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1.  Crevicular Fluid Biomarkers and Periodontal Disease Progression 
Aim
Assess the ability of a panel of gingival crevicular fluid (GCF) biomarkers as predictors of periodontal disease progression (PDP).
Materials and Methods
100 individuals participated in a 12-month longitudinal investigation and categorized into 4 groups according to their periodontal status. GCF, clinical parameters, and saliva were collected bi-monthly. Sub-gingival plaque and serum were collected bi-annually. For 6 months, no periodontal treatment was provided. At 6-months, patients received periodontal therapy and continued participation from 6-12 months. GCF samples were analyzed by ELISA for MMP-8, MMP-9, OPG, CRP and IL-1β. Differences in median levels of GCF biomarkers were compared between stable and progressing participants using Wilcoxon Rank Sum test (p=0.05). Clustering algorithm was used to evaluate the ability of oral biomarkers to classify patients as either stable or progressing.
Results
Eighty-three individuals completed the 6-month monitoring phase. With the exception of GCF C-reactive protein, all biomarkers were significantly higher in the PDP group compared to stable patients. Clustering analysis showed highest sensitivity levels when biofilm pathogens and GCF biomarkers were combined with clinical measures, 74% (95% CI = 61,86).
Conclusions
Signature of GCF fluid-derived biomarkers combined with pathogens and clinical measures provides a sensitive measure for discrimination of PDP (ClinicalTrials.gov NCT00277745).
doi:10.1111/jcpe.12194
PMCID: PMC4247885  PMID: 24303954
Gingival crevicular fluid; biomarkers; periodontitis; longitudinal; disease progression
2.  Myostatin and IGF-I signaling in end-stage human heart failure: a qRT-PCR study 
Background
Myostatin (Mstn) is a key regulator of heart metabolism and cardiomyocyte growth interacting tightly with insulin-like growth factor I (IGF-I) under physiological conditions. The pathological role of Mstn has also been suggested since Mstn protein was shown to be upregulated in the myocardium of end-stage heart failure. However, no data are available about the regulation of gene expression of Mstn and IGF-I in different regions of healthy or pathologic human hearts, although they both might play a crucial role in the pathomechanism of heart failure.
Methods
In the present study, heart samples were collected from left ventricles, septum and right ventricles of control healthy individuals as well as from failing hearts of dilated (DCM) or ischemic cardiomyopathic (ICM) patients. A comprehensive qRT-PCR analysis of Mstn and IGF-I signaling was carried out by measuring expression of Mstn, its receptor Activin receptor IIB (ActRIIB), IGF-I, IGF-I receptor (IGF-IR), and the negative regulator of Mstn miR-208, respectively. Moreover, we combined the measured transcript levels and created complex parameters characterizing either Mstn- or IGF-I signaling in the different regions of healthy or failing hearts.
Results
We have found that in healthy control hearts, the ratio of Mstn/IGF-I signaling was significantly higher in the left ventricle/septum than in the right ventricle. Moreover, Mstn transcript levels were significantly upregulated in all heart regions of DCM but not ICM patients. However, the ratio of Mstn/IGF-I signaling remained increased in the left ventricle/septum compared to the right ventricle of DCM patients (similarly to the healthy hearts). In contrast, in ICM hearts significant transcript changes were detected mainly in IGF-I signaling. In paralell with these results miR-208 showed mild upregulation in the left ventricle of both DCM and ICM hearts.
Conclusions
This is the first demonstration of a spatial asymmetry in the expression pattern of Mstn/IGF-I in healthy hearts, which is likely to play a role in the different growth regulation of left vs. right ventricle. Moreover, we identified Mstn as a massively regulated gene in DCM but not in ICM as part of possible compensatory mechanisms in the failing heart.
doi:10.1186/s12967-014-0365-0
PMCID: PMC4301667  PMID: 25591711
Heart failure; Myostatin; IGF-I; Activin receptor IIB; IGF-I receptor; qRT-PCR; microRNA-208; miRNA
3.  The Superiority of the Time-to-Event Continual Reassessment Method to the Rolling Six Design in Pediatric Oncology Phase I Trials 
Background
The Rolling Six Design (RSD) is currently being used by the Children’s Oncology Group (COG) as their standard design for Phase I trials. Because the COG has large multi-center trials with fast accrual, the motivation for adopting the RSD is to hasten accrual and shorten the duration of their trials. However, trial suspension due to completion of follow-up still cannot be entirely avoided by the RSD. Therefore, a design that allows continuous enrollment of patients throughout the entire trial is needed.
Purpose
To demonstrate the superior performance of the Time-to-Event Continual Reassessment Method (TITE-CRM) with continuous patient recruitment relative to the RSD, in terms of identifying the maximum tolerated dose (MTD) and reducing exposure of patients to toxic doses.
Methods
Using scenarios that were based on an actual pediatric Phase I trial at the University of Michigan, Monte Carlo simulations were used to investigate the operational characteristics of RSD and TITE-CRM.
Results
The TITE-CRM treated all available patients, identified the MTD more accurately than the RSD and did not increase the probability of exposing patients to toxic doses.
Limitations
Both the TITE-CRM and RSD assume that the probability of dose limiting toxicity increases with higher dose level.
Conclusions
The TITE-CRM, which allows for continual enrollment of patients, provides a safe design for pediatric oncology Phase I trials with better accuracy than the RSD.
doi:10.1177/1740774511407533
PMCID: PMC4281887  PMID: 21610004
4.  Phase 1/2 trial of vorinostat plus tacrolimus and mycophenolate to prevent graft versus host disease following related donor reduced intensity conditioning allogeneic hematopoietic stem cell transplantation 
The lancet oncology  2013;15(1):87-95.
BACKGROUND
Acute graft-versus-host disease (GVHD) remains a significant barrier to a more widespread application of allogeneic hematopoietic stem cell transplantation (HSCT). Vorinostat (suberoylanilide hydroxamic acid) is a histone deacetylases (HDAC) inhibitor that has been shown to attenuate GVHD in pre-clinical models. We aimed to study the safety and activity of vorinostat in combination with standard immunoprophylaxis for GVHD prevention in patients undergoing related donor reduced intensity conditioning HSCT.
METHODS
In this prospective, single-arm phase 1/2 study of vorinostat, we recruited patients with high-risk hematologic malignances at two centers in the USA. We enrolled patients aged 18 years or older who were candidates for a reduced intensity conditioning HSCT and had an available 8/8- or 7/8-Human Leukocyte Antigen (HLA) matched related donor. Disease status had to be adequately controlled at the time of transplant. All patients received a conditioning regimen consisting of fludarabine 40 mg/m2 daily for four days (total dose 160 mg/m2) and busulfan 3·2 mg/kg daily for two days (total dose 6·4 mg/kg). GVHD prophylaxis consisted of mycophenolate mofetil 1 gram three times daily from day 0 and through day 28 and tacrolimus beginning on day −3 pre-HSCT and tapered beginning on day 56 and discontinued by day 180 post-HSCT in the absence of GVHD. The investigational agent, vorinostat, was initiated on day −10 through day 100 post-HSCT. The primary endpoint of the study was grade 2–4 acute GVHD by day 100. We expected to reduce the incidence to 25% from 42% based on similarly treated patients from the study centers and published literature. Patients were assessed for both toxicity and the primary endpoint if at least 21 days of vorinostat were administered. Patients who received less than 21 days of therapy were still assessed for toxicity and were replaced in accordance to the protocol. The trial is registered with ClinicalTrials.gov, NCT00810602.
FINDINGS
Between March 2008 and February 2013, we enrolled 50 patients evaluable for both toxicity and response. All patients engrafted neutrophils and platelets at expected times post-HSCT. The median percentages of chimerism in whole-blood at day 100 and 1-year were 98% (interquartile range [IQR], 98–100) and 100% (IQR, 100–100), respectively. The primary endpoint of the study was met with a day 100 cumulative incidence of grade 2–4 acute GVHD of 22% (95% cumulative incidence: 13%, 36%). Eight additional patients enrolled were assessed for toxicity only, in accordance with the protocol, because they received less than 21 days of study drug. The most common non-hematologic adverse events were all grade 3 and included electrolyte disturbances (N=15), hyperglycemia (N=10), infections (N=4), mucositis (N=4), and elevated liver enzymes (N=3). There was one grade 4 hypokalemia event and two grade 4 infections. Non-symptomatic thrombocytopenia which occurred after engraftment was the most common hematologic grade 3 or 4 adverse event (N=9), but was transient and all cases resolved swiftly.
INTERPRETATION
Administration of vorinostat in combination with standard GVHD prophylaxis after related donor reduced intensity conditioning HSCT is safe and appears to reduce severe GVHD. Future studies are needed to assess the effect of vorinostat in the prevention of GVHD in broader HSCT settings.
doi:10.1016/S1470-2045(13)70512-6
PMCID: PMC4103793  PMID: 24295572
GVHD; hematopoietic stem cell transplantation; HDAC inhibitor; vorinostat
5.  The miR-206/133b cluster is dispensable for development, survival and regeneration of skeletal muscle 
Skeletal Muscle  2014;4(1):23.
Background
Three different gene clusters code for the muscle-specific miRNAs miR-206, miR-1 and miR-133a/b. The two miR-1/133a clusters generate identical mature miR-1 and miR-133a miRNAs in heart and skeletal muscle, while the cognate miR-206/133b cluster is exclusively expressed in skeletal muscle. Since sequences of the miRNAs miR-133a and miR-133b are almost identical, it seems likely that they share potential targets. Similarly, miR-1 and miR-206 are structurally related and contain identical seed sequences important for miRNA-target recognition. In the past, different functions of these miRNAs were suggested for development, function and regeneration of skeletal muscle using different in vivo and in vitro models; however, mutants lacking the complete miR-206/133b cluster, which generates a single pri-miRNA constituting a functional unit, have not been analyzed.
Methods
We generated miR-206/133b knock-out mice and analyzed these mice morphologically; at the transcriptome and proteome level to elucidate the contribution of this miRNA cluster for skeletal muscle development, differentiation, regeneration in vivo; and by systematic analysis. In addition, we studied the consequences of a genetic loss of miR-206/133b for expression of Pax7 and satellite cell differentiation in vitro.
Results
Deletion of the miR-206/133b cluster did not reveal any obvious essential function of the miRNA-cluster for development and differentiation of skeletal muscle. Careful examination of skeletal muscles of miR-206/133b mutants revealed no structural alterations or molecular changes at the transcriptome and proteome level. In contrast to previous studies, deletion of the miR-206/133b cluster did not impair regeneration of skeletal muscle in mdx mice. Likewise, differentiation of miR-206/133b deficient satellite cells in vitro was unaffected and no change in Pax7 protein concentration was apparent.
Conclusions
We conclude that the miR-206/133b cluster is dispensable for development, function and regeneration of skeletal muscle, probably due to overlapping functions of the related miR-1/133a clusters, which are strongly expressed in skeletal muscle. We reason that the miR-206/133b cluster alone is not an essential regulator of skeletal muscle regeneration, although more subtle functions might exist that are not apparent under laboratory conditions.
doi:10.1186/s13395-014-0023-5
PMCID: PMC4272821  PMID: 25530839
miR-206; miR-133b; miR-1; MDX; Muscle regeneration; Pax7
6.  Molecular signatures that correlate with induction of lens regeneration in newts: lessons from proteomic analysis 
Human Genomics  2014;8(1):22.
Background
Amphibians have the remarkable ability to regenerate missing body parts. After complete removal of the eye lens, the dorsal but not the ventral iris will transdifferentiate to regenerate an exact replica of the lost lens. We used reverse-phase nano-liquid chromatography followed by mass spectrometry to detect protein concentrations in dorsal and ventral iris 0, 4, and 8 days post-lentectomy. We performed gene expression comparisons between regeneration and intact timepoints as well as between dorsal and ventral iris.
Results
Our analysis revealed gene expression patterns associated with the ability of the dorsal iris for transdifferentiation and lens regeneration. Proteins regulating gene expression and various metabolic processes were enriched in regeneration timepoints. Proteins involved in extracellular matrix, gene expression, and DNA-associated functions like DNA repair formed a regeneration-related protein network and were all up-regulated in the dorsal iris. In addition, we investigated protein concentrations in cultured dorsal (transdifferentiation-competent) and ventral (transdifferentiation-incompetent) iris pigmented epithelial (IPE) cells. Our comparative analysis revealed that the ability of dorsal IPE cells to keep memory of their tissue of origin and transdifferentiation is associated with the expression of proteins that specify the dorso-ventral axis of the eye as well as with proteins found highly expressed in regeneration timepoints, especially 8 days post-lentectomy.
Conclusions
The study deepens our understanding in the mechanism of regeneration by providing protein networks and pathways that participate in the process.
Electronic supplementary material
The online version of this article (doi:10.1186/s40246-014-0022-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s40246-014-0022-y
PMCID: PMC4271507  PMID: 25496664
Regeneration; Lens; Newt; Proteomics; Gene expression; Regeneration program
7.  MiRNA-1/133a Clusters Regulate Adrenergic Control of Cardiac Repolarization 
PLoS ONE  2014;9(11):e113449.
The electrical properties of the heart are primarily determined by the activity of ion channels and the activity of these molecules is permanently modulated and adjusted to the physiological needs by adrenergic signaling. miRNAs are known to control the expression of many proteins and to fulfill distinct functions in the mammalian heart, though the in vivo effects of miRNAs on the electrical activity of the heart are poorly characterized. The miRNAs miR-1 and miR-133a are the most abundant miRNAs of the heart and are expressed from two miR-1/133a genomic clusters. Genetic modulation of miR-1/133a cluster expression without concomitant severe disturbance of general cardiomyocyte physiology revealed that these miRNA clusters govern cardiac muscle repolarization. Reduction of miR-1/133a dosage induced a longQT phenotype in mice especially at low heart rates. Longer action potentials in cardiomyocytes are caused by modulation of the impact of β-adrenergic signaling on the activity of the depolarizing L-type calcium channel. Pharmacological intervention to attenuate β-adrenergic signaling or L-type calcium channel activity in vivo abrogated the longQT phenotype that is caused by modulation of miR-1/133a activity. Thus, we identify the miR-1/133a miRNA clusters to be important to prevent a longQT-phenotype in the mammalian heart.
doi:10.1371/journal.pone.0113449
PMCID: PMC4240597  PMID: 25415383
8.  Using Joint Utilities of the Times to Response and Toxicity to Adaptively Optimize Schedule-Dose Regimes 
Biometrics  2013;69(3):673-682.
A Bayesian two-stage phase I-II design is proposed for optimizing administration schedule and dose of an experimental agent based on the times to response and toxicity in the case where schedules are non-nested and qualitatively different. Sequentially adaptive decisions are based on the joint utility of the two event times. A utility function is constructed by partitioning the two-dimensional positive real quadrant of possible event time pairs into rectangles, eliciting a numerical utility for each rectangle, and fitting a smooth parametric function to the elicited values. We assume that each event time follows a gamma distribution with shape and scale parameters both modeled as functions of schedule and dose. A copula is assumed to obtain a bivariate distribution. To ensure an ethical trial, adaptive safety and efficacy acceptability conditions are imposed on the (schedule, dose) regimes. In stage 1 of the design, patients are randomized fairly among schedules and, within each schedule, a dose is chosen using a hybrid algorithm that either maximizes posterior mean utility or randomizes among acceptable doses. In stage 2, fair randomization among schedules is replaced by the hybrid algorithm. A modified version of this algorithm is used for nested schedules. Extensions of the model and utility function to accommodate death discontinuation of follow up are described. The method is illustrated by an autologous stem cell transplantation trial in multiple myeloma, including a simulation study.
doi:10.1111/biom.12065
PMCID: PMC3963428  PMID: 23957592
Adaptive decision making; Bayesian design; Phase I/II clinical trial; Stem cell transplantation; Utility
9.  Engraftment Syndrome following allogeneic hematopoietic cell transplantation predicts poor outcomes 
Engraftment syndrome (ES), characterized by fever, rash, pulmonary edema, weight gain, liver and renal dysfunction, and/or encephalopathy, occurs at the time of neutrophil recovery following hematopoietic cell transplantation (HCT). In this study, we evaluated the incidence, clinical features, risk factors, and outcomes of ES in children and adults undergoing first-time allogeneic HCT. Among 927 patients, 119 (13%) developed ES at a median of 10 days (interquartile range 9–12) post-HCT. ES patients experienced significantly higher cumulative incidence of grade 2–4 acute GVHD at day 100 (75% vs. 34%, p<0.001) and higher non-relapse mortality at 2 years (38% vs. 19%, p<0.001), compared with non-ES patients, resulting in lower overall survival at 2 years (38% vs. 54%, p<0.001). There was no significant difference in relapse at 2 years (26% vs. 31%, p=0.772). ST2, IL2Rα, and TNFR1 plasma biomarker levels were significantly elevated in ES patients. Our results illustrate the clinical significance and prognostic impact of ES on allogeneic HCT outcomes. Despite early recognition of the syndrome and prompt institution of corticosteroid therapy, outcomes in ES patients were uniformly poor. This study suggests the need for a prospective approach of collecting clinical features combined with correlative laboratory analyses to better characterize ES.
doi:10.1016/j.bbmt.2014.05.022
PMCID: PMC4142041  PMID: 24892262
Engraftment syndrome; hematopoietic cell transplantation; cytokine storm
10.  Etanercept plus topical corticosteroids as initial therapy for grade 1 acute graft-versus-host disease after allogeneic hematopoietic cell transplant 
Clinical diagnosis of grade 1 acute graft-versus-host disease (GVHD) marks the beginning of a potentially progressive and fatal course of GVHD after hematopoietic stem cell transplantation (HSCT). However, interventional studies to treat early GVHD are lacking. We conducted a single-arm prospective phase II trial to test the hypothesis that treatment of newly-diagnosed grade 1 acute GVHD with etanercept and topical corticosteroids would reduce progression to grade 2–4 within 28 days. Study patients (n=34) had a median age of 51 years (range, 10–67 years) and had undergone unrelated (n=22) or related (n=12) donor HSCT. Study patients were treated with etancercept (0.4 mg/kg, maximum 25 mg/dose) twice weekly for 4–8 weeks. Ten of 34 patients (29%) progressed to grade 2–4 acute GVHD within 28 days. The cumulative incidence of grade 2–4 and grade 3–4 acute GVHD at 1-year were 41% and 3%, respectively. Non-relapse mortality was 19% and overall survival was 63% at 2-years. Among a contemporaneous control cohort of patients that were diagnosed with grade 1 acute GVHD and treated with topical corticosteroids but not etanercept during the study period, 12 of 28 patients (43%) progressed to grade 2–4 GVHD within 28 days, with 1-year incidence of grade 2–4 GVHD and grade 3–4 GVHD of 61% (41% vs 61%, p=0.08) and 18% (3% vs 18%, p=0.05), respectively. Patients treated with etanercept also experienced less increase in GVHD plasma biomarkers ST2 (p=0.06) and Reg3α (p=0.01) 28 days after grade 1 acute GVHD diagnosis compared to contemporaneous control patients. This study was terminated early due to poor accrual. Future prospective studies are needed to identify patients with grade 1 acute GVHD at risk of swift progression to more severe GVHD and to establish consensus for the treatment of grade 1 acute GVHD. This trial is registered with ClinicalTrials.gov, number NCT00726375.
doi:10.1016/j.bbmt.2014.05.023
PMCID: PMC4145722  PMID: 24892263
11.  NHLH2: At the intersection of obesity and fertility 
Nescient helix loop helix 2 (NSCL2/NHLH2) is a neuronal transcription factor originally thought to be involved in neuronal development and childhood neuroblastomas. Accumulating evidence has since identified roles for NHLH2 in adult phenotypes of obesity and fertility. Here, we summarize these findings, and attempt to link genotype with phenotype in mouse models and humans. In particular, NHLH2 (Nhlh2 in mice) is one of only two genes that are genetically linked to physical activity levels. Nhlh2 also controls obesity and fertility, with strong sexual dimorphism displayed for both phenotypes by Nhlh2 mutant animals. We propose that Nhlh2 might function as a molecular sensor in different adult hypothalamic neurons to regulate energy balance, leading to normal body weight and reproduction.
doi:10.1016/j.tem.2013.04.003
PMCID: PMC3732504  PMID: 23684566
reproduction; exercise; body weight; sexual behavior; transcription
12.  NOA1, a Novel ClpXP Substrate, Takes an Unexpected Nuclear Detour Prior to Mitochondrial Import 
PLoS ONE  2014;9(7):e103141.
The mitochondrial matrix GTPase NOA1 is a nuclear encoded protein, essential for mitochondrial protein synthesis, oxidative phosphorylation and ATP production. Here, we demonstrate that newly translated NOA1 protein is imported into the nucleus, where it localizes to the nucleolus and interacts with UBF1 before nuclear export and import into mitochondria. Mutation of the nuclear localization signal (NLS) prevented both nuclear and mitochondrial import while deletion of the N-terminal mitochondrial targeting sequence (MTS) or the C-terminal RNA binding domain of NOA1 impaired mitochondrial import. Absence of the MTS resulted in accumulation of NOA1 in the nucleus and increased caspase-dependent apoptosis. We also found that export of NOA1 from the nucleus requires a leptomycin-B sensitive, Crm1-dependent nuclear export signal (NES). Finally, we show that NOA1 is a new substrate of the mitochondrial matrix protease complex ClpXP. Our results uncovered an unexpected, mandatory detour of NOA1 through the nucleolus before uptake into mitochondria. We propose that nucleo-mitochondrial translocation of proteins is more widespread than previously anticipated providing additional means to control protein bioavailability as well as cellular communication between both compartments.
doi:10.1371/journal.pone.0103141
PMCID: PMC4114565  PMID: 25072814
13.  Stem Cell Therapy for Craniofacial Bone Regeneration: A Randomized, Controlled Feasibility Trial 
Cell transplantation  2013;22(5):767-777.
Stem cell therapy offers potential in the regeneration of craniofacial bone defects; however, it has not been studied clinically. Tissue repair cells (TRCs) isolated from bone marrow represent a mixed stem and progenitor population enriched in CD90- and CD14-positive cells. In this phase I/II, randomized, controlled feasibility trial, we investigated TRC cell therapy to reconstruct localized craniofacial bone defects. Twenty-four patients requiring localized reconstruction of jawbone defects participated in this longitudinal trial. For regenerative therapy, patients were randomized to receive either guided bone regeneration (GBR) or TRC transplantation. At 6 or 12 weeks following treatment, clinical and radiographic assessments of bone repair were performed. Bone biopsies were harvested and underwent quantitative micro-computed tomographic (μCT) and bone histomorphometric analyses. Oral implants were installed, subsequently restored, and functionally loaded with tooth restorations. Reconstructed sites were assessed for 1 year following therapy. No study-related, serious adverse events were reported. Following therapy, clinical, radiographic, tomographic, and histological measures demonstrated that TRC therapy accelerated alveolar bone regeneration compared to GBR therapy. Additionally, TRC treatment significantly reduced the need for secondary bone grafting at the time of oral implant placement with a fivefold decrease in implant bony dehiscence exposure (residual bone defects) as compared to GBR-treated sites (p < 0.01). Transplantation of TRCs for treatment of alveolar bone defects appears safe and accelerates bone regeneration, enabling jawbone reconstruction with oral implants. The results from this trial support expanded studies of TRC therapy in the treatment of craniofacial deformities (ClinicalTrials.gov number CT00755911).
doi:10.3727/096368912X652968
PMCID: PMC4100608  PMID: 22776413
Bone regeneration; Craniofacial tissue engineering; Stem cells; Cell therapy; Implants
14.  Phase I/II trial design when response is unobserved in subjects with dose-limiting toxicity 
Statistical methods in medical research  2012;10.1177/0962280212464541.
We propose a Phase I/II trial design in which subjects with dose-limiting toxicity are not followed for response, leading to three possible outcomes for each subject: dose-limiting toxicity, absence of therapeutic response without dose-limiting toxicity, and presence of therapeutic response without dose-limiting toxicity. We define the latter outcome as a ‘success,’ and the goal of the trial is to identify the dose with the largest probability of success. This dose is commonly referred to as the most successful dose. We propose a design that accumulates information on subjects with regard to both dose-limiting toxicity and response conditional on no dose-limiting toxicity. Bayesian methods are used to update the estimates of dose-limiting toxicity and response probabilities when each subject is enrolled, and we use these methods to determine the dose level assigned to each subject. Due to the need to explore doses more fully, each subject is not necessarily assigned the current estimate of the most successful dose; our algorithm may instead assign a dose that is in a neighborhood of the current most successful dose. We examine the ability of our design to correctly identify the most successful dose in a variety of settings via simulation and compare the performance of our design to that of competing approaches.
doi:10.1177/0962280212464541
PMCID: PMC4096077  PMID: 23117408
Dose-finding studies; early-phase clinical trials; most successful dose; adaptive design
15.  Myf5-Positive Satellite Cells Contribute to Pax7-Dependent Long-Term Maintenance of Adult Muscle Stem Cells 
Cell stem cell  2013;13(5):590-601.
SUMMARY
Skeletal muscle contains Pax7-expressing muscle stem or satellite cells, enabling muscle regeneration throughout most of adult life. Here, we demonstrate that induced inactivation of Pax7 in Pax7-expressing cells of adult mice leads to loss of muscle stem cells and reduced heterochromatin condensation in rare surviving satellite cells. Inactivation of Pax7 in Myf5-expressing cells revealed that the majority of adult muscle stem cells originate from myogenic lineages, which express the myogenic regulators Myf5 or MyoD. Likewise, the majority of muscle stem cells are replenished from Myf5-expressing myogenic cells during adult life, and inactivation of Pax7 in Myf5-expressing cells after muscle damage leads to a complete arrest of muscle regeneration. Finally, we demonstrate that a relatively small number of muscle stem cells are sufficient for efficient repair of skeletal muscles. We conclude that Pax7 acts at different levels in a nonhierarchical regulatory network controlling muscle-satellite-cell-mediated muscle regeneration.
doi:10.1016/j.stem.2013.07.016
PMCID: PMC4082715  PMID: 23933088
16.  Reconstitution of the Myocardium in Regenerating Newt Hearts is Preceded by Transient Deposition of Extracellular Matrix Components 
Stem Cells and Development  2013;22(13):1921-1931.
Adult newts efficiently regenerate the heart after injury in a process that involves proliferation of cardiac muscle and nonmuscle cells and repatterning of the myocardium. To analyze the processes that underlie heart regeneration in newts, we characterized the structural changes in the myocardium that allow regeneration after mechanical injury. We found that cardiomyocytes in the damaged ventricle mainly die by necrosis and are removed during the first week after injury, paving the way for the extension of thin myocardial trabeculae, which initially contain only very few cardiomyocytes. During the following 200 days, these thin trabeculae fill up with new cardiomyocytes until the myocardium is fully reconstituted. Interestingly, reconstruction of the newly formed trabeculated network is accompanied by transient deposition of extracellular matrix (ECM) components such as collagen III. We conclude that the ECM is a critical guidance cue for outgrowing and branching trabeculae to reconstruct the trabeculated network, which represents a hallmark of uninjured cardiac tissue in newts.
doi:10.1089/scd.2012.0575
PMCID: PMC3685393  PMID: 23398466
17.  Adaptive Prior Variance Calibration in the Bayesian Continual Reassessment Method 
Statistics in medicine  2012;32(13):2221-2234.
Use of the Continual Reassessment Method (CRM) and other model-based approaches to design in Phase I clinical trials has increased due to the ability of the CRM to identify the maximum tolerated dose (MTD) better than the 3+3 method. However, the CRM can be sensitive to the variance selected for the prior distribution of the model parameter, especially when a small number of patients are enrolled. While methods have emerged to adaptively select skeletons and to calibrate the prior variance only at the beginning of a trial, there has not been any approach developed to adaptively calibrate the prior variance throughout a trial. We propose three systematic approaches to adaptively calibrate the prior variance during a trial and compare them via simulation to methods proposed to calibrate the variance at the beginning of a trial.
doi:10.1002/sim.5621
PMCID: PMC3561509  PMID: 22987660
adaptive design; Bayes factor; dose-finding study; dose-escalation study
18.  Sham Surgery and Inter-Individual Heterogeneity Are Major Determinants of Monocyte Subset Kinetics in a Mouse Model of Myocardial Infarction 
PLoS ONE  2014;9(6):e98456.
Aims
Mouse models of myocardial infarction (MI) are commonly used to explore the pathophysiological role of the monocytic response in myocardial injury and to develop translational strategies. However, no study thus far has examined the potential impact of inter-individual variability and sham surgical procedures on monocyte subset kinetics after experimental MI in mice. Our goal was to investigate determinants of systemic myeloid cell subset shifts in C57BL/6 mice following MI by developing a protocol for sequential extensive flow cytometry (FCM).
Methods and Results
Following cross-sectional multiplex FCM analysis we provide for the first time a detailed description of absolute quantities, relative subset composition, and biological variability of circulating classical, intermediate, and non-classical monocyte subsets in C57BL/6 mice. By using intra-individual longitudinal measurements after MI induction, a time course of classical and non-classical monocytosis was recorded. This approach disclosed a significant reduction of monocyte subset dispersion across all investigated time points following MI. We found that in the current invasive model of chronic MI the global pattern of systemic monocyte kinetics is mainly determined by a nonspecific inflammatory response to sham surgery and not by the extent of myocardial injury.
Conclusions
Application of sequential multiplexed FCM may help to reduce the impact of biological variability in C57BL/6 mice. Furthermore, the confounding influence of sham surgical procedures should always be considered when measuring monocyte subset kinetics in a murine model of MI.
doi:10.1371/journal.pone.0098456
PMCID: PMC4043649  PMID: 24893162
19.  openBEB: open biological experiment browser for correlative measurements 
BMC Bioinformatics  2014;15:84.
Background
New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis.
Results
We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository.
Conclusions
The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the “house keeping” abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated.
doi:10.1186/1471-2105-15-84
PMCID: PMC3987129  PMID: 24666611
20.  Hmga2 is required for canonical WNT signaling during lung development 
BMC Biology  2014;12:21.
Background
The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2 −/− ).
Results
We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2 −/− mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression.
Conclusions
Our results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury.
doi:10.1186/1741-7007-12-21
PMCID: PMC4064517  PMID: 24661562
Branching morphogenesis; HMGA2; GATA6; Lung development; WNT signaling
21.  ST2 as a Marker for Risk of Therapy-Resistant Graft-versus-Host Disease and Death 
The New England journal of medicine  2013;369(6):529-539.
Background
No plasma biomarkers are associated with the response of acute graft-versus-host disease (GVHD) to therapy after allogeneic hematopoietic stem-cell transplantation.
Methods
We compared 12 biomarkers in plasma obtained a median of 16 days after therapy initiation from 10 patients with a complete response by day 28 after therapy initiation and in plasma obtained from 10 patients with progressive GVHD during therapy. The lead biomarker, suppression of tumorigenicity 2 (ST2), was measured at the beginning of treatment for GVHD in plasma from 381 patients and during the first month after transplantation in three independent sets totaling 673 patients to determine the association of this biomarker with treatment-resistant GVHD and 6-month mortality after treatment or transplantation.
Results
Of the 12 markers, ST2 had the most significant association with resistance to GVHD therapy and subsequent death without relapse. As compared with patients with low ST2 values at therapy initiation, patients with high ST2 values were 2.3 times as likely to have treatment-resistant GVHD (95% confidence interval [CI], 1.5 to 3.6) and 3.7 times as likely to die within 6 months after therapy (95% CI, 2.3 to 5.9). Patients with low ST2 values had lower mortality without relapse than patients with high ST2 values, regardless of the GVHD grade (11% vs. 31% among patients with grade I or II GVHD and 14% vs. 67% among patients with grade III or IV GVHD, P<0.001 for both comparisons). Plasma ST2 values at day 14 after transplantation were associated with 6-month mortality without relapse, regardless of the intensity of the conditioning regimen.
Conclusions
ST2 levels measured at the initiation of therapy for GVHD and during the first month after transplantation improved risk stratification for treatment-resistant GVHD and death without relapse after transplantation. (Funded by the National Institutes of Health.)
doi:10.1056/NEJMoa1213299
PMCID: PMC3943357  PMID: 23924003
22.  A Generalized Continual Reassessment Method for Two-Agent Phase I Trials 
doi:10.1080/19466315.2013.767213
PMCID: PMC3891671  PMID: 24436776
23.  Assessing longitudinal quality of life in prostate cancer patients and their spouses: a multilevel modeling approach 
Purpose
This study aimed at examining the relationship between quality of life (QOL) in prostate cancer (PCa) patients and partners and how baseline demographics, cancer-related factors, and time-varying psychosocial and symptom covariates affect their QOL over time.
Methods
Guided by a modified Stress-Coping Model, this study used multilevel modeling to analyze longitudinal data from a randomized clinical trial that tested a family-based intervention to improve QOL in couples managing PCa. Patients and partners from the usual-care control group (N = 134 dyads) independently completed the measurements at baseline, and at 4-, 8-, and 12-month follow-ups.
Results
Correlations of QOL between patients and partners over time were small to moderate. Patients’ lower education level, partners’ older age, higher family income, and localized cancer at baseline were associated with better QOL in couples. Over time, couples’ QOL improved as their social support and cancer-related dyadic communication increased and as couples’ uncertainty, general symptoms, and patients’ prostate cancer-related sexual and hormonal symptoms decreased.
Conclusions
Evidence indicates that couples’ QOL during cancer survivorship is affected by multiple contextual factors (e.g., baseline demographics and time-varying psychosocial factors and symptoms). Intervention research is needed to explore comprehensive strategies to improve couples’ QOL during the continuum of PCa survivorship.
doi:10.1007/s11136-010-9753-y
PMCID: PMC3888242  PMID: 20927648
Prostate cancer; Symptom; Family; Quality of life; Multilevel model; Communication; Uncertainty
24.  Permutation Tests for Random Effects in Linear Mixed Models 
Biometrics  2011;68(2):10.1111/j.1541-0420.2011.01675.x.
Summary
Inference regarding the inclusion or exclusion of random effects in linear mixed models is challenging because the variance components are located on the boundary of their parameter space under the usual null hypothesis. As a result, the asymptotic null distribution of the Wald, score, and likelihood ratio tests will not have the typical χ2 distribution. Although it has been proved that the correct asymptotic distribution is a mixture of χ2 distributions, the appropriate mixture distribution is rather cumbersome and nonintuitive when the null and alternative hypotheses differ by more than one random effect. As alternatives, we present two permutation tests, one that is based on the best linear unbiased predictors and one that is based on the restricted likelihood ratio test statistic. Both methods involve weighted residuals, with the weights determined by the among- and within-subject variance components. The null permutation distributions of our statistics are computed by permuting the residuals both within and among subjects and are valid both asymptotically and in small samples. We examine the size and power of our tests via simulation under a variety of settings and apply our test to a published data set of chronic myelogenous leukemia patients.
doi:10.1111/j.1541-0420.2011.01675.x
PMCID: PMC3883440  PMID: 21950470
Hypothesis testing; Longitudinal data; Variance components
25.  A Phase I Bayesian Adaptive Design to Simultaneously Optimize Dose and Schedule Assignments Both Between and Within Patients 
Journal of the American Statistical Association  2013;108(503):10.1080/01621459.2013.806927.
In traditional schedule or dose-schedule finding designs, patients are assumed to receive their assigned dose-schedule combination throughout the trial even though the combination may be found to have an undesirable toxicity profile, which contradicts actual clinical practice. Since no systematic approach exists to optimize intra-patient dose-schedule assignment, we propose a Phase I clinical trial design that extends existing approaches to optimize dose and schedule solely between patients by incorporating adaptive variations to dose-schedule assignments within patients as the study proceeds. Our design is based on a Bayesian non-mixture cure rate model that incorporates multiple administrations each patient receives with the per-administration dose included as a covariate. Simulations demonstrate that our design identifies safe dose and schedule combinations as well as the traditional method that does not allow for intra-patient dose-schedule reassignments, but with a larger number of patients assigned to safe combinations. Supplementary materials for this article are available online.
doi:10.1080/01621459.2013.806927
PMCID: PMC3821780  PMID: 24222927
Bayesian statistics; Clinical trial; Dose-escalation study; Dynamic treatment regime; Non-mixture cure model

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