bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani
Authors: Zhang Wen-Wei , Matlashewski Greg 
Published: July 2015

ABSTRACT

The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to
Leishmania donovani
, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into
L. donovani
and generated guide RNA (gRNA) expression vectors by using the
L. donovani
rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that
L. donovani
mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in
L. donovani
. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and
GFP
gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (
LdMT
). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in
LdMT
(M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for
L. donovani.

IMPORTANCE
Leishmania donovani
is the causative agent of fatal visceral leishmaniasis. To understand
Leishmania
infection and pathogenesis and identify new drug targets for control of leishmaniasis, more-efficient ways to manipulate this parasite genome are required. In this study, we have implemented CRISPR-Cas9 genome-editing technology in
L. donovani
. Both single- and dual-gRNA expression vectors were developed using a strong RNA polymerase I promoter and ribozymes. With this system, it was possible to generate loss-of-function insertion and deletion mutations and introduce drug selection markers and the
GFP
sequence precisely into the
L. donovani
genome. These methods greatly improved the ability to manipulate this parasite genome and will help pave the way for high-throughput functional analysis of
Leishmania
genes. This study further revealed that double-stranded DNA breaks created by CRISPR-Cas9 were repaired by the homology-directed repair (HDR) pathway and microhomology-mediated end joining (MMEJ) in
Leishmania
.
Deciphering the Regulatory Circuitry That Controls Reversible Lysine Acetylation in Salmonella enterica
Authors: Hentchel Kristy L. , Thao Sandy , Intile Peter J. , Escalante-Semerena Jorge C. 
Published: July 2015

ABSTRACT

In
Salmonella enterica
, the reversible lysine acetylation (RLA) system is comprised of the protein acetyltransferase (Pat) and sirtuin deacetylase (CobB). RLA controls the activities of many proteins, including the acetyl coenzyme A (acetyl-CoA) synthetase (Acs), by modulating the degree of Acs acetylation. We report that IolR, a
myo
-inositol catabolism repressor, activates the expression of genes encoding components of the RLA system.
In vitro
evidence shows that the IolR protein directly regulates
pat
expression. An
iolR
mutant strain displayed a growth defect in minimal medium containing 10 mM acetate, a condition under which RLA function is critical to control Acs activity. Increased levels of Pat, CobB, or Acs activity reversed the growth defect, suggesting the Pat/CobB ratio in an
iolR
strain is altered and that such a change affects the level of acetylated, inactive Acs. Results of quantitative reverse transcription-PCR (qRT-PCR) analyses of
pat
,
cobB
, and
acs
expression indicated that expression of the genes alluded to in the IolR-deficient strain was reduced 5-, 3-, and 2.6-fold, respectively, relative to the levels present in the strain carrying the

iolR
+

allele. Acs activity in cell-free extracts from an
iolR
mutant strain was reduced ~25% relative to that of the
iolR
+
strain. Glucose differentially regulated expression of
pat
,
cobB
, and
acs
. The catabolite repressor protein (Crp) positively regulated expression of
pat
while having no effect on
cobB
.

IMPORTANCE
Reversible lysine acylation is used by cells of all domains of life to modulate the function of proteins involved in diverse cellular processes. Work reported herein begins to outline the regulatory circuitry that integrates the expression of genes encoding enzymes that control the activity of a central metabolic enzyme in C2 metabolism. Genetic analyses revealed effects on reversible lysine acylation that greatly impacted the growth behavior of the cell. This work provides the first insights into the complexities of the system responsible for controlling reversible lysine acylation at the transcriptional level in the enteropathogenic bacterium
Salmonella enterica
.
Impact of cardiovascular comorbidities on COPD Assessment Test (CAT) and its responsiveness to pulmonary rehabilitation in patients with moderate to very severe COPD: protocol of the Chance study
Authors: Smid Dionne E , Wilke Sarah , Jones Paul W , Muris Jean W M , Wouters Emiel F M , Franssen Frits M E , Spruit Martijn A 
Published: July 2015

IntroductionChronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality. Patients with COPD are characterised by a reduced health status, which can be easily assessed by the COPD Assessment Test (CAT). Previous studies show that health status can be worsened by the presence of comorbidities. However, the impact of cardiovascular comorbidities on health status as assessed with CAT is not sufficiently investigated. Therefore, the current study has the following objectives: (1) to study the clinical, (patho)physiological and psychosocial determinants of the CAT, and impact of previously established and/or newly diagnosed cardiovascular comorbidities on health status in tertiary care patients with COPD; (2) to assess the effects of pulmonary rehabilitation on CAT scores in patients with COPD; (3) to develop reference values for the CAT in Dutch elderly patients without COPD; and (4) to validate the CAT in a broad sample of Dutch patients with COPD.Methods and analysisTheCOPD,Health statusandComorbidities (Chance) study is a monocentre study consisting of an observational cross-sectional part and a longitudinal part. Demographic and clinical characteristics will be assessed in primary care, secondary care and tertiary care patients with COPD, and in patients without COPD. To assess health status, the CAT, Clinical COPD Questionnaire (CCQ) and St George's Respiratory Questionnaire (SGRQ) will be used. The longitudinal part consists of a comprehensive pulmonary rehabilitation programme in 500 tertiary care patients. For the cross-sectional part of the study, 150 patients without COPD, 100 primary care patients and 100 secondary care patients will be assessed during a single home visit.Ethics and disseminationThe Medical Ethical Committee of the Maastricht University Medical Centre+ (MUMC+), Maastricht, the Netherlands (METC 11-3-070), has approved this study. The study has been registered at the Dutch Trial Register (NTR 3416).
Access to healthcare and financial risk protection for older adults in Mexico: secondary data analysis of a national survey
Authors: Doubova Svetlana V , Pérez-Cuevas Ricardo , Canning David , Reich Michael R 
Published: July 2015

ObjectivesWhile the benefits of Seguro Popular health insurance in Mexico relative to no insurance have been widely documented, little has been reported on its effects relative to the pre-existing Social Security health insurance. We analyse the effects of Social Security and Seguro Popular health insurances in Mexico on access to healthcare of older adults, and on financial risk protection to their households, compared with older adults without health insurance.SettingSecondary data analysis was performed using the 2012 Mexican Survey of Health and Nutrition (ENSANUT).ParticipantsThe study population comprised 18 847 older adults and 13 180 households that have an elderly member.Outcome measuresThe dependent variables were access to healthcare given the reported need, the financial burden imposed by health expenditures measured through catastrophic health-related expenditures, and using savings for health-related expenditures. Separate propensity score matching analyses were conducted for each comparison. The analysis for access was performed at the individual level, and the analysis for financial burden at the household level. In each case, matching on a wide set of relevant characteristics was achieved.ResultsSeguro Popular showed a protective effect against lack of access to healthcare for older adults compared with those with no insurance. The average treatment effect on the treated (ATET) was ascertained through using the nearest-neighbour matching (−8.1%, t-stat −2.305) analysis. However, Seguro Popular did not show a protective effect against catastrophic expenditures in a household where an older adult lived. Social Security showed increased access to healthcare (ATET −11.3%, t-stat −3.138), and protective effect against catastrophic expenditures for households with an elderly member (ATET −1.9%, t-stat −2.178).ConclusionsSeguro Popular increased access to healthcare for Mexican older adults. Social Security showed a significant protective effect against lack of access and catastrophic expenditures compared with those without health insurance.
Loss of RACK1 Promotes Metastasis of Gastric Cancer by Inducing a miR-302c/IL8 Signaling Loop
Authors: Chen Ling , Min Lingqiang , Wang Xuefei , Zhao Junjie , Chen Hua , Qin Jing , Chen Weidong , Shen Zhenbin , Tang Zhaoqing , Gan Qiangjun , Ruan Yuanyuan , Sun Yihong , Qin Xinyu , Gu Jianxin 
Published: July 2015

Abstract
Gastric cancer remains the third leading cause of cancer-related mortality worldwide, and invasion and metastasis of gastric cancer represent the major reason for its poor prognosis. In this study, we found that loss of the receptor for activated C-kinase 1 (RACK1) promoted the metastasis of gastric cancer by enhancing the autocrine of IL8 in vitro and in vivo. microRNA (miRNA; miR) array identified that RACK1 modulated the expression of a series of miRNAs, including the miR-302 cluster, and RACK1 modulated the IL8 expression and tumor invasion through miRNA-302c. Moreover, upregulation of IL8 in turn decreased the level of miRNA-302c and induced IL8 expression in a feedback manner. Tissue microarray also indicated that RACK1 was correlated with invasion/metastasis phenotype, IL8 expression, as well as 5-year survival in clinical cases of gastric cancer. Together, our results imply that loss of RACK1 in gastric cancer links epigenetics to inflammatory cytokines to promote tumor metastasis. Cancer Res; 75(18); 3832–41. ©2015 AACR.
Genome-wide Gene–Asbestos Exposure Interaction Association Study Identifies a Common Susceptibility Variant on 22q13.31 Associated with Lung Cancer Risk
Authors: Liu Chen-yu , Stücker Isabelle , Chen Chu , Goodman Gary , McHugh Michelle K. , D'Amelio Anthony M. , Etzel Carol J. , Li Su , Lin Xihong , Christiani David C. 
Published: July 2015

Abstract
Background: Occupational asbestos exposure has been found to increase lung cancer risk in epidemiologic studies.
Methods: We conducted an asbestos exposure–gene interaction analyses among several Caucasian populations who were current or ex-smokers. The discovery phase included 833 Caucasian cases and 739 Caucasian controls, and used a genome-wide association study (GWAS) to identify single-nucleotide polymorphisms (SNP) with gene–asbestos interaction effects. The top ranked SNPs from the discovery phase were replicated within the International Lung and Cancer Consortium (ILCCO). First, in silico replication was conducted in those groups that had GWAS and asbestos exposure data, including 1,548 cases and 1,527 controls. This step was followed by de novo genotyping to replicate the results from the in silico replication, and included 1,539 cases and 1,761 controls. Multiple logistic regression was used to assess the SNP–asbestos exposure interaction effects on lung cancer risk.
Results: We observed significantly increased lung cancer risk among MIRLET7BHG (MIRLET7B host gene located at 22q13.31) polymorphisms rs13053856, rs11090910, rs11703832, and rs12170325 heterozygous and homozygous variant allele(s) carriers (P &lt; 5 × 10−7 by likelihood ratio test; df = 1). Among the heterozygous and homozygous variant allele(s) carriers of polymorphisms rs13053856, rs11090910, rs11703832, and rs12170325, each unit increase in the natural log-transformed asbestos exposure score was associated with age-, sex-, smoking status, and center-adjusted ORs of 1.34 [95% confidence interval (CI), 1.18–1.51], 1.24 (95% CI, 1.14–1.35), 1.28 (95% CI, 1.17–1.40), and 1.26 (95% CI, 1.15–1.38), respectively, for lung cancer risk.
Conclusion: Our findings suggest that MIRLET7BHG polymorphisms may be important predictive markers for asbestos exposure–related lung cancer.
Impact: To our knowledge, our study is the first report using a systematic genome-wide analysis in combination with detailed asbestos exposure data and replication to evaluate asbestos-associated lung cancer risk. Cancer Epidemiol Biomarkers Prev; 24(10); 1564–73. ©2015 AACR.
Cellular Senescence Markers p16INK4a and p21CIP1/WAF Are Predictors of Hodgkin Lymphoma Outcome
Authors: Caliò Anna , Zamò Alberto , Ponzoni Maurilio , Zanolin Maria Elisabetta , Ferreri Andrés J.M. , Pedron Serena , Montagna Licia , Parolini Claudia , Fraifeld Vadim E. , Wolfson Marina , Yanai Hagai , Pizzolo Giovanni , Doglioni Claudio , Vinante Fabrizio , Chilosi Marco 
Published: July 2015

AbstractPurpose: There is evidence that Hodgkin Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) could display some molecular and morphologic markers of cellular senescence (CS). We hypothesized that CS mechanisms may have potential prognostic relevance in cHL and investigated whether the expression of the well-established CS biomarkers p21CIP1/WAF1 and p16INK4a by HRS cells might be predictive of the probability of event-free survival (EFS).Experimental Design: The study analyzed a retrospective cohort of 147 patients and the results were validated on a cohort of 91 patients independently diagnosed and treated in a different institution. p16INK4a and p21CIP1/WAF1 were categorized as dichotomous variables (&lt; or ≥ 30% of HRS cells at diagnosis) and evaluated in univariate and multivariate analysis.Results: Both molecules were independent prognostic factors. A positive staining of one of the two molecules in more than 30% HRS cells predicted a better EFS (P &lt; 0.01). p16INK4a/p21CIP1/WAF1 together as a unique categorical variable (both &lt;30%, either &lt;30%, both ≥ 30%) sorted out three prognostic groups with better, intermediate, or worse outcome either overall or within I–II, bulky and advanced stages. The presence or the lack of the robust expression of p21CIP1/WAF1 and/or p16INK4a defined the prognosis in our series.Conclusions: These findings point to (i) the relevance of CS-related mechanisms in cHL, and to (ii) the prognostic value of a simple, reproducible, and low-cost immunohistochemical evaluation of p16INK4a and p21CIP1/WAF1 expression. Clin Cancer Res; 21(22); 5164–72. ©2015 AACR.
Less Can Be More for Gene Dose and Drug Sensitivity
Authors: Gatz Susanne A. , Shipley Janet M. 
Published: July 2015

Abstract
CDK4 is preclinically validated as a therapeutic target in PAX3–FOXO1 fusion gene-positive rhabdomyosarcomas. Pharmacologic targeting showed sensitivity but, contrary to expectation, CDK4 genomic amplification and overexpression associated with 25% of cases that exhibited the lowest sensitivities. This emphasizes the importance of tumor-specific preclinical studies to define and understand drug sensitivity. Clin Cancer Res; 21(21); 4750–2. ©2015 AACR.
See related article by Olanich et al., p. 4947
Clinical Profile of Athletes With Hypertrophic Cardiomyopathy
Authors: Sheikh Nabeel , Papadakis Michael , Schnell Frédéric , Panoulas Vasileios , Malhotra Aneil , Wilson Mathew , Carré François , Sharma Sanjay 
Published: July 2015

Background—
The phenotype of individuals with hypertrophic cardiomyopathy (HCM) who exercise regularly is unknown. This study characterized the clinical profile of young athletes with HCM.

Methods and Results—

The electrical, structural, and functional cardiac parameters from 106 young (14–35 years) athletes with HCM were compared with 101 sedentary HCM patients. A subset of athletes with HCM exhibiting morphologically mild (13–16 mm), concentric disease was compared with 55 healthy athletes with mild physiological left ventricular hypertrophy (LVH). Most athletes with HCM (96%) exhibited T-wave inversion and had milder LVH (15.8±3.4 mm versus 19.7±6.5 mm,
P
<0.001), larger left ventricular cavity dimensions (47.8±6.0 mm versus 44.3±7.7 mm,
P
<0.001), and superior indices of diastolic function (average
E
/
E
′ 7.9±2.4 versus 10.7±3.9,
P
<0.001) compared with sedentary HCM patients. In athletes with HCM, LVH was frequently (36%) confined to the apex and only 15 individuals (14%) exhibited mild concentric LVH mimicking physiological LVH. In these 15 athletes, conventional structural and functional cardiac parameters showed modest sensitivity and specificity for differentiating HCM from physiological LVH: 13% had a left ventricular cavity >54 mm, 87% had a left atrium ≤40, and 100% had an
E
/
E
′ <12.

Conclusions—
Athletes with HCM exhibit less LVH, larger left ventricular cavities, and normal indices of diastolic function compared with sedentary patients. Only a minority of athletes with HCM constitute the conventional gray zone of mild, concentric LVH. In this minority, conventional echocardiographic parameters alone are insufficient to differentiate HCM from physiological LVH and should be complemented by additional structural and functional assessments to minimize the risk of false reassurance.
Hemodynamic Characteristics Including Pulmonary Hypertension at Rest and During Exercise Before and After Heart Transplantation
Authors: Lundgren Jakob , Rådegran Göran 
Published: July 2015

Background

Little is known about the hemodynamic response to exercise in heart failure patients at various ages before and after heart transplantation (
HT
). This information is important because postoperative hemodynamics may be a predictor of survival. To investigate the hemodynamic response to
HT
and exercise, we grouped our patients based on preoperative age and examined their hemodynamics at rest and during exercise before and after
HT
.

Methods and Results

Ninety‐four patients were evaluated at rest prior to HT with right heart catheterization at our laboratory. Of these patients, 32 were evaluated during slight supine exercise before and 1 year after
HT
. Postoperative evaluations were performed at rest 1 week after
HT
and at rest and during exercise at 4 weeks, 3 months, 6 months, and 1 year after
HT
. The exercise patients were divided into 2 groups based on preoperative age of ≤50 or >50 years. There were no age‐dependent differences in the preoperative hemodynamic exercise responses. Hemodynamics markedly improved at rest and during exercise at 1 and 4 weeks, respectively, after
HT
; however, pulmonary and, in particular, ventricular filling pressures remained high during exercise at 1 year after
HT
, resulting in normalized pulmonary vascular resistance response but deranged total pulmonary vascular resistance response.

Conclusions

Our findings suggest that, (1) in patients with heart failure age ≤50 or >50 years may not affect the hemodynamic response to exercise to the same extent as in healthy persons, and (2) total pulmonary vascular resistance may be more adequate than pulmonary vascular resistance for evaluating the exercise response after
HT
.
Nonalcoholic Fatty Liver Disease Is Associated With QT Prolongation in the General Population
Authors: Hung Chi‐Sheng , Tseng Ping‐Huei , Tu Chia‐Hung , Chen Chien‐Chuan , Liao Wei‐Chih , Lee Yi‐Chia , Chiu Han‐Mo , Lin Hung‐Ju , Ho Yi‐Lwun , Yang Wei‐Shiung , Wu Ming‐Shiang , Chen Ming‐Fong 
Published: July 2015

Background

Nonalcoholic fatty liver disease (
NAFLD
) is independently associated with
QT
prolongation among patients with diabetes. It has not yet been determined whether this association remains valid in the general population. We designed an observational study to explore this association.

Methods and Results

We conducted a cross‐sectional analysis of 31 116 consecutive participants in our health management program. Heart rate–corrected
QT
(
QT
c) interval was derived from 12‐lead electrocardiography and by Bazett's formula.
NAFLD
was diagnosed by abdominal ultrasonography and classified as
none, mild, moderate
, or
severe
, according to the ultrasonographic criteria. A multivariable linear regression model was fitted for the association between
QT
c interval and potential predictors (including demographic, anthropometric, biochemical factors, and comorbidities). Multivariable logistic regression analyses were fitted to assess the association between the severity of
NAFLD
and
QT
c prolongation, with the adjustment of significant predictors derived from multivariable linear regression. The mean
QT
c interval was 421.3 ms (
SD
45.4 ms). In the multivariable linear regression analyses, mild, moderate, and severe
NAFLD
were associated with increases of 2.55, 6.59, and 12.13 ms, respectively, in
QT
c interval compared with no
NAFLD
(all
P
<0.001). In the multivariable logistic regression analyses, mild, moderate, and severe
NAFLD
were associated with an increased risk for
QT
c prolongation, with odds ratios of 1.11 (95% CI: 1.01 to 1.21,
P
<0.05), 1.61 (95% CI: 1.36 to 1.9,
P
<0.001), and 1.31 (95% CI: 1.16 to 2.24,
P
<0.01), respectively, in women, and 1.11 (95% CI: 1.01 to 1.21,
P
<0.05), 1.39 (95% CI: 1.22 to 1.59,
P
<0.001), and 1.87 (95% CI: 1.16 to 2.24,
P
<0.001), respectively, in men, after adjusting for predictors known to be associated with the
QT
c interval. The association remained significant among subgroups with or without diabetes.

Conclusions

The severity of
NAFLD
was associated with a higher risk for
QT
c prolongation in the general population with and without diabetes.
Trends in Snow Cover and Related Quantities at Weather Stations in the Conterminous United States
Authors: Knowles Noah 
Published: July 2015

Abstract
Trend tests, linear regression, and canonical correlation analysis were used to quantify changes in National Weather Service Cooperative Observer (COOP) snow depth data and derived quantities, precipitation, snowfall, and temperature over the study period 1950–2010. Despite widespread warming, historical trends in snowfall and snow depth are generally mixed owing to competing influences of trends in precipitation. Trends toward later snow-cover onset in the western half of the conterminous United States and earlier onset in the eastern half and a widespread trend toward earlier final meltoff of snow cover combined to produce trends toward shorter snow seasons in the eastern half of the United States and in the west and longer snow seasons in the Great Plains and southern Rockies. The annual total number of days with snow cover exhibited a widespread decline. Monthly trend patterns show the dominant influence of temperature trends on occurrence of snow cover in the warmer snow-season months and a combination of temperature and precipitation trends in the colder months. A canonical correlation analysis indicated that most trends presented here took hold in the 1970s, consistent with the temporal pattern of global warming during the study period.