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1.  Reversible Antibiotic Tolerance Induced in Staphylococcus aureus by Concurrent Drug Exposure 
mBio  2015;6(1):e02268-14.
ABSTRACT  
Resistance of Staphylococcus aureus to beta-lactam antibiotics has led to increasing use of the glycopeptide antibiotic vancomycin as a life-saving treatment for major S. aureus infections. Coinfection by an unrelated bacterial species may necessitate concurrent treatment with a second antibiotic that targets the coinfecting pathogen. While investigating factors that affect bacterial antibiotic sensitivity, we discovered that susceptibility of S. aureus to vancomycin is reduced by concurrent exposure to colistin, a cationic peptide antimicrobial employed to treat infections by Gram-negative pathogens. We show that colistin-induced vancomycin tolerance persists only as long as the inducer is present and is accompanied by gene expression changes similar to those resulting from mutations that produce stably inherited reduction of vancomycin sensitivity (vancomycin-intermediate S. aureus [VISA] strains). As colistin-induced vancomycin tolerance is reversible, it may not be detected by routine sensitivity testing and may be responsible for treatment failure at vancomycin doses expected to be clinically effective based on such routine testing.
Importance   Commonly, antibiotic resistance is associated with permanent genetic changes, such as point mutations or acquisition of resistance genes. We show that phenotypic resistance can arise where changes in gene expression result in tolerance to an antibiotic without any accompanying genetic changes. Specifically, methicillin-resistant Staphylococcus aureus (MRSA) behaves like vancomycin-intermediate S. aureus (VISA) upon exposure to colistin, which is currently used against infections by Gram-negative bacteria. Vancomycin is a last-resort drug for treatment of serious S. aureus infections, and VISA is associated with poor clinical prognosis. Phenotypic and reversible resistance will not be revealed by standard susceptibility testing and may underlie treatment failure.
Importance  
Commonly, antibiotic resistance is associated with permanent genetic changes, such as point mutations or acquisition of resistance genes. We show that phenotypic resistance can arise where changes in gene expression result in tolerance to an antibiotic without any accompanying genetic changes. Specifically, methicillin-resistant Staphylococcus aureus (MRSA) behaves like vancomycin-intermediate S. aureus (VISA) upon exposure to colistin, which is currently used against infections by Gram-negative bacteria. Vancomycin is a last-resort drug for treatment of serious S. aureus infections, and VISA is associated with poor clinical prognosis. Phenotypic and reversible resistance will not be revealed by standard susceptibility testing and may underlie treatment failure.
doi:10.1128/mBio.02268-14
PMCID: PMC4313918  PMID: 25587013
2.  Increased Persistent Sodium Current Due to Decreased PI3K Signaling Contributes to QT Prolongation in the Diabetic Heart 
Diabetes  2013;62(12):4257-4265.
Diabetes is an independent risk factor for sudden cardiac death and ventricular arrhythmia complications of acute coronary syndrome. Prolongation of the QT interval on the electrocardiogram is also a risk factor for arrhythmias and sudden death, and the increased prevalence of QT prolongation is an independent risk factor for cardiovascular death in diabetic patients. The pathophysiological mechanisms responsible for this lethal complication are poorly understood. Diabetes is associated with a reduction in phosphoinositide 3-kinase (PI3K) signaling, which regulates the action potential duration (APD) of individual myocytes and thus the QT interval by altering multiple ion currents, including the persistent sodium current INaP. Here, we report a mechanism for diabetes-induced QT prolongation that involves an increase in INaP caused by defective PI3K signaling. Cardiac myocytes of mice with type 1 or type 2 diabetes exhibited an increase in APD that was reversed by expression of constitutively active PI3K or intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K. The diabetic myocytes also showed an increase in INaP that was reversed by activated PI3K or PIP3. The increases in APD and INaP in myocytes translated into QT interval prolongation for both types of diabetic mice. The long QT interval of type 1 diabetic hearts was shortened by insulin treatment ex vivo, and this effect was blocked by a PI3K inhibitor. Treatment of both types of diabetic mouse hearts with an INaP blocker also shortened the QT interval. These results indicate that downregulation of cardiac PI3K signaling in diabetes prolongs the QT interval at least in part by causing an increase in INaP. This mechanism may explain why the diabetic population has an increased risk of life-threatening arrhythmias.
doi:10.2337/db13-0420
PMCID: PMC3837031  PMID: 23974924
3.  High Frequency Oscillation and Airway Pressure Release Ventilation in Pediatric Respiratory Failure 
Pediatric pulmonology  2013;49(7):707-715.
Summary
Background
Airway pressure release ventilation (APRV) and high frequency oscillatory ventilation (HFOV) are frequently used in acute lung injury (ALI) refractory to conventional ventilation. Our aim was to describe our experience with APRV and HFOV in refractory pediatric ALI, and to identify factors associated with survival.
Methods
We analyzed 104 patients with hypoxemia refractory to conventional ventilation transitioned to either APRV or HFOV. Demographics, oxygenation index (OI), and PaO2/FiO2 (PF ratio) were recorded before transition to either mode of nonconventional ventilation (NCV) and for every 12 hr after transition.
Results
Relative to APRV, patients on HFOV were younger and had more significant lung disease evidenced by higher OI (28.5 [18.6, 36.2] vs. 21.0 [15.5, 30.0], P = 0.008), lower PF ratios (73 [59,94] vs. 99 [76,131], P = 0.002), and more frequent use of inhaled nitric oxide. In univariate analysis, HFOV was associated with more frequent neuromuscular blockade. Forty-one of 104 patients died on NCV (39.4%). Survivors demonstrated improvement in OI 24 hr after transition to NCV, whereas non-survivors did not (12.9 [8.9, 20.9] vs. 28.1 [17.6, 37.1], P < 0.001). After controlling for immunocompromised status, number of vasopressors, and OI before transition, mode of NCV was not associated with mortality.
Conclusions
In a heterogeneous PICU population with hypoxemia refractory to conventional ventilation transitioned to NCV, improvement in oxygenation at 24 hr was associated with survival. Immunocompromised status, number of vasopressor infusions, and the OI before transition to NCV were independently associated with survival.
doi:10.1002/ppul.22853
PMCID: PMC4092114  PMID: 23853049
mechanical ventilation; high frequency oscillatory ventilation; airway pressure release ventilation; acute respiratory distress syndrome; acute lung injury; pediatric
4.  Calpain‐Dependent Cleavage of Junctophilin‐2 and T‐Tubule Remodeling in a Mouse Model of Reversible Heart Failure 
Background
A highly organized transverse tubule (T‐tubule) network is necessary for efficient Ca2+‐induced Ca2+ release and synchronized contraction of ventricular myocytes. Increasing evidence suggests that T‐tubule remodeling due to junctophilin‐2 (JP‐2) downregulation plays a critical role in the progression of heart failure. However, the mechanisms underlying JP‐2 dysregulation remain incompletely understood.
Methods and Results
A mouse model of reversible heart failure that is driven by conditional activation of the heterotrimeric G protein Gαq in cardiac myocytes was used in this study. Mice with activated Gαq exhibited disruption of the T‐tubule network and defects in Ca2+ handling that culminated in heart failure compared with wild‐type mice. Activation of Gαq/phospholipase Cβ signaling increased the activity of the Ca2+‐dependent protease calpain, leading to the proteolytic cleavage of JP‐2. A novel calpain cleavage fragment of JP‐2 is detected only in hearts with constitutive Gαq signaling to phospholipase Cβ. Termination of the Gαq signal was followed by normalization of the JP‐2 protein level, repair of the T‐tubule network, improvements in Ca2+ handling, and reversal of heart failure. Treatment of mice with a calpain inhibitor prevented Gαq‐dependent JP‐2 cleavage, T‐tubule disruption, and the development of heart failure.
Conclusions
Disruption of the T‐tubule network in heart failure is a reversible process. Gαq‐dependent activation of calpain and subsequent proteolysis of JP‐2 appear to be the molecular mechanism that leads to T‐tubule remodeling, Ca2+ handling dysfunction, and progression to heart failure in this mouse model.
doi:10.1161/JAHA.113.000527
PMCID: PMC4309042  PMID: 24958777
Calpain; G protein; heart failure; junctophilin‐2; T‐tubules
5.  Class IA PI3-kinase p110β subunit promotes autophagy through Rab5 small GTPase in response to growth factor limitation 
Molecular cell  2013;50(1):29-42.
Summary
Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110β catalytic subunit of the Class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes, and increases its interaction with the small GTPase Rab5. This p110β-Rab5 association maintains Rab5 in its GTP-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110β mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110β acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.
doi:10.1016/j.molcel.2013.01.022
PMCID: PMC3628298  PMID: 23434372
6.  Widespread Modulation of Cerebral Perfusion Induced during and after Transcranial Direct Current Stimulation Applied to the Left Dorsolateral Prefrontal Cortex 
The Journal of Neuroscience  2013;33(28):11425-11431.
Noninvasive neuromodulatory techniques such as transcranial direct current stimulation (tDCS) are attracting increasing interest as potential therapies for a wide range of neurological and psychiatric conditions. When targeted to the dorsolateral prefrontal cortex (DLPFC), anodal, facilitatory tDCS has been shown to improve symptoms in a range of domains including working memory, mood, and pain perception (Boggio et al., 2008a; Dockery et al., 2009; Kalu et al., 2012). However, the mechanisms underlying these promising behavioral effects are not well understood. Here, we investigated brain perfusion changes, as assessed using whole-brain arterial spin labeling (ASL), during tDCS applied to the left DLPFC in healthy humans. We demonstrated increased perfusion in regions closely anatomically connected to the DLPFC during anodal tDCS in conjunction with a decreased functional coupling between the left DLPFC and the thalami bilaterally. Despite highly similar effects on cortical excitability during and after stimulation (Nitsche and Paulus, 2000, 2001), cortical perfusion changes were markedly different during these two time periods, with widespread decreases in cortical perfusion being demonstrated after both anodal and cathodal tDCS compared to the period during stimulation. These findings may at least partially explain the different effects on behavior in these time periods described previously in the motor system (Stagg et al., 2011). In addition, the data presented here provide mechanistic explanations for the behavioral effects of anodal tDCS applied to the left DLPFC in terms of modulating functional connectivity between the DLPFC and thalami, as has been hypothesized previously (Lorenz et al., 2003).
doi:10.1523/JNEUROSCI.3887-12.2013
PMCID: PMC3724554  PMID: 23843514
7.  Rapid automated cell quantification on HIV microfluidic devices† 
Lab on a chip  2009;9(23):10.1039/b911882a.
Lab-chip device analysis often requires high throughput quantification of fluorescent cell images, obtained under different conditions of fluorescent intensity, illumination, focal depth, and optical magnification. Many laboratories still use manual counting - a tedious, expensive process prone to inter-observer variability. The manual counting process can be automated for fast and precise data gathering and reduced manual bias. We present a method to segment and count cells in microfluidic chips that are labeled with a single stain, or multiple stains, using image analysis techniques in Matlab and discuss its advantages over manual counting. Microfluidic based cell capturing devices for HIV monitoring were used to validate our method. Captured CD4+ CD3+ T lymphocytes were stained with DAPI, AF488-anti CD4, and AF647-anti CD3 for cell identification. Altogether 4788 (76 × 3 × 21) gray color images were obtained from devices using discarded 10 HIV infected patient whole blood samples (21 devices). We observed that the automatic method performs similarly to manual counting for a small number of cells. However, automated counting is more accurate and more than 100 times faster than manual counting for multiple-color stained cells, especially when large numbers of cells need to be quantified (>500 cells). The algorithm is fully automatic for subsequent microscope images that cover the full device area. It accounts for problems that generally occur in fluorescent lab-chip cell images such as: uneven background, overlapping cell images and cell detection with multiple stains. This method can be used in laboratories to save time and effort, and to increase cell counting accuracy of lab-chip devices for various applications, such as circulating tumor cell detection, cell detection in biosensors, and HIV monitoring devices, i.e. CD4 counts.
doi:10.1039/b911882a
PMCID: PMC3839566  PMID: 19904402
8.  AKT activation promotes PTEN hamartoma tumor syndrome–associated cataract development 
The Journal of Clinical Investigation  2013;123(12):5401-5409.
Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport. Specific loss of PTEN in the lens resulted in cataract. PTEN KO lenses exhibited a progressive age-related increase in intracellular hydrostatic pressure, along with, increased intracellular sodium concentrations, and reduced Na+/K+-ATPase activity. Collectively, these defects lead to lens swelling, opacities and ultimately organ rupture. Activation of AKT was highly elevated in PTEN KO lenses compared to WT mice. Additionally, pharmacological inhibition of AKT restored normal Na+/K+-ATPase activity in primary cultured lens cells and reduced lens pressure in intact lenses from PTEN KO animals. These findings identify a direct role for PTEN in the regulation of lens ion transport through an AKT-dependent modulation of Na+/K+-ATPase activity, and provide a new animal model to investigate cataract development in PHTS patients.
doi:10.1172/JCI70437
PMCID: PMC3859396  PMID: 24270425
9.  Paraneoplastic Autoimmunity Associated with Testicular Myeloid Sarcoma and Chronic Myelomonocytic Leukemia 
Case Reports in Hematology  2013;2013:656543.
Myeloid sarcomas are rare extramedullary solid tumors composed of immature myeloid cells. The clinical presentations of these malignant neoplasms are highly variable, ranging from asymptomatic to localized mass effect. Here, we report an unusual case of myeloid sarcoma of the testis found in association with chronic myelomonocytic leukemia where the presenting symptoms were autoimmune pericarditis and migratory arthralgias and myalgias that preceded testicular enlargement by nearly three months. Treatment with both radical orchiectomy and leukemia-directed chemotherapy led to immediate reductions in symptom severity, suggesting that these early symptoms were paraneoplastic in origin. Review of the literature identified the association between hematological malignancies, including chronic myelomonocytic leukemia, and paraneoplastic autoimmune phenomena with features similar to polymyalgia rheumatica and rheumatoid arthritis. Importantly, rheumatologic symptoms related to these disease entities may be easily dismissed as vague or unrelated complaints or treated as purely rheumatologic conditions, thus delaying the formal diagnoses. Clinicians must recognize the common association between possible paraneoplastic rheumatologic symptoms and hematologic malignancies such as chronic myelomonocytic leukemia.
doi:10.1155/2013/656543
PMCID: PMC3807721  PMID: 24198985
10.  Pathological impact of SMN2 mis-splicing in adult SMA mice 
EMBO Molecular Medicine  2013;5(10):1586-1601.
Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose–response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA.
doi:10.1002/emmm.201302567
PMCID: PMC3799581  PMID: 24014320
adult-onset SMA; pathology; SMN2; spinal muscular atrophy; splicing
11.  Restoration of defective L-type Ca2+ current in cardiac myocytes of type 2 diabetic db/db mice by Akt and PKC-Ι 
Diabetes is associated with increased risk of heart failure and development of a cardiomyopathy whose etiology is only partially understood. Ca2+ entry through the voltage-dependent L-type Ca2+ channel CaV1.2 initiates the contractile cycle in cardiac myocytes. Decreased cardiac contractility and depressed CaV1.2 function have been reported in obese type 2 diabetic db/db mice. Here we demonstrate that a reduction in phosphoinositide 3-kinase (PI3K) signaling is a major contributor to the altered function of CaV1.2 in db/db cardiac myocytes. Using the whole-cell patch clamp technique, we determined that intracellular infusion of cardiac myocytes from db/db mice with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K, increased the L-type Ca2+ current (ICa,L) density nearly to the level seen in wildtype cells. PIP3 also reversed the positive shift in the voltage dependence of steady-state current activation observed in db/db myocytes. Infusion of protein kinases that act downstream of PI3K also affected ICa,L. Akt1 and Akt2 were as effective as PIP3 in restoring ICa,L density in db/db myocytes, but did not affect the voltage dependence of current activation. Infusion of atypical PKC-Ι (the human homolog of mouse PKC-λ) caused a small but significant increase in ICa,L density and completely reversed the shift in voltage dependence of steady-state current activation. These results indicate that a defect in PI3K/PIP3/Akt/PKC-λ signaling is mainly responsible for the depressed CaV1.2 function in the heart of type 2 diabetic db/db mice.
doi:10.1097/FJC.0b013e318228e68c
PMCID: PMC3615892  PMID: 21753738
Diabetes; L-type Ca2+ current; cardiac myocytes; phosphoinositide 3-kinase (PI3K); Akt; PKC
12.  Mammalian PIK3C3/VPS34 
Autophagy  2012;8(4):707-708.
PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.
doi:10.4161/auto.19627
PMCID: PMC3679090  PMID: 22498475
autophagy; cancer; endocytosis; heart; liver; PIK3C3; PtdIns3K; PtdIns3P; Vps34
13.  Allogeneic Hematopoietic Cell Transplantation for Therapy-Related Myeloid Leukemia following Orthotopic Cardiac Transplantation 
Case Reports in Hematology  2013;2013:140138.
Therapy-related myeloid neoplasm (t-MN) is a subtype of acute myeloid leukemia with adverse cytogenetics and poor overall prognosis despite intensive induction chemotherapy and allogeneic hematopoietic cell transplantation (allo-HCT). It is increasingly recognized as a late complication of chronic immunosuppression in patients who have received solid organ transplantation. In this paper, we describe a case of t-MN following orthotopic cardiac transplantation and its treatment with allo-HCT. We discuss molecular and biological challenges and considerations in double solid organ and bone marrow transplantation and review similar cases at our institution. Our experience suggests general feasibility and safety of allo-HCT in patients who have received solid organ transplantation.
doi:10.1155/2013/140138
PMCID: PMC3625539  PMID: 23607004
14.  MicroRNA 
Circulation research  2012;111(7):816-818.
Cardiac excitation-contraction (E-C) coupling links action potentials to muscle contraction and is in essence a process of calcium ion mobilization.1 The central mechanism governing this process in ventricular myocytes is Ca2+- induced Ca2+ release, or CICR. It has been established for more than 20 years that CICR operates in a local control mode, taking place in a restricted junctional space of ≈ 12 to 15 nm between the transverse (T)-tubule and sarcoplasmic reticulum (SR) membranes, namely, the junctional membrane complexes or cardiac dyads.2,3 Within this dyadic “fuzzy space,”4 clusters of ryanodine receptor (RyR) Ca2+ release channels on the SR constitute the calcium release apparatus together with the directly apposed voltage-gated L-type Ca2+ channels (LTCCs) located primarily on the T-tubule membrane. 5 On membrane depolarization, a small amount of Ca2+ influx through the opening of LTCCs locally activates adjacent RyRs to release a much larger (≈10 times) amount of Ca2+ from the SR.6,7 The normal, functional cross-talk between LTCCs and RyRs depends on a stable local ultrastructure— the cardiac dyad.
doi:10.1161/CIRCRESAHA.112.278390
PMCID: PMC3505765  PMID: 22982867
calcium signaling; excitation-contraction coupling; gene transcription; T-tubules
15.  Suppression of Phosphoinositide 3-Kinase Signaling and Alteration of Multiple Ion Currents in Drug-Induced Long QT Syndrome 
Science translational medicine  2012;4(131):131ra50.
Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K+) current IKr. Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K+ currents IKr and IKs, the L-type calcium ion (Ca2+) current ICa,L, and the peak sodium ion (Na+) current INa and increased the persistent Na+ current INaP. Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in INaP. These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.
doi:10.1126/scitranslmed.3003623
PMCID: PMC3494282  PMID: 22539774
16.  VALIDATION OF A GENE EXPRESSION-BASED SUBCLASSIFICATION STRATEGY FOR PEDIATRIC SEPTIC SHOCK 
Critical care medicine  2011;39(11):2511-2517.
Objective
Septic shock heterogeneity has important implications for clinical trial implementation and patient management. We previously addressed this heterogeneity by identifying 3 putative subclasses of children with septic shock based exclusively on a 100-gene expression signature. Here we attempted to prospectively validate the existence of these gene expression-based subclasses in a validation cohort.
Design
Prospective observational study involving microarray-based bioinformatics.
Setting
Multiple pediatric intensive care units in the United States.
Patients
Separate derivation (n=98) and validation (n=82) cohorts of children with septic shock.
Interventions
None other than standard care.
Measurements and Main Results
Gene expression mosaics of the 100 class-defining genes were generated for 82 individual patients in the validation cohort. Using computer-based image analysis, patients were classified into 1 of 3 subclasses (“A”, “B”, or “C”) based on color and pattern similarity relative to reference mosaics generated from the original derivation cohort. After subclassification, the clinical database was mined for phenotyping. Subclass A patients had higher illness severity relative to subclasses B and C, as measured by maximal organ failure, fewer ICU-free days, and a higher PRISM score. Patients in subclass A were characterized by repression of genes corresponding to adaptive immunity and glucocorticoid receptor signaling. Separate subclass assignments were conducted by 21 individual clinicians, using visual inspection. The consensus classification of the clinicians had modest agreement with the computer algorithm.
Conclusions
We have validated the existence of subclasses of children with septic shock based on a biologically relevant, 100-gene expression signature. The subclasses have relevant clinical differences.
doi:10.1097/CCM.0b013e3182257675
PMCID: PMC3196776  PMID: 21705885
microarray; gene expression; stratification; staging; septic shock; pediatrics
17.  Interleukin-27 is a novel candidate diagnostic biomarker for bacterial infection in critically ill children 
Critical Care  2012;16(5):R213.
Introduction
Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children.
Methods
Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis.
Results
Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone.
Conclusions
Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel candidate diagnostic biomarker for predicting bacterial infection in critically ill children. Additional studies will be required to test further the diagnostic performance of IL-27.
The microarray data reported in this article have been deposited in the Gene Expression Omnibus under accession number GSE4607.
doi:10.1186/cc11847
PMCID: PMC3682317  PMID: 23107287
18.  Correlation analyses of clinical and molecular findings identify candidate biological pathways in systemic juvenile idiopathic arthritis 
BMC Medicine  2012;10:125.
Background
Clinicians have long appreciated the distinct phenotype of systemic juvenile idiopathic arthritis (SJIA) compared to polyarticular juvenile idiopathic arthritis (POLY). We hypothesized that gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with each disease would reveal distinct biological pathways when analyzed for significant associations with elevations in two markers of JIA activity, erythrocyte sedimentation rate (ESR) and number of affected joints (joint count, JC).
Methods
PBMC RNA from SJIA and POLY patients was profiled by kinetic PCR to analyze expression of 181 genes, selected for relevance to immune response pathways. Pearson correlation and Student's t-test analyses were performed to identify transcripts significantly associated with clinical parameters (ESR and JC) in SJIA or POLY samples. These transcripts were used to find related biological pathways.
Results
Combining Pearson and t-test analyses, we found 91 ESR-related and 92 JC-related genes in SJIA. For POLY, 20 ESR-related and 0 JC-related genes were found. Using Ingenuity Systems Pathways Analysis, we identified SJIA ESR-related and JC-related pathways. The two sets of pathways are strongly correlated. In contrast, there is a weaker correlation between SJIA and POLY ESR-related pathways. Notably, distinct biological processes were found to correlate with JC in samples from the earlier systemic plus arthritic phase (SAF) of SJIA compared to samples from the later arthritis-predominant phase (AF). Within the SJIA SAF group, IL-10 expression was related to JC, whereas lack of IL-4 appeared to characterize the chronic arthritis (AF) subgroup.
Conclusions
The strong correlation between pathways implicated in elevations of both ESR and JC in SJIA argues that the systemic and arthritic components of the disease are related mechanistically. Inflammatory pathways in SJIA are distinct from those in POLY course JIA, consistent with differences in clinically appreciated target organs. The limited number of ESR-related SJIA genes that also are associated with elevations of ESR in POLY implies that the SJIA associations are specific for SJIA, at least to some degree. The distinct pathways associated with arthritis in early and late SJIA raise the possibility that different immunobiology underlies arthritis over the course of SJIA.
doi:10.1186/1741-7015-10-125
PMCID: PMC3523070  PMID: 23092393
Arthritis; Inflammation; Juvenile idiopathic arthritis (JIA); Systemic JIA; Polyarticular JIA; Transcriptional analysis
19.  The pediatric sepsis biomarker risk model 
Critical Care  2012;16(5):R174.
Introduction
The intrinsic heterogeneity of clinical septic shock is a major challenge. For clinical trials, individual patient management, and quality improvement efforts, it is unclear which patients are least likely to survive and thus benefit from alternative treatment approaches. A robust risk stratification tool would greatly aid decision-making. The objective of our study was to derive and test a multi-biomarker-based risk model to predict outcome in pediatric septic shock.
Methods
Twelve candidate serum protein stratification biomarkers were identified from previous genome-wide expression profiling. To derive the risk stratification tool, biomarkers were measured in serum samples from 220 unselected children with septic shock, obtained during the first 24 hours of admission to the intensive care unit. Classification and Regression Tree (CART) analysis was used to generate a decision tree to predict 28-day all-cause mortality based on both biomarkers and clinical variables. The derived tree was subsequently tested in an independent cohort of 135 children with septic shock.
Results
The derived decision tree included five biomarkers. In the derivation cohort, sensitivity for mortality was 91% (95% CI 70 - 98), specificity was 86% (80 - 90), positive predictive value was 43% (29 - 58), and negative predictive value was 99% (95 - 100). When applied to the test cohort, sensitivity was 89% (64 - 98) and specificity was 64% (55 - 73). In an updated model including all 355 subjects in the combined derivation and test cohorts, sensitivity for mortality was 93% (79 - 98), specificity was 74% (69 - 79), positive predictive value was 32% (24 - 41), and negative predictive value was 99% (96 - 100). False positive subjects in the updated model had greater illness severity compared to the true negative subjects, as measured by persistence of organ failure, length of stay, and intensive care unit free days.
Conclusions
The pediatric sepsis biomarker risk model (PERSEVERE; PEdiatRic SEpsis biomarkEr Risk modEl) reliably identifies children at risk of death and greater illness severity from pediatric septic shock. PERSEVERE has the potential to substantially enhance clinical decision making, to adjust for risk in clinical trials, and to serve as a septic shock-specific quality metric.
doi:10.1186/cc11652
PMCID: PMC3682273  PMID: 23025259
20.  Ablation of PI3K p110-α Prevents High-Fat Diet–Induced Liver Steatosis 
Diabetes  2011;60(5):1483-1492.
OBJECTIVE
To determine whether the phosphoinositide 3-kinase (PI3K) catalytic subunits p110-α and p110-β play a role in liver steatosis induced by a high-fat diet (HFD).
RESEARCH DESIGN AND METHODS
Liver-specific p110-α and p110-β knockout mice and control animals for each group were fed an HFD or normal chow for 8 weeks. Biochemical assays and quantitative real-time PCR were used to measure triglyceride, expression of lipogenic and gluconeogenic genes, and activity of protein kinases downstream of PI3K in liver lysates. Fatty acid uptake and incorporation into triglycerides were assessed in isolated hepatocytes.
RESULTS
Hepatic triglyceride levels in HFD-fed p110-α−/− mice were 84 ± 3% lower than in p110-α+/+ mice, whereas the loss of p110-β did not significantly alter liver lipid accumulation. p110-α−/− livers also showed a reduction in atypical protein kinase C activity and decreased mRNA and protein expression of several lipogenic genes. Hepatocytes isolated from p110-α−/− mice exhibited decreased palmitate uptake and reduced fatty acid incorporation into triglycerides as compared with p110-α+/+ cells, and hepatic expression of liver fatty acid binding protein was lower in p110-α−/− mice fed the HFD as compared with controls. Ablation of neither p110-α nor p110-β ameliorated glucose intolerance induced by the HFD, and genes involved in gluconeogenesis were upregulated in the liver of both knockout animals.
CONCLUSIONS
PI3K p110-α, and not p110-β, promotes liver steatosis in mice fed an HFD. p110-α might exert this effect in part through activation of atypical protein kinase C, upregulation of lipogenesis, and increased uptake of fatty acids.
doi:10.2337/db10-0869
PMCID: PMC3292322  PMID: 21464441
22.  The beta identity of class I PtdIns3K 
Autophagy  2011;7(2):246-247.
Autophagy is critically controlled by phosphatidylinositol 3-kinases (PtdIns3Ks). The common understanding for mammalian autophagy is that class I PtdIns3Ks inhibit autophagy by activating the Akt-TOR kinase cascade, whereas the class III PtdIns3K (Vps34) promotes autophagy by generating the phospholipid PtdIns(3)P. However, direct genetic evidence for a role of class I PtdIns3Ks in autophagy has been lacking. Using mice with a conditional deletion of the class I PtdIns3K catalytic subunit isoform p110α or p110β, we revealed an unexpected function of p110β as a positive regulator of autophagy.
doi:10.4161/auto.7.2.14346
PMCID: PMC3359470  PMID: 21160279
autophagy; PtdIns3K; p110α; p110β; Vps34; PtdIns(3)P
23.  Identification of candidate serum biomarkers for severe septic shock-associated kidney injury via microarray 
Critical Care  2011;15(6):R273.
Introduction
Septic-shock-associated acute kidney injury (SSAKI) carries high morbidity in the pediatric population. Effective treatment strategies are lacking, in part due to poor detection and prediction. There is a need to identify novel candidate biomarkers of SSAKI. The objective of our study was to determine whether microarray data from children with septic shock could be used to derive a panel of candidate biomarkers for predicting SSAKI.
Methods
A retrospective cohort study compared microarray data representing the first 24 hours of admission for 179 children with septic shock with those of 53 age-matched normal controls. SSAKI was defined as a >200% increase of baseline serum creatinine, persistent to 7 days after admission.
Results
Patients with SSAKI (n = 31) and patients without SSAKI (n = 148) were clinically similar, but SSAKI carried a higher mortality (45% vs. 10%). Twenty-one unique gene probes were upregulated in SSAKI patients versus patients without SSAKI. Using leave-one-out cross-validation and class prediction modeling, these probes predicted SSAKI with a sensitivity of 98% (95% confidence interval (CI) = 81 to 100) and a specificity of 80% (95% CI = 72 to 86). Serum protein levels of two specific genes showed high sensitivity for predicting SSAKI: matrix metalloproteinase-8 (89%, 95% CI = 64 to 98) and elastase-2 (83%, 95% CI = 58 to 96). Both biomarkers carried a negative predictive value of 95%. When applied to a validation cohort, although both biomarkers carried low specificity (matrix metalloproteinase-8: 41%, 95% CI = 28 to 50; and elastase-2: 49%, 95% CI = 36 to 62), they carried high sensitivity (100%, 95% CI = 68 to 100 for both).
Conclusions
Gene probes upregulated in critically ill pediatric patients with septic shock may allow for the identification of novel candidate serum biomarkers for SSAKI prediction.
doi:10.1186/cc10554
PMCID: PMC3388679  PMID: 22098946
24.  The Influence of Developmental Age on the Early Transcriptomic Response of Children with Septic Shock 
Molecular Medicine  2011;17(11-12):1146-1156.
Septic shock is a frequent and costly problem among patients in the pediatric intensive care unit (PICU) and is associated with high mortality and devastating survivor morbidity. Genome-wide expression patterns can provide molecular granularity of the host response and offer insight into why large variations in outcomes exist. We derived whole-blood genome-wide expression patterns within 24 h of PICU admission from children with septic shock. We compared the transcriptome between septic shock developmental-age groups defined as neonates (≤28 d, n = 17), infants (1 month to 1 year, n = 62), toddlers (2–5 years, n = 54) and school-age (≥6 years, n = 47) and age-matched controls. Direct intergroup comparisons demonstrated profound changes in neonates, relative to older children. Neonates with septic shock demonstrated reduced expression of genes representing key pathways of innate and adaptive immunity. In contrast to the largely upregulated transcriptome in all other groups, neonates exhibited a predominantly downregulated transcriptome when compared with controls. Neonates and school-age subjects had the most uniquely regulated genes relative to controls. Age-specific studies of the host response are necessary to identify developmentally relevant translational opportunities that may lead to improved sepsis outcomes.
doi:10.2119/molmed.2011.00169
PMCID: PMC3321808  PMID: 21738952
25.  VIP and endothelin receptor antagonist: An effective combination against experimental pulmonary arterial hypertension 
Respiratory Research  2011;12(1):141.
Background
Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy?
Methods
Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival.
Results
Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days.
Conclusions
1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.
doi:10.1186/1465-9921-12-141
PMCID: PMC3210095  PMID: 22029879
Pulmonary Hypertension; Vasoactive Intestinal Peptide; Endothelin Receptor Antagonist; Pulmonary Vascular Remodeling; Monocrotaline

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