bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacodynamics of Cefepime Combined with Tazobactam against Clinically Relevant Enterobacteriaceae in a Neutropenic Mouse Thigh Model
Authors: Melchers Maria J. , van Mil Anita C. , Lagarde Claudia , den Hartigh Jan , Mouton Johan W.
Published: June 2017

The lack of new antibiotics has prompted investigation of the combination of two existing agents—cefepime, a broad-spectrum cephalosporin, and tazobactam—to broaden their efficacy against extended-spectrum beta-lactamase (ESBL)-producing
Enterobacteriaceae
. We determined the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the combination in a murine neutropenic thigh model in order to establish its exposure-response relationships (ERRs). The PK of cefepime were determined for five doses; that of tazobactam was determined in earlier studies (Melchers et al., Antimicrob Agents Chemother 59:3373–3376, 2015,
https://doi.org/10.1128/AAC.04402-14
). The PK were linear for both compounds. The estimated mean (standard deviation [SD]) half-life of cefepime was 0.33 (0.12) h, and that of tazobactam was 0.176 (0.026) h; the volumes of distribution (
V
) were 0.73 liters/kg and 1.14 liters/kg, respectively. PD studies of cefepime administered every 2 h (q2h) with or without tazobactam, including dose fractionation studies of tazobactam, were performed against six ESBL-producing isolates. A sigmoidal maximum-effect (
E
max
) model was fitted to the data. In the dose fractionation study, the q2h regimen was more efficacious than the q4h and q6h regimens, indicating time-dependent activity of tazobactam. The threshold concentration (

C
T

) best correlating with tazobactam efficacy was 0.25 mg/liter, as evidenced by the best fit of the percentage of time above the threshold concentration (%
fT
>

C
T

) and response. A mean %
fT
>

C
T

of 24.6% (range, 11.4 to 36.3%) for a

C
T

of 0.25 mg/liter was required to obtain a bacteriostatic effect. We conclude that tazobactam enhanced the effect of cefepime in otherwise resistant isolates of
Enterobacteriaceae
and that the %
fT
>

C
T

of 0.25 mg/liter best correlated with efficacy. These studies provide the basis for the development of human dosing regimens for this combination.
Genome Evolution to Penicillin Resistance in Serotype 3 Streptococcus pneumoniae by Capsular Switching
Authors: Chiba Naoko , Murayama Somay Y. , Morozumi Miyuki , Iwata Satoshi , Ubukata Kimiko
Published: June 2017

Streptococcus pneumoniae
isolates of serotype 3 were collected from cases of invasive pneumococcal disease (
n
= 124) throughout Japan between April 2010 and March 2013. A penicillin-resistant
S. pneumoniae
(PRSP) isolate from an adult patient, strain KK0981 of serotype 3, was identified among these strains. Whole-genome analysis characterized this PRSP as a recombinant strain derived from PRSP of serotype 23F with the
cps
locus (20.3 kb) replaced by that of a penicillin-susceptible strain of serotype 3.
Modification of a Pollen Trap Design To Capture Airborne Conidia of Entomophaga maimaiga and Detection of Conidia by Quantitative PCR
Authors: Bittner Tonya D. , Hajek Ann E. , Liebhold Andrew M. , Thistle Harold
Published: June 2017

The goal of this study was to develop effective and practical field sampling methods for quantification of aerial deposition of airborne conidia of
Entomophaga maimaiga
over space and time. This important fungal pathogen is a major cause of larval death in invasive gypsy moth (
Lymantria dispar
) populations in the United States. Airborne conidia of this pathogen are relatively large (similar in size to pollen), with unusual characteristics, and require specialized methods for collection and quantification. Initially, dry sampling (settling of spores from the air onto a dry surface) was used to confirm the detectability of
E. maimaiga
at field sites with
L. dispar
deaths caused by
E. maimaiga
, using quantitative PCR (qPCR) methods. We then measured the signal degradation of conidial DNA on dry surfaces under field conditions, ultimately rejecting dry sampling as a reliable method due to rapid DNA degradation. We modified a chamber-style trap commonly used in palynology to capture settling spores in buffer. We tested this wet-trapping method in a large-scale (137-km) spore-trapping survey across gypsy moth outbreak regions in Pennsylvania undergoing epizootics, in the summer of 2016. Using 4-day collection periods during the period of late instar and pupal development, we detected variable amounts of target DNA settling from the air. The amounts declined over the season and with distance from the nearest defoliated area, indicating airborne spore dispersal from outbreak areas.

IMPORTANCE
We report on a method for trapping and quantifying airborne spores of
Entomophaga maimaiga
, an important fungal pathogen affecting gypsy moth (
Lymantria dispar
) populations. This method can be used to track dispersal of
E. maimaiga
from epizootic areas and ultimately to provide critical understanding of the spatial dynamics of gypsy moth-pathogen interactions.
Quantifying Vibrio cholerae Enterotoxicity in a Zebrafish Infection Model
Authors: Mitchell Kristie C. , Breen Paul , Britton Sarah , Neely Melody N. , Withey Jeffrey H.
Published: June 2017

Vibrio cholerae
is the etiological agent of cholera, an acute intestinal infection in humans characterized by voluminous watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the proper infrastructure for proper water and sewage treatment.
Vibrio cholerae
is an aquatic bacterium that inhabits coastal and estuarine areas, and it is known to have several environmental reservoirs, including fish. Our laboratory has recently described the use of the zebrafish as a new animal model for the study of
V. cholerae
intestinal colonization, pathogenesis, and transmission. As early as 6 h after exposure to
V. cholerae
, zebrafish develop diarrhea. Prior work in our laboratory has shown that this is not due to the action of cholera toxin. We hypothesize that accessory toxins produced by
V. cholerae
are the cause of diarrhea in infected zebrafish. In order to assess the effects of accessory toxins in the zebrafish, it was necessary to develop a method of quantifying diarrheal volume as a measure of pathogenesis. Here, we have adapted cell density, protein, and mucin assays, along with enumeration of
V. cholerae
in the zebrafish intestinal tract and in the infection water, to achieve this goal. Combined, these assays should help us determine which toxins have the greatest diarrheagenic effect in fish and, consequently, which toxins may play a role in environmental transmission.

IMPORTANCE
Identification of the accessory toxins that cause diarrhea in zebrafish can help us understand more about the role of fish in the wild as aquatic reservoirs for
V. cholerae
. It is plausible that accessory toxins can act to prolong colonization and subsequent shedding of
V. cholerae
back into the environment, thus perpetuating and facilitating transmission during an outbreak. It is also possible that accessory toxins help to maintain low levels of intestinal colonization in fish, giving
V. cholerae
an advantage when environmental conditions are not optimal for survival in the water. Studies such as this one are critical because fish could be an overlooked source of cholera transmission in the environment.
Structures, Compositions, and Activities of Live Shewanella Biofilms Formed on Graphite Electrodes in Electrochemical Flow Cells
Authors: Kitayama Miho , Koga Ryota , Kasai Takuya , Kouzuma Atsushi , Watanabe Kazuya
Published: June 2017

An electrochemical flow cell equipped with a graphite working electrode (WE) at the bottom was inoculated with
Shewanella oneidensis
MR-1 expressing an anaerobic fluorescent protein, and biofilm formation on the WE was observed over time during current generation at WE potentials of +0.4 and 0 V (versus standard hydrogen electrodes), under electrolyte-flow conditions. Electrochemical analyses suggested the presence of unique electron-transfer mechanisms in the +0.4-V biofilm. Microscopic analyses revealed that, in contrast to aerobic biofilms, current-generating biofilm (at +0.4 V) was thin and flat (∼10 μm in thickness), and cells were evenly and densely distributed in the biofilm. In contrast, cells were unevenly distributed in biofilm formed at 0 V.
In situ
fluorescence staining and biofilm recovery experiments showed that the amounts of extracellular polysaccharides (EPSs) in the +0.4-V biofilm were much smaller than those in the aerobic and 0-V biofilms, suggesting that
Shewanella
cells suppress the production of EPSs at +0.4 V under flow conditions. We suggest that
Shewanella
cells perceive electrode potentials and modulate the structure and composition of biofilms to efficiently transfer electrons to electrodes.

IMPORTANCE
A promising application of microbial fuel cells (MFCs) is to save energy in wastewater treatment. Since current is generated in these MFCs by biofilm microbes under horizontal flows of wastewater, it is important to understand the mechanisms for biofilm formation and current generation under water-flow conditions. Although massive work has been done to analyze the molecular mechanisms for current generation by model exoelectrogenic bacteria, such as
Shewanella oneidensis
, limited information is available regarding the formation of current-generating biofilms over time under water-flow conditions. The present study developed electrochemical flow cells and used them to examine the electrochemical and structural features of current-generating biofilms under water-flow conditions. We show unique features of mature biofilms actively generating current, creating opportunities to search for as-yet-undiscovered current-generating mechanisms in
Shewanella
biofilms. Furthermore, information provided in the present study is useful for researchers attempting to develop anode architectures suitable for wastewater treatment MFCs.
Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis
Authors: Guarnieri Michael T. , Chou Yat-Chen , Salvachúa Davinia , Mohagheghi Ali , St. John Peter C. , Peterson Darren J. , Bomble Yannick J. , Beckham Gregg T.
Published: June 2017

Actinobacillus succinogenes
, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition,
A. succinogenes
is capnophilic, incorporating CO
2
into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO
2
to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in
A. succinogenes
, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe.

IMPORTANCE
Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in
Actinobacillus succinogenes
. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in
A. succinogenes
metabolism.
Rapid, High-Throughput Identification of Anthrax-Causing and Emetic Bacillus cereus Group Genome Assemblies via BTyper, a Computational Tool for Virulence-Based Classification of Bacillus cereus Group Isolates by Using Nucleotide Sequencing Data
Authors: Carroll Laura M. , Kovac Jasna , Miller Rachel A. , Wiedmann Martin
Published: June 2017

The
Bacillus cereus
group comprises nine species, several of which are pathogenic. Differentiating between isolates that may cause disease and those that do not is a matter of public health and economic importance, but it can be particularly challenging due to the high genomic similarity within the group. To this end, we have developed BTyper, a computational tool that employs a combination of (i) virulence gene-based typing, (ii) multilocus sequence typing (MLST), (iii)
panC
clade typing, and (iv)
rpoB
allelic typing to rapidly classify
B. cereus
group isolates using nucleotide sequencing data. BTyper was applied to a set of 662
B. cereus
group genome assemblies to (i) identify anthrax-associated genes in non-
B. anthracis
members of the
B. cereus
group, and (ii) identify assemblies from
B. cereus
group strains with emetic potential. With BTyper, the anthrax toxin genes
cya
,
lef
, and
pagA
were detected in 8 genomes classified by the NCBI as
B. cereus
that clustered into two distinct groups using
k
-medoids clustering, while either the
B. anthracis
poly-γ-
d
-glutamate capsule biosynthesis genes
capABCDE
or the hyaluronic acid capsule
hasA
gene was detected in an additional 16 assemblies classified as either
B. cereus
or
Bacillus thuringiensis
isolated from clinical, environmental, and food sources. The emetic toxin genes
cesABCD
were detected in 24 assemblies belonging to
panC
clades III and VI that had been isolated from food, clinical, and environmental settings. The command line version of BTyper is available at
https://github.com/lmc297/BTyper
. In addition, BMiner, a companion application for analyzing multiple BTyper output files in aggregate, can be found at
https://github.com/lmc297/BMiner
.

IMPORTANCE
Bacillus cereus
is a foodborne pathogen that is estimated to cause tens of thousands of illnesses each year in the United States alone. Even with molecular methods, it can be difficult to distinguish nonpathogenic
B. cereus
group isolates from their pathogenic counterparts, including the human pathogen
Bacillus anthracis
, which is responsible for anthrax, as well as the insect pathogen
B. thuringiensis
. By using the variety of typing schemes employed by BTyper, users can rapidly classify, characterize, and assess the virulence potential of any isolate using its nucleotide sequencing data.
Therapeutic potential of omega-3 fatty acids supplementation in a mouse model of dry macular degeneration
Authors: Prokopiou Ekatherine , Kolovos Panagiotis , Kalogerou Maria , Neokleous Anastasia , Papagregoriou Gregory , Deltas Constantinos , Malas Stavros , Georgiou Tassos
Published: June 2017

Purpose

To evaluate the therapeutic effects of omega-3 (ω-3) and omega-6 (ω-6) fatty acids in the CCL2−/−model of dry age-related macular degeneration (AMD). The blood level of eicosapentaenoic acid (EPA) and arachidonic acid (AA) served to adjust the treatment dosage (AA/EPA=1–1.5).

Methods

Nine-month-old animals were allocated to different groups: (A) C57BL/6 untreated , (B) CCL2−/−untreated, (C) CCL2−/−treated with ω-3+ω-6, and (D) CCL2−/−treated with ω-3. Treatment was daily administered by gavage for 3 months. Fatty acids analysis was performed and retinas were histologically examined. Three-month-old wild type mice were used for comparison purposes. Real-time PCR and Western blot were performed for retinal inflammatory mediators.

Results

Increased EPA and decreased AA levels were observed in both blood and retinas in the treatment groups. The outer nuclear layer thickness was increased in groups C (45.0±3.9 µm) and D (62.8±4.9 µm), compared with groups B (65.6±3.0 µm) and A (71.1±4.2 µm), and in younger mice, it was 98.0±3.9 µm. A decrease in NF-κB expression was noted in the treatment groups. Interleukin (IL) 18 protein levels demonstrated a significant reduction in the ω-3-treated group only.

Conclusion

Supplementation with ω-3+ω-6 or ω-3 alone (AA/EPA=1–1.5) suggests a protective mechanism in the CCL2−/−animal model of dry AMD, with a more beneficial effect when ω-3 are used alone. Our findings indicated that inflammation is not the only determining factor; perhaps a regenerative process might be involved following administration of ω-3 fatty acids.
School zone safety diagnosis using automated conflicts analysis technique
Authors: Reyad Passant , Sayed Tarek , Zaki Mohamed H. , Ibrahim Shewkar E.
Published: June 2017

School safety is a high priority for road safety agencies worldwide due to the growing concerns about student safety and emphasis on community livability. However, the lack of consistent data available at schools makes it challenging for engineers to understand the safety issues faced accurately. Traffic conflicts have been advocated in the literature as a surrogate safety measure due to the advantages it offers for road safety evaluations. This paper demonstrates the capability of automated traffic safety diagnosis at a school using computer vision techniques. The selected school is located in a residential area in the City of Edmonton, Alberta, and is on a main roadway in the neighbourhood. The age group of the school is from 5 to 16 years old (corresponding to Grade 1 to 9 respectively). Data are collected during the fall and winter terms. The severity and frequency of conflicts and traffic violations were analyzed to quantify the safety concerns. These concerns included driving violations, jaywalking violations as well as conflicts between pedestrians and vehicles as well as between vehicles and each other. Hourly and seasonal trends were observed and analyzed to assist in the selection of treatments and recommendations to improve the safety around schools. The results show that pedestrian safety has improved in winter due to lower vehicle speeds.
A-type K+ channels contribute to the prorenin increase of firing activity in hypothalamic vasopressin neurosecretory neurons
Authors: Pitra Soledad , Stern Javier E.
Published: June 2017

Recent studies have supported an important contribution of prorenin (PR) and its receptor (PRR) to the regulation of hypothalamic, sympathetic, and neurosecretory outflows to the cardiovascular system, including systemic release of vasopressin (VP), both under physiological and cardiovascular disease conditions. Still, the identification of precise cellular mechanisms and neuronal/molecular targets remain unknown. We have recently shown that PRR is expressed in VP neurons and that their activation increases neuronal activity. However, the underlying ionic channel mechanisms are undefined. Here, we performed patch-clamp electrophysiology from identified VP neurons in acute hypothalamic slices obtained from enhanced green fluorescent protein-VP transgenic rats. Voltage-clamp recordings showed that PR inhibited the magnitude of A-type K+ current ( IA; ~50% at −25 mV), a subthreshold voltage-dependent current that restrains VP firing activity. PR also increased the inactivation rate of IA and shifted the steady-state voltage-dependent inactivation function toward more hyperpolarized membrane potential (~7 mV shift), thus resulting in less channel availability to be activated at any given membrane potential. PR also inhibited a sustained component of IA (“window” current). PR-mediated changes in action potential waveform and increased firing activity were occluded when IA was blocked by 4-aminopyridine. Finally, PR failed to increase superoxide production within the supraoptic nucleus/paraventricular nucleus, and PR excitatory effects persisted in slices treated with the SOD mimetic tempol. Taken together, these experiments indicated that PR excitatory effects on vasopressin neurons involve inhibition of IA, due, in part, to increases in its voltage-dependent inactivation properties. Moreover, our results indicate that PR effects did not involve an increase in oxidative stress.

NEW & NOTEWORTHY Here, we demonstrate that prorenin/the prorenin receptor is an important signaling unit for the regulation of vasopressin firing activity and, thus, systemic hormonal release. We identified A-type K+ channels as key molecular targets mediating prorenin stimulation of vasopressin neuronal activity, thus standing as a potential therapeutic target for neurohumoral activation in cardiovascular disease.
Sex differences in vascular physiology and pathophysiology: estrogen and androgen signaling in health and disease
Authors: Boese Austin C. , Kim Seong C. , Yin Ke-Jie , Lee Jean-Pyo , Hamblin Milton H.
Published: June 2017

Sex differences between women and men are often overlooked and underappreciated when studying the cardiovascular system. It has been long assumed that men and women are physiologically similar, and this notion has resulted in women being clinically evaluated and treated for cardiovascular pathophysiological complications as men. Currently, there is increased recognition of fundamental sex differences in cardiovascular function, anatomy, cell signaling, and pathophysiology. The National Institutes of Health have enacted guidelines expressly to gain knowledge about ways the sexes differ in both normal function and diseases at the various research levels (molecular, cellular, tissue, and organ system). Greater understanding of these sex differences will be used to steer future directions in the biomedical sciences and translational and clinical research. This review describes sex-based differences in the physiology and pathophysiology of the vasculature, with a special emphasis on sex steroid receptor (estrogen and androgen receptor) signaling and their potential impact on vascular function in health and diseases (e.g., atherosclerosis, hypertension, peripheral artery disease, abdominal aortic aneurysms, cerebral aneurysms, and stroke).
Significant role of the cardiopostural interaction in blood pressure regulation during standing
Authors: Xu Da , Verma Ajay K. , Garg Amanmeet , Bruner Michelle , Fazel-Rezai Reza , Blaber Andrew P. , Tavakolian Kouhyar
Published: June 2017

Cardiovascular and postural control systems have been studied independently despite the increasing evidence showing the importance of cardiopostural interaction in blood pressure regulation. In this study, we aimed to assess the role of the cardiopostural interaction in relation to cardiac baroreflex in blood pressure regulation under orthostatic stress before and after mild exercise. Physiological variables representing cardiovascular control (heart rate and systolic blood pressure), lower limb muscle activation (electromyography), and postural sway (center of pressure derived from force and moment data during sway) were measured from 17 healthy participants (25 ± 2 yr, 9 men and 8 women) during a sit-to-stand test before and after submaximal exercise. The cardiopostural control (characterized by baroreflex-mediated muscle-pump effect in response to blood pressure changes, i.e., muscle-pump baroreflex) was assessed using wavelet transform coherence and causality analyses in relation to the baroreflex control of heart rate. Significant cardiopostural blood pressure control was evident counting for almost half of the interaction time with blood pressure changes that observed in the cardiac baroreflex (36.6–72.5% preexercise and 34.7–53.9% postexercise). Thus, cardiopostural input to blood pressure regulation should be considered when investigating orthostatic intolerance. A reduction of both cardiac and muscle-pump baroreflexes in blood pressure regulation was observed postexercise and was likely due to the absence of excessive venous pooling and a less stressed system after mild exercise. With further studies using more effective protocols evoking venous pooling and muscle-pump activity, the cardiopostural interaction could improve our understanding of the autonomic control system and ultimately lead to a more accurate diagnosis of cardiopostural dysfunctions.

NEW & NOTEWORTHY We examined the interaction between cardiovascular and postural control systems during standing before and after mild exercise. Significant cardiopostural input to blood pressure regulation was shown, suggesting the importance of cardiopostural integration when investigating orthostatic hypotension. In addition, we observed a reduction of baroreflex-mediated blood pressure regulation after exercise.
Impedance Synthesis Based Vibration Analysis of Geared Transmission System
Authors: Ren Yafeng , Chang Shan , Liu Geng , Wu Liyan , Lim Teik C.
Published: June 2017

The severity of gear noise response depends on the sensitivity of geared rotor system dynamics to the transmission error. As gearbox design trending towards lighter weight and lower noise, the influence of housing compliance on system dynamic characteristics cannot be ignored. In this study, a gear-shaft-bearing-housing coupled impedance model is proposed to account for the effect of housing compliance on the vibration of geared transmission system. This proposed dynamic model offers convenient modeling, efficient computing, and ability to combine computed parameters with experimental ones. The numerical simulations on system dynamic characteristics are performed for both a rigid housing configuration and a flexible one. Natural frequencies, dynamic mesh forces, and dynamic bearing reaction loads are computed, and the housing compliance contribution on system dynamic characteristics is analyzed. Results show that increasing housing compliance will decrease the system natural frequencies and will affect the dynamic bearing reaction loads significantly but have very little influence on the dynamic mesh force. Also, the analysis shows that bearing stiffness has significant influence on the degree of housing contribution on system dynamic characteristics.
Health-Related Quality of Life after Pediatric Liver Transplantation: A Qualitative Analysis of the Perspectives of Health Care Providers
Authors: Miserachs Mar , Nicholas David B. , Otley Anthony R. , Ng Vicky Lee
Published: June 2017

With improved survival outcomes after pediatric liver transplantation (LT), health-related quality of life (HRQoL) is an important outcome metric. Understanding the elements contributing to HRQoL after LT in children would enable more targeted strategies towards optimizing best outcomes. This qualitative study aimed to explore health care providers (HCP) perceptions about HRQoL after pediatric LT. Thirteen experienced HCP participated in two focus group discussions. Data analysis via a thematic analysis approach revealed 4 major themes: “LT as a facilitator of better HRQoL,” “coping and adapting to LT,” “living with a transplanted liver,” and “the family context.” HCP identified elements that both enhance (improved physical health, peer relationship, and activities of daily living) and challenge (need for immunosuppression, transplant follow-up, and restrictions) the multidimensional domains of HRQoL. HCP perceived LT to be a stressful life-changing event for children and their families. Patients and their parents’ ability to cope and adjust positively to LT was perceived as a key contributor to better HRQoL. HCP perspective highlights the importance of promoting psychosocial support and a family-centered care delivery model towards the overarching goal of optimizing durable outcomes.
The Inhibitory Effect of WenxinKeli on H9C2 Cardiomyocytes Hypertrophy Induced by Angiotensin II through Regulating Autophagy Activity
Authors: Li Jie , Li Yang , Zhang Ying , Hu Dan , Gao Yonghong , Shang Hongcai , Xing Yanwei
Published: June 2017

Objectives. We investigated the role of cardiomyocyte autophagy and its regulatory mechanisms by WenxinKeli (WXKL) in cells subjected to hypertrophy. Methods. H9C2 cardiomyocytes were divided into 8 groups. Cytoskeletal proteins as well as endogenously expressed autophagy marker proteins were studied by confocal imaging. Western blotting was used to assess the levels of light chain‐3 (LC3) and mechanistic target of rapamycin (mTOR). The cell viability assay was used to detect the content of ATP. Flow cytometry was used to detect apoptotic cardiomyocytes. Results. (1) Compared with the control group, the length and width of cells in the Angiotensin II (AngII) group were significantly increased, while those in the 3‐methyladenine (3‐MA) and the WXKL groups were decreased. (2) Compared with AngII group, the expression of LC3 II/I protein in the 3‐MA and WXKL groups was downregulated, while the expression of mTOR protein was upregulated. (3) Compared with the AngII group, the cardiomyocytes in the WXKL group showed increased ATP and decreased apoptosis rate and number of autophagosome. Conclusions. We propose a novel role of WXKL as a likely inhibitor of cardiac hypertrophy by regulation of pathological autophagy.
Effect of Preoperative Intravitreal Bevacizumab on the Surgical Outcome of Neovascular Glaucoma at Different Stages
Authors: Kwon Junki , Sung Kyung Rim
Published: June 2017

Purpose. To evaluate the effect of preoperative intravitreal bevacizumab (IVB) injection on the surgical outcome of Ahmed glaucoma valve (AGV) implantation according to the angle status in neovascular glaucoma (NVG) eyes. Materials and Methods. This retrospective study included 70 NVG patients who underwent AGV implantation and were followed up for at least 12 months. An IVB injection before AGV implantation was administered to 45 eyes (IVB group), while it was not administered to 25 eyes (control group). Subgroup analyses were done at different stages in terms of the extent of peripheral anterior synechiae (PAS). Results. Mean follow-up period after AGV implantation was 27 ± 15 months. The IVB group showed higher prevalence of the eyes with less than 50% of PAS than that of the control group (78% versus 44%). The overall success rate 1 year postoperatively was 80% and 64% for the IVB and control groups, respectively (P=0.142). When PAS extent was less than 50%, preoperative IVB had a marginally positive effect on surgical outcome (HR = 0.39, P=0.064, per 1-time IVB injection). Conclusions. Preoperative IVB may enhance the success rate of AGV implantation in NVG eyes, before PAS has extensively formed. Further prospective randomized studies controlling the extent of PAS are warranted.
The Burden of Illness Related to Chronic Obstructive Pulmonary Disease Exacerbations in Québec, Canada
Authors: Dang-Tan Tam , Zhang Shiyuan , Tavares Ruben V. , Stutz Melissa , Ismaila Afisi S. , Vaillancourt Julie , Corriveau Diane , Stanford Richard H. , Lin Xiwu , Nadeau Gilbert A. , Simidchiev Alexander , Parsons Daria , Sampalis John S.
Published: June 2017

Background. Chronic obstructive pulmonary disease (COPD) prevalence in Canada has risen over time. COPD-related exacerbations contribute to the increased health care utilization (HCU) in this population. This study investigated the impact of exacerbations on COPD-related HCU. Methods. This retrospective observational cohort study used patient data from the Québec provincial health insurance databases. Eligible patients with a new HCU claim with a diagnostic billing for COPD during 2001–2010 were followed until March 31, 2011. Exacerbation rates and time to first exacerbation were assessed. Unadjusted analyses and multivariable models compared the rate of HCU by exacerbation classification (any [moderate/severe], moderate, or severe). Results. The exacerbation event rate in patients with an exacerbation was 34.3 events/100 patient-years (22.7 for moderate exacerbations and 11.6 for severe exacerbations). Median time to first exacerbation of any classification was 37 months. In unadjusted analyses, COPD-related HCU significantly increased with exacerbation severity. In the multivariable, HCU rates were significantly higher after exacerbation versus before exacerbation (p<0.01) for patients with an exacerbation or moderate exacerbations. For severe exacerbations, general practitioner, respiratory specialist, emergency room, and hospital visits were significantly higher after exacerbation versus before exacerbation (p<0.001). Conclusions. Exacerbations were associated with increased HCU, which was more pronounced for patients with severe exacerbations. Interventions to reduce the risk of exacerbations in patients with COPD may reduce disease burden.
A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220
Authors: Hou Panpan , Wu Chao , Wang Yuchen , Qi Rui , Bhavanasi Dheeraj , Zuo Zhixiang , Dos Santos Cedric , Chen Shuliang , Chen Yu , Zheng Hong , Wang Hong , Perl Alexander , Guo Deyin , Huang Jian
Published: June 2017

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD–positive and –negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR-pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated reactivation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML. Cancer Res; 77(16); 4402–13. ©2017 AACR.
A Naturally Generated Decoy of the Prostate Apoptosis Response-4 Protein Overcomes Therapy Resistance in Tumors
Authors: Hebbar Nikhil , Burikhanov Ravshan , Shukla Nidhi , Qiu Shirley , Zhao Yanming , Elenitoba-Johnson Kojo S.J. , Rangnekar Vivek M.
Published: June 2017

Primary tumors are often heterogeneous, composed of therapy-sensitive and emerging therapy-resistant cancer cells. Interestingly, treatment of therapy-sensitive tumors in heterogeneous tumor microenvironments results in apoptosis of therapy-resistant tumors. In this study, we identify a prostate apoptosis response-4 (Par-4) amino-terminal fragment (PAF) that is released by diverse therapy-sensitive cancer cells following therapy-induced caspase cleavage of the tumor suppressor Par-4 protein. PAF caused apoptosis in cancer cells resistant to therapy and inhibited tumor growth. A VASA segment of Par-4 mediated its binding and degradation by the ubiquitin ligase Fbxo45, resulting in loss of Par-4 proapoptotic function. Conversely, PAF, which contains this VASA segment, competitively bound to Fbxo45 and rescued Par-4–mediated induction of cancer cell–specific apoptosis. Collectively, our findings identify a molecular decoy naturally generated during apoptosis that inhibits a ubiquitin ligase to overcome therapy resistance in tumors. Cancer Res; 77(15); 4039–50. ©2017 AACR.
Acetylation of Mastermind-like 1 by p300 Drives the Recruitment of NACK to Initiate Notch-Dependent Transcription
Authors: Jin Ke , Zhou Wen , Han Xiaoqing , Wang Zhiqiang , Li Bin , Jeffries Shawn , Tao Wensi , Robbins David J. , Capobianco Anthony J.
Published: June 2017

Although it has long been appreciated that p300 acts as a critical Notch coactivator, the mechanistic details of p300 in Notch-mediated transcription remain unclear. We previously demonstrated that PEAK1-related kinase activating pseudokinase 1 (NACK), also known as SGK223, is a critical coactivator of Notch signaling and binds to the Notch1 ternary complex. Herein we report that p300 and CBP acetylate Mastermind-like 1 (Maml1) on amino acid residues K188 and K189 to recruit NACK to the Notch1 ternary complex, which results in the recruitment of RNA polymerase II to initiate transcription. NACK is recruited to the ternary complexes containing Maml1 and Maml3, but not Maml2. Simultaneous inhibition of p300/CBP and Notch has a synergistic effect in esophageal adenocarcinoma. In summary, this study provides a deeper mechanistic understanding of the assembly of the Notch transcriptional complex and provides rationale and proof of concept for a combinatorial therapeutic attack on Notch-dependent cancers. Cancer Res; 77(16); 4228–37. ©2017 AACR.
Intestine-Specific Homeobox Gene ISX Integrates IL6 Signaling, Tryptophan Catabolism, and Immune Suppression
Authors: Wang Li-Ting , Chiou Shyh-Shin , Chai Chee-Yin , Hsi Edward , Yokoyama Kazunari K. , Wang Shen-Nien , Huang Shau-Ku , Hsu Shih-Hsien
Published: June 2017

The intestine-specific homeobox transcription factor intestine-specific homeobox (ISX) is an IL6-inducible proto-oncogene implicated in the development of hepatocellular carcinoma, but its mechanistic contributions to this process are undefined. In this study, we provide evidence that ISX mediates a positive feedback loop integrating inflammation, tryptophan catabolism, and immune suppression. We found that ISX-mediated IL6-induced expression of the tryptophan catabolic enzymes Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase in hepatocellular carcinoma cells, resulting in an ISX-dependent increase in the tryptophan catabolite kynurenine and its receptor aryl hydrocarbon receptor (AHR). Activation of this kynurenine/AHR signaling axis acted through a positive feedback mechanism to increase ISX expression and enhance cellular proliferation and tumorigenic potential. RNAi-mediated attenuation of ISX or AHR reversed these effects. In an IDO1-dependent manner, ectopic expression of ISX induced expression of genes encoding the critical immune modulators CD86 (B7-2) and programmed death ligand-1 (PD-L1), through which ISX conferred a significant suppressive effect on the CD8+ T-cell response. In hepatocellular carcinoma specimens, expression of IDO1, kynurenine, AHR, and PD-L1 correlated negatively with survival. Overall, our results identified a feed-forward mechanism of immune suppression in hepatocellular carcinoma organized by ISX, which involves kynurenine-AHR signaling and PD-L1, offering insights into immune escape by hepatocellular carcinoma, which may improve its therapeutic management. Cancer Res; 77(15); 4065–77. ©2017 AACR.
Potential Susceptibility Loci Identified for Renal Cell Carcinoma by Targeting Obesity-Related Genes
Authors: Shu Xiang , Purdue Mark P. , Ye Yuanqing , Tu Huakang , Wood Christopher G. , Tannir Nizar M. , Wang Zhaoming , Albanes Demetrius , Gapstur Susan M. , Stevens Victoria L. , Rothman Nathaniel , Chanock Stephen J. , Wu Xifeng
Published: June 2017

Background: Obesity is an established risk factor for renal cell carcinoma (RCC). Although genome-wide association studies (GWAS) of RCC have identified several susceptibility loci, additional variants might be missed due to the highly conservative selection.

Methods: We conducted a multiphase study utilizing three independent genome-wide scans at MD Anderson Cancer Center (MDA RCC GWAS and MDA RCC OncoArray) and National Cancer Institute (NCI RCC GWAS), which consisted of a total of 3,530 cases and 5,714 controls, to investigate genetic variations in obesity-related genes and RCC risk.

Results: In the discovery phase, 32,946 SNPs located at ±10 kb of 2,001 obesity-related genes were extracted from MDA RCC GWAS and analyzed using multivariable logistic regression. Proxies (R2 > 0.8) were searched or imputation was performed if SNPs were not directly genotyped in the validation sets. Twenty-one SNPs with P < 0.05 in both MDA RCC GWAS and NCI RCC GWAS were subsequently evaluated in MDA RCC OncoArray. In the overall meta-analysis, significant (P < 0.05) associations with RCC risk were observed for SNP mapping to IL1RAPL2 [rs10521506-G: ORmeta = 0.87 (0.81–0.93), Pmeta = 2.33 × 10−5], PLIN2 [rs2229536-A: ORmeta = 0.87 (0.81–0.93), Pmeta = 2.33 × 10−5], SMAD3 [rs4601989-A: ORmeta = 0.86 (0.80–0.93), Pmeta = 2.71 × 10−4], MED13L [rs10850596-A: ORmeta = 1.14 (1.07–1.23), Pmeta = 1.50 × 10−4], and TSC1 [rs3761840-G: ORmeta = 0.90 (0.85–0.97), Pmeta = 2.47 × 10−3]. We did not observe any significant cis-expression quantitative trait loci effect for these SNPs in the TCGA KIRC data.

Conclusions: Taken together, we found that genetic variation of obesity-related genes could influence RCC susceptibility.

Impact: The five identified loci may provide new insights into disease etiology that reveal importance of obesity-related genes in RCC development. Cancer Epidemiol Biomarkers Prev; 26(9); 1436–42. ©2017 AACR.
The Impact of a Population-Based Screening Program on Income- and Immigration-Related Disparities in Colorectal Cancer Screening
Authors: Kiran Tara , Glazier Richard H. , Moineddin Rahim , Gu Sumei , Wilton Andrew S. , Paszat Lawrence
Published: June 2017

Background: A population-based program promoting the Fecal Occult Blood Test (FOBT) for colorectal cancer screening was introduced in 2008 in Ontario, Canada, where opportunistic screening with colonoscopy had been increasing in frequency. We evaluated the impact of the program on income and immigration-related disparities in screening.

Methods: We used linked administrative data to calculate colorectal cancer screening rates for eligible Ontarians in each year between 2001/02 (n = 2,852,619) and 2013/14 (n = 4,139,304). We quantified disparities using an “inequality ratio” of screening rates in the most disadvantaged group relative to the most advantaged group. We performed segmented logistic regression analyses stratified by screening modality and adjusted for age, sex, rurality, comorbidity, and morbidity.

Results: Between 2001/02 and 2013/14, the income and immigration inequality ratios narrowed from 0.74 to 0.80 and 0.55 to 0.69, respectively. Before the screening program, the income inequality ratio was widening by 1% per year (95% CI 1% to 1%); in the year it was introduced, it narrowed by 4% (95% CI 2% to 7%) and in the years following, it remained stable [0% decrease (95% CI 1% decrease to 0% decrease) per year]. Results were similar for immigration-related disparities. After program introduction, disparities in receiving FOBT were narrowing at a faster rate while disparities in receiving colonoscopy were widening at a slower rate.

Conclusions: Introduction of a population-based screening program promoting FOBT for colorectal cancer was associated with only modest improvements in immigration and income-related disparities.

Impact: Reducing immigration and income-related disparities should be a focus for future research and policy work. Disparities in Ontario seem to be driven by a higher uptake of colonoscopy among more advantaged groups. Cancer Epidemiol Biomarkers Prev; 26(9); 1401–10. ©2017 AACR.
Fractionated Dosing Improves Preclinical Therapeutic Index of Pyrrolobenzodiazepine-Containing Antibody Drug Conjugates
Authors: Hinrichs Mary Jane Masson , Ryan Pauline M. , Zheng Bo , Afif-Rider Shameen , Yu Xiang Qing , Gunsior Michele , Zhong Haihong , Harper Jay , Bezabeh Binyam , Vashisht Kapil , Rebelatto Marlon , Reed Molly , Ryan Patricia C. , Breen Shannon , Patel Neki , Chen Cui , Masterson Luke , Tiberghien Arnaud , Howard Phillip W. , Dimasi Nazzareno , Dixit Rakesh
Published: June 2017

Purpose: To use preclinical models to identify a dosing schedule that improves tolerability of highly potent pyrrolobenzodiazepine dimers (PBDs) antibody drug conjugates (ADCs) without compromising antitumor activity.

Experimental Design: A series of dose-fractionation studies were conducted to investigate the pharmacokinetic drivers of safety and efficacy of PBD ADCs in animal models. The exposure–activity relationship was investigated in mouse xenograft models of human prostate cancer, breast cancer, and gastric cancer by comparing antitumor activity after single and fractionated dosing with tumor-targeting ADCs conjugated to SG3249, a potent PBD dimer. The exposure–tolerability relationship was similarly investigated in rat and monkey toxicology studies by comparing tolerability, as assessed by survival, body weight, and organ-specific toxicities, after single and fractionated dosing with ADCs conjugated to SG3249 (rats) or SG3400, a structurally related PBD (monkeys).

Results: Observations of similar antitumor activity in mice treated with single or fractionated dosing suggests that antitumor activity of PBD ADCs is more closely related to total exposure (AUC) than peak drug concentrations (Cmax). In contrast, improved survival and reduced toxicity in rats and monkeys treated with a fractionated dosing schedule suggests that tolerability of PBD ADCs is more closely associated with Cmax than AUC.

Conclusions: We provide the first evidence that fractionated dosing can improve preclinical tolerability of at least some PBD ADCs without compromising efficacy. These findings suggest that preclinical exploration of dosing schedule could be an important clinical strategy to improve the therapeutic window of highly potent ADCs and should be investigated further. Clin Cancer Res; 23(19); 5858–68. ©2017 AACR.
SLC45A2: A Melanoma Antigen with High Tumor Selectivity and Reduced Potential for Autoimmune Toxicity
Authors: Park Jungsun , Talukder Amjad H. , Lim Seon A. , Kim Kwanghee , Pan Ke , Melendez Brenda , Bradley Sherille D. , Jackson Kyle R. , Khalili Jahan S. , Wang Junmei , Creasy Caitlin , Pan Bih-Fang , Woodman Scott E. , Bernatchez Chantale , Hawke David , Hwu Patrick , Lee Kyung-Mi , Roszik Jason , Lizée Gregory , Yee Cassian
Published: June 2017

Cytotoxic T lymphocyte (CTL)–based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient–derived cell lines, we identified a number of shared HLA class I–bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402–restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618–29. ©2017 AACR.