-L. , Eriksen Jason L. , Tam Vincent H.
Published: December 2015

Despite dose-limiting nephrotoxicity concerns, polymyxin B has resurged as the treatment of last resort for multidrug-resistant Gram-negative bacterial infections. However, the pharmacokinetic, pharmacodynamic, and nephrotoxic properties of polymyxin B still are not thoroughly understood. The objective of this study was to provide additional insights into the overall biodistribution and disposition of polymyxin B in an animal model. Sprague-Dawley rats were dosed with intravenous polymyxin B (3 mg/kg of body weight). Drug concentrations in the serum, urine, bile, and tissue (brain, heart, lungs, liver, spleen, kidneys, and skeletal muscle) samples over time were assayed by a validated methodology. Among all the organs evaluated, polymyxin B distribution was highest in the kidneys. The mean renal tissue/serum polymyxin B concentration ratios were 7.45 (95% confidence interval [CI], 4.63 to 10.27) at 3 h and 19.62 (95% CI, 5.02 to 34.22) at 6 h postdose. Intrarenal drug distribution was examined by immunostaining. Using a ratiometric analysis, proximal tubular cells showed the highest accumulation of polymyxin B (Mander's overlap coefficient, 0.998) among all cell types evaluated. Less than 5% of the administered dose was recovered in urine over 48 h, but all 4 major polymyxin B components were detected in the bile over 4 h. These findings corroborate previous results that polymyxin B is highly accumulated in the kidneys, but the elimination likely is via a nonrenal route. Biliary excretion could be one of the routes of polymyxin B elimination, and this should be further explored. The elucidation of mechanism(s) of drug uptake in proximal tubular cells is ongoing.
Analysis of Hepatitis C Virus Genotype 1b Resistance Variants in Japanese Patients Treated with Paritaprevir-Ritonavir and Ombitasvir
Authors: Krishnan Preethi , Schnell Gretja , Tripathi Rakesh , Beyer Jill , Reisch Thomas , Zhang Xinyan , Setze Carolyn , Rodrigues Lino , Burroughs Margaret , Redman Rebecca , Chayama Kazuaki , Kumada Hiromitsu , Collins Christine , Pilot-Matias Tami
Published: December 2015

Treatment of HCV genotype 1b (GT1b)-infected Japanese patients with paritaprevir (NS3/4A inhibitor boosted with ritonavir) and ombitasvir (NS5A inhibitor) in studies M12-536 and GIFT-I demonstrated high sustained virologic response (SVR) rates. The virologic failure rate was 3% (13/436) across the two studies. Analyses were conducted to evaluate the impact of baseline resistance-associated variants (RAVs) on treatment outcome and the emergence and persistence of RAVs in patients experiencing virologic failure. Baseline paritaprevir resistance-conferring variants in NS3 were infrequent, while Y93H in NS5A was the most prevalent ombitasvir resistance-conferring variant at baseline. A comparison of baseline prevalence of polymorphisms in Japanese and western patients showed that Q80L and S122G in NS3 and L28M, R30Q, and Y93H in NS5A were significantly more prevalent in Japanese patients. In the GIFT-I study, the prevalence of Y93H in NS5A varied between 13% and 21% depending on the deep-sequencing detection threshold. Among patients with Y93H comprising <1%, 1 to 40%, or >40% of their preexisting viral population, the 24-week SVR (SVR
24
) rates were >99% (276/277), 93% (38/41), and 76% (25/33), respectively, indicating that the prevalence of Y93H within a patient's viral population is a good predictor of treatment response. The predominant RAVs at the time of virologic failure were D168A/V in NS3 and Y93H alone or in combination with other variants in NS5A. While levels of NS3 RAVs declined over time, NS5A RAVs persisted through posttreatment week 48. Results from these analyses are informative in understanding the resistance profile of an ombitasvir- plus paritaprevir/ritonavir-based regimen in Japanese GT1b-infected patients.
Modification of the 1-Phosphate Group during Biosynthesis of Capnocytophaga canimorsus Lipid A
Authors: Renzi Francesco , Zähringer Ulrich , Chandler Courtney E. , Ernst Robert K. , Cornelis Guy R. , Ittig Simon J.
Published: December 2015

Capnocytophaga canimorsus
, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (
P
-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the
C. canimorsus
genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1
P
-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an
P
-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of
lpxE
or
eptA
led to mutants lacking the
P
-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of
lpxE-eptA
was similar to that of a single
lpxE
mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.
Mycobacterium-Specific γ9δ2T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity
Authors: Abate Getahun , Spencer Charles T. , Hamzabegovic Fahreta , Blazevic Azra , Xia Mei , Hoft Daniel F.
Published: December 2015

Numerous pathogens, includingMycobacterium tuberculosis, can activate human γ9δ2T cells to proliferate and express effector mechanisms. γ9δ2T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We preparedM. tuberculosis-specific γ9δ2T cell lines to study their direct protective effects and APC functions forM. tuberculosis-specific αβ T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αβ T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ9δ2T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ9δ2T cells enhance the expansion ofM. tuberculosis-specific αβ T cells and increase the ability of αβ T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ9δ2T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ9δ2T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4+αβ T cells and γ9δ2T cells provide similar immune enhancing/APC functions forM. tuberculosis-specific T cells. These effector and helper effects of γ9δ2T cells further indicate that these T cells should be considered important new targets for new TB vaccines.
Mycobacterium tuberculosis Coinfection Has No Impact on Plasmodium berghei ANKA-Induced Experimental Cerebral Malaria in C57BL/6 Mice
Authors: Blank Jannike , Behrends Jochen , Jacobs Thomas , Schneider Bianca E.
Published: December 2015

Cerebral malaria (CM) is the most severe complication of human infection withPlasmodium falciparum. The mechanisms predisposing to CM are still not fully understood. Proinflammatory immune responses are required for the control of blood-stage malaria infection but are also implicated in the pathogenesis of CM. A fine balance between pro- and anti-inflammatory immune responses is required for parasite clearance without the induction of host pathology. The most accepted experimental model to study human CM isPlasmodium bergheiANKA (PbANKA) infection in C57BL/6 mice that leads to the development of a complex neurological syndrome which shares many characteristics with the human disease. We applied this model to study the outcome ofPbANKA infection in mice previously infected withMycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis is coendemic with malaria in large regions in the tropics, and mycobacteria have been reported to confer some degree of unspecific protection against rodentPlasmodiumparasites in experimental coinfection models. We found that concomitantM. tuberculosisinfection did not change the clinical course ofPbANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. The immunological environments in spleen and brain did not differ between singly infected and coinfected animals; instead, the overall cytokine and T cell responses in coinfected mice were comparable to those in animals solely infected withPbANKA. Our data suggest thatM. tuberculosiscoinfection is not able to change the outcome ofPbANKA-induced disease, most likely because the inflammatory response induced by the parasite rapidly dominates in mice previously infected withM. tuberculosis.
Nonredundant Roles of Iron Acquisition Systems in Vibrio cholerae
Authors: Peng Eric D. , Wyckoff Elizabeth E. , Mey Alexandra R. , Fisher Carolyn R. , Payne Shelley M.
Published: December 2015

Vibrio cholerae
, the causative agent of the severe diarrheal disease cholera, thrives in both marine environments and the human host. To do so, it must encode the tools necessary to acquire essential nutrients, including iron, under these vastly different conditions. A number of
V. cholerae
iron acquisition systems have been identified; however, the precise role of each system is not fully understood. To test the roles of individual systems, we generated a series of mutants in which only one of the four systems that support iron acquisition on unsupplemented LB agar, Feo, Fbp, Vct, and Vib, remains functional. Analysis of these mutants under different growth conditions showed that these systems are not redundant. The strain carrying only the ferrous iron transporter Feo grew well at acidic, but not alkaline, pH, whereas the ferric iron transporter Fbp promoted better growth at alkaline than at acidic pH. A strain defective in all four systems (null mutant) had a severe growth defect under aerobic conditions but accumulated iron and grew as well as the wild type in the absence of oxygen, suggesting the presence of an additional, unidentified iron transporter in
V. cholerae
. In support of this, the null mutant was only moderately attenuated in an infant mouse model of infection. While the null mutant used heme as an iron source
in vitro
, we demonstrate that heme is not available to
V. cholerae
in the infant mouse intestine.