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1.  MRD assessed by WT1 and NPM1 transcript levels identifies distinct outcomes in AML patients and is influenced by gemtuzumab ozogamicin 
Oncotarget  2014;5(15):6280-6288.
We analysed the prognostic significance of minimal residual disease (MRD) level in adult patients with acute myeloid leukemia (AML) treated in the randomized gemtuzumab ozogamicin (GO) ALFA-0701 trial.
Levels of WT1 and NPM1 gene transcripts were assessed using cDNA-based real-time quantitative PCR in 183 patients with WT1 overexpression and in 77 patients with NMP1 mutation (NPM1mut) at diagnosis.
Positive WT1 MRD (defined as > 0.5% in the peripheral blood) after induction and at the end of treatment were both significantly associated with a higher risk of relapse and a shorter overall survival (OS). Positive NPM1mut MRD (defined as > 0.1% in the bone marrow) after induction and at the end of treatment also predicted a higher risk of relapse, but did not influence OS. Interestingly, the achievement of a negative NPM1mut MRD was significantly more frequent in patients treated in the GO arm compared to those treated in control arm (39% versus 7% (p=0.006) after induction and 91% versus 61% (p=0.028) at the end of treatment). However, GO did not influence WT1 MRD levels.
Our study supports the prognostic significance of MRD assessed by WT1 and NPM1mut transcript levels and show that NPM1 MRD is decreased by GO treatment.
PMCID: PMC4171629  PMID: 25026287
minimal residual disease; gemtuzumab ozogamicin; acute myeloid leukemia; WT1 expression; NPM1 mutation
2.  Sympathetic dysfunction in vasovagal syncope and the postural orthostatic tachycardia syndrome 
Orthostatic intolerance is the inability to tolerate the upright posture and is relieved by recumbence. It most commonly affects young women and has a major impact on quality of life and psychosocial well-being. Several forms of orthostatic intolerance have been described. The most common one is the recurrent vasovagal syncope (VVS) phenotype which presents as a transient and abrupt loss of consciousness and postural tone that is followed by rapid recovery. Another common type of orthostatic intolerance is the postural orthostatic tachycardia syndrome (POTS) which is characterized by an excessive rise in heart rate upon standing and is associated with symptoms of presyncope such as light-headedness, fatigue, palpitations, and nausea. Maintenance of arterial pressure under condition of reduced central blood volume during the orthostasis is accomplished in large part through sympathetic efferent nerve traffic to the peripheral vasculature. Therefore sympathetic nervous system (SNS) dysfunction is high on the list of possible contributors to the pathophysiology of orthostatic intolerance. Investigations into the role of the SNS in orthostatic intolerance have yielded mixed results. This review outlines the current knowledge of the function of the SNS in both VVS and POTS.
doi:10.3389/fphys.2014.00280
PMCID: PMC4112787  PMID: 25120493
syncope; postural tachycardia; orthostatic response; sympathetic nervous activity; tilt table
3.  Cognitive function, health-related quality of life, and symptoms of depression and anxiety sensitivity are impaired in patients with the postural orthostatic tachycardia syndrome (POTS) 
The Postural Orthostatic Tachycardia Syndrome (POTS) is a condition in which heart rate increases abnormally when the individual assumes an upright position. In addition to the marked tachycardia, presyncope, and syncope, patients with POTS often complain of light-headedness, fatigue, and difficulty in concentrating. The present study assessed individuals with POTS for psychiatric comorbidity, anxiety sensitivity and health related quality of life and examined general cognitive ability. Data was obtained from patients with POTS (n = 15, 12 female, aged 30 ± 3 years) and age matched healthy subjects (n = 30, 21 female, aged 32 ± 2 years). Patients with POTS commonly presented with symptoms of depression, elevated anxiety and increased anxiety sensitivity, particularly with regards to cardiac symptoms, and had a poorer health related quality of life in both the physical and mental health domains. While patients with POTS performed worse in tests of current intellectual functioning (verbal and non-verbal IQ) and in measures of focused attention (digits forward) and short term memory (digits back), test results were influenced largely by years of education and the underlying level of depression and anxiety. Acute changes in cognitive performance in response to head up tilt were evident in the POTS patients. From results obtained, it was concluded that participants with POTS have an increased prevalence of depression and higher levels of anxiety. These underlying symptoms impact on cognition in patients with POTS, particularly in the cognitive domains of attention and short-term memory. Our results indicate that psychological interventions may aid in recovery and facilitate uptake and adherence of other treatment modalities in patients with POTS.
doi:10.3389/fphys.2014.00230
PMCID: PMC4070177  PMID: 25009504
orthostatic intolerance; tachycardia; psychiatric comorbidity; sympathetic nervous system; noradrenaline transporter
4.  Grape Polyphenols Prevent Fructose-Induced Oxidative Stress and Insulin Resistance in First-Degree Relatives of Type 2 Diabetic Patients 
Diabetes Care  2013;36(6):1454-1461.
OBJECTIVE
To assess the clinical efficacy of nutritional amounts of grape polyphenols (PPs) in counteracting the metabolic alterations of high-fructose diet, including oxidative stress and insulin resistance (IR), in healthy volunteers with high metabolic risk.
RESEARCH DESIGN AND METHODS
Thirty-eight healthy overweight/obese first-degree relatives of type 2 diabetic patients (18 men and 20 women) were randomized in a double-blind controlled trial between a grape PP (2 g/day) and a placebo (PCB) group. Subjects were investigated at baseline and after 8 and 9 weeks of supplementation, the last 6 days of which they all received 3 g/kg fat-free mass/day of fructose. The primary end point was the protective effect of grape PPs on fructose-induced IR.
RESULTS
In the PCB group, fructose induced 1) a 20% decrease in hepatic insulin sensitivity index (P < 0.05) and an 11% decrease in glucose infusion rate (P < 0.05) as evaluated during a two-step hyperinsulinemic-euglycemic clamp, 2) an increase in systemic (urinary F2-isoprostanes) and muscle (thiobarbituric acid–reactive substances and protein carbonylation) oxidative stress (P < 0.05), and 3) a downregulation of mitochondrial genes and decreased mitochondrial respiration (P < 0.05). All the deleterious effects of fructose were fully blunted by grape PP supplementation. Antioxidative defenses, inflammatory markers, and main adipokines were affected neither by fructose nor by grape PPs.
CONCLUSIONS
A natural mixture of grape PPs at nutritional doses efficiently prevents fructose-induced oxidative stress and IR. The current interest in grape PP ingredients and products by the global food and nutrition industries could well make them a stepping-stone of preventive nutrition.
doi:10.2337/dc12-1652
PMCID: PMC3661802  PMID: 23275372
5.  Direct Evidences for Sympathetic Hyperactivity and Baroreflex Impairment in Tako Tsubo Cardiopathy 
PLoS ONE  2014;9(3):e93278.
Background
The exact pathophysiology of Tako-Tsubo cardiomyopathy (TTC) remains unknown but a role for sympathetic hyperactivity has been suggested. Up to now, no direct evidence of sympathetic nerve hyperactivity has been established nor involvement of sympathetic baroreflex identified. The aim of our study was to determine, by direct sympathetic nerve activity (SNS) recording if sympathetic nervous system activity is increased and spontaneous baroreflex control of sympathetic activity reduced in patients with TTC.
Methods
We included 13 patients who presented with TTC and compared their SNS activity and spontaneous baroreflex control of sympathetic activity with that of 13 control patients with acutely decompensated chronic heart failure. SNS activity was evaluated by microneurography, a technique assessing muscle sympathetic nerve activity (MSNA). Spontaneous baroreflex control of sympathetic activity was evaluated as the absolute value of the slope of the regression line representing the relationship between spontaneous diastolic blood pressure values and concomitant SNS activity. Control patients were matched for age, sex, left ventricular ejection fraction and creatinine clearance.
Results
The mean age of the patients with TTC was 80 years, all patients were women. There were no significant differences between the two groups of patients for blood pressure, heart rate or oxygen saturation level. TTC patients presented a significant increase in sympathetic nerve activity (MSNA median 63.3 bursts/min [interquartile range 61.3 to 66.0] vs median 55.7 bursts/min [interquartile range 51.0 to 61.7]; p = 0.0089) and a decrease in spontaneous baroreflex control of sympathetic activity compared to matched control patients (spontaneous baroreflex control of sympathetic activity median 0.7%burst/mmHg [interquartile range 0.4 to 1.9] vs median 2.4%burst/mmHg [interquartile range 1.8 to 2.9]; p = 0.005).
Conclusions
We report for the first time, through direct measurement of sympathetic nerve activity, that patients with TTC exhibit elevated SNS activity associated with a decrease in spontaneous baroreflex control of sympathetic activity. These data may explain the pathophysiology and clinical presentation of patient with TTC.
doi:10.1371/journal.pone.0093278
PMCID: PMC3965544  PMID: 24667435
7.  Predicting the Glycemic Response to Gastric Bypass Surgery in Patients With Type 2 Diabetes 
Diabetes Care  2012;36(1):20-26.
OBJECTIVE
To find clinically meaningful preoperative predictors of diabetes remission and conversely inadequate glycemic control after gastric bypass surgery. Predicting the improvement in glycemic control in those with type 2 diabetes after bariatric surgery may help in patient selection.
RESEARCH DESIGN AND METHODS
Preoperative details of 154 ethnic Chinese subjects with type 2 diabetes were examined for their influence on glycemic outcomes at 1 year after gastric bypass. Remission was defined as HbA1c ≤6%. Analysis involved binary logistic regression to identify predictors and provide regression equations and receiver operating characteristic curves to determine clinically useful cutoff values.
RESULTS
Remission was achieved in 107 subjects (69.5%) at 12 months. Diabetes duration <4 years, body mass >35 kg/m2, and fasting C-peptide concentration >2.9 ng/mL provided three independent preoperative predictors and three clinically useful cutoffs. The regression equation classification plot derived from continuous data correctly assigned 84% of participants. A combination of two or three of these predictors allows a sensitivity of 82% and specificity of 87% for remission. Duration of diabetes (with different cutoff points) and C-peptide also predicted those cases in which HbA1c ≤7% was not attained. Percentage weight loss after surgery was also predictive of remission and of less satisfactory outcomes.
CONCLUSIONS
The glycemic response to gastric bypass is related to BMI, duration of diabetes, fasting C-peptide (influenced by insulin resistance and residual β-cell function), and weight loss. These data support and refine previous findings in non-Asian populations. Specific ethnic and procedural regression equations and cutoff points may vary.
doi:10.2337/dc12-0779
PMCID: PMC3526207  PMID: 23033249
8.  Neuroadrenergic Dysfunction Along the Diabetes Continuum 
Diabetes  2012;61(10):2506-2516.
Neuroadrenergic function in type 2 diabetic (T2D) patients without neuropathy is poorly characterized. We therefore compared sympathetic nervous system activity at rest and during an oral glucose tolerance test in obese metabolic syndrome (MetS) subjects classified as glucose intolerant (impaired glucose tolerance [IGT]; n = 17) or treatment-naive T2D (n = 17). Untreated subjects, matched for age (mean 59 ± 1 year), sex, BMI (32.4 ± 0.6 kg/m2), and family history of diabetes were studied. We measured resting muscle sympathetic nerve activity (MSNA) by microneurography, whole-body norepinephrine kinetics by isotope dilution, insulin sensitivity by euglycemic-hyperinsulinemic clamp (steady-state glucose utilization adjusted for fat-free mass and steady-state insulin concentration [M/I]), and MetS components. T2D subjects had higher resting MSNA burst incidence (67 ± 4 versus 55 ± 3 bursts per 100 heartbeats; P = 0.05) and arterial norepinephrine levels (264 ± 33 versus 167 ± 16 pg/mL; P = 0.02), lower plasma norepinephrine clearance (by 17%; P = 0.03), and reduced neuronal reuptake compared with IGT subjects (by 46%; P = 0.04). Moreover, norepinephrine spillover responses to glucose ingestion were blunted in T2D subjects. The M/I value independently predicted whole-body norepinephrine spillover (r = −0.47; P = 0.008), whereas fasting insulin level related to neuronal norepinephrine reuptake (r = −0.35, P = 0.047). These findings demonstrate that progression to T2D is associated with increased central sympathetic drive, blunted sympathetic responsiveness, and altered norepinephrine disposition.
doi:10.2337/db12-0138
PMCID: PMC3447913  PMID: 22664956
9.  General practitioners' management of mental disorders: A rewarding practice with considerable obstacles 
BMC Family Practice  2012;13:19.
Background
Primary care improvement is the cornerstone of current reforms. Mental disorders (MDs) are a leading cause of morbidity worldwide and widespread in industrialised countries. MDs are treated mainly in primary care by general practitioners (GPs), even though the latter ability to detect, diagnose, and treat patients with MDs is often considered unsatisfactory. This article examines GPs' management of MDs in an effort to acquire more information regarding means by which GPs deal with MD cases, impact of such cases on their practices, factors that enable or hinder MD management, and patient-management strategies.
Methods
This study employs a mixed-method approach with emphasis on qualitative investigation. Based on a previous survey of 398 GPs in Quebec, Canada, 60 GPs representing a variety of practice settings were selected for further study. A 10-minute-long questionnaire comprising 27 items was administered, and 70-minute-long interviews were conducted. Quantitative (SPSS) and qualitative (NVivo) analyses were performed.
Results
At least 20% of GP visits were MD-related. GPs were comfortable managing common MDs, but not serious MDs. GPs' based their treatment of MDs on pharmacotherapy, support therapy, and psycho-education. They used clinical intuition with few clinical tools, and closely followed their patients with MDs. Practice features (salary or hourly fees payment; psycho-social teams on-site; strong informal networks), and GPs' individual characteristics (continuing medical education; exposure and interest in MDs; traits like empathy) favoured MD management. Collaboration with psychologists and psychiatrists was considered key to good MD management. Limited access to specialists, system fragmentation, and underdeveloped group practice and shared-care models were impediments. MD management was seen as burdensome because it required more time, flexibility, and emotional investment. Strategies exist to reduce the burden (one-problem-per-visit rule; longer time slots). GPs found MD practice rewarding as patients were seen as grateful and more complying with medical recommendations compared to other patients, generally leading to positive outcomes.
Conclusions
To improve MD management, this study highlights the importance of extending multidisciplinary GP practice settings with salary or hourly fee payment; access to psychotherapeutic and psychiatric expertise; and case-discussion training involving local networks of GPs and MD specialists that encourage both knowledge transfer and shared care.
doi:10.1186/1471-2296-13-19
PMCID: PMC3355055  PMID: 22423592
10.  Advances in Sympathetic Nerve Recording in Humans 
In humans, sympathetic activity is commonly assessed by measuring the efferent traffic in the peroneal nerve. The firing activity is the sum of several active neurons, which have the tendency to fire together in a bursting manner. While the estimation of overall sympathetic nervous activity using this multiunit recording approach has advanced our understanding of sympathetic regulation in health and disease no information is gained regarding the underling mechanisms generating the bursts of sympathetic activity. The introduction of single-unit recording has been a major step forward, enabling the examination of specific sympathetic firing patterns in diverse clinical conditions. Disturbances in sympathetic nerve firing, including high firing probabilities, high firing rates or high incidence of multiple firing, or a combination of both may impact on noradrenaline release and effector response, and therefore have clinical implications with regards to the development and progression of target organ damage. Understanding the mechanisms and consequences of specific firing patterns would permit the development of therapeutic strategies targeting these nuances of sympathetic overdrive.
doi:10.3389/fphys.2012.00011
PMCID: PMC3273724  PMID: 22347191
sympathetic nervous system; microneurography; cardiovascular disease
11.  Effects of Renal Denervation on Sympathetic Activation, Blood Pressure, and Glucose Metabolism in Patients with Resistant Hypertension 
Increased central sympathetic drive is a hallmark of several important clinical conditions including essential hypertension, heart failure, chronic kidney disease, and insulin resistance. Afferent signaling from the kidneys has been identified as an important contributor to elevated central sympathetic drive and increased sympathetic outflow to the kidney and other organs is crucially involved in cardiovascular control. While the resultant effects on renal hemodynamic parameters, sodium and water retention, and renin release are particularly relevant for both acute and long term regulation of blood pressure, increased sympathetic outflow to other vascular beds may facilitate further adverse consequences of sustained sympathetic activation such as insulin resistance, which is commonly associated with hypertension. Recent clinical studies using catheter-based radiofrequency ablation technology to achieve functional renal denervation in patients with resistant hypertension have identified the renal nerves as therapeutic target and have helped to further expose the sympathetic link between hypertension and insulin resistance. Initial data from two clinical trials and several smaller mechanistic clinical studies indicate that this novel approach may indeed provide a safe and effective treatment alternative for resistant hypertension and some of its adverse consequences.
doi:10.3389/fphys.2012.00010
PMCID: PMC3270497  PMID: 22347190
renal denervation; hypertension; sympathetic
12.  Change in Sympathetic Nerve Firing Pattern Associated with Dietary Weight Loss in the Metabolic Syndrome 
Sympathetic activation in subjects with the metabolic syndrome (MS) plays a role in the pathogenesis of cardiovascular disease development. Diet-induced weight loss decreases sympathetic outflow. However the mechanisms that account for sympathetic inhibition are not known. We sought to provide a detailed description of the sympathetic response to diet by analyzing the firing behavior of single-unit sympathetic nerve fibers. Fourteen subjects (57 ± 2 years, nine men, five females) fulfilling ATP III criteria for the MS underwent a 3-month low calorie diet. Metabolic profile, hemodynamic parameters, and multi-unit and single-unit muscle sympathetic nerve activity (MSNA, microneurography) were assessed prior to and at the end of the diet. Patients’ weight dropped from 96 ± 4 to 88 ± 3 kg (P < 0.001). This was associated with a decrease in systolic and diastolic blood pressure (−12 ± 3 and −5 ± 2 mmHg, P < 0.05), and in heart rate (−7 ± 2 bpm, P < 0.01) and an improvement in all metabolic parameters (fasting glucose: −0.302.1 ± 0.118 mmol/l, total cholesterol: −0.564 ± 0.164 mmol/l, triglycerides: −0.414 ± 0.137 mmol/l, P < 0.05). Multi-unit MSNA decreased from 68 ± 4 to 59 ± 5 bursts/100 heartbeats (P < 0.05). Single-unit MSNA indicated that the firing rate of individual vasoconstrictor fibers decreased from 59 ± 10 to 32 ± 4 spikes/100 heart beats (P < 0.05). The probability of firing decreased from 34 ± 5 to 23 ± 3% of heartbeats (P < 0.05), and the incidence of multiple firing decreased from 14 ± 4 to 6 ± 1% of heartbeats (P < 0.05). Cardiac and sympathetic baroreflex function were significantly improved (cardiac slope: 6.57 ± 0.69 to 9.57 ± 1.20 ms·mmHg−1; sympathetic slope: −3.86 ± 0.34 to −5.05 ± 0.47 bursts/100 heartbeats·mmHg−1, P < 0.05 for both). Hypocaloric diet decreased sympathetic activity and improved hemodynamic and metabolic parameters. The sympathoinhibition associated with weight loss involves marked changes, not only in the rate but also in the firing pattern of active vasoconstrictive fibers.
doi:10.3389/fphys.2011.00052
PMCID: PMC3162210  PMID: 21904529
metabolic syndrome; sympathetic nervous system; autonomic function; diet
13.  Sympathetic Neural Adaptation to Hypocaloric Diet With or Without Exercise Training in Obese Metabolic Syndrome Subjects 
Diabetes  2009;59(1):71-79.
OBJECTIVE
Sympathetic nervous system (SNS) overactivity contributes to the pathogenesis and target organ complications of obesity. This study was conducted to examine the effects of lifestyle interventions (weight loss alone or together with exercise) on SNS function.
RESEARCH DESIGN AND METHODS
Untreated men and women (mean age 55 ± 1 year; BMI 32.3 ± 0.5 kg/m2) who fulfilled Adult Treatment Panel III metabolic syndrome criteria were randomly allocated to either dietary weight loss (WL, n = 20), dietary weight loss and moderate-intensity aerobic exercise (WL+EX, n = 20), or no treatment (control, n = 19). Whole-body norepinephrine kinetics, muscle sympathetic nerve activity by microneurography, baroreflex sensitivity, fitness (maximal oxygen consumption), metabolic, and anthropometric measurements were made at baseline and 12 weeks.
RESULTS
Body weight decreased by −7.1 ± 0.6 and −8.4 ± 1.0 kg in the WL and WL+EX groups, respectively (both P < 0.001). Fitness increased by 19 ± 4% (P < 0.001) in the WL+EX group only. Resting SNS activity decreased similarly in the WL and WL+EX groups: norepinephrine spillover by −96 ± 30 and −101 ± 34 ng/min (both P < 0.01) and muscle sympathetic nerve activity by −12 ± 6 and −19 ± 4 bursts/100 heart beats, respectively (both P < 0.01), but remained unchanged in control subjects. Blood pressure, baroreflex sensitivity, and metabolic parameters improved significantly and similarly in the two lifestyle intervention groups.
CONCLUSIONS
The addition of moderate-intensity aerobic exercise training to a weight loss program does not confer additional benefits on resting SNS activity. This suggests that weight loss is the prime mover in sympathetic neural adaptation to a hypocaloric diet.
doi:10.2337/db09-0934
PMCID: PMC2797947  PMID: 19833893
14.  Weight Loss May Reverse Blunted Sympathetic Neural Responsiveness to Glucose Ingestion in Obese Subjects With Metabolic Syndrome 
Diabetes  2009;58(5):1126-1132.
OBJECTIVE
The purpose of this study was to examine the effects of weight loss on sympathetic nervous system responsiveness to glucose ingestion in obese subjects with metabolic syndrome, in whom such responses are reportedly blunted.
RESEARCH DESIGN AND METHODS
Thirty four subjects, 19 insulin resistant and 15 insulin sensitive and aged 55 ± 1 years (mean ± SE) with BMI 31.6 ± 0.6 kg/m2, who fulfilled the Adult Treatment Panel III criteria for metabolic syndrome participated. Simultaneous measurements of whole-body norepinephrine spillover rate, calf blood flow, and intra-arterial blood pressure were made at times 0, 30, 60, 90, and 120 min postglucose (75 g). The experiment was repeated after a 3-month hypocaloric diet with or without an exercise program.
RESULTS
Body weight decreased by 8.1 ± 0.9 and 8.4 ± 1.1 kg and resting norepinephrine spillover by 94 ± 31 and 166 ± 58 ng/min (all P ≤ 0.01) in insulin-resistant and insulin-sensitive subjects, respectively. Weight loss was accompanied by a marked increase in sympathetic responsiveness after glucose but only in insulin-resistant subjects. In this subgroup, comparative increases in norepinephrine spillover rates at baseline and after weight loss averaged −3 ± 25 versus 73 ± 24 ng/min at 30 min (P = 0.039), 36 ± 21 versus 115 ± 28 ng/min at 60 min (P = 0.045), 9 ± 21 versus 179 ± 50 ng/min at 90 min (P < 0.001), and 40 ± 48 versus 106 ± 39 ng/min at 120 min (P = 0.24).
CONCLUSIONS
Weight loss reverses blunted sympathetic responsiveness to glucose ingestion in insulin-resistant subjects with metabolic syndrome, which is relevant to postprandial energy utilization and body weight homeostasis.
doi:10.2337/db08-1595
PMCID: PMC2671050  PMID: 19188428
15.  Autophagy: a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model 
Cancer research  2008;68(19):7966-7974.
Doxorubicin is a genotoxic chemotherapy agent used in treatment of a wide variety of cancers. Significant clinical side-effects, including cardiac toxicity and myelosuppression, severely limit the therapeutic index of this commonly used agent and methods which improve doxorubicin efficacy could benefit many patients. Because doxorubicin cytotoxicity is cell cycle specific, the cell cycle is a rational target to enhance its efficacy.
We examined the direct, cyclin-dependent kinase inhibitor, roscovitine, as a means of enhancing doxorubicin cytotoxicity. This study demonstrated synergistic cytotoxicity between doxorubicin and roscovitine in three sarcoma cell lines: SW-982 (synovial sarcoma); U2OS-LC3-GFP (osteosarcoma) and SK-LMS-1 (uterine leiomyosarcoma), but not the fibroblast cell line, WI38. The combined treatment of doxorubicin and roscovitine was associated with a prolonged G2/M cell cycle arrest in the three sarcoma cell lines. Using three different methods for detecting apoptosis, our results revealed that apoptotic cell death did not account for the synergistic cytotoxicity between doxorubicin and roscovitine. However, morphologic changes observed by light microscopy and increased cytoplasmic LC3-GFP puncta in U20S-LC3-GFP cells after the combined treatment suggested the induction of autophagy. Induction of autophagy was also demonstrated in SW-982 and SK-LMS-1 cells treated with both doxorubicin and roscovitine by acridine orange staining. These results suggest a novel role of autophagy in the enhanced cytotoxicity by cell cycle inhibition following genotoxic injury in tumor cells. Further investigation of this enhanced cytotoxicity as a treatment strategy for sarcomas is warranted.
doi:10.1158/0008-5472.CAN-08-1333
PMCID: PMC2561224  PMID: 18829554
roscovitine; doxorubicin; autophagy; synergy; cell cycle
16.  Incidence, Risk Factors, and Impact of Severe Neutropenia After Hyperthermic Intraperitoneal Mitomycin C 
Annals of Surgical Oncology  2009;16(8):2181-2187.
Background
Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are considered the standard of care for patients with peritoneal dissemination of appendiceal cancer and are increasingly being evaluated for use in patients with carcinomatosis from colon cancer. Mitomycin C (MMC) is one of the most frequently used HIPEC agents in the management of peritoneal-based gastrointestinal malignancies. This study analyzes the incidence and risk factors for developing neutropenia following MMC-HIPEC combined with CRS.
Methods
All patients undergoing CRS and MMC-HIPEC for appendiceal cancer between January 1993 and October 2006 were retrospectively reviewed. Logistic regression was used to identify risk factors for the development of neutropenia, defined as an absolute neutrophil count (ANC) <1,000/mm3.
Results
One hundred and twenty MMC-HIPEC were performed in 117 patients with appendiceal cancer. The incidence of neutropenia was 39%. Neutropenia occurred in 57.6% of female and 21.3% of male patients (p < 0.0001). Female gender and MMC dose per body surface area (BSA) were independent risk factors for neutropenia on multivariable logistic regression [odds ratio (OR) of neutropenia in females = 3.58 (95% confidence interval, CI: 1.52, 8.43); OR for 5 unit (mg/m2) increase in MMC dose per BSA = 3.37 (95% CI: 1.72, 6.63)]. Neutropenia did not increase the risk of mortality, postoperative infection or length of hospital stay.
Conclusion
Neutropenia is a frequent complication associated with MMC-HIPEC. Female sex and MMC dose per BSA are independent risk factors for neutropenia. These differences must be considered in the management of patients undergoing MMC-HIPEC to minimize the toxicity of the procedure.
doi:10.1245/s10434-009-0523-4
PMCID: PMC2711905  PMID: 19475451
17.  Viricidal Capability of Resin-Triiodide Demand-Type Disinfectant † 
Polyoma, Newcastle disease virus, and adenovirus, as well as two coliphages, lambda and T4, were inactivated by strong base quaternary ammonium anion-exchange resin-triiodide. Organic matter interfered with viral inactivation capability of the resin-triiodide. The viruses, as they were being inactivated by the resin disinfectant beads, were not retained or filtered by the beads.
PMCID: PMC242198  PMID: 6297389
18.  Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern 
Developmental biology  2013;386(2):428-439.
Transient receptor potential, melastatin-like 7 (Trpm7) is a combined ion channel and kinase implicated in the differentiation or function of many cell types. Early lethality in mice and frogs depleted of the corresponding gene impedes investigation of the functions of this protein particularly during later stages of development. By contrast, zebrafish trpm7 mutant larvae undergo early morphogenesis normally and thus do not have this limitation. The mutant larvae are characterized by multiple defects including melanocyte cell death, transient paralysis, and an ion imbalance that leads to the development of kidney stones. Here we report a requirement for Trpm7 in differentiation or function of dopaminergic neurons in vivo. First, trpm7 mutant larvae are hypomotile and fail to make a dopamine-dependent developmental transition in swim-bout length. Both of these deficits are partially rescued by the application of levodopa or dopamine. Second, histological analysis reveals that in trpm7 mutants a significant fraction of dopaminergic neurons lack expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Third, trpm7 mutants are unusually sensitive to the neurotoxin 1-methyl-4-phenylpyridinium, an oxidative stressor, and their motility is partially rescued by application of the iron chelator deferoxamine, an anti-oxidant. Finally, in SH-SY5Y cells, which model aspects of human dopaminergic neurons, forced expression of a channel-dead variant of TRPM7 causes cell death. In summary, a forward genetic screen in zebrafish has revealed that both melanocytes and dopaminergic neurons depend on the ion channel Trpm7. The mechanistic underpinning of this dependence requires further investigation.
doi:10.1016/j.ydbio.2013.11.015
PMCID: PMC3971878  PMID: 24291744
Parkinson’s; Zebrafish; Dopamine; TRPM7
19.  Impact of tumor location and pathological discordance on survival of children with midline high-grade gliomas treated on Children’s Cancer Group high-grade glioma study CCG-945 
Journal of neuro-oncology  2014;121(3):573-581.
Children with high-grade glioma (HGG) have a poor prognosis compared to those with low-grade glioma (LGG). Adjuvant chemotherapy may be beneficial, but its optimal use remains undetermined. Histology and extent of resection are important prognostic factors. We tested the hypothesis that patients with midline HGG treated on Children’s Cancer Group Study (CCG) CCG-945 have a worse prognosis compared to the entire group. Of 172 children eligible for analysis, 60 had midline tumors primarily localized to the thalamus, hypothalamus and basal ganglia. Time-to-progression and death were determined from the date of initial diagnosis, and survival curves were calculated. Univariate analyses were undertaken for extent of resection, chemotherapy regimen, anatomic location, histology, proliferation index, MGMT status and p53 over-expression. For the entire midline tumor group, 5-year PFS and OS were 18.3 ± 4.8 and 25 ± 5.4 %, respectively. Many patients only had a biopsy (43.3 %). The sub-groups with near/total resection and hypothalamic location appeared to have better PFS and OS. However, the effect of tumor histology on OS was significant for children with discordant diagnoses on central pathology review of LGG compared to HGG. Proliferative index (MIB-1 > 36 %), MGMT and p53 over-expression correlated with poor outcomes. Children treated on CCG-945 with midline HGG have a worse prognosis when compared to the entire group. The midline location may directly influence the extent of resection. Central pathology review and entry of patients on clinical trials continue to be priorities to improve outcomes for children with HGG.
doi:10.1007/s11060-014-1669-x
PMCID: PMC4323766  PMID: 25431150
Childhood malignant gliomas; Anaplastic astrocytoma; Glioblastoma multiforme; Chemotherapy; Midline tumors; Thalamus; Hypothalamus; Basal ganglia
20.  Improving wear time compliance with a 24-hour waist-worn accelerometer protocol in the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE) 
Background
We compared 24-hour waist-worn accelerometer wear time characteristics of 9–11 year old children in the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE) to similarly aged U.S. children providing waking-hours waist-worn accelerometer data in the 2003–2006 National Health and Nutrition Examination Survey (NHANES).
Methods
Valid cases were defined as having ≥4 days with ≥10 hours of waking wear time in a 24-hour period, including one weekend day. Previously published algorithms for extracting total sleep episode time from 24-hour accelerometer data and for identifying wear time (in both the 24-hour and waking-hours protocols) were applied. The number of valid days obtained and a ratio (percent) of valid cases to the number of participants originally wearing an accelerometer were computed for both ISCOLE and NHANES. Given the two surveys’ discrepant sampling designs, wear time (minutes/day, hours/day) from U.S. ISCOLE was compared to NHANES using a meta-analytic approach. Wear time for the 11 additional countries participating in ISCOLE were graphically compared with NHANES.
Results
491 U.S. ISCOLE children (9.92±0.03 years of age [M±SE]) and 586 NHANES children (10.43 ± 0.04 years of age) were deemed valid cases. The ratio of valid cases to the number of participants originally wearing an accelerometer was 76.7% in U.S. ISCOLE and 62.6% in NHANES. Wear time averaged 1357.0 ± 4.2 minutes per 24-hour day in ISCOLE. Waking wear time was 884.4 ± 2.2 minutes/day for U.S. ISCOLE children and 822.6 ± 4.3 minutes/day in NHANES children (difference = 61.8 minutes/day, p < 0.001). Wear time characteristics were consistently higher in all ISCOLE study sites compared to the NHANES protocol.
Conclusions
A 24-hour waist-worn accelerometry protocol implemented in U.S. children produced 22.6 out of 24 hours of possible wear time, and 61.8 more minutes/day of waking wear time than a similarly implemented and processed waking wear time waist-worn accelerometry protocol. Consistent results were obtained internationally. The 24-hour protocol may produce an important increase in wear time compliance that also provides an opportunity to study the total sleep episode time separate and distinct from physical activity and sedentary time detected during waking-hours.
Trial registration
ClinicalTrials.gov NCT01722500.
Electronic supplementary material
The online version of this article (doi:10.1186/s12966-015-0172-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12966-015-0172-x
PMCID: PMC4328595
Accelerometry; Measurement; Physical activity; Exercise; Sedentary time
21.  Hepatocyte Nuclear Factor 4 Alpha Polymorphisms and the Metabolic Syndrome in French-Canadian Youth 
PLoS ONE  2015;10(2):e0117238.
Objectives
Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor involved in the regulation of serum glucose and lipid levels. Several HNF4A gene variants have been associated with the risk of developing type 2 diabetes mellitus. However, no study has yet explored its association with insulin resistance and the cardiometabolic risk in children. We aimed to investigate the relationship between HNF4A genetic variants and the presence of metabolic syndrome (MetS) and metabolic parameters in a pediatric population.
Design and Methods
Our study included 1,749 French-Canadians aged 9, 13 and 16 years and evaluated 24 HNF4A polymorphisms that were previously identified by sequencing.
Results
Analyses revealed that, after correction for multiple testing, one SNP (rs736824; P<0.022) and two haplotypes (P1 promoter haplotype rs6130608-rs2425637; P<0.032 and intronic haplotype rs736824-rs745975-rs3212183; P<0.025) were associated with the risk of MetS. Additionally, a significant association was found between rs3212172 and apolipoprotein B levels (coefficient: -0.14 ± 0.05; P<0.022). These polymorphisms are located in HNF4A P1 promoter or in intronic regions.
Conclusions
Our study demonstrates that HNF4α genetic variants are associated with the MetS and metabolic parameters in French Canadian children and adolescents. This study, the first exploring the relation between HNF4A genetic variants and MetS and metabolic variables in a pediatric cohort, suggests that HNF4α could represent an early marker for the risk of developing type 2 diabetes mellitus.
doi:10.1371/journal.pone.0117238
PMCID: PMC4325000  PMID: 25671620
22.  Generation of BAC Transgenic Tadpoles Enabling Live Imaging of Motoneurons by Using the Urotensin II-Related Peptide (ust2b) Gene as a Driver 
PLoS ONE  2015;10(2):e0117370.
Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP+ cells in the larvae. More than 98% of the GFP+ cells expressed choline acetyltransferase, while their projections were co-localized with α-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP+ cells were observed. In addition, intracellular electrophysiological recordings of GFP+ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord.
doi:10.1371/journal.pone.0117370
PMCID: PMC4319907  PMID: 25658845
23.  Structural requirements of steroidal agonists of transient receptor potential melastatin 3 (TRPM3) cation channels 
British Journal of Pharmacology  2014;171(4):1019-1032.
Background and Purpose
Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation.
Experimental Approach
Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances.
Key Results
Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels.
Conclusions and Implications
Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.
doi:10.1111/bph.12521
PMCID: PMC3925040  PMID: 24251620
cation membrane channel; ClC-3; dihydropyridine; enantiomer; neurosteroid; pregnenolone sulphate; proton-activated outwardly rectifying anion channel; PAORAC; transient receptor potential; transient receptor potential melastatin
24.  Pharmacological Study of Cefoxitin as an Alternative Antibiotic Therapy to Carbapenems in Treatment of Urinary Tract Infections Due to Extended-Spectrum-β-Lactamase-Producing Escherichia coli 
Cefoxitin could be an alternative to carbapenems in extended-spectrum-beta-lactamase-producing Escherichia coli (ESBL-EC) infections. However, pharmacological and clinical data regarding cefoxitin are limited. Using a recent pharmacological model and the MICs of ESBL-EC collected from pyelonephritis, we determined the probabilities to reach four pharmacological targets: free cefoxitin concentrations above the MIC during 50% and 100% of the administration interval (T>MIC = 50% and T>MIC = 100%, respectively) and free cefoxitin concentrations above 4× MIC during 50% and 100% of the administration interval (T>4MIC = 50% and T>4MIC = 100%, respectively). Cefoxitin could be used to treat ESBL-EC pyelonephritis, but administration modalities should be optimized according to MICs in order to reach pharmacological targets.
doi:10.1128/AAC.02509-14
PMCID: PMC4135984  PMID: 24777104
25.  Acquisition of Human Polyomaviruses in the First 18 Months of Life 
Emerging Infectious Diseases  2015;21(2):365-367.
We investigated the presence of 4 human polyomaviruses (PyVs) (WU, KI, Merkel cell, and Malawi) in respiratory specimens from a community-based birth cohort. These viruses typically were acquired when children were ≈1 year of age. We provide evidence that WU, KI, and Malawi, but not Merkel cell PyVs, might have a role in respiratory infections.
doi:10.3201/eid2102.141429
PMCID: PMC4313659  PMID: 25626138
polyomavirus; viruses; WU polyomavirus; KI polyomavirus; Malawi polyomavirus; Merkel cell polyomavirus; PCR; infectious diseases; community-based study; longitudinal birth cohort

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